thesis submitted for the oeqree of jfi!la
TRANSCRIPT
studies on Plasmid Harbouring Strains of Escherichia coli Isolated form Gastroenteritis and Uro-genitaltract Infections of Man and Animals
THESIS SUBMITTED FOR THE OEQREE OF
jfi!la<B;ter of $l)iloiB!Qptip IN
Agriculture (Microbiology)
To The Aligarh Muslim University Aligarh
BY
IQBAb AHMAD
DIVISION OF MICROBIOLOGY CENTRAL DRUG RESEARCH INSTITUTE
LUCKNOW-226001
1991
2 7 JUN 199A
DS2326
C E R T I F I C A T E
This i s to ce r t i fy t h a t t h e work emboided t h i s t h e s i s en t i t l ed
"Studies on Plasmid Harbouring Strains of Escherichia col i Isolated
from Gastroenteritis and Urogenital tract infections of Man and
Animals" has been c a r r i e d out by MR. IQBAL AHMAD under our
s u p e r v i s i o n s .
He has fulf i l led t h e requi rements for Degree of Master of
Philosophy in Agriculture (Microbiology) of Aligarh Muslim Univer
s i t y , Aligarh, r egard ing nature and pe r iod of inves t iga t iona l work .
The work included in t h i s t h e s i s i s or ig ina l unless s ta ted o t h e r w i s e ,
and has not been submit ted for any o the r d e g r e e .
Dri J .N.S.Yadava, ' J P h . D . , FNA, FNAA,
Scientist Emeritus Division of Microbiology Central Drug Research Inst i tute , Lucknow-226001
Dr. Shamim Ahmad, Ph .D.
Sr. Lecturer in Microbiology Institute of Ophthalmology, JN Medical College, Aligarh Muslim Universi ty , Aligarh-202002
ACKNOWLEDGEMENTS
I welcome t h i s oppor tuni ty to p lace on r eco rd my deep sense of g ra t i t ude to my esteemed t eache r and Research guide Dr. J . N . S . Yadava, FNA, Emeri tus Sc ien t i s t , Division of Microbiology, Centra l Drug- Research In s t i t u t e , Lucknow and Dr. Shamim Ahmad, S r . Lec tu re r , Ins t i tu te of Ophthalmology, J . N . Medical College, Aligarh Muslim Un ive r s i t y , Aligarh for t h e i r omniscient guidance, f a t he r l y behav iou r , helpful c r i t i c i sm , unflagging i n t e r e s t and unflinching s u p p o r t . They in t roduced me with the fascinating realm of p lasmid biology and nur tured in me the ab i l i t y of independent w o r k . I sha l l remain eve r grateful to them for in i t i a l push at t h e infant s tage of my r e s e a r c h c a r e e r .
I offer my deep r e g a r d s to Prof. B.N. Dhawan, FNA, FAMS, Direc to r , Central Drug Research In s t i t u t e , Lucknow and Dr. G.P. Dutta, FNA, Deputy Di rec to r and Head, Division of Microbiology, C D . R . I . , . Lucknow for t h e i r keen in t e r e s t and necessa ry f ac i l i t i e s p r o v i d e d during pu r su i t of t h i s work .
I owe my s ince re thanks to Prof. S.K. Saxena, and Prof. Khalid Kfahmood and (Late) Prof. Israr Ahmad t he p re sen t and former co-ord ina -to r s of Agr icul tura l Cent re , Aligarh Muslim Un ive r s i t y , Aligarh and Prof. M. Saleemuddin, Head Division of B iochemis t ry , AMU, Al igarh , without t h e i r h e l p many h u r d l e s would have become insurmountable .
I a l so e x p r e s s my deep sense of g ra t i tude to a l l my teachers e spec ia l ly Prof. Nafees Ahmad, Dr. Masood Ahmad of Aligarh Muslim Unive r s i ty , Aligarh and Dr. N.B. Singh, Assis tant Di rec to r , C . D . R . I . , Lucknow for t h e i r va luab le suggestions from time to t i m e .
Delighted I feel to convey my s incere thanks to Dr. S.K. Puri, Dr. H.B. Rajani, Dr. K.K. Kamboj, Dr. C.X. George, and Dr. (Mrs) Kavita Gupta, C . D . R . I . , Lucknow for . t h e i r ae s the t i c suggestions and continued encouragements during the course of p r e sen t inves t iga t ion . I a lso acknowledge with thanks the co-opera t ion and h e l p , I rece ived from Dr. Mahipal Singh, National Ins t i tu te of Heal th , Be thesda , MD, U.S.A.
It i s unvanished t ru th t h a t t r e a t i s e would have never ma te r i a l i sed in the absence of my f r iends wi th whom I sha red my joys and sor rows . In naming them I am l i k e l y to miss many of them who contr ibuted in d i f ferent w a y s . N e v e r t h e l e s s , Ozair az iz , N. Rehmani, Alam Jaferi, Sandeep, Apama, Dr. Gh. Mashkoor, Dr. Anser Azim, Dr. M.A. Khan, Dr. Fayyaz and Junaid need to be mentioned.
I a lso thank to (Mrs) Kanchan Duggal, Ms. Savitree and Mr. Mahanand Shukla for immaculate t y p i n g . I feel immense p l ea su re to acknowledge with thanks the technica l a s s i s t a n c e , e spec i a l l y rendered by Shri Chandrika Prasad, Technical off icer , Mrs. Nuzhat Kamal and Mr. Ashok Yadav to accomplish t h i s work .
My acknowledgement would remain incomplete without making a spec ia l mention for unst inted s u p p o r t , I r ece ived from my e l d e r b r o t h e r s Mr. Abu Sufiyan, Mr. Abu-Faiz, Mr. Ehsan Ahmand and Mr. Imran Ahmad. The tremendous co-opera t ion , love and affection which I rece iveed from my mother , s i s t e r s , wife "SEEMA" and o the r family members i s beyond my a b i l i t y to give them w o r d s .
Words go deep into sorrow when I r e c a l l my fa the r who had left me when I was s tudying in graduat ion course but these a re h i s b le s s ings which a lways remain the guiding l igh t of my c a r e e r .
(IQ^AL AHMAD) October, 1991
C O N T E N T S
Page No.
INTRODUCTION
REVIEW OF LITERATURE
MATERIALS AND METHODS
RESULTS
DISCUSSION
BIBLIOGRAPHY
1 - 5
6 - 3 3
34 - 54
55 - 78
79 - 87
88 - -110
I N T R O D U C T I O N
In many countries infant i le mor t i l i t y rate can reach as high
as 220 death, per 1000 chi ldren born. At least 25% of these deaths
are due to diarrhoea! disease only. Thre are at least 25 dif ferent
bacter ia, viruses and parasites that causes acute diarrhoea, but Esch
er ichia col i has emerged as a single most important pathogen causing
upto one quarter of a l l diarrhoeas, special ly in developing countries
(WHO 1980, 1990). This f igure may be as high as 35% or at t imes,
even 50% of a l l the chi ldren born. The average mortal i ty for Asia,
Afr ica and Latin America was estimated about 2 b i l l i ons . The to l l
w i l l probably continue to be high i f the immediate steps are not
taken to prevnt th is scourage.
The impact of E.col i diarrhoea on agr icul tural ly important, domes
t ic and laboratory animals health is also of very high magnitude.
Reports appearing from a l l over the world indicates heavy economic
losses due to the morbidi ty and high mortal i ty in young calves, lambs,
fow ls , piglets e tc . , becuase of white scour and diarrhoea. In USSR,
there is an annual loss of 25.2% and 34.0% in new born calves and
piglets respect ive ly . Simi lar ly in the USA losses due to diarrhoea
are estimated to be A8.6 to 71.2 mi l l ion dol lars annually (House,
1978). However, the estimations of the economic losses in India as
well as in other developing or under developed countriefe is very
d i f f i cu l t because there is no proper reporting system. Moreover, consi
dering the prevai l ing conditions of sanitat ion, poor hygienic conditions,
malnutr i t ion, overcrowding and close association between man and
animals,infection syndrome is expected to be of much higher magnitute.
In India, a survey was made between 1966-70 at dif ferent Government
Buffalo farms in Haryana. The calf mortal i ty rate varied fr*
and the neonatal mortal i ty rate from 6.3-13.6%. The main dise
ponsible for losses were pneumonia (41.3%) and gastroenterit is (32.1%)
(Verma and Kalra, 1974).
Escherichia col i is associated with many disease conditions
in adults such as: gastro-enter i t is (d iarrhoea) , urinary tract infections
septiceamia, materi t is and mastit is in man and animals. In addit ion
to these diseases, E.coli is also producing col ibaci l los is (per i ton i t i s ,
meningitis, en te r i t i s , toxinfections, pye l i t i s , pyelonephr i t is , angiocholi-
t i s , salpingo-oophori t is, appendic i t is , o t i t i s , puerperalseptic etc.)
in adul ts. In a number of cases th is organism is also responsible
for food poisoning (Aksenova and Lisovskaya, 1987). E.coli is the
causative organism in more than 80% of urinary tract infections. E.coli
isolates from UTI, generally possess several factors which contribute
virulence. These virulence factors include adhering factor, serum res is
tance, cytotoxins, specif ic 0 and K antigens and haemolysins. Several
structures and products of E.coli have either a demonstrable or a
potential role in virulence in the gut and other t issues. These structure
includes f lagel la , capsule cel l wall components, and p i l i (fimbrae) ,
the products includes co l i ins , enterotoxins, cytotoxins and haemolysins.
Plasmids are extrachromosomal self repl icat ing circular double
hel ical DNA elements of bacteria that constitute a reasonably stable
but dispensable gene pool . I t has been evident that special characteris
t ics exhib i ted by bacterial strains are often plasmid mediated e.g.
drug resistance, metal resistance, production of aerobactin, haemolysin,
co l i c in , enterotoxin and adhering factors. The wide spread appearence
of drug resistant bacteria to common ant ibacter ial agents has posed
a great hazard to medical and veterinary pract ice. Surveys in a l l
parts of the world have shown that R-factors are now common and
wide spread special ly in gram-negative bacter ia. Resistance to sulfon
amide, tetracycl ine, chloramphenicol, amp ic i l l i n , streptomycin, kana-
mycin, neomycin, gentamicin and tr imethoprim have been found mostly
to be extrachromosomal in nature.
R-factors in Enterobacteriaceae special ly in E.col i are found
to be transferable from E.col i to E.col i and from E.coli to any
other genera of th is family and rendering them resistant to various
drugs and at the same time enabling these recipients also to transmit
thei r R-determinants to other sensit ive bacter ia. Spread of R-factors
in Enterobacteriaceae generally occurs by conjugation and phage
mediated transduction. Although chromosomal resistance gene can
in pr inc ip le be transformed from one cel l to another by sex factor
mediated conjugation, transduction or transformation, but there are
l i t t l e evidences for transfer by the lat ter two processes. In addit ion
to drug resistance, other special characters, mediated by plasmids
of E.coli includes the production of Col ic ins, Haemolysin and Entero-
toxins and some appandages l i ke Adhering factors (CFA-1, CFA-I I ,
CFA-IV) and antigenic moieties l i ke K-88, K-89, K-99 etc. play
an important role in the virulence and pathogenicity of E .co l i .
I t is found that genes encoding haemolysin production are
situated on chromosome and are also mediated by plasmids. Both
chromosomal and plasmid genes have a high degree of sequence homo
logy, /-haemolysin producing gene is found more frequently on trans
ferable H ly-p lasmid. Recent studies have shown that haemolysin
is an important virulence factor associated with pathogenicity and
virulence of E.coli and other bacteria (Blanco, ^ al_. 1990).
Colicin production is also encoded by plasmid called 'Co l -
factor ' and is considered as an indication of the i r virulence because
th is plasmid provides bacteria the means for self establishment
and better surv iva l in the intestinal tract of man and animals by
eradicating the col ic in sensit ive population of micnoflora present
in the intestinal t rac t . Col-factors may be transferable or non-trans
ferable l i ke R 6 Hly- factors , but non-transf enable, Col-factor may
interact with R-factor and become transferable. By now i t has become
evident that col ic in production l i ke many other factors is some
how related to increased invasiveness in producing organism.
The auto-transferable nature of plasmid of E.coli encoding
mult iple drug resistance and other virulence factors have created
an immense c l in ical problem in the treatment of infectious disieases.
Simultaneous transfer of Hly- factor , Ent-factor, Col-factor, Adhering
factor in any possible combination wi th R-factors is much more
dangerous and i t is of great interest to check the rapid and wide
spread of more virulent and mult ip le resistant bacter ia. Unfortuna
te l y , no specif ic treatment is avai lable for infections of R bacteria.
Curing of R-factors with some specif ic DNA inh ib i t ing agents such
as, acridine dyes,eth id ium-bromide, mitomycin-C etc. do not occur
at suff icient high frequency in a l l cases, another problem is that
these compounds can not be used for therapeutic purposes because
of thei r tox ic , carcinogenic and mutagenic propert ies.
The best solution to this problem is to find out to which
drugs the pathogens are s t i l l sensit ive and to use only those drugs
for treatment of such infections. In addit ion to t h i s , a search should
be made to f ind out some curing agents which can ef f ic ient ly eliminate
R-plasmids and can be used with safty in patients.
In the l igh t of above facts the present problem was undertaken
with fol lowing object ives.
Isolation of E.col i from various diseased conditions and thei r ident i
f icat ion on the basis of the i r biochemical characters.
To study thei r ant ib iot ic sensi t iv i ty behaviour with a view to f ind
out i ts resistance patterns and resistance level towards commonly
used ant ib io t ics .
Survey of auto-transferable, R-plasmids among ant ib iot ic resistant
s t ra ins, by using E.col i K12-J62 as recipient indicator s t ra in .
Curing of the R-plasmids present in E.col i s t ra ins, chemical ly,
by using DNA repl icat ion inh ib i tors and surface acting agents.
Screening for the detection of virulence factors l i k e col ic in and
haemolysin.
Finding out some correlation between virulence factors with drug
resistance.
Escherichia co l i belonging to the family of Enterobacteriaceae
in a Gram negative rod measuring about 1-3JLI X O . A - 0 . 7 M J arranged
singly or in pa i r s . I t is moti le by per i t r i chate f lage l la , though
some strains may be non-moti le. Many carbohydrate l i ke monosaccharide
(arabinose, xy lose, rhamnose); disaccharide (sucrose, lactose);
Tr isaccharides ( ra f f inose) ; alcohol ic sugars (adoni to l , . i nos i t o l ,
du l c i t o l , sorb i to l ) and glucosides (sal ic in) are fermented to a v a r i
able degree wi th d i f ferent s t ra ins , wi th the production of acid
and gas or only a c i d . E.col i is Indole and Methyl Red posi t ive
and Vogus Proskaur (VP) and Citrate negative. Gelatin is not l i q u i
f i e d , H S is not formed. Urea is not hydro lysed and growth does
not occur in KCN medium (Edward and Ewing^1972).
E.col i is responsible in causing various disease in man, animal
and pou l t r y . Recent development have made i t possible to determine
wether, certain isolates possess speci f ic propert ies responsible
for pathogenci i ty. Advances in molecular genetics now permit very
f ine d is t inct ion to be made among types of E.col i that are normal
intest inal f lo ra from E.col i that cause enteric and ext ra- in test ina l
diseases (Harnett and Gyles, 1983).
Several structures and products of E.col i have a demonstrated
or potential role in virulence in the gut and other tissues. Such
virulence factors can be summarised as fol lows : -
FLAGELLA:
In Salmonella typhimurium f lagel la appear to promote i n t ra
cel lu lar su rv i va l of the organism. However, non-motile s t ra in of
E.col i are also able to produce disease l i k e a f lagel lar st ra in of
E .co l i and there is l i t t l e evidnece for a role of the f lagel la in
viru lence. (Gyles, 1986).
CAPSULE;
Certain strains of E.col i that cause diarrhoea in calves and
in pigs was found to produce abundant capsular polysaccharide and
form mucoid colonies. There are evidences that capsule or polysacc
har ide formation is an virulence factor . Studies on the ultrastructne
of the capsulated E.col i in association w i th the intestine of calves,
suggest that the capsular material contr ibute to the formation of
adhering factor causing microcolonies attachment to the intest inal
epithel ium (Hadad and Gyles, 1982; Acres, 1985).
Among E.col i strains that causes septicaemia in humans, there
is a high prevalence of strains wi th the Kl-capsular polysaccharide
and i t was reported by Glyles (1986) that K1 strains were more
v i ru lent than K1 strains i n - v i vo animal studies.
PILI (FIMBRIAE);
P i l i / f imbr iae are slender fi laments of protein that project
from the surface of the bacteria and confer adhesive propert ies
on the organisms (Gaastra and De Grraf , 1982). In animal pathogenic
s t ra ins, the wel l characterized p i l i are K88 p i l i (smith and Muggins,
1978; De Grraf , 1982); K99 p i l i found on bovine, ovine and porcine
ETEC. (Gaastra and De Grraf , 1982); 987 P -p i l i present por ic in
6 Bovine ETEC) ; F41 P i l i also frequently occur in addi t ion to K99
p i l i on bovine and porcine ETEC s t ra ins . Various other colonization
factors , such as factors I 6 I I (CFA/1 and CFA/II) are found in
human ETEC strains (Gyles 1986; Saxena 6 Yadava . 1986).
'ENTEROTOXINS:
Smith and Gyles (1970) have reported two classes of E.col i
enterotoxins heat lab i le (LT) and heat stable (ST) enterotoxins which
are responsible for derangements of f l u id and e lect ro ly te movement
across the gut epithel ium result ing in development of d iarrhoea.
Gene encoding for the production of enterotoxins in E.col i is mainly
located on plasmid (Saxena 6 Yadava^^ 1985, 1986)'
Other bacter ia l products l i ke Haemolysin (Shamim & Yadava,
1980; Czi rok, 1985), Colicin (Carbonetti & Wi l l iam, 1984; Yadava
et a l , 1985) Cytotoxin and necrotising toxin (Alonso et_ a]_.,. 1987;
Blanco et_ al_, 1990) and Verotoxin production , or Shiga l i ke toxin
(Smith et_ £l_. 1983) and other newly characterized products of E.col i
also have a potential or demonstrable role in E.col i pathogenici ty.
ASSOCIATION OF E.COLI IN DIARRHOEAL DISEASEOF MAN AND ANIMALS:
The wel l characterized enteric pathogens l i ke Escherichia c o l i ,
Salmonellae, Shigellae, V ibr io cholerae and Campylobacter ju jun i ,
some viruses and protozoan accounts for more than half of the known
cases of infectious d iar rhoea. In recent years, an ever increasing
volume of evidences has impl icated Escherichia co l i as an important
agent of enteric diseases.
Diarrhoeagenic E.col i strains have been c lass i f ied into f i ve
categoriese by Levine (1987). His c lassi f icat ion in mainly based on
d is t inc t virulence proper t ies , d i f ferent interactions wi th intest inal
mucosa, d is t inc t c l in ica l syndromes, differences in epidemiology and
d is t in t 0:K:H serotypes. These categories are as fo l lows:
ENTEORTOXIGENIC E.COLI (ETEC):
ETEC produces enterotoxins and are important cause of diarrhoea
in infants, young ch i l d ren , adults and in neonatal calves as wel l
as in adult animals. ETEC strains tend to f a l l wi th in rest r ic ted 0:H
serotypes. Reis et_ al_. (1979) reported that there could be a cor re la
tion between serotypes and enterotoxin types, for example. 06:H16
usually produces LT/ST whi le 0128:H12 almost always produce ST,
l i ke wise 027:H7, 027:H20, 0128:H12, 0153:H10 are mainly the ST
producers (Orskov and Orskov 1978).
ENTERO INVASIVE E.COLI (E IEC) ;
EIEC cause sporadic food born infection and outbreaks of d i a r r
hoea in a l l ages world w ide. EIEC is s imi lar to Shigella both b io
chemically and sero log ica l ly . Like Shigella they invade and pro l i ferate
wi th in the ep i the l ia l cel ls and cause damage and death of the cel ls
(WHO^ 1990). This groups of E.col i causes dysentery l i k e Shigel la.
The invasive nature of ETEC group is dependent on the presence of
plasmid (Harr is et_ al_. 1932) .
ENTERPATHOGENIC E.COLI (EPEC):
In some urban areas upto 30% of acute diarrhoea cases in young
infants are a t t r ibuted to EPEC. The mechanism of EPEC diarrhoea
is not yet c lear. Enteroadherent and production of a cytotoxin may
be important in th is group. EPEC mainly belongs to certain rest r ic ted
serotypes such as 018, 020, 026, 044, 055, 086, 0111, 0112, 0114,
0125, 0126, 0127, 0128 and 0142 (Orskov 5 Orskov, 1978; Levine,
1987). The EPEC category is fu r ther sub-d iv ided into two classes.
Class-1 EPEC exh ib i t s local ized adherence to Hep-2 cel ls in tissue
cul ture. (Carvioto e^al_.^1979) where as Class-I I EPEC exh ib i t s e i ther
dif fuse adherence or non-adherence at a l l to Hep-2 cel ls (Moon et
_ | | . j 1 9 8 3 ; Nataro et aj^.^1985). The adhering propert ies of class I
10
and class I I EPEC st ra in are mediated by plasmids (Baldin i et^ a l .
1983; Levine e^ al^^. 1985; lev ine, 1987).
i v ) ENTEROADHERENT E.COLI (EAEC):
E .co l i of t h i s group neither produces LT, ST, or sh iga- l i ke
toxin nor invades ep i the l i a l ce i l s . The mechanism involved in the
pathogenesis and serogroups of th is category are yet to be establ ished.
Prel iminary works suggest that the E.col i are ident i f iab le by a p a r t i
cular pattern of adherence to Hep-2 cel ls which is c lear ly d i s t i n
guishable from both local ized as wel l as di f fuse adherence (Mathewson
et al_., 1986). EAEC cause watery diarrhoea in young ch i ld ren which
may become persistant (WHO, 1990).
v) ENTERO-HAEMORRHAGIC E.COLI (EHEC):
EHEC causes sporadic haemorrhagic co l i t i s in North America
and some other countr ies. Occassionally d i rec t person to person trans
mission may occur. EHEC produces a cytotoxin which may be respon
s ib le for Oedema and dif fuse bleeding in the colon result ing in bloody
diarrhoea as wel l as the haemolytic - uraemic syndrome, that some
times develop in ch i ld ren (WHO^ 1990). E.col i sero group 026, Oi l
and 0157 are wel l characterized serogroups of th is category. Among
these, 0157 emerged perhaps the single most important enteric pathogen
of publ ic health importance. Several reports on the epidemiology
of 0157:H7 are appeared in last few years. (Karmali et al_., 1985;
Spika et a l . ^ 1986; Levine, 1987).
In addi t ion to a l l above discussed, diarrhoegenic E .co l i , recently
a putative new category has been described as Entero adherent-aqqre-
g^^^^Q E-coli (EAggEC) ident i f iab le by the character is t ic aggregative
pattern of adherence of bacteria in Hep-2 cel l assay (Nataro e^ a l . ,
1987; Levine e^ 31^.^1988). Bhan e^ al_., (1989) referred th is newly
11
characterised group as entenoaggregative E.col i (EaggftC) and
they found that EAggEC are t yp i ca l l y negative wi th DNA probes
that ident i fy other categories of diarrhoegenic E .co l i , and they
do not f a l l into 0:H serotypes, t yp ica l of these other categories
of diarrhoegenic E.col i and they also reported that EAggEC were
found s igni f icant ly more often in patient wi th persistant diarrhoea
(29.5%) than acute diarrhoea (12.8%).
DIARRHOEA AND OTHER DISEASE CAUSED BY E.COLI IN ANIMAL;
Diarrhoea due to E.col i occurs most commonly in calves
less than 1 week of age but can also be a problem in claves
as old as 2-3 weeks (Acres et_ aJ_.,. 1977), causing heavy morbid i ty
and morta l i ty result ing in heavy economic losses. Bovine ETEC
belong to a l imi ted number of 0:K sero-groups and most strains
are K99"^. Isolates of 08:K25, 08:K85, 09:K30; 09:K35, 0101 :K28
or 0101:K30. Pearson and Longan (1979) have reported that of
ETEC is fed ora l ly to unsuckled calves then lower small intestine
is reported to be the f i r s t region to be colonized.
A dysentery, syndrome in calves have been associated with
non-enterotoxigenic E.col i that adhere to enterocytes and cause
damage to m i c r o - v i l l i as wel l as a mi ld acute inflamation of the
underlying lamina (Gyles , 1986). Pig may also suffer from E.col i
diarrhoea from b i r t h to 12 week of age. The f i r s t type of porcine
ETEC that were recognized, produced ST or ST/LT enterotoxins
and belong to 0 serogroups 8, 48, 138, 141, 147, 149 or 157
and la t ter colonization factor associated to porcine strains
12
were found k99, 987P and F41-Pi l i (Gyiesj 1986).
Chen et_ al_. (1984) reported morb id i ty and morta l i ty rate
among 3659 suckl ing piglets at Tiwan due to E.col i diarrhoea were
56.2% and 24.7% respect ively during the f i r s t week of l i f e and 19.9%
and 3.5% at 2-4 weeks giv ing total rate of 76.1% and 28.2%.
Djonne (1985)' isolated 315 E.col i strains from piglets which
had died from neonatal E.col i diarrhoea or traumatic lesions. Pobel
et a l . (1988) isolated 49 strains of E.col i from piglets suffering
from d iar rhoea, of which 21 possesed the K99 adhesion and they
are s imi lar to K99 strains isolated from calves. E.col i is respon
s ib le for diarrhoea in the young of the wide var ie ty of animal
species including cats, dogs, horses and rabb i ts e tc . However,
characterization of ETEC strains from these species has not almost
occured. Non-enterotoxigenic strains of E.col i have been implicated
as a cause of tympanit is and diarrhoea in rabbi ts (Cantey and Hoster-
man^1979; Moon et_ al_. 1983).
Ugochukawu and Anukam (1988) reported 73% • of E.col i among
35 faecal swabs of Afr ican dwarf goats, suffering from diarrhoea.
Camgulhene and Milon (1989) studied on 575 st ra in of E.col i isolated
from weaned rabb i ts experiencing diarrhoea in 119 French Commercial
Farms. They reported predominance of serogroup 0103 among their
s t ra ins .
Janke et_ aL (1990) reported a form of enteric E.col i infection
among 60 calves from 59 farming operat ions. The E.col i responsible
for these infections p r inc ipa l l y colonized them selves in the colon
13
including a d is t inc t i ve lession described as attaching and ef fect ing,
haemorrhagic entero-col i t is or blood in the faeces was observed
on 40% of the farms. Of affected calves, 86.6% were da i ry calves
(average age 11.8, days ) . F o r t y - f o u r calves were infected con
current ly wi th other enteropathogens.
Other important animal diseases caused by E.col i are septicae
mia, mast i t i s , ur inany- t ract infect ions, co l iobac i l los is , oedema
etc. Septicaemia in calves, p igs, dogs and poul t ry is very common
due to E .co l i . Most stra ins of septicaemic E.col i calves belong
to 0 group 15, 35, 78, 115, 117 or 137. Strains of serogroup 078:K80
are frequently impl icated in Septicaemic conditions (Gyles ,1986).
Mast i t is due to E.coli in Bovine may occur as per acute,
acute, chronic or subcl in ical in fect ion. (Eberhart et_ a l . , . 1979) .
Mastit is control programme that are ef fect ive in reducing masti t is
due to staphylococci and streptococci are generally ineffect ive
in E.col i masti t is as i t is generally not contro led.
Urinary t ract infections due to E.col i are more common and
sometimes severe in human whi le E.col i is the most frequent causes
of UTI in dogs and cats a lso. (Osborne and Klausmer, 1979; oxenford
et aj^.,^1984). Cys t i t i s is the most common form of ur inary tract
in fect ion, but u r e t h r i t i s , u t e r i t i s , p ros ta t i t i s and pyelonephr i t is
also occur in response to E.col i infection (Gyles,1986).
Farid et_ al_. (1976) in Egypt studied the morta l i ty rate among
elves over a per iod of 8 years on d i f ferent farms and showed
that enter i t is was responsible for 11% of Losses among total newborn
14
suckled buffalo calves (representing A1.7% of total death) . While
in Friesian calves the morta l i ty was 2.7% (33.7% of tota l death) .
I t was found that major i ty of calves died due to E.col i co l i bac i l l o -
s i s . Urinary t ract infection among buffaloes has also been reported
due to E.col i which resulted in albuminuria (EL. Rauf. £t_al^: ,1985)
PLASMID ENCODED VIRULENCE FACTORS OF ESCHERICHIA CQLI;
Plasmid DNA is a dynamic genetic domain shared by bacteria
of di f ferent species and even genera which is composed of a pool
of genetic information that act ive ly inter-change, assemble, dissociate
and in general, undergo molecular rearrangements. These rearrange
ments are mainly generated by DNA recombination and t ransposi t ion.
(Campbell, 1981).
Various plasmid mediated propert ies of Escherichia co l i
are reported as production of co l ic in (Ansari 6 Yadava, 1981);
Haemolysin (Smith, 1963), haemagglutinin (Minshew et_ ai_, 1978);
drug resistance (Yadava et_ a\_, 1982); Serum resistance (Goel £t_
a l , 1985) metal resistance (Shamim e; al^, 1988) and enterotoxins
production (Gyles et_ aj_, 1977) have been reported to be closely
associated wi th v irulence and pathogenicity of Escherichia c o l i .
a) Drug Resistance in Escherichia co l i
In recent years the indiscr iminate use of ant ib iot ics has
resulted in increased emergence of drug resistant bacter ia. Microbes
can develop drug resistance by a var ie ty of mechanism (Da vies
15
and Smith, 1978) such as :
( i ) Al terat ion of the target s i te in the ce l l that reduce or
el iminate the binding of the drug to the target s i te
( i i ) Blocking the transport of the ant ib io t ic into the ce l l regard
less of whether or not speci f ic or act ive mechanisms of
drug transport are invo lved , because a change in the t rans
port system can reduce the penetration of the drug into
the cel l
i i i ) Increasing the level of enzyme inact ivat ing the d rug , so
that drug is "Saturated" and t i t ra ted out
( i v ) Decreasing the cel ls metabolic requirement for the pathway
Or reaction inh ib i ted by the drug
(v) Provid ing the ce l l w i th replacement for the metabolic step
that is inh ib i ted by ant imicrobia l agent by pass mechanism
(v i ) Detoxif icat ion or inact ivat ion of the ant ib io t ic wi th increased
productioD o.tienzymes
[ v i i ) Production of a metabolite that can antagonise the inh ib i to ry
effect of the inh ib i to r
Considering these possible mechanisms of resistance we
can say that mechanisms ( i ) , ( i i ) , ( i i i ) and ( i v ) could easi ly
be attained by point mutations in chromosomal, s t ructura l or by
regular genes in the absence of any such events. However, for
many an t ib io t i cs , i t is unl ike ly that mechanisms ( i v ) , ( v ) , or
( v i ) could ar ise by simple mutation of chromosomal genes. In these
cases ent i re ly new ce l lu lar functions are required that could be
provided by the inheritance of new genes in the form of plasmids
(Davies and Smith, 1978).
16
Kimato and coworkers (1956) were the f i r s t to discover
transferable drug resistance in 1955 in Shiegel ia, isolated from
dysentry cases in Japan. I t carr ied drug resistance factors against
sulhonamide (Su) streptomycin (Sm) chloramphenicol (Cm) and te t racy
cl ine (Tc) . In. U.K. i t was f i r s t demostrated by Datta (1962). The
resistance against kamamycin (Km) and neomycin (Nm) in addi t ion
to the above four an t ib io t i cs .
In India the f i r s t survey in th is d i rect ion was conducted
by Yadava (1966) at Central Drug Research Ins t i tu te , Lucknow and
reported mul t ip le drug resistance among E_ co l i strains against
six ant ib iot ics namely penci l l in (Pn), streptomycin (Sm), tetracycl ine
(Tc) ch lorotet racyc l in (CTc) , oxytet racycl ine (OTc ) and chloramphe-
nical (Cm) simultaneously. These strains of E_ co l i were isolated
from buffal lo calf diarrhoea which were found h igh ly resistant
to as many as 6 ant ib iot ics (Pn, Sm, OTc , CTc and Cm). These
animals neither received any ant ib io t ic in the i r feed as addi t ives
or as growth simultants, nor were given any ant ib io t ic therapy
before the isolat ion of these s t ra ins . R-factors confering resistance
against mul t ip le drugs among E_j_ co l i strains were also reported
by various workers (Ochiai £t_ al_, 1959; Akiba et_ ^ , 1960; Smith
and Hal ls , 1966; Datta 1969 and Yadava, 1970).
R-factors have been postulated by Watanabe in (1963)
to consist of a l inear linkages of two genetic determinants ( i ) Drug
resistance determinants and ( i i ) Resistance transfer factor (RTF).
Anderson (1965) demonstrated hat RTF and R-determinants behave
as basical ly independent genetic units which have a f f in i t y to asso-
R-determinant
Non- conjugative R-plasmid
RTF RTF Component
Conjugative R-plasmid
Fig. la: Structure of conjugative and non-conjugative R-plasmids.
RTF Non-conjugative R-plasmid with
TC determinant RTF
Conjugative R-plasmid with Sm determinant
Compound conjugative R-plasmid conferring resistance to Sm and
Tc.
Fig. lb: : Proposed mechanism for the evolution of compound
R-plasmids.
17
present in the same c e l l . The role of piasmids in mediating the
resistance to various ant imicrobia l agents and the i r influence on
bacter ia l evolut ion has been reviewed by several workers (Rilay
and An i l ion is , 1978; Ansari and Yadava, 1984). Occurrence of t rans-
posons both on bacter ia l chromosome and on piasmids has greatly
influenced the spread of drug resistance marker gene (Hedges and
Jacob, 1974; Nuquent et_ al_, 1979; Datta, 1980; Th re l fa l l e; al_,
1983).
Precht et_ a\_. (1975) during a course of 10 years study on
the ant ib io t ic resistance of ur inary pathogens from the patients,
observed E_ co l i as main pathogen (55.6%) and indicated gentamicin
as the most ef fect ive an t ib io t i cs . Tr ishk ina et_ ^ L (1977) determined
sens i t iv i ty of 300 E^ co l i strains (mainly 0141, 035) against seven
ant ib iot ics isolated from claves. Resistance was determined in 75
to 90% of the s t ra ins , of which 64% had mul t ip le drug resistance.
Treatment wi th polymyxin alone or wi th furazolidone was found
most e f fec t ive .
Kar iuk i (1977) reported high incidence of mul t ip le ant ib io t ic
resistance in E_ co l i strains isolated from d iar rhoeic c laves. Most ly,
the strains were resistant to Su, Tc and Sm. None of his s t ra in
was found resistance to furazolidone or Neomycin. Out of 193 s t ra ins,
105 (54.5%) could transfer the i r resistance intrageneric to Salmonella
typhimur ium.
Diwan and Sharma (1978) studied 200 strains of E_ col i
from UTI and reported 175 (87.5%) strains to be resistant to one
or more an t ib io t i cs . They observed maximum resistance against
18
Tc (90.2%); Ap (81%) and Cpm (78.2%). Nearly 55% of the resistant
strains were able to transfer the i r resistance par t i a l l y or completely
to the recip ient E_ co l i K12 by conjugation.
Nakamura et al_. (1978) reported the drug resistance pattern
and R-plasmid d is t r i bu t ion to faecal E_ co l i isolates, 68.8% of
the bovine and 89.9% of the porcine isolates were found resistant
to one or more of the ant ib io t ics (Tc, Cm,: Sm; Km, Pm, Na)
Transferable R-plasmids were carr ied by 57.7% of the bovine and
52.2% of the porcine resistant isolates.
Sarkar et aL, (1979) reported mul t ip le drug resistance in
71% of the strains from 250 Enteropathogenic E_ co l i s t ra ins , isolated
from diarrhoeal diseases in ch i l d ren . The most common pattern
of resistance were Ap, Cm, Km, Nm, Sm, Ap, Cm, Sm and Ap,
Sm. None of the s t ra in was resistant to gentamicin and only nine
were resistant to furazol idone. Adetosye (1980) studied on drug
resistance and d is t r ibu t ion of R-factors among 333 E_ co l i isolates
from c l in i ca l l y healthy and sick animals. Fifteen mul t ip le and three
single ant ib io t ic resistance patterns were found. Of 285 resistant
s t ra ins, 133 (39.73%) t ransferred the i r resistance determinants to
sensit ivee E_ co l i K12 rec ip ien t .
Alsowaygh and S h i b l ( i 9 8 1 ) studied 1818 isolated of entero-
bacteria from faecal specimens and reported 50.2% isolates to be
resistant to one or more of the 10 ant ib io t ics they tested. About
0.8% of E_ co l i isolates were found to be resistant to 6 ant ib io t ics
(Am, Cm, Sm, Tc and Carbenic i l l in) Simultaneously, but none of
them was resistant to gentamicin, and they observed 40% incidence
19
of self transferable R-plasmid among 86 resistant strains tested.
Agarwal et_ aL (1981) isolated 76 strains of E^ co l i from infantsc
and ch i ld ren suffering from acute-diarrhoeal disease for the presence
of auto-transferable plasmids. They demonstrated that 65 (81.5%)
of the resistant strains possessed transferable drug resistance factor .
Ranganekar e^ aL. (1982) studied 181 E_ co l i s t ra ins, isolated from
healthy and hospi tal ised patients and found 79% strains resistant
to one or more drugs. They reported that 84.6% of the resistant
strains carr ied R-factors.
In 1983 Bridge and Manning observed 60% of transferable
R-plasmid among 100 mult ip le resistant E_ Col i . Minton et_ al_. in
the same year studied faecal col i forms of domestic dogs wi th acute
enteric infection and isolated wide range of mul t ip le drug resistant
organisms. They fur ther studied 113 E. col i strains for the i r plasmids
content and found 1-5 plasmids were present in each s t ra in . Four
of the 13 transferred the i r drug resistance to a recipient E^ col i
K12. One of the s t ra in , they observed harboured two conjugal p las
mids of 60 and 133 M dal which coded for resistance to Cm, Tc,
Ap and Cm, Tc and Km respect ively and one c r y p t i c , non-conjugable
plasmid of 29 M-Dal.
Agarwal e^ aJ_ (198A) studied 285 E_ col i strains of human
origin for the i r ant ib iot ic sens i t i v i ty behaviour. Mul t ip le drug
resistance was observed in 76.5% of the s t ra in . They observed
42.6% of thei r strains to have transferable plasmids.
Wise et_ al_. (1985) reported t r imethopr in (Tp) resistance
plasmid among 1572 strains of E_ co l i isolated from cases of d i a r r -
20
hoea in ca t t le , pigs and sheep. Resistant to t r imethopr im was detected
28% (263/956) of bovine isolates, 13% (59/441) of porcine isolates
and 9% (15/177) of ovine isolates. Nearly 45% of resistant isolates
transferred the i r Tp resistance to E_ co l i K12 and 17% strains were
shown to carry non-self transferatj le plasmids which were capable
of being mobil ized to E. co l i K12 by Rp p lasmid. Incompat ib i l i ty
group of 31 autotransferable plasmids were determined.
In a study of 284 strains of enterobacteria from Vel lore,
Young et_ aL (1986) demonstrated 64% of the strains resistant to t r ime
thopr im. They also observed 60% strains to be having transferable
plasmids in E_. co l i and ident i f ied 58 d i f ferent plasmid types. Helin and
Araj (1986) studied 1253 isolates of UTI from ch i ld ren and adults
for the i r sens i t i v i ty against 13 drugs. They observed a high res is
tance among E_ co l i strains against Ap, Su and Cotrimoxazole.
Taku et al^. (1990) studied out of 17 ETEC isolated from
diarrhoeic calves and reported the incidence of transferable nature
of R-plasmids. Two isolates transferred resistance marker for 3-
4 drugs each Ap, Cm, OTc, Sm and Tc, Km, Sm, whereas two isolates
transferred marker enblock for Km, Tc, Sm and four isolates transfe
r red only one resistance marker ie e i ther Ap, Km, or Sm to the
recipient K12. They also reported the co-transfer of K99 p i l i and
streptomycin resistance.
In the same year Lamikanra e\_ aU studied nature of t r imethoprim
resistance among E. co l i s t ra ins , isolated from cases of infant i le
d iar rhoea. Out o 190 isolates, 72 (37.9%) were found resistant
to t r imethopr im and of the 70 isolates tested, 38 (54.27%) transferred
21
high level t r imethopr im resistance (MIC 1000 mg/L) into E_
co l i EC-1005 rec ip ient .
Nandivada and Amyes (1990) studied on 284 c l in ica l isolates
of entero-bacteria for the i r sens i t iv i t ies to a series of B-lactam an t i
b io t i cs . They reported a very high incidence of B-lactam resistance
(Ampic i l l in resistance) 80% and cephalor idine resistance 65%). Seventy
seven percent of E_ co l i isolates carr ied resistance gene for both
ampic i l l in and cephalor idine which were trasnferable e i ther by di rect
conjugation or by mobi l izat ion wi th the X-p lasmid. They also reported
that transferable resistance to ampic i l l i n was not necessarily accompa
nied by resistance to cephalor idine and v ice_versa.
Molecular basis of resistance against B-lactam ant ib iot ics due
to plasmid mediated B-lactamases among members of the family entero-
bacteriaceae have been studied in recent years by many workers. Thomson
et aL (1990) observed resistance to B-lactam-enzyme inh ib i to r combina
tions among 73 strains of enterobacteriaceae. They reported that the
level and type of B-lactamase were important determinants of suscept ib i
l i t y to B-lactam inh ib i to r combination especial ly t r i carc i l l in -Ca lvu lanate .
Jacoby and Sutton (1991) reported some propert ies of plasmids respon
s ib le for the production of extended spectrom-B-lactamase. They exami
ned 15 plasmids. Unlike the average TEM plasmids a l l were large
ranging in size from 80 to 300 Kb. A l l determined resistance mult ip le
drugs ranging from 5 to 1 1 and some conferred resistance to heavy
metals and Uv radiat ion as w e l l .
Recently Mahipal et aJ_ (1991) reported spectrum of drug res is
tance (against 8 drugs) prevalent in 302 strains of E. col i isolated
22
from human and animal population in north Ind ia . They found 63.2%
of the strains were resistant to one or more drugs, of which 41%
strains were mul t i res is tant . Resistant strains showed a wide range
of resistance levels (MIC values 0.012 to 2.30 mg/L) i r respect ive
of the i r source of or ig in and maximum resistance was observed against
Ampic i l l i n (43.5%) fol lowed by tetracycl ine (36.4%) and least for t r ime-
thoprim-sulphonamide (9.3%). Major i ty of the resistance strains were
showing cross resistance among B-lactam ant ib io t ics (Amp ic i l l i n , Amoxy
c i l l i n and Cloxac i l l in ) they also observe lack of cross resistance
among few B-lactam resistant strains and concluded that inab i l i t y of
cross resistace might be due to the di f ferent substrate spec i f ic i ty
of di f ferent B-lactamase enzymes.
ELIMINATION OF EXTRACHROMOSOAL DNA (PLASMID5)
Considerable interest has centered on the experimental e l i m i
nation of plasmids from cel ls by chemicals (Curing agents). Hirota
and l i j i m a (1957) were able to produce F cel ls of Escherichia col i
+ from F by growing i t in the presence of a c r i f l a v i n , and i t was later
- 5
found that acr id ine organge (7.5x10 M) was 100% effect ive in e l i m i
nating F from cul tures. (Hi rota, 1960). Further various workers have
reported the curing capabi l i ty of acr id ine organge without affecting
the chromosomal repl icat ion system (Watanabe and Fukasawa, 1961-
b, Mitsuhashi et aj_, 1961; Jacob, Brener and Cuzin, 1963).
Rownd et_ ai_ (1966) grew several R-factors containing bacterial
strains in the presence of ac r i f l av in and isolated those bacteria which
had lost a l l the i r drug resistance determinants (p lasmids) . That was
the f i r s t c r i t i c a l study of chemical el imination of plasmid determinants
23
which showed that th is effect was the result of a physical el imination
of R-factor from bacteria by a c r i f l a v i n .
As a result of extensive studies, par t i cu la r ly on the mechanism
of plasmid DNA rep l i ca t ion , various approaches to eliminate the plasmid
DNA have been done such as:
i ) Direct inh ib i t ion of DNA synthesis by intercaulating dyes
i i ) Inh ib i t ion of DNA rep l ica t ion by a lky la t ing agents or inh ib i t ion
of synthesis of functional proteins
( i i i ) Dissolution of ce l l surface by surface acting agents
( i v ) Elevation of temperature e .g . (May e^ al_, 1974', Stalder and
Adelberg, 1972-, Singh and Yadav, 1980)
(v) Nutr i t ional starvat ion e .g . (Pinney 6 Smith, 1971-, Bremer et_
31^,1973; Singh and Yadav, 1980)
( v i ) Ultra v io let i r rad ia t ion (Round et_ al_, 1966; Wamburker and i-'anse,
1982).
( v i i ) Incompat ib i l i ty grouping (Toh-E and Wickner, 1981)
( v i i i ) Protoplast formation and regeneration (Novie e t ^ a l , 1980).
None of the above methods mentioned for el imination of plasmid
DNA, is able to cure a l l plasmids of a l l groups. However, DNA in ter
calating agents, surface acting agents and recently introduced compound
in A-quinolone group which have inh ib i to ry effect on DNA gyrase are
supposed to be the agents of choice to eliminate £ . col i plasmids.
Chemical substances which bind to double hel ica l DNA by in ter
calat ion, el iminate plasmid DNA through a molecular mechanism, by
which only episomal DNA (c losed, c i rcu lar ) is converted into unnatural
le f t handed supercoils and can not be repl icated in th is a r t i f i c i a l
24
c o n f o r m a t i o n l e a d i n g to loss of plasmid DNA. I t does have
l i t t l e or no effect on chromosomal DNA repl icat ion system.
Various interalat ing compounds el iminating plasmid DNA have
been reported as acr id ine orange (Bouanchaul and Chabbert, 1971;
Kaur et aj_, 1985; Singh and Yadav, 1980). Ac r i f l av in (Hashimoto e^
a l , 1964) and by ethiodium-bromide (Bon anchand et_ aj_, 1969) etc.
Inh ib i t ion of plasmid DNA repl icat ion has also been reported
by various compounds that e i ther i nh ib i t protein synthesis or inactivate
the functional proteins par t i cu la r l y at various stages of t ranscr ipt ion
and translat ion or block the process of repl icat ion which ult imately
lead to the loss of extrachromosomal DNA in subsequent progenies.
Such compounds are refampicin (Bazzicalupo and Tocchini-Valen-
t i n i , 1972; Obaseki-Ebor, 1984) which binds to the B-submit of l- NA
polymerase and inact ivate i t , thus stopping the t ranscr ip t ion , Mi to
mycin C ( Iyer and Szyba lsk i , 1964; Chakrabar ty , 1972) which insert
occasional a lky la t ing cross l inks between two strands of double hel ica l
DNA. This cross l inkage of DNA blocks t ranscr ip t ion by RNA and
prevents rep l i ca t ion . The coumarines are inh ib i to rs of enzyme DNA
gyrase which is responsible for the introduction of negative supercoils
into relaxed c i rcu lar closed DNA molecule (Gel lert e; aj_, 1976 a)
and subunit A and B having nicking-closing and supercoil ing ac t i v i t y
respect ive ly . Coumarines antagonizes sub unit B (Gel lert et ai,1969
b ) . Subunit A is inh ib i ted by a di f ferent class of chemotherapeutic
agents such as 4-quinolone (Sugino et a l , 1977).
25
In last few years inh ib i t i on of conjugal transfer of plasmids
and el imination of plasmids have been studied by various workers,
using di f ferent agents which acts e i ther on A or B subunits of DNA
gyrase, which resulted the inh ib i t ion of plasmid DNA repl icat ion and
ult imately el iminted in subsequent progenies. Wolfson et_ aL (1982) have
reported that plasmids pBR-322 and pMGIIO could be eliminated wi th
coumermycin A which antagonize B- sub units of DNA gyrase. Michel -
Briand et aL (1983) found that plasmid transfer could be inh ib i ted
in Escherichia co l i by oxinic acid which acts on A subunits of the
DNA gyrase.
Hooper et^ aL (1984) reported the el imination of plasmid PMG
110 from E_^ co l i by novobiocin and other i nh ib i to r of DNA gyrase.
In the same year Hi ra i et_ aL observed that norf loxacin could be used
to i nh ib i t the conjugal transfer of R-plasmid in Pseudomonas aeruginosa.
Wiesser and Wiedeman (1985) found that plasmids could be eliminated
from bacteria by new 4-quinolone which exh ib i t s curing capabi l i ty
s imi lar to the coumarines.
Detail study on the curing capabi l i ty of 4-quinolones have
been studied by Wiesser and Wiedeman (1986,1987). They reported
that the ab i l i t i e s of 4-quinolone anoxacin and of loxac in, inh ib i to rs
of DNA gyrase subunit A to eliminate plasmids from E. c o l i . They
reported that these compounds curved plasmid more ef f ic ient ly at
the highest concentration which s t i l l al lows cel l growth and produced
20% to 100% plasmid free ce l l s , depending upon the plasmid tested.
Higher concentration were required to eliminate plasmids from a
s t ra in , consistant w i th l igher MICs, but maximal curing frequency
26
s were s imi lar to those obtained wi th the na l id i x i c acid sensit ive (NA )
s t ra in . They reported that plasmid loss occcurred by inh ib i t ion of
plasmid rep l i ca t ion . Low or high curing frequency wi th a given curing
agent seems to be property of the plasmid tested. An anionic surface
acting agent, sodium dodecyl sulphate (SDS) has been reported to
cure R and F factors in E_ co l i wi th very high eff ic iency (Tomeda
e^ a]^, 1968; Salisbury e^ aJ_, 1972; Mahipal e^ aJ_' 1988).
I t has been shown that bacter ia l chromosome and plasmids
as repl icons are associated d i rec t l y wi th a segment of bacter ia l membrane,
SDS destroys the cel l wal l and then ce l l membrane completely or par t ia l l y
which ul t imately results in the l ys is of bacter ia. Plasmids are asso
ciated more closely wi th ce l l membrane as rep l icons, than chromosome
thus plasmid would more easi ly be cured by SDS, which may lead
to complete or pa r t ia l loss of plasmid DNA. Some sort of repair may
then fol low and cel ls wi th plasmids pa r t i a l l y or completeley eliminated
might surv ive in cu l ture .
Kaur et aL (1985) reported that s i l ve r ion resistance marker
could be eliminated by acr id ine orange (AO) ethidium bromide (Eth-
Br) and sodium dodecyl sulphate (SDS). They found that maxium curing
rate was 70% by SDS fol lowed by Ethidium Bromide 65% and 40% with
Acr id ine orange.
HAEMOLYSIN PRODUCTION
Haemolysis i . e . the a b i l i t y to cause l ys is of erythrocytes
is a rather wide-spread phenomenon among bacteria and other micro
organism. Extracel lu lar enz oroteases, l ipases and other hyd ro l y t i c
27
enzymes have been shown to be responsible for haemolysis in some
bacteria (Wiseman et_ al^, 1967; Fuj i ta and Koshimura, 1975).
In other bacteria "non-enzymatic" proteins which d is rupt the
membrane of erythrocytes have also been ident i f ied as causative agents
for haemolysis (Freer et_ al^, 1968; Fackre l l and Wiseman, 1975). A l l
these agents are termed as hameolysins. I t has been reported that E^
co l i strains causing more or less severe diarrhoea especial ly in new
born animals and in human infants, u r ina ry - t rac t infections or extra
intest inal infections have frequently the capabi l i ty of haemolysin p ro
duction (Smith, 1969; Ratinere e^ al_., 1976; EUwel l and Sh ip ley , 1980;
Shamin & Yadava, 1980; MuUer et a l . , 1983;Konig et_ al^., 1986).
Smith (1963) employed e ry th rocy t i c agar medium for the recogni
tion of haemolytic Ej_ co l i in faecal sample of d i f ferent man and animals.
He reported a number of haemolytic Ej_ co l i strains also from faecal
sample of catt le (67%) pigs (63%) sheep (53%) and man (198%) in di f ferent
out break of gas t ro-enter i t i s . He also d i f ferent iated c l ea r l y , the cel l
bound (B-haemolysin) and cel l f ree (c/Q- haemolysin) and preparation
containing high concentration of (pC-haemolysin was found toxic for mice,
rabb i ts and guinea p igs . Walton and Smith (1969) found t h i r d type of
haemolysin (Y ) , produced by mutent, resistant to na l id i x i c acid which
does not lyse RBCs of human and rabb i t but lyse RBCs of other species.
Haemolytic ac t i v i t y is a t t r ibuted for increased bacter ia l virulence
and pathogenicity (Shamim and Yadava, 1983; Yadava et a l . , 1986; Hughes
£t^al^,1983; Blanco et a l ,1990) . Chungh and his colleagues (1978) examined
the cultures of E_^ co l i isolates from UTI for haemolyt ic, enterotoxigenic
and necrotic propert ies along wi th the i r 0-sero groupings. The common
28
serogroups present in the urine sample were 02,06,07,08,012 and 075,
whi le strains isolated from faecal samples belonged to serogroups 06,07,
018, 075, 082 and 086. They found same serogroups in faeces and urine
of 6 out of AO pat ients. Cabello (1979) studied on E. co l i strains isolated
from blood and stool for haemolytic a c t i v i t y , co l ic in -v production
and haemugglutination and concluded that bacter ia l factors other than
K-1 antigen influence the pathogenic potential of K-1 s t ra ins .
Deboy et aU (1983) showed that most of the strains of serotype
06 isolates (85%) produced haemolysin, which showed some association
between serotypes and the production of haemolysin. Hughes et_ aL (1983)
reported that Ej_ co l i strains of high virulence for the ur inary t rac t ,
generally are haemolyt ic, serum resistant , toxic for mice and cause
mannose resistant haemagglutination.
Czirok (1985) reported that E_ co l i belonging to antigenic group
04,06 and 018 produced haemolysin and showed mannose resistant haemug-
glut inating capaci ty . Thus these factors are closely associated to
the i r v i ru lence. Marre e^ aU (1986) studied on the role of S-Fimbriae,
haemolysisn and serum-resistance in nephropathogenicity and reported
that these determinants contr ibuted to the mul t i fac tor ia l phenomenon
of E_ col i nephro-pathogenic i ty,
Ushijma et aL (1990) studied the role of E_ co l l haemolysin
in the pathogenic synergy of colonic bacteria in sub-cutaneous abscess
formation in mice. They reported that E_ co l i haemolysin is one factor
that may potentiate pathogenic synergy among colonic bacteria especial ly
between E. co l i and Bacteroides ovatus.
29
The transmissible nature of the haemolytic character in E.
col i was f i r s t described by Smith and Halls (1967) and they reported
that haemolytic phenotype assumed to be encoded by a plasmid designated
' H l y ' l a t te r . Extrachromosomal nature of Hly-encoding gene were also
studied by curing Hly factor from bacter ia l s t ra ins . Mi tche l l and Ken-
worthy (1977) found that Hly is el iminated at high frequency by RNA
inh ib i to rs (Act inomycin- D, r i fampic in and st reovar ic in) as compared
to intercalat ing DNA inh ib i to rs on plasmid rep l icat ion ( l i ke ac r i f l a v i n ,
eth id ium-bromide, daunorubicin and e thy l Violet) which are less ef fect ive.
Mishew et_ a l . (1978) proposed that -^C-hamolysin production by
some strains of E_ co l i are probably chromosomally determined. Richard
et a l . (1980) reported that genes required for haemolysin production
in the ur inary t ract in s t ra in Ej_ co l i J96 reside on the chromosome
rather on p lasmid. Noegel et_ aL (1981) reported that ^ -haemo ly t i c E.
col i strains MM 152 harbour 3 transmissible plasmids which could be
transferred to recip ient s t ra in E_ co l i K-12. With isolated plasmids
they could show that the genetic determinant required for haemolysin
was located ent i re ly on p lasmid. Their data suggests that there are
at least three clustered cistron which are required for haemolysin pro
duct ion.
Berger et_ al_. (1982) and Mullere et aJ_ (1983) reported that
the st ructura l gene (Hly-A) for haemolysin from di f ferent isolates of
haemolytic E_ co l i show some var iat ion in nucleotide sequence. This
suggest that differences in virulence of E^ co l i isolates or tox ic i ty
of the haemolysin molecule might be ref lect ion of amino acid composition
of ind iv idua l nucleotide among such plasmids. Welch and Falkow (1984)
have ident i f ied a region of haemolysin in the chromosome which accounts
30
for quant i tat ive differences in haemolysin production and v i ru lence.
COLICIN PRODUCTION
Colicin may be defined as ant ib io t ic l i ke substances or complexes
or ant ibacter ia l substances, produced by strains of intest inal bacter ia,
which are h igh ly speci f ic and act upon strains of the same or closely
related species (F red r i cq , 1957). They are protein in nature. The genes
for col ic in production are plasmid cal led col- factors which are s imi lar
to R-factors (Wi l l iams, 1978).
Colicin production in bacteria is considered as an indication
of the i r virulence and means of self establishment in the intest inal
t ract of man and animals (Yadava & Gupta, 1971). Some colicinogenic
strains transferred the i r colicinogeny to col strains without any linkage
of the col gene to chromosomal markers. Colicins are the most exten
sively studied of bacteriocins and the f i r s t to be described was the
"Pr inc ip le -V" . This property of bacteria have also been used as a
means of c lassi fy bacter ia l strains in epidemiological studies of pathoge
nic E_ co l i (Nomura, 1967, Yadava and Gupta, 1971, De Alwis 6 Thomlin-
son, 1973).
Arunachalam et a l , (1974) isolated 59 strains associated with
colisepticaemia and enter i t i s in poul t ry and found that 22% of the strains
were col icinogenic. Col ic in ' H ' was produced by a l l the strains and
a l l of these strains were sensit ive to one or more of co l ic ins . Schal
(1975) studied on 103 Ej_ co l i strains isolated from piglets and calves
died of co l ibac i l l os is , of these 45% were col icinogenic. On the other
hand 29% colicinogenic strains were detected among 352 strains isolated
from faecal samples of healty cc' '^s.
31
I t is possible that coiicinogeny may be a factor in the es tab l ish
ment of paihogenic bacteria in the intest inal t rac t . The pathogen may,
therefore, be more colicinogenic than non-pathogens. An assessment in
the respect may be d i f f i c u l t because there may be many other factors
l i ke tox in and haemolysin production and drug resistance associated
wi th gene of co l -p lasmid . Smith and Huggins (1976) found that the asso
ciation of co l ic in-V plasmid in E_ co l i wi th pathogenicity and surv iva l
in the alimentary t rac t , when inoculated intramuscular ly . Col V strains
were more pathogenic for chickens than the corresponding Col V forms.
Obi and Campbell (1978) screened 300 Ej_ co l i strains from healthy
sheep, goats and catt le for the i r coiicinogeny and the highest incidence
was recorded among sheep strains (46%) fol lowed by goats (38%) and
catt le (16%). They could ident i fy only 9 type of co l ic ins .
Njoku-Obi et_ aj^ (1978) studied 419 E_ co l i strains recovered
from human pathological materials in Niger ia , 51-55% strains were found
to be colicinogenic. Colicinogenic type B was the most frequent among
strains of faeces whi le type A predominated among the strains from
UTI, fol lowed by equal d is t r ibu t ion of B, E and I co l ic ins . Binns et a l . z a
(1979) found that el iminat ion of Col-V factor from human, bovine, ovine
and avian strains invar iab ly reduced the i r pathogenicity for experimental
animals. Goel et cil_. (1980) reported an increased surv iva l of E^ col i
K-12 harbouring col-plasmid in the intest inal t ract of mice.
Ansari and Yadava (1981) studied on three hundred and ten
strains of E_ co l i of human and animal o r i g i n , for the production of
co l ic ins . They reported 13.5% strains as col icinogenic. Higher incidence
of coiicinogeny was reported in animal strains as compared to strains
from human o r i g i n . Surpr is ingly 55% of the buffalo strains were recorded
32
as colicinogenic fol lowed by strains from cows (24.6%), mares (4.4%)
and poul t ry (3.6%). Aguero et_al.(1983) reported increased col-V plasmids
and manose resistant haemagglutination (MRHA) in an E_ co l i K1 population
and they discussed that presence of these propert ies may be a role
in the ab i l i t y of some E_ co l i K1 serogroup to invade.
Ansari and Yadava (1984) reported various types of col icins
production among 310 strains of E . c o l i . Out of 310 strains 144 (46.4%)
strains were inh ib i ted by one or more of the 12 standard co l ic ins.
Among colicinogenic s t ra ins , the co l ic in 1 and B+M producer strains
were most prevalent (46.4% and 36.4% respec t i ve l y ) . By using 12 standard
co l ic ins , 51 d i f ferent co l ic in sens i t i v i ty types were repor ted . The maxi
mum inh ib i t o ry ac t i v i t y was exh ib i ted by col ic in F (84%) fol lowed by
col ic in A (56.9%) and E+I (47.2%). Goel and Yadava (1985) studied on
serum resistance mediated by R-plasmids and i ts correlat ion wi th c o l i c i
nogenic and haemolytic fac tors . They reported that major i ty of serum
resistance strains were colicinogenic (84%), haemolytic (80%) and an t i
b iot ic resistant (83%). Transfer of R-plasmids along wi th col ic in and
haemolysin was recorded simultaneously in several s t ra ins .
Yadava et_ aiU (1986) reported that among 152 E_ col i strains
of human or ig in from UTI and GTI, only 14 (9.2%) strains were col ic ino
genic. About 85.7% of colicinogenic strains were found resistant to
one or more of the an t ib io t i cs , they tested. Nearly 64% of colicinogenic
strains were found to have transferable co l - fac tor , whi le only 33,3% of drug
resistant s t ra in could transfer the i r R-factor. They also reported in
two cases the determinants of col ic in production and ant ib io t ic resistance
were transferred simultaneousl- •=•'though both the propert ies were encoded
by separate plasmids.
33
Brennard et_ al^. (1989) reported s ix pathogenic E_ coLi strains
isolated in Braz i l and found resistant to human serum and ant ib iot ics
and produced col ic in and haemolysin. They demonstrated the plasmid
coded propert ies by conjugation and curing experiments. Mahipal et_
a l . (1989) studied on 122 strains of E_ co l i isolated from c l in ica l condi
tions of animals for the production of co l ic in and the i r sens i t iv i ty
behaviour against 16 drugs. 22.1% strains were found colicinogenic and
93% of these col strains were found resistant to one or more of the
drugs tested in various combinations. Transferable nature of R-plasmids
and col-plasmids were observed in (18.5%) strains i n d i v i d u a l l y . Co-
t ransferab i l i ty of R-plasmids and col- factors was reported in one s t ra in .
Blanco et_ aj^. (1990) studied various virulence factors among
37 septicaemic E_^ co l i and compared wi th AO E . co l i isolates from
the faeces of healthy i nd i v idua ls . They reported that co l ic in V production
was neither more frequent in septicaemic strains than in control strains
nor i t was associated wi th le tha l i t y of mice. While the role of Hly
and colonizing factors MRHA and MSHA has been discussed regarding
the increased virulence and pathogenicity of such E. co l i s t ra ins.
MATERIALS AND METHODS
34
Collection of samples:
( i ) Faecal swabs:
Faecal samples from diseased animals showing symptoms of
gastroenter i t is wi th or without septicaemia were collected from labo
ratory animals. The external area of the animals was cleaned wi th
a swab moistened wi th an ant isept ic solut ion. The s te r i le cotton
swab attached to a s t ick molstenea wi th s te r i l i zed peptone water
was introduced into the rectum, rotated ins ide, so as to touch the
mucosa and taken out and transferred into the s te r i le test tube and
brought immediately to the laboratory for isolat ion of the organism.
( i i ) Faeces from human:
Faecal samples of patient showing symptoms of gas t r i t i s or
gastroenteri t is both (males S female) of d i f ferent age groups admitted
in Balrampur Hospital and sporadic d iarrhoeal cases of staff members
of CDRI, Lucknow were collected for microbiological examination.
( i i i ) Urine samples;
Urine samples were col lected in s ter i lzed glass containers from
human patient suffering from urinogenital infecions. For the microbiolo
gical examination of ur ine, the sample was d i lu ted and 0.1 ml was
spreaded over the surface of MacConkey' agar p la te . The colonies
were counted to know the number of IE_ co l i ce l l (C.F.U. ) per ml
and smooth colony was picked up for fur ther studies.
35
ISOLATION AND CHARACTERIZATION OF THE ORGANISM;
Soon after the co l lec t ion, samples were brought to the laboratory,
the faecal swabs from animals were suspended in serum broth and
a loopful of the suspended/di luted material was streaked on MacConkey
agar p lates, whi le the stool samples from human were ser ia l l y d i luted
in s te r i l i zed normal saline solution and 0.1 ml of appropr iate d i lu t ion
were spread on MacConkey agar p lates. A l l plates were incubated
aerobical ly at 37°C for 24 hrs to 48 h r s .
Two to four opague, smooth, lactose fermenting colonies wi th
entire regular edges suspected to hp Escherichia co l i were picked
up form the p la te . Each was given an isolate number. A loopful
of the uniform suspension of an isolated in nutrient broth was streaked
on MacConkey agar medium to obtain pure cu l ture. The isolates were
cultured on nutrient agar slant and stored at 4°C for fur ther s tudy.
One set of culuture was f reez-dr ied and s to red . The culture characte
r i s t i c , morphology and biochemical reactions were studied as described
by Edward 8 Ewing (1972), Breed et al_. (1973) and Cruickshank
et a l ' (1975). Lactose fermenting. Gram negative, f a i r l y rods with
rounded ends or cocco-bac i l la ry , non-sporulat ing, looking l i ke Escher i
chia co l i on microscopic examination were taken for the i r fur ther
biochemical studies.
A total of 48 isolates were obtained from above said sources
as represented in Table N o . 1 .
36
Table-1 : Source of iso lat ion of 48 Escher ichia co l i s t ra ins
1 1 1
Source i C l in ica l ' symptoms i 1 1
Laboratory Animal
UTI (Ur inary /Uroge
n i ta l t ract in fect ion)
GTI (Gastroenter i t is)
GTI (Gastroenter i t is)
Total
Strain numbers
01 ,03,04,05,06.07, 09,10,12,13,14,15, 16,17,19,20,21,22,23, 25,26
02,08,11 ,18,24, 27 29,30,31,32,33,34, 35,36,37,38 '.
101,102,103,104,105, 106,107,108,109,110
Total number of strains
21
17
10
48
37
BIOCHEMICAL REACTIONS;
I n d o l e p r o d u c t i o n , methy r e d (MR) r e a c t i o n s , Voges
Proskauer reaction (VP), u t i l i za t ion of c i t ra te as sole source of
carbon, catalase product ion, growth in KCN medium and H^S production
e t c . ' w e r e studied according to the methods described by Edwards
& Ewing (1972) and Cruickshank et al» (1973).
Methyl red and Voges proskauer reaction (MR 6 VP):
Glucose phosphate peptone water (GPP) medium was inocula
ted wi th test s t ra in and wa5 incubated at 37°C for 48 h r s . To
half of the inoculated mediuhn added few drops of methyl red
reagent (Methy red 0.1 g , e thy l alcohol 300 m l . , d i s t i l l e d water
200 ml) mixed and read the reaction immediately. Posi t ive reaction
was indicated by red colour due to acid production from glucose
metabolism and negative by yellow colour. To another half of the
culture medium, added 0.5 ml of 5% of alcohol ic solution of alpha
naphthol and 0.5 ml of 40% KOH solution containing 0.3% creat inine.
On shaking vigorusly at in tervals a pink or red colour develops,
showing the presence of ace ty l -methy l -carb ino l , confirming a posi t ive
VP react ion.
Indole product ion;
KOVAC's reagent was added to 48 h r s . cultures growth
in peptone water and vigorusly shaken. A layer of red colour
or r ing is formed, indicat ing a posi t ive reaction due to indole
38
formation as a resul t of peptone metabolism (Kovac 's Reagent: Isoamyl ,
a lcoho l , 150 m l , ; p -d ime thy l amino benzaldehyde, 10 g - concentra-
ted-HCl , 50 m l . ) .
Citrate utilization:
Simmon'.s c i t r a te slants were Inoculated heav i l y and incubated at
37°C for 48 h r . The change in colour of slants from green to blue
was a pos i t i ve ind ica t ion of u t i l i s a t i o n of sodium c i t ra te as sole source
of carbon. For media cont ro l K lebs ie l la s t ra in was always included
as pos i t i ve con t ro l . Composit ion of Simmon's c i t ra te agar is given
on page No. A3.
Mitrate reduction test:
For n i t ra te reduct ion test the cu l tu re was inoculated in
the n i t ra te medium (po t . n i t r a t e , 0.2 g . , peptone, 5 g and d i s t i
l led water , 1 l i t . ) and incubated for 96 h r s . For the test , immedia
tely before use, mixed equal volumes of test so lu t ion . 'A ' (con ta in ing
su lpha l in ic a c i d , 8 g ; 5 N, acetic acid 1, l i t ) and test s o l . ' B '
(containing alpha naphthy lamine, 5.0 g ; 5-N acetic a c i d , 1 l i t . ]
and added 0.1 ml of mixed test reagent to test cu l tu re . Development
of red colour w i t h i n few minutes, ind icated the reduct ion of n i t ra te
to n i t r i t e .
Phenylalanine deaminase tes t :
A heavy inoculum of the test cu l ture was inoculated on
39
on to the medium (containing yeast ex t rac t , 3 g; D-L-Phenylala:rune,
1g; disodium hydrogen phosphate 1 g ; sodium ch lor ide 5 g ; agar 2
g; and d i s t i l l e d water 1 l i t r e and incubated for 4 hr or i f des i red,
upto 24 hrs at 37°C.
Pour a few drop of 10% solution of f e r r i c ch lor ide to run down
the growth on the slope. A green colour developed in f lu id and on
the slope showed the deamination of phenylalanine amino ac id , ind ica
ting the presence of phenylalanine deaminase enzyme.
Catalase test :
One ml of the hydrogen peroxide solution (10%) was poured
over a 24 hr nutrient agar slope culture of the test organism and
the tube was held in a slanting pos i t ion. The production of the gas
bubbles from the surface of the sol id culture material indicated a
posi t ive react ion.
Urease test :
Christensen's medium was inoculated wi th the test organism
heavi ly and incubated at 37°C upto 4 days. They were observed d a i l y .
Urease posi t ive culture hydrolyses urea and changes the colour of
the medium to purple or pink indicat ing alkal ine pH due to l iberat ion
of ammonia from urea. Christensen's medium consists of Peptone, 1
g; Sod. ch lor ide 5 g ; dipotassium hydrogenphosphate, 2 g ; Phenol
red (1 in 500 aqueous s o l . ) , 6 m l ; agar, 20 g ; d i s t i l l e d water 1
l i t ; 10 ml of 10 percent s ter i le solution of glucose; 100 ml of s ter i le
solution of 20% urea.
40
Glucose and urea solutions were s te r i l i sed by f i l t r a t i o n . The
basal medium was prepared without glucose and urea. pH was adjusted
to 6.8-5.9 and s te r i l i sed by autoclaving at 121°C for 30 mts. It is
cooled to about 50°C, added glucose and urea and tubes are prepared
as deep slopes.
HQ S production
Hydrogen sulphide production was demonstrated by inoculating
the TSI agar slopes and incubated at 37°C for 24-96 h r s . The blacken
ing of the medium indicated the production of H2.S by the organism.
TSI agar medium consists of beef extract 3 g; yeast ex t rac t , 3 g;
Peptone (Bacto), 15 g , Proteose peptone, g ; lactose 10 g; sucrose,
10 g ; glucose, 1 g ; ferrous sulphate 0.2 g; Sod. ch lo r ide , 5g; Sod.
th iosulphate, 0.3 g ; agar 12 g ; Phenol r e d , 0.024 g ; d i s t i l l ed water,
1000 ml and pH was adjusted to 7.6^ dispense the medium in tubes
and autoclave at 121 °C, slants were made wi th deep but ts .
Gelatin l iquefact ion;
The Gelatin medium was inoculated as stab culture by taking
culture from the agar slants and incubated at 37°C. The negative tests
were detected after 30 days of incubation on ly . The gelatin at the
above concentration melts at about 24°C and is l i qu id at 37°C. Liquefa
ction was tested at in terva ls by removing the nutrient gelatin cultures
from the incubator and holding them at 4°C for 30 minutes before reading
the resu l ts . Gelatin media was prepared by adding 120 g of gelatin
to 1 l i t r e of nutr ient broth and al lowed to stand at 10°C overnight.
I t was warmed to 45°C to dissolve the gelating and pH was adjusted
41
"to 8 .4 . The medium was cooled to 45°C slowly added beaten white
of two eggs. I t was steamed for 30 mts. wi th occasional s t i r r i n g .
F i l tered through hardened f i l t e r paper, pH adjusted to 7.6 and bott led
in 12 ml amounts. These bott les were kept in autoclave in free steam
for 10 mts. fol lowed by 115°C for another 10 minutes. I t was removed
from the autoclave qu ick ly and kept at low temperature. The f ina l
medium was transparent and f i rm in consistancy.
Ammonia product ion:
Inoculated peptone water (pH 7.4) wi th the test organism and
incubated at 30°C for 5 days. In th is culture 5 drops of Nessler 's
reagent were added. The development of a ye l low, orange or brown
colour of the medium indicated posi t ive react ion. A faint yellow colour
was considered as negative.
SUGAR FERMENTATION REACTIONS
For studying the sugar fermentation react ion, the fol lowing
sugars were included-
Monosaccharides Glucose, xylose
Disaccharides Lactose, Maltose
Polysaccharides Inu l in , Glycogen
Alcoholic sugars Sorb i to l , Inos i to l
Preparation of carbohydrate media for fermentation reactions;
Andrade's ind icator : Sodium hydrox ide (IN) was added to 0.5% solution of acid
A2
fuchsin ; in t i l the colour just became yel low.
To one l i t r e of peptone water (pH 7.2) 10 ml of Andrade's
indicator was added s low ly . To th is solution 1% carbohydrate was
added and medium was d is t r ibu ted in sugar tube containing inverted
Durham's tubes. The tubes were s te r i l i sed at 10 I b s / s q . in pressure
for 15 minutes. Disaccharides and monosaccharides were s ter i l i zed
at 5 lbs pressure/sq. i n . for 10 minutes for 3 consecutive days or
or s ter i l i zed by f i l t r a t i o n .
Twelve hours growth of test culture was taken in peptone water
and the culture was inoculated by pasteur pipette into each of these
sugar tubes containing 1 ml of 1% solution of the respect ive sugar.
The tubes were incubated at 37°C for atleast 5 days. Acid and gas
production was recorded d a i l y . Production of gas was indicated by
an empty space at the top of the Durham's tube and change of carbo
hydrate solution to pink colour was ind icat ive of acid product ion.
Tubes showing negative reactions were fur ther incubated for atleast
15 days before they were declared negative to detect any late fe r -
menter.
Serological t yp ing :
The serological typing of E^ co l i s t ra in was not done because
antisera were not avai lable at C D . R . I . Lucknow. However, these
strains have been send to National Escherichia co l l Centre, Central
43
Research Ins t i tu te , Kasauli (H.P.) for the i r serological typing and
the result is awai ted.
Bacterial strains and media:
Forty eight strains of E_ co l i were isolated from the cases
of gastroenter i t is and ur inary/urogeni ta l t ract infections of human
and animals (Tab le-1) . Following indicator strains were also included
in the present s tudy.
( i ) Escherichia co l i B;
An international test s t ra in which is non-colicinogenic, non-
plasmid harbour ing, sensit ive to a l l ant ib io t ics. E_ co l i B. (E.C.B.)
was used as an ant ib io t ic potency control for conducting ant ib io t ic
sens i t iv i ty test .
( i i ) E^ co l i K-12-J 62:
I t is a lactose negative, deficient for p ro l ine , h is t id ine
and t ryp tophan, resistant to na l id i x i c acid and sensit ive to a l l other
ant ib io t ics . This stra in was used as receipient in conjugation e x p e r i
ments.
( i i i ) ^ co l i K-12-J 53:
I t is s imi lar to the above strains but is lactose pos i t i ve ,
( i v ) E_ co l l ROW :
A sensit ive st ra in to almost a l l the col ic ins and used as
an indicator s t ra in for co l ic in test ing. E. co l i B was also included
l\l\
in col ic in studies. A l l above indicator strains were obtained from
C D . R . I . Culture Collection.
COMPOSITION OF MEDIA USED IN THE STUDY
Nutrient Agar;
Peptone, 20 g -, sodium chlor ide 5 g , disodium hydrogen
phosphate, 2.5 g. ; glucose 2.0 g , agar power 2%, d i s t i l l ed water
1000 ml pH = 7.2 ± 0 .2 .
Mac-Conkey Agar;
Sodium taurocholate, 5 mg; lactose, 10 g , peptone, 2.0 g ,
agar powder 20 g , d i s t i l l e d water, 1000 m l , pH = 7.2 ± 0 .2 , to
th is 3.5 ml of neutral red (2% solution in 5% ethanol) was added as
indicator. The medium was s ter i l i zed by autoclaving for 15 minutes
at 15 lb pressor.
Simmon's Citrate Agar;
Sodium ch lo r ide , 5.0 g ; MgSO ; 0.2 g ; NH H PO^, 1.0 g ;
KH PO , 1.0 g ; sodiuir c i t rate . 5.0 g ; agar: 20 g ; Promo-
thymol b lue; 40 ml (0.2% solution) and one l i t r e d i s t i l l ed water
was added, pH was adjusted to 6 .8 . The medium was s ter i l i zed by
autoclaving for 15 mirntes at 10 lb pressure.
Blood agar;
Seven percent sheep erythrocytes were added to s ter i l ized
nutrient agar at 45°C. After shaking, the plates were poured.
A 5
M i n i m a l o r s y n t h e t i c m e d i u m :
NH C I , 1.0 g ; KH PO , 3 . 5 g ; MgSO 7H 0 , 0 . 1 g ; l a c t i c
a c i d - 9 g ; o r 7 . 5 m l ; d i s t i l l e d w a t e r 1000 m l , a g a r p o w d e r , 20 g j
pH 7 . 2 . T h e m e d i u m w a s s t e r i l i z e d b y a u t o c l a v i n g f o r 10 m i n u t e s
a t 10 l b p r e s s u r e .
Yeas t e x t r a c t m e d i u m :
Y e a s t e x t r a c t , 2 . 5 g ; c a s e i n h y d r o l y s a t e , 2 . 5 g' ; p e p t o n e ,
15 g ; s o d i u m c h l o r i d e , 5 g ' d e x t r o s e , 20 g _,' Na HPO , 2 . 5 gj
d i s t i l l e d w a t e r , one l i t r e y p H 7 . 2 a n d s t e r i l i z e d as a b o v e .
A n t i b i o t i c s / C h e m o t h e r a p e u t i c a g e n t s :
F o r c o n d u c t i n g a n t i b i o t i c s e n s i t i v i t y a n d t o f i n d ou t t h e m i n i m u m
i n h i b i t o r y c o n c e n t r a t i o n s , t h e f o l l o w i n g a n t i b i o t i c d i s c s and p o w d e r
w e r e u s e d :
A n t i b i o t i c d i s c Code P o t e n c y ug / d i s c
R e s p e c t i v e f i r m s
S t r e p t o m y c i n Sm
T e t r a c y c l i n e Tc
C h l o r a m p h e n i c a l Cm
A m p i c i l l i n A p
A m o x y l i n A x
K a n a m y c i n Km
10
30
30
10
10
30
H i M e d i a l L a b o r a t o r y
P a s t e u r B i o l o g i c a l L a b ,
A 6
Co- t r imoxazo le
N a l i d i x i c ac i d
N i t r o f u r a n t i o n
Co
Na
Fd
30 Pasteur B io l og i ca l Lab,
30
300
b .
Drug Powder S o l u b i l i t y
T e t r a c y c l i n e (base powder )
S t rep tomyc in Sulphate
A m p i c i l l i n anhydrous
A m o x y l i n anhydrous
N a l i d i x i c ac id
N i t r o f u r a n t i o n
N i t ro fu razone
Ch lo ramphen ica l (Pa rax in Capsule)
N o r f l o x a c i n (250 mg Tab le t )
Co - t r imoxazo le
Water
Water
Water + 0.1 NaOH
Aqueous
I I
100% e thano l
Water + 0 .1 N NaOH
Water
Kanamycin Su lphate Water (Kancin)
Source
Hindustan A n t i b i o t i c s P i m p r i , Pune
Boehr inge r -KnoU L t d .
Sarabha i Chemicals
H i Media Labo ra to r i es
A lemb ic Chemica l Works .
hi
(a) Disc di f fusion method:
Ant ib io t ic sens i t i v i t y behaviour of E_^ co i i strains were deter
mined by disc di f fusion method of Bauer et a l . (1966). Using a sensit ive
control s t ra in E.col i B. Concentrations of ant ib io t ic in each disc were
as stated above.
Nutrient agar plates were prepared by pouring s te r i le nutrient
agar into pe t r id i shes . After the media was s o l d i f i e d , plates were
dr ied before use at 37°C, to remove excess moisture from surface.
Broth cultures were prepared by inoculating 4-5 colonies of a strain
from f resh ly sub-cultured petr ip la tes into 5 ml nutr ient broth and
incubating the culture at 37°C for 3-4 hrs to obtain moderate t u r b i d i t y .
A s te r i l e cotton swab was dipped into d i lu ted cul ture, excess f lu id
was removed from swab by rotating i t at inner side of test tube wal l
and i t was streaked/spread onto agar surface of pe t r ip la tes . Plate
was kept at room temperature for 10 minutes, mounted the ant ib iot ic
discs and incubated the plate at 37°C for over n ight . The plates
were scored for resistance or sens i t i v i t y after 18 hrs by comparing
the chart based on the inh ib i to ry zone diameter as given by the disc
manufacturer.
(b) Determination of resistance level by plate d i lu t ion method:
The minimum inh ib i to ry concentrations (MIC) of ind iv idua l st ra in
was worked out by using f resh ly prepared ant ib io t ic solutions of d i f f e
rent concentrations which were added to s te r i l i zed nutrient agar medium
of pH 7.2, cooled to 45-50°C, mixed well and poured into pet r ip la tes ,
48
(20 ml in each p l a t e j , . A f t e r s o l i d i f i c a t i o n p la tes were s to red in co ld
room and used them nex t d a y . The recommendat ions of WHO (1961)
were f o l l o w e d in conduct ing t he a n t i b i o t i c s e n s i t i v i t y tes ts as adapted
by Yadava and Gupta (1971) .
Each p l a t e was d i v i d e d i n to t w e l v e equal s e c t o r s . A l o o p f u l
(5 mm d iame te r ) of IB h r b r o t h c u l t u r e , d i l u t e d 10 f o l d in normal
sa l ine so lu t i on (NSS) was spot inocu la ted in d u p l i c a t e , i n each sec to r ,
on a n t i b i o t i c media p l a t e s , p r e v i o u s l y d r i e d f o r an h r s at 37°C. A l l
the tes t s t r a i n were a lso inocu la ted on a c o n t r o l p l a t e w i t hou t any
a n t i b i o t i c . Inocu la ted p la tes were a l l owed to d r y and the p la tes were
incubated o v e r n i g h t at 37°C. The p la tes were examined fo r the p r e
sence or absence of g r o w t h on spo t ted area of a n t i b i o t i c p l a t e . The
presence of g r o w t h was i n d i c a t e d by ( + ) , absence of g rowth \ jy ( - ) and
mot t led g r o w t h by ( ± ) . Concent ra t ion of a n t i b i o t i c w h i c h p e r m i t t e d
+ g rowth and beyond wh i ch t h e r e was no g r o w t h was cons idered as
minimum i n h i b i t o r y concent ra t ion ( M I C ) . E . c o l i B cu l t u re was s i m i l a r l y
spot inocu la ted in one of the sector as a n t i b i o t i c potency c o n t r o l .
In t he absence of any s t r i c t - d e f i n i t i o n f o r a n t i b i o t i c res is tance
l e v e l (WHO, 1961), MICs va lue of ECB were taken as s e n s i t i v e l e v e l
and was cons idered as one u n i t . S t ra ins showing MIC values four f o l d s
or more than E_ c o l i B MIC values were cons idered as r es i s t an t l e v e l
(Yadava and Gupta, 1971).
SCREENING OF AUTO-TRANSFERABLE R-PLASMID IN VITRO BY CONJUGAriON EXPERIMENT: ~ "
Drug res is tance t r a n s f e r s tud ies were c a r r i e d out by using the
A9
technique of Watanabe and Fukasawa (1961-b) and Dutta and Nugent
(1984), wi th l i t t l e modif icat ions.
Escherichia co l i s t ra ins , resistant to one on more ant ib iot ics
and sensit ive to na l id i x i c ac i d , were used as donor for transfer of V _ — —
drug resistance markers. E.col i K-12 J62 (Na, Pro , His , Try )
was taken as sensit ive recipient s t ra in .
The donor and recip ient strains were grown in 5 ml nutrient
broth and incubated for 3-..4 hr at 37°C. Donor and recipient growths
were mixed in 1:2 ra t io in 7 ml f resh nutrient broth and incubated
overnight at 37°C. Different d i lu t ions of conjugates mixture were made
in normal saline solution and 0.1 ml of d i lu ted mixture were then plated
on to MacConkey lactose agar plates containing double ant ib iot ics (50
/ug Nal id ix ic acid + 50/ug of drug) to which the donor was res is tant ) .
At least 2 plates were used for each set of mixture.Plates were incubated
at 37°C overnight and mixed broth were kept at room temperature.
I f no colony appears on selection p la tes, more plates were inoculated
with mixtures that have been le f t at room temperature (28°C) day before,
to look for a plasmid that is temperature sensit ive for the i r conjugation
function.
Confirmation of trans-conjugant colonies:
Non-lactose fermenting colonies appearing on selection plates
(double ant ib io t ic plate) were observed. . They were ident i f ied and
confirmed as recipient E . co l i K-12-J 62 on the basis of the i r cu l tu ra l ,
morphological and nutr i t ional characters. Colonies from each type of
plates were repl icated on to 3 sets of plate for confirming the recipient
E. co l i K-12 J62.
50
( i ) minimal agar (Posi t ive control)
( i i ) minimal agar + Pro, H is , Trp (negative control)
( i i i ) minimal agar + Pro, H is , Trp + corresponding ant ib iot ics
colonies appearing on 3rd set of plates were confirmed as K-12 J62
which acquired drug resistance plasmids.
Percent of t ransfer :
Percent of transfer of d i f ferent drug resistant markers was calcu
lated as fo l lows:
Colonial count of recipient strain on double ant ib io t ic plate which acquired resistance from donor.
Percent transfer = Colonial count of recipient strain on na l i d i x i c (PT) acid plate without any drug
SCREENING OF E. COLI STRAINS FOR THEIR HAEMOLYTIC ACTIVITY
A l l the strains of E_ col i under study were tested for the i r
haemolytic behaviour, employing e ry th rocy t i c agar plates using the
method of s imi lar as used by Shamim S Yadava (19^0) and Blanco et
al (1990), Asept ical ly collected sheep blood were taken and about 7%
washed ery throcytes were asept ical ly added in nutrient agar medium
at 45°C, and erythrocytes were mixed by shaking without bubles and
plates were poured asept ical ly inside the laminar f low. Overnight growth
of each strains were appropr ia te ly d i luted in NSS and plated/streaked
d i rec t ly on the blood agar plate in duplicate so as to obtain wel l
isolated colonies. The plates were incubated for 2 4 - 4 8 and even some
times for 72 hr, at 37°C to observe the clear l y s i s . Haemolytic and
non-haemolytic bacter ial strains were always included as controls in
each experiment.
51
COLICIN PRODUCTION:
Colicin production was studied according to the method adopted
by Ansari and Yadava (1984). A l l the strains of E_j_ co l i were grown over
night in nutr ient broth at 37°C. One ml of 18 hrs old culture was added
in 9 ml of yeast extract medium in a test tube and incubated for 30 minutes
at 37°C. The content of the tube was poured in a pe t r id ish to form
2 mm deep layer . The bacteria in pe t r id i sh were exposed to u l t ra violet
l i gh t for 2.5 minutes as standardized ear l ie r at distance of 25 cm from
a portable Honovia Ul t ra v io le t lamp (wavelength, 3000A°), wi th a deep
v io let f i l t e r . The plate was placed on a sheet of card board and shaken
during i r r ad ia t i on . After i r rad ia t ion the content was poured in the tube
and incubated for 38 hrs at 37°C. Few drops of chloroform were added
and the tube shaken vigorously for 15 seconds and allowed to stand at
room temperature for 5 minutes. The contents were then centrifuged at
5000 rpm for 30 minutes. Undiluted supernatant f l u i d was spot inoculated
with 5 mm diameter platinum loop on the indicator s t ra in E_ col i Row.
The l ys is or inh ib i t i on of indicator s t ra in indicate presence of colcin
or lysogenic phage.
To confirm the presence of co l ic in supernatant was d i lu ted 2 fo ld
ser ia l ly and spot inoculated on the indicator strains E. co l i Row by loop
as above. The intensity of i nh ib i t i on of the indicator growth decreases
and an end point is taken as the highest d i lu t ion at which a zone of
is
inh ib i t ion is not c lear ly v i s i b l e o r / h a z y . At high d i lu t ion also the lyso
genic phage produces plaques on the indicator strains and on th is basis
col ic in and phage (lysogenic) can easi ly be d i f fe ren t ia ted .
52
ELIMINATION OF R-PLASMIDS (DETECTION OF PLASMID MEDIATED NON-TRANSFERABLE DRUG RESISTANCE:
Plasmid el iminat ion from ' the host s t ra in by treating the cei ls
wi th curing agents prov ide an addi t ional information about the non
transferable plasmid mediated drug resistance. The method described
by Watanabe and Fukasawa (1966b) and Singh and Yadava (1988) were
used. Three curing agents were used which are as fo l lows:
( i ) Sodium dodecyl-suiphate (SDS)
( i i ) Acr id ine organe (AO)
( i i i ) Norf loxacin (4-quinolone-compound)
Determination of curing agents ac t i v i t y on bacter ia l growth;
Minimum inh ib i to ry concentration (MICs) of curing agents were
determined by plat d i lu t ion techniques as described previous for
ant ib iot ics and then sub- inh ib i to ry concentration which is just below
the MIC concentration were taken to cure plasmid DNA select ively
for acr id ine orange and nor f loxacin, the MIC range ( a s depicted m
Table-2) were selected to cure R-plasmids. Sensi t iv i ty to 10%
SDS were determined against resistant s t ra ins . Strains showing ±
or + growth in d i lu t ion plate method were taken for curing experiments
wi th SDS.
Curing procedure:
Nutrient broth whose pH was adjusted to 7.6 wi th IN NaOH
was dispensed in tubes ( 5 ml each) and autoclaved. Various concentra
tions of s te r i l i zed curing agents (except SDS which was added in
nutrient broth p r i o r to autoclaving) were added into tubes for e l im i -
53
T a b l e - 2 : Range of i n h i b i t o r y concen t ra t i on of cu r i ng agents aga inst i n d i v i d u a l r e s i s t a n t s t r a i n s of E . c o l i
Cur ing agents
N o r f l o x a c i n
Range MIC j^g/m
0.05
0 .1
0 .2
of
1)
S t ra i ns numbers
17
0 2 , 0 7 , 0 9 , 1 1 , 1 2 , 1 3 , 14,29,34
0 3 , 0 4 , 1 6 , 1 8 , 2 3 , 2 6
Range of Concent r a t i o n s - taken f o r cu r i ng i | u g / m l )
0 . 0 1 , 0 . 0 2 , 0 . 0 4 , 0 .05 0 . 0 4 , 0 . 0 8 , 0 . 1
0 . 0 4 , 0 . 0 8 , 0 . 1 5 , 0 .2
0.3 20,21,29,38 0.08,0.1,0.15,0.2, 0.25,0.3
Acridine orange 1600 11,12,13,14,16,18, 800,1000,1600 19,20,29,30,32,24,38
800 35,37 200,400,800
400 17 100,200,400
200 04 50,100,200
Sod ium d o d e c y l s u l p h a t e (SDS) 10% (vv /v ) was used to cure R— d e t e r m i n a n t s . S t r a i n s show ing ± o r + g r o w t h a t 10% SDS were taken f o r c u r i n g
5A
nating the plasmid DNA. Tubes were inoculated wi th 0.1 ml of overnig l i '
culture (d i lu ted to 10 CFD/ml) and incubated at 37"C for 18 h rs .
Culture was then appropr ia te ly d i lu ted in NSS, 0.1 ml of i t was spreaded
on nutr ient agar and then incubated overnight at ?"°C. Resulting colonies
were tested for ant ib io t ic resistance by rep l ica plat ing on nutrient
agar plate incorporated wi th d i f ferent an t ib io t i cs .
Control :
Nutrient broth of pH 7.6 without curing agent was inoculated
wi th the same organism to check for normal growth and for spontaneous
loss of drug resistance markers, i f any.
Procedure fol lowed for rep l ica p la t ing ;
Replica plat ing method of Lederberg and Lederberg (1952),
with s l ight modi f icat ions, was used for testing large number of colonies
for presence or absence of R-determinants. A master plate was prepared
by inoculating the isolated colonies wi th the help of s ter i l i zed tooth
picks on to a fresh media p la te , previously numbered in sectors. Master
plate was incubated overn ight . Resulting colonies were repl icated from
master plate onto ind iv idua l ant ib io t ic plates in corresponding sectors
wi th the help of a tooth pick for each colony. Plates were incubated
over night and scored for presence or absence of R-determinants in
each colony.
Determination of curing frequency: The curing fnsquency was calculated
as fo l lows:
Total colonial count on simple nutrient agar plate Curing Frequency — — . xlOO
Total coloni • count on respective ant ib iot ic plate
R E S U L T S
55
Morphological, Cultural and Biochemical Characters of E. coli strain
Smooth, round, opaque with entire edge and lactose fermenting
colonies appeared on Mac-Conkey agar plate were taken as E^ coli
s t ra in , Gram negative, short rods with rounded end or coccobacillary,
non-sporulating, looking l ike E_ coli on microscopic examinations were
taken for the i r biochemical s tudies . All 48 strains of Ej_ coli isolated
from man and animals were studied for various biochemical reactions,
e .g. methyl red (MR) and voges Proskauer (VP) reactions, production
of inodole, ammonia, catalase, urease and hydrogen sulphide (H s)
gas, reduction of ni trate to n i t r i t e , liquification of gelatin deamination
of phenyl-alanine and utilization of ci t rate as sole source of carbon
and various sugars ( glucose, xylose, lactose, maltose, inulin, glycogen,
inositol and sorbitol) were used to study thei r fermentation behaviour.
All 48 s trains were found posit ive for M.R. reaction, indole
production, ni trate reduction and production of catalase. They were
negative for V .P . , H S, urease, gelatinase, c i t rate and phenyl-alanine
deaminase reactions (Table-3A). All s trains fermented sugars (glucose,
lactose, maltose and sorbitol) with the production of acid and gas .
While none of the strains could ferment inositol , inulin and glycogen
(Table-3B> .
Antibiotic sensi t ivi ty behaviour of Escherichia coli s t rains
In the present investigation a wide spectrum of drug resistant
strains of E_ coli were found from a man and animal srouces. All
48 strains tested for the i r sensi t ivi ty against 10 antibacterial drugs
56
for the determination of the i r minimum inhibitory concentrations (MICs).
The strains is said to be resis tant if a par t icular strain showing their
MIC value equivalent to four folds or more of the i r MIC to E_ coli
B (an internationally accepted antibiotic sensit ive strain which was
isolated in p re -an t ib io t i c e r a ) . Minimum inhibi tory concentration of
all E^ coli s trains have been presented in Table-4 and range of MIC
against various antibiotics have been shown in Table-5. Out of these
48 strains tested for the i r sensi t ivi ty (66.66%) were found to be r e s i s
tant to one or more of the ten ant i -bacter ial drugs . While only 16
(33.34%) strains were found sensitive to all the 10 antibacterial drugs
used. The MICs of different antibacterial drugs (Ap, Ax, Sm, Tc, Cm,
Km, Co, Na, Fd, and Fz) against these strains varied great ly . Their
MIC values ranging from 0.4 to 6400 /Ug/ml for different drugs . Very
high level of drug resistance (even upto 6400 vug/ml) were recorded
against four ant ibiot ics , namely amoxycillin, ampicill in, streptomycin
and kanamycin and total number of s trains showing such resistance
were 12, 10, 3 and 2 respec t ive ly . Six strains were also showing higher
MIC (3200/Ug/ml) against co-trimoxazole. None of the strains was found
to withstand 800/jg/ml of chloramphenicol. Similarly none of the strains
could res is t more than 100/ug/ml of nitrofurantoin, 50^g /ml of nitrofura-
zone and 6.25/ug/ml of nalidixic acid (Table 4,5 and Figure-2) .
The overall incidence of drug resistance e i ther singly or in
combination with other drugs among 48 strains of E. coli have been
showin in Table-6 and 7 and Fig . -3 , . . The maximum number of strains
were resistant to streptomycin (60.40%) followed by Ap (56.25%), Tc
(52%), Ax (50%), Co and Cm (43.75% each) . Km (12.57%) and minimum
57
resistant was recorded against Fd and nitrofurazone (8.33% each) . The
overall sensi t ivi ty pat terns of E^ coli s t rains have been presented
in Table-8 which reveals that multiple drugs resistance were recorded
in 26 (81.56%) out of 32 resistant s t r a ins . Maximum 9 (18.75%) strains
were resistant to six drugs in various combinations. Two strains no.
29 and 39 were found resis tant upto even eight drugs .
The percentage of drug resistance was more in human isolates
(76.31%) as compared to animal isolates (30%) Table-9. Escherichia
coli strains isolated from urinary/urogenital t ract infections were slightly
more resistant (67.19%) as compared to strains isolated from gastroenteri
t i s cases (62.96%)^ Table-1Q. Statist ically th is difference was not found
significant.
Studies on tranferabi l i ty of drug resistance markers
On the basis of the i r MIC values 32 resistant strains representing
both human and animal sources were selected to study the transferable
nature of drug res is tance . Antibiotic resistance markers exhibiting
at least 50 yuglml concentration of individual drugs were taken for R-factor
transfer s tudy.
Auto-transferable R-plasmids
In th is study out of 32 resistant s t rains only 13 (40.62%) could
transfer the i r drug resistance e i ther par t ia l ly or completely in conjuga
tion experiments, while 19 (59.38%) were non-tranferable in nature
(Table-11). It was observed that total incidence of transferable R-
determinants among 32 resis tant s t rains showed great variation. Maximum'
58
•percent of transfer was observed against Km (60%) followed by Ax'
(59.19%), Ap (52.0%), Co (37.5%), Tc (28%) and Cm and Sm (27.77%
and 25% respec t ive ly ) . R-determinants of Fd and FZ were found non
transferable in nature (Table-12). Patterns of drug resistance transfer
varied greatly in different s trains i r respect ive of drug resistance
pattern of donor s t ra in .
Ap/Ax combination were transferred most frequently in all
13 (100%) s t ra ins , followed by transfer of Km (75%), Sm (66%), Tc
(63%), Cm (62%) and least for cotrimoxazole (54.54%). Transfer frequen
cies of donor s t ra ins for ampicillin varied greatly even with the
same recipient s t ra in . This variation was so much that with one strain -2
(34) the transfer frequency was recorded 1.50x10 and with o ther donor
strain (36) it was 0.78x10 only. Remaining transferable strains were
found to have transfer frequencies in order of 10 ( 6 s t ra ins ) , -4 -3 -7
10 (3 s trains) and 10 and 10 (one strain each) Table-13.
Curing/Elimination of R-plasmids;
Ninteen s t ra ins having various resistance patterns did not
transfer the i r resistance markers d i rect ly through direct conjugation
with recipient s t ra in . These strains were sleeted to examine whether
resistance to the drugs were plasmid mediated or chromosomal mediated.
For th is purpose three curing agents namely norfloxacin (4-quniolone,
acting on the A-subunit of DNA gyrase enzyme), sodium dodecyi sulphate
(a surface acting agent) and acridine orange ( a DNA intercalating
dye) were used. Out of these 19 non-conjugable drug resistant strains
only 8 (42.1%) were found to be curved of the i r drug resistance markers
in various combination with other resistant determinants (Table 14).
59
Further ten res is tant s t ra ins (4 possessing non-transferable'
and 6 possessing transferable resistance factors) were chosen to de ter
mine curing frequency of par t icular markers using norfloxacin as curing
agent. It was observed that Ap and Ax markers were eliminated most
frequently, followed by Tc, Sm, Co, Km and Cm (Table-15).
Comparative curing efficacy of all three curing agents were
determined against 25 resis tant s trains (Nineteen non-transferable and
6 t ransferable) . It was observed that norfloxacin was a bet ter curing
agent for E. coli R-plasmids, eliminating 48% of R-determinants followed
by SDS and acridine orange eliminating 24% and 20% resistance markers
from resis tant s t rains respect ively (Table-16). It was also observed
that curing concentrations of acridine orange and norfloxacin just below
the MIC levels was most effective in eliminating R-determinants.
Presence of virulence factors (Colicin and Haemolysin) among E.coli strains
All the 48 strains of E_ coli under study were tested for
the i r production of colicin and haemolysin. Incidence of colicinogeny
was recorded as 27.1% (Table-17). It was observed that the production
of haemolysin and colicin among UTI isolates were much higher (47.6%
and 42.85% respect ively) as compared to GTI isolates which was recorded
as 11.11% for haemolysin and 14.43% for colicin (Table- I8) .
Five s trains (3 from UTI, 2 from GTI) produced both haemoly
sin as well as colicin and surprisingly four strains were also resistant
to one or more of the ant ib iot ics . There was great affinity of R-plasmids
with haemolysin and colicin p lasmids , as the incidences of drug re s i s -
^tance was 78.5% among haemolytic s t rains and 76.9% among colicinogenic
strains (Table-19).
60
Table-3A: Biochemical reactions of 48 isolates of E. coli .
Biochemical reactions Strain posit ive Strain negative
Number % Number
Indole
Methyl Red
Voges Proskauer
Citrate
Nitrate
Ammonia
Catalase
Hydrogen sulphide
Urease
Gelatin (22 C)
Phenyl alanine deam.inase
48
48
-
-
48
48
48
-
-
-
_
100
100
-
-
100
100
100
-
-
-
_
—
-
48
48
-
-
-
48
48
48
48
—
-
100
100
-
-
-
11)0
100
100
100
6]
Table-SB: Sugar fermentat ion by 48 i s o l a t e s of E_ co l i .
C a r b o h y d r a t e P o s i t i v e s t r a i n Negative s t r a i n % of pos i t i ve s t r a i n s for fermentation
Glucose 48 - 100
Lactose 48 - 100
Xylose 48 - 100
Maltose .48 - 100
Sorb i to l 48 - 100
Inosi tol - 48
Inulin - 48
Glycogen - 48
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73
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65
Table-5 : Range of minimum inh ib i to ry concentrations (MIC) (ug/ml) of ant ibacter ia l drugs against 48 E.col i strains
Cone, of Total number of strains resistant to ant ibacter ia l drug (ug/ml) Ap Ax Tc Sm Cm Co Km Na Fd Fz
6400
3200
1500
800
400
200
100
50
25
12.5
6.25
3.125
1.56
0.8
0.4
10
05
02
02
01
02
-
03
02
11
*
09
01
-
-
12
02
02
03
-
03
-
-
02
01
10*
07
04
01
-
-
01
01
03
09
05
05
-
-
01
03*
08
09
02
-
03
03
07
03
01
04
01
01
06
16
03*
-
-
-
-
-
-
-
-
08
06
04
-
02
-
-
05*
13
07
03
-
06
06
02
-
02
02
01
04
17*
08
-
-
0
0
02
-
01
01
-
-
01
-
01
25
12*
05
-
0
0
-
-
-
-
-
-
-
-
-
-
10
23*
11
03
01
-
-
-
-
-
-
02
02
11
25*
08
-
-
-
-
-
-
-
-
-
-
04
19
11*
14
-
-
-
-
*= MIC va lues of E . co l i B.
F-4
E
?
U
2
6400 "
3200-
1600 -
800-
400^
200-
100-
50.-
25 •
12.5--
6.25-.
3.125--
1.5&-
0.78 -•
0.39 1
T" 02
02
03
-
03
-
-
02 -
10
*10
.07
04
01
-
-- 1(
OS
02
02
01
02
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03
02
11
*-
09
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06
16
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01
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-
01
-
01
25
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05
=
-
-
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01
03
09
06
05
-
-
01
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08
09
02
•
-' 06
06
02
-
02
02
01
-
M7
08
-
-
-
-
- •
- 08-|
06
04
-
-
02
-
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13
07
03
-02-
02
11
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08
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-
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19
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14
-
-
-
- I
-'"1 =•=23
11
03
01
Ax Ap Sm Km Tc Co Cm Fd Fz Na
antibacterial drugs
Fig. 2 : MICs of 48 strains of E_ co i l
(a) Figures in bars indicate number of strains showing corresponding MIC values.
( b ) * Corresponding MIC values indicate MIC value of ant ib iot ic potency control st ra in E. col i B.
66
Table - 6: Frequency of resistance among 48 strains of E. coli against various antibacterial drugs .
Antibacterial agents Resistant-strains Number Percentage(I!
Streptomycin
Ampicillin
Tetracycline
Amoxycillin
Chloramphenicol
Co-trimoxazole
Kanamycin
Nitrofurantoin
Nitrofurazone
Nalidixic acid
29
27
25
24
20
19
06
4
4
Nil
60.41
56.25
52.83
50.00
41.66
39.54
12.50
8.33
8.33
Nil
60 -
55 -
50 -
45 -
40 -
35 •
0) 30 -o c
2 If) 25
•r* m <u '- 20 -0
S«! 15 -
10 -
05 -
Sm Ap Tc Ax Cm Co Km Fd Fz
Antibacterial drugs
Fig. 3 : Comparative resistance percentage among E. coi i strains against various an t i
bacter ial drugs.
67
T a b l e - 7 : Frequency of i n d i v i d u a l an t i b i o t i c r e s i s t a n c e e i t h e r s ingly or in combination wi th o t h e r d rugs among 32 drug r e s i s t a n t s t r a i n s of E . c o l i .
SI .No. An t ibac t e r i a l d rugs Total number of Percentage (%) r e s i s t a n t s t r a i n s
1. S t reptomycin 29 90.62
2 . Ampici l l in 27 84.37
3 . . Amoxycil l in Z4 73.00
4 . T e t r a c y c l i n e 25 77.50
5 . Chloramphenico l 20 62.50
6. Co- t r imoxazole . 1 9 '-34
7. Kanamycin 06 18.75
8. Nitrofurantoin 04 l/:-50
9. Nitrofurazone 04 12.50
10. Na l id ix i c ac id
68
T a b l e - 8 : S e n s i t i v i t y of 48 E . c o l i s t r a i n s aga ins t 10 a n t i b a c t e r i a l d rugs
Resistance p a t t e r n of s t r a i n s S t r a i n numbers To ta l •no. of s t r a i n s
(A) S e n s i t i v e to a l l d rugs
(B) Res is tant to
(a) Single Drug
Sm Tc
(b ) Double Drugs :
Sm, Tc Sm,Ap
(c) Th ree Drugs ;
A p , A x , F 2
(d) Four Drugs :
A p , S m , T c , C o Ap ,Ax ,Sm ,Cm
, A p , A x , S m , T c A p , A x , S m , C o
0 1 , 0 5 , 0 6 , 1 0 , 1 5 , 2 7 , 3 1 , 3 3 , 3 5 , 1 0 1 , 1 0 2 , 1 0 3 , 1 0 5 , 106,107,108
16
09 ,25 08
03,22 13
104
2 3 , 24 04 32 14
(e) F i v e Drugs :
A p , A x ,Sm,' T o , Cm A p , A x , T c , C m , C o
( f ) S ix Drugs :
A p , A x , S m , T c , C o , F z A p , A x , S m , C m , F d , F z A p , A x ,Sm , T c , C m , K m A p , A x ,Sm ,Tc ,Cm,Co .
(g) Seven Drugs :
A p , A x ,Sm ,Tc ,Cm ,Km,Co A p , A x , S m , T c , C m , C o , Fd
(h ) E igh t Drugs :
07 ,02 ,37 28
110 18 11 13 ,17 ,19 ,26 ,30 ,109
16,20 21 ,34,36
69
T a b l e - 9 : Incidence of drug r e s i s t a n c e among human and animal i so la t e s of E . co l i
Source Total no. of s t r a i n
Total no . of r e s i s t a n t s t r a i n
% of r e s i s t a n t s t r a i n
Human
Animal
38
10
29
3
76.31
30
Table-10: Incidence of drug r e s i s t a n c e among UTI and GTI s t r a i n s of E . col i
Source
Urine (UTI) Human
Stool (GTI) Human
Laboratory-animal (GTI)
Total no . s t r a i n s
21
17
10
of Res is tan t to one or more an t ib io t i c s
16
13
3
% of r e s i s t a n t s t r a i n s
76.19
76.46
30
70
Tab le-11 : Incidence of t ransferable and non-transferable drug resistance among 32 resistant stra ins of E.co l i
Nature Strains numbers Total no. Percentage
A u t o - t r a n s f e r a b l e 1 2 , 1 4 , 1 6 , 1 7 , 1 8 , 1 9 , 13 40.62 2 0 , 3 0 , 3 2 , 3 4 , 3 6 , 1 0 9 , 101
Non-transferable 02,03,04,07,08,09, 19 59.38 1 1 , 1 3 , 2 1 , 2 2 , 2 3 , 2 4 , 2 5 , 2 6 , 2 8 , 2 9 , 3 7 , 3 8 , 104
71
Table-12: Incidence of transferable R-determinants among resistant s t rains of E. col i .
SI.No, R-determinants Resistant s t rains at 50 ug/ml cone.
Total no. of t rans ferable strains
Percent of
transfer
1.
2 .
3 .
4 .
5 .
6.
7.
8.
9.
Kanamycin
Amoxycillin
Ampidllin
Co-trimoxazole
Tetracycline
Chloramphenicol
Streptomycin
Nitrofurantoin
Nitrofurazone
05
22
25
16
25
18
24
04
04
03
13
13
06
07
05
06
60.0
59.19
52.0
37.5
28.0
27.77
25.0
60
55 .
50 -
0 (fl C to c
o 0)
(0
c U L.
a.
45
40
35
30
25
20
15
10
05
Km Ax Ap Co Tc Cm Sm
Ant ibacter ia l drugs
F i g . 4 : T rans fe r f requency of va r ious R-de te rminan ts .
72
Table -13 : Patterns of drug resistance transfer (by conjugation) in 13 c l i n i ca l isolates of E.co l i
Strain Drug resistance pattern Drug resistance markers % of No. of donar t ransfer red to K12-J62I transfer
(at 5Qug/ml cone.) frequency
12
14
16
17
18
19
20
30
32
34
35
109
no
A p , A x , T c , C m , C o
A p , A x , Co
Ap , A x , S m , T c , C m , K m , C o
A p , A x , T c , C o
A p , A x , F d
Ap , Ax ,Sm ,Tc ,Cm ,Co
Ap ,Ax ,Sm ,Tc ,Cm ,Km ,Co
Ap , Ax ,Sm ,Tc ,Cm ,Km,Co
A p , A x , S m , T c
Ap , A x , S m , T c , C m , K m , C o
Ap , Ax ,Sm , T c , C m , C o
Ap ,Ax ,Sm ,Tc ,Cm ,Co
A p , A x , S m , T c , C o
A p , A x , T c , C o
A p , A x
A p , A x , C m , To,Km,Sm
A p , A x , C o
A p , A x
A p , A x ,
Ap ,Axm ,5m ,Tc ,Cm ,Km ,Co
A p , A x
Ap ,Ax ,Sm ,Tc
Ap ,Ax ,Sm ,Tc ,Cm ,Km ,Co
A p , Ax ,Tc ,Sm,Cm
Ap ,Ax ,Tc,Sm ,Cm ,Co
A p , A x , C o
0 .88x10
2.27x10~
0 .3x10
0.13x10
0 .33x10" ' '
0 .45x10
1.38x10""^
0.68x10~
1 .9x10~
1 .52x10~^
0.78x10~^
0.8x10"' '^
2 . 4 x 1 0 " ^
73
Tab le \^^ Pa t te rns of e l i m i n a t i o n of d r u g res i s tance m a r k e r s among 25 res is tant s t ra ins of E.coli by using sub-minimum inhibitory concentrations of curing agents .
S t r a i n Res is tan t p a t t e r n Drug r es i s t ance m a r k e r s cured No . by f o l l o w i n g cu r i ng agents
N o r f l o x a c i n Sodium d o d e c y l A c r i d i n e s u l p h a t e orange
02 A p , A x , S m , T c , C m A p , A x , S m , C m
03 Sm,Tc Sm,Tc , Sm,Tc
04 A p , A x , S m , C m A p , A x , C m
07 A p , A x , S m , C m - -
08 Tc - -
09 Sm - -
11 Ap ,Ax ,Sm ,Tc ,Cm ,Km Sm -
13 Ap,Sm - -
14* A p , A x , S m , C o A p , A x - A p , A x
16* Ap ,Ax ,Sm , T c , C m , A p , A x , S m , K m , Tc,Cm A p , A x , S m , K m , Km,Co COjTCjCm Km,Co ,Tc ,Cm
18* A p , A x ,Sm ,Cm ,Fd - -
19 A p , A x , S m , T c , C m , A p , A x , T c " Tc Km,Co
20* A p , A x , S m , T c , C m , A p , A x , S m , T c , A p , A x A p , A x Km,Co Cm,Km,Co
21 Ap ,Ax ,Sm ,Tc ,Cm A p , A x , T c - A p , A x , S m
22
23
24
25
26
28
29
34* A p , A x ,Sm , T c , C m , A p , A x , S m , T c , A p , A x , S m , T c , A p , A x , S m , T c , Cm,Km,Co Cm,Km,Co
37 Ap ,Ax ,Sm , T c , C m , - Tc ,Sm,Cm
38 Ap ,Ax ,Sm ,Tc ,Cm ,Co - - -
104 A p , A x , F z - - -
Ap ,Ax ,Sm ,Tc, Km,Co
Tc
Ap,Ax,Co
Ap,Sm,Tc,Co
Sm
Ap ,Ax ,Sm ,Tc,
Ap,Ax,Tc,Cm
Ap ,Ax ,Sm ,Tc, Km ,Co
Ap,Ax ,Sm ,Tc, Km ,Co
iCm Ap,Ax,Tc
-
-
-
-
,Cm,Co -
,Co Cm
,Cm, Tc
,Cm, Ap,Ax,Sm,Tc, Cm ,Km ,Co
74
Table -15 •* Curing frequency of various antibiotic resistance markers among 10 E.coli strains using norfloxacin as curing agent.
Strain Resistance patterns Antibiotic resistant markers no. Ap Ax Tc Sm Km Cm Co
02 A p , A x , S m , T c , C m 86 86 - 100 - 30 -
03 Sm,Tc - - 100 50 - - -
04 Ap ,Ax ,Sm,Cm - - 100 60 - 100
14* Ap ,Ax ,Co ,Sm 100 100 - -
16* A p , A x , S m , T c , C m , K m , C o 100 100 100 100 100 100 100
18* A p , A x , S m , C m , F d , F z . J _ _ _ _ _
19* A p , A x , S m , T c , C m , C o 100 100 60 -
20* A p , A x , S m , T c , C m , K m , C o 100 100 - -
21 A p , A x , S m , T c , K m , C m , C o 100 100 58 -
34* A p , A x , S m , T c , C m , K m , C o 100 100 100 100 100 100 100
*StrainSr bearing transferable plasmids .
75,
l ao ie - i b : comparative curing eff icacy of curing agents
Curing agents Total st ra in treated
Non-transfera (NTF) = 19
Transferable (TF) = 6
NTF
TF
NTF
TF
Total strain cured
ble 7
5
2
4
1
h.
Total % of cured strain
Norfloxacin
SDS
Acridine orange
12
48%
24%
20%
76
Table- 17. Incidence of col ic inogenic and haemoly t i c s t r a i n s among 48 E . col i i s o l a t e s .
St ra in number Total Number
Colicinogenic 02, 05 , 09, 13 14 29
19, 22, 23 , 25 ,
26, 27, 32, 33,
37, 108
Haemolytic 0 1 , 02, 03 , 04, 13 27.1
05, 07, 09, 10,
2 1 , 23 , 24, 26,
28
77
Table - ig
Occurrence of virulence factors among E_^ coli s trains of UTI and GTI origin.
Virulence Factors
Haemolytic strains
Colicinogenic s trains
UTI isolates n=21
10 (47.6%)
9 (42.85%)
GTI isolates (n=27)
3 (11.11%)
4 (14.43%)
78
T a b l e - I 9 i 0 c c u r r e n c o of v i r u l e n c e f a c t o r s w i t h or w i t h o u t d rug r p s i s -tance ma i ' ke rs among E . c o l i s t r a i n s
S t r a i n No. To ta l number
To ta l s e n s i t i v e s t r a i n s
To ta l r e s i s tant s t r a i n s
% of d rug res is tance w i t h v i r u lence f ac to r s
Co l i c i nogen ic s t r a i n s
0 2 , 0 5 , 0 9 , 1 3 , 1 9 , 2 2 , 2 3 , 2 5 , 2 5 , 2 7 , 3 2 , 3 3 , 37,108
14 11 78.5%
Haemo ly t i c s t r a i n
0 1 , 0 2 , 0 3 , 0 4 , 0 5 , 0 7 , 0 9 , 1 0 , 2 1 , 2 3 , 24 ,25 ,28
13 10 75.9%
Co l i c inogen ic & h a e m o l y t i c s t r a i n s
0 2 , 2 3 , 0 9 , 2 5 . 0 5 iO%
D I S C U S S I O N
79
A total of 48 strains of Escherichia coli isolated from gastroenterit is
(GTI) and urinary/urogenital t ract infections (UTI) of man and animals
were identified on the basis of the i r morphological and biochemical charac
t e r s . These reactions are not only useful for identification of the organisms
but also to study the epidemiology and the pathogenicity of the organism
in relation to the diseases they cause. All 48 s t rains of E^ coli gaVe
typical biochemical reactions as reported by ear l ier workers (Edward
and Ewing, 1962; Yadava and Gupta, 1969; Rajani, 1981). Although these
48 strains of E_ coli could be grouped in different biotypes after studying
thei r fermentation behaviour against some more differentiating sugars (such
as sucrose, sal icin, raffinose and dulicitol e tc . ) as reported by Yadava
and Gupta (1969). However, I could not use the differentiating sugars
due to the i r non-avai labi l i ty .
In the present investigation a large number of E^ coli strains (66.66%)
isolated from GTI and UTI infections of man and animals were recorded
to be resistant to one or more of the ten antibacterial drugs tested, e i ther
singly, or in various combinations. Multiple drug resistance was observed
in majority (81.56%) of the resis tant s t ra ins . This is an indication of
plasmid mediated drug resistance (infectious drug resistance) because
host chromosome can not afford for such an ext load of various drug
resistance gene pool.
The maximum number of s t rains were found resis tant against Sm(
60.41%) followed by Ap (56.26%), Tc (52%), Ax (50%), Co and Cm (- .75?
each) . Km (12.5%) and minimum resistance was recorded against Fd and
Fz (8.33% each) . This emergence of multiple drug resistance in increasing
frequency might be. because of wide and non-judicial use of these drugs
80
to and consequent development of resistance d ue/selection of resistant bacteria
under drug p ressure . Our findings are comparable with the reports of other
workers . Trishkina et_ al . (1977) reported upto 75 to 90% resistance among
diarrhoeal isolates of Ej_ coli against commonly used seven antibiotic in
human chemotherapy. They found 64% of the i r strains were multiple drug
resistance. Ranganekar et_ a l . (1982) also reported 79% of E_ coli strains
resistant to one or more of the drugs . Agarwal et_ al» (1984) also reported
76.5% of multiple drug resistance among 285 E_ coli isolates from human
origin. Similarly Diwan and Sharma (1978), Young (1986), Yadava (1986),
Nandivada and Amyes (1990) have also reported high frequency of drug
resistane among pathogenic E. coli s t r a ins . This variation from study to
study reflecting both differences in local ecology and pressure of drugs
in a community as well as differences in the methodology employed for
examination of drug resistance and also differs due to the different standards
adopted for measuring the level of drug resistance because no uniform standard
for resistance level has been fixed as yet .
In the present study drug resistance among human isolates of E.
coli were found more (76.13%) as compared to laboratory animal isolates
(30%). This might have been due to the more use of antibiotics in human
chemotherapy as compared to lesser exposure of animals to these drugs. Ttiis
indicate the use of antibiotics might be creating t he selection pressure and
t h i s may select a drug resistant mutant or multi—resistant plasmids in E.
coli which may spread through different ecological modes resulting in the
emergence of high frequency drug resistant isolates among intestinal flora.
The abi l i ty of a strain to to lera te concentration depends upon the efficacy
of expression of the gene encoding for resistance to that drug and the gene
dosage. Because plasmids are small repliconc where expression is quite
efficient that is why, generally the plasmid mediated drug resistance strains
can tolerate higher levels of drug concentrations. Therefore, these strains
tolerating higher concentrations of drug suggest mediation of plasmids in
such type of res is tance .
The minimum inhibitory concentrations against 10 antibacterial dnif s usod in
th is study showed great variat ion. Level of resistance varied with different
antibiotics, for example maximum MIC values (6400 Aig/ml) were recorded
against 4 antibiotics namely amoxycillin, ampicillin, sterptomycin and kanamy-
cin, where 12, 10 and 3 strains respect ively were found to tolerate this
high amount of drugs. Six s trains were found to tolerate (3200yug/ml) drug
concentration of cotri-moxazole and eight s trains were showing MIC value
800/ug/ml for chloramphenicol. Our finding of higher MIC for various drugs
suggest the plasmid mediated drug res is tance. Plasmid encoded B-lactamase
enzyme is mainly responsible for resistance against B-lactam ant ibiot ics .
This might be the reason in above 12 strains showing very high MIC (6400
/ug/ml) against Ap/Ax.
Our findings are in agreement with the repor ts of Larlier et a l ,
1988; Paul e^ a]_, 1989; Jacoby and Sutton, 1991 and Mahipal
£t_ aX,^ 1991, who reported various types of ^-lactamases responsible for r e s i s
tance against various J3-Iactam ant ib iot ics . Another possibi l i ty of higher
level drug resistance is that sometime:; repeat i t ive sequences of the same
nucleotide clusters encoding for biosynthesis of an enhanced amount of enzyme
which can degrade the ant ib iot ics .
Auto-transferable nature of drug resistance
The prevalence of infectious R-plasmids encoding for multiple drug
resistance is the serious problem since such plasmid of enterobacteriaceae
82
especially of E_ coli easily transferred the i r resistance markers by celJ
to cell contact to other susceptible bacteria l ike Shigella ^ Salmonella, Entero-
bacter e t c . and making them resis tant to various drugs at a time. This
has made it a subject of great concern to clinicians, part icularly in curing
the disease caused by enteric bacteria both in human and animals.
In the present study nearly 40.62% E. coli strains showed transferable
R-plasmids by conjugation with E_ coli KlZ-JbZl as recipient while
59.38% of resistant s trains were found non-conjugable in nature. Thus the
treatment of bacterial infections becomes complicated due to the presence
of highly resistant strains par t icular ly among cases of GTI and UTI infections
of man and animals. Similar observation were also made by Adetosy (1980),
,4Isowagh' and Shibl (1981) and Agarwal et_ aL- (1984). They reported
39.73%, 40% and 42.6% incidences of transferable R-plasmids respect ively ,
among different studies on pathogenic E_ coli strains of animals and human
origin. However, our resul ts are at variance when compared with such
studies for example Wise et_ ah (1985), Yong et_ ah (1986) and Lamikanra
e ^ a l . ( 1 9 9 0 ) , they reported 81.5%, 60% and 54.27% incidences of transferable
R-plasmids respect ively among pathogenic strains of E_ coli isolated from
various clinical conditions of man and animals. These observations may
be due to the isolates of E. coli , taken from different sources. Further
frequency of transfer of various antibiotic resistant markers were studied
and i t was found that maximum transfer was recorded for Ap/Ax followed
by Km, Sm, Tc and Co-trimoxazole resis tant determinants, which signifies
the clinical importance of these drugs . These markers are located mostly
on plasmids or transponsons.
Elimination of drug resistance determinants in E. coli
Elimination of plasmid mediated drug resistance is of practical signi-
ficane both in chemotherapy of bacterial infections and in microbial genetics.
Loss of genetic markers from a bacter ial cell on treating it with curing
agents is an apparent indication of involvement of plasmid DNA mediating
genetic markers . Because plasmids being the smaller repl icons, are selectively
sensitive to the concentrations of curing agents than the bigger and complex
replicons l ike chromosome. This character is t ic is exploited by microbial
geneticists in studying the prevalent plasmid populations in clinical bacteria .
In the present study three curing agents namely norfloxacin (acting
on A subunit of DNA gyrase enzymes) sodium dodecyl sulphate (Surface acting
agent) and acridine orange (an intercalating agent) were used to cure non
transferable drug resistance determinants and comparative efficacy of norfloxa
cin with SDS and acridine orange was determined against both transferable
and non-transferable resis tant s t r a ins . Various concentration of norfloxacin
and acr idine orange were taken for curing of R-plasmids while 10% SDS
was used for the same. It was observed that sub-inhibi tory concentration
of norfloxacin and acridine orange could eliminate efficiently the drug r e s i s
tance markers . Our resul ts showed that norfloxacin was a bet ter curing
agent for eliminating the E^ coli R-plasmids.
R-determinants among 48% of resis tant strains were eliminated by norfloxacin
while SDS and AO could eliminate only 24% and 20% plasmids respect ively .
It was also observed that Ap and Ax were eliminated more efficiently by
noxfloxacin while Tc, Sm, Km, Co and Cm resistance markers were elimi
nated with varying frequencies.
Our finding can be correlated with Wiesser and Wiedeman (1985, 86).
They reported that 4-quinolones compounds cured plasmids more efficiently
at the highest concentration which s t i l l allows cell growth and produced
20 to 100% plasmid free ce l l s . While Hopper et al^, 1984 also reported the
abi l i t ies of 4-quinolones to eliminate R-plasmids.
84
Our findings that SDS is better curing agent than acridine orange
are supported by the reports of Kaur et_ al; (1985) and Singh & Yadava
(1988). They also reported the similar observations in the i r plasmid
curing s tudies . Out of 19 resis tant s trains possessing non-transferable
drug resistance by conjugation, 42.1% were found to be cured of their
drug resistance markers by the above curing agents. Strains resistant
to single drug did not show elimination. Maximum elimination were
observed from multiple drug resistant s t rains as compared to single
or double antibiotic res is tant s trains which shows that multiple drug
resistance is of plasmid encoded which may be transferable or non
transferable . The data revealed that the resis tant determinants for
(Ap, Ax, Sm, Tc, Cm, Km, and Co) could be eliminated in various
combinations ei ther by one or more of the curing agents. Though r e s i s
tant determinants from seven multiple drug resistance strains (7, 18,
23, 24, 26, 38, 104) bearing non-transferable drug resistance markers
could not be eliminated by any of these 3 curing agents. The nature
of drug resis tance, whether chromosomal or plasmid mediated could
not be interpreted in these strains because of the existence of many
plasmids of E_ coli which can not be eliminated by these curing agents,
as the curing depends upon the nature of the nucleotide sequence
of plasmid DNA (Wiesser and Wiedeman, 1976 and 1987; Singh and
Yadava, 1988).
The term 'pathogenicity ' denotes the abi l i ty of a parasite
to cause d iseases . Pathogenicity is a taxonomical a t t r ibute , being
the property of a species . The individual s trains of bacterial species
may, however, vary widely in the i r abi l i ty to harm the host species
and th is relat ive pathogenicity is termed as virulence. Virulence is
accordinly an at t r ibute uf a s t ra in , not a species , one may speak
bf a highly virulent , a weakly virulent or even an avirulent s t ra in . In
general, the virulence of a pathogenic species is determined by two
factors i t s invasiveness or abi l i ty to proliferate in the body of host
and i ts toxigenicitv or abi l i ty to produce chemical substances or
toxins that damage the t issue of the hos t s . But the research in past
decades about bacterial pathogenicity have been reached at th is conclu
sion that some strains are non-invasive and non-enterotoxigenic, yet
they are pathogenic and virulent . It means some other factors are
also involved in bacter ial pathogenicity.
Some plasmids mediated proper t ies of E_ coli eg. the production
of colicin (Ansari and Yadava, 1981), haemolysin (Ahmad and Yadava,
1980), haemugglutination (Minshew et_ aj_., 1978), drug resistance (Yadava
et a l . , 1982) and serum resistance (Yadava et_ al_., 1982) have been
reported to be closely associated with virulence of E_ coli s t ra ins . Coli
cin plasmids provides bacteria the means for self establishment against
a competitive struggle for existence in the environment of intestinal
t ract of man and animals by eradicating colicin sensitive microflora.
By now it is evident that l ike many other pathogenic factors, colicin
is also somehow related to increased invasiveness of the colicinogenic
organism.
In the present study 29.1% of 48 E^ col i . s trains were found
to be colicinogenic. It was observed that production of colicin among
the cases of UTI and GTI were 42.85% and 14.43% respect ive ly . The
production of haemolysins or cytolysins which are extracel lular protein- .
86
toxins, that d isrupt the membrane of erythrocytes is a property of
many bacter ia l species , including E^ col i . The production of haemolysin
is an important factor contributing to the pathogenicity of E. coli .
Gene responsible haemolysin production were located on chromosome
(K4uner et a l . ,M4AJ;^ and also on plasmids (Noegal et_ aj^, 1981). We
observed haemolysin production in 27.1% of s t r a ins . The production
of haemolysin among UTI isolates were higher (47.6%) as compared to
GTI isolates which was recorded as (11.11%). Similar findings with
varying frequency of occurrence of haemolysin have also been reported
by Smith, 1969; Elwell and Shipley, 1980; Shamim and Yadava, 1980;
Konig, 1986; Blanco et_ a]_, 1990).
Nearly 80% of the UTI infections have been reported due to
E. coli s t r a ins . Therefore, haemolysin production by E . coli of UTI
origin can-direct ly Be correlated wi th the role of haemolysin in increased
pathogencity of such strainSt
Correlation between virulence factors and drug resistance
Haemolytic act ivi ty is well known to be associated with the
pathogenicity and virulence of E^ coli s t ra ins , while colicinogenicity
help in the establishment of pathogenic bacteria in the intestinal tract
of man and animals and thus helps in the survival and multiplication
of bacter ia .
Association of haemolytic proper ty or/and colicinogenic property
with drug resistance further exaggerates the bacterial pathogenicity.
In the present investigation out of 14 haemolytic s t rains 11 (78.5%)
were found to be resis tant to one or more of the drugs . Out of 13*
87
fcolicinogenic strains 10 (76.9%) were resis tant to one or more of the
drugs. In addition to above, five s trains produced both haemolysin
and colicin and surpris ingly of t h e s e , four s t rains were also resistant
to one or more ant ib io t ics . However, very high incidences of drug
resistance was recorded among haemolytic and colicinogenic strains which
can be summarized as 78.5% and 77% respec t ive ly . Therefore, some
correlation seems to exist between drug resistance and these virulence
factors .
The abi l i ty of the E_^ coli s trains to produce haemolysins and
colicin with drug resistance is very dangerous and further complicates
the problem of chemotherapy in man as well as in animals.
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