Showing papers by "Alessandra Carattoli published in 2009"

Resistance Plasmid Families in Enterobacteriaceae

Abstract: Bacteria carry extrachromosomal, self-replicating genetic elements called plasmids. A plasmid is defined as a double-stranded, circular DNA molecule capable of autonomous replication. By definition, plasmids do not carry genes essential for the growth of host cells under nonstressed conditions ([109

Complete nucleotide sequences of plasmids pEK204, pEK499, and pEK516, encoding CTX-M enzymes in three major Escherichia coli lineages from the United Kingdom, all belonging to the international O25:H4-ST131 clone.

Abstract: We determined the complete nucleotide sequences of three plasmids that encode CTX-M extended-spectrum beta-lactamases (ESBLs) in pulsed-field gel electrophoresis-defined United Kingdom variants (strains A, C, and D) of the internationally prevalent Escherichia coli O25:H4-ST131 clone. Plasmid pEK499 (strain A; 117,536 bp) was a fusion of type FII and FIA replicons and harbored the following 10 antibiotic resistance genes conferring resistance to eight antibiotic classes: bla(CTX-M-15), bla(OXA-1), bla(TEM-1,) aac6'-Ib-cr, mph(A), catB4, tet(A), and the integron-borne dfrA7, aadA5, and sulI genes. pEK516 (strain D; 64,471 bp) belonged to incompatibility group IncFII and carried seven antibiotic resistance genes: bla(CTX-M-15), bla(OXA-1), bla(TEM-1), aac6'-Ib-cr, catB4, and tet(A), all as in pEK499. It also carried aac3-IIa, conferring gentamicin resistance, and was highly related to pC15-1a, a plasmid encoding the CTX-M-15 enzyme in Canada. By contrast, pEK204 (strain C; 93,732 bp) belonged to incompatibility group IncI1 and carried only two resistance genes, bla(CTX-M-3) and bla(TEM-1). It probably arose by the transposition of Tn3 and ISEcp1-bla(CTX-M-3) elements into a pCOLIb-P9-like plasmid. We conclude that (i) United Kingdom variants of the successful E. coli ST131 clone have acquired different plasmids encoding CTX-M ESBLs on separate occasions, (ii) the bla(CTX-M-3) and bla(CTX-M-15) genes on pEK204 and pEK499/pEK516 represent separate escape events, and (iii) IncFII plasmids harboring bla(CTX-M-15) have played a crucial role in the global spread of CTX-M-15 ESBLs in E. coli.

Characterization of plasmids harbouring qnrS1, qnrB2 and qnrB19 genes in Salmonella

Abstract: Objectives: The aim of this study was to identify and characterize plasmids carrying qnrS1, qnrB2 and qnrB19 genes identified in Salmonella strains from The Netherlands. The identification of plasmids may help to follow the dissemination of these resistance genes in different countries and environments. Methods: Plasmids from 33 qnr-positive Salmonella strains were transferred to Escherichia coli and analysed by restriction, Southern blot hybridization, PCR and sequencing of resistance determinants. They were also assigned to incompatibility groups by PCR-based replicon typing, including three additional PCR assays for the IncU, IncR and ColE groups. The collection included isolates from humans and one from chicken meat. Results: Five IncN plasmids carrying qnrS1, qnrB2 and qnrB19 genes were identified in Salmonella enterica Bredeney, Typhimurium PT507, Kentucky and Saintpaul. qnrS1 genes were also located on three further plasmid types, belonging to the ColE (in Salmonella Corvallis and Anatum), IncR (in Salmonella Montevideo) and IncHI2 (in Salmonella Stanley) groups. Conclusions: Multiple events of mobilization, transposition and replicon fusion generate the complexity observed in qnr-positive isolates that are emerging worldwide. Despite the fact that the occurrence of qnr genes in bacteria from animals is scarcely reported, these genes are associated with genetic elements and located on plasmids that are recurrent in animal isolates.

Conjugative Transferability of the A/C Plasmids from Salmonella enterica Isolates That Possess or Lack blaCMY in the A/C Plasmid Backbone

Abstract: The objective of this study was to understand the conjugative transmissibility of resistance plasmids present in 205 Salmonella enterica isolates from bovine sources. Polymerase chain reaction (PCR)–based replicon typing was used to type plasmid replicons. Conjugation experiments were preformed in triplicate at 30°C and 37°C on solid medium. PCR mapping of the A/C transfer gene operon was done on 17 Salmonella Newport isolates that were only positive for A/C. Eighty-six percent (n = 177) of the Salmonella isolates were multidrug resistant (MDR) with resistance to 3–12 antimicrobial agents. Of these, 82% (n = 146) were resistant to extended-spectrum cephalosporins and possessed a blaCMY gene. A/C was the predominant replicon detected, present in 90% (n = 160) of the MDR isolates. Twenty-three percent (n = 37) of the A/C-positive strains were positive for a second replicon. Replicons coresident with A/C included I1, N, B/O, HI1, and HI2. Only 31% (n = 54) of the MDR isolates produced transconjugant...

First Report of Plasmid-Mediated Quinolone Resistance Determinant qnrS1 in an Escherichia coli Strain of Animal Origin in Italy

Abstract: A qnrS1-positive strain of Escherichia coli was detected among 73 poultry isolates showing ciprofloxacin MICs of ≥0.125 μg/ml. The qnrS1 gene was associated with a Tn3-like transposon, as previously described to occur in a Salmonella enterica serovar Infantis strain of animal origin, but the plasmid scaffold carrying this element resembled that of a plasmid previously identified in Salmonella enterica serovar Dublin. These elements suggest genetic exchanges among Salmonella and E. coli and a potential animal reservoir for the qnr genes.

Novel genetic environment of plasmid-mediated quinolone resistance gene qnrB2 in Salmonella Bredeney from poultry.

Abstract: Sir, Plasmid-mediated quinolone resistance (PMQR) in Enterobacteriaceae of human origin is an emerging problem in Europe. A variety of qnrA, qnrB and qnrS genes are commonly isolated from Enterobacteriaceae of human origin, and qnr genes have been detected in several Salmonella enterica serotypes in Europe, the USA, Africa, Australia and Asia. However, few reports describe the occurrence of PMQR among bacteria from food-producing animals. – 5 The presence of these genetic traits in bacteria isolated from food-producing animals may constitute a public health concern, since the transfer of resistant bacteria or mobile resistance determinants between animals and humans via the food chain or by direct contact has been suggested. Recently, we reported a Salmonella Bredeney strain 137.25 of animal origin, with reduced susceptibility to fluoroquinolones encoded by a plasmid-mediated qnrB2 gene. This strain was the only qnr-positive strain from animal sources identified among the collection of the Central Veterinary Institute in Lelystad, the Netherlands, which included 15011 Salmonella isolates collected from humans, poultry, pigs, cattle and other sources between 1999 and 2006. This isolate originated from a Dutch broiler chicken, and showed a ciprofloxacin MIC of 0.25 mg/L and a nalidixic acid MIC of 16 mg/L. Since little information is available on strains of animal origin carrying qnr genes, we investigated the genetic environment surrounding the qnrB2 gene in the IncN p137.25 plasmid. PCR assays were initially performed, as previously described, and amplicons of 600, 3000, 2500 and 5700 bp were obtained with the 50CS-30CS, ORF513FW-qnrB2FW, 50CS-ORF513RV and 50CS-qnrB2FW primers and partially sequenced. A class 1 integron carrying the trimethoprim resistance gene dfrA25 as the unique gene cassette was identified and the qnrB2 gene was located adjacent to the 30CS of the integron, within an ISCR1 element. Two PstI fragments of 2200 and 2900 bp containing the qnrB2 gene and the region flanking the ISCR1 were cloned from plasmid p137.25 into the PstI-pZErO-2 kanamycinresistant vector (Invitrogen, Milan, Italy) and fully sequenced (Figure 1; EMBL accession number FJ844401). DNA sequence comparison showed that ISCR1 carried the sapA-like, orf2, qnrB2 and pspF open reading frames, followed by a second copy of the qacED-sul1 30CS (Figure 1). The same structure was previously described for five plasmids of human origin: pSE936/ 05 from Salmonella Enteritidis from Taiwan; pKP1334/05-1 from Klebsiella pneumoniae from Taiwan; a plasmid of Salmonella Keurmassar from France; pJIBE401 from K. pneumoniae from Australia; and pEC-IMPQ from Enterobacter cloacae from Taiwan. – 11 Part of this structure was also identified in pB1004 from Salmonella Bredeney from Spain. Plasmid p137.25 shows a novel genetic environment, which is, however, closely related to that of Salmonella pSE936/05 from Taiwan, since both contain the same class 1 integron carrying the dfrA25 gene (Figure 1). Two open reading frames encoding hypothetical proteins (ORF5 and ORF6) and the insertion element IS6100 were located after the second 30CS of the ISCR1 element. This region has been previously described at the 30-end of several ISCR1 elements, including that located on another IncN plasmid, pKP96, identified in K. pneumoniae isolated in China in 2002 (Figure 1). Plasmid pKP96 differed from p137.25, being positive for qnrA1 instead of qnrB2 and also carrying the aac(60)-Ib-cr gene as the integron-borne gene cassette (EU195449). The genetic environments of the other qnrB2 plasmids were different: pJIBE401 showed the insertion of IS4321-orf97-orf98 and the deletion of orf6; and a second ISCR1 was identified in the plasmid from Salmonella Keurmassar and in pEC-IMPQ (the DNA sequence of this region was not determined for pSE936/05 and pKP1334/05-1 plasmids). In the pEC-IMPQ plasmid, the acquisition of the qnrB2 gene occurred by integration of the ISCR1 rolling circular intermediate of replication, carrying the qnrB2-qacED1-sul1 resistance genes, into the pEC-IMP highly related plasmid. The qnrB2 gene was identified on an IncFII plasmid carrying the blaCTX-M-15 extended-spectrum b-lactamase gene in Escherichia coli isolated from a dog in Portugal. Plasmids carrying blaCTX-M-15 and belonging to the IncFII group have been reported worldwide in E. coli of human origin, but none of these plasmids carried the qnrB2 gene. This evidence suggests that the acquisition of this resistance determinant might have occurred in Enterobacteriaceae originating from an animal host. The presence of the qnrB2 gene in animal isolates and zoonotic pathogens opens the possibility that genetic exchange and plasmid acquisition of the qnrB2 gene could occur in the faecal flora of the animals. Interestingly, p137.25 belongs to the IncN plasmid family that is able to replicate in different enterobacterial strains, but also seems prevalent in faecal flora from animals. In fact, a study performed on a large collection of E. coli from the USA demonstrated that the prevalence of IncN plasmids is high in avian E. coli (10%–16%) but negative in E. coli from faeces of healthy humans. This evidence supports the hypothesis that the Salmonella 137.25 strain acquired the qnrB2 gene on an IncN plasmid circulating in avian bacterial flora. This strain could cause infections in humans through the food chain Journal of Antimicrobial Chemotherapy doi:10.1093/jac/dkp356