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Lactococcal conjugative plasmids are poorly characterized compared to those harbored by numerous other Gram-positive bacteria, despite their significance in dairy fermentations and starter culture development. Furthermore, the transcriptional landscape of these lactococcal conjugation systems and their regulation have not been studied in any detail. Lactococcal plasmids pNP40 and pUC11B possess two genetically distinct and prevalent conjugation systems. Here, we describe the detailed transcriptional analysis of the pNP40 and pUC11B conjugation-associated gene clusters, revealing three and five promoters, respectively, for which the corresponding transcriptional start sites were identified. Regulation of several of these promoters, and therefore conjugation, is shown to involve the individual or concerted activities of the corresponding relaxase and transcriptional repressor(s) encoded by each conjugative plasmid. This work highlights how the conjugative potential of these systems may be unlocked, with significant implications for the starter culture and food fermentation industry.

With the development of multidrug resistance in Salmonella spp. in recent years, ciprofloxacin, ceftriaxone, and azithromycin have become the principal antimicrobial agents used for the treatment of Salmonella infections. The underlying mechanisms of plasmid-mediated ciprofloxacin and ceftriaxone resistance have attracted extensive research interest, but not much is focused on azithromycin resistance in Salmonella. In this study, we investigated the genetic features of two conjugative plasmids and a non-transferable virulence plasmid that encode azithromycin resistance in food-borne Salmonella strains. We showed that the azithromycin resistance phenotype of these strains was conferred by erm(B) gene and/or the complete genetic structure IS26-mph(A)-mrx-mphR-IS6100. Comparative genetic analysis showed that these conjugative plasmids might originate from Escherichia coli and play a role in the rapid dissemination of azithromycin resistance in Salmonella. These conjugative plasmids may also serve as a reservoir of antimicrobial resistance (AMR) genes in Salmonella in which these AMR genes may be acquired by the virulence plasmids of Salmonella via genetic transposition events. Importantly, the formation of a novel macrolide-resistance and virulence-encoding plasmid, namely pS1380-118 kb, was observed in this study. This plasmid was found to exhibit transmission potential and pose a serious health threat as the extensive transmission of azithromycin resistant and virulent Salmonella strains would further compromise the effectiveness of treatment for salmonellosis. Further surveillance and research on the dissemination and evolution routes of pS1380-118kb-like plasmids in potential human pathogens of the family of Enterobacteriaceae are necessary.

Bacterial cells often suffer a fitness cost after conjugative plasmids' entry because these cells replicate slower than plasmid-free cells. Compensatory mutations may appear after tens of or a few hundred generations, reducing or eliminating this cost. A previous work based on a mathematical model and computer simulations has shown that plasmid-bearing cells already adapted to the plasmid may gain a fitness advantage when plasmids transfer into neighboring plasmid-free cells because these cells are still unadapted to the plasmid. These slow-growing transconjugants use fewer resources, which can benefit donor cells. However, opportunities for compensatory mutations in transconjugants increase if these cells become numerous (through replication or conjugation). Moreover, transconjugants also gain an advantage when transferring the plasmid, but the original donors may be too distant from conjugation events to gain an advantage. To understand which consequence prevails, we performed further computer simulations allowing versus banning transfer from transconjugants. The advantage to donors is higher if transconjugants do not transfer plasmids, mainly when donors are rare and when the plasmid transfer rate (from donors) is high. These results show that conjugative plasmids are efficient biological weapons even if the transconjugant cells are poor plasmid donors. After some time, conjugative plasmids gain other host-benefit genes, such as virulence and drug-resistance. [ABSTRACT FROM AUTHOR]

ArdB, ArdA, and Ocr proteins inhibit the endonuclease activity of the type I restriction-modification enzymes (RMI). In this study, we evaluated the ability of ArdB, ArdA, and Ocr to inhibit different subtypes of Escherichia coli RMI systems (IA, IB, and IC) as well as two Bacillus licheniformis RMI systems. Furthermore we explored, the antirestriction activity of ArdA, ArdB, and Ocr against a type III restriction-modification system (RMIII) EcoPI and BREX. We found that DNA- mimic proteins, ArdA and Ocr exhibit different inhibition activity, depending on which RM system tested. This effect might be linked to the DNA mimicry nature of these proteins. In theory, DNA-mimicmight competitively inhibit any DNA-binding proteins; however, the efficiency of inhibition depend on the ability to imitate the recognition site in DNA or its preferred conformation. In contrast, ArdB protein with an undescribedmechanismof action, demonstrated greater versatility against various RMI systems and provided similar antirestriction efficiency regardless of the recognition site. However, ArdB protein could not affect restriction systems that are radically different from the RMI such as BREX or RMIII. Thus, we assume that the structure of DNA-mimic proteins allows for selective inhibition of any DNA-binding proteins depending on the recognition site. In contrast, ArdB-like proteins inhibit RMI systems independently of the DNA recognition site. [ABSTRACT FROM AUTHOR]

Background: Bacterial cells often harbor multiple plasmids, but the interaction between these plasmids is poorly understood. Pseudomonas putida ND6, one of the typical naphthalene-degrading bacteria, contains two annotated plasmids, pND6-1 and pND6-2. The conjugative plasmid pND6-2 can be used as a helper to mobilize the naphthalene-degrading plasmid pND6-1. The role of pND6-1 in functions other than naphthalene degradation has never been explored. Therefore, it is of great significance to study the possible effect of pND6-1 on the transfer rate or maintenance of pND6-2. Results: To investigate the interaction between pND6-1 and pND6-2, we successfully cured plasmid pND6-1 using the incompatibility method, generated the pND6-1-null strain ND6dp1, and first classified it into the IncP-7 group by curing experimental evidence. Subsequently, the pND6-1-cured mutant showed an insignificant increase in cell growth compared with the wild ND6, indicating that a lower cost was imposed on the host by pND6-1. The influences of pND6-1 on the stability and conjugation of plasmid pND6-2 were subsequently explored. The results showed that the presence of pND6-1 enhanced the stability of pND6-2 in natural host ND6 and heterologous host P. putida KT2440. In addition, the presence of pND6-1 in wild-type hosts promoted intra-specific and inter-specific conjugative transfer efficiency of pND6-2 by 3.9–6.3-fold with P. putida, P. resinovans, and P. fluorescens as the recipients, respectively. Conclusions: Collectively, these results suggest that the plasmid pND6-1 improves the pND6-2 plasmid's stability and transfer rate in the ND6 strain with the selected recipient strains.How to cite: Wang S, Liu M, Wang D, et al. Plasmid pND6-1 enhances the stability and conjugative transfer of co-resident companion plasmid pND6-2 in the naphthalene-degradative Pseudomonas putida strain ND6. Electron J Biotechnol 2022;59. https://doi.org/10.1016/j.ejbt.2022.08.002.

Abstract OXA‐23 is the predominant carbapenemase in carbapenem‐resistant Acinetobacter baumannii. The co‐evolutionary dynamics of A. baumannii and OXA‐23‐encoding plasmids are poorly understood. Here, we transformed A. baumannii ATCC 17978 with pAZJ221, a blaOXA−23‐containing plasmid from clinical A. baumannii isolate A221, and subjected the transformant to experimental evolution in the presence of a sub‐inhibitory concentration of imipenem for nearly 400 generations. We used population sequencing to track genetic changes at six time points and evaluated phenotypic changes. Increased fitness of evolving populations, temporary duplication of blaOXA−23 in pAZJ221, interfering allele dynamics, and chromosomal locus‐level parallelism were observed. To characterize genotype‐to‐phenotype associations, we focused on six mutations in parallel targets predicted to affect small RNAs and a cyclic dimeric (3′ → 5′) GMP‐metabolizing protein. Six isogenic mutants with or without pAZJ221 were engineered to test for the effects of these mutations on fitness costs and plasmid kinetics, and the evolved plasmid containing two copies of blaOXA−23 was transferred to ancestral ATCC 17978. Five of the six mutations contributed to improved fitness in the presence of pAZJ221 under imipenem pressure, and all but one of them impaired plasmid conjugation ability. The duplication of blaOXA−23 increased host fitness under carbapenem pressure but imposed a burden on the host in antibiotic‐free media relative to the ancestral pAZJ221. Overall, our study provides a framework for the co‐evolution of A. baumannii and a clinical blaOXA−23‐containing plasmid in the presence of imipenem, involving early blaOXA−23 duplication followed by chromosomal adaptations that improved the fitness of plasmid‐carrying cells.

ArdB, ArdA, and Ocr proteins inhibit the endonuclease activity of the type I restriction-modification enzymes (RMI). In this study, we evaluated the ability of ArdB, ArdA, and Ocr to inhibit different subtypes of Escherichia coli RMI systems (IA, IB, and IC) as well as two Bacillus licheniformis RMI systems. Furthermore we explored, the antirestriction activity of ArdA, ArdB, and Ocr against a type III restriction-modification system (RMIII) EcoPI and BREX. We found that DNA-mimic proteins, ArdA and Ocr exhibit different inhibition activity, depending on which RM system tested. This effect might be linked to the DNA mimicry nature of these proteins. In theory, DNA-mimic might competitively inhibit any DNA-binding proteins; however, the efficiency of inhibition depend on the ability to imitate the recognition site in DNA or its preferred conformation. In contrast, ArdB protein with an undescribed mechanism of action, demonstrated greater versatility against various RMI systems and provided similar antirestriction efficiency regardless of the recognition site. However, ArdB protein could not affect restriction systems that are radically different from the RMI such as BREX or RMIII. Thus, we assume that the structure of DNA-mimic proteins allows for selective inhibition of any DNA-binding proteins depending on the recognition site. In contrast, ArdB-like proteins inhibit RMI systems independently of the DNA recognition site.

Gene transfer in crenarchaea has been observed within natural and experimental populations of Sulfolobus. However, the molecular factors that govern how gene transfer and recombination manifest themselves in these populations is still unknown. In this study, we examine a plasmid-mediated mechanism of gene transfer in S. islandicus that results in localized high frequency recombination within the chromosome. Through chromosomal marker exchange assays with defined donors and recipients, we find that while bidirectional exchange occurs among all cells, those possessing the integrated conjugative plasmid, pM164, mobilize a nearby locus at a significantly higher frequency when compared to a more distal marker. We establish that traG is essential for this phenotype and that high frequency recombination can be replicated in transconjugants after plasmid transfer. Mapping recombinants through genomic analysis, we establish the distribution of recombinant tracts with decreasing frequency at increasing distance frompM164.We suggest the bias in transfer is a result of an Hfr (high frequency recombination)-like conjugation mechanism in this strain. In addition, we find recombinants containing distal non-selected recombination events, potentially mediated by a different host- encoded marker exchange (ME) mechanism. [ABSTRACT FROM AUTHOR]

Haishan Mo,1,* Xu Lin,1,* Ming Liu,2 Hui Liang1 1Department of General Surgery, Zhuhai People’s Hospital (Zhuhai Hospital Affiliated with Jinan University), Zhuhai, People’s Republic of China; 2College of Oceanography, Hohai University, Nanjing, People’s Republic of China*These authors contributed equally to this workCorrespondence: Hui Liang, Department of General Surgery, Zhuhai People’s Hospital (Zhuhai Hospital affiliated with Jinan University), Zhuhai, People’s Republic of China, Email zhlianghui@126.comAbstract: Cephalosporins and azithromycin are clinical antibiotics used to treat infections. Co-resistance to cephalosporins and azithromycin has been observed in some Enterobacterales, but it has only rarely been reported in Vibrio species. In this study, we isolated a cephalosporin- and azithromycin-resistant V. furnissii strain, VFN3, from hospital sewage. Whole-genome sequencing results showed that the strain VFN3 possesses an IncA/C2 plasmid, pVFN3-blaOXA-193K. This conjugative plasmid carries several clinically relevant drug resistance genes, including mph(A) and blaOXA-1. We also found that in the strain VFN3, mph(A) and blaOXA-1 are surrounded by insertion sequences and class I integrons, respectively. These data suggest that mobile elements mediate the transfer of mph(A) and blaOXA-1. This is the first reported Vibrio species that possesses an mph(A)- and blaOXA-1-bearing conjugative plasmid. The emergence of this conjugative multi-drug-resistance plasmid is of great concern to public health.Keywords: mph(A), blaOXA-1, conjugative plasmid, Vibrio furnissii

Abstract Background BEC-producing Clostridium perfringens is a causative agent of foodborne gastroenteritis. It was first reported in 2014, and since then, several isolates have been identified in Japan and the United Kingdom. The novel binary ADP-ribosylating toxin BEC, which consists of two components (BECa and BECb), is encoded on a plasmid that is similar to pCP13 and harbours a conjugation locus, called Pcp, encoding homologous proteins of the type 4 secretion system. Despite the high in vitro conjugation frequency of pCP13, its dissemination and that of related plasmids, including bec-harbouring plasmids, in the natural environment have not been characterised. This lack of knowledge has limited our understanding of the genomic epidemiology of bec-harbouring C. perfringens strains. Results In this study, we determined the complete genome sequences of five bec-harbouring C. perfringens strains isolated from 2009 to 2019. Each isolate contains a ~ 3.36 Mbp chromosome and 1–3 plasmids of either the pCW3-like family, pCP13-like family, or an unknown family, and the bec-encoding region in all five isolates was located on a ~ 54 kbp pCP13-like plasmid. Phylogenetic and SNP analyses of these complete genome sequences and the 211 assembled C. perfringens genomes in GenBank showed that although these bec-harbouring strains were split into two phylogenetic clades, the sequences of the bec-encoding plasmids were nearly identical (>99.81%), with a significantly smaller SNP accumulation rate than that of their chromosomes. Given that the Pcp locus is conserved in these pCP13-like plasmids, we propose a mechanism in which the plasmids were disseminated by horizontal gene transfer. Data mining showed that strains carrying pCP13-like family plasmids were unexpectedly common (58/216 strains) and widely disseminated among the various C. perfringens clades. Although these plasmids possess a conserved Pcp locus, their ‘accessory regions’ can accommodate a wide variety of genes, including virulence-associated genes, such as becA/becB and cbp2. These results suggest that this family of plasmids can integrate various foreign genes and is transmissible among C. perfringens strains. Conclusion This study demonstrates the potential significance of pCP13-like plasmids, including bec-encoding plasmids, for the characterisation and monitoring of the dissemination of pathogenic C. perfringens strains.

Gene transfer in crenarchaea has been observed within natural and experimental populations of Sulfolobus. However, the molecular factors that govern how gene transfer and recombination manifest themselves in these populations is still unknown. In this study, we examine a plasmid-mediated mechanism of gene transfer in S. islandicus that results in localized high frequency recombination within the chromosome. Through chromosomal marker exchange assays with defined donors and recipients, we find that while bidirectional exchange occurs among all cells, those possessing the integrated conjugative plasmid, pM164, mobilize a nearby locus at a significantly higher frequency when compared to a more distal marker. We establish that traG is essential for this phenotype and that high frequency recombination can be replicated in transconjugants after plasmid transfer. Mapping recombinants through genomic analysis, we establish the distribution of recombinant tracts with decreasing frequency at increasing distance from pM164. We suggest the bias in transfer is a result of an Hfr (high frequency recombination)-like conjugation mechanism in this strain. In addition, we find recombinants containing distal non-selected recombination events, potentially mediated by a different host-encoded marker exchange (ME) mechanism.

Pyrococcus furiosus is a hyperthermophilic archaeon with three effector CRISPR complexes (types I-A, I-B, and III-B) that each employ crRNAs derived from seven CRISPR arrays. Here, we investigate the CRISPR adaptation response to a newly discovered and self-transmissible plasmid, pT33.3. Transconjugant strains of Pyrococcus furiosus exhibited dramatically elevated levels of new spacer integration at CRISPR loci relative to the strain harboring a commonly employed, laboratory-constructed plasmid. High-throughput sequence analysis demonstrated that the vast majority of the newly acquired spacers were preferentially selected from DNA surrounding a particular region of the pT33.3 plasmid and exhibited a bi-directional pattern of strand bias that is a hallmark of primed adaptation by type I systems. We observed that one of the CRISPR arrays of our Pyrococcus furiosus laboratory strain encodes a spacer that closely matches the region of the conjugative plasmid that is targeted for adaptation. The hyper-adaptation phenotype was found to strictly depend both on the presence of this single matching spacer as well as the I-B effector complex, known to mediate primed adaptation. Our results indicate that Pyrococcus furiosus naturally encountered this conjugative plasmid or a related mobile genetic element in the past and responds to reinfection with robust primed adaptation. [ABSTRACT FROM AUTHOR]

Background: The carbapenem-resistant K. pneumoniae (CRKP) poses a serious threat to antibiotic applicability and public health. During treatment, K. pneumoniae (KP) frequently exhibits shifts in drug-resistant phenotypes, complicating clinical treatment as it transitions from sensitivity to resistance. In this study, we analyzed the clinical and molecular characteristics of drug resistance changes in KP strains isolated from a single patient, and the potential mechanisms underlying these resistance changes., Methods: Antimicrobial susceptibility test and string test were conducted to evaluate the resistant and virulent characterization of the strains. Pulsed field gel electrophoresis (PFGE) was used to investigate the homology relationship between the strains. The whole genome sequencing and phylogenetic analysis of 9 representative isolates was also performed. The transfer ability of the drug-resistant plasmid was studied by plasmid conjugation experiment. The transconjugants were verified by PCR amplification of specific genes, antimicrobial susceptibility test and PFGE., Results: Our results revealed that 9 KP strains, isolated from the same patient, exhibited 'resistance-sensitivity-resistance-sensitivity' alternately to carbapenems. The differences in DNA fingerprint bands among the nine KP isolates were ≤ 3, which can be classified as the same PFGE type. Phylogenetic analysis showed that these 9 strains constituted a distinct branch within the phylogenetic tree. All nine KP strains belonged to the ST15-KL19 clone. Six of the strains were classified as CRKP, all of which carried eleven drug resistance genes: oqxB, oqxA, fosA6, aac(3)-lld, bla SHV-28 , bla KPC-2 , bla OXA-1 , mph(A), tet(A), catB3 and aac(6')-lb-cr, mediating drug resistance to quinolones, fosfomycin, aminoglycosides, β-lactam, carbapenems, macrolides and chloramphenicol, belonging to multi-drug resistant bacteria. The carbapenem-resistant plasmid p2-KP3762-1 was found to transfer within species, from CRKP to hypervirulent K. pneumoniae (hvKP) RJF293HA, carbapenem-sensitive K. pneumoniae (CSKP) KP3657 and E. coli C600 at a frequency of (1.19±1.58) ×10 -6 , (1.09±1.38) ×10 -7 and (10.9±9.53) ×10 -6 respectively, resulting in the dissemination of carbapenem resistance genes., Conclusions: In this study, KP strains isolated from a single patient exhibited an alternating phenotype of resistant-sensitive-resistant-sensitive to carbapenems. The 9 KP isolates share a high degree of genetic similarity. The plasmid p2-KP3762-1, harboring the carbapenem resistance gene bla KPC-2 , may undergo inter-strain and inter-clone transfer via conjugation in the patient during treatment. Furthermore, our findings suggest that the pathogens in this patient are likely to have a common ancestral origin., (Copyright © 2024. Published by Elsevier Ltd.)

This retrospective investigation (2019-2022) identified two plasmid-mediated mcr-10 from 6800 food samples in Shanghai, China and localized in a conjugative plasmid (pEC1918-mcr10) in Escherichia kobei from ready-to-eat food with high-level polymyxin B resistance, and a nonconjugative plasmid (pEC2001-mcr10) in E. coli from chicken. These genes were adjacent to IS Ec36 . This report highlights the emergence of mcr-10 from food samples in Shanghai, China. Active surveillance of vital resistance genes along food production chain should be performed.

Multidrug-resistant (MDR) Pseudomonas aeruginosa poses a great challenge to clinical treatment. In this study, we characterized a ST768 MDR P. aeruginosa strain, Pa150, that was isolated from a diabetic foot patient. The minimum inhibitory concentration (MIC) assay showed that Pa150 was resistant to almost all kinds of antibiotics, especially aminoglycosides. Whole genome sequencing revealed multiple antibiotic resistant genes on the chromosome and a 437-Kb plasmid (named pTJPa150) that harbors conjugation-related genes. A conjugation assay verified its self-transmissibility. On the pTJPa150 plasmid, we identified a 16S rRNA methylase gene, rmtB, that is flanked by mobile genetic elements (MGEs). The transfer of the pTJPa150 plasmid or the cloning of the rmtB gene into the reference strain, PAO1, significantly increased the bacterial resistance to aminoglycoside antibiotics. To the best of our knowledge, this is the first report of an rmtB-carrying conjugative plasmid isolated from P. aeruginosa, revealing a novel possible transmission mechanism of the rmtB gene. [ABSTRACT FROM AUTHOR]

Reports on multidrug-resistant hypervirulent Klebsiella pneumoniae (MDR-hvKP) in recent years indicate the wide-spreading trend of MDR-hvKP. The co-occurrence of hypervirulence and drug resistance poses a great challenge to clinical treatment. In this study, molecular characteristics of an MDR strain hvKP247 and 30 clinically isolated hvKP strains were characterized. The whole genome of hvKP247 belonging to sequence type (ST) 5214 and capsule serotype K1 was sequenced and analyzed. Conjugation experiments were performed to evaluate transferability of the plasmids in hvKP247. We found two new STs among our isolates, ST5570 and ST5571. The ST5214 hvKP247 contained two transferable plasmids: a hybrid virulence plasmid (pHvKP247-vir) carrying transfer-related modules that had self-transferable ability, and a drug-resistant plasmid (pHvKP247-MDR) that could be indirectly transferred with the help of pHvKP247-vir. The virulence-related genes were located on the pHvKP247-vir and chromosomal ICEKp1 mobile genetic element. In conclusion, the hybrid virulence plasmid and the drug-resistant plasmid are co-transferred, which emphasizes the importance of raising public awareness of the simultaneous spread of virulence and resistance genes of MDR-hvKP strains. [ABSTRACT FROM AUTHOR]

The main mechanism of virulence in Klebsiella pneumoniae is the acquisition of K. pneumoniae virulence plasmids (KpVPs), which include two dominant types, namely, KpVP-1 (carrying iuc1, iro1, rmpA, and rmpA2) and KpVP-2 (carrying iuc2, iro2, and rmpA). Both are non-conjugative and associated with different hypervirulent clones. In contrast to KpVP-1 reported in K1, K2, and other serotypes of K. pneumoniae, KpVP-2 was only reported in K2 strains and rarely characterized. In this study, we identified a conjugative KpVP-2-type virulence plasmid from a clinical hypervirulent K. pneumoniae strain. This plasmid was generated by the integration of conjugative transfer genes into the KpVP-2-type plasmid Kp52.145 II and could be readily conjugated to Escherichia coli strain EC600 and K. pneumoniae strains of various types which are clinically existing, mediating hypervirulence. Furthermore, this kind of conjugative KpVP-2-type virulence plasmid has been disseminated in clinical settings in Hong Kong and other regions of the world. The generation of conjugative virulence plasmid may promote its transmission and explain the evolution of this type of virulence plasmid. [ABSTRACT FROM AUTHOR]

OXA‐23 is the predominant carbapenemase in carbapenem‐resistant Acinetobacter baumannii. The co‐evolutionary dynamics of A. baumannii and OXA‐23‐encoding plasmids are poorly understood. Here, we transformed A. baumannii ATCC 17978 with pAZJ221, a blaOXA−23‐containing plasmid from clinical A. baumannii isolate A221, and subjected the transformant to experimental evolution in the presence of a sub‐inhibitory concentration of imipenem for nearly 400 generations. We used population sequencing to track genetic changes at six time points and evaluated phenotypic changes. Increased fitness of evolving populations, temporary duplication of blaOXA−23 in pAZJ221, interfering allele dynamics, and chromosomal locus‐level parallelism were observed. To characterize genotype‐to‐phenotype associations, we focused on six mutations in parallel targets predicted to affect small RNAs and a cyclic dimeric (3′ → 5′) GMP‐metabolizing protein. Six isogenic mutants with or without pAZJ221 were engineered to test for the effects of these mutations on fitness costs and plasmid kinetics, and the evolved plasmid containing two copies of blaOXA−23 was transferred to ancestral ATCC 17978. Five of the six mutations contributed to improved fitness in the presence of pAZJ221 under imipenem pressure, and all but one of them impaired plasmid conjugation ability. The duplication of blaOXA−23 increased host fitness under carbapenem pressure but imposed a burden on the host in antibiotic‐free media relative to the ancestral pAZJ221. Overall, our study provides a framework for the co‐evolution of A. baumannii and a clinical blaOXA−23‐containing plasmid in the presence of imipenem, involving early blaOXA−23 duplication followed by chromosomal adaptations that improved the fitness of plasmid‐carrying cells. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii is a major cause of nosocomial infection, and the incidence of extensively drug-resistant A. baumannii (XDRAB) infections has dramatically increased worldwide. In this study, we aimed to explore the complete genome sequence of XDRAB 329, ST1166/98 (Oxford/Pasteur), which is an outbreak clone from a hospital in Thailand. Whole-genome sequencing (WGS) was performed using short-read Illumina and long-read PacBio sequencing, and a conjugation assay of its plasmid was performed. The complete genome sequence of A. baumannii AB329 revealed a circular chromosome 3,948,038 bp in length with 39% GC content. Antibiotic resistance genes (ARGs), including beta-lactam resistance (blaOXA-51, blaADC-25, blaOXA-23, blaTEM-1D), aminoglycoside resistance (aph(30)-Ia, aph(300)-Ib, aph(6)-Id, armA), tetracycline resistance (tet (B), tet (R)), macrolide resistance (mph(E), msr(E)), and efflux pumps, were found. Mobile genetic elements (MGEs) analysis of A. baumannii AB329 revealed two plasmids (pAB329a and pAB329b), three prophages, 19 genomic islands (GIs), and 33 insertion sequences (ISs). pAB329a is a small circular plasmid of 8,731 bp, and pAB329b is a megaplasmid of 82,120 bp. aph(30)-VIa was detected in pAB329b, and a major facilitator superfamily (MFS) transporter was detected in the prophage. Acinetobacter baumannii resistance island 4 (AbaR4) harboring tetracycline and aminoglycoside resistance was detected in the genome of A. baumannii AB329. pAB329b, which belongs to Rep-type GR6 (plasmid lineage LN_1), is a conjugative plasmid with the ability to transfer an aminoglycoside resistance gene to sodium azide-resistant A. baumannii. This study provides insights into the features of the MGEs of XDRAB, which are the main reservoir and source of dissemination of ARGs. [ABSTRACT FROM AUTHOR]

ABSTRACT: Objectives: We sequenced two IncA/C plasmids harbouring blaCTX-M-2 in Klebsiella pneumoniae clinical isolates and compared their antibiotic resistance islands. Methods: Transconjugants were obtained from two clinical K. pneumoniae isolates harbouring blaCTX-M-2. Plasmid DNA from transconjugants underwent short-read whole-genome sequencing, reads were assembled, and gaps were closed by PCR and sequencing. Determination of plasmid replicons, antibiotic resistance genes, identification and characterisation of insertion sequence (IS) elements, and comparison with publicly available plasmid sequences were performed. Results: blaCTX-M-2 was located in a complex class 1 integron In35::ISCR1::blaCTX-M-2, inserted in two different transposons designated Tn7057 and Tn7058, that reside in the resistance islands of plasmids pUR-KP0923 and pUR-KP1025, respectively. The general modules of both transposons were In35::ISCR1::blaCTX-M-2–Tn1000-like–Tn2*–ISKpn11-12-13 variable module–ΔTn21. In Tn7057 there was ΔIS10R–catA2 associated with an additional ISKpn13. Both plasmids belonged to IncC type 2 and ST3. pUR-KP0923 was 167 138 bp in length and had a 37 926-bp resistance island at position 4 (RI-4). Plasmid pUR-KP1025 was 168 128 bp with a RI-4 of 36 222 bp. Conclusion: This report describes the molecular nature of two transposons (Tn7057 and Tn7058) harbouring blaCTX-M-2 that reside in IncC type 2 ST3 plasmids. These transposons mediate resistance to oxyimino-cephalosporins, gentamicin and, in the case of Tn7057, chloramphenicol. CTX-M-2 is an important extended-spectrum β-lactamase (ESBL) to South American epidemiology. It is remarkable that despite being only two plasmid sequences, the information revealed here could contribute to a better understanding of the resistance islands from IncC type 2 plasmids.

Acinetobacter baumannii is a major cause of nosocomial infection, and the incidence of extensively drug-resistant A. baumannii (XDRAB) infections has dramatically increased worldwide. In this study, we aimed to explore the complete genome sequence of XDRAB 329, ST1166/98 (Oxford/Pasteur), which is an outbreak clone from a hospital in Thailand. Whole-genome sequencing (WGS) was performed using short-read Illumina and long-read PacBio sequencing, and a conjugation assay of its plasmid was performed. The complete genome sequence of A. baumannii AB329 revealed a circular chromosome 3,948,038 bp in length with 39% GC content. Antibiotic resistance genes (ARGs), including beta-lactam resistance (blaOXA-51, blaADC-25, blaOXA-23, blaTEM-1D), aminoglycoside resistance (aph(3′)-Ia, aph(3″)-Ib, aph(6)-Id, armA), tetracycline resistance (tet(B), tet (R)), macrolide resistance (mph(E), msr(E)), and efflux pumps, were found. Mobile genetic elements (MGEs) analysis of A. baumannii AB329 revealed two plasmids (pAB329a and pAB329b), three prophages, 19 genomic islands (GIs), and 33 insertion sequences (ISs). pAB329a is a small circular plasmid of 8,731 bp, and pAB329b is a megaplasmid of 82,120 bp. aph(3′)-VIa was detected in pAB329b, and a major facilitator superfamily (MFS) transporter was detected in the prophage. Acinetobacter baumannii resistance island 4 (AbaR4) harboring tetracycline and aminoglycoside resistance was detected in the genome of A. baumannii AB329. pAB329b, which belongs to Rep-type GR6 (plasmid lineage LN_1), is a conjugative plasmid with the ability to transfer an aminoglycoside resistance gene to sodium azide-resistant A. baumannii. This study provides insights into the features of the MGEs of XDRAB, which are the main reservoir and source of dissemination of ARGs.

Plasmids orchestrate bacterial adaptation across diverse environments and facilitate lateral gene transfer within bacterial communities. Their presence can perturb host metabolism, creating a competitive advantage for plasmid-free cells. Plasmid stability hinges on efficient replication and partition mechanisms. While plasmids commonly encode histone-like nucleoid-structuring (H-NS) family proteins, the precise influence of plasmid-encoded H-NS proteins on stability remains elusive. In this study, we examined the conjugative plasmid pMBL6842, harboring the hns gene, and observed its positive regulation of parAB transcription, critical for plasmid segregation. Deletion of hns led to rapid plasmid loss, which was remedied by hns complementation. Further investigations unveiled adverse effects of hns overexpression on the bacterial host. Transcriptome analysis revealed hns 's role in regulating numerous bacterial genes, impacting both host growth and swimming motility in the presence of the hns gene. Therefore, our study unveils the multifaceted roles of H-NS in both plasmid stability and host physiology, underscoring its biological significance and paving the way for future inquiries into the involvement of H-NS in horizontal gene transfer events. [ABSTRACT FROM AUTHOR]

Bacterial cells often suffer a fitness cost after conjugative plasmids’ entry because these cells replicate slower than plasmid-free cells. Compensatory mutations may appear after tens of or a few hundred generations, reducing or eliminating this cost. A previous work based on a mathematical model and computer simulations has shown that plasmid-bearing cells already adapted to the plasmid may gain a fitness advantage when plasmids transfer into neighboring plasmid-free cells because these cells are still unadapted to the plasmid. These slow-growing transconjugants use fewer resources, which can benefit donor cells. However, opportunities for compensatory mutations in transconjugants increase if these cells become numerous (through replication or conjugation). Moreover, transconjugants also gain an advantage when transferring the plasmid, but the original donors may be too distant from conjugation events to gain an advantage. To understand which consequence prevails, we performed further computer simulations allowing versus banning transfer from transconjugants. The advantage to donors is higher if transconjugants do not transfer plasmids, mainly when donors are rare and when the plasmid transfer rate (from donors) is high. These results show that conjugative plasmids are efficient biological weapons even if the transconjugant cells are poor plasmid donors. After some time, conjugative plasmids gain other host-benefit genes, such as virulence and drug-resistance.

The Burkholderia cepacia complex (Bcc) is a group of increasingly multi-drug resistant opportunistic bacteria. This resistance is driven through a combination of intrinsic factors and the carriage of a broad range of conjugative plasmids harbouring virulence determinants. Therefore, novel treatments are required to treat and prevent further spread of these virulence determinants. In the search for phages infective for clinical Bcc isolates, CSP1 phage, a PRD1-like phage was isolated. CSP1 phage was found to require pilus machinery commonly encoded on conjugative plasmids to facilitate infection of Gram-negative bacteria genera including Escherichia and Pseudomonas. Whole genome sequencing and characterisation of one of the clinical Burkholderia isolates revealed it to be Burkholderia contaminans. B. contaminans 5080 was found to contain a genome of over 8 Mbp encoding multiple intrinsic resistance factors, such as efflux pump systems, but more interestingly, carried three novel plasmids encoding multiple putative virulence factors for increased host fitness, including antimicrobial resistance. Even though PRD1-like phages are broad host range, their use in novel antimicrobial treatments shouldn't be dismissed, as the dissemination potential of conjugative plasmids is extensive. Continued survey of clinical bacterial strains is also key to understanding the spread of antimicrobial resistance determinants and plasmid evolution. [ABSTRACT FROM AUTHOR]

Cephalosporins and azithromycin are clinical antibiotics used to treat infections. Co-resistance to cephalosporins and azithromycin has been observed in some Enterobacterales, but it has only rarely been reported in Vibrio species. In this study, we isolated a cephalosporin- and azithromycin-resistant V. furnissii strain, VFN3, from hospital sewage. Whole-genome sequencing results showed that the strain VFN3 possesses an IncA/C2 plasmid, pVFN3-blaOXA-193K. This conjugative plasmid carries several clinically relevant drug resistance genes, including mph(A) and blaOXA-1. We also found that in the strain VFN3, mph(A) and blaOXA-1 are surrounded by insertion sequences and class I integrons, respectively. These data suggest that mobile elements mediate the transfer of mph(A) and blaOXA-1. This is the first reported Vibrio species that possesses an mph(A)- and blaOXA-1-bearing conjugative plasmid. The emergence of this conjugative multi-drug-resistance plasmid is of great concern to public health. [ABSTRACT FROM AUTHOR]

Campylobacter jejuni is a foodborne pathogen causing bacterial gastroenteritis, with the highest incidence reported in Europe. The prevalence of antibiotic resistance in C. jejuni, as well as in many other bacterial pathogens, has increased over the last few years. In this report, we describe the presence of a plasmid in a multi-drug-resistant C. jejuni strain isolated from a gastroenteritis patient. Mating experiments demonstrated the transference of this genetic element (pCjH01) among C. jejuni by plasmid conjugation. The pCjH01 plasmid was sequenced and assembled, revealing high similarity (97% identity) with pTet, a described tetracycline resistance encoding plasmid. pCjH01 (47.7 kb) is a mosaic plasmid composed of a pTet backbone that has acquired two discrete DNA regions. Remarkably, one of the acquired sequences carried an undescribed variant of the aadE-sat4-aphA-3 gene cluster, providing resistance to at least kanamycin and gentamycin. Aside from the antibiotic resistance genes, the cluster also carries genes coding for putative regulators, such as a sigma factor of the RNA polymerase and an antisigma factor. Homology searches suggest that Campylobacter exchanges genetic material with distant G-positive bacterial genera. [ABSTRACT FROM AUTHOR]

ABSTRACT Type IV pili (T4P) are common bacterial surface appendages involved in different biological processes such as adherence, motility, competence, pathogenesis, and conjugation. In this work, we describe the T4P of TP114, an IncI2 enterobacterial conjugative plasmid recently shown to disseminate at high rates in the mouse intestinal tract. This pilus is composed of the major PilS and minor PilV pilins that are both important for conjugation in broth and in the gut microbiota but not on a solid support. The PilV-coding sequence is part of a shufflon and can bear different C-terminal domains. The shufflon is a multiple DNA inversion system containing many DNA cassettes flanked by recombination sites that are recognized by a shufflon-specific tyrosine recombinase (shufflase) promoting the recombination between DNA segments. The different PilV variants act as adhesins that can modify the affinity for different recipient bacteria. Eight PilV variants were identified in TP114, including one that has not been described in other shufflons. All PilV variants allowed conjugative transfer with different recipient Escherichia coli strains. We conclude that the T4P carried by TP114 plays a major role in mating pair stabilization in broth as well as in the gut microbiota and that the shufflon acts as a biological switch modifying the conjugative host range specificity. IMPORTANCE Conjugative plasmids are involved in horizontal gene transfer in the gut microbiota, which constitutes an important antibiotic resistance gene reservoir. However, the molecular mechanisms used by conjugative plasmids to select recipient bacteria and transfer at high rates in the mouse gut microbiota remain poorly characterized. We studied the type IV pilus carried by TP114 and demonstrated that the minor pilin PilV acts as an adhesin that can efficiently select target cells for conjugative transfer. Moreover, the pilV gene can be rapidly modified by a shufflon, hence modulating the nature of the recipient bacteria during conjugation. Our study highlights the role of mating pair stabilization for conjugation in broth as well as in the gut microbiome and explains how the host spectrum of a plasmid can be expanded simply by remodeling the PilV adhesin.

Background: BEC-producing Clostridium perfringens is a causative agent of foodborne gastroenteritis. It was first reported in 2014, and since then, several isolates have been identified in Japan and the United Kingdom. The novel binary ADP-ribosylating toxin BEC, which consists of two components (BECa and BECb), is encoded on a plasmid that is similar to pCP13 and harbours a conjugation locus, called Pcp, encoding homologous proteins of the type 4 secretion system. Despite the high in vitro conjugation frequency of pCP13, its dissemination and that of related plasmids, including bec-harbouring plasmids, in the natural environment have not been characterised. This lack of knowledge has limited our understanding of the genomic epidemiology of bec-harbouring C. perfringens strains. Results: In this study, we determined the complete genome sequences of five bec-harbouring C. perfringens strains isolated from 2009 to 2019. Each isolate contains a ~ 3.36 Mbp chromosome and 1–3 plasmids of either the pCW3-like family, pCP13-like family, or an unknown family, and the bec-encoding region in all five isolates was located on a ~ 54 kbp pCP13-like plasmid. Phylogenetic and SNP analyses of these complete genome sequences and the 211 assembled C. perfringens genomes in GenBank showed that although these bec-harbouring strains were split into two phylogenetic clades, the sequences of the bec-encoding plasmids were nearly identical (>99.81%), with a significantly smaller SNP accumulation rate than that of their chromosomes. Given that the Pcp locus is conserved in these pCP13-like plasmids, we propose a mechanism in which the plasmids were disseminated by horizontal gene transfer. Data mining showed that strains carrying pCP13-like family plasmids were unexpectedly common (58/216 strains) and widely disseminated among the various C. perfringens clades. Although these plasmids possess a conserved Pcp locus, their 'accessory regions' can accommodate a wide variety of genes, including virulence-associated genes, such as becA/becB and cbp2. These results suggest that this family of plasmids can integrate various foreign genes and is transmissible among C. perfringens strains. Conclusion: This study demonstrates the potential significance of pCP13-like plasmids, including bec-encoding plasmids, for the characterisation and monitoring of the dissemination of pathogenic C. perfringens strains. [ABSTRACT FROM AUTHOR]

Abstract Background Carbapenem resistant Acinetobacter species have caused great difficulties in clinical therapy in the worldwide. Here we describe an Acinetobacter johnsonii M19 with a novel bla OXA-23 containing transposon Tn6681 on the conjugative plasmid pFM-M19 and the ability to transferand carbapenem resistance. Methods A. johnsonii M19 was isolated under selection with 8 mg/L meropenem from hospital sewage, and the minimum inhibitory concentrations (MICs) for the representative carbapenems imipenem, meropenem and ertapenem were determined. The genome of A. johnsonii M19 was sequenced by PacBio RS II and Illumina HiSeq 4000 platforms. A homologous model of OXA-23 was generated, and molecular docking models with imipenem, meropenem and ertapenem were constructed by Discovery Studio 2.0. Type IV secretion system and conjugation elements were identified by the Pathosystems Resource Integration Center (PATRIC) server and the oriTfinder. Mating experiments were performed to evaluate transfer of OXA-23 to Escherichia coli 25DN. Results MICs of A. johnsonii M19 for imipenem, meropenem and ertapenem were 128 mg/L, 48 mg/L and 24 mg/L, respectively. Genome sequencing identified plasmid pFM-M19, which harbours the carbapenem resistance gene bla OXA-23 within the novel transposon Tn6681. Molecular docking analysis indicated that the elongated hydrophobic tunnel of OXA-23 provides a hydrophobic environment and that Lys-216, Thr-217, Met-221 and Arg-259 were the conserved amino acids bound to imipenem, meropenem and ertapenem. Furthermore, pFM-M19 could transfer bla OXA-23 to E. coli 25DN by conjugation, resulting in carbapenem-resistant transconjugants. Conclusions Our investigation showed that A. johnsonii M19 is a source and disseminator of bla OXA-23 and carbapenem resistance. The ability to transfer bla OXA-23 to other species by the conjugative plasmid pFM-M19 raises the risk of spread of carbapenem resistance. Graphic abstract The carbapenem resistance gene bla OXA-23 is disseminated by a conjugative plasmid containing the novel transposon Tn6681 in Acinetobacter johnsonii M19.

Introduction Aminoglycosides are formidable broad-spectrum antibiotics used in clinical settings; woefully their usage has been reduced by the emergence and distribution of resistance mainly due to aminoglycoside modifying enzymes (AME). Purpose This study was performed to determine the diverse prevalence of AME and their pattern of occurrence in the clinical isolates of gram-negative bacteria. This study also aimed to detect the presence of AMEs that are prevalent in gram-positive bacteria, among gram negatives. Materials and Methods A total number of 386 clinical isolates were included in this study. Polymerase chain reaction revealed the prevalence rate of AMEs screened [aac(6′)-lb, aac(3′)-I, aac(3′)-II, aac(3′)-VI, ant(2′)-I, ant(4′)-IIb, aac(3′)-III, aac(3′)-IV, aph(2′)-Ib, aph(2′)-Ic, aph(2′)-Id, aac (6′)-Ie- aph(2′)-Ia, and aph(3′)-IIIa]. Conjugation experiment was performed for the clinical isolates which harbored any one of the AME which was prevalent in gram-positive bacteria [aph(3′)-IIIa, aac(6′)-Ie-aph(2′)-Ia]. Results aac(6′)-lb is the most prevalent AME, followed by aac(3′)-I, aph(3′)-VI, aac(3′)-VI, and aac(3′)-II. The AMEs such as ant (2′)-I, ant(4′)-IIb, aac(3′)-III, aac(3′)-IV, aph(2′)-Ib, aph(2′)-Ic, and aph(2′)-Id were not established in our study isolates. The rate of prevalence of aph(3′)-IIIa, aac(6′)-Ie-aph(2′)-Ia—the AMEs encountered in gram-positive and their co-existence was 19.68% and the conjugation experiment revealed their transfer via plasmids. Conclusion This is the first report from India revealing the presence and prevalence of AMEs which are often encountered among gram-positive bacteria in gram negatives and their presence on conjugative plasmids.

ABSTRACT The spread of resistance genes via horizontal plasmid transfer plays a significant role in the formation of multidrug-resistant (MDR) Pseudomonas aeruginosa strains. Here, we identified a megaplasmid (ca. 513 kb), designated pPAG5, which was recovered from a clinical multidrug-resistant P. aeruginosa PAG5 strain. The pPAG5 plasmid belonged to the IncP-2 incompatibility group. Two large multidrug resistance regions (MDR-1 and MDR-2) and two heavy metal resistance operons (merEDACPTR and terZABCDE) were identified in the pPAG5 plasmid. Genetic analysis demonstrated that the formation of MDR regions was mediated by several homologous recombination events. Further conjugation assays identified that pPAG5 could be transferred to P. aeruginosa but not Escherichia coli. Antimicrobial susceptibility testing on transconjugants demonstrated that pPAG5 was capable of transferring resistance genes to transconjugants and producing a multidrug-resistant phenotype. Comparative analysis revealed that pPAG5 and related plasmids shared an overall similar backbone, including genes essential for replication (repA), partition (par), and conjugal transfer (tra). Further phylogenetic analysis showed that pPAG5 was closely related to plasmids pOZ176 and pJB37, both of which are members of the IncP-2-type plasmid group. IMPORTANCE The emergence and spread of plasmid-associated multidrug resistance in bacterial pathogens is a key global threat to public health. It is important to understand the mechanisms of the formation and evolution of these plasmids in patients, hospitals, and the environment. In this study, we detailed the genetic characteristics of a multidrug resistance IncP-2 megaplasmid, pPAG5, and investigated the formation of its MDR regions and evolution. To the best of our knowledge, plasmid pPAG5 is the largest multidrug resistance plasmid ever sequenced in the Pseudomonas genus. Our results may provide further insight into the formation of multidrug resistance plasmids in bacteria and the molecular evolution of plasmids.

This study reported the involvement of a gene cluster from a conjugative plasmid in the biofilm formation of Escherichia coli. We used a novel EZ-Tn5 transposon technique to generate a transposon library and used arbitrarily primed PCR to detect the insertion sites in biofilm formation-deficient mutants. To validate the function of candidate biofilm formation genes, the genes were cloned into plasmid pBluescript II SK (+) and transformed into E. coil DH5α. Biofilm production from the transformants was then assessed by phenotypic biofilm formation using Crystal Violet staining and microscopy. A total of 3,000 transposon mutants of E. coli DH5α-p253 were screened, of which 28 were found to be deficient in biofilm formation. Further characterization revealed that 24/28 mutations were detected with their insertions in chromosome, while the remaining 4 mutations were evidenced that the functional genes for biofilm formation were harbored in the plasmid. Interestingly, the plasmid sequencing showed that these four transposon mutations were all inserted into a fimbriae-associated gene cluster (fim-cluster). This fim-cluster is a hybrid segment spanning a 7,949 bp sequence, with a terminal inverted repeat sequence and two coding regions. In summary, we performed a high-efficiency screening to a library constructed with the EZ-Tn5-based transposon approach and identified the gene clusters responsible for the biofilm production of E. coli, especially the genes harbored in the plasmid. Further studies are needed to understand the spread of this novel plasmid-mediated biofilm formation gene in clinical E. coli isolates and the clinical impacts. [ABSTRACT FROM AUTHOR]

ABSTRACTCeftriaxone and ciprofloxacin are the drugs of choice in treatment of invasive Salmonella infections. This study discovered a novel type of plasmid, pSa44-CIP-CRO, which was recovered from a S. London strain isolated from meat product and comprised genetic determinants that encoded resistance to both ciprofloxacin and ceftriaxone. This plasmid could be resolved into two daughter plasmids and co-exist with such daughter plasmids in a dynamic form in Salmonella; yet it was only present as a single plasmid in Escherichia coli. One daughter plasmid, pSa44-CRO, was found to carry the blaCTX-M-130 gene, which encodes resistance to ceftriaxone, whereas the other plasmid, pSa44-CIP, carried multiple PMQR genes such as qnrB6-aac(6’)-Ib-cr, which mediated resistance to ciprofloxacin. These two daughter plasmids could be integrated into one single plasmid through ISPa40 mediated homologous recombination. Mouse infection and treatment experiments showed that carriage of plasmid, pSa44-CIP-CRO by S. typhimurium led to the impairment of treatment by ciprofloxacin or cefitiofur, a veterinary drug with similar properties as ceftriaxone. In conclusion, dissemination of such conjugative plasmids impairs current choices of treatment for life-threatening Salmonella infection and hence constitutes a serious public health threat.

Multidrug-resistant (MDR) Pseudomonas aeruginosa poses a great challenge to clinical treatment. In this study, we characterized a ST768 MDR P. aeruginosa strain, Pa150, that was isolated from a diabetic foot patient. The minimum inhibitory concentration (MIC) assay showed that Pa150 was resistant to almost all kinds of antibiotics, especially aminoglycosides. Whole genome sequencing revealed multiple antibiotic resistant genes on the chromosome and a 437-Kb plasmid (named pTJPa150) that harbors conjugation-related genes. A conjugation assay verified its self-transmissibility. On the pTJPa150 plasmid, we identified a 16S rRNA methylase gene, rmtB, that is flanked by mobile genetic elements (MGEs). The transfer of the pTJPa150 plasmid or the cloning of the rmtB gene into the reference strain, PAO1, significantly increased the bacterial resistance to aminoglycoside antibiotics. To the best of our knowledge, this is the first report of an rmtB-carrying conjugative plasmid isolated from P. aeruginosa, revealing a novel possible transmission mechanism of the rmtB gene.

lsa(E) is a pleuromutilin, lincosamide, and streptogramin A (PLSA phenotype) resistance gene that was first described in S. aureus and was thought to have been transferred from Enterococcus sp. This study aimed to elucidate the prevalence of the lsa(E) gene among E. faecium isolates at a tertiary teaching hospital and to evaluate the transferability of the lsa(E) gene from E. faecium to S. aureus in vitro. A total of 96 E. faecium strains isolated from one hospital in Beijing in 2013 were analysed for quinupristin-dalfopristin (QDA) resistance genes, and multilocus sequence typing (MLST) was performed. The transferability of QDA resistance between ten E. faecium strains and four S. aureus strains was determined by filter mating. Genome sequencing of the transconjugant was performed. A total of 46 E. faecium isolates (46/96, 47.92%) tested positive for lsa(E), while two isolates (2/96, 2.08%) tested positive for lsa(A). Thirty-six lsa(E)-positive strains (36/46, 78.3%) belonged to ST78. Among 40 mating tests, lsa(E) was successfully transferred through one conjugation at a frequency of 1.125 × 10–7 transconjugants per donor. The QDA resistance of the transconjugant N7435-R3645 was expressed at a higher level (MIC = 16 mg/L) than that of the parent S. aureus strain (MIC = 0.38 mg/L). Next-generation sequencing (NGS) analysis of the transconjugant N7435-R3645 showed that the complete sequence of the lsa(E)-carrying plasmid pN7435-R3645 had a size of 92,396 bp and a G + C content of 33% (accession no. MT022086). The genetic map of pN7435-R3645 had high nucleotide similarity and shared the main open reading frame (ORF) features with two plasmids: E. faecium pMG1 (AB206333.1) and E. faecium LS170308 (CP025078.1). The rep gene of pN7435-R3645 showed 100% identity with that of pMG1, although it did not belong to the rep1-19 family but instead a unique rep family. Multiple antibiotic resistance genes, including lsa(E), aadE and lnu(B), erm(B), ant6-Ia, and lnu(B), were present on the plasmid. In conclusion, an lsa(E)-carrying plasmid that can be transferred by conjugation from E. faecium to S. aureus in vitro was identified. This multidrug resistance (MDR) pMG1-like plasmid may act as a vector in the dissemination of antimicrobial resistance among species.

Plasmid pNP40, which was first identified nearly 40 years ago in Lactococcus lactis subsp. lactis biovar diacetylactis DRC3, encodes functions such as heavy metal-, bacteriophage-, and nisin-resistance, as well as plasmid transfer ability by conjugation. Here, we report an optimized conjugation protocol for this plasmid, yielding a transfer frequency that is approximately 4,000-fold higher than those previously reported in literature, while we also observed high-frequency plasmid co-mobilization. Individual mutations in 18 genes that encompass the presumed conjugation cluster of pNP40 were generated using ssDNA recombineering to evaluate the role of each gene in the conjugation process. A possible transcriptional repressor of this conjugation cluster, the product of the traR gene, was identified in this manner. This mutational analysis, paired with bioinformatic predictions as based on sequence and structural similarities, allowed us to generate a preliminary model of the pNP40 conjugation machinery.

lsa (E) is a pleuromutilin, lincosamide, and streptogramin A (PLSA phenotype) resistance gene that was first described in S. aureus and was thought to have been transferred from Enterococcus sp. This study aimed to elucidate the prevalence of the lsa (E) gene among E. faecium isolates at a tertiary teaching hospital and to evaluate the transferability of the lsa (E) gene from E. faecium to S. aureus in vitro. A total of 96 E. faecium strains isolated from one hospital in Beijing in 2013 were analysed for quinupristin-dalfopristin (QDA) resistance genes, and multilocus sequence typing (MLST) was performed. The transferability of QDA resistance between ten E. faecium strains and four S. aureus strains was determined by filter mating. Genome sequencing of the transconjugant was performed. A total of 46 E. faecium isolates (46/96, 47.92%) tested positive for lsa (E), while two isolates (2/96, 2.08%) tested positive for lsa (A). Thirty-six lsa (E)-positive strains (36/46, 78.3%) belonged to ST78. Among 40 mating tests, lsa (E) was successfully transferred through one conjugation at a frequency of 1.125 × 10–7 transconjugants per donor. The QDA resistance of the transconjugant N7435-R3645 was expressed at a higher level (MIC = 16 mg/L) than that of the parent S. aureus strain (MIC = 0.38 mg/L). Next-generation sequencing (NGS) analysis of the transconjugant N7435-R3645 showed that the complete sequence of the lsa (E)-carrying plasmid pN7435-R3645 had a size of 92,396 bp and a G + C content of 33% (accession no. MT022086). The genetic map of pN7435-R3645 had high nucleotide similarity and shared the main open reading frame (ORF) features with two plasmids: E. faecium pMG1 (AB206333.1) and E. faecium LS170308 (CP025078.1). The rep gene of pN7435-R3645 showed 100% identity with that of pMG1, although it did not belong to the rep 1-19 family but instead a unique rep family. Multiple antibiotic resistance genes, including lsa (E), aadE and lnu (B), erm (B), ant6 -Ia, and lnu (B), were present on the plasmid. In conclusion, an lsa (E)-carrying plasmid that can be transferred by conjugation from E. faecium to S. aureus in vitro was identified. This multidrug resistance (MDR) pMG1-like plasmid may act as a vector in the dissemination of antimicrobial resistance among species. [ABSTRACT FROM AUTHOR]

Plasmid pNP40, which was first identified nearly 40 years ago in Lactococcus lactis subsp. lactis biovar diacetylactis DRC3, encodes functions such as heavy metal-, bacteriophage-, and nisin-resistance, as well as plasmid transfer ability by conjugation. Here, we report an optimized conjugation protocol for this plasmid, yielding a transfer frequency that is approximately 4,000-fold higher than those previously reported in literature, while we also observed high-frequency plasmid co-mobilization. Individual mutations in 18 genes that encompass the presumed conjugation cluster of pNP40 were generated using ssDNA recombineering to evaluate the role of each gene in the conjugation process. A possible transcriptional repressor of this conjugation cluster, the product of the tra R gene, was identified in this manner. This mutational analysis, paired with bioinformatic predictions as based on sequence and structural similarities, allowed us to generate a preliminary model of the pNP40 conjugation machinery. [ABSTRACT FROM AUTHOR]

Escherichia coli is one major cause of bacterial infections and can horizontally acquire antimicrobial resistance and virulence genes through conjugation. Because conjugative plasmids can rapidly spread among bacteria of different species, the plasmids carrying both antimicrobial resistance and virulence genes may pose a significant threat to public health. Therefore, the identification and characterization of these plasmids may facilitate a better understanding of E. coli pathogenesis and the development of new strategies against E. coli infections. Because iron uptake ability is a potential virulence trait of bacteria, we screened for E. coli conjugative plasmids able to confer both iron uptake ability and ampicillin resistance. The plasmid pEC41, which was derived from the bacteremia clinical isolate EC41, was identified. EC41, which carried the fimH 27 allele, belonged to sequence type (ST) 405 and phylogroup D. According to the sequencing analyses, pEC41 was 86 kb in size, and its backbone structure was almost identical to that of another highly conjugative plasmid, pCTX-M3, in which the extended-spectrum β-lactamase gene bla CTX–M–3 was originally identified. pEC41 carried bla CTX–M–3 and bla TEM–1. The ferric citrate uptake (fec) system was identified in pEC41 and was responsible for conferring iron uptake ability. The fec system contributes to the pathogenesis of EC41 in systemic infections but not in urinary tract infections (UTIs). However, this system promoted competitive fitness of a cystitis-associated clinical isolate to colonize urinary tracts. Additionally, the distribution of the fec system was related to E. coli isolates associated with human bacteremia and UTIs. In summary, the present study identified a novel conjugative plasmid, pEC41, which conferred both antimicrobial resistance and an extra iron uptake ability to E. coli. The iron uptake ability was encoded in the fec system and contributed to E. coli pathogenesis. This study is the first to show that the fec system is a virulence factor in E. coli. [ABSTRACT FROM AUTHOR]

Linezolid is a last-resort antibiotic for the treatment of severe infections caused by multidrug-resistant Gram-positive organisms; although linezolid resistance remains uncommon, the number of linezolid-resistant enterococci has increased in recent years due to worldwide spread of acquired resistance genes (cfr, optrA, and poxtA) in clinical, animal, and environmental settings. In this study, we investigated the occurrence of linezolid-resistant enterococci in marine samples from two coastal areas in Italy. Isolates grown on florfenicol-supplemented Slanetz-Bartley agar plates were investigated for their carriage of optrA, poxtA, and cfr genes; optrA was found in one Enterococcus faecalis isolate, poxtA was found in three Enterococcus faecium isolates and two Enterococcus hirae isolates, and cfr was not found. Two of the three poxtA-carrying E. faecium isolates and the two E. hirae isolates showed related pulsed-field gel electrophoresis (PFGE) profiles. Two E. faecium isolates belonged to the new sequence type 1710, which clustered in clonal complex 94, encompassing nosocomial strains. S1 PFGE/hybridization assays showed a double (chromosome and plasmid) location of poxtA and a plasmid location of optrA. Whole-genome sequencing revealed that poxtA was contained in a Tn6657-like element carried by two plasmids (pEfm-EF3 and pEh-GE2) of similar size, found in different species, and that poxtA was flanked by two copies of IS1216 in both plasmids. In mating experiments, all but one strain (E. faecalis EN3) were able to transfer the poxtA gene to E. faecium 64/3. The occurrence of linezolid resistance genes in enterococci from marine samples is of great concern and highlights the need to improve practices aimed at limiting the transmission of linezolid-resistant strains to humans from environmental reservoirs. [ABSTRACT FROM AUTHOR]

Background: This study aimed to characterize carbapenem-nonsusceptible Acinetobacter (CNSA) isolated from patients with bacteremia from 1997 to 2015., Methods: A total of 173 CNSA (12.3%) was recovered from 1403 Acinetobacter isolates. The presence of selected β-lactamase genes in CNSA was determined by PCR amplification. The conjugation test was used to determine the transferability of metallo-β-lactamase (MBL)-carrying plasmids. Whole genome sequencing in combination with phenotypic assays was carried out to characterize MBL-plasmids., Results: In general, a trend of increasing numbers of CNSA was observed. Among the 173 CNSA, A. baumannii (54.9%) was the most common species, followed by A. nosocomialis (23.1%) and A. soli (12.1%). A total of 49 (28.3%) CNSA were extensively drug-resistant, and all were A. baumannii. The most common class D carbapenemase gene in 173 CNSA was bla OXA-24-like (32.4%), followed by ISAba1-bla OXA-51-like (20.8%), ISAba1-bla OXA-23 (20.2%), and IS1006/IS1008-bla OXA-58 (11.6%). MBL genes, bla VIM-11, bla IMP-1 , and bla IMP-19 were detected in 9 (5.2%), 20 (11.6%), and 1 (0.6%) CNSA isolates, respectively. Transfer of MBL genes to AB218 and AN254 recipient cells was successful for 7 and 6 of the 30 MBL-plasmids, respectively. The seven AB218-derived transconjugants carrying MBL-plasmids produced less biofilm but showed higher virulence to larvae than recipient AB218., Conclusions: Our 19-year longitudinal study revealed a stable increase in CNSA during 2005-2015. bla OXA-24-like , ISAba1-bla OXA-51-like , and ISAba1-bla OXA-23 were the major determinants of Acinetobacter carbapenem resistance. MBL-carrying plasmids contribute not only to the carbapenem resistance but also to A. baumannii virulence., Competing Interests: Declaration of competing interest The authors report that there are no competing interests to declare., (Copyright © 2024. Published by Elsevier B.V.)

Campylobacter jejuni is a foodborne pathogen causing bacterial gastroenteritis, with the highest incidence reported in Europe. The prevalence of antibiotic resistance in C. jejuni, as well as in many other bacterial pathogens, has increased over the last few years. In this report, we describe the presence of a plasmid in a multi-drug-resistant C. jejuni strain isolated from a gastroenteritis patient. Mating experiments demonstrated the transference of this genetic element (pCjH01) among C. jejuni by plasmid conjugation. The pCjH01 plasmid was sequenced and assembled, revealing high similarity (97% identity) with pTet, a described tetracycline resistance encoding plasmid. pCjH01 (47.7 kb) is a mosaic plasmid composed of a pTet backbone that has acquired two discrete DNA regions. Remarkably, one of the acquired sequences carried an undescribed variant of the aadE-sat4-aphA-3 gene cluster, providing resistance to at least kanamycin and gentamycin. Aside from the antibiotic resistance genes, the cluster also carries genes coding for putative regulators, such as a sigma factor of the RNA polymerase and an antisigma factor. Homology searches suggest that Campylobacter exchanges genetic material with distant G-positive bacterial genera.

Background: Linezolid is an antibiotic used to treat infections caused by multi-drug-resistant Gram-positive bacteria. Linezolid resistance in enterococci has been reported with increasing frequency, with a recent rise in resistance encoded by optrA, poxtA or cfr.Aim: To investigate a hospital outbreak of linezolid- and vancomycin-resistant Enterococcus faecium (LVREfm) using whole-genome sequencing (WGS).Methods: Thirty-nine VREfm from patient screening (19 isolates, 17 patients) and environmental sites (20 isolates) recovered in October 2019 were investigated. Isolates were screened using polymerase chain reaction for optrA, poxtA and cfr, and underwent Illumina MiSeq WGS. Isolate relatedness was assessed using E. faecium core genome multi-locus sequence typing (cgMLST). One LVREfm underwent MinION long-read WGS (Oxford Nanopore Technologies) and hybrid assembly with MiSeq short-read sequences to resolve an optrA-encoding plasmid.Findings: Twenty isolates (51.3%) were LVREfm and optrA-positive, including the LVREfm from the index patient. A closely related cluster of 28 sequence type (ST) 80 isolates was identified by cgMLST, including all 20 LVREfm and eight linezolid-susceptible VREfm, with an average allelic difference of two (range 0-10), indicating an outbreak. Nineteen (95%) LVREfm harboured a 56,684-bp conjugative plasmid (pEfmO_03). The remaining LVREfm exhibited 44.1% sequence coverage to pEfmO_03. The presence of pEfmO_03 in LVREfm and the close relatedness of the outbreak cluster isolates indicated the spread of a single strain. The outbreak was terminated by enhanced infection prevention and control (IPC) and environmental cleaning measures, ceasing ward admissions and ward-dedicated staff.Conclusion: WGS was central in investigating an outbreak of ST80 LVREfm. The rapid implementation of enhanced IPC measures terminated the outbreak. [ABSTRACT FROM AUTHOR]

The increasing incidence of phenotypic resistance to carbapenems in recent years is mainly attributed to acquisition of mobile carbapenemase‐encoding genetic elements by major bacterial pathogens. Here, a novel carbapenemase known as Vibrio metallo‐β‐lactamase 1 (VMB‐1), which is encoded by a gene (blaVMB‐1) located in an integron‐bearing, highly transmissible IncC type plasmid, namely pVB1796, is identified and characterized, both genetically and functionally. Recovered from a foodborne Vibrio alginolyticus strain that exhibits resistance to all known β‐lactam antibiotics, pVB1796 is found to possess a hybrid backbone that exhibits unique features of both type 1 and type 2 IncC elements. VMB‐1 exhibits 94% sequence homology with several recently reported but poorly characterized metallo‐β‐lactamases (MBLs) produced by the marine organisms Alteromonadaceae, Glaciecola, and Thalassomonas actiniarum. Sequence alignment analysis shows that VMB‐1 shares a structurally identical active site with subclass B1 MBLs. Importantly, pVB1796 is found to be efficiently transferred from Vibrio to other Gram‐negative bacterial pathogens, including Salmonella typhimurium, Klebsiella pneumoniae, and Acinetobacter baumanni, via conjugation. These findings suggest that blaVMB‐1‐bearing plasmids have the potential to be disseminated to other Gram‐negative bacterial pathogens in the near future and render carbapenems useless in treatment of multidrug resistant infections. [ABSTRACT FROM AUTHOR]

In this study, antibiotic resistance and virulence genes and clonal relationships among Escherichia coli isolates from poultry litter and healthy poultry were investigated on a farm with previous outbreak of avian pathogenic E. coli (APEC) colisepsis, in order to establish a system for evaluating management practice and antibiotic use on poultry farms. Multidrug resistant strains were found in 78.3% (116/148) of tested cloacal swabs and in 100% (5/5) poultry litter E. coli isolates from each of the five poultry houses. The highest rate of resistance was to ampicillin and tetracycline, determined by presence of blaTEM and tet(A) genes. Conjugative transfer of resistance and virulence genes and the presence of integrons and integrases proved that the horizontal gene transfer was involved in the spread of multiresistant phenotypes among E. coli in poultry. Multidrug resistant transconjugant from E. coli isolate H4 from poultry litter carried iroN, ompT, hlyF, iss, iutA virulence genes, as well as blaTEM, aadA1, aadA2, sul1 and dfrA5/14 resistance genes and class 1 integron. Pulsed field gel electrophoresis (PFGE) typing distinguished multiple clones. This study demonstrated that poultry litter can serve as a reservoir of multidrug resistant E. coli, and resistotyping and virulence typing of poultry litter and/or isolates from poultry could be used to assess the quality of the management practice on poultry farms. Therefore, we propose a scoring system based on the number and ratio of resistance and virulence genes that can be used for evaluation of the quality of management practices on farms. [ABSTRACT FROM AUTHOR]

Introduction  Aminoglycosides are formidable broad-spectrum antibiotics used in clinical settings; woefully their usage has been reduced by the emergence and distribution of resistance mainly due to aminoglycoside modifying enzymes (AME). Purpose  This study was performed to determine the diverse prevalence of AME and their pattern of occurrence in the clinical isolates of gram-negative bacteria. This study also aimed to detect the presence of AMEs that are prevalent in gram-positive bacteria, among gram negatives. Materials and Methods  A total number of 386 clinical isolates were included in this study. Polymerase chain reaction revealed the prevalence rate of AMEs screened [aac(6′)-lb, aac(3′)-I, aac(3′)-II, aac(3′)-VI, ant(2′)-I, ant(4′)-IIb, aac(3′)-III, aac(3′)-IV, aph(2′)-Ib, aph(2′)-Ic, aph(2′)-Id, aac (6′)-Ie- aph(2′)-Ia, and aph(3′)-IIIa]. Conjugation experiment was performed for the clinical isolates which harbored any one of the AME which was prevalent in gram-positive bacteria [aph(3′)-IIIa, aac(6′)-Ie-aph(2′)-Ia]. Results  aac(6′)-lb is the most prevalent AME, followed by aac(3′)-I, aph(3′)-VI, aac(3′)-VI, and aac(3′)-II. The AMEs such as ant (2′)-I, ant(4′)-IIb, aac(3′)-III, aac(3′)-IV, aph(2′)-Ib, aph(2′)-Ic, and aph(2′)-Id were not established in our study isolates. The rate of prevalence of aph(3′)-IIIa, aac(6′)-Ie-aph(2′)-Ia—the AMEs encountered in gram-positive and their co-existence was 19.68% and the conjugation experiment revealed their transfer via plasmids. Conclusion  This is the first report from India revealing the presence and prevalence of AMEs which are often encountered among gram-positive bacteria in gram negatives and their presence on conjugative plasmids. [ABSTRACT FROM AUTHOR]

ESC-resistant E. coli isolates were collected from broiler chickens, a slaughterhouse, and retail meat to assess their dispersion and their involvement in cross-contamination. ESBL-/AmpC-producing E. coli were isolated during the slaughter process of all six investigated chicken flocks from scalding, feather removal, first conveyor, evisceration, second washing, third conveyor, and third washing areas, and from handling workers in the slaughterhouse. ESC-resistant E. coli isolates with the same pulsed-field gel electrophoresis type were found in the same site (scalding) on different sampling days. ESBL/AmpC-producing E. coli isolates were absent in the lairage area in the slaughterhouse, but present in the retail markets in 36.8% (7/19) of the chicken flocks. The blaCTX-M genes and blaCMY-2 were conjugated to recipient E. coli J53 in 67.5% (27/40) and 56.1% (23/41) of ESBL-producing and AmpC-producing E. coli isolates, respectively. The presence of the same conjugative plasmids was found in genetic diversity ESC-resistant E. coli colonies collected on different sampling days. Our study emphasizes that cross-contamination of ESBL/AmpC-producing E. coli in slaughterhouse has a crucial impact on the occurrence of ESC resistance in retail chicken meat.