Detection of plasmid-borne extended-spectrum β-lactamase (ESBL) genes in Escherichia coli isolates from bovine mastitis.

Extended-spectrum β-lactamase (ESBL)-producing isolates have been increasingly reported during recent years. The aims of this study were to characterize ESBL-producing Escherichia coli from bovine mastitis as well as their ESBL gene-carrying plasmids. A culture collection of E. coli isolated from bovine quarter milk samples (2009-2013), was screened for ESBL production using ESBL selective agar plates. Putative ESBL producers (n=16) were investigated by phenotypic confirmatory tests and were characterized by the detection/sequencing of ESBL genes, XbaI macrorestriction analysis, multilocus sequence typing (MLST), phylotyping and antimicrobial susceptibility testing. ESBL gene-carrying plasmids were investigated by transfer experiments, PCRs for the detection of co-located antimicrobial resistance genes, PCR-based replicon typing and S1-nuclease pulsed-field gel electrophoresis. Twelve ESBL-producing isolates were found. They showed eleven different XbaI patterns and were distributed among eight MLST types [ST10 (n=3), ST117 (n=2), ST361 (n=1), ST362 (n=1), ST540 (n=1), ST1431 (n=2), ST1508 (n=1), and the novel ST5447 (n=1)] and the phylogenetic groups A (n=6), B1 (n=2), B2 (n=1) and D (n=3). ESBL genes blaCTX-M-1 (n=5), blaCTX-M-2 (n=2), blaCTX-M-14 and blaCTX-M-15 (n=4) were found on conjugative plasmids (35-225kb) of diverse incompatibility groups (e.g. IncF, IncI1 or HI2+P). Co-located resistance to sulfonamides, tetracycline, trimethoprim, and chloramphenicol/florfenicol was detected on five ESBL gene-carrying plasmids, but seven plasmids conferred solely resistance to β-lactam antibiotics. The presence of additional resistance genes on the ESBL gene-carrying plasmids suggests that co-selection of ESBL genes may occur even in the absence of β-lactam antibiotics.

Freitag C ，Michael GB ，Kadlec K ，Hassel M ，Schwarz S ... -  《-》

Detection and characterization of extended-spectrum β-lactamase-producing Escherichia coli from chicken production chains in Nigeria.

A total of 405 Escherichia coli from the chicken production chains in Nigeria were investigated for ESBL-production and 4 isolates were identified as ESBL producers. They were characterized by XbaI-PFGE, multilocus sequence typing (MLST), phylotyping, sequencing of porin and regulatory genes and of the regulatory region of chromosomal ampC genes. Transformed ESBL gene-carrying plasmids were characterized by S1-nuclease, replicon typing, conjugation, digestion and PCRs for detection of the genetic environment of ESBL genes. Susceptibility testing, PCRs for the resistance genes, integrons, and the DNA microarray were performed with both, the original isolates and the transformants. All ESBL-producing isolates harboured blaCTX-M-15 genes located on non-conjugative plasmids (120-155kb). Three isolates with closely related/indistinguishable XbaI-patterns belonged to phylogroup A, and MLST sequence type ST10 and the fourth to phylogroup D and ST405. Resistance to aminoglycosides, sulfonamides/trimethoprim, quinolones, and tetracyclines were seen in all isolates. Incompatibility group IncFIB blaCTX-M-15-carrying plasmids were detected in the three related isolates which carried also a class 1 integron (aadA2-orfF-dfrA12) and the resistance genes blaOXA-1, blaTEM-1, aac(3')-IIa, aac(6')-Ib-cr, sul1, sul2, and tet(A). The IncFIA-IncFIB-IncI1 blaCTX-M-15-carrying plasmid harboured additionally the resistance genes aac(3')-IIa and tet(B). The blaCTX-M-15 genes were associated with ISEcp1 and Δorf477. ESBL-producing isolates showed elevated MICs to cefoxitin (16-64mg/L) and ertapenem MICs (0.5-2.0mg/L) mainly due to alterations in the porin genes. The virulence genes astA and prfB were detected. Although a low prevalence of ESBL-producing isolates was found, co-located resistance genes on the ESBL gene-carrying plasmids may facilitate the dissemination of them.

Ojo OE ，Schwarz S ，Michael GB 《-》

Occurrence and characterisation of ESBL-encoding plasmids among Escherichia coli isolates from fresh vegetables.

Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates have been increasingly reported in different reservoirs. The aims of this study were to investigate the presence of ESBL-producing E. coli in fresh vegetables and to characterise their ESBL gene-carrying plasmids. Among the 245 samples from vegetables investigated during 2011-2013, seven putative ESBL-producing E. coli (salad n = 2, sprouts n = 5) were found. They were subjected to ESBL phenotypic confirmatory tests, detection/sequencing of ESBL genes, antimicrobial susceptibility testing (AST), phylotyping, XbaI-macrorestriction analysis, multilocus sequence typing and transformation. Transformants were characterised by AST, S1-nuclease PFGE, replicon typing, conjugation and investigated for co-located antimicrobial resistance genes. Two ESBL gene-carrying plasmids were sequenced using a HiSeq 2500 system. The seven isolates were confirmed as ESBL producers, displayed unrelated XbaI-patterns and unique sequence types (STs) and belonged to the phylogroups A, B1 or D. The ESBL genes were located on plasmids. Two plasmids carrying blaCTX-M-14 genes (incompatibility group IncK or IncHI2) were seen in isolates from salad (ST973) and sprout (ST527). Two blaCTX-M-15- (IncFIB; non-typeable) and the IncN blaCTX-M-65- and IncHI2 blaCTX-M-125-carrying plasmids were found in isolates from sprouts (ST410, ST847, ST10, ST542). All plasmids were conjugative, except for the IncFIA-FIB blaCTX-M-2-carrying plasmid. Sequence analysis of two plasmids identified the ESBL genes in close location to other resistance genes: sulfonamide resistance gene sul2, streptomycin resistance genes strA and strB, the plasmid-mediated quinolone resistance gene qnrS1 and blaTEM-1 (sul2-strA-strB-IS66-blaTEM-1-tnpR-ΔtnpA-ISEcp1-blaCTX-M-15-Δorf477-ΔtnpA-qnrS1) or the fosfomycin resistance gene fosA3 (ΔISEcp1-blaCTX-M-125-ΔIS903B-fosA3). These observations underline the importance of vegetables as reservoirs for multidrug resistant ESBL-producing E. coli.

Freitag C ，Michael GB ，Li J ，Kadlec K ，Wang Y ，Hassel M ，Schwarz S ... -  《-》

High Heterogeneity of Escherichia coli Sequence Types Harbouring ESBL/AmpC Genes on IncI1 Plasmids in the Colombian Poultry Chain.

Castellanos LR ，Donado-Godoy P ，León M ，Clavijo V ，Arevalo A ，Bernal JF ，Timmerman AJ ，Mevius DJ ，Wagenaar JA ，Hordijk J ... -  《PLoS One》

IncI1 plasmids carrying bla(CTX-M-1) or bla(CMY-2) genes in Escherichia coli from healthy humans and animals in Tunisia.

Ben Sallem R ，Ben Slama K ，Rojo-Bezares B ，Porres-Osante N ，Jouini A ，Klibi N ，Boudabous A ，Sáenz Y ，Torres C ... -  《-》