izabela maria kern-zdanowicz‚ącznik nr... · 2018-12-12 · izabela kern-zdanowicz załącznik...

Izabela Kern-Zdanowicz Załącznik nr 3 Autoreferat w jęz. angielskim

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SUMMARY OF PROFESSIONAL ACCOMPLISHMENTS

1. Name

Izabela Maria Kern-Zdanowicz

2. Education and obtained scientific titles

1997 – Doctor of Philosophy degree biochemistry

Institute of Biochemistry and Biophysics Polish Academy of Sciences

PhD thesis titled: „Secretion of streptokinase of Streptococcus equisimilis H46A in

bacterial homologous and heterologous systems, Supervisor: Piotr Cegłowski D. Sc.

Ph. D.

1986 – Master of Science title in biology/ specialization: molecular biology, Faculty of

Biology, University of Warsaw; Supervisor: prof. Ewa Bartnik

3. Employment in scientific institutions

2007 – till now employment as a research assistant in the Institute of Biochemistry

and Biophysics Polish Academy of Sciences in Warsaw,

1997 – 2007 employment as a research adjunct in the Institute of Biochemistry and

Biophysics Polish Academy of Sciences in Warsaw,

1992 – 1997 employment as a research assistant in the Institute of Biochemistry and

Biophysics Polish Academy of Sciences in Warsaw,

1990 – 1992 employment as a research assistant in Department of Pharmaceutical

Microbiology of Warsaw Medical University of

1986 – 1990 a biologist, Research and Development Center of Biotechnology in

Warsaw

4. Scientific achievement* according to the current regulations (article 16,paragraph 2 of the bill

enacted on March 14, 2003, about scientific degrees and a scientific title as well as degrees and a title in arts (Dz. U. 2016 r. poz. 882 ze zm. w Dz. U. z 2016 r. poz. 1311.):

a) the title of the scientific achievement :

The structure, dissemination, and evolution of plasmids conferring antibiotic resistance

b) Publications comprising scientific achievement: IF, impact factor is shown for the publication year The impact factor publications comprising scientific achievement: IF 19,073


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1. Dmowski M., Gołębiewski M., Kern-Zdanowicz I.#. 2018. Characteristics of the conjugative transfer system of the IncM plasmid pCTX-M3 and identification of its putative regulators. J. Bacteriol. 200 (18): e00234-18, #

corresponding author

IF2016/2017 3,143

2. Wasyl D *, Kern-Zdanowicz I.*, Domańska-Blicharz K, Zając M, Hoszowski A. 2015. High-level fluoroquinolone resistant Salmonella enterica serovar Kentucky ST198 epidemic clone with IncA/C conjugative plasmid carrying blaCTX-M-25 gene. Vet Microbiol.;175(1):85-91. * joint first authorship

IF2015 2,564

3. Zienkiewicz M., Kern-Zdanowicz I., Carattoli A., Gniadkowski M., Cegłowski P. 2013.Tandem multiplication of the IS26-flanked amplicon with the blaSHV-5 gene within plasmid p1658/97. FEMS Microbiol Lett. 341:27-36. IF2013 2,723

4. Zienkiewicz M., Kern-Zdanowicz I., Gołębiewski M., Żylińska J., Mieczkowski P.,

Gniadkowski M., Bardowski J. and Cegłowski P. 2007. Mosaic structure of p1658/97, a 125-kilobase plasmid harbouring an active amplicon with the extended-spectrum β-lactamase gene blaSHV-5. Antimicrob Agents Chemother. 51:1164-1171. IF2007 4,39

5. Gołębiewski M., Kern-Zdanowicz I. #, Zienkiewicz M., Adamczyk M., Żylińska J., Baraniak A., Gniadkowski M., Bardowski J. and Cegłowski P.2007. Complete nucleotide sequence of the pCTX-M3 plasmid and its involvement in spread of the extended-spectrum β-lactamase (ESBL) gene blaCTX-M-3. Antimicrob Agents Chemother. 51: 3789-3795. #


IF2007 4,39

6. Nowakowska B., Kern-Zdanowicz I. #, Zielenkiewicz U. and Cegłowski P. 2005. Characterization of Bacillus subtilis clones surviving overproduction of Zeta, a pSM19035 plasmid-encoded toxin. Acta Biochim Polon., 52: 99–107. #


IF2005 1,862

b) Discussion of the scientific goals of the publications comprising the scientific achievement and potential of its further use

The papers included in the achievement are indicated in bold, those which I was a co-author and are cited only, are indicated with bolded numbers. .

Infections caused by strains of bacteria which are resistant to antibacterials, is now a

major problem of contemporary medicine. Increasing resistance to various groups of

therapeutics and the dissemination of the resistance in bacterial populations pushed the

World Health Organization (WHO) to establish the global priority list of antibiotic-resistant


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bacteria. This list helps to guide research, discovery, and development of new antibiotics and

also the improvement of already existing drugs (1). The surveillance of bacteria resistant to

antibiotics originated from infections associated with health care and with animal husbandry

is systematically on-going. Also, the use of antibiotics is monitored. It is estimated that if a

fundamental change in the discovery of new antibiotics or in the development of an

alternative antibacterial therapy is not made by 2050, ten million lives a year can be lost due

to the infections of practically incurable infections caused by drug-resistant bacteria (2). To

the group of the highest priority, which is the group of the critically important pathogens,

are included: carbapenem-resistant Acinetobacter baumannii and Pseudomonas aeruginosa,

as well as carbapenem-resistant or 3rd generation cephalosporin-resistant bacteria of the

Enterobacteriaceae family.

This study includes a set of six papers. Their main aim was the characterization of the

plasmids isolated from the bacterial strains which originated from hospital infections. Three

of the four plasmids analyzed (p1658/97, pCTX-M3 and p1643_10) confer resistance to 3rd

generation cephalosporin. These were isolated from representatives of the

Enterobacteriaceae family and classified as critically important pathogens according to the

WHO priority list. The research conducted on the fourth plasmid, pSM19035, chronologically

the earliest isolated one, was concentrated on selected aspects of its biology which could be

used in the search for new antibacterial therapies.

Plasmids, extrachromosomal DNA molecules, able to autonomously replicate are

important elements of the mobile gene pool, they enable bacteria the quick variability and

adaptability to the changing environment. Evolution of bacterial strains is constantly on-

going but selection of these which are resistant to antibiotics results in acceleration of

bacterial evolution. Plasmids are common in genomes of bacteria and archea (3, 4), where

they can constitute even up to 45% of genomic DNA (5). They are also found in eukaryotic

cells – in mitochondria and cytoplasm of fungi and in mitochondria of plants (6, 7). Plasmids

participate in horizontal gene transfer (HGT) – one of its mechanisms is a conjugative

plasmid transfer. One of the consequences of the latter is a dissemination of

chemotherapeutic resistance genes in the bacterial population. Of main importance are

conjugative plasmids which reside in cells of bacteria living in hospital environments or on

animal farms, i.e. wherever selective pressure in the form of antibiotics is used more

frequently and strongly than in other ecological niches. From such environments, large

plasmids bearing genes conferring resistance to many antibacterials are usually isolated and

these are the environments where the spread of antibiotic-resistant bacteria resistance is

monitored (8, 9).

Majority of plasmids exists in bacterial cells as covalently closed circles; however,

linear plasmids are also known (10). Plasmids significantly differ in size – there are small,

cryptic plasmids that comprise only a region responsible for plasmid replication, a replicon,

and also large plasmids, 100 – 200 kb in size. The largest are megaplasmids present in

Streptomyces - up to 1,8 Mb in size (11). Plasmid replication is precisely regulated in the host

cell and the copy number in which the plasmid exists in the host cell and the range of hosts


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where the plasmid can replicate are fundamental characteristics for each plasmid. The

general rule is that small plasmids are present in a host cell in a high copy number and large

plasmids have a low number of copies (< 10). The plasmid can have a narrow host range

when its hosts are closely related bacteria or a broad host range when it can replicate in

phylogenetically distant bacteria (12, 13). Plasmids with the same replicon cannot stably co-

exist in a cell in the absence of external selection. One cell may contain a few plasmids if

they bear different replicons i.e. belong to different incompatibility groups (Inc).

Large plasmids are present in the cell in a low number of copies; however, they are

stably inherited in bacterial population due to encoded maintenance systems (14, 15). The

simplest, site-specific recombination system resolves plasmid oligomers, raised during

replication, into monomers, thus increasing the number of plasmid units to be segregated

into dividing cells. The second is a partition system, which leads to a physical movement of

plasmid copies to the cell regions which will the centers of the daughter cells after the cell

division (16). The third system, the addiction system, addicts the plasmid host cell from the

plasmid. In its classical form it comprises the stable toxin and the labile antidote, both

plasmid encoded (17). The loss of the plasmid results in the lack of de novo synthesis of

plasmid encoded proteins. The antidote still present in the cytoplasm of a bacterium is

degraded, this results in the release of the toxin from the complex in which it was inactive or

enables synthesis of the toxin. The toxin interacts with the cellular target, which leads to cell

death and the elimination of the plasmid-free cells from the population.

The fourth accessory system which confers stability of the plasmid in bacterial

population is a conjugative transfer system. This system recognizes each plasmid-free cell as

a potential recipient of the plasmid. This system is even more important – it is responsible

for the spread of the plasmids among bacteria. The majority of large plasmids present in

bacteria are conjugative ones. Only in Actinobacteria the dsDNA is transported in

conjugation from the donor to the recipient and the single protein participates in the

transfer (18). In other bacteria, and in archea, DNA is transported as a single strand, in a

complex with proteins. The transporter is constituted of the large protein complex, Mpf

(mating pair formation), responsible for formation of mating pair between the donor and

recipient. The Mpf complex is evolutionary related to the type IV protein secretion system

(T4SS), which is involved in the transport of proteins important in the virulence of some

pathogenic gram-negative bacteria into eukaryotic cells (19). DNA processing, necessary for

transfer, involves the nicking of one strand of the plasmid DNA at the specific nic site within

the origin of transfer (oriT) by relaxase. This enzyme remains covalently bound to the 5’ end

of DNA and together they are transferred to the recipient cell. The action of relaxase and the

Mpf complex are connected by the coupling protein (CP) (20).

Among T4SS, including plasmid Mpf systems, two phylogenetic groups, IVA and IVB,

can be distinguished. The first group, IVA, includes, for example, the conjugation system of

the F plasmid of the IncF group, which shows evolutionary similarities to the IVA prototype,

namely, the oncogenic T-DNA transfer system of Agrobacterium tumefaciens. However, the

second group, IVB, includes those which are homologous to Dot/Icm proteins constituting


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the transport system of proteins involved in a virulence of Legionella pneumophila, the

causative agent of the Legionnaires' disease. This group are includes the conjugation system

of the IncI1 group plasmids, such as R64 or ColIb-P9, and the conjugation system of pCTX-

M3, I am analyzing, which will be described below. T4BSS systems are less thoroughly

analyzed than T4ASS. Another classification of the Mpf systems of conjugative plasmids

distinguished eight MPF groups and it is based on phylogeny of the VirB4 protein, the only

protein homologues of which were found in all known conjugation systems (21).

To be transferred during conjugation, the plasmid must comprise the oriT sequence

as well as both complexes, the relaxase and Mpf, and also the coupling protein have to be

synthesized in the host cell. Conjugative plasmids code for all these elements. However,

there are also other mobilizable plasmids which can utilize Mpf or Mpf and CP of the

conjugative plasmid co-residing in one host cell, so the conjugative plasmid serves as a

helper plasmid to the mobilizable one. Plasmids which are mobilized to transfer may

comprise the oriT only if it is compatible with the conjugation system of the helper plasmid

present within the bacterial cell (22). Similarly, the structures present in genomes of

bacteria, such as genomic island or pathogenicity islands, can be mobilized when they

comprise the oriT region compatible with conjugative plasmids or ICEs (integrative

conjugative elements, formerly called conjugative transposons) co-existing in the cell (23,

24).

In the plasmid, genetic modules which encode the ability to replicate, to be stably

maintained in the bacterial population, and to be conjugatively transferred, constitute the

plasmid backbone which is conserved in evolution. The variable, accessory regions which are

acquired during the evolution of the plasmid are called a load (25). This load, even if it

causes the metabolic burden in the host cell, plays an important role in increasing the

adaptability of the host.

As a load, plasmids bear genes conferring resistance to major antibacterials such as

β-lactams, aminoglycosides, tetracyclines, chloramphenicol, sulphonamides, trimethoprim,

macrolides, and quinolones (26). Also, virulence factors, which increase the selective

advantage of the host cell, are encoded in plasmids, frequently within plasmid-encoded

pathogenic islands (27, 28). Simultaneously, other mobile genetic elements, ISs (insertion

sequences) and Tns (transposons) can integrate into plasmids, which are present in the cell.

These elements are responsible for mobilization of antibiotic resistance genes from bacterial

chromosomes (29). Also, integrons should be mentioned as structures which „gather” gene

cassettes. The integrons have properties of specific, natural expression systems, which

together with other mobile elements, are responsible for the spreading of the whole sets of

resistance genes (30, 31).

History of discovery of plasmid-encoded resistance genes dates back to the early

1950s in Japan. After the war, there were outbreaks of dysentery caused by Shigella

dysenteriae, which were in 80% of cases insensitive to sulphonamides, the antibacterials

used from 1937 (32). And in the mid-fifties, 10% of these strains were additionally resistant

to streptomycin, chloramphenicol, and tetracycline, antibiotics discovered only 10 years


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earlier. In 1959 in Japan it was shown that these resistances could be transferred to other

representatives of Enterobacteriaceae, and this phenomenon was linked to episomes (33).

Thus, it was shown that plasmids are important in the dissemination of antibiotic resistance

among bacteria, and that this happens via conjugation in the same way as the transfer of the

F plasmid, discovered by Tatum and Lederberg in 1946.

In 1928, when Fleming discovered penicillin, the “golden era” of antibiotics began

and lasted until the end of the 1970s. Antibiotics were undeniably the most effective

chemotherapeutics introduced to treatment in the 20th century. Unfortunately, from 1980s,

a 90% decline in new approvals of systemic antibiotics by the US Food and Drug

Administration has been observed (2). It is worth mentioning that even penicillin could not

be produced in the large scale when, in the 1940s, the first strain producing penicillin-

degrading enzyme was isolated, and, in 1963, the E. coli strain producing plasmid-encoded

TEM (TEM-1) penicillinase was isolated (34).

After its discovery in the early 1950s, erythromycin, a macrolide antibiotic, began to

be used for treatment of cases of infections where it was impossible to use penicillin.

Consequently, this led to emergence and selection of macrolide - resistant gram-positive

strains. In streptococci, enterococci, and staphylococci, the genes conferring resistance to

macrolides, lincosamides, and streptogramines (cross resistance MLSB) and also to

vancomycin, chloramphenicol, and aminoglycosides (kanamycin and streptomycin) is

encoded on plasmids of the Inc18 group, such as pIP501, pAMβ1, pRE25, pSM19035 and

others (35, 36). These plasmids are a broad-host-range and replicate in gram-positive

bacteria of low G+C content in their chromosomal DNA. Some of the Inc18 plasmids are

conjugative. Bacteria bearing the Inc18 plasmids were and still are isolated from hospital

environments, from animal farms, and from employees of these farms (36–38). Interestingly,

plasmids of this group are involved in the transfer of vancomycin resistance to

Staphylococcus aureus: the vanA gene, present in Tn1546, was most likely transferred to

methicillin-resistant S. aureus (MRSA) by vancomycin-resistant enterococci, Enterococcus

faecalis and Enterococcus faecium (VRE) (39, 40). In VRE, Tn1546 is encoded in the Inc18

plasmids (35, 41, 42). It should be mentioned that vancomycin is the last resort antibiotic

used to treat infections caused by MRSA, VRE, and penicillin-resistant pneumococci. In

recent years (2013-2016), an increase of invasive (isolated from blood, cerebrospinal fluid,

and other physiologically sterile parts of the body) vancomycin-resistant enterococci strains

has been observed worldwide (8).

Resistance of bacteria to clinically important antibiotics is a growing problem. Since

the discovery of TEM-1, the first β-lactamase, more than 1300 β-lactamase variants were

described. 167 variants of β-lactamases from the TEM family were isolated

(http://www.mbled.uni-stuttgart.de/), also β-lactamases of several dozen other families

emerged, some in numerous variants, of which the most disseminated are SHV, CTX-M, KPC,

NDM. Besides the amino acid sequences, they differ mainly in the spectrum of the β-lactam

groups they hydrolyze (43). β-lactamases able to hydrolyze penicillins, cephalosporins, and

monobactams, so virtually all β-lactams, except cephamycins and carbapenems, such as CTX-


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M family β-lactamases, constitute the group of extended spectrum enzymes (ESBL). Genes

coding for ESBLs and carbapenemases, such as KPC and metallo-β-lactamase NDM-1,

observed more and more often since 2009, are found on plasmids of various Inc groups: a

broad-host-range plasmids, as IncA/C, or a narrow-host-range plasmids, as IncF, IncL/M,

IncN and IncI1 (44, 45). The most frequently isolated plasmid worldwide is the IncF plasmid

bearing the blaCTX-M-15 in the virulent E. coli O25:H4-ST131 strain (26, 46). The ESBL

production is so far the most important clinically and the most common mechanism of

resistance of bacteria of the Enterobacteriaceae family (47).

Multidrug resistant (MDR) bacteria, especially those with resistance encoded on

conjugative plasmids, are one of the current threats to public health (1). For example, the

bacteria which are NDM-1 producers are often resistant to all known antibiotics used for

treatment, though the blaNDM-1 gene confers resistance to all β-lactams, except aztreonam;

blaNDM-1 is located on plasmids that co-harbor genes encoding multiple resistance

determinants such as ESBLs or other carbapenemases, or genes conferring resistance to

aminoglycosides or other chemotherapeutics (48). In the case of infection caused by NDM-1-

or carbapenemase-producing Enterobacteriaceae which are MDR, colistin is the last resort

antibiotic. However, after isolating the E. coli strain with a plasmid with the mcr-1 gene in a

pig farm in 2011 in China, conferring colistin-resistance, and dissemination of plasmids with

this gene in many countries worldwide, the epidemiological situation became dramatic (49,

50).

From the beginning of research on bacterial resistance to antibiotics, one of the

trends concerned R (resistance) factors, thus plasmids which bore antibiotic resistance

genes. Biology of such plasmids quickly became the hot topic for microbiologists.

The pSM19035 plasmid (28 975 bp) of the Inc18 group was one of the first plasmids

isolated from erythromycin-resistant (MLSB) Streptococcus pyogenes (strain 19035). This

plasmid was the object of my mentor's, Piotr Cegłowski (Ph. D., D. Sc.), research interest at

the time I joint his group and became interested in plasmids. pSM19035 was interesting

structurally, it looked like being caught in the course of evolution: in 80% this plasmid

consisted of long inverted repeats, which coded for replication and maintenance modules.

The first unique region comprised the ermB gene and the sequence of the second unique

region was unknown. The research on biology of this plasmid was done on stable,

spontaneous pSM19035 derivative, the pDB101 plasmid, and also on pBT233, the plasmid

comprising the single arm of repeated sequence (51, 52). I sequenced the missing part of

pSM19035 and assembled its whole sequence (GenBank AY357120.1). I found that the

conjugative operon was not complete, comprised only of 10 out of the 15 genes present in

the conjugative plasmids of the Inc18 group (pIP501 and pAMβ1). Also, the oriT sequence

was missing, so pSM19035 was not a mobiliazable plasmid. The pSM19035 plasmid

comprised the maintenance systems of atypical structure: the ω gene, encoding the DNA

binding protein, an element of the partition system δ-ω, was located in the other

transcriptional unit than the δ gene, but in one operon with the ε and ζ genes encoding a

toxin-antidote system (TA) (53, 54). The Zeta toxin, the product of the ζ gene, had a


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bacteriostatic effect on gram-negative bacteria and bactericidal on gram-positives of the

type Firmicutes (55). However, the mechanism of this action was unknown, and the available

Zeta crystal structure was unique among other structures.

On the base of knowledge on of the other TA systems functioning, it was

hypothesized that the Bacillus subtilis cells, which would survive induction of the Zeta toxin

production, with unchanged sequence of the ζ gene, would bear the mutation in a gene

whose product was either the toxin target or was involved in the toxin action. For this

purpose, the B. subtilis strain, with the ζ gene under control of the xylose operon promoter,

integrated into the its genome, was used in this experiment. Also, the assumption was made

that mutations obtained within the ζ gene would be located in regions important for the

activity of this toxin. Recognition of cellular proteins which interact with the Zeta toxin

would allow identification of the new, important physiological processes of bacterial cells,

different from those already known. This would be the target of the new antibacterial

therapy – it would be a response to the problem of increasing antibiotic resistance among

bacteria. The results of this experiment were presented within the paper Nowakowska et

al., 2005, pos. 6 (56). Among several hundred analyzed B. subtilis mutants which survived

the induction of toxin production, none had the unchanged sequences of the ζ gene, and the

xylose promoter. All mutations were in the 5’ part of the ζ gene, therefore, the conclusion

about the significance of the amino terminus for Zeta toxicity was made. One of the isolated

mutants (mutation in ζ A248G resulting in the substitution Y83C in the protein) produced

Zeta with lowered toxicity and such a mutant was used in the experiments on functioning of

the Epsilon-Zeta TA system (57).

The decreasing effectiveness of antibacterial therapies, associated with increasing

resistance to chemotherapeutics of the strains recovered from hospitals, interested many

microbiologists about the huge impact the plasmids made on bacteria. In 1999, the research

group working on biology of plasmids was established, under the leadership of Piotr

Cegłowski (Ph. D., D. Sc.). In the co-operation with prof. Waleria Hryniewicz and Marek

Gniadkowski (Ph. D.) from the National Medicines Institute, the plasmid - bearing ESBL

producer Enterobacteriaceae strains originating from atypical hospital outbreaks were

chosen. After the untimely death of my mentor in 2004, the plasmid genomics group

continued and extended its research under my supervision. The plasmids p1657/98 and

pCTX-M3 were sequenced and analyzed ((58) papers Zienkiewicz et al., 2007, pos. 4; (59)

and Gołębiewski et al., 2007, pos. 5), and biology of each plasmid described ((60)

Zienkiewicz et al., 2013, pos. 3; (61) Dmowski et al., 2018, pos. 1), these papers were

included in this set and will be discussed below.

The p1658/97 plasmid was isolated from one of the twelve E. coli strains producing

ESBL of the SHV family, collected during a hospital outbreak in 1997. Interestingly, among

these 12 strains, including two recovered from one patient, two groups could be

distinguished: “resistant” and “sensitive”, which differed in the β-lactams resistance levels

but were equally resistant to aminoglycosides. The RLFP (Restriction Fragment Length

Polymorphism) analysis with the XbaI restrictase indicated the identity of total DNAs of both


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groups, except that of a single band. It was also shown that in the “resistant” group, the

transcript level of blaSHV was much higher than in the “sensitive” group and this was the

result of the increase of the blaSHV copies (62). The sequence of p1658/97 of the „sensitive”

group was determined and its structure analysed. The results were presented in the paper

Zienkiewicz et al., 2007, pos. 4 (58). p1658/97 was a conjugative plasmid, 125 491 bp in size,

it comprised two active replication systems, FII and FIB. In its sequence there was an

integron, which cassettes covered the whole spectrum of clinically relevant aminoglycosides.

The β-lactamase encoded by blaSHV was the SHV-5 variant, which hydrolysed majority of β-

lactams including newer cephalosporins and monobactams. On the basis of sequence

analysis, the history of plasmid evolution leading to p1658/97 was proposed, the plasmid

was a mosaic of the IncF plasmids (F, R100), and beyond the plasmid backbone – also of

other plasmids - IncM (pSEM, pACM1) and IncI1 (R64). blaSHV-5 was located between genes

naturally present in the Klebsiella pneumoniae chromosome, and this segment was flanked

by two IS26 copies; this module was 8817 bp in size (including the single IS26). The IS26

sequences were probably responsible for mobilization of this fragment from K. pneumoniae

chromosome. The whole segment was able to undergo tandem multiplication, increasing its

number of copies to even more than 10. So, the size of the plasmid increased of ca. 100 kb.

Moreover, this phenomenon was independent of the recA gene of the plasmid host ((60)

paper Zienkiewicz et al., 2013, pos. 3). Multiplication of the region flanked by the IS26

copies (called the amplicon) led to the β-lactam hyper-resistance: for ceftazidime (3rd

generation cephalosporin) from 4 µg/ml to 128 µg/ml. The hyper-resistant clones were

generated spontaneously. A deletion or insertion within the amplicon completely abolished

multiplication and even the deletion of only 10% of the amplicon length cannot be

complemented in trans. The amplicon could be moved to another plasmid via recombination

when this plasmid comprised the IS26 copy, however, there, the multiplication did not

occur, suggesting the importance of topology of the plasmid bearing the amplicon. The

phenomenon of multiplication of the amplicon observed in p1658/97 turned out not to be

unique: in pSEM of Salmonella enterica subsp. enterica serovar Typhimurium of the IncM

group, an amplicon almost identical to that analysed was present (99% identity of sequence

located between the IS26 copies, and 100% identity of IS26). This one also underwent

multiplication, what was shown and discussed in the Zienkiewicz et al., 2013, (60). One

should emphasize that, considering these results and common presence of IS26 in

Enterobacteriaceae genomes, multiplication of the module comprising blaSHV-5 can play an

important role in dissemination of this gene among bacteria.

The pCTX-M3 plasmid of Citrobacter freundii recovered in 1996, was a vector of

blaCTX-M-3, encoding new ESBL, then CTX-M-3. blaCTX-M-3 was located on large plasmids of the

similar RAPD (Random Amplification of Polymorphic DNA) profiles. It was found in

representatives of seven Enterobacteriaceae species in different hospitals in Poland (63),

and quickly became the most common variant encoding ESBL in our country (64). In other

European countries, in which surveillance of ESBL producers was carried on, just like

anywhere in the world, the most common variant was β-lactamase CTX-M-15 encoded in the


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IncF group plasmids. The sequence of pCTX-M3 was determined and analyzed ((59) paper

Gołębiewski et al., 2007, pos. 5). This plasmid (89 468 bp) of the IncM group (previously

IncL/M) was a conjugative one. The genes coding for its conjugation system were located in

two physically distant regions (tra and trb), which showed synteny, and 30 - 60% identity

with genes of the conjugative plasmid R64 of the IncI1 group. So, the Mpf encoded by these

genes belonged to the poorly characterized T4SS group and the MPFI group. In pCTX-M3,

upstream blaCTX-M-3, ISEcp1 was located. The blaCTX-M-3 probably originated from the

chromosome of the enteric bacterium, Kluyvera ascorbata, from which it was mobilized to a

plasmid by ISEcp1. pCTX-M3 encoded many genes conferring resistance to various classes of

antibiotics, some of them constituted an integron. There was also the structure comprising

armA, the gene coding for aminoglycoside resistance. The pCTX-M3-like plasmids were

shown to be responsible for dissemination of blaCTX-M-3: they were of the IncM group, and

coded for the conjugative transfer genes, even if the plasmid was unable to be transferred

((59) paper Gołębiewski et al., 2007, pos. 5). Interestingly, in other representatives of

Enterobacteriaceae, the pCTX-M3 related plasmids were fund, the analysis of their

sequence could reveal the history of evolution leading to the rise of pCTX-M3: pEL60 and

pCTX-M360. pEL60 of Erwinia amylovora from Lebanon, comprised only the pCTX-M3

backbone, i.e. the replicon and the genes encoding the maintenance systems and the

conjugation system, and it did not code for any resistance genes. The pCTX-M360 plasmid of

Klebsiella pneumoniae recovered in China, in addition to the plasmid backbone, comprised

the ISEcp1 with blaCTX-M-3. It is worth noting that in Hong Kong, the E. coli strain was

recovered, which bore pNDM-HK, the plasmid almost identical to pCTX-M3, however,

instead of the pCTX-M3 integron there was blaNDM-1, the metallo-β-lactamase NDM-1

encoding gene. The results of the pCTX-M structure analysis showed that formation of the

region which separated the tra and trb genes and comprising the replicon and all insertion

sequences and transposons and their remnants, as well as all the resistance genes present in

this plasmid, except ISEcp1 and blaCTX-M-3, was initiated by the integration of Tn1/Tn3, which

brought blaTEM-1 next to the pCTX-M3 replicon.

Taking into consideration that the pCTX-M3 conjugation system belonged to the

poorly characterized T4BSS group, the question was raised whether all tra and trb genes are

important for conjugative transfer or mobilization in which pCTX-M3 participated. The

plasmids bearing mutations in each gene of the conjugative transfer system of pCTX-M3 and

also in the leading region (this which as the first enters a recipient cell) were constructed.

The abilities to conjugative transfer of the mutated plasmids and complementation of these

mutations with relevant genes were analyzed (paper Dmowski et al., 2018, pos. 1 (61)). All

genes coding for Mpf of pCTX-M3 were essential for transfer, so this system was different

from conjugative systems of the already mentioned group IncI1 plasmid R64, where

inactivation of some genes resulted only in the reduction of the conjugative transfer

efficiency. These suggested the difference in organization and functioning of the Mpf

complexes encoded by these two plasmids. In pCTX-M3, only deletion of three genes did

not result in changes of the conjugative transfer efficiency: orf35 of the leading region,


11

orf36 of tra, and orf46 of trb. Simultaneously, the deletion of orf35 and orf36 caused an

increase of efficiencies of plasmid mobilization. The orf35 deletion resulted in the increase of

the level of transcription of genes located in the tra region - nikA, nikB, traH and probably

also the downstream genes. The orf36 deletion resulted in the increase of the level of

transcription of traH and probably also the downstream genes, but neither nikA nor nikB.

Additionally, in amino acid sequences predicted from orf35 and orf36 no known DNA binding

motifs were fund, therefore, the mechanism of the regulation is unknown and requires

further study. Interestingly, there was the difference in the range of hosts in which pCTX-M3

was able to replicate and the range of recipients into which the pCTX-M3 conjugation system

was able to transfer mobilizable plasmids. The replicon host range is limited to

representatives of Enterobacteriaceae, while the range of recipients in the conjugative

transfer comprise of representatives of Alfa-, Beta- and Gammaproteobacteria ((61),

Dmowski et al., 2018, pos. 1). Conjugative plasmids transiently generate ssDNA during

conjugation, they induce the SOS response and homologous recombination and also induce

the production of integrases, finally leading to DNA rearrangements. I postulate therefore,

that the conjugative plasmids, such as pCTX-M3, which encode the broad-host-range Mpfs

and the integrases in the integrons and do not encode the anti-SOS systems, may have a

greater impact on bacterial populations than was previously estimated. I am continuing the

research on the regulation of gene expression and on the protein interactions of the pCTX-

M3 conjugation system. The results can increase the state of knowledge on functioning of

T4BSS.

Simultaneously, on the basis of knowledge gathered of plasmid mobilization by the

pCTX-M3 with deleted orf35, the S14 strain, the helper strain for plasmid mobilization, was

constructed. This strain is currently the subject of the PL400718 patent application to the

Polish Patent Office.

In 2013, I initiated the cooperation with Dariusz Wasyl Ph. D. from the National

Veterinary Institute, who conducted a survey on 3rd generation cephalosporin-resistant

Salmonella (according to WHO critically important pathogens) and analyzed a collection of

MDR Salmonella enterica subsp. enterica serovar Kentucky recovered from turkey flocks in

Poland in 2009. The strains belonged to the epidemic ST198 S. Kentucky strain, which was

fluoroquinolone-resistant. This strain comprised the SGI1 variant of the pathogenicity island,

with a class I integron, and in the mobility of this island the broad-host-range plasmids of the

IncA/C group were involved. I analyzed the plasmids present in these strains and the types of

plasmid replicons were determined the with the PBRT method. Except for IncI1 plasmids,

typically present in bacteria of genus Salmonella, in a single strain a plasmid of the IncA/C

group was detected (paper Wasyl et al., 2015, pos. 2 (65)). This plasmid, p1643_10, was

sequenced and its structure was analyzed. The plasmid was conjugative, 167 779 bp in size,

and 75% of its sequence, the plasmid backbone and several additional genes, was of

identical structure to already known pTC2 plasmid of Providentia stuartii. The p1643_10

unique part was the region of ca. 40 kb of a mosaic structure. Analysis of this structure

revealed the complex process of evolution leading to the rise of p1643_10. In this region all


12

resistance genes and all insertion sequences and transposons and their remnants present in

p1643_10 were grouped. In the plasmid, there was an integron of a new type, comprising

the blaOXA-21 cassette, and also several cassettes covering the entire spectrum of clinically

relevant aminoglycosides. The p1643_10 bore ESBL-encoding blaCTX-M-25, never detected in S.

Kentucky, or in IncA/C plasmids, or in Poland. After the next inspection done a few months

later in the same turkey farm from which the analyzed S. Kentucky samples were collected,

no CTX-M-25 producing E. coli commensal strain was recovered. This suggested that

p1643_10 did not give bacteria the selective advantage.

In conclusion, my achievements were:

1/ indication that the amino terminus is important for the toxicity of Zeta, the toxin of the

toxin-antidote system of pSM19035,

2/ demonstration of the structural variability of the p1658/97 plasmid, relying on the

multiplication of the region comprising blaSHV-5 (the amplicon) and characterization of the

process of the amplicon multiplication,

3/ demonstration that multiplication of the blaSHV-5-containing amplicon is a phenomenon

much more common than initially considered, because it take place in pSEM of the IncM

group,

4/ demonstration that the phenomenon of the blaSHV-5-containing amplicon multiplication

can have a great impact on dissemination of blaSHV-5, based on the observation that the

amplicon could jump to other IS26-containing plasmids,

5/ showing that pCTX-M3 and pCTX-M3-related plasmids were responsible for

dissemination of blaCTX-M-3 among representatives of Enterobacteriaceae isolated from

hospital infections in 1996 in Poland,

6/ discovery of the regulators of the expression of the tra genes from the IncM group

conjugative pCTX-M3 plasmid belonging to the poorly characterized T4BSS group;

discovery that deletion of the genes coding for these regulators, orf35 and orf36, in the

helper plasmid, resulted in an increase of the efficiency of plasmid mobilization,

7/ discovery of the difference in the host ranges between the pCTX-M3 replicon –

representatives of Enterobacteriaceae, and the pCTX-M3 conjugative transfer system –

the recipients are representatives of Alfa-, Beta- and Gammproteobacteria,


13

8/ discovery for the first time, and, for the moment, the only one of blaCTX-M-25 in the

IncA/C plasmid, this gene in Salmonella Kentucky, and this gene in Poland, as a result of

the analysis and characterization of p1643_10 isolated from S. Kentucky.

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