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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.

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.

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.

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.

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.)

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.

Summary: Objectives: To investigate the epidemiology and clinical features of infections caused by Enterobacteria producing plasmid-mediated AmpC β-lactamases (pAmpC), which are emerging as a cause of resistance to extended-spectrum cephalosporins. Methods: A prospective multicentre cohort of patients with infection/colonisation due to pAmpC-producing Enterobacteriaceae was performed in 7 Spanish hospitals from February throughout July 2009. pAmpCs were characterised by PCR and sequencing. Results: 140 patients were included; organisms isolated were Escherichia coli (n = 100), Proteus mirabilis (n = 20), Klebsiella pneumoniae (n = 17), and others (n = 3). Overall, 90% had a chronic underlying condition. The acquisition was nosocomial in 43%, healthcare-associated in 41% (14% of those were nursing home residents), and community in 16%. Only 5% of patients had no predisposing feature for infection with multidrug-resistant bacteria. Nineteen percent of patients were bacteraemic. Inappropriate empirical therapy was administered to 81% of bacteraemic patients, who had a crude mortality rate of 48%. The most frequent enzyme was CMY-2 (70%, predominantly in E. coli and P. mirabilis) followed by DHA-1 (19%, predominantly in K. pneumoniae). Conclusion: pAmpC-producing Enterobacteriaceae caused nosocomial, healthcare-associated and community infections mainly in predisposed patients. Invasive infections were associated with high mortality which might be partly related to inappropriate empirical therapy. [Copyright &y& Elsevier]

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 β-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 blaDHA-1 and qnrB genes on the same IncL/M-MOBP13 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 β-lactams. The presence of both resistance genes on the same plasmid and the reported increase in PMQR could perhaps explain the widespread distribution of blaDHA-1 genes. [ABSTRACT FROM AUTHOR]

Escherichia coli and Salmonella enterica isolates carrying the blaCTX-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 blaCTX-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 blaCTX-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. [ABSTRACT FROM AUTHOR]

Investigation of an acute gastroenteritis outbreak involving >100 persons at a summer camp in Girona, Spain, in June 2002 led to the detection of Salmonella enterica and extended-spectrum cephalosporin-resistant Escherichia coil (ESCREC). Stool cultures were performed for 22 symptomatic campers, three asymptomatic food handlers, and 10 healthy household members. Of the 22 campers, 19 had Salmonella enterica, 9 had an ESCREC strain carrying an extended-spectrum β-lactamase, and 2 had a second ESCREC strain carrying a plasmidic cephamycinase. Related ESCREC were detected in two (salmonella-negative) asymptomatic food handlers and in none of the healthy household members. Fecal ESCREC and its β-lactamases and plasmids were extensively characterized. Three of the five ESCREC clones were recovered from multiple hosts. The apparent dissemination of ESCREC suggests a food or water vehicle. The observed distribution of resistance plasmids and β-lactamase genes in several clones indicates a high degree of horizontal transfer. Heightened vigilance and increased efforts must be made to discover the reservoirs and vehicles for community dissemination of ESCREC. [ABSTRACT FROM AUTHOR]

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 blaNDM-7 gene was located on a 50 kb IncX4 plasmid (pEc188-NDM7), both in the original and transconjugant strains. In addition, blaCTX-M-15 was located on a 150 kb IncFIA plasmid and blaCMY-2 on a 95 kb undetermined plasmid type, only in the wild-type strain. The immediate genetic surroundings of blaNDM-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. [ABSTRACT FROM AUTHOR]

Escherichia coli recovered from three hospitals in Barcelona (Spain) were studied to determine the prevalence of isolates with acquired AmpC (ac-AmpC) and/or overproduced chromosomal AmpC (c-AmpC). Mechanisms involved in bla c-AmpC overexpression, bla ac-AmpC and the plasmids associated with their distribution as well as the prevalence of plasmid-mediated quinolone resistance (PMQR) in AmpC-producing isolates were also determined. Isolates were selected according to their resistance phenotype. bla ac-AmpC , alterations in the bla c-AmpC promoter/attenuator, and PMQR genes [ qnrA , qnrB , qnrS , aac(6′)-Ib -cr and qepA ] were characterised by PCR and sequencing. bla c-AmpC expression was determined by qRT-PCR. Population structure analysis was performed using PFGE, MLST and phylogenetic group PCR. Plasmids carrying bla ac-AmpC were characterised by PCR-based replicon typing and S1-PFGE. IncI1 and IncF plasmids were also analysed by plasmid MLST and replicon sequence typing, respectively. Among 21 563 E. coli isolates, 240 (1.1%) overproduced AmpC β-lactamases, including 180 (75.0%) harbouring ac-AmpC (132 CMY-2 variants and 48 DHA-1) and 60 (25.0%) c-AmpC enzymes. Three mutation profiles in the bla c-AmpC promoter/attenuator were associated with a 72.5-, 19.9- and 5.8-fold increased expression, respectively. Moreover, 63.3% of ac-AmpC and 43.3% of c-AmpC isolates belonged to B2, D, E or F phylogenetic groups. PMQR was found in 31% of ac-AmpC isolates [38 qnrB4 , 8 aac(6′)-Ib -cr, 6 qnrS1 and 3 qnrB19 ] and in 10% of c-AmpC isolates [5 aac(6′)-Ib -cr and 1 qnrS1 ]. IncI1-ST12 and IncF were associated with bla CMY-2 and bla DHA-1 , respectively. These results suggest that ac-AmpC β-lactamases were the main mechanism of AmpC production. Isolates and plasmids both showed high genetic diversity. [ABSTRACT FROM AUTHOR]