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The article presents a report on emergence of Escherichia coli co-producing New Delhi metallo-b-lactamase-1(NDM-1) and Klebsiella pneumoniae carbapenemase 2 (KPC-2) from a retail vegetable, China. Topics discussed include use of agar dilution method for determining of minimum inhibitory concentration of antimicrobial agents; its resistance to imipenem, ceftazidime, cefotaxime; and a table depicting characteristics of E. coli TS79e and its transconjugants.

The purpose of this study was to examine the occurrence of fosfomycin-resistant Escherichia coli from chickens and to characterize the plasmids carrying fosA3. A total of 661 E. coli isolates of chicken origin collected from 2009 to 2011 were screened for plasmid-mediated fosfomycin resistance determinants by PCR. Plasmids were characterized using PCR-based replicon typing, plasmid multilocus sequence typing, and restriction fragment length polymorphisms. Associated addiction systems and resistance genes were identified by PCR. PCR-mapping was used for analysis of the genetic context of fosA3. Fosfomycin resistance was detected in 58 isolates that also carried the fosA3 gene. Fifty-seven, 17, and 52 FosA3-producers also harbored blaCTX-M, rmtB, and floR genes, respectively. Most of the 58 fosA3-carrying isolates were clonally unrelated, and all fosA3 genes were located on plasmids belonged to F33:A-:B- (n = 18), IncN-F33:A-: B- (n = 7), IncHI2/ST3 (n = 10), IncI1/ST71 (n = 3), IncI1/ST108 (n = 3), and others. The genetic structures, IS26-ISEcp1-blaCTX M bla -55-orf477TEM-1-IS26-fosA -1758bp-IS26 and ISEcp1-blaCTX-M-65-IS903-iroN-IS26-fosA3-536bp-IS26 were located on highly similar F33:A-:B- plasmids. In addition, blaCTX-M-14-fosA3-IS26 was frequently present on similar IncHI2/ST3 plasmids. IncFII plasmids had a significantly higher frequency of addiction systems (mean 3.5) than other plasmids. Our results showed a surprisingly high prevalence of fosA3 gene in E. coli isolates recovered from chicken in China. The spread of fosA3 can be attributed to horizontal dissemination of several epidemic plasmids, especially F33:A-:B- plasmids. Since coselection by other antimicrobials is the major driving force for the diffusion of the fosA3 gene, a strict antibiotic use policy is urgently needed in China. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the trends and the diversity of CTX-M types of extended-spectrum β-lactamase (ESBL) in Escherichia coli isolated from food animals in China over a ten-year period. From 2003 to 2012, 2815 E. coli isolates collected from diseased animals (chickens, pigs, and waterfowl) were screened for the prevalence of CTX-M genes. CTX-M-positive isolates were tested for their susceptibilities to 10 antimicrobial agents and the clonal relationship of CTX-M-producing E. coli isolates was also assessed. Overall, 677 (20.1%) of the 2815 E. coli isolates carried CTX-M genes. Eighteen different types of CTX-M ESBLs were identified, with CTX-M-14, CTX-M-55, and CTX-M-65 being the most dominant genotypes. The occurrence of CTX-M-producing E. coli increased significantly from 5.7% in 2003-2005 to 35.3% in 2009-2012 (p < 0.0001). High genetic heterogeneities were observed in the CTX-M-producing E. coli isolates. Most CTX-M-producing strains were also resistant to other classes of antimicrobials. Compared to isolates carrying CTX-M-9 subgroup of ESBLs, isolates carrying CTX-M-1 subgroup ESBLs showed significantly higher resistance rates to ceftazidime, amikacin, and fosfomycin (p < 0.01). The study reported the dramatic increase of CTX-M ESBLs in E. coli isolated from animals overtime in China. The increasing incidence of CTX-M-55 with high hydrolytic activity against ceftazidime and the widely spread co-resistance in CTX-M-producing isolates alarm the serious antimicrobial resistance situation in China and highlight the need for urgent control strategies to limit the dissemination of those resistant genes in China. [ABSTRACT FROM AUTHOR]

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A gene encoding a putative multicopper oxidase (MCO) was cloned from the soil bacterium Klebsiella sp. 601 and its corresponding enzyme was overexpressed in an Escherichia coli strain. Klebsiella sp. 601 MCO is composed of 536 amino acids with a molecular mass of 58.2 kDa. Theoretical calculation gave a pI value of 6.11. The amino acid sequence of Klebsiella sp. 601 MCO is strongly homologous to that of E. coli CueO with a similarity of 90% and an identity of 78%. Unlike E. coli CueO, Klebsiella sp. 601 MCO contains an extra 20 amino acids close to its C-terminus. The enzyme was purified to homogeneity by Ni-affinity chromatography. The purified enzyme was capable of using DMP (2,6-dimethoxyphenol), ABTS (2,2′-azino-bis(3-ethylbenzthiazolinesulfonic acid)), and SGZ (syringaldazine) as substrates with an optimal pH of 8.0 for DMP, 3.0 for ABTS, and 7.0 for SGZ. Klebsiella sp. 601 MCO was quite stable at pH 7.0 in which its activity was constant for 25 h without any significant change. Kinetic studies gave Km, kcat, and kcat//Km values of 0.49 mmol/L, 1.08 × 103 s-1, and 2.23 × 103 s-1·mmol/L-1, respectively, for DMP, 5.63 mmol/L, 6.64 × 103 s-1, and 1.18 × 103 s-1·mmol/L-1 for ABTS, and 0.023 mmol/L, 11 s-1, and 4.68 × 102 s-1·mmol/L-1 for SGZ. Un gène codant une oxydase multi-cuivre (OMC) présumée a été cloné chez la bactérie du sol Klebsiella sp. 601 et l’enzyme correspondante a été surexprimée chez Escherichia coli. L’OMC de Klebsiella sp. 601 est composée de 536 acides aminés et possède une masse moléculaire de 58,2 kDa. Selon un calcul théorique, la valeur de pI est de 6,11. La séquence en acides aminés de l’OMC de Klebsiella sp. 601 est fortement homologue à la séquence de CueO de E. coli, avec une similarité de 90 % et une identité de 78 %. Contrairement à la CueO d’E. coli, l’OMC de Klebsiella sp. 601 contient 20 acides aminés supplémentaires à proximité de l’extrémité C-terminale. L’enzyme a été purifiée à homogénéité par chromatographie d’affinité sur Ni. L’enzyme purifiée pouvait utiliser le DMP (2,6-dimethoxyphenol), l’ABTS (2,2′-azino-bis(3-ethylbenzthiazolinesulfonic acid)) et le SGZ (syringaldazine) comme substrats avec un pH optimal de 8,0 pour le DMP, de 3,0 pour l’ABTS et de 7,0 pour le SGZ. L’OMC de Klebsiella sp. 601 était relativement stable à pH 7,0 où son activité était constante pendant 25 h, sans changement significatif. Des études cinétiques ont généré des valeurs de Km, kcat et Km/kcat de 0,49 mmol/L, 1,08 × 103 s-1 et 2,23 × 103 s-1·mmol/L-1 respectivement pour le DMP, 5,63 mmol/L, 6,64 103 s-1 et 1,18 × 103 s-1·mmol/L-1 pour l’ABTS et de 0,023 mmol/L, 11 s-1 et 4,68 × 102 s-1·mmol/L-1 pour le SGZ. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]