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July 7, 2019  |  

Stability of the encoding plasmids and surface expression of CS6 differs in enterotoxigenic Escherichia coli (ETEC) encoding Different heat-stable (ST) enterotoxins (STh and STp).

Enterotoxigenic Escherichia coli (ETEC), one of the most common reasons of diarrhea among infants and children in developing countries, causes disease by expression of either or both of the enterotoxins heat-labile (LT) and heat-stable (ST; divided into human-type [STh] and porcine-type [STp] variants), and colonization factors (CFs) among which CS6 is one of the most prevalent ETEC CFs. In this study we show that ETEC isolates expressing CS6+STh have higher copy numbers of the cssABCD operon encoding CS6 than those expressing CS6+STp. Long term cultivation of up to ten over-night passages of ETEC isolates harboring CS6+STh (n = 10) or CS6+STp (n = 15) showed instability of phenotypic expression of CS6 in a majority of the CS6+STp isolates, whereas most of the CS6+STh isolates retained CS6 expression. The observed instability was a correlated with loss of genes cssA and cssD as examined by PCR. Mobilization of the CS6 plasmid from an unstable CS6+STp isolate into a laboratory E. coli strain resulted in loss of the plasmid after a single over-night passage whereas the plasmid from an CS6+STh strain was retained in the laboratory strain during 10 passages. A sequence comparison between the CS6 plasmids from a stable and an unstable ETEC isolate revealed that genes necessary for plasmid stabilization, for example pemI, pemK, stbA, stbB and parM, were not present in the unstable ETEC isolate. Our results indicate that stable retention of CS6 may in part be affected by the stability of the plasmid on which both CS6 and STp or STh are located.


July 7, 2019  |  

Characterization of an IncA/C multidrug resistance plasmid in Vibrio alginolyticus.

Cephalosporin-resistant Vibrio alginolyticus were firstly isolated from food products with ß-lactamases, blaPER-1, blaVEB-1 and blaCMY-2, being the major mechanisms mediating cephalosporin resistance. The complete sequence of a multidrug resistance plasmid, pVAS3-1, harboring the blaCMY-2 and qnrVC4 genes was decoded in this study. Its backbone exhibited genetic homology to known IncA/C plasmids recoverable from Enterobacteriaceae species, suggesting its possible origin from Enterobacteriaceae. Copyright © 2016, American Society for Microbiology. All Rights Reserved.


July 7, 2019  |  

Dissemination of the mcr-1 colistin resistance gene.

Since our first report on plasmid- mediated colistin resistance gene mcr-1,1 strains previously collected in seven countries (Denmark, the Netherlands, Laos, Nigeria, Thailand, France, and the UK) have been found to carry mcr-1.2–6 Furthermore, the sequences in GenBank show that mcr-1 might also be circulating in Portugal and Malaysia. The earliest mcr-1- positive strain was collected from cattle in France in 2008 (GenBank accession number LMBK01000308). These findings confirm our initial concern that mcr-1 could have already disseminated worldwide.


July 7, 2019  |  

Mechanisms involved in acquisition of blaNDM genes by IncA/C2 and IncFIIY plasmids.

blaNDM genes confer carbapenem resistance and have been identified on transferable plasmids belonging to different incompatibility (Inc) groups. Here we present the complete sequences of four plasmids carrying a blaNDM gene, pKP1-NDM-1, pEC2-NDM-3, pECL3-NDM-1 and pEC4-NDM-6, from four clinical samples originating from four different patients. Different plasmids carry segments that align to different parts of the blaNDM region found on Acinetobacter plasmids. pKP1-NDM-1 and pEC2-NDM-3, from Klebsiella pneumoniae and Escherichia coli, respectively, were identified as type 1 IncA/C2 plasmids with almost identical backbones. Different regions carrying blaNDM are inserted in different locations in the antibiotic resistance island known as ARI-A and ISCR1 may have been involved in acquisition of blaNDM-3 by pEC2-NDM-3. pECL3-NDM-1 and pEC4-NDM-6, from Enterobacter cloacae and E. coli, respectively, have similar IncFIIY backbones but different regions carrying blaNDM are found in different locations. Tn3-derived Inverted-repeat Transposable Elements (TIME) appear to have been involved in acquisition of blaNDM-6 by pEC4-NDM-6 and the rmtC 16S rRNA methylase gene by IncFIIY plasmids. Characterisation of these plasmids further demonstrates that even very closely related plasmids may have acquired blaNDM genes by different mechanisms. These findings also illustrate the complex relationships between antimicrobial resistance genes, transposable elements and plasmids and provide insights into the possible routes for transmission of blaNDM genes amongst species of the Enterobacteriaceae family. Copyright © 2016, American Society for Microbiology. All Rights Reserved.


July 7, 2019  |  

Complete nucleotide sequence of pH11, an IncHI2 plasmid conferring multi-antibiotic resistance and multi-heavy metal resistance genes in a clinical Klebsiella pneumoniae isolate.

The complete 284,628bp sequence of pH11, an IncHI2 plasmid, was determined through single-molecule, real-time (SMRT) sequencing. Harbored by a clinical Klebsiella pneumoniae strain H11, and isolated in Beijing, this plasmid contains multiple antibiotic resistance genes, including catA2, aac(6′)-Ib, strB, strA, dfrA19, blaTEM-1, blaSHV-12, sul1, qacE delta 1, ereA, arr2, and aac3. The aac(6′)-Ib is carried by a class I integron. Plasmid pH11 also carries several genes associated with resistance to heavy metals, such as tellurium, mercury, cobalt, zinc, nickel, copper, lead and cadmium. This plasmid exhibits numerous characteristics, including HipBA and RelBE toxin-antitoxin systems, two major transfer (Tra) regions closely related to those of Salmonella enterica serovar plasmid pRH-R27, a type II restriction modification system (EcoRII R-M system), several methyltransferases and methylases and genes encoding Hha and StpA. These characteristics suggest that pH11 may adapt to various hosts and environments. Multiple insertion sequence elements, transposases, recombinases, resolvases and integrases are scattered throughout pH11. The presence of these genes may indicate that horizontal gene transfer occurs frequently in pH11 and thus may facilitate the dissemination of antimicrobial resistance determinants. Our data suggest that pH11 is a chimera gradually assembled through the integration of different horizontally acquired DNA segments via transposition or homologous recombination. Copyright © 2016 Elsevier Inc. All rights reserved.


July 7, 2019  |  

New Delhi metallo-ß-lactamase-1-producing Klebsiella pneumoniae, Florida, USA(1).

New Delhi metallo-ß-lactamase (NDM)–producing Enterobacteriaceae have swiftly spread worldwide since an initial report in 2008 from a patient who had been transferred from India back home to Sweden (1). Epidemiologically, the global diffusion of NDM-1 producers has been associated with the Indian subcontinent and the Balkan region, which are considered the primary and secondary reservoirs of these pathogens, respectively (1). However, recent reports suggest that countries in the Middle East may constitute another potential reservoir for NDM-1 producers (1). More than 100 NDM-producing isolates have been reported in the United States, most of which were associated with recent travel from the Indian subcontinent (2,3). We report an NDM-1–producing Klebsiella pneumoniae strain that was recovered from a patient who had been transferred from Iran to a hospital in Florida, United States.


July 7, 2019  |  

Survival and evolution of a large multidrug resistance plasmid in new clinical bacterial hosts.

Large conjugative plasmids are important drivers of bacterial evolution and contribute significantly to the dissemination of antibiotic resistance. Although plasmid borne multidrug resistance is recognized as one of the main challenges in modern medicine, the adaptive forces shaping the evolution of these plasmids within pathogenic hosts are poorly understood. Here we study plasmid-host adaptations following transfer of a 73?kb conjugative multidrug resistance plasmid to naïve clinical isolates of Klebsiella pneumoniae and Escherichia coli. We use experimental evolution, mathematical modelling and population sequencing to show that the long-term persistence and molecular integrity of the plasmid is highly influenced by multiple factors within a 25?kb plasmid region constituting a host-dependent burden. In the E. coli hosts investigated here, improved plasmid stability readily evolves via IS26 mediated deletions of costly regions from the plasmid backbone, effectively expanding the host-range of the plasmid. Although these adaptations were also beneficial to plasmid persistence in a naïve K. pneumoniae host, they were never observed in this species, indicating that differential evolvability can limit opportunities of plasmid adaptation. While insertion sequences are well known to supply plasmids with adaptive traits, our findings suggest that they also play an important role in plasmid evolution by maintaining the plasticity necessary to alleviate plasmid-host constrains. Further, the observed evolutionary strategy consistently followed by all evolved E. coli lineages exposes a trade-off between horizontal and vertical transmission that may ultimately limit the dissemination potential of clinical multidrug resistance plasmids in these hosts.© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.


July 7, 2019  |  

Genetic characterization of a blaVEB-2-carrying plasmid in Vibrio parahaemolyticus.

This study reports the first detection of blaVEB-2 gene in Vibrio parahaemolyticus strain isolated from a shrimp sample. The blaVEB-2 was carried on a novel Inc type plasmid, was likely to originate from aquatic organisms upon comparison with other known genetic elements in the GenBank. However, the plasmid contains resistance elements usually harbored by members of Enterobacteriaceae, suggesting that gene transfer events occurred and contributed to the formation of this multidrug resistance-encoding plasmid. Copyright © 2016, American Society for Microbiology. All Rights Reserved.


July 7, 2019  |  

Use of WGS data for investigation of a long-term NDM-1-producing Citrobacter freundii outbreak and secondary in vivo spread of blaNDM-1 to Escherichia coli, Klebsiella pneumoniae and Klebsiella oxytoca.

An outbreak of NDM-1-producing Citrobacter freundii and possible secondary in vivo spread of blaNDM-1 to other Enterobacteriaceae were investigated.From October 2012 to March 2015, meropenem-resistant Enterobacteriaceae were detected in 45 samples from seven patients at Aalborg University Hospital, Aalborg, Denmark. In silico resistance genes, Inc plasmid types and STs (MLST) were obtained from WGS data from 24 meropenem-resistant isolates (13 C. freundii, 6 Klebsiella pneumoniae, 4 Escherichia coli and 1 Klebsiella oxytoca) and 1 meropenem-susceptible K. oxytoca. The sequences of the meropenem-resistant C. freundii isolates were compared by phylogenetic analyses. In vitro susceptibility to 21 antimicrobial agents was tested. Furthermore, in vitro conjugation and plasmid characterization was performed.From the seven patients, 13 highly clonal ST18 NDM-1-producing C. freundii were isolated. The ST18 NDM-1-producing C. freundii isolates were only susceptible to tetracycline, tigecycline, colistin and fosfomycin (except for the C. freundii isolates from Patient 2 and Patient 7, which were additionally resistant to tetracycline). The E. coli and K. pneumoniae from different patients belonged to different STs, indicating in vivo transfer of blaNDM-1 in the individual patients. This was further supported by in vitro conjugation and detection of a 154 kb IncA/C2 plasmid with blaNDM-1. Patient screenings failed to reveal any additional cases. None of the patients had a history of recent travel abroad and the source of the blaNDM-1 plasmid was unknown.To our knowledge, this is the first report of an NDM-1-producing C. freundii outbreak and secondary in vivo spread of an IncA/C2 plasmid with blaNDM-1 to other Enterobacteriaceae.© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.


July 7, 2019  |  

Full-length nucleotide sequences of mcr-1-harboring plasmids isolated from extended- spectrum-ß-lactamase-producing Escherichia coli isolates of different origins.

Here, we present the full sequences of three mcr-1-carrying plasmids isolated from extended-spectrum-ß-lactamase (ESBL)-producing Escherichia coli The plasmids belong to three different replicon types and are 34,640 bp, 209,401 bp, and 247,885 bp in size. We describe for the first time a composite transposon containing mcr-1 localized on a multidrug-resistant (MDR) IncHI2 plasmid harboring additional determinants of resistance to six different classes of antibiotics, including the ESBL gene blaCTX-M-1, and heavy metal resistance. Copyright © 2016, American Society for Microbiology. All Rights Reserved.


July 7, 2019  |  

Plasmids from Shiga toxin-producing Escherichia coli strains with rare enterohemolysin gene (ehxA) subtypes reveal pathogenicity potential and display a novel evolutionary path.

Most Shiga toxin-producing Escherichia coli (STEC) strains associated with severe disease, such as hemolytic-uremic syndrome (HUS), carry large enterohemolysin-encoding (ehxA) plasmids, e.g., pO157 and pO103, that contribute to STEC clinical manifestations. Six ehxA subtypes (A through F) exist that phylogenetically cluster into eae-positive (B, C, F), a mix of eae-positive (E) and eae-negative (A), and a third, more distantly related, cluster of eae-negative (D) STEC strains. While subtype B, C, and F plasmids share a number of virulence traits that are distinct from those of subtype A, sequence data have not been available for subtype D and E plasmids. Here, we determined and compared the genetic composition of four subtype D and two subtype E plasmids to establish their evolutionary relatedness among ehxA subtypes and define their potential role in pathogenicity. We found that subtype D strains carry one exceptionally large plasmid (>200 kbp) that carries a variety of virulence genes that are associated with enterotoxigenic and enterohemorrhagic E. coli, which, quite possibly, enables these strains to cause disease despite being food isolates. Our data offer further support for the hypothesis that this subtype D plasmid represents a novel virulence plasmid, sharing very few genetic features with other plasmids; we conclude that these plasmids have evolved from a different evolutionary lineage than the plasmids carrying the other ehxA subtypes. In contrast, the 50-kbp plasmids of subtype E (pO145), although isolated from HUS outbreak strains, carried only few virulence-associated determinants, suggesting that the clinical presentation of subtype E strains is largely a result of chromosomally encoded virulence factors.Bacterial plasmids are known to be key agents of change in microbial populations, promoting the dissemination of various traits, such as drug resistance and virulence. This study determined the genetic makeup of virulence plasmids from rare enterohemolysin subtype D and E Shiga toxin-producing E. coli strains. We demonstrated that ehxA subtype D plasmids represent a novel E. coli virulence plasmid, and although subtype D plasmids were derived from nonclinical isolates, they encoded a variety of virulence determinants that are associated with pathogenic E. coli In contrast, subtype E plasmids, isolated from strains recovered from severely ill patients, carry only a few virulence determinants. The results of this study reemphasize the plasticity and vast diversity among E. coli plasmids. This work demonstrates that, although E. coli strains of certain serogroups may not be frequently associated with disease, they should not be underestimated in protecting human health and food safety. Copyright © 2016, American Society for Microbiology. All Rights Reserved.


July 7, 2019  |  

IncHI2 plasmids are the key vectors responsible for oqxAB transmission among Salmonella species.

This study reported and analysed the complete sequences of two oqxAB-bearing IncHI2 plasmids harboured by a clinical S. Typhimurium strain and an S. Indiana strain of animal origin, respectively. Particularly, pA3T recovered from S. Indiana comprised the resistance determinants oqxAB, aac(6′)Ib-cr, fosA3 and blaCTX-M-14 Further genetic screening of 63 oqxAB-positive Salmonella spp. isolates revealed that the majority carried IncHI2 plasmids, confirming that such plasmids play a pivotal role in dissemination of oqxAB in Salmonella spp.. Copyright © 2016, American Society for Microbiology. All Rights Reserved.


July 7, 2019  |  

Complete sequence of a F33:A-:B- conjugative plasmid carrying the oqxAB, fosA3, and blaCTX-M-55 elements from a foodborne Escherichia coli strain.

This study reports the complete sequence of pE80, a conjugative IncFII plasmid recovered from an Escherichia coli strain isolated from chicken meat. This plasmid harbors multiple resistance determinants including oqxAB, fosA3, blaCTX-M-55, and blaTEM-1, and is a close variant of the recently reported p42-2 element, which was recovered from E. coli of veterinary source. Recovery of pE80 constitutes evidence that evolution or genetic re-arrangement of IncFII type plasmids residing in animal-borne organisms is an active event, which involves acquisition and integration of foreign resistance elements into the plasmid backbone. Dissemination of these plasmids may further compromise the effectiveness of current antimicrobial strategies.


July 7, 2019  |  

Evolution and comparative genomics of F33:A-:B- plasmids carrying blaCTX-M-55 or blaCTX-M-65 in Escherichia coli and Klebsiella pneumoniae isolated from animals.

To understand the underlying evolution process of F33:A-:B- plasmids among Enterobacteriaceae isolates of various origins in China, the complete sequences of 17 blaCTX-M-harboring F33:A-:B- plasmids obtained from Escherichia coli and Klebsiella pneumoniae isolates from different sources (animals, animal-derived food, and human clinics) in China were determined. F33:A-:B- plasmids shared similar plasmid backbones comprising replication, leading, and conjugative transfer regions and differed by the numbers of repeats in yddA and traD and by the presence of group II intron, except that pHNAH9 lacked a large segment of the leading and transfer regions. The variable regions of F33:A-B- plasmids were distinct and were inserted downstream of the addiction system pemI/pemK, identified as the integration hot spot among F33:A-B- plasmids. The variable region contained resistance genes and mobile elements or contained segments from other types of plasmids, such as IncI1, IncN1, and IncX1. Three plasmids encoding CTX-M-65 were very similar to our previously described pHN7A8 plasmid. Four CTX-M-55-producing plasmids contained multidrug resistance regions related to that of F2:A-B- plasmid pHK23a from Hong Kong. Five plasmids with IncN and/or IncX replication regions and IncI1-backbone fragments had variable regions related to those of pE80 and p42-2. The remaining five plasmids with IncN replicons and an IncI1 segment also possessed closely related variable regions. The diversity in variable regions was presumably associated with rearrangements, insertions, and/or deletions mediated by mobile elements, such as IS26 and IS1294 IMPORTANCE Worldwide spread of antibiotic resistance genes among Enterobacteriaceae isolates is of great concern. F33:A-:B- plasmids are important vectors of resistance genes, such as blaCTX-M-55/-65, blaNDM-1, fosA3, and rmtB, among E. coli isolates from various sources in China. We determined and compared the complete sequences of 17 F33:A-:B- plasmids from various sources. These plasmids appear to have evolved from the same ancestor by mobile element-mediated rearrangement, acquisition, and/or loss of resistance modules and similar IncN1, IncI1, and/or IncX1 plasmid backbone segments. Our findings highlight the evolutionary potential of F33:A-:B- plasmids as efficient vectors to capture and diffuse clinically relevant resistance genes. Copyright © 2018 Wang et al.


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