Molecular characteristics of extended-spectrum beta-lactamases and qnr determinants in Enterobacter species from Japan

The incidence of extended-spectrum β-lactamases (ESBLs) has been increasing worldwide, but screening criteria for detection of ESBLs are not standardized for AmpC-producing Enterobacteriaceae such as Enterobacter species. In this study, we investigated the prevalence of ESBLs and/or AmpC β-lactamases in Japanese clinical isolates of Enterobacter spp. and the association of plasmid-mediated quinolone resistance (PMQR) determinants with ESBL producers. A total of 364 clinical isolates of Enterobacter spp. collected throughout Japan between November 2009 and January 2010 were studied. ESBL-producing strains were assessed by the CLSI confirmatory test and the boronic acid disk test. PCR and sequencing were performed to detect CTX-M, TEM, and SHV type ESBLs and PMQR determinants. For ESBL-producing Enterobacter spp., pulsed-field gel electrophoresis (PFGE) was performed using XbaI restriction enzyme. Of the 364 isolates, 22 (6.0%) were ESBL producers. Seven isolates of Enterobacter cloacae produced CTX-M-3, followed by two isolates producing SHV-12. Two isolates of Enterobacter aerogenes produced CTX-M-2. Of the 22 ESBL producers, 21 had the AmpC enzyme, and six met the criteria for ESBL production in the boronic acid test. We found a significant association of qnrS with CTX-M-3-producing E. cloacae. The 11 ESBL-producing Enterobacter spp. possessing bla(CTX-M), bla(SHV), or bla(TEM) were divided into six unique PFGE types. This is the first report about the prevalence of qnr determinants among ESBL-producing Enterobacter spp. from Japan. Our results suggest that ESBL-producing Enterobacter spp. with qnr determinants are spreading in Japan.     

Extended-Spectrum β-Lactamases and/or Carbapenemases-Producing Enterobacteriaceae Isolated from Retail Chicken Meat in Zagazig,Egypt

Objectives

The aim of the present study was to determine the prevalence and to characterize extended-spectrum β-lactamases- and/or carbapenemases-producing Enterobacteriaceae among Enterobacteriaceae isolated from retail chicken meat in Zagazig, Egypt.

Methods

One hundred and six Enterobacteriaceae isolates were collected from retail chicken meat samples purchased in Zagazig, Egypt in 2013. Species identification was done by MALDI-TOF MS. Screening for ESBL-E was performed by inoculation of isolates recovered from meat samples onto the EbSA (Cepheid Benelux, Apeldoorn, the Netherlands) selective screening agar. ESBL production was confirmed by combination disc diffusion test with clavulanic acid (Rosco, Taastrup, Denmark). Carbapenemases production was confirmed with double disk synergy tests. Resistance genes were characterized by PCR with specific primers for TEM, SHV, and CTX-M and carbapenemases (KPC, NDM, OXA-48, IMP and VIM). PCR products of CTX-M genes were purified and sequenced. Phylogenetic grouping of E. coli was performed by a PCR-based method.

Results

Of these 106 isolates 69 (65.09%) were ESBL producers. Twelve (11.32%) of these isolates were also phenotypically class B carbapenemases producer. TEM genes were detected in 61 (57.55%) isolates. 49 (46.23%) isolates harbored CTX-M genes, and 25 (23.58%) carried genes of the SHV family. All CPE belonged to the NDM group. The predominant CTX-M sequence type was CTX-M-15 (89.80%). The majority (80%) of the ESBL-EC belonged to low virulence phylogroups A and B1.

Conclusions

This is the first study from Egypt reporting high rates of ESBLs and carbapenemases (65.09% and 11.32%, respectively) in Enterobacteriaceae isolated from retail chicken meat. These results raise serious concerns about public health and food safety as retail meat could serve as a reservoir for these resistant bacteria which could be transferred to humans through the food chain.     

Detection of plasmid-mediated quinolone resistance in clinical isolates of Enterobacteriaceae strains in Hamadan,West of Iran

Plasmid mediated quinolone resistance (PMQR) determinants have arisen as a significant concern in recent years. The aim of this study was screening of resistant-clinical isolates to fluoroquinolone antibiotics and detection of qnr and aac(6′)-Ib-cr genes.For this purpose we collected 100 fluoroquinolone-resistant Enterobacteriaceae which were from 3 hospitals in Hamadan, west provinces of Iran, between October 2012 and June 2013. The all samples were identified by biochemical tests and confirmed by PCR method. Antimicrobial susceptibility to 14 antimicrobial agents including levofloxacin and ciprofloxacin were determined by disk diffusion methods and ciprofloxacin MIC was obtained by broth microdilution method as Clinical Laboratory Standards Institute (CLSI) recommendations. The isolates were screened for the presence of qnrA, qnrB, qnrS and aac(6′)-Ib-cr genes using PCR assay. Among the screened isolates, 64 strains (64%) of Escherichia coli, 23 strains (23%) of Klebsiella pneumoniae, 13 strains (13%) of Proteus mirabilis were collected as quinolone-resistant isolates. out of 100 isolates, two (2%) were positive for qnrS, seventeen (17%) isolates were positive for qnrB and we did not find qnrA gene in any of the isolates. There were also 32 positive isolates for aac(6′)-Ib-cr determinant. We described the prevalence of qnr and aac(6′)-Ib-cr genes in fluoroquinolone-resistant Enterobacteriaceae in Hamadan city. The carriage rate of multidrug-resistant Enterobacteriaceae in healthy people in Hamadan City is extremely high. Moreover, genes encoding transferable quinolones, in particular aac(6′)-Ib-cr, are highly prevalent in these strains.     

Development of an immunochromatographic assay for diagnosing the production of IMP-type metallo-β-lactamases that mediate carbapenem resistance in Pseudomonas

Rapid and reliable detection of carbapenem-resistant bacteria is an important infection-control measure and a crucial aspect of antimicrobial chemotherapy. IMP-type metallo-β-lactamase (MBL) is an emzyme that mediate carbapenem resistance in bacteria. Here, an immunochromatographic assay was newly developed using novel monoclonal antibodies (mAbs) recognizing IMP-type MBL. Epitope mapping of mAbs and mutational analysis of the epitope region in IMP antigen suggested that the mAbs could react to all known subtypes of IMP-type MBL. Evaluation of the assay using Pseudomonas aeruginosa strains (n = 248) showed that the results of the immunochromatographic detection of the IMP-type MBLs were fully consistent with those of the PCR analysis for bla_IMP genes, showing false positives and negatives. All positive strains were resistant to carbapenem (MIC ≥ 16 μg/ml). The assay also accurately distinguished the production of IMP-type MBLs in Pseudomonas putida, Acinetobacter baumannii, and Alcaligenes xylosoxidans. The detection limit of the assay was 5.7 × 10⁴ cfu per test. Taken together, these data suggest that the developed assay can be used for rapid and reliable diagnosis of the production of IMP-type MBLs in Gram-negative bacteria.     

Extended-Spectrum β-Lactamase- and Carbapenemase-Producing Enterobacteriaceae Isolated from Egyptian Patients with Suspected Blood Stream Infection

ObjectivesThe aim of the study was to investigate the prevalence of extended-spectrum β-lactamase and carbapenemase production among Enterobacteriaceae isolated from Egyptian patients with suspected blood stream infection.MethodsNinety-four Enterobacteriaceae blood culture isolates from Egyptian patients with suspected blood stream infection were collected, one isolate per patient. Identification of bacterial isolates was performed with MALDI-TOF (MS-based Vitek MS system, bioMerieux). Screening for ESBLs and carbapenemases production was done with the Vitek 2 system (bioMérieux). ESBL production was confirmed using the combined disk diffusion method for cefotaxime, ceftazidime, and cefepime, all with and without clavulanic acid (Rosco). Real-time PCR and sequencing were used to characterize the resistance genes. The phylogenetic groups of E. coli were identified by a PCR-based method.ResultsOf the 94 Enterobacteriaceae isolates 46 (48.93%) showed an ESBL phenotype. One Enterobacter spp isolate was ESBL-producer and meropenem-resistant. The genetic analysis showed that CTX-M was present in 89.13% (41/46) of the ESBL-producing Enterobacteriaceae, whereas TEM and SHV were detected in 56.52% (26/46) and 21.74% (10/46) respectively (47.83%) of the ESBL-producing isolates were multidrug resistant (MDR). Eleven out of 30 ESBL-producing E-coli isolates were assigned to phylogroup B2, followed by groups B1 (8 isolates), A (6 isolates) and D (5 isolates).ConclusionsThe high ESBL-E rates (48.93%) found in this study together with the identification of one carbapenem-resistant Enterobacter spp isolate is worrisome. Our results indicate that systems for monitoring and detection of ESBL-producing bacteria in Egyptian hospitals have to be established. Also strict hospital infection control policies with the restriction of the consumption of extended-spectrum cephalosporins are necessary.     

Characterization of Plasmid-Mediated Quinolone Resistance (PMQR) Genes in Extended-Spectrum β-Lactamase-Producing Enterobacteriaceae Pediatric Clinical Isolates in Mexico

This work describes the characterization of plasmid-mediated quinolone-resistance (PMQR) genes from a multicenter study of ESBL-producing Enterobacteriaceae pediatric clinical isolates in Mexico. The PMQR gene-positive isolates were characterized with respect to ESBLs, and mutations in the GyrA and ParC proteins were determined. The phylogenetic relationship was established by PFGE and the transfer of PMQR genes was determined by mating assays. The prevalence of the PMQR genes was 32.1%, and the rate of qnr-positive isolates was 15.1%; 93.3% of the latter were qnrB and 6.4% were qnrA1. The distribution of isolates in terms of bacterial species was as follows: 23.5% (4/17) corresponded to E. cloacae, 13.7% (7/51) to K. pneumoniae, and 13.6% (6/44) to E. coli. In addition, the prevalence of aac(6’)-Ib-cr and qepA was 15.1% and 1.7%, respectively. The molecular characteristics of qnr- and qepA-positive isolates pointed to extended-spectrum β-lactamase (ESBL) CTX-M-15 as the most prevalent one (70.5%), and to SHV-12 in the case of aac(6’)-Ib-cr-positive isolates. GyrA mutations at codons Ser-83 and Asp-87, and ParC mutations at codons Ser-80 were observed in 41.1% and 35.2% of the qnr-positive isolates, respectively. The analysis of the transconjugants revealed a co-transmission of bla_CTX-M-15 with the qnrB alleles. In general, the prevalence of PMQR genes (qnr and aac(6’)-Ib-cr) presented in this work was much lower in the pediatric isolates, in comparison to the adult isolates in Mexico. Also, ESBL CTX-M-15 was the main ESBL identified in the pediatric isolates, whereas in the adult ones, ESBLs corresponded to the CTX-M and the SHV families. In comparison with other studies, among the PMQR-genes identified in this study, the qnrB-alleles and the aac(6’)-Ib-cr gene were the most prevalent, whereas the qnrS1, qnrA1 and qnrB-like alleles were the most prevalent in China and Uruguay.     

“Occurrence of blaCTX-MGp1 and blaCTX-MGp26 in third generation cephalosporin-resistant and carbapenem- resistant bacterial isolates from southwest region of Saudi Arabia-a preliminary study”

This study was intended to identify the genes responsible for ESBL- and carbapenemase-producing bacterial isolates obtained from Jizan region. A hospital-based cross-sectional study was conducted over a period of 3 months (15th November 2018–15th February 2019). Fifty non-duplicate, 3rd-generation cephalosporin and carbapenem-resistant isolates were collected from microbiology lab of a tertiary care hospital in Jizan province and were screened for ESBLs and MBLs by phenotypic methods (CDT). The positive isolates (by phenotypic method) were then scanned for the presence of bla_ESBLs and bla_NDM-₁ genes, respectively, by PCR.As a result, 10% isolates showed imipenem-cephalosporin co-resistance whereas 92% (46/50) of isolates were found to be ESBL producers by CDT. The maximum occurrence was observed for bla_CTX-M (70%), followed by bla_SHV (16%) and least occurrence was noted for bla_TEM (12%). Moreover, 97% isolates (34/35) were of bla_CTX-MGroup1 but one isolate showed the presence of bla_CTX-M Group26. Despite the co-resistance of cephalosporin and carbapenem, 14% (7/50) were found to be MBL producer on phenotypic detection by Combination Disc Test (CDT), whereas all the isolates were found to be negative for bla_NDM-1. Hence bla_CTX-MGroup1 is present in quite high fraction followed by bla_SHV in the bacterial isolates of Jizan region. Moreover, the occurrence of bla_CTX-M Group1 and bla_CTX-M Group26 in clinical isolates from the Jizan region of Saudi Arabia has been reported for the first time.     

Pathogenic and commensal Escherichia coli from irrigation water show potential in transmission of extended spectrum and AmpC β‐lactamases determinants to isolates from lettuce

There are few studies on the presence of extended-spectrum β-lactamases and AmpC β-lactamases (ESBL/AmpC) in bacteria that contaminate vegetables. The role of the production environment in ESBL/AmpC gene transmission is poorly understood. The occurrence of ESBL/AmpC in Escherichia coli (n = 46) from lettuce and irrigation water and the role of irrigation water in the transmission of resistant E. coli were studied. The presence of ESBL/AmpC, genetic similarity and phylogeny were typed using genotypic and phenotypic techniques. The frequency of β-lactamase gene transfer was studied in vitro. ESBLs/AmpC were detected in 35 isolates (76%). Fourteen isolates (30%) produced both ESBLs/AmpC. Prevalence was highest in E. coli from lettuce (90%). Twenty-two isolates (48%) were multi-resistant with between two and five ESBL/AmpC genes. The major ESBL determinant was the CTX-M type (34 isolates). DHA (33% of isolates) were the dominant AmpC β lactamases. There was a high conjugation efficiency among the isolates, ranging from 3.5 × 10⁻² to 1 × 10⁻² ± 1.4 × 10⁻¹ transconjugants per recipient. Water isolates showed a significantly higher conjugation frequency than those from lettuce. A high degree of genetic relatedness between E. coli from irrigation water and lettuce indicated possible common ancestry and pathway of transmission.     

“Occurrence of blaCTX-MGp1 and blaCTX-MGp26 in third generation cephalosporin-resistant and carbapenem- resistant bacterial isolates from southwest region of Saudi Arabia-a preliminary study”

This study was intended to identify the genes responsible for ESBL- and carbapenemase-producing bacterial isolates obtained from Jizan region. A hospital-based cross-sectional study was conducted over a period of 3 months (15th November 2018–15th February 2019). Fifty non-duplicate, 3rd-generation cephalosporin and carbapenem-resistant isolates were collected from microbiology lab of a tertiary care hospital in Jizan province and were screened for ESBLs and MBLs by phenotypic methods (CDT). The positive isolates (by phenotypic method) were then scanned for the presence of bla_ESBLs and bla_NDM-₁ genes, respectively, by PCR.As a result, 10% isolates showed imipenem-cephalosporin co-resistance whereas 92% (46/50) of isolates were found to be ESBL producers by CDT. The maximum occurrence was observed for bla_CTX-M (70%), followed by bla_SHV (16%) and least occurrence was noted for bla_TEM (12%). Moreover, 97% isolates (34/35) were of bla_CTX-MGroup1 but one isolate showed the presence of bla_CTX-M Group26. Despite the co-resistance of cephalosporin and carbapenem, 14% (7/50) were found to be MBL producer on phenotypic detection by Combination Disc Test (CDT), whereas all the isolates were found to be negative for bla_NDM-1. Hence bla_CTX-MGroup1 is present in quite high fraction followed by bla_SHV in the bacterial isolates of Jizan region. Moreover, the occurrence of bla_CTX-M Group1 and bla_CTX-M Group26 in clinical isolates from the Jizan region of Saudi Arabia has been reported for the first time.     

Prevalence of Plasmid-Mediated Quinolone Resistance and Aminoglycoside Resistance Determinants among Carbapeneme Non-Susceptible Enterobacter cloacae

Background

Simultaneous resistance to aminoglycosides and fluoroquinolones in carbapeneme non-susceptible (CNS) isolates will inevitably create problems. The present study was performed to characterize the prevalence of the plasmid-mediated quinolone resistance determinants (QRDs) and aminoglycoside resistance determinants (ARDs) among the CNS Enterobacter cloacae (E. cloacae) isolates in a Chinese teaching hospital, and to acquire their molecular epidemiological characteristics.

Methods

The β-lactamases genes (including class A carbapenemase genes bla_KPC and bla_SME, metallo-β-lactamase genes (MBLs) bla_IMP, bla_VIM and bla_NDM, and extended spectrum β-lactamases (ESBLs),bla_CTX-M, bla_TEM and bla_SHV), QRDs (including qnrA, qnrB, qnrS and aac(6′)-Ib-cr) and ARDs (including aac(6′)-Ib, armA and rmtB) of these 35 isolates were determined by PCR and sequenced bidirectionally. The clonal relatedness was investigated by pulsed-field gel electrophoresis (PFGE).

Results

Of the 35 isolates, 9 (25.7%) harbored a carbapenemase gene; 23 (65.7%) carried ESBLs; 24 (68.6%) were QRD positive; and 27 (77.1%) were ARD positive. Among the 5 bla_IMP-8 positive strains, 4 (80%) contained both ESBL and QRD genes, and all the 5 (100%) harbored ARD genes. Of the 23 ESBLs positive isolates, 6 (26.1%) were carbapenemase positive, 14 (60.9%) were QRD positive, and 18 (78.3%) were ARD positive. PFGE revealed genetic diversity among the 35 isolates, indicating that the high prevalence of CNS E. cloacae isolates was not caused by clonal dissemination.

Conclusion

QRD and ARD genes were highly prevalent among the CNS E. cloacae isolates. Multiple resistant genes were co-expressed in the same isolates. The CNS E. cloacae isolate co-expressing bla_NDM-1, bla_IMP-26, qnrA1 and qnrS1 was first reported.     

Molecular testing of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> contaminating tissue allografts recovered from deceased donors

Microbiological screening of tissue allografts is crucial to prevent the transmission of bacterial and fungal infections to transplant recipients. Klebsiella was the most prevalent and resistant contaminating microorganism observed in our setting in the Iranian Tissue Bank. This study was conducted to determine the presence of extended-spectrum β-lactamase (ESBL) genes, antimicrobial resistance patterns of Klebsiella pneumoniae isolates, and their clonal relationships in allograft materials. K. pneumoniae contaminating bone and other tissue allografts recovered from deceased donors were identified and ESBL isolates were detected using a phenotypic confirmatory method. Antimicrobial susceptibility testing was carried out using the disk diffusion method. Distribution of ESBL genes and molecular typing were performed using polymerase chain reaction (PCR) and Repetitive-element (rep-PCR) methods. Of 3828 donated tissues, 51 (1.3%) were found contaminated by K. pneumoniae isolates. Compared to tissue allografts from brain-dead, heart-beating tissue donors, allografts from donors with circulatory cessation were associated with a higher risk of K. pneumoniae contamination [odds ratio (OR), 1.2 (CI 95% 0.9–2.3) (P value < 0.001)]. Half of the isolates produced ESBL, and the rate of susceptibility to cephalosporins was 51%. Among isolates, 22 (43.1%) harbored CTX-M, 31 (60.8%) SHV, and 9 (17.6%) harbored TEM types. The rep-dendrogram indicated that clones having identical or related strains with a similar antibiotype were isolated in the same period. This study provides evidence that a single clone of K. pneumoniae contaminated tissue allografts recovered from many different donors. A single clone found on tissues from several donors suggests contamination of tissues from a single source such as the tissue recovery process and environment. Genomic DNA testing and clonality of contaminating bacteria using molecular methods can focus the epidemiologic investigation on the tissue allograft recovery process including a search for contamination of the tissue recovery room environment, recovery staff, recovery equipment, reagents, solutions and supplies.     

Emergence of carbapenem non‐susceptible multidrug resistant Acinetobacter baumannii strains of clonal complexes 103B and 92B harboring OXA‐type carbapenemases and metallo‐β‐lactamases in Southern India

The molecular epidemiology and carbapenem resistance mechanisms of clinical isolates of Acinetobacter baumannii obtained from a south Indian tertiary care hospital were investigated by repetitive extragenic palindromic sequence PCR (REP‐PCR) and multi‐locus sequence typing (MLST). Analysis of resistant determinants was achieved by PCR screening for the presence of genes encoding OXA‐carbapenemases, metallo‐β‐lactamases (MBLs) and efflux pumps. REP‐PCR generated around eight clusters of high heterogeneity; of these, two major clusters (I and V) appeared to be clonal in origin. Analysis of representative isolates from different clusters by MLST revealed that most of the isolates belonged to sequence type 103 of CC103^B. Second most prevalent ST belonged to clonal complex (CC) 92^B which is also referred to as international clone II. Most of the isolates were multi‐drug resistant, being susceptible only to polymyxin‐B and newer quinolones. Class D β‐lactamases such as bla_{OXA‐51‐like} (100%), bla_{OXA‐23‐like} (56.8%) and bla_{OXA‐24‐like} (14.8%) were found to be predominant, followed by a class B β‐lactamase, namely bla_IMP‐1 (40.7%); none of the isolates had bla_OXA‐58 like, bla_NDM‐1 or bla_SIM‐1. Genes of efflux‐pump adeABC were predominant, most of isolates being biofilm producers that were PCR‐positive for autoinducer synthase gene (>94%). Carbapenem non‐susceptible isolates were highly diverse and present throughout the hospital irrespective of type of ward or intensive care unit. Although previous reports have documented diverse resistant mechanisms in A. baumannii, production of MBL and OXA‐type of carbapenamases were found to be the predominant mechanism(s) of carbapenem resistance identified in strains isolated from Southern India.     

Detection of extended-spectrum beta-lactamase-producing Escherichia coli isolates in faecal samples of Iberian lynx

Aims: To characterize the diversity of extended‐spectrum beta‐lactamase (ESBL)‐producing Escherichia coli isolates recovered within the faecal microbiota of Iberian lynx. The identification of other associated resistance genes and the analysis of clonal relationship were also focused in this study. Methods and Results: From 2008 to 2010, 128 faecal samples of Iberian lynx (wild and captive animals) were collected. Eleven tested samples contained cefotaxime‐resistant E. coli isolates (all belonging to captive animals) and 10 ESBL‐producing isolates were showed. CTX‐M‐14 and SHV‐12 ESBL‐types were detected and seven different patterns were identified by pulsed‐field gel electrophoresis analysis. Conclusions: The occurrence of unrelated multiresistant E. coli in faecal flora of captive specimens of Iberian lynx, including the presence of ESBLs, resistant genes in integrons and virulence determinants was showed in this study. Significance and Impact of the Study: The results obtained in this study highlight the environmental problem as future reintroductions of Iberian lynx could lead to a spread of resistant bacteria. Additionally, ESBL‐producing bacteria can represent a health problem for this endangered species.     

Profile and multidrug resistance determinants of Chryseobacterium indologenes from seawater and marine fauna

The aim of this study was to investigate the prevalence and genetic basis of multidrug resistance in Chryseobacterium indologenes from seawater and marine invertebrates used for human consumption, in Ka?tela Bay, Adriatic Sea, Croatia. Out of 16 samples of seawater, Mediterranean mussel (Mytilus galloprovincialis Lam.), Rayed Mediterranean limpets (Patella caerulea L.) and Purple sea urchins (Paracentrotus lividus Lam.) collected, 15 were positive for C. indologenes. In total, 41 isolates were randomly selected and tested for antibiotic susceptibility by disc-diffusion and broth microdilution methods. PCR was used to detect alleles encoding extended-spectrum (ESBLs) and metallo-β-lactamases (MBLs). The clonality of β-lactamase-producing strains was evaluated by random amplified polymorphic DNA (RAPD) analysis. All C. indologenes isolates showed multiple resistance to at least 9 out of 16 antibiotics tested. Lowest resistance rates were found for piperacillin (9.7 %) and ciprofloxacin (24.4 %), whereas only piperacillin/tazobactam and trimethoprim/sulfamethoxazole showed 100 % activity. More than half of isolates carried bla _IND-type gene, including 2 isolates carrying bla _IND-2 and 21 carrying bla _IND-7, that was identified as a major MBL genotype in isolates from Adriatic Sea. RAPD typing of IND-producing isolates revealed 6 major groups with no predominant clone in population. The presence of multidrug resistant and IND-producing C. indologenes in marine environment, including marine fauna, pose a risk for transmitting this opportunistic pathogen to humans through recreation or consummation of seafood. In addition, the antibiotic susceptibility test results have practical relevance for empirical treatment of C. indologenes infections.     

Occurrence of the genes encoding carbapenemases,ESBLs and class 1 integron-integrase among fermenting and non-fermenting bacteria from retail goat meat

The present study was planned to detect the genes encoding carbapenemases, ESBLs and class 1 integron-integrase among bacteria obtained from retail goat meat. Fermenting and non-fermenting bacterial isolates (n = 57), recovered from 61 goat meat samples, were identified by 16S rRNA gene sequencing. Antimicrobial susceptibility of isolates was tested by the broth dilution method using ceftazidime, cefotaxime, meropenem and imipenem. Plasmids were isolated and tested for their physical characters. Plasmids were subjected to screening of carbapenemase, ESBL and intI1 gene. Conjugation assay was performed using bla_NDM-positive isolates as the donor, and Escherichia coli HB101 as the recipient. Isolates showed the high rates of resistance to ceftazidime (77·2%), cefotaxime (70·2%), meropenem (22·8%) and imipenem (17·5%). They showed variability in number and size (~1 to >20 kb) of plasmids. Among all, 1, 4, 13 and 31 isolates showed the bla_KPC, bla_NDM, bla_SHV and bla_TEM genes, respectively. The bla_KPC-2 gene was observed in one E. coli isolate. The bla_NDM-1 gene was detected in Stenotrophomonas maltophilia (n = 2), Acinetobacter baumannii (n = 1) and Ochrobactrum anthropi (n = 1) isolates. These isolates co-harboured the bla_TEM and bla_SHV genes. The intI1 gene was detected in 22 (38·6%) isolates, and 16 of these isolates showed the carbapenemase and/or ESBL genes. The conjugative movement of bla_NDM gene could not be proved after three repetitive mating experiments. The presence of genes encoding carbapenemases and ESBLs in bacteria from goat meat poses public health risks.     

Occurrence of qnr-positive clinical isolates in Klebsiella pneumoniae producing ESBL or AmpC-type beta-lactamase from five pediatric hospitals in China.

The plasmid-mediated quinolone resistance qnr genes in clinical isolates in adults have been described in different countries; however, the frequency of their occurrence has not been detected in pediatric patients. A total of 410 clinical isolates of Klebsiella pneumoniae, identified as producers of an extended-spectrum beta-lactamase (ESBL), or AmpC beta-lactamase, were collected from five children's hospitals in China during 2005-2006. The isolates were screened for the presence of the qnrA, qnrB, and qnrS genes, and then the harboring qnr gene isolates were detected for a bla gene coding for the TEM, SHV, CTX-M, and plasmid-mediated ampC gene by a PCR experiment. Ninety-two isolates (22.7%) were positive for the qnr gene, including 10 of qnrA (2.4%), 25 of qnrB (6.1%), and 62 of qnrS (15.1%). Eighty-one of the 92 (88.0%) qnr-positive isolates carried at least one bla gene for TEM, SHV, CTX-M, or DHA-1. The ciprofloxacin resistance increased 16-256-fold and oflaxacin resistance increased 2-32-fold in transconjugants, respectively. These results indicated that the plasmid-mediated qnr quinolone resistance gene was qnrS, followed by qnrB and qnrA. Most of the isolates also carried a bla gene coding ESBL or ampC gene coding DHA-1 among Klebsiella pneumoniae isolated from Chinese pediatric patients.     

Expression of ESBL-Like Activity in Infrequently Encountered Members of the Family <Emphasis Type="Italic">Enterobacteriaceae</Emphasis>

A collection of 94 unusual members of the Enterobacteriaceae were screened for the presence of extended spectrum β-lactamases (ESBLs) using the MicroScan ESβL plus dried confirmation panel. Presumptively positive strains were then confirmed for the presence of an ESBL by double disk diffusion, E-test strips (AB Biodisk, Solna, Sweden) and PCR for SHV, TEM, and CTX-M2 genes. Of the 18 strains initially positive on the ESβL panel only three strains (Leminorella grimontii, Klebsiella ozaenae, and Kluyvera ascorbata) were positive by confirmation methods. These results suggest laboratories should be cautious regarding the methodology employed in screening for the presence of ESBLs in enteric bacteria. However, it should be noted that of the 94 strains, 29 were found to be resistant to two or more of the antibiotics present in the MicroScan ESβL plus panel indicating that there are potential treatment issues with these organisms despite their lack of ESBLs.     

Occurrence of ESBL-Producing Escherichia coli in Livestock and Farm Workers in Mecklenburg-Western Pomerania,Germany

In recent years, extended-spectrum β-lactamases (ESBL) producing bacteria have been found in livestock, mainly as asymptomatic colonizers. The zoonotic risk for people working in close contact to animal husbandry has still not been completely assessed. Therefore, we investigated the prevalence of ESBL-producing Escherichia spp. in livestock animals and workers to determine the potential risk for an animal-human cross-transmission.In Mecklenburg-Western Pomerania, northeast Germany, inguinal swabs of 73 individuals with livestock contact from 23 different farms were tested for ESBL-producing Escherichia spp. Two pooled fecal samples per farm of animal origin from 34 different farms (17 pig farms, 11 cattle farms, 6 poultry farms) as well as cloacal swabs of 10 randomly selected broilers or turkeys were taken at each poultry farm. For identification, selective chromogenic agar was used after an enrichment step. Phenotypically ESBL-producing isolates (n = 99) were tested for CTX-M, OXA, SHV and TEM using PCR, and isolates were further characterized using multilocus sequence typing (MLST). In total, 61 diverse isolates from different sources and/or different MLST/PCR results were acquired. Five farm workers (three from cattle farms and two from pig farms) harbored ESBL-producing E. coli. All human isolates harbored the CTX-M β-lactamase; TEM and OXA β-lactamases were additionally detected in two, resp. one, isolates. ESBL-producing Escherichia spp. were found in fecal samples at pig (15/17), cattle (6/11) and poultry farms (3/6). In total, 70.6% (24/36) of the tested farms were ESBL positive. Furthermore, 9 out of 60 cloacal swabs turned out to be ESBL positive. All isolated ESBL-producing bacteria from animal sources were E. coli, except for one E. hermanii isolate. CTX-M was the most prevalent β-lactamase at cattle and pig farms, while SHV predominated in poultry. One human isolate shared an identical MLST sequence type (ST) 3891 and CTX-M allele to the isolate found in the cattle fecal sample from the same farm, indicating a zoonotic transfer. Two other pairs of human-pig and human-cattle E. coli isolates encoded the same ESBL genes but did not share the same MLST ST, which may indicate horizontal resistance gene transfer. In summary, the study shows the high prevalence of ESBL-producing E.coli in livestock in Mecklenburg- Western Pomerania and provides the risk of transfer between livestock and farm workers.     

Molecular characterization of the β‐lactamases in Escherichia coli and Klebsiella pneumoniae from a tertiary care hospital in Riyadh,Saudi Arabia

The widespread use of antimicrobials has increased the occurrence of multidrug resistant microbes. The commonest mechanism of antimicrobial resistance in Enterobacteriaceae is production of β‐lactamases such as metallo‐β‐lactamases (MBL) and extended spectrum β‐lactamases (ESBL). Few studies have used a molecular approach to characterize the prevalence of β‐lactamases. Here, the prevalence of different β‐lactamases was characterized by performing three multiplex PCRs targeting genes similar to those described in earlier publications. Antimicrobial susceptibility tests for all isolates were performed using the agar dilution method. β‐lactamase was detected in 72% of the isolates, the detection rate being 64% in 2011 and 75% in 2012. The isolates were highly resistant to carbapenems such as meropenem and imipenem and susceptible to colistin and tigecycline. In this study, 22% of isolates contained both MBL and ESBL. ESBL was detected more frequently in Escherichia coli isolates, whereas carbapenemase was detected more frequently in Klebsiella pneumoniae isolates. These findings suggest the spread of multi‐resistant ESBL and MBL producers in the community. Our results have implications for patient treatment and also indicate the need for increased surveillance and molecular characterization of isolates.     

Extended-spectrum beta-lactamase-producing Escherichia coli in turkey meat production farms in the Czech Republic: national survey reveals widespread isolates with bla(SHV-12) genes on IncFII plasmids

Aim: The occurrence and epidemiology of extended‐spectrum beta‐lactamase (ESBL)‐producing Escherichia coli in the environment of turkey farms in the Czech Republic were studied. Methods and Results: Extended‐spectrum beta‐lactamase‐producing E. coli isolates were found on 8 (20%) of 40 turkey farms surveyed. A total of 200 environmental smears were examined, and a total of 25 ESBL‐producing E. coli were isolated. These isolates were analysed using XbaI pulsed‐field gel electrophoresis and divided into nine pulsotypes. Most of the isolates harboured the gene bla_SHV‐12 on a 40‐kb plasmid of the IncFII group with an identical EcoRV restriction profile. Indistinguishable or clonally related SHV‐12‐producing isolates belonging to the same pulsotypes were found at some unrelated farms. Conclusions: Widespread occurrence of ESBL‐producing E. coli isolates with bla_SHV‐12 carried on IncFII plasmids in meat production flocks in the Czech Republic was demonstrated. Significance and Impact of the Study: Results indicate vertical transmission of ESBL‐producing E. coli within the turkey production pyramid. The study shows the risk of multiresistant ESBL‐producing bacteria and antibiotic‐resistance genes being transmitted to humans via the food chain.