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In this study, the plasmid content of clinical and commensal strains was analyzed and compared. The replicon profile was similar in both populations, except for L, M, A/C, and N (detected only in clinical strains) and HI1 (only in commensal strains). Although I1 and F were the most frequent replicons, only IncI1, sequence type 12 (ST12) was associated with bla in both populations. In contrast, the widespread resistant IncF plasmids were not linked to a single epidemic plasmid.CMY-2 in both populations. In contrast, the widespread resistant IncF plasmids were not linked to a single epidemic plasmid., (Copyright © 2020 American Society for Microbiology.)

This study describes the molecular characterization of an NDM-7 carbapenemase-producing Escherichia coli strain Ec188, recovered from a rectal swab of a male patient who had travelled to Pakistan before his hospitalization at the Hospital del Mar in Barcelona, Spain. The Ec188 isolate, assigned to a new multilocus sequence type ST679, was resistant to all beta-lactams, aminoglycosides (gentamicin, tobramycin, and with reduced susceptibility to amikacin), and ciprofloxacin. The bla NDM-7 gene was located on a 50 kb IncX4 plasmid (pEc188-NDM7), both in the original and transconjugant strains. In addition, bla CTX-M-15 was located on a 150 kb IncFIA plasmid and bla CMY-2 on a 95 kb undetermined plasmid type, only in the wild-type strain. The immediate genetic surroundings of bla NDM-7 included the bleo, trpf, and dsbC genes, and it was flanked by the insertion sequences IS26 and ISAba125, which appeared interrupted by IS5. The res and parA genes were found in the same orientation downstream of the IS26 element. To our knowledge, this is the first report of an NDM-7-carbapenemase carried on an IncX4 plasmid, as well as the first E. coli strain belonging to ST679 harboring an NDM β-lactamase, possibly associated with previous travel to Pakistan. In addition, this study highlights the dissemination of NDM variants accompanied by IncX-type plasmids.

We examined the genetic context of 74 acquired ampC genes and 17 carbapenemase genes from 85 of 640 Enterobacteriaceae isolates collected in 2009. Using S1 pulsed-field gel electrophoresis and Southern hybridization, 37 of 74 bla AmpC genes were located on large plasmids of different sizes belonging to six incompatibility groups. We used sequencing and PCR mapping to investigate the regions flanking the acquired ampC genes. The bla CMY-2-like genes were associated with ISEcp1; the surrounding bla DHA genes were similar to Klebsiella pneumoniae plasmid pTN60013 associated with IS26 and the psp and sap operons; and the bla ACC-1 genes were associated with IS26 elements inserted into ISEcp1. All of the carbapenemase genes (bla VIM-1, bla IMP-22, and bla IMP-28) were located in class 1 integrons. Therefore, although plasmids are the main cause of the rapid dissemination of ampC genes among Enterobacteriaceae, we need to be aware that other mobile genetic elements, such as insertion sequences, transposons, or integrons, can be involved in the mobilization of these genes of chromosomal origin. Additionally, three new integrons (In846 to In848) are described in this study., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

A total of 788 clinical Enterobacteriaceae were collected to describe the aminoglycoside-modifying genes (AME genes) and to characterize the plasmids that carry these genes. Among the 788 strains collected, 330 (41.8%) were aminoglycoside-resistant: 264 Escherichia coli (80%), 33 Proteus mirabilis (10%), 10 Klebsiella pneumoniae (3%), six K. oxytoca (1.8%), five Enterobacter cloacae (1.5%), three Morganella morganii (0.9%), three Providencia stuartii (0.9%), two Salmonella enterica (0.6%), and one each Citrobacter freundii, C. koseri, Proteus vulgaris, and Shigella sonnei. The most affected aminoglycoside was streptomycin (92.7%), followed by kanamycin (26.3%), gentamicin (18%), tobramycin (16.9%), netilmicin (3.6%), and amikacin (1.5%). The AME genes found were aph(3″)-Ib (65.4%), ant(3″)-Ia (37.5%), aph(3')-Ia (13.9%), aac(3)-IIa (12.4%), aac(6')-Ib (4.2%), ant(2″)-Ia (3.6%), and aph(3')-IIa (1.2%). Thirty-four percent of the strains showed more than one enzyme. The most frequent association was ant(3″)-Ia plus aph(3″)-Ib (35 strains). From 66 selected AME genes, 24 were plasmid located: 12 aac(3)-IIa, six aph(3')-Ia, three ant(3″)-Ia, two ant(2″)-Ia, and one aac(6')-Ib. These genes were located in plasmids belonging to incompatibility groups F, FIA, FIB, or HI2. In conclusion, the AME genes involved in aminoglycoside-clinical resistance were aac(3)-IIa, aac(6')-Ib, and ant(2″)-Ia, genes that confer resistance to tobramycin, gentamicin, and amikacin.

Objectives: To gain insights into ampC transmission between bacterial strains., Methods: We examined the genetic context of 117 acquired ampC genes from 27119 Enterobacteriaceae collected between 1999 and 2007. Plasmid analysis was carried out by PCR-based replicon or relaxase typing, S1-PFGE and Southern hybridization. I-CeuI/PFGE was used for isolates not characterized by plasmid analysis. PCR reactions were used to map the genetic organization of the ampC genes., Results: Among the isolates studied, 81.2% of ampC genes were located on plasmids of known Inc/MOB groups, 7.7% were chromosomally located and 11.1% were not determined. A/C, I1 and K were the most commonly found replicons in plasmids carrying bla(CMY-2), while L/M replicons were associated with bla(DHA-1). bla(ACC-1) was linked to I1 and MOB(F11) plasmids; bla(CMY-27) was associated with IncF and MOB(P12) plasmids; the plasmid carrying bla(CMY-25) could not be typed, and bla(CMY-40) was chromosomally located. All 87 isolates carrying bla(CMY-2), bla(CMY-4), bla(CMY-25), bla(CMY-27), bla(CMY-40) or bla(ACC-1) displayed the transposon-like structures ISEcp1/ΔISEcp1-bla(CMY)-blc-sugE or ΔISEcp1-bla(ACC-1)-gdha. The most prevalent structure in bla(DHA-1) (93.3% of cases) was identical to that described in the Klebsiella pneumoniae pTN60013 plasmid. Remarkably, in three isolates containing chromosomal bla(CMY-2), this gene was mobilized by conjugation., Conclusions: Although plasmids are the main cause of the rapid dissemination of ampC genes among bacteria, we need to be aware that other mobile genetic elements such as integrative and conjugative elements (ICEs) can be involved in the mobilization of these genes.

A collection of 30 DHA-1-Enterobacteriaceae producers was examined for the presence of qnr genes. PCR-based replicon typing, plasmid profile and Southern hybridisation analyses revealed that all isolates co-harboured bla(DHA-1) and qnrB genes on the same plasmid. All but one of these plasmids belonged to the L/M group. Genetic organization analyses of a randomly selected isolate revealed the co-localization of both genes on an IS26-composite transposon. As plasmids carrying both genes seem to have a high prevalence and a worldwide distribution, care should be taken when quinolones are used to treat infections caused by DHA-1 producers., (© 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.)

Objectives: To characterize the vectors involved in the dissemination of bla(CMY-2) genes in clinical isolates of Proteus mirabilis collected between 1999 and 2007., Methods: Plasmid analysis of 19 P. mirabilis carrying ampC genes was performed by PCR-based replicon typing, S1-PFGE and Southern hybridization with ampC and replicon probes. Isolates that could not be characterized were examined for the presence of SXT/R391-like elements. To demonstrate the involvement of these elements in the dissemination of bla(CMY-2), we performed a PCR amplification of the integrase (int) and toxin/antitoxin (TA) genes from SXT/R391-like integrative conjugative elements (ICEs). Later on, I-Ceu-I PFGE gels and hybridization with bla(CMY-2), int and prfC probes were performed. The genetic organization of bla(CMY-2) was also studied., Results: ampC genes were located on large conjugative plasmids in 11 of the 19 (58%) P. mirabilis studied. However, in eight of these isolates a plasmid was not involved in the mobilization of ampC genes. I-Ceu-I PFGE and hybridization analyses revealed that bla(CMY-2) were chromosomally located in these eight P. mirabilis isolates. The genetic organization of bla(CMY-2) and hybridization analyses revealed that bla(CMY-2) was carried by an ICE almost identical to ICEPmiJpan1 in seven out of these eight isolates., Conclusions: The prevalence of ICEs carrying bla(CMY-2) was surprisingly high [37% (7 out of 19)]. This is the first study giving prevalence data on ICEs carrying bla(CMY-2) genes. These results suggest the need to study these mobile genetic elements in the context of dissemination of acquired AmpC β-lactamases and also of other β-lactamases, such as extended-spectrum β-lactamases and carbapenemases.

The localisation and genetic organisation of bla(CTX-M-15) were studied in 37 CTX-M-15-producing Klebsiella pneumoniae isolates collected from 2005 to 2008 within the Barcelona metropolitan area. Polymerase chain reaction (PCR)-based replicon typing and Southern hybridisations were used to identify the bla(CTX-M-15) location. The genetic environment was analysed by PCR mapping and sequencing, and transferability of bla(CTX-M-15) was evaluated by conjugation and transformation assays. The majority of the 37 isolates carried bla(CTX-M-15) in a plasmid location, frequently associated with the aac(6')-Ib-cr gene. Plasmids encoding bla(CTX-M-15) carried three distinct replicons, i.e. IncFII, IncR and IncFIIk, the latter two not having been described previously in association with bla(CTX-M-15). Several of these plasmids were not self-transferable. Furthermore, in all isolates belonging to sequence type ST-1, bla(CTX-M-15) was found integrated into the K. pneumoniae chromosome. In all the studied isolates, the mobile element ISEcp1 was found upstream of bla(CTX-M-15), whereas IS26 was found inserted within ISEcp1 in several isolates, in previously unreported positions. In conclusion, these findings indicate that among K. pneumoniae strains isolated in the Barcelona metropolitan area, bla(CTX-M-15) is associated with diverse genetic elements, including the IncR and IncFIIk replicons, as reported for the first time here, and the chromosome., ((c) 2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

We report a Serratia marcescens and an Escherichia coli isolate simultaneously detected in the same patient. Both isolates showed susceptibility patterns suggestive of harbouring a plasmid-mediated AmpC beta-lactamase (pACBL) and a plasmid-encoded quinolone resistance (PMQR). PCR-based replicon, MOB typing, plasmid profile and Southern hybridization analyses revealed that both isolates coharboured bla(DHA-1) and qnrB genes on the same IncL/M-MOB(P13) plasmid approximately 70 kb in size. Together with the fact that both plasmids were conjugative in the laboratory, these results strongly suggest that a horizontal transfer event could take place in vivo. This is the first report of an isolate of S. marcescens harbouring a pACBL. The only phenotypic method that suggests the presence of a pACBL in an isolate harbouring an inducible chromosomal AmpC enzyme is the observation of scattered colonies near the edge of the inhibition zones of some beta-lactams. The presence of both resistance genes on the same plasmid and the reported increase in PMQR could perhaps explain the widespread distribution of bla(DHA-1) genes.

A qnrS2 gene was identified in an Aeromonas caviae isolate (MICs of ciprofloxacin, norfloxacin and ofloxacin >32 mg/L) from a stool sample collected from a patient with gastroenteritis. The analysis of the gyrA and parC genes revealed amino acid substitutions Ser83-Ile and Ser80-Thr, respectively. In addition, five out of 41 nalidixic acid-resistant Aeromonas isolates studied (26 identified as Aeromonas veronii bv sobria and 15 identified as A. caviae) showed ciprofloxacin resistance. The identification of plasmid-mediated qnr genes outside of the Enterobacteriaceae underlines a possible diffusion of these resistance determinants among Gram-negative rods. This emphasizes the importance of monitoring the emergence of these determinants as well as their dissemination among the Aeromonadaceae.

Objectives: We describe 12 VIM-1-producing strains (7 Enterobacter cloacae, 2 Klebsiella pneumoniae and 3 clonal Klebsiella oxytoca strains) detected among clinically relevant Enterobacteriaceae isolates from routine cultures at the Hospital del Mar (Barcelona, Spain) from December 2006 to May 2007., Methods: Susceptibility to carbapenems was evaluated with the MicroScan system. beta-Lactamases were identified by PCR and sequencing. Clonal relationships between the isolates were analysed by PFGE. Transferability of the enzymes was tested by conjugation. Plasmid characterization was performed by PCR-based replicon typing and PFGE with S1 nuclease digestion of whole genomic DNA. The PFGE gels were then transferred and hybridized., Results: The disc diffusion method correctly identified five of the seven E. cloacae isolates as intermediate or resistant strains. All isolates produced the VIM-1 enzyme. Three E. cloacae and three K. oxytoca strains were also CTX-M-9-producing strains, and one E. cloacae was also a CTX-M-3-producing strain. The plasmids carrying the bla(VIM) gene, of unknown incompatibility group, had a size of approximately 75 kb (eight strains) or 40 kb (three strains) and also contained the qnrS and the aac(6')-Ib-cr genes. In the remaining strain the bla(VIM-1) gene was found in an HI2 plasmid of 290 kb together with bla(CTX-M-9), qnrA, qnrS and the aac(6')-Ib-cr genes., Conclusions: The results showed a linkage between the bla(VIM-1) and the qnrS and the aac(6')-Ib-cr genes, and between the bla(CTX-M-9) and the qnrA genes.

Escherichia coli and Salmonella enterica isolates carrying the bla(CTX-M-9) gene located on plasmids prevailed at the Hospital de la Santa Creu i Sant Pau, Barcelona, Spain in the 1996-1999 period. The bla(CTX-M-9)-carrying plasmids showed a great variability in size, suggesting the mobilization of the gene among different plasmid scaffolds. The aim of the present work was to identify and better characterize the plasmids involved in the spread of the bla(CTX-M-9) gene. Results showed that the majority of these strains carried plasmids belonging to the IncHI2 incompatibility group. The IncHI2 plasmids were further characterized, and found to be related to the reference IncHI2 plasmid R478.

• Plasmid conjugation of tet (O) carrying plasmids in Campylobacte r is thermoregulated. • Conjugation of tet (O) carrying plasmids is promoted at high temperature. • Transcriptional expression of putative conjugative genes is temperature dependent. Campylobacter is the causal agent of campylobacteriosis in humans, a self-limiting gastroenteritis. Campylobacteriosis is a zoonosis, commonly transmitted from contaminated chicken meat by either direct consumption or cross contamination during food manipulation. Presence of plasmids encoding for resistance to antibiotics such as tetracycline is common among Campylobacter isolates. In this report, we studied the effect of the temperature in the conjugation frequency of several tet (O) carrying plasmids, providing tetracycline resistance to the recipient cells. The conjugation frequency from donor cells carrying three previously characterized plasmids (pCjA13, pCjA9 and pTet) and from two clinical isolates was determined. Two temperatures, 37 and 42 °C, mimicking the conditions encountered by C. jejuni in the human and broiler chicken gastrointestinal tracts, respectively, were assessed. Our results clearly indicate that the conjugation process is promoted at high temperature. Accordingly, the transcriptional expression of some putative conjugative apparatus genes is thermoregulated, being induced at 42 °C. The two plasmids present in the clinical isolates were sequenced and assembled. Both plasmids are highly related among them and to the pTet plasmid. The high identity of the genes putatively involved in the conjugation process among the plasmids is in agreement with the similar behavior regarding the temperature dependency of the conjugative process. This report suggest that conjugation of plasmids carrying antibiotic resistance genes occurs preferentially at temperatures that resemble the gastrointestinal tract of birds, the main reservoir of C. jejuni. [ABSTRACT FROM AUTHOR]