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Objectives The objective of this study was to characterize New Delhi metallo-β-lactamase (NDM)-producing Enterobacteriaceae isolates reported in Poland in 2012-14. Methods Representative isolates were typed by PFGE and MLST. NDM and other β-lactamase genes were amplified and sequenced. Plasmids with blaNDM genes were analysed by nuclease S1 plus hybridization profiling, by transfer assays and by PCR-based replicon typing. The blaNDM genetic context was studied by PCR mapping assays. Results Of 374 cases of infection/colonization with NDM-positive Enterobacteriaceae identified in 2012-14, 370 cases in 40 hospitals, 10 outpatient clinics and 1 nursing home were associated with a Klebsiella pneumoniae outbreak with epicentres in Poznan and Warsaw. The outbreak strain of K. pneumoniae ST11 was similar to an isolate from the Czech Republic from 2013. Like the Czech strain, many of the isolates had two blaNDM-1-carrying IncFII- and IncR-type plasmids of variable size, sharing a blaNDM-1-containing segment. The early isolates also produced CTX-M-15 co-encoded by the IncR-type plasmids, and differentiated later by extensive plasmid rearrangements. Four other NDM cases were reported in 2013, three being associated with arrivals from Montenegro, India or Afghanistan. The Indian Escherichia coli ST448 NDM-5 isolate revealed similarity to a recent isolate from Spain, including the blaNDM genetic context observed previously in E. coli strains in Poland and France (of Congolese and Indian origins, respectively). The Afghani Proteus mirabilis was the second isolate of this species with a chromosomal blaNDM-1 location. Conclusions The largest NDM outbreak in a non-endemic country has been observed, being an alarming phenomenon in resistance epidemiology in Poland.

Poland's first Enterobacteriaceae isolate producing the New Delhi metallo-β-lactamase (NDM) was identified in August 2011. Escherichia coli sequence type ST410 NDM-1 was cultured from a critically ill patient who had been transferred directly from the Congo. The bla NDM-1 gene was carried by conjugative IncFII-type plasmid pMC-NDM (87,619 bp), which showed structural similarity to plasmid pGUE-NDM, which was identified earlier in France in an E. coli ST131 isolate of Indian origin.

Objectives In 2008-09, the KPC carbapenemase epidemiology in Poland was dominated by a Klebsiella pneumoniae ST258 KPC-2 outbreak in Warsaw and its administrative region. The aim of this study was to analyse the situation in 2010-14, with a focus on new outbreaks in other parts of the country. Methods KPCs were detected in all suspected isolates by PCR. The detailed study was performed on 173 isolates from 2010 to 2012, and included PFGE and MLST, PCR identification of K. pneumoniae clonal group CG258 clades and potential specificity markers ( pilv-1 , IS 66 and prp ), PCR mapping of Tn 4401 transposons, and plasmid analysis by nuclease S1 profiling and PCR-based replicon typing. Results Six hundred and eight KPC cases were identified in Poland in 2010-14, almost half of which occurred in the Warsaw region, and another half in four other areas. The new outbreaks were caused by four K. pneumoniae CG258 genotypes, different from each other and from the organisms spreading in Warsaw. The new lineages were ST258 or ST512 of clade II, and had specific compositions of potential ST258/ST512 clonal markers. The isolates produced KPC-3 encoded by Tn 4401 a or Tn 4401 b elements on plasmids with single or multiple replicons, including I2, FII K (+/-FIB K ), 'FII Y -like', X3 and R. Of other species, Citrobacter freundii ST17 and Enterobacter cloacae ST254 with KPC-2 were identified in a Warsaw hospital. Conclusions The study showed remarkable changes in the KPC epidemiology in Poland in 2010-14, which, following the localized regional spread in the early phase, has converted into multiregional dissemination.

CTX-M-producing Escherichia coli isolates from three Croatian hospitals were analyzed. All bla CTX-M-15 genes and one bla CTX-M-3a gene resided in widely spread IS Ecp1 transposition modules, but other bla CTX-M-3a genes were in a new configuration with two IS 26 copies, indicating a new event of gene mobilization from a Kluyvera ascorbata genome. The study confirmed the role of the E. coli ST131 clonal group with IncFII-type plasmids in the spread of bla CTX-M-15 and of IncL/M pCTX-M3-type plasmids in the dissemination of bla CTX-M-3a .

Penicillin-binding proteins (PBPs) in representatives of two Streptococcus pneumoniae clonal groups that are prevalent in Poland, Poland 23F -16 and Poland 6B -20, were investigated by PBP profile analysis, antibody reactivity pattern analysis, and DNA sequence analysis of the transpeptidase (TP) domain-encoding regions of the pbp2x , pbp2b , and pbp1a genes. The isolates differed in their MICs of β-lactam antibiotics. The majority of the 6B isolates were intermediately susceptible to penicillin (penicillin MICs, 0.12 to 0.5 μg/ml), whereas all 23F isolates were penicillin resistant (MICs, ≥2 μg/ml). The 6B isolates investigated had the same sequence type (ST), determined by multilocus sequence typing, as the Poland 6B -20 reference strain (ST315), but in the 23F group, isolates with three distinct single-locus variants (SLVs) in the ddl gene (ST173, ST272, and ST1506) were included. None of the isolates showed an identical PBP profile after labeling with Bocillin FL and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and only one pair of 6B isolates and one pair of 23F isolates (ST173 and ST272) each contained an identical combination of PBP 2x, PBP 2b, and PBP 1a TP domains. Some 23F isolates contained PBP 3 with an apparently higher electrophoretic mobility, and this feature also did not correlate with their STs. The data document a highly variable pool of PBP genes as a result of multiple gene transfer and recombination events within and between different clonal groups.

Objectives To perform the first multinational Enterobacter cloacae clonality study, using the MLST scheme newly developed in Japan. Methods The analysis included 195 rectal carriage E. cloacae isolates resistant to expanded-spectrum cephalosporins (ESCs), collected from patients in 12 hospital units across Europe and Israel. All of the isolates were typed by PFGE and 173 isolates were subjected to MLST. ESC resistance was analysed phenotypically; genes encoding ESBLs and carbapenemases were identified by PCR and sequencing. Results MLST distinguished 88 STs, which correlated with the PFGE data. PFGE was more discriminatory, producing 129 pulsotypes (169 patterns). Numerous STs were observed in several countries each. The most widespread were ST66, ST78, ST108 and ST114, each having at least 10 isolates from three to five countries, diversified into multiple pulsotypes, with clusters of related isolates in one or more centres. Analysis of the STs against the MLST database revealed several epidemic clonal complexes, such as those with central genotypes ST74 (including ST78) or ST114 (including ST66). ESC resistance was equally related to overexpression of the AmpC cephalosporinase and to ESBL production. Among ESBL producers some spreading subclones were identified, including specific ST66, ST78 and ST114 pulsotypes, associated with CTX-M-15 production. Several isolates produced carbapenemase VIM-1 or KPC-2. Conclusions Together with the information available in the MLST database, our results suggest that, like Escherichia coli and Klebsiella pneumoniae, E. cloacae harbours clonal lineages of increased epidemic potential that may be associated with resistance spread.

The CTX-M-15 extended-spectrum beta-lactamase (ESBL) was recently identified in Enterobacteriaceae isolates in India, and demonstrated significant hydrolytic activity against ceftazidime, in contrast to the majority of CTX-M enzymes. CTX-M-15 differs from CTX-M-3, which is one of the most prevalent ESBLs in Poland, by only a single amino acid change (Asp-240-->Gly). Three cefotaxime- and ceftazidime-resistant Enterobacteriaceae isolates, recovered during 1998-2000 in two Polish hospitals, were found to produce CTX-M-15. Similar to those from India, the isolates contained the ISEcp1 insertion sequence located upstream of the bla(CTX-M-15) gene, which has been recently demonstrated to mobilize 3'-adjacent genes to transfer between DNA replicons. However, its different position with respect to the beta-lactamase gene indicated the independent selection of the ESBL gene in the two countries.

Twenty-two Klebsiella pneumoniae and two K. oxytoca extended-spectrum β-lactamase (ESBL)-producing isolates were collected in 1996 from patients in two pediatric wards of the University Hospital in Wrocław, Poland. Molecular typing has revealed that the K. pneumoniae isolates represented four different epidemic strains. Three kinds of enzymes with ESBL activity (pI values of 5.7, 6.0, and 8.2) were identified. The pI 6.0 β-lactamases belonged to the TEM family, and sequencing of the bla TEM genes amplified from representative isolates revealed that these enzymes were TEM-47, previously identified in K. pneumoniae isolates from pediatric hospitals in Łódź and Warsaw. One of the TEM-47-producing strains from Wrocław was very closely related to the isolates from the other cities, and this indicated countrywide spread of the epidemic strain. The pI 5.7 β-lactamase was produced by a single K. pneumoniae isolate for which, apart from oxyimino-β-lactams, the MICs of β-lactam–inhibitor combinations were also remarkably high. Sequencing revealed that this was a novel TEM β-lactamase variant, TEM-68, specified by the following combination of mutations: Gly238Ser, Glu240Lys, Thr265Met, and Arg275Leu. The new enzyme has most probably evolved from TEM-47 by acquiring the single substitution of Arg275, which before was identified only twice in enzymes with inhibitor resistance (IR) activity. TEM-68 was shown to be a novel complex mutant TEM β-lactamase (CMT-2) which combines strong ESBL activity with relatively weak IR activity and, when expressed in K. pneumoniae , is able to confer high-level resistance to a wide variety of β-lactams, including inhibitor combinations. This data confirms the role of the Arg275Leu mutation in determining IR activity and documents the first isolation of K. pneumoniae producing the complex mutant enzyme.

After the first report in May 2008, the National Reference Center for Susceptibility Testing confirmed 113 cases of infection or colonization by KPC-producing members of the family Enterobacteriaceae in Poland by the end of 2009. The vast majority of patients were found in 18 hospitals; three patients were diagnosed at outpatient clinics. Most of the institutions were in the Warsaw area, including three hospitals with the highest numbers of cases. When available, the data on previous hospitalizations often indicated that these hospitals were the probable acquisition sites; one patient arrived from New York. The group of 119 unique isolates consisted of Klebsiella pneumoniae ( n = 114), followed by Klebsiella oxytoca ( n = 3), and Escherichia coli ( n = 2). The K. pneumoniae isolates were dominated by the clone sequence type 258 (ST258) ( n = 111); others were ST11 and ST23. The ST258 group was heterogeneous, with 28 pulsed-field gel electrophoresis (PFGE) subtypes, ∼25 plasmid profiles, and nine β-lactamase patterns differing by KPC variants (KPC-2 mainly), and SHV-12, CTX-M-3, and TEM-1-like enzymes. Plasmids carrying bla KPC genes varied in size (∼48 to 250 kb), structure, and conjugation potential. Transferable IncFII K plasmids of ∼110 to 160 kb, probably pKpQIL or its derivatives, were observed in all K. pneumoniae clones and in K. oxytoca . Also prevalent were nontypeable pETKp50-like plasmids of ∼50 kb, found in K. pneumoniae ST258 and E. coli isolates (ST93 and ST224). Two K. pneumoniae-E. coli pairs from single patients might represent the in vivo transfer of such plasmids. The striking diversity of KPC producers at the early stage of dissemination could result from several introductions of these bacteria into the country, their multidirectional evolution during clonal spread, and transfer of the plasmids.

Proteus mirabilis isolates obtained in 1999 to 2008 from three European countries were analyzed; all carried chromosomal AmpC-type cephalosporinase bla CMY genes from a Citrobacter freundii origin ( bla CMY-2 -like genes). Isolates from Poland harbored several bla CMY genes ( bla CMY-4 , bla CMY-12 , bla CMY-14 , bla CMY-15 , and bla CMY-38 and the new gene bla CMY-45 ), while isolates from Italy and Greece harbored bla CMY-16 only. Earlier isolates with bla CMY-4 or bla CMY-12 , recovered in France from Greek and Algerian patients, were also studied. All isolates showed striking similarities. Their bla CMY genes resided within IS Ecp1 transposition modules, named Tn 6093 , characterized by a 110-bp distance between IS Ecp1 and bla CMY , and identical fragments of both C. freundii DNA and a ColE1-type plasmid backbone. Moreover, these modules were inserted into the same chromosomal site, within the pepQ gene. Since ColE1 plasmids carrying IS Ecp1 with similar C. freundii DNA fragments (Tn 6114 ) had been identified earlier, it is likely that a similar molecule had mediated at some stage this DNA transfer between C. freundii and P. mirabilis . In addition, isolates with bla CMY-12 , bla CMY-15 , and bla CMY-38 genes harbored a second bla CMY copy within a shorter IS Ecp1 module (Tn 6113 ), always inserted downstream of the ppiD gene. Sequence analysis of all mobile bla CMY-2 -like genes indicated that those integrated in the P. mirabilis chromosome form a distinct cluster that may have evolved by the stepwise accumulation of mutations. All of these observations, coupled to strain typing data, suggest that the bla CMY genes studied here may have originated from a single IS Ecp1 -mediated mobilization-transfer-integration process, followed by the spread and evolution of a P. mirabilis clone over time and a large geographic area.

Plasmidic KPC β-lactamases in gram-negative pathogens are of the highest clinical and epidemiologic concern, conferring resistance to all β-lactams, including carbapenems (15, 18). The main KPC producer is Klebsiella pneumoniae, but other species are being identified as well (15). Strains with KPCs spread rapidly and cause outbreaks; in hospitals on the east coast of the United States and in Israel, they have already become endemic (2, 3, 10, 14, 17). Recently, more countries have reported the presence of these organisms, in some cases as a result of importation (15).

The frequency of tetracycline resistance in Streptococcus pneumoniae isolates in Poland is one of the highest in Europe. The aim of this study was to analyze the clonal diversity and resistance determinants of tetracycline-nonsusceptible S. pneumoniae isolates identified in Poland and to investigate the effect of tetracycline resistance on their susceptibilities to tigecycline, doxycycline, and minocycline. We have analyzed 866 pneumococcal isolates collected from 1998 to 2003 from patients with respiratory tract diseases, and 242 of these (27.9%) were found to be resistant to tetracycline. All of the resistant isolates were characterized by testing of their susceptibilities to other antimicrobials, serotyping, pulsed-field gel electrophoresis (PFGE), and identification of tetracycline resistance genes and transposons. Selected isolates representing the main PFGE types were analyzed by multilocus sequence typing. Among the isolates investigated, 27 serotypes and 146 various PFGE patterns, grouped into 90 types, were discerned. The most common PFGE type, corresponding to serotype 19F and sequence type 423, was represented by 22.3% of all of the tetracycline-resistant isolates. The tet (M) gene was the sole resistance gene in the group of isolates studied, and in over 96% of the isolates, the Tn 916 family of tet (M)-containing conjugative transposons was detected. Several isolates contained specific variants of the transposons, the Tn 1545 -like, Tn 3872 -like, or Tn 2009 -like element. The correlation between the MICs of tetracycline, doxycycline, and minocycline was revealed, whereas no cross-resistance to tetracycline and tigecycline was observed.

We have analyzed 40 metallo-β-lactamase (MBL)-producing isolates of Pseudomonas aeruginosa ( n = 38), Pseudomonas putida ( n = 1), and Acinetobacter genospecies 3 ( n = 1) from 17 hospitals in 12 cities in Poland that were identified in 2000 to 2004. Pulsed field gel electrophoresis typing classified the P. aeruginosa isolates into eight types, with two types differentiated further into subtypes. Each of the types was specific either to a given center or to several hospitals of the same or neighboring geographic area. Almost all of the organisms produced β-lactamase VIM-2; the only exceptions were several P. aeruginosa isolates from two centers which expressed VIM-4. The bla VIM genes resided exclusively within class 1 integrons, and these were located in either chromosomal or plasmid DNA. PCR-restriction fragment length polymorphism study of the variable regions of the integrons, followed by DNA sequencing, revealed the presence of eight different, mostly novel gene cassette arrays, six of which contained bla VIM-2 and two of which contained bla VIM-4 . The occurrence of the integron variants correlated well with the geographic distribution of the MBL-producing organisms, and this suggested that their emergence in particular parts of the country had been likely due to a number of independent events. The following regional dissemination of MBL producers could be attributed to various phenomena, including their clonal spread, horizontal transmission of resistance determinants, or both. All of the data collected in this study revealed that even at this early stage of detection, the epidemiological situation concerning MBL producers in Poland has already been complex and very dynamic.

Eighty-four clinical isolates of the family Enterobacteriaceae , recovered from 1998 to 2000 in 15 hospitals in 10 Polish cities, were analyzed. All the isolates produced β-lactamases with pIs of 8.4 and 5.4, and the pI 8.4 enzymes were demonstrated to hydrolyze cefotaxime but not ceftazidime in the in vitro bioassay. PCR analysis and DNA sequencing have revealed that in all cases the pI 8.4 β-lactamase was probably the CTX-M-3 extended-spectrum β-lactamase (ESBL) variant, which was originally identified in 1996 in Praski Hospital in Warsaw. In the majority of isolates, bla CTX-M-3 genes resided within large conjugative plasmids with similar fingerprints, which, in the context of the high degree of diversity of the randomly amplified polymorphic DNA types of the isolates, suggested that horizontal transfer of plasmids was likely the main mechanism of CTX-M-3 spread. The dissemination of plasmids was probably preceded by the center-to-center transmission of several strains, as indicated by the identification by pulsed-field gel electrophoresis of closely related or possibly related Klebsiella pneumoniae , Escherichia coli , and Citrobacter freundii isolates in five different hospitals. CTX-M-3-producing organisms revealed a very high degree of diversity in β-lactam resistance levels and patterns. This was attributed to several factors, such as the production of other β-lactamases including additional ESBLs, possible quantitative variations in CTX-M-3 expression, segregation of AmpC derepressed mutants, and permeability alterations.