S1 Nuclease Pulsed-field Gel Electrophoresis Research Articles

BlaCTX-M-₁₅-carrying Escherichia coli and Salmonella isolates from livestock and food in Germany.

The characterization of CTX-M-₁₅ β-lactamase-producing Escherichia coli and Salmonella isolates originating mainly from German livestock and food. E. coli (526, mainly commensals) and Salmonella (151) non-human isolates resistant to third-generation cephalosporins, originating from routine and monitoring submissions (2003-12) to the Federal Institute for Risk Assessment and different national targeted studies (2011-12), were examined for the presence of blaCTX-M-₁₅ genes by PCR amplification/sequencing. Additional resistance and virulence genes were screened by DNA microarray and PCR amplification. E. coli isolates with blaCTX-M-₁₅ were characterized by phylogenetic grouping, PFGE and multilocus sequence typing (MLST). The blaCTX-M-15 plasmids were analysed by replicon typing, plasmid MLST, S1 nuclease PFGE and Southern blot hybridization experiments. Twenty-one E. coli (livestock, food and a toy; 4.0%) and two Salmonella (horse and swine; 1.3%) isolates were CTX-M-₁₅ producers. E. coli isolates were mainly ascribed to three clonal lineages of sequence types ST678 (German outbreak with enteroaggregative Shiga-toxin-producing E. coli O104:H4; salmon, cucumber and a toy), ST410 (poultry, swine and cattle farms) and ST167/617 (swine farms and turkey meat). The blaCTX-M-₁₅ genes were located on IncI1 and multireplicon IncF plasmids or on the chromosome of E. coli ST410 isolates. The prevalence of CTX-M-₁₅-producing isolates from non-human sources in Germany is still low. The blaCTX-M-₁₅ gene is, however, present in multidrug-resistant E. coli clones with pathogenic potential in livestock and food. The maintenance of the blaCTX-M-₁₅ gene due to chromosomal carriage is noteworthy. The possibility of an exchange of CTX-M-₁₅-producing isolates or plasmids between livestock and humans (in both directions) deserves continuous surveillance.

IncA/C plasmid-mediated spread of CMY-2 in multidrug-resistant Escherichia coli from food animals in China.

ObjectivesTo obtain a broad molecular epidemiological characterization of plasmid-mediated AmpC β-lactamase CMY-2 in Escherichia coli isolates from food animals in China.MethodsA total of 1083 E. coli isolates from feces, viscera, blood, drinking water, and sub-surface soil were examined for the presence of CMY-2 β-lactamases. CMY-2-producing isolates were characterized as follows: the bla CMY-2 genotype was determined using PCR and sequencing, characterization of the bla CMY-2 genetic environment, plasmid sizing using S1 nuclease pulsed-field gel electrophoresis (PFGE), PCR-based replicon typing, phylogenetic grouping, XbaI-PFGE, and multi-locus sequence typing (MLST).ResultsAll 31 CMY-2 producers were only detected in feces, and presented with multidrug resistant phenotypes. All CMY-2 strains also co-harbored genes conferring resistance to other antimicrobials, including extended spectrum β-lactamases genes (bla CTX-M-14 or bla CTX-M-55), plasmid-mediated quinolone resistance determinants (qnr, oqxA, and aac-(6′)-Ib-cr), floR and rmtB. The co-transferring of bla CMY-2 with qnrS1 and floR (alone and together) was mainly driven by the Inc A/C type plasmid, with sizes of 160 or 200 kb. Gene cassette arrays inserted in the class 1 or class 2 integron were amplified among 12 CMY-2 producers. CMY-2 producers belonged to avirulent groups B1 (n = 12) and A (n = 11), and virulent group D (n = 8). There was a good correlation between phylogenetic groups and sequence types (ST). Twenty-four STs were identified, of which the ST complexes (STC) 101/B1 (n = 6), STC10/A (n = 5), and STC155/B1 (n = 3) were dominant.ConclusionsCMY-2 is the dominant AmpC β-lactamase in food animals and is associated with a transferable replicon IncA/C plasmid in the STC101, STC10, and STC155 strains.

Chromosomal location of blaCTX-M genes in clinical isolates of Escherichia coli from Germany, The Netherlands and the UK

This study aimed to detect and characterise clinical Escherichia coli isolates suspected of carrying chromosomally encoded CTX-M enzymes. Escherichia coli (n=356) obtained in Germany, The Netherlands and the UK (2005–2009) and resistant to third-generation cephalosporins were analysed for the presence of ESBL-/AmpC-encoding genes within the European SAFEFOODERA-ESBL project. β-Lactamases and their association with IS26 and ISEcp1 were investigated by PCR. Isolates were typed by phylogenetic grouping, MLST and PFGE. Plasmids were visualised by S1 nuclease PFGE, and the location of blaCTX-M genes was determined by Southern hybridisation of XbaI-, S1- and I-CeuI-digested DNA. ESBL enzymes could not be located on plasmids in 17/356 isolates (4.8%). These 17 isolates, from different countries and years, were ascribed to phylogenetic groups D (9), B2 (6) and B1 (2), and to seven sequence types, with ST38 being the most frequent (7 phylogroup D isolates). Eleven isolates produced CTX-M-15. blaCTX-M-15 genes were associated with ISEcp1. The remaining isolates expressed the CTX-M group 9 β-lactamases CTX-M-14 (4), CTX-M-9 (1) and CTX-M-51 (1). blaCTX-M probes hybridised with I-CeuI- and/or XbaI-digested DNA, but not with S1-digested DNA, corroborating their chromosomal location. To summarise, only 4.8% of a large collection of ESBL-producing E. coli isolates harboured chromosomal blaCTX-M genes. These isolates were of human origin and belonged predominantly to ST38 and ST131, which possibly indicates the role of these sequence types in this phenomenon. However, heterogeneity among isolates was found, suggesting that their spread is not only due to the dispersion of successful E. coli clones.

Emergence and maintenance of multidrug-resistant Escherichia coli of canine origin harbouring a blaCMY-2-IncI1/ST65 plasmid and topoisomerase mutations

To characterize the mechanisms implicated in fluoroquinolone (FQ) and expanded-spectrum cephalosporin (ESC) resistance in three clinical and seven faecal multidrug-resistant (MDR; resistant to at least three antimicrobial classes) Escherichia coli isolates from a dog with atopic dermatitis, also suffering from recurrent otitis, that had already been exposed to prolonged antimicrobial treatment and colonized for a long period. MICs of FQs, ESCs and other antimicrobials were determined by the broth microdilution method. Phenotypic tests (efflux pump inhibition and combination disc tests) and isoelectric focusing were combined with genotypic analyses [PCRs, sequencing, conjugation, S1 nuclease PFGE, PCR-based replicon typing, plasmid multilocus sequence typing (pMLST) and PCR mapping] to characterize the molecular basis of FQ and ESC resistance. Isolates were further characterized by MLST and PFGE. Three otitis and five faecal isolates with enrofloxacin MICs of 32 to >128 mg/L displayed the GyrA:S83L+D87N/ParC:E62K/ParE:G545D pattern harbouring novel ParC and ParE substitutions, whereas the two remaining faecal isolates were susceptible or borderline resistant single-step mutants (GyrA:S83L pattern) and carried qnrS1. Efflux pump overexpression also contributed to FQ resistance and the MDR phenotype. The three otitis and five faecal isolates also exhibited cefoxitin/ceftazidime MICs of 32-64 mg/L and harboured blaCMY-2, adjusted to ISEcp1, on an IncI1/ST65 conjugative plasmid, previously described in Salmonella Heidelberg from poultry. Interestingly, all isolates shared an identical MLST type (ST212), with the otitis isolates showing indistinguishable patterns with the high-level resistant faecal E. coli isolates. The long-term maintenance of FQ- and ESC-resistant clones harbouring topoisomerase mutations and a blaCMY-2-IncI1/ST65 plasmid in canine commensal flora after prolonged antimicrobial use may contribute to the dissemination of multidrug resistance.

Diversity of Plasmids Encoding Virulence and Resistance Functions in Salmonella enterica subsp. enterica Serovar Typhimurium Monophasic Variant 4,[5],12:i:- Strains Circulating in Europe

Plasmids encoding resistance and virulence properties in multidrug resistant (MDR) Salmonella enterica (S.) serovar Typhimurium monophasic variant 4,[5],12:i:- isolates recovered from pigs and humans (2006-2008) in Europe were characterised. The isolates were selected based on the detection by PCR-amplification of S. Typhimurium virulence plasmid pSLT genes and were analysed by multi-locus sequence typing (MLST). The resistance genes present in the isolates and the association of these genes with integrons, transposons and insertion sequences were characterised by PCR-sequencing, and their plasmid location was determined by alkaline lysis and by S1-nuclease pulsed-field gel electrophoresis (PFGE) Southern-blot hybridisation. Plasmids were further analysed by replicon typing, plasmid MLST and conjugation experiments. The 10 S. 4,[5],12,i:- selected isolates belonged to ST19. Each isolate carried a large plasmid in which MDR with pSLT-associated virulence genes were located. After analysis, eight different plasmids of three incompatibility groups (IncA/C, IncR and IncF) were detected. Two IncA/C plasmids represented novel variants within the plasmid family of the S. 4,[5],12:i:- Spanish clone, and carried an empty class 1 integron with a conventional qacEΔ1-sul1 3′ conserved segment or an In-sul3 type III with estX-psp-aadA2-cmlA1-aadA1-qacH variable region linked to tnpA440-sul3, part of Tn2, Tn21 and Tn1721 transposons, and ISCR2. Four newly described IncR plasmids contained the resistance genes within In-sul3 type I (dfrA12-orfF-aadA2-cmlA1-aadA1-qacH/tnpA440-sul3) and part of Tn10 [tet(B)]. Two pSLT-derivatives with FIIs-ST1+FIB-ST17 replicons carried cmlA1-[aadA1-aadA2]-sul3-dfrA12 and bla TEM-1 genes linked to an In-sul3 type I integron and to Tn2, respectively. In conclusion, three emerging European clones of S. 4,[5],12:i:- harboured MDR plasmids encoding additional virulence functions that could contribute significantly to their evolutionary success.

High diversity of plasmids harbouring blaCMY-2 among clinical Escherichia coli isolates from humans and companion animals in the upper Midwestern USA

To determine the population structure and genetic relatedness of plasmids encoding CMY-2 β-lactamase in clinical Escherichia coli from humans and companion animals within a defined geographical area. In total, 42 human and 73 companion animal isolates displaying an AmpC phenotype were isolated at a regional diagnostic reference laboratory in the upper Midwestern USA during 2009-11. Following PCR screening for transferable AmpC genes and plasmid transformation, blaCMY-2-positive plasmids were characterized by S1 nuclease PFGE, PCR-based replicon typing, antimicrobial susceptibility testing of transformants, conjugation experiments, plasmid multilocus sequence typing and restriction fragment length polymorphism. blaCMY-2 occurred in 6 (14%), 56 (86%) and 6 (75%) isolates from humans, dogs and cats, respectively. Usually plasmids carrying blaCMY-2 were conjugative (78%) and did not contain additional resistance genes (82%). The replicon types were IncI1 (52%), IncA/C (13%), IncFII (10%), IncI2 (5%), IncL/M (3%), IncB/O (2%) or non-typeable (15%). Related IncI1/ST12 plasmids were detected in one human and five canine isolates, while the remaining plasmids did not show similarity across host species. A novel epidemiological linkage of blaCMY-2 with IncL/M plasmids and a new CMY gene variant (blaCMY-108) were found in human isolates. This study is one of the first One Health attempts to compare plasmids encoding CMY-2 β-lactamase among clinical isolates from humans and companion animals in the same region. The results indicate an unforeseen heterogeneity of plasmid backgrounds and suggest limited exchange between the two populations, in which blaCMY-2 occurred at very different frequencies and was harboured by distinct plasmid types.

NDM-1–producing Strains, FamilyEnterobacteriaceae,in Hospital, Beijing, China

NDM-1–producing Strains, Family<i>Enterobacteriaceae,</i>in Hospital, Beijing, China

Epidemiological Characteristics of blaNDM-1 in Enterobacteriaceae and Acinetobacter calcoaceticus–Acinetobacter baumannii Complex in China from 2011 to 2012

Objectives: The study aimed to investigate the prevalence and the epidemiological characteristics of blaNDM-1 in Enterobacteriaceae and Acinetobacter calcoaceticus–Acinetobacter baumannii complex (ABC) in China from July 2011 to June 2012. Methods: All organisms studied were screened for the presence of blaNDM-1 using PCR. For those blaNDM-1- positive strains, 16S rRNA along with API strips were performed to validate the bacterial genus and species. The ABCs were reconfirmed by PCR detection of blaOXA-51-like. The antibiotic susceptibilities were assessed by determining minimum inhibitory concentration (MIC) of them using two-folder agar dilution test recommended by the Clinical and Laboratory Standards Institute (CLSI). Molecular typing was performed using pulsed-field gel electrophoresis (PFGE). An S1 nuclease PFGE (S1-PFGE) and Southern blot hybridization were conducted to ascertain the gene location of blaNDM-1. Results: Among 2170 the family Enterobacteriaceae and 600 ABCs, seven Enterobacteriaceae strains and two A. calcoaceticus isolates from five different provinces carried the blaNDM-1 gene. The seven Enterobacteriaceae strains were four Klebsiella pneumoniae, one Enterobacter cloacae, one Enterobacter aerogen and one Citrobacter freundii, respectively. All of them showed non-susceptible to any agent of imipenem, meropenem, panipenem and ertapenem. Two A. calcoaceticus were both resistant to imipenem and meropenem. Three K. pneumoniae showed the same PFGE profiles. Eight blaNDM-1 genes were located on plasmids and one on chromosome. Conclusions: Compared with the previous reports, the numbers and species of the blaNDM-1 in Enterobacteriaceae have been significantly increased in China and most of them can disseminate which should be drawn great attention. Consecutive surveillance should be implemented and focused on the dissemination of blaNDM-1 among gram-negative clinical isolates as well.

Dissemination and Characterization of Plasmids Carrying oqxAB-blaCTX-M Genes in Escherichia coli Isolates from Food-Producing Animals

BackgroundThe association of PMQR and ESBLs in negative-bacteria isolates has been of great concern. The present study was performed to investigate the prevalence of co-transferability of oqxAB and bla CTX-M genes among the 696 Escherichia coli (E. coli) isolates from food-producing animals in South China, and to characterize these plasmids.MethodsThe ESBL-encoding genes (bla CTX-M, bla TEM and bla SHV), and PMQR (qnrA, qnrB, qnrS, qnrC, qnrD, aac(6’)-Ib-cr, qepA, and oqxAB) of these 696 isolates were determined by PCR and sequenced directionally. Conjugation, S1 nuclease pulsed-field gel electrophoresis (PFGE) and Southern blotting experiments were performed to investigate the co-transferability and location of oqxAB and bla CTX-M. The EcoRI digestion profiles of the plasmids with oqxAB-bla CTX-M were also analyzed. The clonal relatedness was investigated by PFGE.ResultsOf the 696 isolates, 429 harbored at least one PMQR gene, with oqxAB (328) being the most common type; 191 carried bla CTX-M, with bla CTX-M-14 the most common. We observed a significant higher prevalence of bla CTX-M among the oqxAB-positive isolates (38.7%) than that (17.4%) in the oqxAB-negative isolates. Co-transferability of oqxAB and bla CTX-M was found in 18 of the 127 isolates carrying oqxAB-bla CTX-M. These two genes were located on the same plasmid in all the 18 isolates, with floR being on these plasmids in 13 isolates. The co-dissemination of these genes was mainly mediated by F33:A-: B- and HI2 plasmids with highly similar EcoRI digestion profiles. Diverse PFGE patterns indicated the high prevalence of oqxAB was not caused by clonal dissemination.Conclusion bla CTX-M was highly prevalent among the oqxAB-positive isolates. The co-dissemination of oqxAB-bla CTX-M genes in E. coli isolates from food-producing animals is mediated mainly by similar F33:A-: B- and HI2 plasmids. This is the first report of the co-existence of oqxAB, bla CTX-M, and floR on the same plasmids in E. coli.

Characterisation of plasmids implicated in the mobilisation of extended-spectrum and AmpC β-lactamase genes in clinical Salmonella enterica isolates and temporal stability of the resistance genotype

Plasmids implicated in the mobilisation of β-lactamase genes in extended-spectrum β-lactamase (ESBL)- and AmpC-producing Salmonella enterica isolates recovered from three Spanish hospitals were characterised. The temporal stability of these plasmids and of the resistance phenotype without antimicrobial pressure was also assessed in the laboratory setting. The resistance determinants and their genetic environments were characterised by PCR sequencing, and their genomic location was analysed by S1 nuclease pulsed-field gel electrophoresis (PFGE) and I-CeuI PFGE, followed by Southern blot hybridisation. The 11 S. enterica studied strains carried blaCTX-M-9 (serovar Virchow, 2 isolates), blaCTX-M-10 (Virchow, 2), blaCTX-M-14 (Enteritidis, 1), blaCTX-M-15 (Gnesta and S. enterica group C, 2), blaSHV-2 (Livingstone, 1), blaSHV-12 (Enteritidis, 1) and blaCMY-2 (Bredeney, 2). The ISEcp1–blaCTX-M-14–IS903 and ISEcp1–blaCTX-M-15–orf477 genetic structures were detected. IncI1 and IncA/C plasmids carried blaCTX-M-14, blaCTX-M-15, blaSHV-2, blaSHV-12 and blaCMY-2 genes. blaCTX-M-9 included in an In60 complex integron and blaCTX-M-10 linked to a phage-related element were found in non-typeable plasmids. Conjugation and temporal stability experiments were performed in vitro through daily passages (100 days) in the absence of antimicrobial pressure. In the stability experiments, 5 of the 11 tested isolates lost the ESBL or AmpC plasmidic genes and this was associated with concomitant loss of the whole or partial plasmid. In conclusion, successful plasmids belonging to different Inc groups mobilise ESBL- and AmpC-encoding genes in S. enterica. Loss of ESBL/AmpC genes in the absence of antimicrobial pressure might explain the low prevalence of these β-lactamases among Salmonella isolates.

Unique Class 1 Integron and Multiple Resistance Genes Co-located on IncHI2 Plasmid Is Associated with the Emerging Multidrug Resistance of Salmonella Indiana Isolated from Chicken in China

The objective of this study was to clarify the molecular antimicrobial resistance mechanisms of Salmonella enterica serovar Indiana isolated from chickens in China. A total of 327 chicken intestinal content and feces were collected in Shandong, China in 2009. Isolates were serotyped and antimicrobial susceptibility testing was performed. Thirty-five (10.7%) Salmonella isolates were recovered, and 16 (45.7%) were Salmonella enterica serovar Indiana, which were resistant to at least 14 of 15 antimicrobial agents. The 16 Salmonella enterica serovar Indiana detected and other 13 Salmonella enterica serovar Indiana that were selected from 133 Salmonella enterica serovar Indiana isolated in 2008 were subjected to pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Then class 1 integron and drug resistance genes were detected by polymerase chain reaction. Linkage between plasmids and resistance components was determined by conjugation, electrotransformation, S1 nuclease-PFGE, polymerase chain reaction-based replicon typing and Southern blot assays. Regions flanking integrons were sequenced by modified random primer walking strategy. PFGE and MLST suggested that all the 29 Salmonella enterica serovar Indiana isolates that shared >78% similarity in PFGE patterns were of the same MLST type, ST17. Two kinds of class 1 integrons had different integrase genes and the same variable region (dfrA7/aadA4/IS26/aac(6')-Ib/blaOXA-1/catB3/arr-3), and additional antimicrobial resistance genes such as blaCTX-M-24, floR, and so on were detected on IncHI2 plasmids in 29 Salmonella enterica serovar Indiana, and seven plasmids were conjugative. Analysis of the genetic environment of the integrons suggested that these integrons might have been formed by the help of IS26. To our knowledge, the variable region in class 1 integrons is the longest reported in Salmonella to date. The unique integrons and multiple resistance genes co-located on the IncHI2 plasmid contributed to the dissemination of multidrug resistance.

Cfr-Mediated Linezolid-Resistance among Methicillin-Resistant Coagulase-Negative Staphylococci from Infections of Humans

Four methicillin-resistant coagulase-negative staphylococci (MRCoNS), one Staphylococcus haemolyticus and three Staphylococcus cohnii, from infections of humans collected via the Ministry of Health National Antimicrobial Resistance Surveillance Net (Mohnarin) program in China were identified as linezolid-resistant. These four isolates were negative for the 23S rRNA mutations, but positive for the gene cfr. Mutations in the gene for the ribosomal protein L3, which resulted in the amino acid exchanges Gly152Asp and Tyr158Phe, were identified in S. haemolyticus 09D279 and S. cohnii NDM113, respectively. In each isolate, the cfr gene was located on a plasmid of ca. 35.4 kb, as shown by S1 nuclease pulsed-field gel electrophoresis and Southern blotting experiments. This plasmid was indistinguishable from the previously described plasmid pSS-02 by its size, restriction pattern, and a sequenced 14-kb cfr-carrying segment. Plasmid pSS-02 was originally identified in staphylococci isolated from pigs. This is the first time that a cfr-carrying plasmid has been detected in MRCoNS obtained from intensive care patients in China. Based on the similarities to the cfr-carrying plasmid pSS-02 from porcine coagulase-negative staphylococci, a transmission of this cfr-carrying plasmid between staphylococci from pigs and humans appears to be likely.

Molecular characterization of blaESBL–harboring conjugative plasmids identified in multi-drug resistant Escherichia coli isolated from food-producing animals and healthy humans

Background: Extended-spectrum β-lactamase (ESBL)-encoding genes are frequently mapped to plasmids, yet few of these structures have been characterized at the molecular level, to date.Methods: Eighty-seven ESBL-producing Escherichia coli were isolated from fecal samples of food-producing animals and healthy humans in Switzerland from 2009 to 2011. Plasmid DNA of all isolates was purified. Broth mating assays were carried out individually for 32 isolates to determine if the ESBL marker could be transferred by conjugation. The plasmid sizes were determined by S1-nuclease pulsed-field gel electrophoresis (PFGE) and the plasmids were typed by PCR-based replicon typing. Susceptibility tests by disk diffusion followed with a re-analysis S1-nuclease PFGE and PCRs were performed to confirm plasmid transfer. Microarray was performed to detect additional antibiotic resistance markers and multi-locus sequence typing was also performed in selected donor strains. The phylotypes were identified by triplex PCR.Results: About half (n = 46) of the 87 isolates carried small (<20-kb) plasmids. All selected 32 isolates contained large plasmids (ranging in sizes from 20- to 600-kb). Eleven plasmid replicon types were detected. Of these, IncFIA (n = 5), IncFIB (n = 9), and IncK/B (n = 4) were common. Nine isolates demonstrated the ability to transfer their cefotaxime resistance marker at high transfer rates. Plasmid profile re-analysis of these transconjugants identified 16 plasmids. IncFIB and IncI1 were the most prevalent replicon types. Phylogenetic grouping showed that five of the nine donor strains belonged to phylogroup B1. Nine different sequence types were identified in nine tested donor strains.Conclusion: Characterization of these ESBL-encoding conjugative plasmids extends our understanding on these resistance markers in multi-drug resistant E. coli cultured from healthy human and animal sources.

Non-ST131 Escherichia coli from cattle harbouring human-like blaCTX-M-15-carrying plasmids

To characterize bla(CTX-M-15)-carrying plasmids and lineages of nine strains of Escherichia coli from cattle. Plasmid DNA was analysed using PCR-based replicon typing and plasmid sub-typing schemes, restriction fragment length polymorphism, S1 nuclease-PFGE and Southern hybridization. Strains were characterized by PFGE, multilocus sequence typing, phylogenetic grouping and B2-O25b:H4-ST131 (where ST stands for sequence type) clone screening. Susceptibilities to antimicrobials were determined by agar diffusion and resistance genes were characterized by PCR and sequencing. The bla(CTX-M-15) gene was found on F31:A4:B1/IncFII and F2:A-:B-/IncFII plasmids, which have been reported abundantly in humans. On F31:A4:B1/IncFII plasmids, the bla(CTX-M-15) gene was associated with the bla(TEM-1), bla(OXA-1) and aac(6')-Ib-cr resistance genes. The bla(CTX-M-15) gene was also found on IncI1 plasmids of the CC31 clonal complex, recently identified in the human epidemic and virulent E. coli clone O104:H4. None of the cattle isolates belonged to the human and widespread clone B2-O25b:H4/ST131, but were mostly of new STs and of the phylogenetic groups A (n=4), B1 (n=3) or D (n=2). The E. coli isolates harbouring the bla(CTX-M-15)-carrying plasmids were genetically diverse, and were recovered from different geographical locations and farms and at different times. This study demonstrates that bla(CTX-M-15)-carrying plasmids from cattle-derived non-ST131 E. coli isolates were highly similar to those found in ST131 E. coli isolates commonly reported in humans. It also exemplifies the key role of plasmids versus clonal dissemination in the spread of the bla(CTX-M-15) gene among cattle, and possibly between E. coli isolates detected in humans and cattle.

Sequence types and plasmid carriage of uropathogenic Escherichia coli devoid of phenotypically detectable resistance

Plasmids play a major role in the dissemination of antibiotic resistance, and several studies have shown the association between specific resistance mechanisms and certain plasmid types and/or Escherichia coli lineages. This study describes the distribution of plasmids, replicon types, sequence types (STs) and ST complexes (STCs) of E. coli devoid of phenotypic resistance to 24 antibiotics. Eighty E. coli isolates from urinary tract infections from four European countries were selected because of their lack of phenotypically detectable antibiotic resistance. The isolates were characterized to the phylogenetic group level using PCR and to ST by multilocus sequence typing. Plasmid carriage was assessed using S1 nuclease PFGE profiling and PCR-based replicon typing. Plasmids were detected in only 38/80 (47%) of the isolates; one (n = 18), two (n = 14), three (n = 5) and four (n = 1) plasmids. Six different replicon types were identified, the most common being a combination of IncFII and IncFIB. Most isolates belonged to phylogenetic group B2 and STC73 (n = 20), STC95 (n = 7) and ST420 (n = 6). A high proportion of STC73 isolates (75%) was devoid of plasmids. No association could be found between specific STs and replicon type. A large proportion of E. coli strains phenotypically devoid of antibiotic resistance were plasmid naive. Those isolates that harboured plasmids displayed replicon types similar to those of resistant isolates, but the distributions of STs and STCs were different. This may indicate chromosomally encoded mechanisms important for the stabilization of plasmids harbouring antibiotic resistance.

Complete sequencing of the FII plasmid pHK01, encoding CTX-M-14, and molecular analysis of its variants among Escherichia coli from Hong Kong

We characterized plasmids encoding CTX-M-14 β-lactamase originating from Escherichia coli isolates recovered from patients with uncomplicated cystitis or individuals with faecal colonization in Hong Kong from 2002 to 2004. Plasmids carrying CTX-M-14 were studied by conjugation, replicon typing, S1 nuclease-PFGE and plasmid PCR-restriction fragment length polymorphism (RFLP). The complete sequence of pHK01, a 70 kb plasmid encoding CTX-M-14 from an E. coli strain, was determined and the results compared with reference plasmids and aligned with GenBank data. The blaCTX-M-14 plasmids could be transferred in 23 of 44 E. coli strains tested. Among the 23 transconjugants, the replicon types of the CTX-M-14-encoding plasmid were FII (n=13), I1-Iγ (n=4), F1B (n=2), FII and I1-Iγ (n=1), K (80 kb, n=1) and undetermined (n=2). Plasmid pHK01 (FII replicon) shares a high degree of homology with R100 except mainly for a 11 kb variable region containing blaCTX-M-14 (with an upstream ISEcp1 and a downstream truncated IS903), an iron transport system, an outer membrane protein (malB, maltoporin) and a putative toxin-antitoxin plasmid stability system (yacABC). It was highly related to blaCTX-M-14 (pKF3-70) and blaCTX-M-24 (pEG356) plasmids reported from mainland China in 2006 and Vietnam in 2007, respectively. Subtyping by a plasmid PCR-RFLP scheme showed that 10 of the 13 FII plasmids originating from isolates collected by multiple laboratories exhibited either identical or highly similar profiles. This study showed that narrow host-range FII plasmids play important roles in the dissemination of CTX-M-14. FII plasmids closely related to pHK01 have disseminated widely in the Hong Kong community.

Characterization of multidrug-resistant, qnrB2-positive and extended-spectrum- -lactamase-producing Salmonella Concord and Salmonella Senftenberg isolates

To characterize plasmids and resistance genes of multidrug-resistant (MDR) Salmonella Senftenberg and Salmonella Concord isolated from patients in the Netherlands. The resistance genes of four MDR Salmonella isolates (three Salmonella Concord and one Salmonella Senftenberg) were identified by miniaturized microarray, PCR and sequencing. Plasmids were characterized by S1 nuclease-PFGE and PCR-based replicon typing (PBRT). Linkage between plasmids and genes was determined by conjugation experiments and microarray analysis. The genetic relationship between the three Salmonella Concord isolates was determined by XbaI-PFGE. A large variety of resistance genes was detected, including qnrB2 and the beta-lactamase genes bla(TEM-1) and bla(SHV-12) in all isolates; moreover all Salmonella Concord isolates also harboured bla(CTX-M-15). Salmonella Senftenberg harboured a large IncHI2 plasmid. The three Salmonella Concord isolates harboured two large plasmids typed as IncHI2 and IncA/C. We detected the first plasmid-mediated MDR Salmonella isolates in the Netherlands harbouring both qnr and extended-spectrum beta-lactamase (ESBL) genes. In Salmonella Senftenberg one large plasmid (IncHI2) and in Salmonella Concord two large plasmids (IncHI2 and IncA/C) were responsible for the multidrug resistance.