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176300.8882Multidrug Resistance Genes Carried by a Novel Transposon Tn7376 and a Genomic Island Named MMGI-4 in a Pathogenic Morganella morganii Isolate. Antimicrobial resistance in Morganella morganii is increasing in recent years, which is mainly introduced via extra genetic and mobile elements. The aim of our study is to analyze the multidrug resistance (MDR) and characterize the mobile genetic elements (MGEs) in M. morganii isolates. Here, we report the characteristic of a pathogenic M. morganii isolate containing multidrug resistance genes that are mainly carried by a novel transposon Tn7376 and a genomic island. Sequence analysis suggested that the Tn7376 could be generated through homologous recombination between two different IS26-bounded translocatable units (TUs), namely, module A (IS26-Hp-IS26-mph(A)-mrx(A)-mphR-IS6100-chrA-sul1-qacEΔ1) and module B (ISCR1-sul1-qacEΔ1-cmlA1-aadA1-aadB-intI1-IS26), and the genomic island named MMGI-4 might derive from a partial structure of different original genomic islands that also carried IS26-mediated TUs. Notably, a 2,518-bp sequence linked to the module A and B contains a 570-bp dfrA24 gene. To the best of our knowledge, this is the first report of the novel Tn7376 possessing a complex class 1 integron that carried an infrequent gene dfrA24 in M. morganii. IMPORTANCE Mobile genetic elements (MGEs), especially for IS26-bounded translocatable units, may act as a reservoir for a variety of antimicrobial resistance genes in clinically important pathogenic bacteria. We expounded this significant genetic characteristic by investigating a representative M. morganii isolate containing multidrug resistance genes, including the infrequent dfrA24. Our study suggested that these acquired resistance genes were mainly driven by IS26-flanked important MGEs, such as the novel Tn7376 and the MMGI-4. We demonstrated that IS26-related MGEs contributed to the emergence of the extra gene dfrA24 in M. morganii through some potential genetic events like recombination, transposition, and integration. Therefore, it is of importance to investigate persistently the prevalence these MEGs in the clinical pathogens to provide risk assessment of emergence and development of novel resistance genes.202235510850
153510.8851Complete 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
303020.8818Mobile Genomic Island GEI-FN1A in Aeromonas salmonicida FN1 Contributes to the Spread of Antibiotic-Resistance Genes. Antibiotics are used to treat severe bacterial infections. However, owing to excessive antibiotic use, bacteria under high selective pressure for antibiotics develop resistance through spontaneous mutation or by acquiring antibiotic-resistance genes (ARGs) through horizontal gene transfer (HGT). Horizontal transfer of ARGs among bacteria in the environment can lead to the emergence of multidrug-resistant (MDR) bacteria that infect animals and humans, thus causing disease outbreaks. In this study, MDR strain FN1 was isolated from a feces-contaminated soil sample from a chicken farm under pressure from the antibiotic florfenicol (16 mg/L) and identified as Aeromonas salmonicida. Whole-genome sequencing and analysis revealed the 86.8-kb antibiotic-resistant genomic island, GEI-FN1A, in the FN1 genome. Genome annotation revealed that GEI-FN1A carried several ARGs, including two tetracycline-resistance genes [tetR and tet(A)], three aminoglycoside-resistance genes [aph(6), aph(3"), and aac(3)], one trimethoprim-resistance gene (dfrB4), two chloramphenicol/florfenicol-resistance genes (catB3 and floR), three macrolide-resistance genes [mphR(A), mrx(A), and mph(A)] and two sul1 genes. GEI-FN1A also contained genes encoding integrase, transposase, and recombinase, which mediate the horizontal transfer of MDR genes. These findings suggest that GEI-FN1A in A. salmonicida FN1 can potentially spread ARGs among environmental bacteria.202540553200
300030.8797A 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
299940.8796Integrative and conjugative elements in streptococci can act as vectors for plasmids and translocatable units integrated via IS1216E. Mobile genetic elements (MGEs), such as integrative and conjugative elements (ICEs), plasmids and translocatable units (TUs), are important drivers for the spread of antibiotic resistance. Although ICEs have been reported to support the spread of plasmids among different bacteria, their role in mobilizing resistance plasmids and TUs has not yet been fully explored. In this study, a novel TU bearing optrA, a novel non-conjugative plasmid p5303-cfrD carrying cfr(D) and a new member of the ICESa2603 family, ICESg5301 were identified in streptococci. Polymerase chain reaction (PCR) assays revealed that three different types of cointegrates can be formed by IS1216E-mediated cointegration between the three different MGEs, including ICESg5301::p5303-cfrD::TU, ICESg5301::p5303-cfrD, and ICESg5301::TU. Conjugation assays showed that ICEs carrying p5303-cfrD and/or TU successfully transferred into recipient strains, thereby confirming that ICEs can serve as vectors for other non-conjugative MGEs, such as TUs and p5303-cfrD. As neither the TU nor plasmid p5303-cfrD can spread on their own between different bacteria, their integration into an ICE via IS1216E-mediated cointegrate formation not only increases the plasticity of ICEs, but also furthers the dissemination of plasmids and TUs carrying oxazolidinone resistance genes.202336933870
175250.8794Genetic Characterization of a Linezolid- and Penicillin-Resistant Enterococcus hirae Isolate Co-Harboring poxtA and pbp5fm. Linezolid and penicillin are critical for treating multidrug resistant (MDR) Gram-positive infections, but the emergence of resistance to both seriously threatens public health. Here, we first report the cocarrying poxtA (oxazolidinone resistance) and pbp5fm (β-lactam resistance) genes by the plasmid in a strain of Enterococcus hirae HDC14-2 derived from porcine. The isolate also exhibits MDR phenotypes to phenicols, oxazolidinones, tetracyclines, β-lactams, aminoglycosides, macrolides, and lincosamides. Whole-genome sequencing (WGS) revealed these resistance genes, along with tet(L), tet(M), catA, erm(B), aac(6)-aph(2"), aadE, spw, lsa(E), lnu(B), sat4, and aphA3, were clustered in a novel MDR region flanked by IS1216 elements on plasmid pHDC14-2.133K. This IS1216-bounded MDR region formed translocatable units (TUs), including an IS1216-poxtA TU that was also identified on a secondary plasmid, pHDC14-2.27K. Functional assays demonstrated the excisability and mobility of these TUs, indicating its potential ability integration into other plasmids or chromosomes. Critically, electrotransformation confirmed the transfer of pHDC14-2.27K (poxtA-carrying) to Enterococcus faecalis JH2-2, with retained TU activity and minimal fitness cost. This study provides the evidence of colocalized poxtA and pbp5fm on plasmids in enterococci, highlighting their role in disseminating pan-resistance among bacteria. Although E. hirae is not an important pathogenic bacterium to humans and animals, but its potential risk to horizontally spread of these resistance genes important in medicine still cannot be ignored.202540692874
175360.8793Characterization 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
997770.8792IncC conjugative plasmids and SXT/R391 elements repair double-strand breaks caused by CRISPR-Cas during conjugation. Bacteria have evolved defence mechanisms against bacteriophages. Restriction-modification systems provide innate immunity by degrading invading DNAs that lack proper methylation. CRISPR-Cas systems provide adaptive immunity by sampling the genome of past invaders and cutting the DNA of closely related DNA molecules. These barriers also restrict horizontal gene transfer mediated by conjugative plasmids. IncC conjugative plasmids are important contributors to the global dissemination of multidrug resistance among pathogenic bacteria infecting animals and humans. Here, we show that IncC conjugative plasmids are highly resilient to host defence systems during entry into a new host by conjugation. Using a TnSeq strategy, we uncover a conserved operon containing five genes (vcrx089-vcrx093) that confer a novel host defence evasion (hde) phenotype. We show that vcrx089-vcrx090 promote resistance against type I restriction-modification, whereas vcrx091-vcxr093 promote CRISPR-Cas evasion by repairing double-strand DNA breaks via recombination between short sequence repeats. vcrx091, vcrx092 and vcrx093 encode a single-strand binding protein, and a single-strand annealing recombinase and double-strand exonuclease related to Redβ and λExo of bacteriophage λ, respectively. Homologous genes of the integrative and conjugative element R391 also provide CRISPR-Cas evasion. Hence, the conserved hde operon considerably broadens the host range of large families of mobile elements spreading multidrug resistance.202032556263
303180.8792Novel Mobilizable Genomic Island GEI-D18A Mediates Conjugational Transfer of Antibiotic Resistance Genes in the Multidrug-Resistant Strain Rheinheimera sp. D18. Aquatic environments act as reservoirs of antimicrobial-resistant bacteria and antimicrobial resistance (AMR) genes, and the dissemination of antibiotic resistance from these environments is of increasing concern. In this study, a multidrug-resistant bacterial strain, identified as Rheinheimera sp. D18, was isolated from the sea water of an industrial maricultural system in the Yellow Sea, China. Whole-genome sequencing of D18 revealed the presence of a novel 25.8 kb antibiotic resistance island, designated GEI-D18A, which carries several antibiotic resistance genes (ARGs), including aadA1, aacA3, tetR, tet(B), catA, dfrA37, and three sul1 genes. Besides, integrase, transposase, resolvase, and recombinase encoding genes were also identified in GEI-D18A. The transferability of GEI-D18A was confirmed by mating experiments between Rheinheimera sp. D18 and Escherichia coli 25DN, and efflux pump inhibitor assays also suggested that tet(B) in GEI-D18A was responsible for tetracycline resistance in both D18 and the transconjugant. This study represents the first characterization of a mobilizable antibiotic resistance island in a species of Rheinheimera and provides evidence that Rheinheimera spp. could be important reservoirs and vehicles for ARGs in the Yellow Sea area.202032318052
199690.8789Conjugation of plasmid harboring bla (NDM-1) in a clinical Providencia rettgeri strain through the formation of a fusion plasmid. Providencia rettgeri has recently gained increased importance owing to the New Delhi metallo-β-lactamase (NDM) and other β-lactamases produced by its clinical isolates. These enzymes reduce the efficiency of antimicrobial therapy. Herein, we reported the findings of whole-genome sequence analysis and a comprehensive pan-genome analysis performed on a multidrug-resistant P. rettgeri 18004577 clinical strain recovered from the urine of a hospitalized patient in Shandong, China, in 2018. Providencia rettgeri 18004577 was found to have a genome assembly size of 4.6 Mb with a G + C content of 41%; a circular plasmid p18004577_NDM of 273.3 Kb, harboring an accessory multidrug-resistant region; and a circular, stable IncT plasmid p18004577_Rts of 146.2 Kb. Additionally, various resistance genes were identified in its genome, including bla (NDM-1), bla (OXA-10), bla (PER-4), aph(3')-VI, ant(2'')-Ia, ant(3')-Ia, sul1, catB8, catA1, mph(E), and tet. Conjugation experiments and whole-genome sequencing revealed that the bla (NDM-1) gene could be transferred to the transconjugant via the formation of pJ18004577_NDM, a novel hybrid plasmid. Based on the genetic comparison, the main possible formation process for pJ18004577_NDM was the insertion of the [ΔISKox2-IS26-ΔISKox2]-aph(3')-VI-bla (NDM-1) translocatable unit module from p18004577_NDM into plasmid p18004577_Rts in the Russian doll insertion structure (ΔISKox2-IS26-ΔISKox2), which played a role similar to that of IS26 using the "copy-in" route in the mobilization of [aph(3')-VI]-bla (NDM-1). The array, multiplicity, and diversity of the resistance and virulence genes in this strain necessitate stringent infection control, antibiotic stewardship, and periodic resistance surveillance/monitoring policies to preempt further horizontal and vertical spread of the resistance genes. Roary analysis based on 30 P. rettgeri strains pan genome identified 415 core, 756 soft core, 5,744 shell, and 12,967 cloud genes, highlighting the "close" nature of P. rettgeri pan-genome. After a comprehensive pan-genome analysis, representative biological information was revealed that included phylogenetic distances, presence or absence of genes across the P. rettgeri bacteria clade, and functional distribution of proteins. Moreover, pan-genome analysis has been shown to be an effective approach to better understand P. rettgeri bacteria because it helps develop various tailored therapeutic strategies based on their biological similarities and differences.202236687647
1515100.8785A novel transposon Tn7540 carrying bla(NDM-9) and fosA3 in chromosome of a pathogenic multidrug-resistant Salmonella enterica serovar Indiana isolated from human faeces. OBJECTIVES: Emergence of multidrug-resistant (MDR) Salmonella enterica serovar Indiana has raised global concern. Mobile genetic elements (MGEs) play vital roles in accelerating the dissemination of resistance genes in bacteria communities. The study aims to improve our understanding of the underlying resistance mechanisms and characterize the MGEs in a MDR S. Indiana isolate. METHODS: Here, we report the characteristics of a MDR pathogenic S. Indiana isolate. The antimicrobial susceptibility pattern of S. Indiana QT6365 was determined. The genomic structure of the chromosome and the plasmid, serotype, and multi-locus sequence type were analysed by whole genome sequencing. The circular form derived from IS26-flanked transposon was confirmed by reverse polymerase chain reaction and sequencing. RESULTS: S. Indiana QT6365 exhibited resistance to all tested antimicrobials except for aztreonam, amikacin, polymyxin, and tigecycline, was defined as MDR, and belonged to ST17. S. Indiana QT6365 was closely related with food resource S. Indiana C629 with similar resistance gene profiles. Multiple resistance genes are mainly carried by a novel transposon Tn7540 located on the chromosome and an IncHI2/HI2A/N plasmid. Sequence analysis and the formed circular intermediate suggested Tn7540 might be generated through homologous recombination by IS26-bounded translocatable units (IS26-fosA-IS26-intI1-dfrA12-aadA2-sul1-ISCR1-bla(NDM-9)-IS26). CONCLUSIONS: To the best of our knowledge, this is the first report of the novel chromosomal transposon possessing bla(NDM-9) and fosA3 in S. Indiana isolated from human specimen, which might facilitate the dissemination of resistance genes and should arouse serious awareness.202336854357
3023110.8785ICEAplChn1, a novel SXT/R391 integrative conjugative element (ICE), carrying multiple antibiotic resistance genes in Actinobacillus pleuropneumoniae. SXT/R391 integrative conjugative elements (ICEs) are capable of self-transfer by conjugation and highly prevalent in various aquatic bacteria and Proteus species. In the present study, a novel SXT/R391 ICE, named ICEAplChn1, was identified in the multidrug resistant (MDR) Actinobacillus pleuropneumoniae strain app6. ICEAplChn1 was composed of the typical SXT/R391 backbone and insertion DNA at eight hotspots, including HS1, HS2, HS3, HS4, HS5, VRII, VRIII and a new variation region VRVI. Many of the insertion contents were not present in other reported SXT/R391 family members, including ICEApl2, a recently identified SXT/R391 ICE from a clinical isolate of A. pleuropneumoniae. Remarkably, the VRIII region had accumulated seven resistance genes tet(A), erm(42), floR, aphA6, strB (two copies), strA and sul2. Of them, erm(42) and aphA6 emerged for the first time not only in the SXT/R391 elements but also in A. pleuropneumoniae. Phylogenetic analysis showed considerable variation of the backbone sequence of ICEAplChn1, as compared to those of other SXT/R391 ICEs. A circular intermediate form of ICEAplChn1 was detected by nested PCR. However, the conjugation experiments using different bacteria as recipients failed. These findings demonstrated that SXT/R391 ICEs are able to adapt to a broader range of host bacterial species. The presence of the MDR gene cluster in ICEAplChn1 underlines that SXT/R391 ICE could serve as an important vector for the accumulation of antibiotic resistance genes.201829885796
1793120.8783Comparative Genome Analysis of an Extensively Drug-Resistant Isolate of Avian Sequence Type 167 Escherichia coli Strain Sanji with Novel In Silico Serotype O89b:H9. Extensive drug resistance (XDR) is an escalating global problem. Escherichia coli strain Sanji was isolated from an outbreak of pheasant colibacillosis in Fujian province, China, in 2011. This strain has XDR properties, exhibiting sensitivity to carbapenems but no other classes of known antibiotics. Whole-genome sequencing revealed a total of 32 known antibiotic resistance genes, many associated with insertion sequence 26 (IS26) elements. These were found on the Sanji chromosome and 2 of its 6 plasmids, pSJ_255 and pSJ_82. The Sanji chromosome also harbors a type 2 secretion system (T2SS), a type 3 secretion system (T3SS), a type 6 secretion system (T6SS), and several putative prophages. Sanji and other ST167 strains have a previously uncharacterized O-antigen (O89b) that is most closely related to serotype O89 as determined on the basis of analysis of the wzm-wzt genes and in silico serotyping. This O89b-antigen gene cluster was also found in the genomes of a few other pathogenic sequence type 617 (ST617) and ST10 complex strains. A time-scaled phylogeny inferred from comparative single nucleotide variant analysis indicated that development of these O89b-containing lineages emerged about 30 years ago. Comparative sequence analysis revealed that the core genome of Sanji is nearly identical to that of several recently sequenced strains of pathogenic XDR E. coli belonging to the ST167 group. Comparison of the mobile elements among the different ST167 genomes revealed that each genome carries a distinct set of multidrug resistance genes on different types of plasmids, indicating that there are multiple paths toward the emergence of XDR in E. coli. IMPORTANCE E. coli strain Sanji is the first sequenced and analyzed genome of the recently emerged pathogenic XDR strains with sequence type ST167 and novel in silico serotype O89b:H9. Comparison of the genomes of Sanji with other ST167 strains revealed distinct sets of different plasmids, mobile IS elements, and antibiotic resistance genes in each genome, indicating that there exist multiple paths toward achieving XDR. The emergence of these pathogenic ST167 E. coli strains with diverse XDR capabilities highlights the difficulty of preventing or mitigating the development of XDR properties in bacteria and points to the importance of better understanding of the shared underlying virulence mechanisms and physiology of pathogenic bacteria.201930834329
515130.8783The Streptomyces peucetius dpsY and dnrX genes govern early and late steps of daunorubicin and doxorubicin biosynthesis. The Streptomyces peucetius dpsY and dnrX genes govern early and late steps in the biosynthesis of the clinically valuable antitumor drugs daunorubicin (DNR) and doxorubicin (DXR). Although their deduced products resemble those of genes thought to be involved in antibiotic production in several other bacteria, this information could not be used to identify the functions of dpsY and dnrX. Replacement of dpsY with a mutant form disrupted by insertion of the aphII neomycin-kanamycin resistance gene resulted in the accumulation of UWM5, the C-19 ethyl homolog of SEK43, a known shunt product of iterative polyketide synthases involved in the biosynthesis of aromatic polyketides. Hence, DpsY must act along with the other components of the DNR-DXR polyketide synthase to form 12-deoxyaklanonic acid, the earliest known intermediate of the DXR pathway. Mutation of dnrX in the same way resulted in a threefold increase in DXR production and the disappearance of two acid-sensitive, unknown compounds from culture extracts. These results suggest that dnrX, analogous to the role of the S. peucetius dnrH gene (C. Scotti and C. R. Hutchinson, J. Bacteriol. 178:73167321, 1996), may be involved in the metabolism of DNR and/or DXR to acid-sensitive compounds, possibly related to the baumycins found in many DNR-producing bacteria.19989573189
3019140.8780Identification 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
3001150.8778IS26 and the IS26 family: versatile resistance gene movers and genome reorganizers. SUMMARYIn Gram-negative bacteria, the insertion sequence IS26 is highly active in disseminating antibiotic resistance genes. IS26 can recruit a gene or group of genes into the mobile gene pool and support their continued dissemination to new locations by creating pseudo-compound transposons (PCTs) that can be further mobilized by the insertion sequence (IS). IS26 can also enhance expression of adjacent potential resistance genes. IS26 encodes a DDE transposase but has unique properties. It forms cointegrates between two separate DNA molecules using two mechanisms. The well-known copy-in (replicative) route generates an additional IS copy and duplicates the target site. The recently discovered and more efficient and targeted conservative mechanism requires an IS in both participating molecules and does not generate any new sequence. The unit of movement for PCTs, known as a translocatable unit or TU, includes only one IS26. TU formed by homologous recombination between the bounding IS26s can be reincorporated via either cointegration route. However, the targeted conservative reaction is key to generation of arrays of overlapping PCTs seen in resistant pathogens. Using the copy-in route, IS26 can also act on a site in the same DNA molecule, either inverting adjacent DNA or generating an adjacent deletion plus a circular molecule carrying the DNA segment lost and an IS copy. If reincorporated, these circular molecules create a new PCT. IS26 is the best characterized IS in the IS26 family, which includes IS257/IS431, ISSau10, IS1216, IS1006, and IS1008 that are also implicated in spreading resistance genes in Gram-positive and Gram-negative pathogens.202438436262
5209160.8777Complete Nucleotide Sequence of pGA45, a 140,698-bp IncFIIY Plasmid Encoding bla IMI-3-Mediated Carbapenem Resistance, from River Sediment. Plasmid pGA45 was isolated from the sediments of Haihe River using Escherichia coli CV601 (gfp-tagged) as recipients and indigenous bacteria from sediment as donors. This plasmid confers reduced susceptibility to imipenem which belongs to carbapenem group. Plasmid pGA45 was fully sequenced on an Illumina HiSeq 2000 sequencing system. The complete sequence of plasmid pGA45 was 140,698 bp in length with an average G + C content of 52.03%. Sequence analysis shows that pGA45 belongs to IncFIIY group and harbors a backbone region which shares high homology and gene synteny to several other IncF plasmids including pNDM1_EC14653, pYDC644, pNDM-Ec1GN574, pRJF866, pKOX_NDM1, and pP10164-NDM. In addition to the backbone region, plasmid pGA45 harbors two notable features including one bla IMI-3-containing region and one type VI secretion system region. The bla IMI-3-containing region is responsible for bacteria carbapenem resistance and the type VI secretion system region is probably involved in bacteria virulence, respectively. Plasmid pGA45 represents the first complete nucleotide sequence of the bla IMI-harboring plasmid from environment sample and the sequencing of this plasmid provided insight into the architecture used for the dissemination of bla IMI carbapenemase genes.201626941718
1511170.8774Characterization of an Extensively Drug-Resistant Salmonella Kentucky ST198 Co-Harboring cfr, mcr-1 and tet(A) Variant from Retail Chicken Meat in Shanghai, China. The emergence of extensively drug-resistant (XDR) foodborne pathogens poses grave threats to food safety. This study characterizes the genome of an XDR Salmonella Kentucky isolate (Sal23C1) co-harboring cfr, mcr-1 and tet(A) from Shanghai chicken meat in 2022, which was the only isolate co-harboring these three key resistance genes among 502 screened Salmonella isolates. Genomic analysis revealed that the multidrug resistance gene cfr, which confers resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins and streptogramin A, was identified within a Tn3-IS6-cfr-IS6 structure on the transferable plasmid p3Sal23C1 (32,387 bp), showing high similarity to the Citrobacter braakii plasmid pCE32-2 (99% coverage, 99.98% identity). Concurrently, the mcr-1 gene resided in a pap2-mcr-1 structure on the transferable IncI2 plasmid p2Sal23C1 (63,103 bp). Notably, both genes could be co-transferred to recipient bacteria via conjugative plasmids at frequencies of (1.15 ± 0.98) × 10(-6). Furthermore, a novel ~79 kb multidrug resistance region (MRR) chromosomally inserted at the bcfH locus was identified, carrying fosA3, mph(A), rmtB, qnrS1 and bla(CTX-M-55). Additionally, a novel Salmonella Genomic Island 1 variant (SGI1-KI) harbored aadA7, qacEΔ1, sul1 and the tet(A) variant. The acquisition of these antibiotic resistance genes in this isolate enhanced bacterial resistance to 21 antimicrobials, including resistance to the critical last-resort antibiotics tigecycline and colistin, which left virtually no treatment options for potential infections. Taken together, this is the first comprehensive genomic report of an XDR poultry-derived Salmonella Kentucky isolate co-harboring cfr, mcr-1 and the tet(A) variant. The mobility of these resistance genes, facilitated by IS6 elements and conjugative plasmids, underscores significant public health risks associated with such isolates in the food chain.202540941142
1536180.8774Complete Genetic Analysis of Plasmids Carried by Two Nonclonal bla(NDM-5)- and mcr-1-Bearing Escherichia coli Strains: Insight into Plasmid Transmission among Foodborne Bacteria. Our objective was to characterize the genetic features of plasmids harbored by two genetically related, MCR-1 and NDM-5-producing Escherichia coli strains recovered from a chicken meat sample. The genetic profiles of all plasmids harbored by the two test strains, namely, 1106 and 1107, were determined by whole-genome sequencing, S1-pulsed-field gel electrophoresis (PFGE), Southern hybridization, and bioinformatics analysis. The transferability of plasmids harbored by the two strains was assessed by filter mating assay. Strains 1106 and 1107 were resistant to almost all the antibiotics, including colistin and fosfomycin, but remained susceptible to amikacin and tigecycline. The plasmids of p1107-NDM-5 and p1106-NDM-5 both contain a class I integron which lacks the ISAba125 element. The backbone of p1106-IncFII exhibited a high degree of similarity with that of p1106-NDM-5 and p1107-NDM-5, implying that events of plasmid fusion and resolution were involved in the formation of the two plasmids. The plasmids p1106-IncHI2MCR and p1107-IncHI2MCR belong to an IncHI2 replicon type, with three copies of ISApl1 being observed in p1106-IncHI2MCR, implying that the mcr-1 gene was transferable among bacteria that reside in the same food matrix. In this study, p1106-IncFIB, p1107-99K, p1107-111K, and p1107-118K were all found to be phage-like plasmids, with p1106-IncFIB and p1107-118K containing several virulence genes, including iroBCDEN, iucABCD, sitABCD, hlyF, and iss. Surprisingly, resistance genes such as aph(3')-Ia, sul3, and aac(3')-IId could also be found in p1107-118K, but resistance genes were not detected in other phage-like plasmids. In conclusion, enhanced surveillance is required to monitor and control the dissemination of various resistance determinants among foodborne pathogens. IMPORTANCE Carbapenem and colistin are last-resort antibiotics used to treat serious clinical infections caused by multidrug-resistant (MDR) bacterial pathogens. Plasmids encoding resistance to carbapenems and colistin have been reported in clinical pathogens in recent years, and yet few studies reported cocarriage of mcr and bla(NDM) genes in Escherichia coli strains of food origin. How plasmids encoding these two important resistance determinants are being evolved and transmitted in bacterial pathogens is not well understood. In this study, we investigated the genetic features of plasmids harbored by two nonclonal, mcr-1- and bla(NDM-5)-bearing E. coli strains (1106 and 1107) recovered from a fresh chicken meat sample to understand and provide evidence of the level and dynamics of MDR plasmid transmission. Our data confirmed that active plasmid fusion and resolution events were involved in the formation of plasmids that harbor multiple resistance genes, which provide insights into the further control of plasmid evolution in bacterial pathogens.202134468190
9069190.8771Pdif-mediated antibiotic resistance genes transfer in bacteria identified by pdifFinder. Modules consisting of antibiotic resistance genes (ARGs) flanked by inverted repeat Xer-specific recombination sites were thought to be mobile genetic elements that promote horizontal transmission. Less frequently, the presence of mobile modules in plasmids, which facilitate a pdif-mediated ARGs transfer, has been reported. Here, numerous ARGs and toxin-antitoxin genes have been found in pdif site pairs. However, the mechanisms underlying this apparent genetic mobility is currently not understood, and the studies relating to pdif-mediated ARGs transfer onto most bacterial genera are lacking. We developed the web server pdifFinder based on an algorithm called PdifSM that allows the prediction of diverse pdif-ARGs modules in bacterial genomes. Using test set consisting of almost 32 thousand plasmids from 717 species, PdifSM identified 481 plasmids from various bacteria containing pdif sites with ARGs. We found 28-bp-long elements from different genera with clear base preferences. The data we obtained indicate that XerCD-dif site-specific recombination mechanism may have evolutionary adapted to facilitate the pdif-mediated ARGs transfer. Through multiple sequence alignment and evolutionary analyses of duplicated pdif-ARGs modules, we discovered that pdif sites allow an interspecies transfer of ARGs but also across different genera. Mutations in pdif sites generate diverse arrays of modules which mediate multidrug-resistance, as these contain variable numbers of diverse ARGs, insertion sequences and other functional genes. The identification of pdif-ARGs modules and studies focused on the mechanism of ARGs co-transfer will help us to understand and possibly allow controlling the spread of MDR bacteria in clinical settings. The pdifFinder code, standalone software package and description with tutorials are available at https://github.com/mjshao06/pdifFinder.202336470841