CTX-M Enzymes Research Articles

Differences in ESBL Genes between E. coli, Klebsiella spp. and Enterobacter Cloacae Strains

Background: Resistance to beta-Lactam antibiotics in Enterobacteriaceae is increasing worldwide; however, information about the different beta-Lactamase genes in diverse regions is scarce. We aimed to identify beta-lactamse genes in presumably ESBL positive isolates of Enterobacteriaceae. Methods: A nonredundant collection of 757 strains of E. coli, Klebsiella pneumonia, Klebsiella oxytoca and Enterobacter cloacae resistant to third generation cephalosporins and ESBL positive by VITEK II analysis was analyzed by PCR for presence of beta-lactamase genes of the TEM, SHV, CTX-M and ampC families. Results: Distribution of patient age was bimodal, with peaks at 1 ESBL positive bacterial species, 71% may have been repeatedly colonized by different strains, while in 29%, plasmid transfer may have occurred after a single colonization event. Conclusions: The molecular epidemiology of beta-Lactamase genes is complex even if analyzed in a single institution. CTX-M enzymes dominate, and co-occurrence of ampC enzymes is rare. Plasmid transfer between species of Enterobacteriaceae seems to occur in vivo in some patients.

CTX-M-15-H30Rx-ST131 subclone is one of the main causes of healthcare-associated ESBL-producingEscherichia colibacteraemia of urinary origin in Spain

The objective of this study was to assess the prevalence and molecular epidemiology of ESBL-producing Escherichia coli causing healthcare-associated (HCA) and community-associated (CA) bacteraemia of urinary origin (BUO) in Spain. An observational cohort study was conducted at eight hospitals from different Spanish geographical areas (2010-11). BUO episodes (n = 425) were classified as HCA (n = 215) and CA (n = 210), and one blood isolate per episode was collected. Susceptibility testing was performed, ESBLs were screened by double-disc diffusion test and ESBL and OXA-1 genes were characterized (PCR and sequencing). Population structure (phylogenetic groups, XbaI-PFGE and MLST) and ST131 subtyping (PCR) were determined. Virulence genes were detected by PCR and virulence score, profiles and extraintestinal pathogenic E. coli (ExPEC) status calculated. ESBL-producing E. coli prevalence was 9.2% (39/425). ESBL-producing E. coli episodes were significantly associated with HCA-BUO episodes [14% (30/215) versus 4.3% (9/210); P = 0.001]. The highest non-susceptibility proportions corresponded to ciprofloxacin (97.4%), amoxicillin/clavulanate (74.4%), co-trimoxazole (69.2%) and tobramycin (61.5%). Of the 39 ESBL-producing E. coli isolates, 34 produced CTX-M enzymes (21 CTX-M-15, 11 CTX-M-14 and 2 CTX-M-1). Fifteen STs were identified, the B2-ST131 clone being the most prevalent (54%; 21/39). All ST131 isolates were ExPEC and had the highest virulence scores, but they showed less diversity in virulence profiles than other STs. The H30Rx subclone accounted for most ST131 isolates (20/21), co-produced CTX-M-15 (20/20) and OXA-1 (19/20) enzymes and was associated with HCA episodes (16/20). The CTX-M-15-ST131-H30Rx subclone is a relevant MDR pathogen causing BUO, mainly HCA episodes. The dominance of this subclone with comparatively less diversity of virulence profiles reflects the spread of a successful and MDR ESBL ST131 lineage in Spain.

CTX-M extended-spectrum β-lactamase-producing Klebsiella spp, Salmonella spp, Shigella spp and Escherichia coli isolates in Iranian hospitals.

This study was conducted in Iran in order to assess the distribution of CTX-M type ESBLs producing Enterobacteriaceae. From January 2012 to December 2013, totally 198 E. coli, 139 Klebsiella spp, 54 Salmonella spp and 52 Shigella spp from seven hospitals of six provinces in Iran were screened for resistance to extended-spectrum cephalosporins. After identification and susceptibility testing, isolates presenting multiple-drug resistance (MDR) were evaluated for ESBL production by the disk combination method and by Etest using (cefotaxime and cefotaxime plus clavulanic acid). All isolates were also screened for blaCTX-M using conventional PCR. A total of 42.92%, 33.81%, 14.81% and 7.69% of the E. coli, Klebsiella spp, Salmonella spp and Shigella spp isolates were MDR, respectively. The presence of CTX-M enzyme among ESBL-producing isolates was 85.18%, 77.7%, 50%, and 66.7%, in E. coli, Klebsiella spp, Salmonella spp and Shigella spp respectively. The overall presence of CTX-M genes in Enterobacteriaceae was 15.4% and among the resistant isolates was 47.6%. This study indicated that resistance to β-lactams mediated by CTX-M enzymes in Iran had similar pattern as in other parts of the world. In order to control the spread of resistance, comprehensive studies and programs are needed.

Detection of OXA-48-like and NDM carbapenemases producing Klebsiella pneumoniae in Jordan: A pilot study.

Little is known of carbapenemase producing Klebsiella pneumoniae (CPK) in Jordan. This study aimed to determine the prevalence of CPK in a major hospital in Amman, Jordan in 2012-2013 and to characterize the isolates and detect the types of carbapenemase(s) they produced. For the 296 isolates investigated, species identification and antimicrobial susceptibilities were determined (Vitek II, bioMérieux). Isolates with decreased ertapenem susceptibility were tested for carbapenemase production using the Modified Hodge Test. Isolates with a carbapenemase-positive phenotype were characterized further via multiplex PCRs for extended-spectrum β-lactamase and carbapenemase genes and by Pulsed Field Gel Electrophoresis (PFGE). Seven of 296 K. pneumoniae isolated in 2012-2013 (2.4%) were carbapenemase producers, five produced class D carbapenemases (OXA-48-like) and two produced a NDM metallo-beta-lactamase. All seven isolates also encoded CTX-M enzymes; CTX-M-1-like enzymes were detected in five isolates (two co-producing NDM enzymes and three co-producing OXA-48-like enzymes), CTX-M-9 was found in the two remaining OXA-48-like producers. PFGE revealed five genetically distinct types amongst the seven carbapenemase producing K. pneumoniae, with two pairs of identical isolates associated with patients treated on the same wards. The emergence of OXA-48-like and NDM carbapenemases associated with multi-drug resistant (MDR) isolates in Jordan is concerning. The strict implementation of infection control practices will help to disrupt the spread of MDR carbapenemase producers in Jordanian hospitals.

Prevalence of CTX-M enzyme and qnrA, qnrB, qnrC, qnrS, aac-(6)-Ib genes among ESBL (Extended spectrum beta lactamase)-positive isolates in patients undergoing transrectal needle prostate biopsy in Turkey.

Fecal carriage is one of the most important reasons for extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-PE) causing infections. We aimed to demonstrate epidemiological features for subtype of ESBL-PE-encoding TEM, SHV and CTX-M as well as for qnrA, qnrB, qnrC, qnrS, aac-(6)-Ib genes through polymerase chain reaction (PCR) in patients undergoing transrectal needle prostate biopsy (TRNBP). Between October 2008 and February 2010, we collected 400 fecal swabs from patients prior to TRNBP in four separate centers. After detecting ESBL- PE isolates in the material, we further analyzed TEM, SHV and CTX-M enzymes, as well as three types of qnr genes of qnrA, qnrB, qnrC and aac-(6)-Ib by PCR. We detected 80 ESBL-PE isolates in 400 fecal samples. Of the 80 isolates; blaSHV, blaTEM and blaCTX-M were observed in 12, 46 and 79 isolates, respectively. All three genes were present in eight isolates. Resistance to Quinolone was identified in 67 (83.7%) isolates, resistance to aminoglycoside in 52 (65%) isolates, and resistance to both antibiotics in 46 (57.5%) isolates. Subsequently, we determined qnrB, qnrS and aac-(6)-Ib genes in 7 (8.8%), 11 (14%) and 60 (76%) isolates, respectively. qnrA and qnrC were not detected in any of the isolates. CTX-M-producing ESBL-PE is the most common pathogen responsible for fecal carriage in the community. Plasmid mediated quinolone resistance genes (qnr, aac-(6)-Ib) are the reason behind the dissemination of ESBLs.

Complex class 1 integrons harboring CTX-M-2-encoding genes in clinical Enterobacteriaceae from a hospital in Brazil.

CTX-M enzymes are the most prevalent extended-spectrum beta-lactamases (ESBLs) in Brazil and around the world. The spread of CTX-M lies in their ability to be mobilized by insertion sequences and integrons. This study aimed to identify the mobile genetic structures associated with bla(CTX-M) genes from clinical Enterobacteriaceae strains. Twenty-eight clinical non-clonal Enterobacteriaceae were screened by PCR for the presence of bla(CTX-M) genes and class 1 integrase (int1), and for the association of bla(CTX-M) with class 1 integrons. Plasmid incompatibility groups were assessed by PBRT. Wild-type plasmids were transformed into electrocompetent E. coli, and the S1-PFGE technique was used to verify the presence of high-molecular-weight plasmids in both wild-type strains and E. coli transformants. Sequencing showed that strains carried bla(CTX-M-2) (n = 25) and bla(CTX-M-59) (n = 3) genes inserted into the 3'-end of complex class 1 integrons. Thirteen strains also carried bla(TEM) and bla(SHV) genes. CTX-M-2/59-containing complex class 1 integrons were also present in E. coli transformants. The most frequent Inc groups were IncA/C (n = 10) and IncF (n = 8). Heavy plasmids were observed in both wild-type strains and E. coli transformants. The presence of the same bla(CTX-M-2-group)-containing genetic structure in seven Enterobacteriaceae species isolated at seven hospital wards shows the great mobility potential of complex class 1 integrons. Also, this is the first report of TEM-15, SHV-45, and SHV-55 in Latin America. The genetic environment of bla(CTX-M-2) accounts for their maintenance and spread among Gram-negative bacteria.

Characterization of the global stabilizing substitution A77V and its role in the evolution of CTX-M β-lactamases.

The widespread use of oxyimino-cephalosporin antibiotics drives the evolution of the CTX-M family of β-lactamases that hydrolyze these drugs and confer antibiotic resistance. Clinically isolated CTX-M enzymes carrying the P167S or D240G active site-associated adaptive mutation have a broadened substrate profile that includes the oxyimino-cephalosporin antibiotic ceftazidime. The D240G substitution is known to reduce the stability of CTX-M-14 β-lactamase, and the P167S substitution is shown here to also destabilize the enzyme. Proteins are marginally stable entities, and second-site mutations that stabilize the enzyme can offset a loss in stability caused by mutations that enhance enzyme activity. Therefore, the evolution of antibiotic resistance enzymes can be dependent on the acquisition of stabilizing mutations. The A77V substitution is present in CTX-M extended-spectrum β-lactamases (ESBLs) from a number of clinical isolates, suggesting that it may be important in the evolution of antibiotic resistance in this family of β-lactamases. In this study, the effects of the A77V substitution in the CTX-M-14 model enzyme were characterized with regard to the kinetic parameters for antibiotic hydrolysis as well as enzyme expression levels in vivo and protein stability in vitro. The A77V substitution has little effect on the kinetics of oxyimino-cephalosporin hydrolysis, but it stabilizes the CTX-M enzyme and compensates for the loss of stability resulting from the P167S and D240G mutations. The acquisition of global stabilizing mutations, such as A77V, is an important feature in β-lactamase evolution and a common mechanism in protein evolution.

Epidemiology and risk factors for faecal extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E) carriage derived from residents of seven nursing homes in western Shanghai, China.

Nursing homes (NHs) have been implicated as significant reservoirs of antibiotic-resistant organisms causing severe infectious disease. We investigated the prevalence and molecular epidemiology of, and risk factors for, faecal carriage of extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E). A multicentre cross-sectional study was conducted in seven NHs in Shanghai between March 2014 and May 2014. Antimicrobial susceptibility testing and polymerase chain reaction were used to detect genes coding for ESBLs and carbapenemases. NH records at individual-resident level and facility level were examined for potential risk factors. Four hundred and fifty-seven Enterobacteriaceae isolates were collected of which 183 (46·92%) were colonized by ESBL-E. CTX-M enzymes (198/200, 99%) predominated, with CTX-M-14 (84/200, 42%) the most common types. Two carbapenemase producers harboured blaKPC-2. Resistance rates to carbapenems, TZP, AK, FOS, CL and TGC were low. History of invasive procedures [odds ratio (OR) 2·384, 95% confidence interval (CI) 1·318-4·310, P = 0·004], narrow-spectrum cephalosporins (OR 1·635, 95% CI 1·045-2·558, P = 0·031) and broad-spectrum cephalosporins (OR 3·276, 95% CI 1·278-8·398, P = 0·014) were independently associated with ESBL-E carriage. In conclusion, NH residents have a very high prevalence of faecal carriage of ESBL-E. Continuous and active surveillance is important, as are prudent infection control measures and antibiotic use to prevent and control the spread of these antibiotic-resistant strains.

Residues Distal to the Active Site Contribute to Enhanced Catalytic Activity of Variant and Hybrid β-Lactamases Derived from CTX-M-14 and CTX-M-15.

A variety of CTX-M-type extended-spectrum β-lactamases (ESBLs), including hybrid ones, have been reported in China that are uncommon elsewhere. To better characterize the substrate profiles and enzymatic mechanisms of these enzymes, we performed comparative kinetic analyses of both parental and hybrid CTX-M enzymes, including CTX-M-15, -132, -123, -64, -14 and -55, that are known to confer variable levels of β-lactam resistance in the host strains. All tested enzymes were susceptible to serine β-lactamase inhibitors, with sulbactam exhibiting the weakest inhibitory effects. CTX-M-55, which differs from CTX-M-15 by one substitution, A(77)V, displayed enhanced catalytic activity (kcat/Km) against expanded-spectrum cephalosporins (ESCs). CTX-M-55 exhibits higher structure stability, most likely by forming hydrophobic interactions between A(77)V and various key residues in different helices, thereby stabilizing the core architecture of the helix cluster, and indirectly contributes to a more stable active site conformation, which in turn shows higher catalytic efficiency and is more tolerant to temperature change. Analyses of the hybrids and their parental prototypes showed that evolution from CTX-M-15 to CTX-M-132, CTX-M-123, and CTX-M-64, characterized by gradual enhancement of catalytic activity to ESCs, was attributed to introduction of different substitutions to amino acids distal to the active site of CTX-M-15. Similarly, the increased hydrolytic activities against cephalosporins and sensitivity to β-lactamase inhibitors, clavulanic acid and sulbactam, of CTX-M-64 were partly due to the amino acids that were different from CTX-M-14 and located at both the C and N termini of CTX-M-64. These data indicate that residues distal to the active site of CTX-Ms contributed to their enhanced catalytic activities to ESCs.

Emergence and spread of O16-ST131 and O25b-ST131 clones among faecal CTX-M-producing Escherichia coli in healthy individuals in Hunan Province, China.

ObjectivesThe objectives of this study were to determine CTX-M-producing Escherichia coli ST131 strain prevalence in stool specimens from healthy subjects in central China and to molecularly characterize clonal groups.MethodsFrom November 2013 to January 2014, stool specimens from healthy individuals in Hunan Province were screened for ESBL-producing E. coli using chromogenic medium and CTX-M genotypes and phylogenetic groups were determined. ST131 clonal groups were detected by PCR and characterized for antibiotic resistance, fimH, gyrA and parC alleles, plasmid-mediated quinolone resistance determinants, virulence genotypes and PFGE patterns.ResultsAmong 563 subjects, 287 (51.0%) exhibited the presence of faecal ESBL-producing E. coli, all of which produced CTX-M enzymes. The most common CTX-M genotypes were CTX-M-14 (48.4%), CTX-M-15 (27.5%) and CTX-M-27 (15.0%). Of the 287 CTX-M-producing isolates, 32 (11.1%) belonged to the ST131 clone. O16-ST131 isolates were dominant (75%) and contained the fimH41 allele. The remaining eight (25%) ST131 isolates were of the O25b subgroup and contained fimH30 or fimH41. Ciprofloxacin resistance was found in 100% of the O25b-ST131 isolates, whereas only 8% of the O16-ST131 isolates were resistant. All of the O25b-ST131 isolates except one showed gyrA1AB and parC1aAB mutations; most of the O16-ST131 isolates had gyrA1A and parC1b mutations. The virulence genotypes of O16-ST131 resembled those of the O25b-ST131 isolates. The 32 ST131 isolates formed one large group at the 64% similarity level. They comprised 15 PFGE groups (defined at ≥85% similarity).ConclusionsO16-ST131 isolates have emerged as the predominant type of ST131 isolate in faecal CTX-M-producing E. coli in healthy individuals in China.

The prevalence of Escherichia coli strains with extended spectrum beta-lactamases isolated in China.

The extended-spectrum-lactamases-producing Escherichia coli has rapidly spread worldwide. Escherichia coli has been becoming much more resistant to β-lactam antibiotics and other commonly available antimicrobials. We investigated the prevalence, resistance, and probable gene type of extended spectrum beta-lactamases (ESBLs) using minimum inhibitory concentrations (MICs) testing and polymerase chain reaction (PCR). We have collected 289 single-patient E. coli Isolates based on samples of China from July 2013 to August 2014. This article explored that the prevalence of ESBL-producing Isolates showed multi-resistant to antimicrobials such as fluoroquinolones, trimethoprim, tetracycline and aminoglycosides, and so on. The frequencies of resistance in Isolates were as follows: Ciprofloxacin, 74%, gentamicin, 69.5%, levofloxacin, 63%, tobramycin, 39%, and minocycline, 7.9%. According to our results, 197(68.2%) of the total 289 Isolates were ESBL-producing strains; further, 172 (87.3%) producers contained genes encoding CTX-M enzymes and 142(72.1%) producers contained genes encoding TEM enzymes. Most ESBL-producing Escherichia coli has produced more than one type of β-lactamase. Nucleotide sequence analysis has revealed the diversity of ESBLs types: CTX-M -15 is in the majority and TEM-135, CTX-M-3, CTX-M-98, CTX-M-14, CTX-M-142, CTX-M-65, CTX-M-55, CTX-M-27, and CTX-M-123 have been recovered. The results confirm that ESBL producers which are common in hospital strains of Escherichia coli are resistant to cephalosporins and other antibiotics in China. It is important to monitor such strains closely and provide scientific evidence of rational application of antibiotics to prevent their spread.

Characterization of antimicrobial resistance and extended-spectrum β-lactamase genes in Escherichia coli isolated from chickens.

Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli have been frequently isolated from food-producing animals and pose a serious threat to human health. This study collected 195 ESBL-producing E. coli isolates from 20 chicken farms and 3 live-bird markets located in Northeast China (Heilongjiang, Liaoning, Jilin) and Jiangsu province from February 2011 to October 2013. ESBL genes, including blaCTX-M, blaTEM, and blaSHV, were detected and characterized, and the susceptibilities of these strains to various antimicrobial agents were determined. One hundred ninety-one of these isolates carried 1 or more bla genes. blaCTX-M, blaTEM-1, and blaSHV-5 were identified in 183, 121, and 2 isolates, respectively. The most common blaCTX-M genes were blaCTX-M-15 (68 strains), blaCTX-M-65 (41 strains), blaCTX-M-55 (35 strains), blaCTX-M-14 (32 strains), followed by blaCTX-M-3, blaCTX-M-13, blaCTX-M-79, and blaCTX-M-101, as well as the chimeric genes blaCTX-M-64, blaCTX-M-123, and blaCTX-M-132. Fifteen strains (7.7%) co-harboring CTX-M-1 group and CTX-M-9 group genes were detected in 195 ESBL-producing strains. Pulsed-field gel electrophoresis of 45 strains showed that these CTX-M-producing isolates belonged to 34 different types. To our knowledge, this is the first study to report the blaSHV-5 gene in E. coli isolated from chickens in China. Conjugation experiments demonstrated that the blaCTX-M and blaTEM genes could be transferred to E. coli strain J53, while conjugative transfer of the blaSHV-5 gene from two isolates was not detectable. blaCTX-M genes are carried by many kinds of transferable and untypable plasmids. Our findings demonstrate that the CTX-M enzymes are predominant in both type and quantity.

Application of Molecular Diagnostics in Primary Detection of ESBL Directly from Clinical Specimens.

The infections caused by extended-spectrum β-lactamase (ESBL)-producing organisms are associated with increased mortality. The real-time polymerase chain reaction (PCR) method, which enables detection of ESBLs directly from patients' clinical material, was developed. This study focused on blaCTX-M and blaSHV determination in endotracheal aspirates. Each sample was identified with standard microbiological procedures and simultaneously analyzed for the presence of nucleic acids, which encode CTX-M and SHV ESBL enzymes using real-time PCR. A total of 341 samples were investigated. In the set, 27 ESBL-positive samples were identified by phenotypic methods, while 60 positive samples were identified by the PCR method. Of the 60 PCR-positive samples, 58 were positive for the blaCTX-M. In two samples, the ESBL blaSHV-ESBL gene was detected. One phenotypically positive sample was PCR negative. The real-time PCR assay does not require a cultivation step and therefore enables detection of ESBL in 6 hours. The rapid method is necessary for early and adequate antimicrobial treatment.

Colonization with extended-spectrum beta-lactamase-producing and carbapenemase-producing Enterobacteriaceae in international travelers returning to Germany

Two hundred and twenty-five healthy German volunteers traveling to 53 different countries (mostly in Asia, Africa and South America) were enrolled in a prospective cohort study. Stool samples and data on potential travel-associated risk factors (such as type of travel, nutritional habits, occurrence of gastroenteritis) were collected before and after traveling. Screening for extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-PE) and carbapenemase-producing Enterobacteriaceae (CPE) was performed using selective media (CHROMagar™ ESBL/CPE plates). Isolates with confirmed ESBL-phenotype were examined for the presence of blaCTX-M, blaTEM, blaSHV, and blaVIM, blaIMP, blaNDM, blaKPC, blaOXA-48 genes by PCR amplification and sequencing. Antimicrobial susceptibility testing was performed using conventional microbroth dilution. Pre-travel analysis of 205 fully evaluable participants revealed an ESBL-PE prevalence rate of 6.8% (14/205). Among 191 participants that were ESBL-negative before travel, 58 (30.4%) were colonized by ESBL-producing Escherichia coli, and 5 (8.6%) additionally carried ESBL-producing Klebsiella pneumoniae upon return. However, no carbapenem-resistant Enterobacteriaceae were detected. ESBL-genotyping revealed that 52/54 (96.6%) E. coli and 4/4 (100%) K. pneumoniae strains available for sequencing produced CTX-M enzymes, mostly CTX-M-15 (33/56, 58.9%), and 2/54 (3.7%) E. coli strains produced SHV-12 enzymes. Travel to India was associated with the highest ESBL-PE acquisition rate (11/15, 73.3%; p=0.015), followed by South East Asia (22/46, 47.8%; p=0.038). Evaluation of travel-associated risk factors demonstrated significance for the occurrence of gastroenteritis (p=0.011). Strictly practiced hand hygiene and exclusive consumption of packaged beverages showed no protective effect. The ESBL-PE persistence rate after 6 months was 8.6% (3/35). We conclude that global efforts are needed to address the further spread of ESBL-PE in the community. Active surveillance and contact isolation precautions may be recommended at admission to medical facilities especially for patients who traveled to India and South East Asia in the previous 6 months.

Prevalence of extended-spectrum-β-lactamase-producing Escherichia coli from pigs at slaughter in the UK in 2013.

To determine the prevalence and types of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli occurring in pigs at slaughter in the UK in 2013. Caecal samples from 637 pigs, sampled via a UK-wide monitoring programme in 2013, were enriched overnight in buffered peptone water, before plating to CHROMagar CTX and Oxoid Brilliance ESBL agar. Presumptive ESBL-producing E. coli from both media were tested for ESBL phenotype using MAST ESβL ID discs. Isolates with an ESBL phenotype were examined for the presence of blaCTX-M, blaOXA, blaSHV and blaTEM genes using a multiplex PCR. All blaCTX-M and blaSHV genes identified by PCR were sequenced. A total of 23.4% (95% CI 19.2-27.6) of pigs were positive for ESBL-producing E. coli; 22% (95% CI 17.8-26.1) of the pigs carried E. coli producing CTX-M enzymes [comprising enzyme types 1 (18.7% of pigs), 3 (0.2%), 14 (0.5%), 15 (1.4%), 27 (0.5%), 32 (0.5%) and 55 (0.3%)] and 2.2% (95% CI 0.8-3.6) of the pigs carried E. coli producing SHV-12. Five pigs carried both CTX-M- and SHV-12-producing E. coli as different isolates. There were no statistically significant differences observed between the two medium types in terms of the proportions of each CTX-M enzyme type isolated. In this UK study, 23.4% of pigs were found to be positive for ESBL-producing E. coli using selective culture media. The use of two different commercially available ESBL isolation media was found to improve the detection of ESBL-producing E. coli.

Characterization of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli obtained from Danish pigs, pig farmers and their families from farms with high or no consumption of third- or fourth-generation cephalosporins.

To compare and characterize extended-spectrum β-lactamase (ESBL)-producing Escherichia coli from pigsties, pig farmers and their families on farms with previous high or no use of third- or fourth-generation cephalosporins. Twenty farms with no third- or fourth-generation cephalosporin use and 19 herds with previous frequent use were included. The ESBL-producing isolates detected in humans and pigs were characterized by ESBL genotype, PFGE, susceptibility to non-β-lactam antibiotics and phylotype, and selected isolates were characterized by multilocus sequence typing (MLST). Furthermore, transferability of bla(CTX-M-)1 from both human and pig isolates was studied and plasmid incompatibility groups were defined. The volunteers answered a questionnaire including epidemiological risk factors for carriage of ESBL-producing E. coli. ESBL-producing E. coli was detected in pigs on 79% of the farms with high consumption of cephalosporins compared with 20% of the pigs on farms with no consumption. ESBL-producing E. coli was detected in 19 of the 195 human participants and all but one had contact with pigs. The genes found in both humans and pigs at the same farms were blaCTX-M-1 (eight farms), bla(CTX-M-14) (one farm) and bla(SHV-12) (one farm). At four farms ESBL-producing E. coli isolates with the same CTX-M enzyme, phylotype, PFGE type and MLST type were detected in both pigs and farmers. The majority of the plasmids with bla(CTX-M-1) were transferable by conjugation and belonged to incompatibility group IncI1, IncF, or IncN. The present study shows an increased frequency of ESBL-producing E. coli on farms with high consumption of third- or fourth-generation cephalosporins and indicates transfer of either ESBL-producing E. coli or plasmids between pigs and farmers.

Wastewater treatment plants release large amounts of extended-spectrum β-lactamase-producing Escherichia coli into the environment.

The determinants of the spread of extended-spectrum β-lactamase-producing Escherichia coli (ESBLEC) in the community remain unclear. To evaluate its dissemination in the environment, we analyzed the ESBLEC population throughout an urban wastewater network. Samples were collected weekly, over a 10-week period, from 11 sites throughout the wastewater network of Besançon city (France). Total E. coli and ESBLEC loads were determined for each sample. As a control, we analyzed 51 clinical ESBLEC isolates collected at our hospital. We genotyped both environmental and clinical ESBLEC by pulsed-field gel electrophoresis and multilocus sequence typing and identified their blaESBL genes by sequencing. The E. coli load was higher in urban wastewater than in hospital wastewater (7.5 × 10(5) vs 3.5 × 10(5) CFU/mL, respectively). ESBLEC was recovered from almost all the environmental samples and accounted for 0.3% of total E. coli in the untreated water upstream from the wastewater treatment plant (WWTP). The ESBLEC load was higher in hospital wastewater than in community wastewater (27 × 10(3) vs 0.8 × 10(3) CFU/mL, respectively). Treatment by the WWTP eliminated 98% and 94% of total E. coli and ESBLEC, respectively. The genotyping revealed considerable diversity within both environmental and clinical ESBLEC and the overrepresentation of some clonal complexes. Most of the sequence types displayed by the clinical isolates were also found in the environment. CTX-M enzymes were the most common enzymes whatever the origin of the isolates. The treatment at the WWTP led to the relative enrichment of ESBLEC. We estimated that >600 billion of ESBLEC are released into the river Doubs daily and the sludge produced by the WWTP, used as fertilizer, contains 2.6 × 10(5) ESBLEC per gram.

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.

Activity of Ceftaroline-Avibactam Tested Against Contemporary Enterobacteriaceae Isolates Carrying β-Lactamases Prevalent in the United States

Contemporary (2012) β-lactamase-producing isolates (n=493) from U.S. hospitals were tested against ceftaroline-avibactam. Klebsiella spp., Escherichia coli and Proteus mirabilis isolates displaying the Clinical and Laboratory Standards Institute (CLSI) screening criteria for extended spectrum β-lactamase (ESBL) production were evaluated. Isolates carried genes encoding CTX-M (n=316, CTX-M-14-like and -15-like), KPC (n=45), CMY-2-like (n=54), or SHV enzyme with ESBL activity (n=78). Ceftaroline-avibactam inhibited 98.2% of the isolates at ≤0.5 μg/mL, and all strains were inhibited by ≤2 μg/mL of this novel β-lactamase-inhibitor combination. These results confirm that ceftaroline-avibactam could be a useful therapeutic option for Enterobacteriaceae isolates producing β-lactamases that are prevalent in the United States.

The Mediterranean Region : A Reservoir for CTX - M - ESBL - Producing Enterobacteriacae

The incidence of ESBL-producing bacteria is increasing worldwide, which represents a challenge for the healthcare systems. More recently, the emergence of the CTX-M group has been frequently reported as being associated with both nosocomial and community acquired infections. CTX-M-15 was identified as being the most prevalent in a large geographic area including Europe and the Middle East, suggesting the presence of a community reservoir of CTX-M enzymes disseminating in the Mediterranean area. Thus, this review will focus on the Mediterranean region to highlight the increasing prevalence of CTX-M-ESBL producing Enterobacteriacae.