IncF Group Research Articles

A Newly Incompatibility F Replicon Allele (FIB81) in Extensively Drug-Resistant Escherichia coli Isolated from Diseased Broilers.

Multiple drug resistance (MDR) has gained pronounced attention among Enterobacterales. The transfer of multiple antimicrobial resistance genes, frequently carried on conjugative incompatibility F (IncF) plasmids and facilitating interspecies resistance transmission, has been linked to Salmonella spp. and E. coli in broilers. In Egypt, the growing resistance is exacerbated by the limited clinical efficacy of many antimicrobials. In this study, IncF groups were screened and characterized in drug-resistant Salmonella spp. and E. coli isolated from broilers. The antimicrobial resistance profile, PCR-based replicon typing of bacterial isolates pre- and post-plasmid curing, and IncF replicon allele sequence typing were investigated. Five isolates of E. coli (5/31; 16.13%) and Salmonella spp. (5/36; 13.89%) were pan-susceptible to the examined antimicrobial agents, and 85.07% of tested isolates were MDR and extensively drug-resistant (XDR). Twelve MDR and XDR E. coli and Salmonella spp. isolates were examined for the existence of IncF replicons (FII, FIA, and FIB). They shared resistance to ampicillin, ampicillin/sulbactam, amoxicillin/clavulanate, doxycycline, cefotaxime, and colistin. All isolates carried from one to two IncF replicons. The FII-FIA-FIB+ and FII-FIA+FIB- were the predominant replicon patterns. FIB was the most frequently detected replicon after plasmid curing. Three XDR E. coli isolates that were resistant to 12-14 antimicrobials carried a newly FIB replicon allele with four nucleotide substitutions: C99→A, G112→T, C113→T, and G114→A. These findings suggest that broilers are a significant reservoir of IncF replicons with highly divergent IncF-FIB plasmid incompatibility groups circulating among XDR Enterobacterales. Supporting these data with additional comprehensive epidemiological studies involving replicons other than the IncF can provide insights for implementing efficient policies to prevent the spreading of new replicons to humans.

Private and well drinking water are reservoirs for antimicrobial resistant bacteria

Water quality testing does not recognise antimicrobial resistance (AMR) and is often limited to indicators of faecal contamination Escherichia coli and Enterococcus species. In Europe, data on AMR in drinking water is scarce. In Ireland, as in many countries, household drinking water is supplied via mains or via private wells or water schemes. Using citizen science, we identified Irish private drinking water supplies as reservoirs of antimicrobial resistant bacteria (ARB). Gram-negative (n = 464) and Gram-positive (n = 72) bacteria were isolated. We identified instances of potentially opportunistic ARB such as Enterobacter cloacae, Acinetobacter baumannii and Enterococcus species. We report reservoirs of multidrug resistance in Enterococcus casseliflavus, E. cloacae, E. coli, Stenotrophomonas maltophilia, and Serratia rubidaea. We also identified linezolid-resistant Enterococcus in Irish drinking water. Linezolid is a last-resort antibiotic used to treat vancomycin-resistant Enterococcus sp. Additionally, we identified mobile AMR in three water samples, two of which were carried on IncF group, one on IncQ and five on Col-like plasmids. Our work suggests that private drinking water is a potential sink and source of AMR pathogens. This highlights a value of drinking water surveillance in a One Health framework as the surveillance would provide information regarding the movement and persistence of ARB and ARGs that are able to survive in drinking water and subsequently have the opportunity to be mobilised through humans; linking the environment to the human and potentially threatening human health.

Genomic characterization of a multidrug-resistant uropathogenic <i>Escherichia coli</i> and evaluation of <i>Echeveria</i> plant extracts as antibacterials

<abstract> <p>Uropathogenic <italic>Escherichia coli</italic> (UPEC) is the most common bacterial agent associated with urinary tract infections, threatening public health systems with elevated medical costs and high morbidity rates. The successful establishment of the infection is associated with virulence factors encoded in its genome, in addition to antibacterial resistance genes, which could limit the treatment and resolution of the infection. In this sense, plant extracts from the genus <italic>Echeveria</italic> have traditionally been used to treat diverse infectious diseases. However, little is known about the effects of these extracts on bacteria and their potential mechanisms of action. This study aims to sequence a multidrug-resistant UPEC isolate (UTI-U7) and assess the multilocus sequence typing (MLST), virulence factors, antimicrobial resistance profile, genes, serotype, and plasmid content. Antimicrobial susceptibility profiling was performed using the Kirby-Bauer disk diffusion. The antibacterial and anti-adherent effects of the methanol extracts (ME) of <italic>Echeveria</italic> (<italic>E. craigiana</italic>, <italic>E. kimnachii</italic>, and <italic>E. subrigida</italic>) against UTI-U7 were determined. The isolate was characterized as an O25:H4-B2-ST2279-CH40 subclone and had resistant determinants to aminoglycosides, β-lactams, fluoroquinolones/quinolones, amphenicols, and tetracyclines, which matched with the antimicrobial resistance profile. The virulence genes identified encode adherence factors, iron uptake, protectins/serum resistance, and toxins. Identified plasmids belonged to the IncF group (IncFIA, IncFIB, and IncFII), alongside several prophage-like elements. After an extensive genome analysis that confirmed the pathogenic status of UTI-U7 isolate, <italic>Echeveria</italic> extracts were tested to determine their antibacterial effects; as an extract, <italic>E. subrigida</italic> (MIC, 5 mg/mL) displayed the best inhibitory effect. However, the adherence between UTI-U7 and HeLa cells was unaffected by the ME of the <italic>E. subrigida</italic> extract.</p> </abstract>

Comparative genomics analysis and characterization of Shiga toxin-producing Escherichia coli O157:H7 strains reveal virulence genes, resistance genes, prophages and plasmids

Escherichia coli O157:H7 is a foodborne pathogen that has been linked to global disease outbreaks. These diseases include hemorrhagic colitis and hemolytic uremic syndrome. It is vital to know the features that make this strain pathogenic to understand the development of disease outbreaks. In the current study, a comparative genomic analysis was carried out to determine the presence of structural and functional features of O157:H7 strains obtained from 115 National Center for Biotechnology Information database. These strains of interest were analysed in the following programs: BLAST Ring Image Generator, PlasmidFinder, ResFinder, VirulenceFinder, IslandViewer 4 and PHASTER. Five strains (ECP19–198, ECP19–798, F7508, F8952, H2495) demonstrated a great homology with Sakai because of a few regions missing. Five resistant genes were identified, however, Macrolide-associated resistance gene mdf(A) was commonly found in all genomes. Majority of the strains (97%) were positive for 15 of the virulent genes (espA, espB, espF, espJ, gad, chuA, eae, iss, nleA, nleB, nleC, ompT, tccP, terC and tir). The plasmid analysis demonstrated that the IncF group was the most prevalent in the strains analysed. The prophage and genomic island analysis showed a distribution of bacteriophages and genomic islands respectively. The results indicated that structural and functional features of the many O157:H7 strains differ and may be a result of obtaining mobile genetic elements via horizontal gene transfer. Understanding the evolution of O157:H7 strains pathogenicity in terms of their structural and functional features will enable the development of detection and control of transmission strategies.

Antimicrobial resistance, virulence associated genes and phylogenetic background versus plasmid replicon types: the possible associations in avian pathogenic Escherichia coli (APEC)

BackgroundAntimicrobial resistance (AMR) in bacterial isolates from food producing animals not only challenges the preventive and therapeutic strategies in veterinary medicine, but also threatens public health. Genetic elements placed on both chromosome and plasmids could be involved in AMR. In the present study, the associations of genomic backbone and plasmids with AMR were evaluated. We also provided some primary evidences that which genetic lineages potentially host certain groups of plasmids.ResultsIn the current study, 72 avian pathogenic Escherichia coli (APEC) strains were examined. Isolates resistant to tetracycline and trimethoprim-sulfamethoxazole (87.5%; each), and harboring blaTEM (61.1%) were dominant. Moreover, phylogroup D was the most prevalent phylogroup in total (23.6%), and among multidrug-resistant (MDR) isolates (14/63). The most prevalent Inc-types were also defined as follows: IncP (65.2%), IncI1 (58.3%), and IncF group (54.1%). Significant associations among phylogroups and AMR were observed such as group C to neomycin (p = 0.002), gentamicin (p = 0.017) and florfenicol (p = 0.036). Furthermore, group D was associated with blaCTX. In terms of associations among Inc-types and AMR, resistance to aminoglycoside antibiotics was considerably linked with IncP (p = 0.012), IncI1 (p = 0.038) and IncA/C (p = 0.005). The blaTEM and blaCTX genes presence were connected with IncI1 (p = 0.003) and IncFIC (p = 0.013), respectively. It was also shown that members of the D phylogroup frequently occured in replicon types FIC (8/20), P (13/47), I1 (13/42), HI2 (5/14) and L/M (3/3).ConclusionsAccorging to the results, it seems that group D strains have a great potential to host a variety of plasmids (Inc-types) carrying different AMR genes. Thus, based on the results of the current study, phyogroup D could be a potential challenge in dealing with AMR in poultry. There were more strong correlations among Inc-types and AMR compared to phylotypes and AMR. It is suggested that in epidemiological studies on AMR both genomic backbone and major plasmid types should be investigated.

Dissemination of bla NDM- 1 Gene Among Several Klebsiella pneumoniae Sequence Types in Mexico Associated With Horizontal Transfer Mediated by IncF-Like Plasmids.

Nosocomial infections caused by multidrug-resistant (MDR) Klebsiella pneumoniae are a major health problem worldwide. The aim of this study was to describe NDM-1-producing K. pneumoniae strains causing bacteremia in a tertiary referral hospital in Mexico. MDR K. pneumoniae isolates were screened by polymerase chain reaction for the presence of resistance genes. In resistant isolates, plasmids were identified and conjugation assays were performed. Clonal diversity and the sequence types were determined by pulsed-field gel electrophoresis and multilocus sequence typing. A total of 80 K. pneumoniae isolates were collected from patients with bacteremia over a 1-year period. These isolates showed a level of resistance of 59% (47/80) to aztreonam, 56–60% (45–48/80) to cephalosporins, 54% (43/80) to colistin and 12.5% (10/80) to carbapenems. The carbapenem resistant isolates were blaNDM–1 carriers and negative for blaKPC, blaNDM, blaIMP, blaVIM and blaOXA–48–like carbapenemases genes. Conjugative plasmids IncFIIA and IncF group with sizes of 82–195 kbp were carriers of blaNDM–1, blaCTX–M–15, blaTEM–1, aac(6′)-Ib and/or aac(3′)-IIa. Clonal variability and nine different multilocus sequence types were detected (ST661, ST683, ST1395, ST2706, ST252, ST1198, ST690, ST1535, and ST3368) for the first time in the isolates carrying blaNDM–1 in Mexico. This study demonstrates that blaNDM–1 has remained within this hospital in recent years and suggests that it is currently the most prevalent carbapenemase among K. pneumoniae MDR strains causing bacteremia in Mexico. The horizontal transfer of blaNDM–1 gene through IncF-like plasmids among different clones demonstrates the dissemination pathway of antimicrobial resistance and underscore the need for strong and urgent joint measures to control the spread of NDM-1 carbapenemase in the hospital.

Dissemination of IncF group F1:A2:B20 plasmid-harbouring multidrug-resistant Escherichia coli ST131 before the acquisition of blaCTX-M in Japan

ObjectivesThe Escherichia coli O25-ST131 clone is responsible for global dissemination of the blaCTX-M gene. However, the prevalence of this clone in the digestive tract, devoid of antimicrobial selection, and its molecular epidemiology remain unclear. In this study, we examined the origin of blaCTX-M-positive E. coli O25-ST131 and its distribution. MethodsWe separately sequenced the chromosomal and plasmid genomes of 50 E. coli O25 isolates obtained from faecal samples of patients with diarrhoea in Japan. ResultsAlthough 36 (72%) of 50 E. coli O25 isolates were ST131, only 6 harboured blaCTX-M. According to the fimH and ybbW sequences and fluoroquinolone susceptibility, H30R1 isolates were dominant (27/36; 75%) and possessed IncFII-FIA-FIB with FAB formula subtype F1:A2:B20 plasmids at a high frequency (24/27; 89%). The F1:A2:B20 plasmids possessed more resistance genes such as blaTEM-1, aminoglycoside resistance genes and trimethoprim/sulfamethoxazole resistance genes compared with non-F1:A2:B20 plasmids. In contrast, only one blaCTX-M-14 gene was located on the F1:A2:B20 plasmids, whereas the other three were located on IncFII (F4:A-:B-) (n = 1) and IncZ (n = 2) plasmids. Two H30Rx-ST131 isolates harboured blaCTX-M-15: one was on the chromosome and the other on the IncFIA-R plasmid. The stability and conjugation ability of the F1:A2:B20 plasmids were compared with those of non-F1:A2:B20 plasmids, which revealed higher stability but lower conjugative ability. ConclusionsThese results suggest that E. coli H30R1-ST131 is a multidrug-resistant clone containing several resistance genes in the F1:A2:B20 plasmid, which were widely distributed before the acquisition of blaCTX-M.

Plasmid Replicon Typing of Antibiotic-Resistant Escherichia coli From Clams and Marine Sediments.

Unlike human isolates, environmental Escherichia coli isolates have not been thoroughly investigated for the diversity and transferability of antibiotic-resistant plasmids. In this study, antibiotic-resistant strains from marine sediment (n = 50) and clams (n = 53) were analyzed (i) for their plasmid content using a PCR-based plasmid replicon typing (PBRT) kit and (ii) for the transferability of plasmid-associated antibiotic resistance (AR) traits by mating experiments. Fifteen of the thirty replicons targeted by the PBRT kit were detected in the isolates; 8/15 were identified in both sediment and clam isolates, although at different frequencies. The most frequent replicons in sediment (74%) and in clam strains (66%) alike, were FIA, FIB, or FII, which are associated with the IncF group, followed by the I1α replicon, which was more frequent in clam (24.5%) than in sediment (10%) strains. More than 50% of the strains contained multiple replicons; although 15 were untypable, S1-PFGE analysis demonstrated that 14/15 carried no plasmids. All cryptic strains were successfully typed and were positive for IncF or IncI replicons. Antibiotic-resistant strains accounted for 63% of all isolates and were significantly (p < 0.05) more frequent in phylogroup A. Most (35%) multidrug-resistant (MDR) strains belonged to phylogroup A, too. Although 25/26 MDR strains were positive for IncF plasmids (the exception being a clam strain), the FII-FIB rep combination was predominant (63%) among the sediment isolates, whereas most clam isolates (40%) carried the FII replicon alone. In mating experiments, selected MDR strains carrying FIB, FII, and I1α replicons, used as the donors, transferred multiple ARs together with the IncF or IncI plasmids at high frequency. Since IncI plasmids are common in E. coli and Salmonella enterica isolates from poultry, our findings suggest an animal origin to the E. coli clam strains carrying IncI plasmids. They also suggest a role for IncI plasmids in the spread of ARs among environmental Enterobacteriaceae and, through the food chain, to human isolates. In conclusion, the PBRT kit proved to be a useful tool to identify plasmids carrying antibiotic-resistant genes and to shed light on the factors underpinning their diffusion.

Antibiotic resistant and extended-spectrum β-lactamase producing faecal coliforms in wastewater treatment plant effluent

Wastewater treatment plants (WWTPs) provide optimal conditions for the maintenance and spread of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). In this work we describe the occurrence of antibiotic resistant faecal coliforms and their mechanisms of antibiotic resistance in the effluent of two urban WWTPs in Ireland. This information is critical to identifying the role of WWTPs in the dissemination of ARB and ARGs into the environment. Effluent samples were collected from two WWTPs in Spring and Autumn of 2015 and 2016. The bacterial susceptibility patterns to 13 antibiotics were determined. The phenotypic tests were carried out to identify AmpC or extended-spectrum β-lactamase (ESBL) producers. The presence of ESBL genes were detected by PCR. Plasmids carrying ESBL genes were transformed into Escherichia coli DH5α recipient and underwent plasmid replicon typing to identify incompatibility groups. More than 90% of isolated faecal coliforms were resistant to amoxicillin and ampicillin, followed by tetracycline (up to 39.82%), ciprofloxacin (up to 31.42%) and trimethoprim (up to 37.61%). Faecal coliforms resistant to colistin (up to 31.62%) and imipenem (up to 15.93%) were detected in all effluent samples. Up to 53.98% of isolated faecal coliforms expressed a multi-drug resistance (MRD) phenotype. AmpC production was confirmed in 5.22% of isolates. The ESBL genes were confirmed for 11.84% of isolates (9.2% of isolates carried blaTEM, 1.4% blaSHV-12, 0.2% blaCTX-M-1 and 1% blaCTX-M-15). Plasmids extracted from 52 ESBL isolates were successfully transformed into recipient E. coli. The detected plasmid incompatibility groups included the IncF group, IncI1, IncHI1/2 and IncA/C. These results provide evidence that treated wastewater is polluted with ARB and MDR faecal coliforms and are sources of ESBL-producing, carbapenem and colistin resistant Enterobacteriaceae.

Spread of CTX-Type Extended-Spectrum β-Lactamase-Producing Escherichia coli Isolates of Epidemic Clone B2-O25-ST131 Among Dogs and Cats in Japan.

This study was performed to investigate the carriage rates of CTX-M-type extended-spectrum β-lactamase (ESBL)-producing Escherichia coli among ill companion animals in Japan. Among the 178 nonrepetitive E. coli isolates, including 131 from dogs and 47 from cats, collected between September and November 2015, 42 (23.6%) isolates from 29 dogs and 13 cats were identified as ESBL producers. The antimicrobial susceptibility, O serotype, phylogenetic group, β-lactamase genotype, plasmid replicon type, and sequence type (ST) of each isolate were analyzed. The major ESBL types were CTX-M-14 (26.8%), CTX-M-15 (24.4%), CTX-M-27 (19.5%), and CTX-M-55 (19.5%); predominant replicon types of blaCTX-M-carrying plasmid were IncF group and IncI1-Iγ. The most prevalent STs were ST131 (n = 15, 35.7%), followed by ST38, ST10, and ST410. The 15 isolates of ST131 belonged to B2-O25. E. coli B2-O25-ST131 isolates harboring blaCTX-M-15 or blaCTX-M-27 were resistant to ceftazidime and ciprofloxacin. In particular, CTX-M-15 producers showed multidrug resistance. Our results demonstrated that the CTX-M-producing pandemic E. coli clone B2-O25-ST131 has already spread in Japanese companion animals as well. Moreover, the similarity of genotypes, serotypes, phylogenetic groups, and STs of the isolates from companion animals to those from humans suggested probable transmission of resistant bacteria between pets and humans.

Molecular Characterization of β-Lactamase-Producing Escherichia coli Collected from 2001 to 2011 from Pigs in Korea.

To survey the diversity of plasmid-mediated β-lactamases, and the replicon types of the plasmids in Escherichia coli in the community, 95 multidrug-resistant E. coli isolates from stool samples of pigs in Korea from 2001 to 2011 were analyzed, and all isolates were confirmed as β-lactamase-producing bacteria, which mostly carried genes encoding TEM (100%), ACC (95%), SHV (62%), OXA (28%), CTX-M (26%), and NDM-1 (15%). Moreover, each isolate contained at least three β-lactamases genes on its plasmid. The polymerase chain reaction-based replicon typing method revealed that among the 18 replicon types, 15 types were identified in this work, and IncF groups (FIB; 100%, Frep; 99%) and HI1 (98%) type were predominant. Other replicon types such as Y, B/O, A/C, P, FIA, N, HI2, I1, and W were also detected; moreover at least three types were identified in each organism. In conclusion, a high prevalence of various types of β-lactamases genes were identified in E. coli from pigs in Korea, and these genes appeared to be mainly associated with IncF group, IncHI1, and IncY plasmids.

Dissemination of multiresistant Enterobacter cloacae isolates producing OXA-48 and CTX-M-15 in a Spanish hospital

Twenty-one multiresistant Enterobacter cloacae isolates producing OXA-48 (n=10), CTX-M-15 (n=7) or both (n=4) β-lactamases were detected in a Spanish hospital during a 1-year period (June 2013 to June 2014). The isolates were also resistant to non-β-lactam antimicrobials, further complicating the therapeutic options. Genotyping of the isolates identified two major clones (ST74 and ST66) that caused prolonged outbreaks in different buildings of the hospital as well as some sporadic isolates (ST78, ST45 and ST295). Isolates belonging to clone 1 (n=7) were carbapenem-resistant and carried the blaOXA-48 gene on a conjugative IncL/M plasmid of ca. 65kb. Clone 2 isolates (n=11) were resistant to cefepime and harboured the blaCTX-M-15 gene on an ca. 150-kb, non-conjugative plasmid of the IncF group, co-harbouring the qnrB and aac(6′)-Ib-cr genes encoding quinolone resistance. Four clone 2 isolates were also resistant to carbapenems owing to the co-production of OXA-48. Most of the isolates were recovered from critically ill patients and were admitted to intensive care units; a single patient was transferred from another Spanish hospital. Intrahospital and interhospital dissemination of multiresistant E. cloacae isolates is of major clinical concern as it could lead to endemic nosocomial situations.

Complete nucleotide sequences of bla(CTX-M)-harboring IncF plasmids from community-associated Escherichia coli strains in the United States.

Community-associated infections due to Escherichia coli producing CTX-M-type extended-spectrum β-lactamases are increasingly recognized in the United States. The bla(CTX-M) genes are frequently carried on IncF group plasmids. In this study, bla(CTX-M-15)-harboring plasmids pCA14 (sequence type 131 [ST131]) and pCA28 (ST44) and bla(CTX-M-14)-harboring plasmid pCA08 (ST131) were sequenced and characterized. The three plasmids were closely related to other IncFII plasmids from continents outside the United States in the conserved backbone region and multiresistance regions (MRRs). Each of the bla(CTX-M-15)-carrying plasmids pCA14 and pCA28 belonged to F31:A4:B1 (FAB [FII, FIA, FIB] formula) and showed a high level of similarity (92% coverage of pCA14 and 99% to 100% nucleotide identity), suggesting a possible common origin. The blaC(TX-M-14)-carrying plasmid pCA08 belonged to F2:A2:B20 and was highly similar to pKF3-140 from China (88% coverage of pCA08 and 99% to 100% nucleotide identity). All three plasmids carried multiple antimicrobial resistance genes and modules associated with virulence and biochemical pathways, which likely confer selective advantages for their host strains. The bla(CTX-M)-carrying IncFII-IA-IB plasmids implicated in community-associated infections in the United States shared key structural features with those identified from other continents, underscoring the global nature of this plasmid epidemic.

Emergence of Escherichia coli Sequence Type 131 (ST131) and ST3948 with KPC-2, KPC-3 and KPC-8 carbapenemases from a Long-Term Care and Rehabilitation Facility (LTCRF) in Northern Italy.

Aim of the study was to characterize KPC-producing Escherichia coli (KPC-Ec) clinical isolates among a Northern Italy Long-Term Care and Rehabilitation Facility (LTCRF) residents. Thirteen consecutive non repeated MDR E. coli isolates showing ertapenem Minimum Inhibitory Concentrations (MICs) >0.5mg/L, collected during the period March 2011 - May 2013 from ASP "Redaelli" inpatients, were investigated. The bla KPC/CTX-M/SHV/TEM/OXA genes were identified by PCR and sequencing. KPC-Ec isolates underwent phylotyping, Pulsed-Field Gel Electrophoresis (PFGE), multilocus sequence typing (MLST) and repetitive sequence-based PCR (rep-PCR) profiling. Incompatibility groups analysis and conjugation were also performed. Eleven out of 13 isolates, resulted bla KPC-type positive, were consistently resistant to third generation cephalosporins, fluoroquinolones and trimethoprim-sulphametoxazole (84.6%), retaining susceptibility to colistin (EUCAST guidelines). At least n = 4/11 of KPC-Ec patients received ≥48h of meropenem therapy. Sequencing identified 9 bla KPC-2, 1 bla KPC-3 and 1 bla KPC-8 determinants. KPC-Ec plasmids belonged to IncF group (FIIk replicon); conjugation confirmed bla KPC/TEM-1/OXA-9 genes transferability for 10 KPC-Ec. Although three pulsotypes (A, B, C) were identified, all KPC-Ec belonged to phylogenetic group B2. Clone B (B-B5) caused an outbreak of infection involving nine inpatients at five wards. Rep-PCR showed relatedness for seven representative KPC-Ec isolates. Here we report a LTCRF outbreak caused by a ST131-B2 E. coli associated with bla KPC-2 and bla KPC-8 genes, and the emergence of the new ST3948. Elderly people with co-morbidities are at risk for ST131 colonization. KPC-Ec clones local monitoring appears essential both to avoid their spreading among healthcare settings, and to improve therapeutic choices for LTCRF residents.

High prevalence of ST131 among CTX-M-producing Escherichia coli from community-acquired infections, in the Czech Republic.

A total of 2,683 nonrepetitive Escherichia coli isolates were collected from microbiological laboratories covering all regions of the Czech Republic, during April 2011. Antimicrobial susceptibility patterns of E. coli were assessed. All 38 cefotaxime-resistant (CTX-R) isolates were found to be extended-spectrum β-lactamase (ESBL)-positive by the double-disc synergy test. Thirty-two of those isolates produced enzymes of CTX-M-1 family, five of CTX-M-9 family, and one isolate both CTX-M types. Genotyping by multilocus sequence typing classified all ESBL-producing isolates into 13 sequence types (STs). ST131 was the most prevalent and was exclusively correlated with E. coli belonging to the more-virulent phylogroup B2. blaCTX-M-15 and blaCTX-M-9-like genes were mainly carried by plasmids belonging to the IncF group, while replicon I1 was predominant among CTX-M-1-encoding plasmids. Additionally, 63% of the ESBL-producing isolates were also resistant to ciprofloxacin. Sequence analysis of quinolone resistance-determining regions of gyrA and parC revealed the presence of amino acid substitutions in 22 out of 23 ciprofloxacin-resistant isolates. The acc(6')-Ib-cr and qnrB1 plasmid-mediated quinolone resistance genes were also detected in some of the isolates. This is the first report on the emergence and spread of CTX-M-producing E. coli in the community of the Czech Republic, indicating the high prevalence of ST131 clone among CTX-M producers.

Copy Number Change of the NDM-1 Sequence in a Multidrug-Resistant Klebsiella pneumoniae Clinical Isolate

The genetic features of the antimicrobial resistance of a multidrug resistant Klebsiella pneumoniae strain harboring bla NDM-1 were investigated to increase our understanding of the evolution of NDM-1. The strain, KPX, came from a Taiwanese patient with a hospitalization history in New Delhi. Complete DNA sequencing was performed; and the genes responsible for antimicrobial resistance were systematically examined and isolated by library screening. KPX harbored two resistance plasmids, pKPX-1 and pKPX-2, which are 250-kb and 141-kb in size, respectively, with bla NDM-1 present on pKPX-1. The plasmid pKPX-1 contained genes associated with the IncR and IncF groups, while pKPX-2 belonged to the IncF family. Each plasmid carried multiple antimicrobial resistance genetic determinants. The gene responsible for resistance to carbapenems was found on pKPX-1 and that for resistance to aztreonam was found on pKPX-2. To our surprise, we discovered that bla NDM-1 exists on pKPX-1 as multiple copies in the form of tandem repeats. Amplification of bla NDM-1 was found to occur by duplication of an 8.6-kb unit, with the copy number of the repeat varying from colony to colony. This repeat sequence is identical to that of the pNDM-MAR except for two base substitutions. The copy number of bla NDM-1 of colonies under different conditions was assessed by Southern blotting and quantitative PCR. The bla NDM-1 sequence was maintained in the presence of the antimicrobial selection; however, removal of antimicrobial selection led to the emergence of susceptible bacterial populations with a reduced copy number or even the complete loss of the bla NDM-1 sequence. The dynamic nature of the NDM-1 sequence provides a strong argument for judicious use of the broad-spectrum antimicrobials in order to reduce the development and spread of antimicrobial resistance among pathogens.

Genetic and Functional Properties of the Self-Transmissible Yersinia enterocolitica Plasmid pYE854, Which Mobilizes the Virulence Plasmid pYV

Yersinia strains frequently harbor plasmids, of which the virulence plasmid pYV, indigenous in pathogenic strains, has been thoroughly characterized during the last decades. Yet, it has been unknown whether the nonconjugative pYV can be transferred by helper plasmids naturally occurring in this genus. We have isolated the conjugative plasmids pYE854 (95.5 kb) and pYE966 (70 kb) from a nonpathogenic and a pathogenic Yersinia enterocolitica strain, respectively, and demonstrate that both plasmids are able to mobilize pYV. The complete sequence of pYE854 has been determined. The transfer proteins and oriT of the plasmid reveal similarities to the F factor. However, the pYE854 replicon does not belong to the IncF group and is more closely related to a plasmid of gram-positive bacteria. Plasmid pYE966 is very similar to pYE854 but lacks two DNA regions of the larger plasmid that are dispensable for conjugation.

Characterizing the DNA Contacts and Cooperative Binding of F Plasmid TraM to Its Cognate Sites at oriT

TraM is a DNA binding protein required for conjugative transfer of the self-transmissible IncF group of plasmids, including F, R1, and R100. F TraM binds to three sites in F oriT: two high affinity binding sites, sbmA and sbmB, which are direct repeats of nearly identical sequence involved in the autoregulation of the traM gene; and a lower affinity site, sbmC, an inverted repeat important for transfer, which is situated nearest to the nic site where transfer originates. TraM bound cooperatively to its binding sites at oriT; the presence of sbmA and sbmB increased the affinity for sbmC 10-fold. Bending of oriT DNA by TraM was minimal, suggesting that TraM, a tetramer, was able to loop the DNA when bound to sbmA and sbmB simultaneously. Hydroxyl radical footprinting of DNA of sbmA and sbmC revealed that TraM contacted the DNA within a region previously delineated by DNase I footprinting. TraM protected the CT bases within the sequence CTAG, which occurred at 12-base intervals on the top and bottom strand of sbmA, most consistently with other protected bases. The footprint on sbmC revealed that the predicted inverted repeats were protected by TraM with a pattern that began at the center of the repeats and radiated outward at 11-12 base intervals toward the 5'-ends of either strand. At high protein concentrations, this pattern extended beyond the footprint defined by DNase I, suggesting that the DNA was wrapped around the protein forming a nucleosome-like structure, which could aid in preparing the DNA for transfer.

Replicon typing of virulence plasmids of enterotoxigenic Escherichia coli isolates from cattle.

Plasmid DNA hybridization with probes for virulence factors used for basic replicons of plasmids was used to identify the virulence plasmids of a collection of enterotoxigenic Escherichia coli isolates from cattle. The virulence probes were derived from the genes coding for the heat-stable enterotoxin STaP and for the F5 (K99) and F41 fimbrial adhesins. The replicon probes were derived from 16 different basic replicons of plasmids (probes repFIA, repFIB, repFIC, repFIIA, repI1, repHI1, repHI2, repL/M, repN, repP, repQ, repT, repU, repW, repX, and repY). The virulence genes coding for the STaP enterotoxin and for the F5 adhesin were located on a single plasmid band in each isolate. The sizes of most of these virulence plasmids were from 65 to 95 MDa. The F41 probe failed to hybridize with any plasmid band. The virulence plasmids had multireplicon types typical of plasmids of the IncF groups. The most common basic replicon association was the triple RepFIA-RepFIB-RepFIC family association.

Presentation of foreign antigenic determinants at the bacterial cell surface using the TraT lipoprotein

The TraT protein is an oligomeric outer membrane lipoprotein, specified by plasmids of the IncF group, that is very highly exposed at the bacterial cell surface. We have investigated the feasibility of using the protein as a carrier of foreign antigenic determinants by genetic insertion of the C3 epitope of type 1 poliovirus into defined sites in the protein. Several of the hybrid proteins constructed had features characteristic of the native protein and one in particular retained the ability to function in surface exclusion and genetic suppression assays as well as to assemble into oligomers. Our results suggest that the TraT protein can be used to transport and present foreign antigenic determinants at the cell surface.