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Microbiological Study of Neonatal Sepsis Using Automated

Blood Culture System at a Tertiary Health Care Centre

226

Original Article

1 2 3 4 5Vaibhav V. Rajhans , Sharmila S. Raut , Sanjay R. More , Vimal S. Rathod , Vivek M. Gujar

ABSTRACT

Introduction: Sepsis in neonates is responsible for highly considerable mortality, especially in

developing countries like India, where it is contributing 19% of neonatal deaths. Present study was

undertaken to study aerobic bacterial profile of neonatal septicemia by automated blood culture

systems, using BacT/ALERT 3D 60 blood culture system and miniAPI bacterial identification and

antibiotic susceptibility testing system, considering the importance of urgent and accurate

identification of the pathogen and to reduce the unnecessary therapeutic load on patients as well as

clinicians.

Aim: To isolate and identify bacterial pathogens in neonatal sepsis and to know their antibiotic

susceptibility pattern using BacT/ALERT and miniAPI systems.

Material and Methods: 157 blood samples from neonates suspected to be in sepsis were studied

during the study period using BacT/ALERT 3D 60 systems for blood culture and miniAPI system

for identification and antibiotic susceptibility of isolates.

Results: 70 blood cultures were flagged positive by BacT/ALERT. E.coli was the most common i.e

16 (22.85%); followed by Coagulase negative Staphylococcus (CoNS), isolated in 15 (21.42%),

followed by Staphylococcus aureus isolated in 14 (20%).

Conclusion: Total blood culture positivity rate was 44.58%. E. coli was the most common pathogen

isolated. By using BacT/ALERT 3D 60, 65.71% blood culture positivity was observed within 24

hours incubation and bottles flagged positive within 48 hours were 59 (84.28%). Both, the

BacT/ALERT 3D 60 and the miniAPI systems, were efficient, less time consuming and labor saving

which is essential for the clinician and more importantly for the patient.

Key words : Neonatal sepsis, Bacterial sepsis, automated blood culture, BacT/ALERT, miniAPI

INTRODUCTION:

eonatal sepsis is responsible for 30- 50%

of the total neonatal deaths in developing Ncountries. It is estimated that up to 20%

of the neonates develop sepsis and approximately

1% die of sepsis related causes. According to the

data from National Neonatal Perinatal Database,

sepsis is found to be one of the commonest causes

of neonatal mortality in India contributing to 19%

of all neonatal deaths.

There is an urgent need to know whether the baby

has sepsis to institute treatment as quickly as

possible. Confirmation of the diagnosis may take

time, and diagnostic tests are used to obtain a rapid

Address for correspondence:

Dr. Vaibhav V. Rajhans, Assistant Professor, Department of Microbiology, Government Medical College, Aurangabad,

Maharashtra - 431 001 Email id: [email protected] Ph.No: 9766003534

1Assistant Professor, Department of Microbiology, Government Medical College, Aurangabad, Maharashtra.2Professor and Head, Department of Microbiology, I.G.G.M.C. Nagpur, Maharashtra.3Professor and Head, Department of Microbiology,S.R.T.R.G.M.C. Ambajogai, Maharashtra.4Associate professor, Department of Microbiology, Dr.S.C.G.M.C. Nanded, Maharashtra.5Associate professor, Department of Microbiology, Shri V.N.G.M.C. Yavatmal, Maharashtra.

National Journal of Basic Medical Sciences | Volume 7 | Issue 4 | 2017

227National Journal of Basic Medical Sciences | Volume 7 | Issue 4 | 2017

indication of the infection status. These tests are not

perfect. Some real cases of infection will produce

negative test results, whereas some babies without

infection will test positive.

The gold standard for diagnosis of septicemia is the 1isolation of bacterial agents from the blood culture.

Newer methods of detecting bacteremia through

blood culture studies have emerged and are proven

to be most promising with better sensitivity and 2

specificity; while not sacrificing time. Continuous

monitoring demonstrates that most positive blood

cultures are detected in a relatively short period of 3time, the majority within 24 hours.

BacT/ALERT 3D is an automated culture system,

which continuously monitors for any growth in

every 10min in each bottle independently. The

equipment works on the principle of colorimetry

and gives a signal as soon as any trace of growth is 4encountered based on inbuilt set of algorithms.

The API system includes 32 wells containing

dehydrated reagent that corresponds to various

biochemical reactions using which most of the

microorganisms can be processed using rapid strips 5

in just 4-5 hours.

Adjusting antibiotic therapy according to the

results of blood culture findings leads to a

narrowing of antibiotic therapy. Thereby, the

emergence of antibiotic resistance can be avoided

or delayed. Antibiotic resistance may be reversed if

the antibiotic use is decreased.

Hence, present study was undertaken to study

aerobic bacterial profile of neonatal septicemia by

automated blood culture systems, using

BacT/ALERT 3D 60 blood culture system and

miniAPI (Figure 1) bacterial identification and

antibiotic susceptibility testing system, both by

Biomerieux.

Figure 1. The BacT/ALERT 3D 60 system and

miniAPI system

Dr. Vaibhav V.Rajhans, et.al : A bacteriological study of sepsis in neonates using BacT/ALERT and miniAPI at a tertiary care centre


MATERIAL AND METHODS:

Study Design:

Blood culture sample of neonates were subjected

for processing under automated blood culture

system BacT/ALERT 3D 60 and positive blood

culture samples were further processed by using

miniAPI system for bacterial identification and

antibiotic susceptibility testing of isolates.

This Observational study was conducted in the

microbiology laboratory of a tertiary care hospital

over a period of one and a half year (January 13 to

June 14). Blood samples from clinically diagnosed

or suspected neonates with septicemia (0-28 days

of age) of both the sexes during the study period

were collected and processed.

61-2 ml of blood sample from the each neonate was

collected and inoculated in special pediatric blood

culture media bottle - The BacT/Alert PF bottle

containing 20ml broth. Collection of blood was 7

done using all standard aseptic precautions. After

blood was collected in the syringe using the needle;

same needle was used to inoculate BacT/Alert 6bottles.

The Bact/Alert bottles possess

a plastic top cap which is to be

removed by breaking just

before inoculation. Under the

plastic cap, there is a rubber

septum which is not sterile

and hence was cleansed with

70% alcohol or iodine

solution and allowed to dry

before inoculation. Blood was

introduced into the bottle only

after the drying of rubber 8septum.

The inoculated bottles were loaded into the

BacT/Alert 3D/60 system incubator and incubated 9,10

for a maximum period of 5 days. The bottles

contain media and atmosphere which promote the

recovery of a wide variety of microorganisms

without venting. Standard BacT/Alert software

provided with the system was used for recording

the results. Bottles flagged as negative after the

maximum period of 5 days were unloaded from

incubator and subcultured for a final confirmation.

Bottles flagged as positive were unloaded from the

system and processed further as follows -

Appropriate amount of fluid was aspirated from the

bottles with sterile precautions. This fluid was used

for smear preparation for Gram staining and

subculture; for which Nutrient agar, 5% Blood agar,

MacConkey agar were inoculated and incubated.

Based on colony morphology of the growth on

subcultured media plates, Gram staining of the

smear from colony, Catalase test and Oxidase test;

selection and processing of miniAPI strips for

identification and antibiotic susceptibility of that

organism was done as per manufacturer's

instructions. (Figure 2)

Figure 2.For selection of miniAPI strips.



The miniAPI strips are basically made up of plastic having 32 wells or depressions; called as cupules, in each strip. The ID strips are for the identification of the organism and ATB strips are for the antibiotic susceptibility of that isolate. ID strips are provided with biochemicals and other reagents in dried and preserved form in the cupules. ATB strips are provided with the antibiotics in dried preserved form in the cupules.

After the selection, the ID strips as well as the ATB strips were inoculated according to the guidelines provided by the manufacturer.

The miniAPI strips used are

Identification strips (ID strips)Rapid ID 32 E, ID 32 GN, ID 32 STAPH, rapid ID 32 STREP.

Antimicrobial susceptibility testing strips (ATB strips)Rapid ATB E 4, ATB PSE 5, ATB STAPH 5, ATB STREP 5, ATB ENTEROC 5.

The strips were then read using the miniAPI system.

OBSERVATION AND RESULTS:

Out of total 157 blood samples, 99 (63.05%) samples were collected from male and 58 (36.94%) were from female neonates. Out of them, 70 (44.58%) were flagged as positive by BacT/ALERT 3D 60 system and 87 (55.41%) were negative. (Figure 3)

Figure 3. Blood culture report by BacT/ALERT

As far as Time duration taken by BacT/ALERT 3D

60 is concerned, of the total 70 culture positive

samples, 46 (65.71%) were flagged positive within

first 24 hours of incubation. Additional 13

(18.57%) were flagged between 24-48 hours and 11

(15.71%) samples were flagged positive after 48

hours. In total, 59 (84.28%) positive blood samples

were detected within 48 hours of incubation.

(Figure 4)

Figure 4. Time taken to flag the bottles as

positive by BacT/ALERT 3D 60 system

Sex wise distribution of culture positive cases

revealed positivity more in male, i.e. 44 (62.85%)

out of 70; while Female neonates comprised of 26

(37.14%). The male to Female ratio observed was

1.69:1. (Figure 5)

Figure 5. Sex wise distribution of culture

positive cases

Of the total 70 isolates, E.coli was the most

common i.e 16 (22.85%); followed by Coagulase



negative Staphylococcus (CoNS), isolated in 15

(21.42%), followed by Staphylococcus aureus

isolated in 14 (20%), Klebsiella pneumoniae in 10

(14.28%), Pseudomonas aeruginosa in 6 (8.57%),

En t e rococcus f aeca l i s -3 (4 .28%) and

Streptococcus pyogenes in 2 (2.85%). (Figure 6)

Figure 6. Organism wise distribution of isolates

from positive blood samples

Antibiotic susceptibility patterns:

Among Coagulase negative Staphylococci

(CoNS), maximum susceptibility was found to the

Quinupr i s t in -Da l fopr i s t in (100%) and

Vancomycin (100%) followed by Minocycline

(93.33%) and Levofloxacin (86.66%). Least

susceptibil i ty by CoNS was shown to

Erythromycin (40%) and to Penicillin (13.33%).

Staphylococcus aureus showed maximum

susceptibility to the Quinupristin-Dalfopristin

(100%) and Vancomycin (100%) followed by

Tetracycline (85.71%) and Rifampicin (78.57%).

Cotri-moxazole and Penicillin were the drugs to

which Staphylococcus aureus isolates were least

susceptible, 42.85% and 21.42%, respectively.

E.coli isolates were most susceptible to Piperacillin

+ Tazobactum (93.75%) and Nitrofurantoin

(93.75%), followed by Ticarcillin + Clavulanic

acid (87.5%). Least susceptibility of E.coli isolates

was observed to Cefepime (18.75%) and

Ampicillin (12.50%).

Among Klebsiella pneumoniae isolates, maximum

susceptibility was shown to Piperacillin +

Tazobactum (90%) and Piperacillin (80%)

followed by Ciprofloxacin (70%) and Ceftriaxone

(70%). Least susceptibility was shown to

Ampicillin (20%), Cefoxitin (20%) and Cefazolin

(10%).

Pseudomonas aeruginosa were most susceptible to

Imipenem (100%), Meropenem (100%) and

Piperacillin + Tazobactum (83.33%) followed by

Amikacin (66.66%) and Gentamicin (66.66%).

These isolates were least susceptible to Cefepime

(33.33%) and Ticarcillin (16.66%)

Among the 70 neonates who were diagnosed to be

having septicemia by positive blood cultures, 58

(82.85%) improved and were discharged from the

wards. 12 (17.14%) neonates succumbed to their

illness.

DISCUSSION:

Globally, an estimated 4 million babies die in the

first 4 weeks of life (the neonatal period) every 11

year and half of them die in their 1st 24 hours. It

accounts for 40% of under 5 mortality. 98% of these 12

deaths occur in developing countries.

In India, the Sample Registration System estimates

of neonatal mortality for the year 2010 is about 25

per 1000 live births in early neonatal period (0-7

days) with 28 for rural areas and 15 for urban 12areas.

In our study, out of 157 blood samples, 44.58% i.e.

70 blood samples were confirmed as positive. 13Ghanshyamet al (2002) in their study, observed

42 .00% pos i t i v i ty i n b lood cu l tu re s .



14Ananthakrishnanet al (2009) in their study

concluded that 40.6% samples were positive in 15blood culture. According to Gandhi S et al (2013)

out of 238 samples studied, blood culture was

positive in 76 cases (32%).

16In the study by Wadud A et al (2009) , out of 4915

sample, 707 (14.38 %) were positive by

BacT/ALERT system. In the study by Kennedy GT 3

et al (1995) , Four thousand bottles were analyzed,

of which 477 (11.92%) were positive by

BacT/ALERT system. The percentage of positivity

in our study is higher as compared to the above

mentioned studies where BacT/ALERT system

was used.

Out of 70, for11 (15.71%) samples the

BacT/ALERT system needed more than 48 hours to

detect as positive while 59 (84.28%) positive

samples were detected within 48 hours of

incubation.

3Kennedy GT et al (1995) mentioned in their study

that, 29% positive blood cultures were detected in

less than 12 hours, 68% by 24 hours, 81% by 48

hours, and 87% by 72 hours.

Our observations about detecting 84.28% positive

samples in less than 48 hours duration is

comparable with the study by Kennedy GT et al.

7In the study by Jyothi P et al1 , among the culture

positive cases, there were 86 (65.5%) male and 45

(34.5%) female neonates with the male-to-female

ratio of 1.9:1.

18In the study by HaseebM et al (2014), analysis

with respect to sex of the baby showed that 65 cases

(61.9%) were males and 40 cases (38.09%) were

females with a male: female ratio of 1.62: 1.

19Gheibiet al (2008) observed the male to female

ratio as 1.67:1 in a study of 227 cases. Our 19

observation is similar to that of Gheibiet al .

20Khatuaet al postulated that the factors regulating

the synthesis of gamma globulins are probably

situated on the X chromosome. Presence of one X

chromosome in the male infant thus confers less

immunological protection compared to female 21counterpart. Singh stated that male infants are

around 4 times at increased risk to develop sepsis

compared to females. Term male infants have an

approximately two fold higher incidence of

septicemia than term females

17According to the study by Jyothi P et al , etiology

of the 131 isolates included Gram-negative bacilli

(73/131, 55.7%) and Gram-positive cocci (58/131,

44.3%). Klebsiella spp. (30.5%) and coagulase-

negative Staphylococci (CONS) (27.5%) were the

most common Gram-negative and Gram-positive

organisms respectively.

1Gandhi S et al studies observations are that E.coli

(21.25%) was the most common isolate,

Staphylococcus aureus (20%) and Klebsiella

pneumoniae (20%) were isolated in equal

proportion. The next isolate was CoNS (11.25%).

The observations of present study correlate well 1 22

with the study by Gandhi S et al . Freeman et al

reported a rise in CONS septicemia in neonates

from 25% to 68.8% during 1975-1982 at their

hospital. Data from a tertiary care hospital in India

for year 2001-2002 revealed that CONS were the

commonest gram positive bacteria isolated from 23

cases of neonatal septicemia.

24Although in a study by Baumgart S et al , CONS

were found to be the most frequent blood 25contaminant; Hammerberg et al stated that after

the careful cleaning of venipuncture site, the

growth of CONS in blood culture of specimens of

premature neonates indicates bacteremia rather

than the skin contaminant in the vast majority of

cases.



In the present study, 6 (8.57%) strains of

Pseudomonas aeruginosa (P. aeruginosa) were 26 27isolated. Guhaet al and Movahedianet al isolated

16.2% and 36% P. aeruginosa in their studies;

respectively. Pseudomonas infection is rarely 28

perinatally acquired however, it is among the

more common gram-negative organisms causing 29nosocomial sepsis in NICU patients.

Prior to antibiotic era, the mortality from

septicemia was 90% but it declined to 24%-58% 30after antibiotics came into use. The varying

microbiological pattern of neonatal septicemia

warrants the need for an outgoing review of

causative organisms and their antibiotic sensitivity

pattern. The inability to be certain of infection,

coupled with non- specific signs of life threatening

illness in neonates have resulted in widespread use

of antibiotics, aggravating the problem of antibiotic

resistance.

17Jyothi P et al observed that Gram-positive isolates

had sensitivity of 91% to Linezolid, 68% to

Tetracycline, 64% to Piperacillin / Tazobactam,

Erythromycin, and 52% to Ciprofloxacin.

13Ghanshyamet al reported that 50% of the

Klebsiellaand E. coli isolates were sensitive to 27

Cefotaxime. A study by Movahedianet al revealed

a very high degree of resistance in gram negative

organisms not only to commonly used antibiotics,

but also predominantly to broad spectrum 19cephalosporins. Gheibiet al reported high

resistance to Cefotaxime (67.5%), Ceftriaxone

(65.3%) and Ceftazidime (64.3%) among gram

negative organisms.

17Jyothi P et al observed that best overall sensitivity

among Gram-negative isolates was to Imipenem

(93%), followed by Amikacin (52%) and

Netilmicin (41%).

Although positive blood culture is the gold

standard in the diagnosis of neonatal sepsis; in the

absence of proof of sepsis, many clinicians and

even some neonatologists feel obliged to continue

antibiotics for a full 10 days course. If the baby is

not infected he or she is being subjected to

unnecessary treatment. There is also danger of

removing useful or susceptible organisms and

encouraging resistant ones. If this occurs we shall

reach a stage to go on to use more expensive

antibiotics, or we have no more chance to use new 27

drug combinations.

Strict infection control in neonatal units, hand

washing combined with judicious policy for

antibiotic therapy are the main solution to this

problem. It will be important, however, to continue

surveillance of neonatal sepsis in order to follow

closely changes in trends and risk factors, to obtain

information for empiric antibiotic therapy and to

react rapidly in case of major changes in

susceptibility patterns and occurrence of 27outbreaks.

LIMITATION OF THE STUDY:

This is an observational study. As the study group

was restricted to neonates only, sample volume for

blood culture was a crucial issue. Neonates

admitted in the hospital were subjected to various

investigations under biochemistry, microbiology

and pathology (hematology) lab. As sample

collection is not that easy in neonates, mostly due to

small size of blood vessels and most neonates being

investigated are underweight. Multiple pricking is

not advisable hence blood collected in single prick

was distributed for three labs as biochemistry,

microbiology and pathology (hematology). As

sample received in microbiology was less in

volume, it was subjected only for automated blood

culture system; conventional blood culture system

being excluded from the study. And for the very

same reason, comparison of blood culture by

conventional and modern system was not possible.



CONCLUSION:

Total blood culture positivity rate was 44.58%. By

using BacT/ALERT 3D 60, 65.71% blood culture

positivity was observed within 24 hours incubation

and bottles flagged positive within 48 hours were

84.28%.

All 70 positive blood cultures were monobacterial.

Amongst gram positive bacteria CoNS and in Gram

negative bacteria E.coli were predominant. E.coli

isolates showed maximum susceptibility to

Piperacillin + Tazobactum combination and least

susceptible to Ampicillin. CoNS were maximum

susceptible to Vancomycin and Quinupristin -

Dalfopristin while least susceptible to Penicillin.

Time for detection of positivity by BacT/ALERT in

blood cultures was short. Both these systems, the

BacT/ALERT 3D 60 and the miniAPI, were

efficient, less time consuming and labor saving

which is essential for the clinician and more

importantly for the patient.

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Received on 28/02/2017 Revised on 06/04/2017 Accepted on 06/06/2017

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