Epidemic Territorial Spread of IncP-2-Type VIM-2 Carbapenemase-Encoding Megaplasmids in Nosocomial Pseudomonas aeruginosa Populations.

In 2003 to 2004, the first five VIM-2 metallo-ß-lactamase (MBL)-producing Pseudomonas aeruginosa (MPPA) isolates with an In4-like integron, In461 (aadB-blaVIM-2-aadA6), on conjugative plasmids were identified in three hospitals in Poland. In 2005 to 2015, MPPA expanded much in the country, and as many as 80 isolates in a collection of 454 MPPA (∼18%) had In461, one of the two most common MBL-encoding integrons. The organisms occurred in 49 hospitals in 33 cities of 11/16 main administrative regions. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) classified them into 55 pulsotypes and 35 sequence types (STs), respectively, revealing their remarkable genetic diversity overall, with only a few small clonal clusters. S1 nuclease/hybridization assays and mating of 63 representative isolates showed that ∼85% of these had large In461-carrying plasmids, ∼350 to 550 kb, usually self-transmitting with high efficiency (∼10-1 to 10-2 per donor cell). The plasmids from 19 isolates were sequenced and subjected to structural and single-nucleotide-polymorphism (SNP)-based phylogenetic analysis. These formed a subgroup within a family of IncP-2-type megaplasmids, observed worldwide in pseudomonads from various environments and conferring resistance/tolerance to multiple stress factors, including antibiotics. Their microdiversity in Poland arose mainly from acquisition of different accessory fragments, as well as new resistance genes and multiplication of these. Short-read sequence and/or PCR mapping confirmed the In461-carrying plasmids in the remaining isolates to be the IncP-2 types. The study demonstrated a large-scale epidemic spread of multidrug resistance plasmids in P. aeruginosa populations, creating an epidemiological threat. It contributes to the knowledge on IncP-2 types, which are interesting research objects in resistance epidemiology, environmental microbiology, and biotechnology.

Activity of temocillin against ESBL-, AmpC-, and/or KPC-producing Enterobacterales isolated in Poland.

We evaluated the in vitro effectiveness of temocillin and several commonly used antimicrobials against Enterobacterales bacteria in isolates from Polish patients. We tested 400 isolates: 260 extended-spectrum ß-lactamase (ESBL)- and/or ampC ß-lactamase (AmpC)-producing isolates; 40 Klebsiella pneumoniae carbapenemase (KPC)-producing isolates; and 100 ESBL-, AmpC-, and KPC-negative isolates. The minimal inhibitory concentrations (MICs) of temocillin and 16 other antimicrobials were determined by reference microdilution. We also determined the activities of fosfomycin and ceftazidime/avibactam in KPC-producing isolates. The antibiotic sensitivities were interpreted according to EUCAST, BSAC, and CLSI criteria. Overall, 91% of the isolates were susceptible to temocillin using the urinary tract infection breakpoint (≤ 32 mg/L), and 61.8% were susceptible using the systemic infection breakpoint (≤ 8 mg/L). Meropenem and imipenem were the most active drugs (MIC50 values of 0.06 and 0.5 mg/L, respectively). Colistin and ertapenem (both MIC50 = 0.12 mg/L) were less active than meropenem or imipenem, but some strains were 77% susceptible to each of them. Among the KPC-producing isolates, 42.5% had MIC values of ≤ 32 mg/L (urinary tract infection breakpoint), but 100% were resistant to temocillin (systemic infection breakpoint). Ceftazidime/avibactam was active against 100% of the KPC-producing isolates, and fosfomycin was active against 40%. The empirical susceptibility rate observed among the urinary isolates suggests that temocillin may be considered as an alternative to carbapenems in the absence of KPC-producing bacteria. With regard to isolates from other sources, temocillin might be useful as a documented therapy agent or an empirical treatment in hospitals with a low prevalence of ESBL/AmpC-producing strains.

Multiregional dissemination of KPC-producing Klebsiella pneumoniae ST258/ST512 genotypes in Poland, 2010-14.

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.

Genomic Epidemiology of MBL-Producing Pseudomonas putida Group Isolates in Poland.

INTRODUCTION: Pseudomonas putida group are described as low-incidence opportunistic pathogens, but also as a significant reservoir of antimicrobial resistance (AMR) genes, including those of metallo-ß-lactamases (MBLs). Our objective was the molecular and genomic characterization of MBL-producing P. putida (MPPP) group isolates from Poland, focusing on population structures, successful genotypes and MBL-encoding integrons. METHODS: During a country-wide MBL surveillance in Pseudomonas spp., 59 non-duplicate MPPP isolates were collected from 36 hospitals in 23 towns from 2003 to 2016. All of the isolates were subjected to whole-genome sequencing (WGS), followed by species identification, multi-locus sequence typing (MLST), single-nucleotide polymorphism (SNP)-based phylogenetic/clonality analysis, resistome determination, and susceptibility testing. RESULTS: The study collection comprised 12 species, of which P. alloputida (n = 19), P. monteilii (n = 15), and P. asiatica (n = 11) prevailed, while the others were P. kurunegalensis, P. putida, P. soli, P. mosselii, P. juntendi, and four potentially new species. MLST classified the isolates into 23 sequence types (STs) of which 21 were new, with three main clones, namely P. alloputida ST69, P.monteilii ST95 and P. asiatica ST15. The isolates produced VIM-like MBLs only, largely VIM-2 (n = 40), encoded by 24 different class 1 integrons (ten new), a number of which occurred also in P. aeruginosa and/or Enterobacterales in Poland. The plasmid pool was dominated by IncP-9, IncP-2, and pMOS94-like types. Multiple isolates were extensively drug-resistant. CONCLUSIONS: This study, being one of the most comprehensive analyses of MPPP so far, has shown high diversity of the isolates in general, with three apparently international lineages, each internally diversified by MBL-encoding structures.

Pathogenic Factors Correlate With Antimicrobial Resistance Among Clinical Proteus mirabilis Strains.

Proteus mirabilis is the third most common etiological factor of urinary tract infection. It produces urease, which contributes to the formation of a crystalline biofilm, considered to be one of the most important virulence factors of P. mirabilis strains, along with their ability to swarm on a solid surface. The aim of this study was to analyze the pathogenic properties of two selected groups of clinical P. mirabilis isolates, antimicrobial susceptible and multidrug resistant (MDR), collected from hospitals in different regions in Poland. The strains were examined based on virulence gene profiles, urease and hemolysin production, biofilm formation, and swarming properties. Additionally, the strains were characterized based on the Dienes test and antibiotic susceptibility patterns. It turned out that the MDR strains exhibited kinship more often than the susceptible ones. The strains which were able to form a stronger biofilm had broader antimicrobial resistance profiles. It was also found that the strongest swarming motility correlated with susceptibility to most antibiotics. The correlations described in this work encourage further investigation of the mechanisms of pathogenicity of P. mirabilis.

blaCTX-M genes in escherichia coli strains from Croatian Hospitals are located in new (blaCTX-M-3a) and widely spread (blaCTX-M-3a and blaCTX-M-15) genetic structures.

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 ISEcp1 transposition modules, but other bla(CTX-M-3a) genes were in a new configuration with two IS26 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).

Molecular survey of beta-lactamases conferring resistance to newer beta-lactams in Enterobacteriaceae isolates from Polish hospitals.

The first national survey of resistance to newer beta-lactams in nosocomial populations of Enterobacteriaceae in Poland was performed. The study covered all nonrepetitive enterobacterial isolates cultured from specimens from inpatients in 13 regional secondary-care hospitals from November 2003 to January 2004. Among 2,388 isolates, the predominant species was Escherichia coli (59.6%), followed by Proteus mirabilis (14.5%) and Klebsiella spp. (8.5%). The frequency of extended-spectrum beta-lactamases (ESBLs) was very high, with ESBLs present in 11.1% of all isolates and 40.4% of Klebsiella pneumoniae isolates, the latter value greatly exceeding that for E. coli (2.5%). The contribution of outbreak isolates was significant, resulting, for example, in a particularly high rate of ESBL producers among Serratia marcescens isolates (70.8%). The pool of ESBL types was overwhelmingly dominated (81.7%) by CTX-M-like beta-lactamases CTX-M-3 (80.6%) and CTX-M-15, with SHV types (17.5%; SHV-2, SHV-5, and SHV-12) and sporadic TEM-like enzymes (0.7%; TEM-19 and TEM-48) being the next most frequent. Acquired AmpC-type cephalosporinases were observed exclusively in P. mirabilis, in 20.5% of the isolates of this species (compared with the frequency of ESBL producers of 11.5% of P. mirabilis isolates). All these cephalosporinases (CMY-12, CMY-15, and a novel variant, CMY-38) originated from Citrobacter freundii. Four isolates of E. coli (two isolates), K. pneumoniae (one isolate), and P. mirabilis (one isolate) produced class A inhibitor-resistant beta-lactamases (TEM-30, TEM-32, TEM-37, and SHV-49), being the first of such producers identified in Poland. The survey documented both specific and more global characteristics of the epidemiology of the beta-lactamase-mediated resistance in enterobacteria from Polish hospitals and demonstrated that the ESBL frequency has reached an alarming level.

Four variants of the Citrobacter freundii AmpC-Type cephalosporinases, including novel enzymes CMY-14 and CMY-15, in a Proteus mirabilis clone widespread in Poland.

Twenty-nine Proteus mirabilis isolates from 17 Polish hospitals were analyzed. The isolates were resistant to a variety of antimicrobials, and their patterns of resistance to beta-lactams resembled those of the constitutive class C cephalosporinase (AmpC) producers. Indeed, beta-lactamases with a pI of approximately 9.0 were found in all of the isolates, and they were subsequently identified as four AmpC-type cephalosporinases, CMY-4, -12, -14, and -15, of which the two last ones were novel enzyme variants. The enzymes were of Citrobacter freundii origin and were closely related to each other, with CMY-4 likely being the evolutionary precursor of the remaining ones. The bla(CMY) genes were located exclusively in chromosomal DNA, within EcoRI restriction fragments of the same size of approximately 10 kb. In the CMY-12- and -15-producing isolates, an additional fragment of approximately 4.5 kb hybridized with the bla(CMY) probe as well, which could have arisen from a duplication event during the evolution of the genes. In all of the isolates, the ISEcp1 mobile element, which most probably is involved in mobilization of the C. freundii ampC gene, was placed at the same distance from the 5' ends of the bla(CMY) genes, and sequences located between them were identical in isolates carrying each of the four genes. These data suggested that a single chromosome-to-chromosome transfer of the ampC gene from C. freundii to P. mirabilis could have initiated the spread and evolution of the AmpC-producing P. mirabilis in Poland. The hypothesis seems to be confirmed by pulsed-field gel electrophoresis typing, which revealed several cases of close relatedness between the P. mirabilis isolates from distant centers and showed an overall similarity between the majority of the multiresistant isolates.