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1.  Longitudinal Farm Study of Extended-Spectrum β-Lactamase-Mediated Resistance 
Journal of Clinical Microbiology  2006;44(5):1630-1634.
Extended-spectrum β-lactamase (ESBL)-mediated resistance is of considerable importance in human medicine. Recently, such enzymes have been reported in bacteria from animals. We describe a longitudinal study of a dairy farm suffering calf scour with high mortality rates. In November 2004, two Escherichia coli isolates with resistance to a wide range of β-lactams (including amoxicillin-clavulanate and cefotaxime) were isolated from scouring calves. Testing by PCR and sequence analysis confirmed the isolates as being both blaCTX-M14/17 and blaTEM-35 (IRT-4) positive. They had indistinguishable plasmid and pulsed-field gel electrophoresis (PFGE) profiles. Transferability studies demonstrated that blaCTX-M was located on a conjugative 65-MDa IncK plasmid. Following a farm visit in December 2004, 31/48 calves and 2/60 cows were positive for E. coli with blaCTX-M. Also, 5/48 calf and 28/60 cow samples yielded blaCTX- and blaTEM-negative E. coli isolates that were resistant to cefotaxime, and sequence analysis confirmed that these presented mutations in the promoter region of the chromosomal ampC gene. Fingerprinting showed 11 different PFGE types (seven in blaCTX-M-positive isolates). Six different PFGE clones conjugated the same blaCTX-M-positive IncK plasmid. One clone carried a different-sized, blaCTX-M-positive, transformable plasmid. This is the first report of blaCTX-M from livestock in the United Kingdom, and this report demonstrates the complexity of ESBL epidemiology. Results indicate that horizontal plasmid transfer between strains as well as horizontal gene transfer between plasmids have contributed to the spread of resistance. We have also shown that some clones can persist for months, suggesting that clonal spread also contributes to the perpetuation of resistance.
doi:10.1128/JCM.44.5.1630-1634.2006
PMCID: PMC1479190  PMID: 16672386
2.  Commensal Enterobacteriaceae as reservoirs of extended-spectrum beta-lactamases, integrons, and sul genes in Portugal 
Bacteria colonizing the human intestine have a relevant role in the spread of antimicrobial resistance. We investigated the faecal carriage of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in healthy humans from Portugal and analyzed the distribution of sul genes and class 1 and 2 integrons. Faecal samples (n = 113) were recovered from healthy persons (North/Centre of Portugal, 2001–2004) and plated on MacConkey agar with and without ceftazidime (1 mg/L) or cefotaxime (1 mg/L). Isolates representing different morphotypes/plate and antibiotic susceptibility patterns (n = 201) were selected. Isolates resistant to sulfonamides and/or streptomycin, gentamicin, and trimethoprim were screened (PCR and sequencing) for sul genes (sul1, sul2, sul3) and class 1 and 2 integrons. Presence of ESBLs was inferred using the double disk synergy test (DDST) and further confirmed by PCR and sequencing. ESBL producers were selected for clonal analysis, plasmid characterization and conjugation assays by standard methods. ESBL-producing isolates were found in 1.8% (2/113) of samples, corresponding to Escherichia coli of phylogroups A (n = 1) and B1 (n = 1) carrying transferable blaCTX-M-14 and the new blaTEM-153, respectively. A 80kb IncK plasmid bearing blaCTX-M-14 was found, being highly related to that widely spread among CTX-M-14 producers of humans and animals from Portugal and other European countries. sul genes were found in 88% (22/25; sul2-60%, sul1-48%, sul3-4%) of the sulfonamide resistant isolates. Class 1 integrons were more frequently found than class 2 (7%, 14/201 vs. 3%, 6/201). Interestingly, gene cassette arrangements within these platforms were identical to those commonly observed among Enterobacteriaceae from Portuguese food-producing animals, although aadA13 is here firstly described in Morganella morganii. These results reinforce the relevance of human commensal flora as reservoir of clinically relevant antibiotic resistance genes including blaESBLs, and highly transferable genetic platforms as IncK epidemic plasmids.
doi:10.3389/fmicb.2013.00080
PMCID: PMC3619534  PMID: 23579192
ESBLs; CTX-M-14; TEM-153; class 1 and class 2 integrons; healthy volunteers
3.  Host-Specific Patterns of Genetic Diversity among IncI1-Iγ and IncK Plasmids Encoding CMY-2 β-Lactamase in Escherichia coli Isolates from Humans, Poultry Meat, Poultry, and Dogs in Denmark 
Applied and Environmental Microbiology  2016;82(15):4705-4714.
ABSTRACT
CMY-2 is the most common plasmid-mediated AmpC β-lactamase in Escherichia coli isolates of human and animal origin. The aim of this study was to elucidate the epidemiology of CMY-2-producing E. coli in Denmark. Strain and plasmid relatedness was studied in 93 CMY-2-producing clinical and commensal E. coli isolates collected from 2006 to 2012 from humans, retail poultry meat, broilers, and dogs. Multilocus sequence typing (MLST), antimicrobial susceptibility testing, and conjugation were performed in conjunction with plasmid replicon typing, plasmid multilocus sequence typing (pMLST), restriction fragment length polymorphism (RFLP), and sequencing of selected blaCMY-2-harboring plasmids. MLST revealed high strain diversity, with few E. coli lineages occurring in multiple host species and sample types. blaCMY-2 was detected on plasmids in 83 (89%) isolates. Most (75%) of the plasmids were conjugative and did not (96%) cotransfer resistance to antimicrobials other than cephalosporins. The main replicon types identified were IncI1-Iγ (55%) and IncK (39%). Isolates from different host species mainly carried distinct plasmid subtypes. Seven of the 18 human isolates harbored IncI1-Iγ/sequence type 2 (ST2), IncI1-Iγ/ST12, or IncK plasmids highly similar to those found among animal isolates, even though highly related human and animal plasmids differed by nonsynonymous single nucleotide polymorphisms (SNPs) or insertion sequence elements. This study clearly demonstrates that the epidemiology of CMY-2 can be understood only by thorough plasmid characterization. To date, the spread of this β-lactam resistance determinant in Denmark is mainly associated with IncK and IncI1-Iγ plasmids that are generally distributed according to host-specific patterns. These baseline data will be useful to assess the consequences of the increasing human exposure to CMY-2-producing E. coli via animal sources.
IMPORTANCE CMY-2 is the most common plasmid-mediated AmpC β-lactamase in Escherichia coli. This β-lactamase is poorly inhibited by clavulanic acid and confers resistance to cephamycins, third-generation cephalosporins, and aztreonam. Furthermore, resistance to carbapenems has been reported in E. coli as a result of production of plasmid-encoded CMY-2 β-lactamase in combination with decreased outer membrane permeability. The gene encoding CMY-2 generally resides on transferable plasmids belonging to different incompatibility groups. The prevalence of CMY-2-mediated cephalosporin resistance in E. coli varies significantly depending on the geographical region and host. This study demonstrates that the epidemiology of CMY-2 can be understood only by thorough plasmid characterization. To date, the spread of this β-lactam resistance determinant in Denmark is mainly associated with IncK and IncI1-Iγ plasmids, which are generally distributed according to host-specific patterns. These data will be useful to assess the consequences of the increasing human exposure to CMY-2-producing E. coli via animal sources.
doi:10.1128/AEM.00495-16
PMCID: PMC4984282  PMID: 27235431
4.  Dissemination of Cephalosporin Resistance Genes between Escherichia coli Strains from Farm Animals and Humans by Specific Plasmid Lineages 
PLoS Genetics  2014;10(12):e1004776.
Third-generation cephalosporins are a class of β-lactam antibiotics that are often used for the treatment of human infections caused by Gram-negative bacteria, especially Escherichia coli. Worryingly, the incidence of human infections caused by third-generation cephalosporin-resistant E. coli is increasing worldwide. Recent studies have suggested that these E. coli strains, and their antibiotic resistance genes, can spread from food-producing animals, via the food-chain, to humans. However, these studies used traditional typing methods, which may not have provided sufficient resolution to reliably assess the relatedness of these strains. We therefore used whole-genome sequencing (WGS) to study the relatedness of cephalosporin-resistant E. coli from humans, chicken meat, poultry and pigs. One strain collection included pairs of human and poultry-associated strains that had previously been considered to be identical based on Multi-Locus Sequence Typing, plasmid typing and antibiotic resistance gene sequencing. The second collection included isolates from farmers and their pigs. WGS analysis revealed considerable heterogeneity between human and poultry-associated isolates. The most closely related pairs of strains from both sources carried 1263 Single-Nucleotide Polymorphisms (SNPs) per Mbp core genome. In contrast, epidemiologically linked strains from humans and pigs differed by only 1.8 SNPs per Mbp core genome. WGS-based plasmid reconstructions revealed three distinct plasmid lineages (IncI1- and IncK-type) that carried cephalosporin resistance genes of the Extended-Spectrum Beta-Lactamase (ESBL)- and AmpC-types. The plasmid backbones within each lineage were virtually identical and were shared by genetically unrelated human and animal isolates. Plasmid reconstructions from short-read sequencing data were validated by long-read DNA sequencing for two strains. Our findings failed to demonstrate evidence for recent clonal transmission of cephalosporin-resistant E. coli strains from poultry to humans, as has been suggested based on traditional, low-resolution typing methods. Instead, our data suggest that cephalosporin resistance genes are mainly disseminated in animals and humans via distinct plasmids.
Author Summary
The rapid global rise of infections caused by Escherichia coli that are resistant to clinically relevant antimicrobials, including third-generation cephalosporins, is cause for concern. The intestinal tract of livestock, in particular poultry, is an important reservoir for drug resistant E. coli, but it is unknown to what extent these bacteria can spread to humans. Food is thought to be an important source because drug-resistant E. coli have been detected in animals raised for meat consumption and in meat products. Previous studies that used traditional, low-resolution, genetic typing methods found that drug resistant E. coli present in humans and poultry were indistinguishable from each other, suggesting dissemination of these bacteria through the food-chain to humans. However, by applying high-resolution, whole-genome sequencing methods, we did not find evidence for such transmission of bacteria through the food-chain. Instead, by employing a novel approach for the reconstruction of mobile genetic elements from whole-genome sequence data, we discovered that genetically unrelated E. coli isolates from both humans and animal sources carried nearly identical plasmids that encode third-generation cephalosporin resistance determinants. Our data suggest that cephalosporin resistance is mainly disseminated via the transfer of mobile genetic elements between animals and humans.
doi:10.1371/journal.pgen.1004776
PMCID: PMC4270446  PMID: 25522320
5.  The Sudden Dominance of blaCTX–M Harbouring Plasmids in Shigella spp. Circulating in Southern Vietnam 
Background
Plasmid mediated antimicrobial resistance in the Enterobacteriaceae is a global problem. The rise of CTX-M class extended spectrum beta lactamases (ESBLs) has been well documented in industrialized countries. Vietnam is representative of a typical transitional middle income country where the spectrum of infectious diseases combined with the spread of drug resistance is shifting and bringing new healthcare challenges.
Methodology
We collected hospital admission data from the pediatric population attending the hospital for tropical diseases in Ho Chi Minh City with Shigella infections. Organisms were cultured from all enrolled patients and subjected to antimicrobial susceptibility testing. Those that were ESBL positive were subjected to further investigation. These investigations included PCR amplification for common ESBL genes, plasmid investigation, conjugation, microarray hybridization and DNA sequencing of a blaCTX–M encoding plasmid.
Principal Findings
We show that two different blaCTX-M genes are circulating in this bacterial population in this location. Sequence of one of the ESBL plasmids shows that rather than the gene being integrated into a preexisting MDR plasmid, the blaCTX-M gene is located on relatively simple conjugative plasmid. The sequenced plasmid (pEG356) carried the blaCTX-M-24 gene on an ISEcp1 element and demonstrated considerable sequence homology with other IncFI plasmids.
Significance
The rapid dissemination, spread of antimicrobial resistance and changing population of Shigella spp. concurrent with economic growth are pertinent to many other countries undergoing similar development. Third generation cephalosporins are commonly used empiric antibiotics in Ho Chi Minh City. We recommend that these agents should not be considered for therapy of dysentery in this setting.
Author Summary
Shigellosis is a disease caused by bacteria belonging to Shigella spp. and is a leading cause of bacterial gastrointestinal infections in infants in unindustrialized countries. The Shigellae are dynamic and capable of rapid change when placed under selective pressure in a human population. Extended spectrum beta lactamases (ESBLs) are enzymes capable of degrading cephalosporins (a group of antimicrobial agents) and the genes that encode them are common in pathogenic E. coli and other related organisms in industrialized countries. In southern Vietnam, we have isolated multiple cephalosporin-resistant Shigella that express ESBLs. Furthermore, over two years these strains have replaced strains isolated from patients with shigellosis that cannot express ESBLs. Our work describes the genes responsible for this characteristic and we investigate one of the elements carrying one of these genes. These finding have implications for treatment of shigellosis and support the growing necessity for vaccine development. Our findings also may be pertinent for other countries undergoing a similar economic transition to Vietnam's and the corresponding effect on bacterial populations.
doi:10.1371/journal.pntd.0000702
PMCID: PMC2882334  PMID: 20544028
6.  blaCTX-M-1/9/1 Hybrid Genes May Have Been Generated from blaCTX-M-15 on an IncI2 Plasmid 
Three hybrid CTX-M β-lactamases, CTX-M-64, CTX-M-123, and CTX-M-132, with N and C termini matching CTX-M-1 group enzymes and centers matching CTX-M-9 group enzymes, have been identified. The hybrid gene sequences suggested recombination between blaCTX-M-15 and blaCTX-M-14, the two most common blaCTX-M variants worldwide. However, blaCTX-M-64 and blaCTX-M-123 are found in an ISEcp1-blaCTX-M transposition unit with a 45-bp “spacer,” rather than the 48 bp usually associated with blaCTX-M-15, and 112 bp of IncA/C plasmid backbone. This is closer to the context of blaCTX-M-55, which has one nucleotide difference from blaCTX-M-15, on IncI2 plasmid pHN1122-1. Here, we characterized an IncI2 plasmid carrying blaCTX-M-15 with a 45-bp spacer (pHNY2-1) by complete sequencing and also sequenced IncI2 plasmids carrying blaCTX-M-64 (pHNAH46-1) or blaCTX-M-132 (pHNLDH19) and an IncI1 plasmid carrying blaCTX-M-123 (pHNAH4-1). pHNY2-1 has the same ISEcp1-blaCTX-M-IncA/C insertion as pHN1122-1, pHNAH46-1, and pHNLDH19, and all four plasmid backbones are almost identical. pHNAH4-1 (IncI1 sequence type 108 [ST108]) carries a transposition unit that includes a 2,720-bp fragment of the IncI2 backbone, suggesting ISEcp1-mediated transfer of blaCTX-M-IncA/C-IncI2 to an IncI1 plasmid. All three hybrid blaCTX-M genes may have resulted from recombination between blaCTX-M-14 and blaCTX-M-15 with a 45-bp spacer on an IncI2 plasmid. Five additional Escherichia coli isolates of different sequence types from different provinces, farms, and/or animals had blaCTX-M-64 on a pHNAH46-1-like IncI2 plasmid and 9 had blaCTX-M-123 on a pHNAH4-1-like IncI1 ST108 plasmid. Thus, epidemic IncI plasmids may be responsible for the spread of blaCTX-M-64 and blaCTX-M-123 between different animals and different locations in China.
doi:10.1128/AAC.00501-15
PMCID: PMC4505238  PMID: 25987615
7.  CTX-M-27 Producing Salmonella enterica Serotypes Typhimurium and Indiana Are Prevalent among Food-Producing Animals in China 
Salmonella spp. is one of the most important food-borne pathogens causing digestive tract and invasive infections in both humans and animals. Extended-spectrum β-lactamases (ESBLs) especially the CTX-M-type ESBLs are increasingly being reported worldwide and in China. These studies seldom focused on Salmonella isolates from food-producing animals. The aim of this study was to characterize the antimicrobial resistance profiles, serotypes and ESBLs and in particular, CTX-M producing Salmonella isolates from chickens and pigs in China. Salmonella isolates were identified by API20E system and polymerase chain reaction (PCR) assay; serotypes were determined using slide agglutination with hyperimmune sera; antimicrobial susceptibility was tested using the ager dilution method; the prevalence of ESBLs and PMQR genes were screened by PCR; CTX-M-producing isolates were further characterized by conjugation along with genetic relatedness and plasmid replicon type. In total, 159 Salmonella strains were identified, among which 95 strains were Salmonella enterica serovar Typhimurium, 63 strains were S. enterica serovar Indiana, and 1 strain was S. enterica serovar Enteritidis. All of these isolates presented multi-drug resistant phenotypes. Forty-five isolates carried blaCTX-M genes, the most common subtype was CTX-M-27(34), followed by CTX-M-65(7) and CTX-M-14(4). Most blaCTX-M genes were transmitted by non-typeable or IncN/IncFIB/IncP/IncA/C/IncHI2 plasmids with sizes ranging from 80 to 280 kb. In particular, all the 14 non-typeable plasmids were carrying blaCTX-M-27 gene and had a similar size. PFGE profiles indicated that CTX-M-positive isolates were clonally related among the same serotype, whilst the isolates of different serotypes were genetically divergent. This suggested that both clonal spread of resistant strains and horizontal transmission of the resistance plasmids contributed to the dissemination of blaCTX-M-9G-positive Salmonella isolates. The presence and spread of CTX-M, especially the CTX-M-27 in S. enterica serovars Typhimurium and Indiana from food-producing animals poses a potential threat for public health. Control strategies to limit the dissemination of these strains through the food chain are necessary.
doi:10.3389/fmicb.2016.00436
PMCID: PMC4814913  PMID: 27065989
Salmonella; food-producing animals; serotype; antimicrobial resistance; CTX-M
8.  Characterization of Extended-Spectrum β-Lactamase Genes Found among Escherichia coli Isolates from Duck and Environmental Samples Obtained on a Duck Farm 
Applied and Environmental Microbiology  2012;78(10):3668-3673.
In this study, we focused on evaluating the occurrence of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in fecal samples of healthy ducks and environmental samples from a duck farm in South China. Duck cloacal swabs and pond water samples were cultivated on MacConkey agar plates supplemented with ceftiofur. Individual colonies were examined for ESBL production. Bacteria identified as E. coli were screened for the presence of ESBL and plasmid-borne AmpC genes. The genetic relatedness, plasmid replicon type, and genetic background were determined. Of 245 samples analyzed, 123 had E. coli isolates with ceftiofur MICs higher than 8 μg/ml (116 [50.4%] from 230 duck samples and 7 [46.7%] from 15 water samples). blaCTX-M, blaSHV-12, blaCMY-2, and blaDHA-1 were identified in 108, 5, 9, and 1 isolates, respectively. The most common blaCTX-M genes were blaCTX-M-27 (n = 34), blaCTX-M-55 (n = 27), blaCTX-M-24e (n = 22), and blaCTX-M-105 (n = 20), followed by blaCTX-M-14a, blaCTX-M-14b, blaCTX-M-24a, and blaCTX-M-24b. Although most of the CTX-M producers had distinct pulsotypes, clonal transmission between duck and water isolates was observed. blaCTX-M genes were carried by transferable IncN, IncF, and untypeable plasmids. The novel CTX-M gene blaCTX-M-105 was flanked by two hypothetical protein sequences, partial ISEcp1 upstream and truncated IS903D, iroN, orf1, and a Tn1721-like element downstream. It is suggested that the horizontal transfer of blaCTX-M genes mediated by mobile elements and the clonal spread of CTX-M-producing E. coli isolates contributed to the dissemination of blaCTX-M in the duck farm. Our findings highlight the importance of ducks for the dissemination of transferable antibiotic resistance genes into the environment.
doi:10.1128/AEM.07507-11
PMCID: PMC3346353  PMID: 22407683
9.  Characterization of CTX-M-14-producing Escherichia coli from food-producing animals 
Bacterial resistance to the third-generation cephalosporin antibiotics has become a major concern for public health. This study was aimed to determine the characteristics and distribution of blaCTX-M-14, which encodes an extended-spectrum β-lactamase, in Escherichia coli isolated from Guangdong Province, China. A total of 979 E. coli isolates isolated from healthy or diseased food-producing animals including swine and avian were examined for blaCTX-M-14 and then the blaCTX-M-14 -positive isolates were detected by other resistance determinants [extended-spectrum β-lactamase genes, plasmid-mediated quinolone resistance, rmtB, and floR] and analyzed by phylogenetic grouping analysis, PCR-based plasmid replicon typing, multilocus sequence typing, and plasmid analysis. The genetic environments of blaCTX-M-14 were also determined by PCR. The results showed that fourteen CTX-M-14-producing E. coli were identified, belonging to groups A (7/14), B1 (4/14), and D (3/14). The most predominant resistance gene was blaTEM (n = 8), followed by floR (n = 7), oqxA (n = 3), aac(6′)-1b-cr (n = 2), and rmtB (n = 1). Plasmids carrying blaCTX-M-14 were classified to IncK, IncHI2, IncHI1, IncN, IncFIB, IncF or IncI1, ranged from about 30 to 200 kb, and with insertion sequence of ISEcp1, IS26, or ORF513 located upstream and IS903 downstream of blaCTX-M-14. The result of multilocus sequence typing showed that 14 isolates had 11 STs, and the 11 STs belonged to five groups. Many of the identified sequence types are reported to be common in E. coli isolates associated with extraintestinal infections in humans, suggesting possible transmission of blaCTX-M-14 between animals and humans. The difference in the flanking sequences of blaCTX-M-14 between the 2009 isolates and the early ones suggests that the resistance gene context continues to evolve in E. coli of food producing animals.
doi:10.3389/fmicb.2015.01136
PMCID: PMC4606122  PMID: 26528278
Escherichia coli; CTX-M-14; plasmids; MLST; cephalosporin
10.  Dissemination and Characterization of Plasmids Carrying oqxAB-blaCTX-M Genes in Escherichia coli Isolates from Food-Producing Animals 
PLoS ONE  2013;8(9):e73947.
Background
The 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 blaCTX-M genes among the 696 Escherichia coli (E. coli) isolates from food-producing animals in South China, and to characterize these plasmids.
Methods
The ESBL-encoding genes (blaCTX-M, blaTEM and blaSHV), 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 blaCTX-M. The EcoRI digestion profiles of the plasmids with oqxAB-blaCTX-M were also analyzed. The clonal relatedness was investigated by PFGE.
Results
Of the 696 isolates, 429 harbored at least one PMQR gene, with oqxAB (328) being the most common type; 191 carried blaCTX-M, with blaCTX-M-14 the most common. We observed a significant higher prevalence of blaCTX-M among the oqxAB-positive isolates (38.7%) than that (17.4%) in the oqxAB-negative isolates. Co-transferability of oqxAB and blaCTX-M was found in 18 of the 127 isolates carrying oqxAB-blaCTX-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
blaCTX-M was highly prevalent among the oqxAB-positive isolates. The co-dissemination of oqxAB-blaCTX-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, blaCTX-M, and floR on the same plasmids in E. coli.
doi:10.1371/journal.pone.0073947
PMCID: PMC3767592  PMID: 24040123
11.  Plasmid and Host Strain Characteristics of Escherichia coli Resistant to Extended-Spectrum Cephalosporins in the Norwegian Broiler Production 
PLoS ONE  2016;11(4):e0154019.
Escherichia coli resistant to extended-spectrum cephalosporins have been detected in the Norwegian broiler production, despite the fact that antimicrobial agents are rarely used. The genetic mechanism responsible for cephalosporin resistance is mainly attributed to the presence of the blaCMY-2 gene encoding a plasmid-mediated AmpC-beta-lactamase (pAmpC). The aim of this study was to characterize and compare blaCMY-2 containing Escherichia coli isolated from the intestinal flora of broilers and retail chicken meat (fillets) to identify possible successful clones and/or resistance plasmids widespread in the Norwegian broiler production. Methods used included PCR based phylotyping, conjugation experiments, plasmid replicon typing, pulsed-field gel electrophoresis, multiple locus variable-number tandem-repeats analysis and whole genome sequencing. The nucleotide sequence of an IncK plasmid carrying blaCMY-2 was determined. Intestinal isolates displayed a higher degree of genetic diversity than meat isolates. A cluster of genetically related isolates belonging to ST38, phylogroup D, carrying blaCMY-2 containing IncK plasmids was identified. Furthermore, genes encoding plasmid stability systems (relBE/stbDE and pndAC) were identified on the IncK plasmid. Single nucleotide polymorphism (SNP) analysis of a subset of isolates confirmed a close genetic relationship within the two most prevalent STs. The IncK plasmids within these two STs also shared a high degree of similarity. Cephalosporin-resistant E. coli with the same genetic characteristics have been identified in the broiler production in other European countries, and the IncK plasmid characterized in this study showed close homology to a plasmid isolated from retail chicken meat in the Netherlands. The results indicate that both clonal expansion and horizontal transfer of blaCMY-2 containing plasmids contribute to dissemination of cephalosporin resistant E. coli in the broiler production. The presence of plasmid stability systems may explain why the IncK plasmid containing blaCMY-2 is maintained and disseminated in the Norwegian broiler production in absence of selection pressure from the use of antimicrobial agents.
doi:10.1371/journal.pone.0154019
PMCID: PMC4844124  PMID: 27111852
12.  Spread of blaCTX-M-14 Is Driven Mainly by IncK Plasmids Disseminated among Escherichia coli Phylogroups A, B1, and D in Spain▿  
Antimicrobial Agents and Chemotherapy  2009;53(12):5204-5212.
Since its first description in 2000, CTX-M-14 has become one of the most widespread extended-spectrum β-lactamases in Spain. In the present Escherichia coli multilevel population genetic study involving the characterization of phylogroups, clones, plasmids, and genetic platforms, 61 isolates from 16 hospitalized patients and 40 outpatients and healthy volunteers recovered from 2000 to 2005 were analyzed. Clonal relatedness (XbaI pulsed-field gel electrophoresis [PFGE] type, phylogenetic group, multilocus sequence type [MLST]) was established by standard methods. Analysis of transferred plasmids (I-CeuI; S1 nuclease; restriction fragment length polymorphism analysis; and analysis of RNA interference, replicase, and relaxase) was performed by PCR, sequencing, and hybridization. The genetic environment of blaCTX-M-14 was characterized by PCR on the basis of known associated structures (ISEcp1, IS903, ISCR1). The isolates were mainly recovered from patients in the community (73.8%; 45/61) with urinary tract infections (62.2%; 28/45). They were clonally unrelated by PFGE and corresponded to phylogenetic groups A (36.1%), D (34.4%), and B1 (29.5%). MLST revealed a high degree of sequence type (ST) diversity among phylogroup D isolates and the overrepresentation of the ST10 complex among phylogroup A isolates and ST359/ST155 among phylogroup B1 isolates. Two variants of blaCTX-M-14 previously designated blaCTX-M-14a (n = 59/61) and blaCTX-M-14b (n = 2/61) were detected. blaCTX-M-14a was associated with either ISEcp1 within IncK plasmids (n = 27), ISCR1 linked to an IncHI2 plasmid (n = 1), or ISCR1 linked to IncI-like plasmids (n = 3). The blaCTX-M-14b identified was associated with an ISCR1 element located in an IncHI2 plasmid (n = 1) or with ISEcp1 located in IncK (n = 1). The CTX-M-14-producing E. coli isolates in our geographic area are frequent causes of community-acquired urinary tract infections. The increase in the incidence of such isolates is mostly due to the dissemination of IncK plasmids among E. coli isolates of phylogroups A, B1, and D.
doi:10.1128/AAC.01706-08
PMCID: PMC2786348  PMID: 19786598
13.  Detection and Molecular Characterization of Escherichia coli CTX-M-15 and Klebsiella pneumoniae SHV-12 β-Lactamases from Bovine Mastitis Isolates in the United Kingdom 
Recent reports raised concerns about the role that farm stock may play in the dissemination of extended-spectrum β-lactamase (ESBL)-producing bacteria. This study characterized the ESBLs in two Escherichia coli and three Klebsiella pneumoniae subsp. pneumoniae isolates from cases of clinical bovine mastitis in the United Kingdom. Bacterial culture and sensitivity testing of bovine mastitic milk samples identified Gram-negative cefpodoxime-resistant isolates, which were assessed for their ESBL phenotypes. Conjugation experiments and PCR-based replicon typing (PBRT) were used for characterization of transferable plasmids. E. coli isolates belonged to sequence type 88 (ST88; determined by multilocus sequence typing) and carried blaCTX-M-15 and blaTEM-1, while K. pneumoniae subsp. pneumoniae isolates carried blaSHV-12 and blaTEM-1. Conjugation experiments demonstrated that blaCTX-M-15 and blaTEM-1 were carried on a conjugative plasmid in E. coli, and PBRT identified this to be an IncI1 plasmid. The resistance genes were nontransferable in K. pneumoniae subsp. pneumoniae isolates. Moreover, in the E. coli isolates, an association of ISEcp1 and IS26 with blaCTX-M-15 was found where the IS26 element was inserted upstream of both ISEcp1 and the blaCTX-M promoter, a genetic arrangement highly similar to that described in some United Kingdom human isolates. We report the first cases in Europe of bovine mastitis due to E. coli CTX-M-15 and also of bovine mastitis due to K. pneumoniae subsp. pneumoniae SHV-12 β-lactamases in the United Kingdom. We also describe the genetic environment of blaCTX-M-15 and highlight the role that IncI1 plasmids may play in the spread and dissemination of ESBL genes, which have been described in both human and cattle isolates.
doi:10.1128/AAC.00752-13
PMCID: PMC3910873  PMID: 24247146
14.  Widespread distribution of CTX-M and plasmid-mediated AmpC β-lactamases in Escherichia coli from Brazilian chicken meat 
Memórias do Instituto Oswaldo Cruz  2015;110(2):249-254.
The dissemination of plasmid-mediated antimicrobial resistance genes may pose a substantial public health risk. In the present work, the occurrences of blaCTX-M and plasmid-mediated ampC and qnr genes were investigated in Escherichia coli from 16 chicken carcasses produced by four commercial brands in Brazil. Of the brands tested, three were exporters, including one of organic chicken. Our study assessed 136 E. coli isolates that were grouped into 77 distinct biotypes defined by their origin, resistance profiling, the presence of β-lactamase and plasmid-mediated quinolone resistance genes and enterobacterial repetitive intergenic consensus-polimerase chain reaction typing. The blaCTX-M-15, blaCTX-M-2 and blaCTX-M-8 genes were detected in one, 17 and eight different biotypes, respectively (45 isolates). Twenty-one biotypes (46 isolates) harboured blaCMY-2. Additionally, blaCMY-2 was identified in isolates that also carried either blaCTX-M-2 or blaCTX-M-8. The qnrB and/or qnrS genes occurred in isolates carrying each of the four types of β-lactamase determinants detected and also in oxyimino-cephalosporin-susceptible strains. Plasmid-mediated extended-spectrum β-lactamase (ESBL) and AmpC determinants were identified in carcasses from the four brands tested. Notably, this is the first description of blaCTX-M-15 genes in meat or food-producing animals from South America. The blaCTX-M-8, blaCTX-M-15 and blaCMY-2 genes were transferable in conjugation experiments. The findings of the present study indicate that plasmid-mediated ESBL and AmpC-encoding genes are widely distributed in Brazilian chicken meat.
doi:10.1590/0074-02760140389
PMCID: PMC4489457  PMID: 25946250
ESBL; plasmid AmpC; qnr; chicken meat; Escherichia coli
15.  Predictors of blaCTX-M-15 in varieties of Escherichia coli genotypes from humans in community settings in Mwanza, Tanzania 
BMC Infectious Diseases  2016;16:187.
Background
Extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae commonly cause infections worldwide. BlaCTX-M-15 has been commonly detected in hospital isolates in Mwanza, Tanzania. Little is known regarding the faecal carriage of ESBL isolates and blaCTX-M-15 allele among humans in the community in developing countries.
Methods
A cross-sectional study involving 334 humans from the community settings in Mwanza City was conducted between June and September 2014. Stool specimens were collected and processed to detect ESBL producing enterobacteriaceae. ESBL isolates were confirmed using disc approximation method, commercial ESBL plates and VITEK-2 system. A polymerase chain reaction and sequencing based allele typing for CTX-M ESBL genes was performed to 42 confirmed ESBL isolates followed by whole genome sequence of 25 randomly selected isolates to detect phylogenetic groups, sequence types plasmid replicon types.
Results
Of 334 humans investigated, 55 (16.5 %) were found to carry ESBL-producing bacteria. Age, history of antibiotic use and history of admission were independent factors found to predict ESBL-carriage. The carriage rate of ESBL-producing Escherichia coli was significantly higher than that of Klebsiella pneumoniae (15.1 % vs. 3.8 %, p = 0.026). Of 42 ESBL isolates, 37 (88.1 %) were found to carry the blaCTX-M-15 allele. Other transferrable resistance genes were aac(6’)Ib-cr, aac(3)-IIa, aac(3)-IId, aadA1, aadA5, strA, strB and qnrS1. Eight multi-locus sequence types (ST) were detected in 25 E. coli isolates subjected to genome sequencing. ST-131 was detected in 6 (24 %), ST-38 in 5 (20 %) and 5 (20 %) clonal complex − 10(ST-617, ST-44) of isolates. The pathogenic phylogenetic groups D and B2 were detected in 8/25 (32 %) and 6/25 (24 %) of isolates respectively. BlaCTX-M-15 was found to be located in multiple IncY and IncF plasmids while in 13/25(52 %) of cases it was chromosomally located.
Conclusion
The overlap of multi-drug resistant bacteria and diversity of the genotypes carrying CTX-M-15 in the community and hospitals requires an overall approach that addresses social behaviour and activity, rationalization of the antibiotic stewardship policy and a deeper understanding of the ecological factors that lead to persistence and spread of such alleles.
doi:10.1186/s12879-016-1527-x
PMCID: PMC4850702  PMID: 27129719
16.  Predominance of CTX-M-15 among ESBL Producers from Environment and Fish Gut from the Shores of Lake Victoria in Mwanza, Tanzania 
Extended-Spectrum Beta-Lactamase (ESBL)-producing bacteria are a common cause of healthcare and community-associated infections worldwide. The distribution of such isolates in the environment and their presence in fish as a result of sewage contamination is not well-studied. Here we examined fish and environmental samples from Mwanza city for the presence of ESBL-producing bacteria. From 196 fish sampled from local markets, 26 (13.3%) contained lactose-fermenting ESBL-producing bacteria, while 39/73 (53.4%) environmental samples from the same area were ESBL producers. Antibiotic resistance genes, multi locus sequence types (MLST) and plasmid replicon types in 24 selected isolates from both populations were identified with whole genome sequencing using Illumina MiSeq. Nine of eleven sequenced fish isolates had the blaCTX-M-15 gene whereas 12/13 from environment carried blaCTX-M-15. Antibiotic resistance genes encoding resistance to sulfonamides (sul1/sul2), tetracyclines [tet(A)/tet(B)] fluoroquinolones [e.g., aac(6′)-Ib-cr, qnrS1], aminoglycosides [e.g., aac(3)-lld, strB, strA,] and trimethoprim (e.g., dfrA14) were detected. E. coli sequence type ST-38 (2) and ST-5173 (2) were detected in isolates both from the environment and fish. IncY plasmids carrying blaCTX-M-15, qnrS1, strA, and strB were detected in five environmental E. coli isolates and in one E. coli isolate from fish. Our data indicate spillage of resistant environmental isolates into Lake Victoria through the sewage system. Persistence of blaCTX-M-15 in the Mwanza city environment is complex, and involves both clonal spread of resistant strains as well as dissemination by commonly occurring IncY plasmids circulating in isolates present in humans, the environment as well as in the food chain.
doi:10.3389/fmicb.2016.01862
PMCID: PMC5130978  PMID: 27990135
blaCTX-M-15; fish; environment
17.  Three Cefotaximases, CTX-M-9, CTX-M-13, and CTX-M-14, among Enterobacteriaceae in the People's Republic of China 
Of 15 extended-spectrum β-lactamase (ESBL)-producing isolates of the family Enterobacteriaceae collected from the First Municipal People's Hospital of Guangzhou, in the southern part of the People's Republic of China, 9 were found to produce CTX-M ESBLs, 3 produced SHV-12, and 3 produced both CTX-M and SHV-12. Eleven isolates produced either TEM-1B or SHV-11, in addition to an ESBL. Nucleotide sequence analysis of the 12 isolates carrying blaCTX-M genes revealed that they harbored three different blaCTX-M genes, blaCTX-M-9 (5 isolates), blaCTX-M-13 (1 isolate), and blaCTX-M-14 (6 isolates). These genes have 98% nucleotide homology with blaToho-2. The blaCTX-M genes were carried on plasmids that ranged in size from 35 to 150 kb. Plasmid fingerprints and pulsed-field gel electrophoresis showed the dissemination of the blaCTX-M genes through transfer of different antibiotic resistance plasmids to different bacteria, suggesting that these resistance determinants are highly mobile. Insertion sequence ISEcp1, found on the upstream region of these genes, may be involved in the translocation of the blaCTX-M genes. This is the first report of the occurrence of SHV-12 and CTX-M ESBLs in China. The presence of strains with these ESBLs shows both the evolution of blaCTX-M genes and their dissemination among at least three species of the family Enterobacteriaceae, Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae, isolated within a single hospital. The predominance of CTX-M type enzymes seen in this area of China appears to be similar to that seen in South America but is different from those seen in Europe and North America, suggesting different evolutionary routes and selective pressures. A more comprehensive survey of the ESBL types from China is urgently needed.
doi:10.1128/AAC.46.3.630-637.2002
PMCID: PMC127467  PMID: 11850241
18.  Molecular epidemiology of extended-spectrum beta-lactamase-producing Escherichia coli in Tunisia and characterization of their virulence factors and plasmid addiction systems 
BMC Microbiology  2013;13:147.
Background
Extended-spectrum β-lactamases (ESBLs), particularly CTX-M- type ESBLs, are among the most important resistance determinants spreading worldwide in Enterobacteriaceae. The aim of this study was to characterize a collection of 163 ESBL-producing Escherichia coli collected in Tunisia, their ESBL-encoding plasmids and plasmid associated addiction systems.
Results
The collection comprised 163 ESBL producers collected from two university hospitals of Sfax between 1989 and 2009. 118 isolates harbored blaCTX-M gene (101 blaCTX-M-15 gene and 17 blaCTX-M-14 gene). 49 isolates carried blaSHV-12 gene, 9 blaSHV-2a gene and only 3 blaTEM-26 gene. 16 isolates produced both CTX-M and SHV-12. The 101 CTX-M-15-producing isolates were significantly associated to phylogroup B2 and exhibiting a high number of virulence factors. 24 (23.7%) of the group B2 isolates belonged to clonal complex ST131. Pulsed-field gel electrophoresis (PFGE) typing revealed a genetic diversity of the isolates. 144 ESBL determinants were transferable mostly by conjugation. The majority of plasmid carrying blaCTX-M-15 genes (72/88) were assigned to various single replicon or multireplicon IncF types and had significantly a higher frequency of addiction systems, notably the VagCD module.
Conclusion
This study demonstrates that the dissemination of CTX-M-15 producing E. coli in our setting was due to the spread of various IncF-type plasmids harboring multiple addiction systems, into related clones with high frequency of virulence determinants.
doi:10.1186/1471-2180-13-147
PMCID: PMC3701463  PMID: 23800277
E. coli; ESBL; CTX-M-15; Plasmid; Addiction systems; Virulence
19.  Molecular Characterization of Escherichia coli Strains Isolated from Retail Meat That Harbor blaCTX-M and fosA3 Genes 
A total of 55 cefotaxime-resistant Escherichia coli isolates were obtained from retail meat products purchased in Shenzhen, China, during the period November 2012 to May 2013. Thirty-seven of these 55 isolates were found to harbor a blaCTX-M gene, with the blaCTX-M-1 group being the most common type. blaCMY-2 was detected in 16 isolates, alone or in combination with other extended-spectrum β-lactamase (ESBL) determinants. Importantly, the fosA3 gene, which encodes fosfomycin resistance, was detected in 12 isolates, with several being found to reside in the conjugative plasmid that harbored the blaCTX-M gene. The insertion sequence IS26 was observed upstream of some of the blaCTX-M-55 and fosA3 genes. Conjugation experiments showed that blaCTX-M genes from 15 isolates were transferrable, with Inc I1 and Inc FII being the most prevalent replicons. High clonal diversity was observed among the blaCTX-M producers, suggesting that horizontal transfer of the blaCTX-M genes among E. coli strains in retail meats is a common event and that such strains may constitute an important reservoir of blaCTX-M genes, which may be readily disseminated to other potential human pathogens.
doi:10.1128/AAC.03101-15
PMCID: PMC4808224  PMID: 26856843
20.  CTX-M-1 and CTX-M-15-producing Escherichia coli in dog faeces from public gardens 
Background
Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli are increasingly reported in dogs. The objective of this study was to provide data on the prevalence of ESBL-producing E. coli in dog faecal deposits in public gardens.
Results
A total of 209 faecal deposits collected in nine public gardens in Copenhagen, Denmark were screened by selective enrichment followed by plating on MacConkey agar supplemented with cefotaxime. Presumptive ESBL-producing E. coli were confirmed by MALDI-TOF MS and polymerase chain reaction (PCR) for detection of common cefotaxime resistance determinants (blaTEM, blaSHV, blaCTX-M and blaCMY-2). ESBL-producers were further characterized by multilocus sequence typing (MLST) and antimicrobial susceptibility testing using broth microdilution. Plasmids harbouring ESBL genes were characterized by S1 nuclease pulsed field gel electrophoresis, PCR-based replicon typing, and pMLST. Cefotaxime-resistant E. coli were detected in four (1.9 %) samples. Three samples harboured CTX-M-1-producing isolates, and one sample contained two CTX-M-15-producing isolates displaying distinct colony morphology. All isolates belonged to distinct sequence types (STs), including one E. coli lineage previously associated to a human-specific pathotype (ST59). blaCTX-M-1 was carried on IncI1 plasmids classified as ST3 or ST58 by pMLST, whereas blaCTX-M-15 was located on IncF/Y and non-typeable plasmids in the two strains isolated from the same sample.
Conclusions
The study shows that dog faeces are a vector for dissemination of CTX-M-producing E. coli within urban areas. The risk derived from human exposure to dog faeces in public gardens depends on the prevalence of these bacteria in the local dog population as well as on the owners’ practice to remove and dispose their dog’s faeces.
doi:10.1186/s13028-015-0174-3
PMCID: PMC4660786  PMID: 26608707
ESBL; Canine; Environment; Antibiotic resistance; Zoonoses; CTX-M
21.  blaCTX-M-152, a Novel Variant of CTX-M-group-25, Identified in a Study Performed on the Prevalence of Multidrug Resistance among Natural Inhabitants of River Yamuna, India 
Natural environment influenced by anthropogenic activities creates selective pressure for acquisition and spread of resistance genes. In this study, we determined the prevalence of Extended Spectrum β-Lactamases producing gram negative bacteria from the River Yamuna, India, and report the identification and characterization of a novel CTX-M gene variant blaCTX-M-152. Of the total 230 non-duplicate isolates obtained from collected water samples, 40 isolates were found positive for ESBL production through Inhibitor-Potentiation Disc Diffusion test. Based on their resistance profile, 3% were found exhibiting pandrug resistance (PDR), 47% extensively drug resistance (XDR), and remaining 50% showing multidrug resistant (MDR). Following screening and antimicrobial profiling, characterization of ESBLs (blaTEM and blaCTX-M), and mercury tolerance determinants (merP, merT, and merB) were performed. In addition to abundance of blaTEM-116 (57.5%) and blaCTX-M-15 (37.5%), bacteria were also found to harbor other variants of ESBLs like blaCTX-M-71 (5%), blaCTX-M-3 (7.5%), blaCTX-M-32 (2.5%), blaCTX-M-152 (7.5%), blaCTX-M-55 (2.5%), along with some non-ESBLs; blaTEM-1 (25%) and blaOXY (5%). Additionally, co-occurrence of mercury tolerance genes were observed among 40% of isolates. In silico studies of the new variant, blaCTX-M-152were conducted through modeling for the generation of structure followed by docking to determine its catalytic profile. CTX-M-152 was found to be an out-member of CTX-M-group-25 due to Q26H, T154A, G89D, P99S, and D146G substitutions. Five residues Ser70, Asn132, Ser237, Gly238, and Arg273 were found responsible for positioning of cefotaxime into the active site through seven H-bonds with binding energy of -7.6 Kcal/mol. Despite small active site, co-operative interactions of Ser237 and Arg276 were found actively contributing to its high catalytic efficiency. To the best of our knowledge, this is the first report of blaCTX-M-152 of CTX-M-group-25 from Indian subcontinent. Taking a note of bacteria harboring such high proportion of multidrug and mercury resistance determinants, their presence in natural water resources employed for human consumption increases the chances of potential risk to human health. Hence, deeper insights into mechanisms pertaining to resistance development are required to frame out strategies to tackle the situation and prevent acquisition and dissemination of resistance determinants so as to combat the escalating burden of infectious diseases.
doi:10.3389/fmicb.2016.00176
PMCID: PMC4762991  PMID: 26941715
antibiotics; ESBL; mechanisms of resistance; polluted environment; resistance genes
22.  Strain Diversity of CTX-M-Producing Enterobacteriaceae in Individual Pigs: Insights into the Dynamics of Shedding during the Production Cycle 
Applied and Environmental Microbiology  2014;80(21):6620-6626.
The aim of this study was to evaluate the population dynamics of CTX-M-producing Enterobacteriaceae in individual pigs on a farm positive for CTX-M-14-producing Escherichia coli. Fecal samples were collected once around the farrowing time from five sows and four times along the production cycle from two of their respective offspring. Multiple colonies per sample were isolated on cefotaxime-supplemented MacConkey agar with or without prior enrichment, resulting in 98 isolates identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry and tested for blaCTX-M. CTX-M-positive isolates (n = 86) were typed by pulsed-field gel electrophoresis (PFGE). Plasmids harboring blaCTX-M were characterized in 22 representative isolates by replicon typing and restriction fragment length polymorphism. Based on the PFGE results, all individuals shed unrelated CTX-M-14-producing E. coli strains during the course of life. Concomitant shedding of CTX-M-2/97-producing Proteus mirabilis or Providencia rettgeri was observed in two sows and two offspring. At least two genetically unrelated CTX-M-producing E. coli strains were isolated from approximately one-fourth of the samples, with remarkable differences between isolates obtained by enrichment and direct plating. A clear decrease in strain diversity was observed after weaning. Dissemination of blaCTX-M-14 within the farm was attributed to horizontal transfer of an IncK plasmid that did not carry additional resistance genes and persisted in the absence of antimicrobial selective pressure. Assessment of strain diversity was shown to be influenced by the production stage from which samples were collected, as well as by the isolation method, providing useful information for the design and interpretation of future epidemiological studies of CTX-M-producing Enterobacteriaceae in pig farms.
doi:10.1128/AEM.01730-14
PMCID: PMC4249051  PMID: 25128344
23.  Characterization of the genetic environment of blaESBL genes, integrons and toxin-antitoxin systems identified on large transferrable plasmids in multi-drug resistant Escherichia coli 
Objectives: Previously 14 conjugative plasmids from multi-drug resistant (MDR) Escherichia coli from healthy humans and food-producing animals in Switzerland were sequenced. The aim of this study was to extend the genetic characterization of these plasmids with a focus on blaESBL genes including blaCTX-M-1 and blaTEM, class 1 integrons and toxin-antitoxin (TA) systems contained therein.
Methods: The nucleotide sequences and subsequent annotation therein of 14 conjugative plasmids were previously determined from their corresponding transconjugants. The TA loci were confirmed by RASTA-Bacteria.
Results: Eight of the conjugative plasmids identified were found to encode genes expressing ESBLs. Structural heterogeneity was noted in the regions flanking both the blaCTX-M-1 and blaTEM genes. The blaCTX-M-1 genes were associated with the common insertion sequences ISEcp1 and IS26, and uniquely with an IS5 element in one case; while blaTEM genes were found to be associated with IS26 and Tn2. A new blaTEM-210 gene was identified. Seven class 1 integrons were also identified and assigned into 3 groups, denoted as In54, In369 and In501. Sixteen TA loci belonging to 4 of the TA gene families (relBE, vapBC, ccd and mazEF) were identified on 11 of these plasmids.
Conclusions: Comparative sequence analysis of these plasmids provided data on the structures likely to contribute to sequence diversity associated with these accessory genes, including IS26, ISEcp1 and Tn2. All of them contribute to the dissemination of the corresponding resistance genes located on the different plasmids. There appears to be no association between β-lactam encoding genes and TA systems.
doi:10.3389/fmicb.2014.00716
PMCID: PMC4285173  PMID: 25610429
bla genes; plasmid sequencing; CTX-M; TEM; accessory genes
24.  IncH-Type Plasmid Harboring blaCTX-M-15, blaDHA-1, and qnrB4 Genes Recovered from Animal Isolates 
The whole sequence of plasmid pENVA carrying the extended-spectrum β-lactamase gene blaCTX-M-15 was determined. It was identified from a series of clonally related Klebsiella pneumoniae sequence type 274 strains recovered from companion animals. This plasmid was 253,984 bp in size and harbored, in addition to blaCTX-M-15, a large array of genes encoding resistance to many antibiotic molecules, including β-lactams (blaTEM-1, blaDHA-1), aminoglycosides (aacA2, aadA1), tetracycline (tetA), quinolones (qnrB4), trimethoprim (dfrA15), and sulfonamides (two copies of sul1). In addition, genes encoding resistance to mercury, tellurium, nickel, and quaternary compounds were identified. It also carried genes encoding DNA damage protection and mutagenesis repair and a locus for a CRISPR system, which corresponds to an immune system involved in protection against bacteriophages and plasmids. Comparative analysis of the plasmid scaffold showed that it possessed a structure similar to that of only a single plasmid, which was pNDM-MAR encoding the carbapenemase NDM-1 and identified from human K. pneumoniae isolates. Both plasmids possessed two replicons, namely, those of IncFIB-like and IncHIB-like plasmids, which were significantly different from those previously characterized. The blaCTX-M-15 gene, together with the other antibiotic resistance genes, was part of a large module likely acquired through a transposition process. We characterized here a new plasmid type carrying the blaCTX-M-15 gene identified in a K. pneumoniae isolate of animal origin. The extent to which this plasmid type may spread efficiently and possibly further enhance the dissemination of blaCTX-M-15 among animal and human isolates remains to be determined.
doi:10.1128/AAC.02695-14
PMCID: PMC4068538  PMID: 24752252
25.  Molecular Epidemiological Analysis of Escherichia coli Sequence Type ST131 (O25:H4) and blaCTX-M-15 among Extended-Spectrum-β-Lactamase-Producing E. coli from the United States, 2000 to 2009 
Escherichia coli sequence type ST131 (from phylogenetic group B2), often carrying the extended-spectrum-β-lactamase (ESBL) gene blaCTX-M-15, is an emerging globally disseminated pathogen that has received comparatively little attention in the United States. Accordingly, a convenience sample of 351 ESBL-producing E. coli isolates from 15 U.S. centers (collected in 2000 to 2009) underwent PCR-based phylotyping and detection of ST131 and blaCTX-M-15. A total of 200 isolates, comprising 4 groups of 50 isolates each that were (i) blaCTX-M-15 negative non-ST131, (ii) blaCTX-M-15 positive non-ST131, (iii) blaCTX-M-15 negative ST131, or (iv) blaCTX-M-15 positive ST131, also underwent virulence genotyping, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis (PFGE). Overall, 201 (57%) isolates exhibited blaCTX-M-15, whereas 165 (47%) were ST131. ST131 accounted for 56% of blaCTX-M-15-positive- versus 35% of blaCTX-M-15-negative isolates (P < 0.001). Whereas ST131 accounted for 94% of the 175 total group B2 isolates, non-ST131 isolates were phylogenetically distributed by blaCTX-M-15 status, with groups A (blaCTX-M-15-positive isolates) and D (blaCTX-M-15-negative isolates) predominating. Both blaCTX-M-15 and ST131 occurred at all participating centers, were recovered from children and adults, increased significantly in prevalence post-2003, and were associated with molecularly inferred virulence. Compared with non-ST131 isolates, ST131 isolates had higher virulence scores, distinctive virulence profiles, and more-homogeneous PFGE profiles. blaCTX-M-15 was associated with extensive antimicrobial resistance and ST131 with fluoroquinolone resistance. Thus, E. coli ST131 and blaCTX-M-15 are emergent, widely distributed, and predominant among ESBL-positive E. coli strains in the United States, among children and adults alike. Enhanced virulence and antimicrobial resistance have likely promoted the epidemiological success of these emerging public health threats.
doi:10.1128/AAC.05824-11
PMCID: PMC3346636  PMID: 22354301

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