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301900.9764Identification and Characterization of New Resistance-Conferring SGI1s (Salmonella Genomic Island 1) in Proteus mirabilis. Salmonella genomic island 1 (SGI1) is a resistance-conferring chromosomal genomic island that contains an antibiotic resistance gene cluster. The international spread of SGI1-containing strains drew attention to the role of genomic islands in the dissemination of antibiotic resistance genes in Salmonella and other Gram-negative bacteria. In this study, five SGI1 variants conferring multidrug and heavy metal resistance were identified and characterized in Proteus mirabilis strains: SGI1-PmCAU, SGI1-PmABB, SGI1-PmJN16, SGI1-PmJN40, and SGI1-PmJN48. The genetic structures of SGI1-PmCAU and SGI1-PmABB were identical to previously reported SGI1s, while structural analysis showed that SGI1-PmJN16, SGI1-PmJN40, and SGI1-PmJN48 are new SGI1 variants. SGI1-PmJN16 is derived from SGI1-Z with the MDR region containing a new gene cassette array dfrA12-orfF-aadA2-qacEΔ1-sul1-chrA-orf1. SGI1-PmJN40 has an unprecedented structure that contains two right direct repeat sequences separated by a transcriptional regulator-rich DNA fragment, and is predicted to form two different extrachromosomal mobilizable DNA circles for dissemination. SGI1-PmJN48 lacks a common ORF S044, and its right junction region exhibits a unique genetic organization due to the reverse integration of a P. mirabilis chromosomal gene cluster and the insertion of part of a P. mirabilis plasmid, making it the largest known SGI1 to date (189.1 kb). Further mobility functional analysis suggested that these SGIs can be excised from the chromosome for transfer between bacteria, which promotes the horizontal transfer of antibiotic and heavy metal resistance genes. The identification and characterization of the new SGI1 variants in this work suggested the diversity of SGI1 structures and their significant roles in the evolution of bacteria.201830619228
987410.9731Genomic islands related to Salmonella genomic island 1; integrative mobilisable elements in trmE mobilised in trans by A/C plasmids. Salmonella genomic island 1 (SGI1), an integrative mobilisable element (IME), was first reported 20 years ago, in the multidrug resistant Salmonella Typhimurium DT104 clone. Since this first report, many variants and relatives have been found in Salmonella enterica and Proteus mirabilis. Thanks to whole genome sequencing, more and more complete sequences of SGI1-related elements (SGI1-REs) have been reported in these last few years among Gammaproteobacteria. Here, the genetic organisation and main features common to SGI1-REs are summarised to help to classify them. Their integrases belong to the tyrosine-recombinase family and target the 3'-end of the trmE gene. They share the same genetic organisation (integrase and excisionase genes, replicase module, SgaCD-like transcriptional activator genes, traN, traG, mpsB/mpsA genes) and they harbour AcaCD binding sites promoting their excision, replication and mobilisation in presence of A/C plasmid. SGI1-REs are mosaic structures suggesting that recombination events occurred between them. Most of them harbour a multiple antibiotic resistance (MAR) region and the plasticity of their MAR region show that SGI1-REs play a key role in antibiotic resistance and might help multiple antibiotic resistant bacteria to adapt to their environment. This might explain the emergence of clones with SGI1-REs.202133582118
153520.9729Complete Genome Sequencing of Acinetobacter baumannii AC1633 and Acinetobacter nosocomialis AC1530 Unveils a Large Multidrug-Resistant Plasmid Encoding the NDM-1 and OXA-58 Carbapenemases. Carbapenem-resistant Acinetobacter spp. are considered priority drug-resistant human-pathogenic bacteria. The genomes of two carbapenem-resistant Acinetobacter spp. clinical isolates obtained from the same tertiary hospital in Terengganu, Malaysia, namely, A. baumannii AC1633 and A. nosocomialis AC1530, were sequenced. Both isolates were found to harbor the carbapenemase genes bla(NDM-1) and bla(OXA-58) in a large (ca. 170 kb) plasmid designated pAC1633-1 and pAC1530, respectively, that also encodes genes that confer resistance to aminoglycosides, sulfonamides, and macrolides. The two plasmids were almost identical except for the insertion of ISAba11 and an IS4 family element in pAC1633-1, and ISAba11 along with relBE toxin-antitoxin genes flanked by inversely orientated pdif (XerC/XerD) recombination sites in pAC1530. The bla(NDM-1) gene was encoded in a Tn125 composite transposon structure flanked by ISAba125, whereas bla(OXA-58) was flanked by ISAba11 and ISAba3 downstream and a partial ISAba3 element upstream within a pdif module. The presence of conjugative genes in plasmids pAC1633-1/pAC1530 and their discovery in two distinct species of Acinetobacter from the same hospital are suggestive of conjugative transfer, but mating experiments failed to demonstrate transmissibility under standard laboratory conditions. Comparative sequence analysis strongly inferred that pAC1633-1/pAC1530 was derived from two separate plasmids in an IS1006-mediated recombination or transposition event. A. baumannii AC1633 also harbored three other plasmids designated pAC1633-2, pAC1633-3, and pAC1633-4. Both pAC1633-3 and pAC1633-4 are cryptic plasmids, whereas pAC1633-2 is a 12,651-bp plasmid of the GR8/GR23 Rep3-superfamily group that encodes the tetA(39) tetracycline resistance determinant in a pdif module.IMPORTANCE Bacteria of the genus Acinetobacter are important hospital-acquired pathogens, with carbapenem-resistant A. baumannii listed by the World Health Organization as the one of the top priority pathogens. Whole-genome sequencing of carbapenem-resistant A. baumannii AC1633 and A. nosocomialis AC1530, which were isolated from the main tertiary hospital in Terengganu, Malaysia, led to the discovery of a large, ca. 170-kb plasmid that harbored genes encoding the New Delhi metallo-β-lactamase-1 (NDM-1) and OXA-58 carbapenemases alongside genes that conferred resistance to aminoglycosides, macrolides, and sulfonamides. The plasmid was a patchwork of multiple mobile genetic elements and comparative sequence analysis indicated that it may have been derived from two separate plasmids through an IS1006-mediated recombination or transposition event. The presence of such a potentially transmissible plasmid encoding resistance to multiple antimicrobials warrants vigilance, as its spread to susceptible strains would lead to increasing incidences of antimicrobial resistance.202133504662
302030.9724Combining sequencing approaches to fully resolve a carbapenemase-encoding megaplasmid in a Pseudomonas shirazica clinical strain. Horizontal transfer of plasmids plays a pivotal role in dissemination of antibiotic resistance genes and emergence of multidrug-resistant bacteria. Plasmid sequencing is thus paramount for accurate epidemiological tracking in hospitals and routine surveillance. Combining Nanopore and Illumina sequencing allowed full assembly of a carbapenemase-encoding megaplasmid carried by multidrug-resistant clinical isolate FFUP_PS_41. Average nucleotide identity analyses revealed that FFUP_PS_41 belongs to the recently proposed new species Pseudomonas shirazica, related to the P. putida phylogenetic group. FFUP_PS_41 harbours a 498,516-bp megaplasmid (pJBCL41) with limited similarity to publicly-available plasmids. pJBCL41 contains genes predicted to encode replication, conjugation, partitioning and maintenance functions and heavy metal resistance. The |aacA7|blaVIM-2|aacA4| cassette array (resistance to carbapenems and aminoglycosides) is located within a class 1 integron that is a defective Tn402 derivative. This transposon lies within a 50,273-bp region bound by Tn3-family 38-bp inverted repeats and flanked by 5-bp direct repeats (DR) that composes additional transposon fragments, five insertion sequences and a Tn3-Derived Inverted-Repeat Miniature Element. The hybrid Nanopore/Illumina approach allowed full resolution of a carbapenemase-encoding megaplasmid from P. shirazica. Identification of novel megaplasmids sheds new light on the evolutionary effects of gene transfer and the selective forces driving antibiotic resistance.201931381486
302140.9722Sequencing and comparative analysis of IncP-1α antibiotic resistance plasmids reveal a highly conserved backbone and differences within accessory regions. Although IncP-1 plasmids are important for horizontal gene transfer among bacteria, in particular antibiotic resistance spread, so far only three plasmids from the subgroup IncP-1α have been completely sequenced. In this study we doubled this number. The three IncP-1α plasmids pB5, pB11 and pSP21 were isolated from bacteria of two different sewage treatment plants and sequenced by a combination of next-generation and capillary sequencing technologies. A comparative analysis including the previously analysed IncP-1α plasmids RK2, pTB11 and pBS228 revealed a highly conserved plasmid backbone (at least 99.9% DNA sequence identity) comprising 54 core genes. The accessory elements of the plasmid pB5 constitute a class 1 integron interrupting the parC gene and an IS6100 copy inserted into the integron. In addition, the tetracycline resistance genes tetAR and the ISTB11-like element are located between the klc operon and the trfA-ssb operon. Plasmid pB11 is loaded with a Tn5053-like mercury resistance transposon between the parCBA and parDE operons and contains tetAR that are identical to those identified in plasmid pB5 and the insertion sequence ISSP21. Plasmid pSP21 harbours an ISPa7 element in a Tn402 transposon including a class 1 integron between the partitioning genes parCBA and parDE. The IS-element ISSP21 (99.89% DNA sequence identity to ISSP21 from pB11), inserted downstream of the tetR gene and a copy of ISTB11 (identical to ISTB11 on pTB11) inserted between the genes pncA and pinR. On all three plasmids the accessory genes are almost always located between the backbone modules confirming the importance of the backbone functions for plasmid maintenance. The striking backbone conservation among the six completely sequenced IncP-1α plasmids is in contrast to the much higher diversity within the IncP-1β subgroup.201121115076
987550.9722Antibiotic Resistance in Vibrio cholerae: Mechanistic Insights from IncC Plasmid-Mediated Dissemination of a Novel Family of Genomic Islands Inserted at trmE. Cholera remains a formidable disease, and reports of multidrug-resistant strains of the causative agent Vibrio cholerae have become common during the last 3 decades. The pervasiveness of resistance determinants has largely been ascribed to mobile genetic elements, including SXT/R391 integrative conjugative elements, IncC plasmids, and genomic islands (GIs). Conjugative transfer of IncC plasmids is activated by the master activator AcaCD whose regulatory network extends to chromosomally integrated GIs. MGIVchHai6 is a multidrug resistance GI integrated at the 3' end of trmE (mnmE or thdF) in chromosome 1 of non-O1/non-O139 V. cholerae clinical isolates from the 2010 Haitian cholera outbreak. In the presence of an IncC plasmid expressing AcaCD, MGIVchHai6 excises from the chromosome and transfers at high frequency. Herein, the mechanism of mobilization of MGIVchHai6 GIs by IncC plasmids was dissected. Our results show that AcaCD drives expression of GI-borne genes, including xis and mobI(M) , involved in excision and mobilization. A 49-bp fragment upstream of mobI(M) was found to serve as the minimal origin of transfer (oriT) of MGIVchHai6. The direction of transfer initiated at oriT was determined using IncC plasmid-driven mobilization of chromosomal markers via MGIVchHai6. In addition, IncC plasmid-encoded factors, including the relaxase TraI, were found to be required for GI transfer. Finally, in silico exploration of Gammaproteobacteria genomes identified 47 novel related and potentially AcaCD-responsive GIs in 13 different genera. Despite sharing conserved features, these GIs integrate at trmE, yicC, or dusA and carry a diverse cargo of genes involved in phage resistance.IMPORTANCE The increasing association of the etiological agent of cholera, Vibrio cholerae serogroup O1 and O139, with multiple antibiotic resistance threatens to deprive health practitioners of this effective tool. Drug resistance in cholera results mainly from acquisition of mobile genetic elements. Genomic islands conferring multidrug resistance and mobilizable by IncC conjugative plasmids were reported to circulate in non-O1/non-O139 V. cholerae clinical strains isolated from the 2010 Haitian cholera outbreak. As these genomic islands can be transmitted to pandemic V. cholerae serogroups, their mechanism of transmission needed to be investigated. Our research revealed plasmid- and genomic island-encoded factors required for the resistance island excision, mobilization, and integration, as well as regulation of these functions. The discovery of related genomic islands carrying diverse phage resistance genes but lacking antibiotic resistance-conferring genes in a wide range of marine dwelling bacteria suggests that these elements are ancient and recently acquired drug resistance genes.202032848007
987260.9719pCTX-M3-Structure, Function, and Evolution of a Multi-Resistance Conjugative Plasmid of a Broad Recipient Range. pCTX-M3 is the archetypic member of the IncM incompatibility group of conjugative plasmids (recently referred to as IncM2). It is responsible for the worldwide dissemination of numerous antibiotic resistance genes, including those coding for extended-spectrum β-lactamases and conferring resistance to aminoglycosides. The IncM plasmids acquired during evolution diverse mobile genetic elements found in one or two multiple resistance regions, MRR(s), grouping antibiotic resistance genes as well as mobile genetic elements or their remnants. The IncM plasmids can be found in bacteria inhabiting various environments. The information on the structure and biology of pCTX-M3 is integrated in this review. It focuses on the functional modules of pCTX-M3 responsible for its replication, stable maintenance, and conjugative transfer, indicating that the host range of the pCTX-M3 replicon is limited to representatives of the family Enterobacteriaceae (Enterobacterales ord. nov.), while the range of recipients of its conjugation system is wide, comprising Alpha-, Beta-, and Gammaproteobacteria, and also Firmicutes.202133925677
301570.9715Genetic structure and biological properties of the first ancient multiresistance plasmid pKLH80 isolated from a permafrost bacterium. A novel multidrug-resistance plasmid, pKLH80, previously isolated from Psychrobacter maritimus MR29-12 found in ancient permafrost, was completely sequenced and analysed. In our previous studies, we focused on the pKLH80 plasmid region containing streptomycin and tetracycline resistance genes, and their mobilization with an upstream-located ISPpy1 insertion sequence (IS) element. Here, we present the complete sequence of pKLH80 and analysis of its backbone genetic structure, including previously unknown features of the plasmid's accessory region, notably a novel variant of the β-lactamase gene blaRTG-6. Plasmid pKLH80 was found to be a circular 14 835 bp molecule that has an overall G+C content of 40.3 mol% and encodes 20 putative ORFs. There are two distinctive functional modules within the plasmid backbone sequence: (i) the replication module consisting of repB and the oriV region; and (ii) the mobilization module consisting of mobA, mobC and oriT. All of the aforementioned genes share sequence identities with corresponding genes of different species of Psychrobacter. The plasmid accessory region contains antibiotic resistance genes and IS elements (ISPsma1 of the IS982 family, and ISPpy1 and ISAba14 of the IS3 family) found in environmental and clinical bacterial strains of different taxa. We revealed that the sequences flanking blaRTG-6 and closely related genes from clinical bacteria are nearly identical. This fact suggests that blaRTG-6 from the environmental strain of Psychrobacter is a progenitor of blaRTG genes of clinical bacteria. We also showed that pKLH80 can replicate in different strains of Acinetobacter and Psychrobacter genera. The roles of IS elements in the horizontal transfer of antibiotic resistance genes are examined and discussed.201425063046
306080.9713Integron mobilization unit as a source of mobility of antibiotic resistance genes. Antibiotic resistance genes are spread mostly through plasmids, integrons (as a form of gene cassettes), and transposons in gram-negative bacteria. We describe here a novel genetic structure, named the integron mobilization unit (IMU), that has characteristics similar to those of miniature inverted transposable elements (MITEs). Two IMUs (288 bp each) were identified from a carbapenem-resistant Enterobacter cloacae isolate that formed a composite structure encompassing a defective class 1 integron containing the carbapenem resistance gene bla(GES-5). This beta-lactamase gene was located on a 7-kb IncQ-type plasmid named pCHE-A, which was sequenced completely. The plasmid pCHE-A was not self conjugative but was mobilizable, and it was successfully transferred from E. cloacae to Pseudomonas aeruginosa. The in silico analysis of the extremities of the IMU elements identified similarities with those of insertion sequence ISSod9 from Shewanella oneidensis MR-1. The mobilization of the IMU composite structure was accomplished by using the transposase activity of ISSod9 that was provided in trans. This is the first identification of MITE-type structures as a source of gene mobilization, implicating here a clinically relevant antibiotic resistance gene.200919332679
300990.9711Identification of a novel conjugative plasmid carrying the multiresistance gene cfr in Proteus vulgaris isolated from swine origin in China. The multiresistance gene cfr has a broad host range encompassing both Gram-positive and Gram-negative bacteria, and can be located on the chromosomes or on plasmids. In this study, a novel conjugative plasmid carrying cfr, designated as pPvSC3, was characterized in a Proteus vulgaris strain isolated from swine in China. Plasmid pPvSC3 is 284,528 bp in size and harbors 10 other antimicrobial resistance genes, making it a novel plasmid that differs from all known plasmids due to its unique backbone and repA gene. BLAST analysis of the plasmid sequence shows no significant homology to any known plasmid backbone, but shows high level homology to Providencia rettgeri strain CCBH11880 Contig_9, a strain isolated from surgical wound in Brazil, 2014. There are two resistance-determining regions in pPvSC3, a cfr-containing region and a multidrug-resistant (MDR) region. The cfr-containing region is flanked by IS26, which could be looped out via IS26-mediated recombination. The MDR region harbors 10 antimicrobial resistance genes carried by various DNA segments that originated from various sources. Plasmid pPvSC3 could be successfully transferred to Escherichia coli by conjugation. In summary, we have characterized a novel conjugative plasmid pPvSC3 carrying the multiresistance gene cfr and 10 other antimicrobial resistance genes, and consider that this novel type of plasmid deserves attention.201931499097
9853100.9711The master activator of IncA/C conjugative plasmids stimulates genomic islands and multidrug resistance dissemination. Dissemination of antibiotic resistance genes occurs mostly by conjugation, which mediates DNA transfer between cells in direct contact. Conjugative plasmids of the IncA/C incompatibility group have become a substantial threat due to their broad host-range, the extended spectrum of antimicrobial resistance they confer, their prevalence in enteric bacteria and their very efficient spread by conjugation. However, their biology remains largely unexplored. Using the IncA/C conjugative plasmid pVCR94ΔX as a prototype, we have investigated the regulatory circuitry that governs IncA/C plasmids dissemination and found that the transcriptional activator complex AcaCD is essential for the expression of plasmid transfer genes. Using chromatin immunoprecipitation coupled with exonuclease digestion (ChIP-exo) and RNA sequencing (RNA-seq) approaches, we have identified the sequences recognized by AcaCD and characterized the AcaCD regulon. Data mining using the DNA motif recognized by AcaCD revealed potential AcaCD-binding sites upstream of genes involved in the intracellular mobility functions (recombination directionality factor and mobilization genes) in two widespread classes of genomic islands (GIs) phylogenetically unrelated to IncA/C plasmids. The first class, SGI1, confers and propagates multidrug resistance in Salmonella enterica and Proteus mirabilis, whereas MGIVmi1 in Vibrio mimicus belongs to a previously uncharacterized class of GIs. We have demonstrated that through expression of AcaCD, IncA/C plasmids specifically trigger the excision and mobilization of the GIs at high frequencies. This study provides new evidence of the considerable impact of IncA/C plasmids on bacterial genome plasticity through their own mobility and the mobilization of genomic islands.201425340549
3028110.9711Novel macrolide resistance module carried by the IncP-1beta resistance plasmid pRSB111, isolated from a wastewater treatment plant. The macrolide resistance plasmid pRSB111 was isolated from bacteria residing in the final effluents of a wastewater treatment plant. The 47-kb plasmid confers resistance to azithromycin, clarithromycin, erythromycin, roxithromycin, and tylosin when it is carried by Pseudomonas sp. strain B13 and is very similar to prototype IncP-1beta plasmid pB3, which was previously isolated from an activated-sludge bacterial community of a wastewater treatment plant. The two plasmids differ in their accessory regions, located downstream of the conjugative transfer module gene traC. Nucleotide sequence analysis of the pRSB111 accessory region revealed that it contains a new macrolide resistance module composed of the genes mphR(E), mph(E), and mrx(E), which putatively encode a transcriptional regulator, a macrolide phosphotransferase, and a transmembrane transport protein, respectively. Analysis of the contributions of the individual genes of the macrolide resistance module revealed that mph(E) and mrx(E) are required for high-level macrolide resistance. The resistance genes are flanked by two insertion sequences, namely, ISPa15 and ISRSB111. Two truncated transposable elements, IS6100 and remnants of a Tn3-like transposon, were identified in the vicinity of ISRSB111. The accessory element of pRSB111 apparently replaced the Tn402-like element present on the sister plasmid, pB3, as suggested by the conservation of Tn402-specific terminal inverted repeats on pRSB111.200717101677
3000120.9710A large conjugative Acinetobacter baumannii plasmid carrying the sul2 sulphonamide and strAB streptomycin resistance genes. Acinetobacter baumannii is an important nosocomial pathogen that often complicates treatment because of its high level of resistance to antibiotics. Though plasmids can potentially introduce various genes into bacterial strains, compared to other Gram-negative bacteria, information about the unique A. baumannii plasmid repertoire is limited. Here, whole genome sequence data was used to determine the plasmid content of strain A297 (RUH875), the reference strain for the globally disseminated multiply resistant A. baumannii clone, global clone 1(GC1). A297 contains three plasmids. Two known plasmids were present; one, pA297-1 (pRAY*), carries the aadB gentamicin, kanamycin and tobramycin resistance gene and another is an 8.7kb cryptic plasmid often found in GC1 isolates. The third plasmid, pA297-3, is 200kb and carries the sul2 sulphonamide resistance gene and strAB streptomycin resistance gene within Tn6172 and a mer mercuric ion resistance module elsewhere. pA297-3 transferred sulphonamide, streptomycin and mercuric ion resistance at high frequency to a susceptible A. baumannii recipient, and contains several genes potentially involved in conjugative transfer. However, a relaxase gene was not found. It also includes several genes encoding proteins involved in DNA metabolism such as partitioning. However, a gene encoding a replication initiation protein could not be found. pA297-3 includes two copies of a Miniature Inverted-Repeat Transposable Element (MITE), named MITE-297, bracketing a 77.5kb fragment, which contains several IS and the mer module. Several plasmids related to but smaller than pA297-3 were found in the GenBank nucleotide database. They were found in different A. baumannii clones and are wide spread. They all contain either Tn6172 or a variant in the same position in the backbone as Tn6172 in pA297-3. Some related plasmids have lost the segment between the MITE-297 copies and retain only one MITE-297. Others have segments of various lengths between two MITE-297 copies, and these can be derived from the region in pA297-3 via a deletion adjacent to IS related to IS26 such as IS1007 or IS1007-like. pA297-3 and its relatives represent a third type of conjugative Acinetobacter plasmid that contributes to the dissemination of antibiotic resistance in this species.201627601280
1533130.9710A Transferable IncC-IncX3 Hybrid Plasmid Cocarrying bla(NDM-4), tet(X), and tmexCD3-toprJ3 Confers Resistance to Carbapenem and Tigecycline. Tigecycline is a last-resort antimicrobial against carbapenemase-producing Enterobacterales (CPE). However, mobile tigecycline resistance genes, tet(X) and tmexCD-toprJ, have emerged in China and have spread possibly worldwide. Tet(X) family proteins function as tigecycline-inactivating enzymes, and TMexCD-TOprJ complexes function as efflux pumps for tigecycline. Here, to the best of our knowledge we report a CPE isolate harboring both emerging tigecycline resistance factors for the first time. A carbapenem- and tigecycline-resistant Klebsiella aerogenes strain, NUITM-VK5, was isolated from an urban drainage in Vietnam in 2021, and a plasmid, pNUITM-VK5_mdr, cocarrying tet(X) and tmexCD3-toprJ3 along with the carbapenemase gene bla(NDM-4) was identified in NUITM-VK5. pNUITM-VK5_mdr was transferred to Escherichia coli by conjugation and simultaneously conferred high-level resistance against multiple antimicrobials, including carbapenems and tigecycline. An efflux pump inhibitor reduced TMexCD3-TOprJ3-mediated tigecycline resistance, suggesting that both tigecycline resistance factors independently and additively contribute to the high-level resistance. The plasmid had the IncX3 and IncC replicons and was estimated to be a hybrid of plasmids with different backbones. Unlike IncX3 plasmids, IncC plasmids are stably maintained in an extremely broad range of bacterial hosts in humans, animals, and the environment. Thus, the future global spread of multidrug resistance plasmids such as pNUITM-VK5_mdr poses a public health crisis. IMPORTANCE Tigecycline is important as a last-resort antimicrobial and effective against antimicrobial-resistant bacteria, such as carbapenem-producing Enterobacterales (CPE), whose infections are difficult to treat with antimicrobials. Since 2019, mobile tigecycline resistance genes, tet(X) and tmexCD-toprJ, and their variants have been reported mainly from China, and it has become important to understand their epidemiological situation and detailed genetic mechanisms. In this study, we identified a bacterial isolate coharboring tet(X) and tmexCD-toprJ on the same plasmid. A Klebsiella aerogenes isolate in Vietnam carried both these tigecycline resistance genes on a transferable plasmid leading to high-level resistance to multiple clinically important antimicrobials, including carbapenem and tigecycline, and could actually transfer the plasmid to other bacteria. The spread of such a multidrug resistance plasmid among bacterial pathogens should be of great concern because there are few antimicrobials to combat bacteria that have acquired the plasmid.202134346701
3008140.9710Sequence of conjugative plasmid pIP1206 mediating resistance to aminoglycosides by 16S rRNA methylation and to hydrophilic fluoroquinolones by efflux. Self-transferable IncFI plasmid pIP1206, isolated from an Escherichia coli clinical isolate, carries two new resistance determinants: qepA, which confers resistance to hydrophylic fluoroquinolones by efflux, and rmtB, which specifies a 16S rRNA methylase conferring high-level aminoglycoside resistance. Analysis of the 168,113-bp sequence (51% G+C) revealed that pIP1206 was composed of several subregions separated by copies of insertion sequences. Of 151 open reading frames, 56 (37%) were also present in pRSB107, isolated from a bacterium in a sewage treatment plant. pIP1206 contained four replication regions (RepFIA, RepFIB, and two partial RepFII regions) and a transfer region 91% identical with that of pAPEC-O1-ColBM, a plasmid isolated from an avian pathogenic E. coli. A putative oriT region was found upstream from the transfer region. The antibiotic resistance genes tet(A), catA1, bla(TEM-1), rmtB, and qepA were clustered in a 33.5-kb fragment delineated by two IS26 elements that also carried a class 1 integron, including the sulI, qacEDelta1, aad4, and dfrA17 genes and Tn10, Tn21, and Tn3-like transposons. The plasmid also possessed a raffinose operon, an arginine deiminase pathway, a putative iron acquisition gene cluster, an S-methylmethionine metabolism operon, two virulence-associated genes, and a type I DNA restriction-modification (R-M) system. Three toxin/antitoxin systems and the R-M system ensured stabilization of the plasmid in the host bacteria. These data suggest that the mosaic structure of pIP1206 could have resulted from recombination between pRSB107 and a pAPEC-O1-ColBM-like plasmid, combined with structural rearrangements associated with acquisition of additional DNA by recombination and of mobile genetic elements by transposition.200818458128
9855150.9707Conjugative IncC Plasmid Entry Triggers the SOS Response and Promotes Effective Transfer of the Integrative Antibiotic Resistance Element SGI1. The broad-host-range IncC plasmid family and the integrative mobilizable Salmonella genomic island 1 (SGI1) and its derivatives enable the spread of medically important antibiotic resistance genes among Gram-negative pathogens. Although several aspects of the complex functional interactions between IncC plasmids and SGI1 have been recently deciphered regarding their conjugative transfer and incompatibility, the biological signal resulting in the hijacking of the conjugative plasmid by the integrative mobilizable element remains unknown. Here, we demonstrate that the conjugative entry of IncC/IncA plasmids is detected at an early stage by SGI1 through the transient activation of the SOS response, which induces the expression of the SGI1 master activators SgaDC, shown to play a crucial role in the complex biology between SGI1 and IncC plasmids. Besides, we developed an original tripartite conjugation approach to directly monitor SGI1 mobilization in a time-dependent manner following conjugative entry of IncC plasmids. Finally, we propose an updated biological model of the conjugative mobilization of the chromosomal resistance element SGI1 by IncC plasmids. IMPORTANCE Antimicrobial resistance has become a major public health issue, particularly with the increase of multidrug resistance (MDR) in both animal and human pathogenic bacteria and with the emergence of resistance to medically important antibiotics. The spread between bacteria of successful mobile genetic elements, such as conjugative plasmids and integrative elements conferring multidrug resistance, is the main driving force in the dissemination of acquired antibiotic resistances among Gram-negative bacteria. Broad-host-range IncC plasmids and their integrative mobilizable SGI1 counterparts contribute to the spread of critically important resistance genes (e.g., extended-spectrum β-lactamases [ESBLs] and carbapenemases). A better knowledge of the complex biology of these broad-host-range mobile elements will help us to understand the dissemination of antimicrobial resistance genes that occurred across Gammaproteobacteria borders.202336472437
3007160.9706Analysis of the complete nucleotide sequence of an Actinobacillus pleuropneumoniae streptomycin-sulfonamide resistance plasmid, pMS260. pMS260 is an 8.1-kb non-conjugative but mobilizable plasmid that was isolated from Actinobacillus pleuropneumoniae and encodes streptomycin (SM) and sulfonamide (SA) resistances. The analysis of the complete nucleotide sequence of the plasmid revealed a high degree of similarity between pMS260 and the broad-host-range IncQ family plasmids. pMS260 had a single copy of an origin of vegetative replication (oriV). This sequence was identical to a functional oriV of the IncQ-like plasmid pIE1130 that had been exogenously isolated from piggery manure. However, pMS260 did not carry the second IncQ plasmid RSF1010-like oriV region present in pIE1130. A pIE1130-identical transfer origin was also found in pMS260. In addition, the deduced amino acid sequences from 10 open reading frames identified in pMS260 were entirely or nearly identical to those from genes for the replication, mobilization, and SM-SA resistance of pIE1130, indicating that pMS260 belongs to the IncQ-1 gamma subgroup. pMS260 is physically indistinguishable from pIE1130 apart from two DNA regions that contain the chloramphenicol and kanamycin resistance genes (catIII and aphI, respectively) and the second oriV-like region of pIE1130. The codon bias analysis of each gene of pIE1130 and the presence of potential recombination sites in the sulII-strA intergenic regions suggest that pIE1130 seems to have acquired the catIII and aphI genes more recently than the other genes of pIE1130. Therefore, pMS260 may be the ancestor of pIE1130. Information regarding the broad-host-range replicon of pMS260 will be useful in the development of genetic systems for a wide range of bacteria including A. pleuropneumoniae.200414711528
3017170.9706The ancient small mobilizable plasmid pALWED1.8 harboring a new variant of the non-cassette streptomycin/spectinomycin resistance gene aadA27. The small mobilizable plasmid pALWED1.8 containing a novel variant of the streptomycin/spectinomycin resistance gene aadA27 was isolated from the permafrost strains of Acinetobacter lwoffii. The 4135bp plasmid carries mobА and mobC genes that mediate its mobilization by conjugative plasmids. The nucleotide sequences of mobА and mobC are similar to those of mobilization genes of the modern plasmid pRAY* and its variants, which contain aadB gene, and are widespread among the pathogenic strains of Acinetobacter baumannii. Almost identical pALWED1.8 variants were detected in modern environmental Аcinetobacter strains. A highly similar plasmid was revealed in a strain of Acinetobacter parvus isolated from mouse intestine. Furthermore, we discovered six previously unidentified variants of plasmids related to pALWED1.8 and pRAY* in public databases. In contrast to most known variants of aadA which are cassette genes associated with integrons, the aadA27 variant harbored by pALWED1.8 is a non-cassette, autonomously transcribed gene. Non-cassette aadA genes with 96% sequence identity to aadA27 were detected in the chromosomes of Acinetobacter gyllenbergii and several uncharacterized strains of Аcinetobacter sp. Moreover, we revealed that the autonomous aadA-like genes are present in the chromosomes of many gram-positive and gram-negative bacteria. The phylogenetic analysis of amino acid sequences of all identified AadA proteins showed the following: (i) cassette aadA genes form a separate monophyletic group and mainly reside on plasmids and (ii) chromosomal non-cassette aadA genes are extremely diverse and can be inherited both vertical and via horizontal gene transfer.201626896789
1753180.9705Characterization of a Linezolid- and Vancomycin-Resistant Streptococcus suis Isolate That Harbors optrA and vanG Operons. Linezolid and vancomycin are among the last-resort antimicrobial agents in the treatment of multidrug-resistant Gram-positive bacterial infections. Linezolid- and vancomycin-resistant (LVR) Gram-positive bacteria may pose severe threats to public health. In this study, three optrA- and vanG-positive Streptococcus suis strains were isolated from two farms of different cities. There were only 1 and 343 single-nucleotide polymorphisms in coding region (cSNPs) of HCB4 and YSJ7 to YSJ17, respectively. Mobilome analysis revealed the presence of vanG, erm(B), tet(O/W/32/O), and aadE-apt-sat4-aphA3 cluster on an integrative and conjugative element, ICESsuYSJ17, and erm(B), aphA3, aac(6')-aph(2″), catpC(194), and optrA on a prophage, ΦSsuYSJ17-3. ICESsuYSJ17 exhibited a mosaic structure and belongs to a highly prevalent and transferable ICESa2603 family of Streptococcus species. ΦSsuYSJ17-3 shared conserved backbone to a transferable prophage Φm46.1. A novel composite transposon, IS1216E-araC-optrA-hp-catpC(194)-IS1216E, which can be circulated as translocatable unit (TU) by IS1216E, was integrated on ΦSsuYSJ17-3. Vancomycin resistance phenotype and vanG transcription assays revealed that the vanG operon was inducible. The LVR strain YSJ17 exhibited moderate virulence in a zebrafish infection model. To our knowledge, this is the first report of LVR isolate, which is mediated by acquired resistance genes optrA and vanG operons in Gram-positive bacteria. Since S. suis has been recognized as an antimicrobial resistance reservoir in the spread of resistance genes to major streptococcal pathogens, the potential risks of disseminating of optrA and vanG from S. suis to other Streptococcus spp. are worrisome and routine surveillance should be strengthened.201931551963
3018190.9705The large Bacillus plasmid pTB19 contains two integrated rolling-circle plasmids carrying mobilization functions. Plasmid pTB19 is a 27-kb plasmid originating from a thermophilic Bacillus species. It was shown previously that pTB19 contains an integrated copy of the rolling-circle type plasmid pTB913. Here we describe the analysis of a 4324-bp region of pTB19 conferring resistance to tetracycline. The nucleotide sequence of this region revealed all the characteristics of a second plasmid replicating via the rolling-circle mechanism. This sequence contained (i) the tetracycline resistance marker of pTB19, which is highly similar to other tetL-genes of gram-positive bacteria; (ii) a hybrid mob gene, which bears relatedness to both the mob-genes of pUB110 and pTB913; (iii) a palU type minus origin identical to those of pUB110 and pTB913; and (iv) a plus origin of replication similar to that of pTB913. A repB-type replication initiation gene sequence identical to that of pTB913 was present, which lacked the middle part (492 bp), thus preventing autonomous replication of this region. The hybrid mob gene was functional in conjugative mobilization of plasmids between strains of Bacillus subtilis.19911946749