Large Conjugative Plasmid Research Articles

A two-component system serves as a central hub for connecting energy metabolism and plasmid dissemination in bacteria.

Mobile genetic elements such as conjugative plasmids play a key role in the acquisition of antibiotic resistance by pathogenic bacteria. Resistance genes on plasmids can be transferred between bacteria using specialized conjugation machinery. Acinetobacter baumannii, the most common bacterium associated with nosocomial infections, harbors a large conjugative plasmid that encodes a type IV secretion system (T4SS). Feng et al. recently found that the A. baumannii T4SS is specialized for plasmid transfer, suggesting that it may be involved in multidrug resistance (Z. Feng, L. Wang, Q. Guan, X. Chu, and Z.-Q. Luo, mBio e02276-23, 2023, https://doi.org/10.1128/mbio.02276-23), T4SS-encoding genes are shown to be controlled by a versatile GacA/S two-component regulatory system. GacA/S is also found to regulate genes involved in central metabolism. The coordinated regulation of metabolism and plasmid conjugation may be a bacterial strategy for adapting to selective pressure from antibiotics.

Genetic background of neomycin resistance in clinical Escherichia coli isolated from Danish pig farms.

Neomycin is the first-choice antibiotic for the treatment of porcine enteritis caused by enterotoxigenic Escherichia coli. Resistance to this aminoglycoside is on the rise after the increased use of neomycin due to the ban on zinc oxide. We identified the neomycin resistance determinants and plasmid contents in a historical collection of 128 neomycin-resistant clinical E. coli isolates from Danish pig farms. All isolates were characterized by whole-genome sequencing and antimicrobial susceptibility testing, followed by conjugation experiments and long-read sequencing of eight selected representative strains. We detected 35 sequence types (STs) with ST100 being the most prevalent lineage (38.3%). Neomycin resistance was associated with two resistance genes, namely aph(3')-Ia and aph(3')-Ib, which were identified in 93% and 7% of the isolates, respectively. The aph(3')-Ia was found on different large conjugative plasmids belonging to IncI1α, which was present in 67.2% of the strains, on IncHI1, IncHI2, and IncN, as well as on a multicopy ColRNAI plasmid. All these plasmids except ColRNAI carried genes encoding resistance to other antimicrobials or heavy metals, highlighting the risk of co-selection. The aph(3')-Ib gene occurred on a 19 kb chimeric, mobilizable plasmid that contained elements tracing back its origin to distantly related genera. While aph(3')-Ia was flanked by either Tn903 or Tn4352 derivatives, no clear association was observed between aph(3')-Ib and mobile genetic elements. In conclusion, the spread of neomycin resistance in porcine clinical E. coli is driven by two resistance determinants located on distinct plasmid scaffolds circulating within a highly diverse population dominated by ST100. IMPORTANCE Neomycin is the first-choice antibiotic for the management of Escherichia coli enteritis in pigs. This work shows that aph(3')-Ia and to a lesser extent aph(3')-Ib are responsible for the spread of neomycin resistance that has been recently observed among pig clinical isolates and elucidates the mechanisms of dissemination of these two resistance determinants. The aph(3')-Ia gene is located on different conjugative plasmid scaffolds and is associated with two distinct transposable elements (Tn903 and Tn4352) that contributed to its spread. The diffusion of aph(3')-Ib is mediated by a small non-conjugative, mobilizable chimeric plasmid that likely derived from distantly related members of the Pseudomonadota phylum and was not associated with any detectable mobile genetic element. Although the spread of neomycin resistance is largely attributable to horizontal transfer, both resistance determinants have been acquired by a predominant lineage (ST100) associated with enterotoxigenic E. coli, which accounted for approximately one-third of the strains.

A Brief Evaluation of a Copper Resistance Mobile Genetic Island in the Bacterial Leaf Spot Pathogen Xanthomonas euvesicatoria pv. perforans.

Due to the continuous use of copper containing bactericides without effective alternative bactericides, copper resistance has become more prevalent in plant pathogens, including Xanthomonas euvesicatoria pv. perforans (formerly Xanthomonas perforans), a predominant cause of bacterial leaf spot disease of tomato and pepper in the Southeastern United States. Previously, reports of copper resistance have been associated with a large conjugative plasmid. However, we have characterized a copper resistance genomic island located within the chromosome of multiple X. euvesicatoria pv. perforans strains. The island is distinct from a previously described chromosomally encoded copper resistance island in X. vesicatoria strain XVP26. Computational analysis revealed the genomic island to contain multiple genes associated with genetic mobility, including both phage-related genes and transposase. Among copper-tolerant strains of X. euvesicatoria pv. perforans isolated from Florida, the majority of strains were found to have the copper resistance chromosomally encoded rather than plasmid borne. Our results suggest that this copper resistance island may have two modes of horizontal gene transfer and that chromosomally encoded copper resistance genes may provide a fitness advantage over plasmid-borne resistance.

Genomic features of Klebsiella isolates from artisanal ready-to-eat food production facilities

Increasing reports on K. pneumoniae strains with antimicrobial resistance and virulence traits from food and farm animals are raising concerns about the potential role of Klebsiella spp. as a foodborne pathogen. This study aimed to report and characterize Klebsiella spp. isolates from two artisanal ready-to-eat food (soft cheese and salami) producing facilities, and to track similar genotypes in different ecological niches. Over 1170 samples were collected during the whole production chain of different food batches. The overall Klebsiella prevalence was 6%. Strains were classified into the three Klebsiella species complexes: K. pneumoniae (KpSC, n = 17), K. oxytoca (KoSC, n = 38) and K. planticola (KplaSC, n = 18). Despite high genetic diversity we found in terms of known and new sequence types (STs), core genome phylogeny revealed clonal strains persisting in the same processing setting for over 14 months, isolated from the environment, raw materials and end-products. Strains showed a natural antimicrobial resistance phenotype-genotype. K. pneumoniae strains showed the highest virulence potential, with sequence types ST4242 and ST107 strains carrying yersiniabactin ybt16 and aerobactin iuc3. The latter was detected in all K. pneumoniae from salami and was located on a large conjugative plasmid highly similar (97% identity) to iuc3+ plasmids from human and pig strains circulating in nearby regions of Italy. While identical genotypes may persist along the whole food production process, different genotypes from distinct sources in the same facility shared an iuc3-plasmid. Surveillance in the food chain will be crucial to obtain a more comprehensive picture of the circulation of Klebsiella strains with pathogenic potential.

Compensatory mutations reducing the fitness cost of plasmid carriage occur in plant rhizosphere communities.

Plasmids drive bacterial evolutionary innovation by transferring ecologically important functions between lineages, but acquiring a plasmid often comes at a fitness cost to the host cell. Compensatory mutations, which ameliorate the cost of plasmid carriage, promote plasmid maintenance in simplified laboratory media across diverse plasmid-host associations. Whether such compensatory evolution can occur in more complex communities inhabiting natural environmental niches where evolutionary paths may be more constrained is, however, unclear. Here we show a substantial fitness cost of carrying the large conjugative plasmid pQBR103 in Pseudomonas fluorescens SBW25 in the plant rhizosphere. This plasmid fitness cost could be ameliorated by compensatory mutations affecting the chromosomal global regulatory system gacA/gacS, which arose rapidly in plant rhizosphere communities and were exclusive to plasmid carriers. These findings expand our understanding of the importance of compensatory evolution in plasmid dynamics beyond simplified lab media. Compensatory mutations contribute to plasmid survival in bacterial populations living within complex microbial communities in their environmental niche.

Spread of blaCTX-M-9 and Other Clinically Relevant Resistance Genes, Such as mcr-9 and qnrA1, Driven by IncHI2-ST1 Plasmids in Clinical Isolates of Monophasic Salmonella enterica Serovar Typhimurium ST34

The monophasic 4,[5],12:i:-variant of Salmonella enterica serovar Typhimurium with sequence type ST34 has become one of the most prevalent non-typhoidal salmonellae worldwide. In the present study, we thoroughly characterized seven isolates of this variant detected in a Spanish hospital and selected based on cefotaxime resistance and cefoxitin susceptibility, mediated by blaCTX-M-9. For this, conventional microbiological techniques, together with whole genome sequencing performed with the Illumina platform, were applied. All selected isolates carried the resistance region RR or variants therein, and most also contained the SGI-4 genomic island. These chromosomal elements, typically associated with monophasic S. Typhimurium ST34, confer resistance to traditional antibiotics (ampicillin, streptomycin, sulfonamides, and tetracycline) and tolerance to heavy metals (mercury, silver, and copper). In addition, each isolate carried a large IncHI2-ST1 conjugative plasmid containing additional or redundant resistance genes. All harbored the blaCTX-M-9 gene responsible for cefotaxime resistance, whereas the qnrA1 gene mediating fluoroquinolone resistance was detected in two of the plasmids. These genes were embedded in ISCR1-bearing complex class 1 integrons, specifically In60-like and In36-like. The mcr-9 gene was present in all but one of the IncHI2-ST1 plasmids found in the analyzed isolates, which were nevertheless susceptible to colistin. Most of the resistance genes of plasmid origin clustered within a highly complex and variable region. The observed diversity results in a wide range of resistance phenotypes, enabling bacterial adaptation to selective pressure posed by the use of antimicrobials.

Role of megaplasmids and chromosomal integration in acquisition of CTX-M-encoding genes by Pseudomonas aeruginosa.

The genes that encode CTX-M-type ESBLs have diffused extensively among Enterobacterales over the past decades.1 Despite this epidemic success at the global level, few of these have been detected (blaCTX-M-1,-2,-3,-14,-15and-43) in the distantly related opportunistic pathogen Pseudomonas aeruginosa.1–4 The narrow host range of plasmids carrying the blaCTX-M determinants in Enterobacterales (mainly of incompatibility group IncF) plausibly accounts for the low prevalence of these resistance genes in P. aeruginosa.5 On the other hand, little is known about the mobile elements that enable the acquisition of CTX-M enzymes by clinical P. aeruginosa strains. One plasmid close to broad-host-range incompatibility group IncP2 has been found to carry the gene blaCTX-M-2, while another one belonging to IncQ harboured blaCTX-M-3.6,7 The present report describes the structure of a large conjugative plasmid determining a novel CTX-M variant, named CTX-M-206, and the genetic environment of a chromosomally located blaCTX-M-15 gene in a P. aeruginosa isolate present in a second strain.

Sequence analysis and plasmid mobilization of a 6.6-kb kanamycin resistance plasmid, pSNC3-Kan, from a Salmonella enterica serotype Newport isolate

Research on the transfer of antibiotic resistance plasmids has been mainly focused on the large multi-drug resistance conjugative plasmids, while the transmission of small mobilizable plasmids remains under-investigated. A series of diverse ColE-like kanamycin resistance plasmids (“KanR plasmids”) from Salmonella enterica were characterized previously. In this study, the 6.6-kb pSNC3-Kan from a Salmonella enterica serotype Newport isolate was investigated. It possessed highly conserved RNA I/II and Tn602 (IS903-aph-IS903) regions to two other KanR plasmids pSe-Kan and pSBardo-Kan, but carried a mobC-mobA/BD operon. The mobilization proteins encoded by the mob operon of pSNC3-Kan showed high sequence identity (~95%) to those of an E. coli plasmid pEC34B, except that MobE was not present; and were much less conserved to those of another KanR plasmid pSN11/00Kan (43% - 86% identity). Four structurally different KanR plasmids were investigated for their ability to be mobilized by the conjugal transfer (tra) genes from F and IncP plasmids. Transfer genes derived from IncP plasmids can efficiently mobilize KanR plasmids possessing the mob operons (mobC-mobA/BD), such as pSNC3-Kan and pSN11/00Kan, in bi-parental mating experiments. On the other hand, F tra genes were able to mobilize pU302S, pSNC3-Kan and pSe-Kan, but not pSN11/00Kan. A plasmid-borne mob operon was not required for mobilization of the oriT(F)-bearing pSe-Kan by the F tra genes. This study underscores the complexity of plasmid interaction and the importance of how small mobilizable plasmids may contribute to the spread of antibiotic resistance genes.

Healthcare-associated infections caused by chlorhexidine-tolerant Serratia marcescens carrying a promiscuous IncHI2 multi-drug resistance plasmid in a veterinary hospital.

The bacterium Serratia marcescens can cause opportunistic infections in humans and in animals. In veterinary settings, the diversity, reservoirs and modes of transmission of this pathogen are poorly understood. The phenotypes and genotypes of Serratia spp. isolated from dogs, cats, horses, a bird and a rabbit examined at an Australian veterinary hospital between 2008 and 2019 were characterised. The isolates were identified as S. marcescens (n = 15) or S. ureilytica (n = 3) and were placed into four distinct phylogenetic groups. Nine quasi-clonal isolates associated with post-surgical complications in different patients displayed high levels of resistance to the antimicrobials fluoroquinolones, cephalosporins, aminoglycosides, and to the disinfectant chlorhexidine. A Serratia sp. with a similar resistance profile was also isolated from chlorhexidine solutions used across the Hospital, suggesting that these infections had a nosocomial origin. A genomic island encoding a homolog of the Pseudomonas MexCD-OprJ biocide efflux system was detected in the chlorhexidine-tolerant Serratia. The nine multi-drug resistant Serratia isolates also possessed a Ser-83-Ile mutation in GyrA conferring fluoroquinolone resistance, and carried a large IncHI2 conjugative plasmid encoding antimicrobial and heavy metal resistances. This replicon was highly similar to a plasmid previously detected in a strain of Enterobacter hormaechei recovered from the Hospital environment. IncHI2 plasmids are commonly found in Enterobacteriaceae, but are rarely present in Serratia spp., suggesting that this plasmid was acquired from another organism. A chlorhexidine-tolerant Serratia isolate which lacked the IncHI2 plasmid was used in mating experiments to demonstrate the transfer of multi-drug resistance from a E. hormaechei donor. This study illustrates the importance of environmental surveillance of biocide-resistance in veterinary hospitals.

Characterization of a mcr-1 and CRISPR-Cas System Co-harboring Plasmid in a Carbapenemase-Producing High-Risk ST11 Klebsiella pneumoniae Strain.

We set out to study the prevalence of the mcr-1 gene in carbapenemase-producing Klebsiella pneumoniae (CPKP) strains, and to determine whether its presence is associated with a fitness cost. A total of 234 clinical CPKP isolates were collected from a tertiary medical center in Taiwan from January 2018 to January 2019. The mcr-1 and carbapenemase genes were detected by polymerase chain reaction (PCR) followed by Sanger sequencing. The mcr-1-positive carbapenemase-producing strain was characterized by whole genome sequencing, a plasmid stability test and a conjugation assay. In vitro growth rate and an in vivo virulence test were compared between the parental mcr-1-positive strain and its mcr-1 plasmid-cured strain. We identified only one mcr-1 positive strain (KP2509), co-harboring blaKPC–2 and blaOXA–48, among 234 (1/234, 0.43%) CPKP strains. KP2509 and its Escherichia coli mcr-1 transconjugant showed moderate colistin resistance (MIC = 8 mg/L). The mcr-1 is located on a large conjugative plasmid (317 kb), pKP2509-MCR, with three replicons, IncHI, IncFIB, and IncN. Interestingly, a complete Type IV-A3 CRISPR-Cas system was identified in pKP2509-MCR. Plasmid pKP2509-MCR was highly stable in KP2509 after 270 generation of passage, and the pKP2509-MCR cured strain PC-KP2509 showed similar growth rate and in vivo virulence in comparison to KP2509. The prevalence of mcr-1 in CPKP strains remains low in our center. Notably, we identified a large plasmid with multiple replicons containing both the mcr-1 and the Type IV-3A CRISPR-Cas genes. The further spread of this highly stable plasmid raises concern that it may promote the increase of mcr-1 prevalence in CPKP.

Genomic Comparison of Conjugative Plasmids from Salmonella enterica and Escherichia coli Encoding Beta-Lactamases and Capable of Mobilizing Kanamycin Resistance Col-like Plasmids

Salmonella enterica and Escherichia coli are important human pathogens that frequently contain plasmids, both large and small, carrying antibiotic resistance genes. Large conjugative plasmids are known to mobilize small Col plasmids, but less is known about the specificity of mobilization. In the current study, six S. enterica and four E. coli strains containing large plasmids were tested for their ability to mobilize three different kanamycin resistance Col plasmids (KanR plasmids). Large conjugative plasmids from five isolates, four S. enterica and one E. coli, were able to mobilize KanR plasmids of various types. Plasmids capable of mobilizing the KanR plasmids were either IncI1 or IncX, while IncI1 and IncX plasmids with no evidence of conjugation had disrupted transfer regions. Conjugative plasmids of similar types mobilized similar KanR plasmids, but not all conjugative plasmid types were capable of mobilizing all of the KanR plasmids. These data describe some of the complexities and specificities of individual small plasmid mobilization.

Plasmid-Encoded H-NS Controls Extracellular Matrix Composition in a Modern Acinetobacter baumannii Urinary Isolate

Acinetobacter baumannii is emerging as a multidrug-resistant (MDR) nosocomial pathogen of increasing threat to human health worldwide. The recent MDR urinary isolate UPAB1 carries the plasmid pAB5, a member of a family of large conjugative plasmids (LCPs). LCPs encode several antibiotic resistance genes and repress the type VI secretion system (T6SS) to enable their dissemination, employing two TetR transcriptional regulators. Furthermore, pAB5 controls the expression of additional chromosomally encoded genes, impacting UPAB1 virulence. Here, we show that a pAB5-encoded H-NS transcriptional regulator represses the synthesis of the exopolysaccharide PNAG and the expression of a previously uncharacterized three-gene cluster that encodes a protein belonging to the CsgG/HfaB family. Members of this protein family are involved in amyloid or polysaccharide formation in other species. Deletion of the CsgG homolog abrogated PNAG production and chaperone-usher pathway (CUP) pilus formation, resulting in a subsequent reduction in biofilm formation. Although this gene cluster is widely distributed in Gram-negative bacteria, it remains largely uninvestigated. Our results illustrate the complex cross-talks that take place between plasmids and the chromosomes of their bacterial host, which in this case can contribute to the pathogenesis of Acinetobacter. IMPORTANCE The opportunistic human pathogen Acinetobacter baumannii displays the highest reported rates of multidrug resistance among Gram-negative pathogens. Many A. baumannii strains carry large conjugative plasmids like pAB5. In recent years, we have witnessed an increase in knowledge about the regulatory cross-talks between plasmids and bacterial chromosomes. Here, we show that pAB5 controls the composition of the bacterial extracellular matrix, resulting in a drastic reduction in biofilm formation. The association between biofilm formation, virulence, and antibiotic resistance is well documented. Therefore, understanding the factors involved in the regulation of biofilm formation in Acinetobacter has remarkable therapeutic potential.

Modern Acinetobacter baumannii clinical isolates replicate inside spacious vacuoles and egress from macrophages.

Multidrug-resistant Acinetobacter baumannii infections are increasing at alarming rates. Therefore, novel antibiotic-sparing treatments to combat these A. baumannii infections are urgently needed. The development of these interventions would benefit from a better understanding of this bacterium's pathobiology, which remains poorly understood. A. baumannii is regarded as an extracellular opportunistic pathogen. However, research on Acinetobacter has largely focused on common lab strains, such as ATCC 19606, that have been isolated several decades ago. These strains exhibit reduced virulence when compared to recently isolated clinical strains. In this work, we demonstrate that, unlike ATCC 19606, several modern A. baumannii clinical isolates, including the recent clinical urinary isolate UPAB1, persist and replicate inside macrophages within spacious vacuoles. We show that intracellular replication of UPAB1 is dependent on a functional type I secretion system (T1SS) and pAB5, a large conjugative plasmid that controls the expression of several chromosomally-encoded genes. Finally, we show that UPAB1 escapes from the infected macrophages by a lytic process. To our knowledge, this is the first report of intracellular growth and replication of A. baumannii. We suggest that intracellular replication within macrophages may contribute to evasion of the immune response, dissemination, and antibiotic tolerance of A. baumannii.

The proficiency of the original host species determines community-level plasmid dynamics.

Plasmids are common in natural bacterial communities, facilitating bacterial evolution via horizontal gene transfer. Bacterial species vary in their proficiency to host plasmids: whereas plasmids are stably maintained in some species regardless of selection for plasmid-encoded genes, in other species, even beneficial plasmids are rapidly lost. It is, however, unclear how this variation in host proficiency affects plasmid persistence in communities. Here, we test this using multispecies bacterial soil communities comprising species varying in their proficiency to host a large conjugative mercury resistance plasmid, pQBR103. The plasmid reached higher community-level abundance where beneficial and when introduced to the community in a more proficient host species. Proficient plasmid host species were also better able to disseminate the plasmid to a wider diversity of host species. These findings suggest that the dynamics of plasmids in natural bacterial communities depend not only upon the plasmid's attributes and the selective environment but also upon the proficiency of their host species.

A Novel Family of Acinetobacter Mega-Plasmids Are Disseminating Multi-Drug Resistance Across the Globe While Acquiring Location-Specific Accessory Genes.

Acinetobacter species are emerging as major nosocomial pathogens, aided by their ability to acquire resistance to all classes of antibiotics. A key factor leading to their multi-drug resistance phenotypes is the acquisition of a wide variety of mobile genetic elements, particularly large conjugative plasmids. Here, we characterize a family of 21 multi-drug resistance mega-plasmids in 11 different Acinetobacter species isolated from various locations across the globe. The plasmid family exhibits a highly dynamic and diverse accessory genome, including 221 antibiotic resistance genes (ARGs) that confer resistance to 13 classes of antibiotics. We show that plasmids isolated within the same geographic region are often evolutionarily divergent members of this family based on their core-genome, yet they exhibit a more similar accessory genome. Individual plasmids, therefore, can disseminate to different locations around the globe, where they then appear to acquire diverse sets of accessory genes from their local surroundings. Further, we show that plasmids from several geographic regions were enriched with location-specific functional traits. Together, our findings show that these mega-plasmids can transmit across species boundaries, have the capacity for global dissemination, can accumulate a diverse suite of location-specific accessory genes, and can confer multi-drug resistance phenotypes of significant concern for human health. We therefore highlight this previously undescribed plasmid family as a serious threat to healthcare systems worldwide. These findings also add to the growing concern that mega-plasmids are key disseminators of antibiotic resistance and require global surveillance.

Colonization of a hand washing sink in a veterinary hospital by an Enterobacter hormaechei strain carrying multiple resistances to high importance antimicrobials

BackgroundHospital intensive care units (ICUs) are known reservoirs of multidrug resistant nosocomial bacteria. Targeted environmental monitoring of these organisms in health care facilities can strengthen infection control procedures. A routine surveillance of extended spectrum beta-lactamase (ESBL) producers in a large Australian veterinary teaching hospital detected the opportunistic pathogen Enterobacter hormaechei in a hand washing sink of the ICU. The organism persisted for several weeks, despite two disinfection attempts. Four isolates were characterized in this study.MethodsBrilliance-ESBL selective plates were inoculated from environmental swabs collected throughout the hospital. Presumptive identification was done by conventional biochemistry. Genomes of multidrug resistant Enterobacter were entirely sequenced with Illumina and Nanopore platforms. Phylogenetic markers, mobile genetic elements and antimicrobial resistance genes were identified in silico. Antibiograms of isolates and transconjugants were established with Sensititre microdilution plates.ResultsThe isolates possessed a chromosomal Tn7-associated silver/copper resistance locus and a large IncH12 conjugative plasmid encoding resistance against tellurium, arsenic, mercury and nine classes of antimicrobials. Clusters of antimicrobial resistance genes were associated with class 1 integrons and IS26, IS903 and ISCR transposable elements. The blaSHV-12, qnrB2 and mcr-9.1 genes, respectively conferring resistance to cephalosporins, quinolones and colistin, were present in a locus flanked by two IS903 copies. ESBL production and enrofloxacin resistance were confirmed phenotypically. The isolates appeared susceptible to colistin, possibly reflecting the inducible nature of mcr-9.1.ConclusionsThe persistence of this strain in the veterinary hospital represented a risk of further accumulation and dissemination of antimicrobial resistance, prompting a thorough disinfection of the ICU. The organism was not recovered from subsequent environmental swabs, and nosocomial Enterobacter infections were not observed in the hospital during that period. This study shows that targeted routine environmental surveillance programs to track organisms with major resistance phenotypes, coupled with disinfection procedures and follow-up microbiological cultures are useful to control these risks in sensitive areas of large veterinary hospitals.

Coexistence of blaKPC-2–IncN and mcr-1–IncX4 plasmids in a ST48 Escherichia coli strain in China

ObjectivesThe aim of this study was to explore the genomic content of a blaKPC-2- and mcr-1-harbouring Escherichia coli strain and to clarify the molecular mechanism for the transmission of multidrug resistance. MethodsAntimicrobial susceptibility testing was conducted by the broth microdilution method to determine the resistance profile. Filter-mating conjugation assays were performed to confirm the plasmid transfer ability. Whole-genome sequence data were acquired by a combination of Illumina paired-end reads and Nanopore long-read sequencing. ResultsEscherichia coli strain QE11−421 was an mcr-1-positive colistin-resistant isolate that co-harboured the blaKPC-2 gene conferring carbapenem resistance. Genome sequence data confirmed QE11−421 as a sequence type 48 (ST48) E. coli that harboured five large conjugative plasmids encoding several multidrug resistance genes. The blaKPC-2 gene was located on a Tn3–Tn4401 composite transposon, which is part of a 65-kb multidrug-resistant IncN plasmid. The mcr-1 gene was harboured on another 33-kb IncX4 plasmid that was more conserved and presented few antimicrobial resistance determinants. No copies of insertion sequence ISApl1 were found flanking the mcr-1 gene, decreasing the mobility of mcr-1 based on its original Tn6330 transposon. ConclusionsHorizontal transfer of multidrug resistance plasmids or resistance-related transposon units was responsible for the emergence of this notorious superbug. The coexistence of blaKPC-2–IncN and mcr-1–IncX4 plasmids in a ST48 E. coli strain in humans poses a great threat.

Antimicrobial Resistance in Wildlife in Guadeloupe (French West Indies): Distribution of a Single blaCTX–M–1/IncI1/ST3 Plasmid Among Humans and Wild Animals

Limited data are available on the contribution of wildlife to the spread of antibacterial resistance. We determined the prevalence of resistance to antibiotics in Escherichia coli isolates collected from wild animals in 2013 and 2014 and the genetic basis for resistance to third-generation cephalosporin in Guadeloupe. We recovered 52 antibiotic-resistant (AR) E. coli strains from 48 of the 884 (5.4%) wild animals tested (46 iguanas, 181 birds, 289 anoles, and 368 rodents at 163 sampling sites). Rodents had higher rates of carriage (n = 38, 10.3%) than reptiles and birds (2.4% and 1.1%, respectively, p < 0.001). A significant association (p < 0.001) was found between the degree of anthropization and the frequency of AR E. coli carriage for all species. The carriage rate of ciprofloxacin- and cefotaxime-resistant isolates was 0.7% (6/884) and 1.5% (13/884), respectively. Most (65.4%) AR E. coli were multi-drug resistant, and the prevalence of extended-spectrum beta-lactamase (ESBL)-producing E. coli was low (n = 7, 0.8%) in all species. Eight ESBL-producing E. coli were recovered, two genetically unrelated isolates being found in one bird. These isolates and 20 human invasive ESBL E. coli isolates collected in Guadeloupe during the same period were investigated by whole genome sequencing. blaCTX–M–1 was the only ESBL gene shared by three animal classes (humans, n = 2; birds, n = 2; rodents, n = 2). The blaCTX–M–1 gene and most of the antimicrobial resistance genes were present in a large conjugative IncI1 plasmid that was highly similar (>99% nucleotide identity) to ESBL-carrying plasmids found in several countries in Europe and in Australia. Although the prevalence of ESBL-producing E. coli isolates was very low in wild animals, it is of concern that the well-conserved IncI1 plasmid-carrying blaCTX–M–1 is widespread and occurs in various E. coli strains from animals and humans.

Large plasmids encoding antibiotic resistance and localized-like adherence in atypical enteropathogenic Escherichia coli strains

BackgroundIn previous studies, we have shown that atypical enteropathogenic Escherichia coli (aEPEC) strains are important diarrheal pathogens among Brazilian children. In the characterization of a collection of 126 aEPEC strains, we identified 29 strains expressing the localized-like adherence (LAL) pattern on HEp-2 cells and harboring large plasmids in the range of 60 to 98 MDa. In this study, we examined 18 of these strains for their ability to transfer the LAL phenotype to a E. coli K-12 C600 strain.ResultsIn conjugation experiments, using eight strains which were resistant to one or more antimicrobials and positive for F-pili genes (traA), we were able to cotransfer antimicrobial resistance markers along with adhesion genes. By transforming E. coli DH5α with plasmid DNA from strains A46 (pIS46), A66 (pIS66) and A102 (pIS102), we were able to demonstrate that genes encoding ampicillin, tetracycline and LAL were encoded on a 98-MDa conjugative plasmid. To identify a gene responsible for LAL, we constructed a transposon mutant library of A102 strain. Among 18 mutants that did not adhere to HeLa cells, four carried insertions within fimbrial genes (fimA and traJ) and agglutinin genes (tia and hek). Using these Tn5 mutants as donors, we were able to obtain kanamycin-resistant E. coli MA3456 transconjugants. Sequence analysis of the plasmid genes revealed a region exhibit to 80 and 73% amino acid similarities to the agglutinins Tia and Hek, respectively.ConclusionIn this study, we have identified three large conjugative plasmids, pIS46, pIS66 and pIS102, coding for antimicrobial resistance and localized-like adherence (LAL) to HeLa cells. In addition, we identified a tia/hek homolog encoded on the pIS102 plasmid, which seems to be involved in adhesion of A102 strain.

Highly Transferable pAQU-Related Plasmids Encoding Multidrug Resistance Are Widespread in the Human and Fish Pathogen Photobacterium damselae subsp. damselae in Aquaculture Areas in the Black Sea.

The marine bacterium Photobacterium damselae subsp. damselae is a pathogen that causes disease in diverse marine animals, and is also a serious opportunistic human pathogen that can cause fatal infections. Strains of this pathogen isolated from diseased European sea bass in aquaculture facilities in the Turkish coast of the Black Sea were found to exhibit reduced sensitivity to multiple antimicrobials. Selected representative strains were subjected to complete genome sequencing and plasmid characterization. It was found that multidrug resistant (MDR) isolates harboured large conjugative plasmids sharing part of their sequence backbone with pAQU-group plasmids, hitherto reported exclusively in China and Japan. Four new pAQU-group versions of plasmids were identified in the present study, containing distinct combinations of the resistance determinants tetB, floR, sul2, qnrVC, dfrA and strAB. Conjugative transfer of pPHDD2-OG2, a representative plasmid of 170,998 bp, occurred at high frequencies (2.2 × 10-2 transconjugants per donor cell), to E. coli and to pathogenic P. damselae subsp. damselae and subsp. piscicida strains. Upon transfer, pPHDD2-OG2 conferred reduced susceptibility to a number of antimicrobials to the recipient strains. Comparative genomics analysis of host strains suggested that these MDR plasmids of the pAQU-group were acquired by different genetic lineages of Pdd. This study provides evidence that P. damselae subsp. damselae isolated from diseased fish constitute a reservoir for conjugative MDR pAQU-group plasmids in the Mediterranean basin, and have the potential to spread to diverse bacterial species.