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300000.9973A 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
177210.9973Molecular Characterization and Comparative Genomics of IncQ-3 Plasmids Conferring Resistance to Various Antibiotics Isolated from a Wastewater Treatment Plant in Warsaw (Poland). As small, mobilizable replicons with a broad host range, IncQ plasmids are widely distributed among clinical and environmental bacteria. They carry antibiotic resistance genes, and it has been shown that they confer resistance to β-lactams, fluoroquinolones, aminoglycosides, trimethoprim, sulphonamides, and tetracycline. The previously proposed classification system divides the plasmid group into four subgroups, i.e., IncQ-1, IncQ-2, IncQ-3, and IncQ-4. The last two subgroups have been poorly described so far. The aim of this study was to analyze five newly identified IncQ-3 plasmids isolated from a wastewater treatment plant in Poland and to compare them with all known plasmids belonging to the IncQ-3 subgroup whose sequences were retrieved from the NCBI database. The complete nucleotide sequences of the novel plasmids were annotated and bioinformatic analyses were performed, including identification of core genes and auxiliary genetic load. Furthermore, functional experiments testing plasmid mobility were carried out. Phylogenetic analysis based on three core genes (repA, mobA/repB, and mobC) revealed the presence of three main clusters of IncQ-3 replicons. Apart from having a highly conserved core, the analyzed IncQ-3 plasmids were vectors of antibiotic resistance genes, including (I) the qnrS2 gene that encodes fluoroquinolone resistance and (II) β-lactam, trimethoprim, and aminoglycoside resistance genes within integron cassettes.202032957637
452520.9972Integrative and Conjugative Elements (ICEs) in Pasteurellaceae Species and Their Detection by Multiplex PCR. Strains of the Pasteurellaceae bacteria Pasteurella multocida and Mannheimia haemolytica are major etiological agents of bovine respiratory disease (BRD). Treatment of BRD with antimicrobials is becoming more challenging due to the increasing occurrence of resistance in infecting strains. In Pasteurellaceae strains exhibiting resistance to multiple antimicrobials including aminoglycosides, beta-lactams, macrolides and sulfonamides, the resistance determinants are often chromosomally encoded within integrative and conjugative elements (ICEs). To gain a more comprehensive picture of ICE structures, we sequenced the genomes of six strains of P. multocida and four strains of M. haemolytica; all strains were independent isolates and eight of them were multiple-resistant. ICE sequences varied in size from 49 to 79 kb, and were comprised of an array of conserved genes within a core region and varieties of resistance genes within accessory regions. These latter regions mainly account for the variation in the overall ICE sizes. From the sequence data, we developed a multiplex PCR assay targeting four conserved core genes required for integration and maintenance of ICE structures. Application of this assay on 75 isolates of P. multocida and M. haemolytica reveals how the presence and structures of ICEs are related to their antibiotic resistance phenotypes. The assay is also applicable to other members of the Pasteurellaceae family including Histophilus somni and indicates how clustering and dissemination of the resistance genes came about.201829997583
579330.9971Association between Escherichia coli with NotI-restriction resistance and urinary tract infections. BACKGROUND: Escherichia coli is the most common cause of urinary tract infections (UTIs). It is widely accepted that uropathogenic E. coli (UPEC) mainly emerge from the distal gut microbiota. Identification of bacterial characteristics that are able to differentiate UPEC from fecal commensal strains will facilitate the development of novel strategies to detect and monitor the spread of UPEC. METHODS: Fifty fecal commensal, 83 UTI-associated and 40 biliary tract infection (BTI)-associated E. coli isolates were analyzed. The NotI restriction patterns of chromosomal DNA in the isolates were determined by pulse-field gel electrophoresis. The phylogenetic types and the presence of 9 known virulence genes of each isolate were determined by PCR analyses. Additionally, the susceptibilities of the isolates to antibiotics were revealed. Then the associations of NotI resistance with UTI-associated isolates, phylotypes, and antibiotic resistance were assessed. RESULTS: NotI resistance was correlated with UTI-associated isolates, compared to the fecal isolates. Consistently, NotI-resistant isolates harbored a greater number of virulence factors and mainly belonged to phylotype B2. Additionally NotI resistance was correlated with chloramphenicol resistance among the bacteria. Among the fecal, UTI-associated and BTI-associated groups, the distribution of NotI-resistant group B2 isolates was correlated with UTI-associated bacteria. CONCLUSION: NotI resistance alone is a potential marker for distinguishing fecal strains and UPEC, while the combination of NotI resistance and B2 phylogeny is a candidate marker to differentiate UPEC from fecal and other extraintestinal pathogenic E. coli. Additionally, NotI resistance may be valuable for assessing the potential of chloramphenicol resistance of E. coli.202234963576
179540.9971Accessory genome of the multi-drug resistant ocular isolate of Pseudomonas aeruginosa PA34. Bacteria can acquire an accessory genome through the horizontal transfer of genetic elements from non-parental lineages. This leads to rapid genetic evolution allowing traits such as antibiotic resistance and virulence to spread through bacterial communities. The study of complete genomes of bacterial strains helps to understand the genomic traits associated with virulence and antibiotic resistance. We aimed to investigate the complete accessory genome of an ocular isolate of Pseudomonas aeruginosa strain PA34. We obtained the complete genome of PA34 utilising genome sequence reads from Illumina and Oxford Nanopore Technology followed by PCR to close any identified gaps. In-depth genomic analysis was performed using various bioinformatics tools. The susceptibility to heavy metals and cytotoxicity was determined to confirm expression of certain traits. The complete genome of PA34 includes a chromosome of 6.8 Mbp and two plasmids of 95.4 Kbp (pMKPA34-1) and 26.8 Kbp (pMKPA34-2). PA34 had a large accessory genome of 1,213 genes and had 543 unique genes not present in other strains. These exclusive genes encoded features related to metal and antibiotic resistance, phage integrase and transposons. At least 24 genomic islands (GIs) were predicated in the complete chromosome, of which two were integrated into novel sites. Eleven GIs carried virulence factors or replaced pathogenic genes. A bacteriophage carried the aminoglycoside resistance gene (AAC(3)-IId). The two plasmids carried other six antibiotic resistance genes. The large accessory genome of this ocular isolate plays a large role in shaping its virulence and antibiotic resistance.201930986237
156750.9971Chromosomal Amplification of the blaOXA-58 Carbapenemase Gene in a Proteus mirabilis Clinical Isolate. Horizontal gene transfer may occur between distantly related bacteria, thus leading to genetic plasticity and in some cases to acquisition of novel resistance traits. Proteus mirabilis is an enterobacterial species responsible for human infections that may express various acquired β-lactam resistance genes, including different classes of carbapenemase genes. Here we report a Proteus mirabilis clinical isolate (strain 1091) displaying resistance to penicillin, including temocillin, together with reduced susceptibility to carbapenems and susceptibility to expanded-spectrum cephalosporins. Using biochemical tests, significant carbapenem hydrolysis was detected in P. mirabilis 1091. Since PCR failed to detect acquired carbapenemase genes commonly found in Enterobacteriaceae, we used a whole-genome sequencing approach that revealed the presence of bla(OXA-58) class D carbapenemase gene, so far identified only in Acinetobacter species. This gene was located on a 3.1-kb element coharboring a bla(AmpC)-like gene. Remarkably, these two genes were bracketed by putative XerC-XerD binding sites and inserted at a XerC-XerD site located between the terminase-like small- and large-subunit genes of a bacteriophage. Increased expression of the two bla genes resulted from a 6-time tandem amplification of the element as revealed by Southern blotting. This is the first isolation of a clinical P. mirabilis strain producing OXA-58, a class D carbapenemase, and the first description of a XerC-XerD-dependent insertion of antibiotic resistance genes within a bacteriophage. This study revealed a new role for the XerC-XerD recombinase in bacteriophage biology.201727855079
468260.9971Revealing antimicrobial resistance profile of the novel probiotic candidate Faecalibacterium prausnitzii DSM 17677. Faecalibacterium prausnitzii, a resident anaerobic bacterium commonly found in healthy gut microbiota, has been proposed as a next generation probiotic with high potential for application in food matrices and pharmaceutical formulations. Despite its recognized health benefits, detailed information regarding its antimicrobial susceptibility profile is still lacking. However, this information is crucial to determine its safety, since the absence of acquired antimicrobial resistance is required to qualify a probiotic candidate as safe for human and animal consumption. Herein, the antimicrobial susceptibility profile of F. prausnitzii DSM 17677 strain was evaluated by integrating both phenotypic and in silico data. Phenotypic antimicrobial susceptibility was evaluated by determining minimum inhibitory concentrations of 9 antimicrobials using broth microdilution and E-test® methods. Also, the whole genome of F. prausnitzii DSM 17677 was analysed, using several databases and bioinformatics tools, to identify possible antibiotic resistance genes (ARG), genomic islands (GI) and mobile genetic elements (MGE). With exception of erythromycin, the same classification (susceptible or resistant) was obtained in both broth microdilution and E-test® methods. Phenotypic resistance to ampicillin, gentamycin, kanamycin and streptomycin were detected, which was supported by the genomic context. Other ARG were also identified but they seem not to be expressed under the tested conditions. F. prausnitzii DSM 17677 genome contains 24 annotated genes putatively involved in resistance against the following classes of antimicrobials: aminoglycosides (such as gentamycin, kanamycin and streptomycin), macrolides (such as erythromycin), tetracyclines and lincosamides. The presence of putative ARG conferring resistance to β-lactams could only be detected using a broader homology search. The majority of these genes are not encoded within GI or MGE and no plasmids were reported for this strain. Despite the fact that most genes are related with general resistance mechanisms, a streptomycin-specific ARG poses the only potential concern identified. This specific ARG is encoded within a GI and a MGE, meaning that it could have been laterally acquired and might be transferred to other bacteria present in the same environment. Thus, our findings provide relevant insights regarding the phenotypic and genotypic antimicrobial resistance profiles of the probiotic candidate F. prausnitzii DSM 17677.202234953344
907170.9971RAC: Repository of Antibiotic resistance Cassettes. Antibiotic resistance in bacteria is often due to acquisition of resistance genes associated with different mobile genetic elements. In Gram-negative bacteria, many resistance genes are found as part of small mobile genetic elements called gene cassettes, generally found integrated into larger elements called integrons. Integrons carrying antibiotic resistance gene cassettes are often associated with mobile elements and here are designated 'mobile resistance integrons' (MRIs). More than one cassette can be inserted in the same integron to create arrays that contribute to the spread of multi-resistance. In many sequences in databases such as GenBank, only the genes within cassettes, rather than whole cassettes, are annotated and the same gene/cassette may be given different names in different entries, hampering analysis. We have developed the Repository of Antibiotic resistance Cassettes (RAC) website to provide an archive of gene cassettes that includes alternative gene names from multiple nomenclature systems and allows the community to contribute new cassettes. RAC also offers an additional function that allows users to submit sequences containing cassettes or arrays for annotation using the automatic annotation system Attacca. Attacca recognizes features (gene cassettes, integron regions) and identifies cassette arrays as patterns of features and can also distinguish minor cassette variants that may encode different resistance phenotypes (aacA4 cassettes and bla cassettes-encoding β-lactamases). Gaps in annotations are manually reviewed and those found to correspond to novel cassettes are assigned unique names. While there are other websites dedicated to integrons or antibiotic resistance genes, none includes a complete list of antibiotic resistance gene cassettes in MRI or offers consistent annotation and appropriate naming of all of these cassettes in submitted sequences. RAC thus provides a unique resource for researchers, which should reduce confusion and improve the quality of annotations of gene cassettes in integrons associated with antibiotic resistance. DATABASE URL: http://www2.chi.unsw.edu.au/rac.201122140215
987580.9971Antibiotic 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
586690.9970tet(L)-mediated tetracycline resistance in bovine Mannheimia and Pasteurella isolates. OBJECTIVES: Tetracycline-resistant Mannheimia and Pasteurella isolates, which were negative for the tetracycline resistance genes (tet) commonly detected among these bacteria, were investigated for other tet genes present and their location. METHODS: Mannheimia and Pasteurella isolates were investigated for their MICs of tetracycline and their plasmid content. Identification of tet genes was achieved by PCR. Plasmids mediating tetracycline resistance were identified by transformation and hybridization experiments. Plasmid pCCK3259 from Mannheimia haemolytica was sequenced completely and analysed for its structure and organization. RESULTS: All tetracycline-resistant isolates carried the gene tet(L) either on plasmids or on the chromosome. Two M. haemolytica isolates and one Mannheimia glucosida isolate harboured a common 5.3 kb tet(L) plasmid, designated pCCK3259. This plasmid was similar to the tet(B)-carrying tetracycline resistance plasmid pHS-Tet from Haemophilus parasuis and the streptomycin/spectinomycin resistance plasmid pCCK647 from Pasteurella multocida in the parts coding for mobilization functions. The tet(L) gene was closely related to that of the Geobacillus stearothermophilus plasmid pTB19. However, the translational attenuator responsible for the tetracycline-inducible expression of tet(L) was missing in plasmid pCCK3259. A recombination site was identified downstream of tet(L), which might explain the integration of the tet(L) gene region into a basic pCCK3259 replicon. CONCLUSION: A tet(L) gene was shown for the first time to be responsible for tetracycline resistance in Mannheimia and Pasteurella isolates. This report demonstrates a lateral transfer of a tetracycline efflux gene in Gram-negative bovine respiratory tract pathogens, probably originating from Gram-positive bacteria.200515972309
3010100.9970Identification of mcr-1 and a novel chloramphenicol resistance gene catT on an integrative and conjugative element in an Actinobacillus strain of swine origin. The aim of this study was to characterize a mcr-1-carrying integrative and conjugative element (ICE) in a novel Pasteurellaceae-like bacteria of swine origin. The mcr-1-positive GY-402 strain, recovered from a pig fecal sample, was subjected to whole genome sequencing with the combination of Illumina Hiseq and MinION platforms. Genome-based taxonomy revealed that strain GY-402 exhibited highest ANI value (84.89 %) to Actinobacillus succinogenes, which suggested that it represented a novel Actinobacillus species. Sequence analysis revealed that mcr-1 was clustered with eight other resistance genes in the MDR region of a novel ICE element, named ICEAsp1. Inverse PCR and mating assays showed that ICEAsp1 is active and transferrable. In addition, six circular forms mediated by four ISApl1 elements were detected with different inverse PCR sets, indicating that flexible composite transposons could be formed by pairwise combinations of multiple IS copies. Cloning experiment and phylogenetic analysis revealed that the novel Cat protein, designated CatT, belongs to type-A family and confers resistance to chloramphenicol. In conclusion, this is, to the best of our knowledge, the first report of mcr-1 gene on ICE structure and also in Pasteurellaceae bacteria. The diverse composite transposons mediated by multicopy IS elements may facilitate the dissemination of different resistance genes.202133486327
2013110.9970Identification and characterization of class 1 integrons in bacteria from an aquatic environment. In a survey of 3000 Gram-negative bacteria isolated from an estuarine environment over a 2 month period, the incidence of class 1 integrons was determined to be 3.6%. Of 85 integrons studied further, 11 lacked both the qacEdelta1 and sull genes usually present in the 3' conserved segment of the integron. The qacEdelta1 and sull genes were identified in the 3' conserved segment of 36 integrons. The remaining 38 integrons lacked a sull gene but contained a qacE gene. The variable region of 74 integrons was characterized by PCR and sequence analysis. Forty of the integrons were found to lack integrated gene cassettes, although 21 of these 'empty' integrons were shown to contain inserted DNA which has been tentatively identified as a novel insertion sequence (IS) element. Of the 34 integrons which contained inserted gene cassettes, the aadA1a gene was found to be the most prevalent (74%). Nineteen integrons contained additional or other gene cassettes in their variable region, including those encoding resistance to trimethoprim (dfr1a, dfrIIc, dfrV, dfrVII, dfrXII), chloramphenicol (catB3, catB5), aminoglycosides (aadA2, aacA4, aacC1), beta-lactamases (oxa2) and erythromycin (ereA). This study confirms the occurrence of integrons in bacteria from a natural habitat and suggests that in the absence of continued antibiotic selective pressures, integrons which persist appear to preferentially exist without integrated antibiotic resistance gene cassettes.199910459805
5810120.9970Evolutionary conservation of motifs within vanA and vanB of vancomycin-resistant enterococci. BACKGROUND AND AIM: Global Health is threatened by the rapid emergence of multidrug-resistant bacteria. Antibiotic resistomes rapidly evolve, yet conserved motifs elucidated in our study have the potential for future drug targets for precision medicine. This study aimed to identify conserved genetic sequences and their evolutionary pathways among vancomycin-resistant Enterococcus species such as Enterococcus faecium and Enterococcus faecalis. MATERIALS AND METHODS: We retrieved a total of 26 complete amino acid and nucleotide sequences of resistance determinant genes against vancomycin (vanA and vanB), streptomycin (aac-aah), and penicillin (pbp5) from the publicly available genetic sequence database, GenBank. The sequences were comprised of bacteria classified under the genera of Enterococcus, Staphylococcus, Amycolatopsis, Ruminococcus, and Clostridium. Sequences were aligned with Clustal Omega Multiple Sequence Alignment program and Percent Identity Matrices were derived. Phylogenetic analyses to elucidate evolutionary relationships between sequences were conducted with the neighbor-end joining method through the Molecular Evolutionary Genetics Analysis (MEGAX) software, developed by the Institute of Molecular Evolutionary Genetics at Pennsylvania State University. Subsequent network analyses of the resistance gene, vanB, within E. faecium were derived from ScanProsite and InterPro. RESULTS: We observed the highest nucleotide sequence similarity of vanA regions within strains of E. faecium (100%) and E. faecalis (100%). Between Enterococcus genera, we continued to observe high sequence conservation for vanA and vanB, up to 99.9% similarity. Phylogenetic tree analyses suggest rapid acquisition of these determinants between strains within vanA and vanB, particularly between strains of Enterococcus genera, which may be indicative of horizontal gene transfer. Within E. faecium, Adenosine 5'-Triphosphate (ATP)-Grasp and D-ala-D-ala ligase (Ddl) were found as conserved domains of vanA and vanB. We additionally found that there is notable sequence conservation, up to 66.67%, between resistomes against vancomycin and streptomycin among E. faecium. CONCLUSION: Resistance genes against vancomycin have highly conserved sequences between strains of Enterococcus bacteria. These conserved sequences within vanA and vanB encode for ATP-Grasp and Ddl motifs, which have functional properties for maintaining cell wall integrity. High sequence conservation is also observed among resistance genes against penicillin and streptomycin, which can inform future drug targets for broader spectrum therapies.202236425127
9968130.9970Antibiotic Resistance, Core-Genome and Protein Expression in IncHI1 Plasmids in Salmonella Typhimurium. Conjugative plasmids from the IncHI1 incompatibility group play an important role in transferring antibiotic resistance in Salmonella Typhimurium. However, knowledge of their genome structure or gene expression is limited. In this study, we determined the complete nucleotide sequences of four IncHI1 plasmids transferring resistance to antibiotics by two different next generation sequencing protocols and protein expression by mass spectrometry. Sequence data including additional 11 IncHI1 plasmids from GenBank were used for the definition of the IncHI1 plasmid core-genome and pan-genome. The core-genome consisted of approximately 123 kbp and 122 genes while the total pan-genome represented approximately 600 kbp. When the core-genome sequences were used for multiple alignments, the 15 tested IncHI1 plasmids were separated into two main lineages. GC content in core-genome genes was around 46% and 50% in accessory genome genes. A multidrug resistance region present in all 4 sequenced plasmids extended over 20 kbp and, except for tet(B), the genes responsible for antibiotic resistance were those with the highest GC content. IncHI1 plasmids therefore represent replicons that evolved in low GC content bacteria. From their original host, they spread to Salmonella and during this spread these plasmids acquired multiple accessory genes including those coding for antibiotic resistance. Antibiotic-resistance genes belonged to genes with the highest level of expression and were constitutively expressed even in the absence of antibiotics. This is the likely mechanism that facilitates host cell survival when antibiotics suddenly emerge in the environment.201627189997
1562140.9970Detection of an IMI-2 carbapenemase-producing Enterobacter asburiae at a Swedish feed mill. Occurrence of multidrug resistant Enterobacteriaceae in livestock is of concern as they can spread to humans. A potential introduction route for these bacteria to livestock could be animal feed. We therefore wanted to identify if Escherichia spp., Enterobacter spp., Klebsiella spp., or Raoutella spp. with transferable resistance to extended spectrum cephalosporins, carbapenems or colistin could be detected in the environment at feed mills in Sweden. A second aim was to compare detected isolates to previous described isolates from humans and animals in Sweden to establish relatedness which could indicate a potential transmission between sectors and feed mills as a source for antibiotic resistant bacteria. However, no isolates with transferable resistance to extended-cephalosporins or colistin could be identified, but one isolate belonging to the Enterobacter cloacae complex was shown to be carbapenem-resistant and showing carbapenemase-activity. Based on sequencing by both short-read Illumina and long-read Oxford Nanopore MinIon technologies it was shown that this isolate was an E. asburiae carrying a bla (IMI-2) gene on a 216 Kbp plasmid, designated pSB89A/IMI-2, and contained the plasmid replicons IncFII, IncFIB, and a third replicon showing highest similarity to the IncFII(Yp). In addition, the plasmid contained genes for various functions such as plasmid segregation and stability, plasmid transfer and arsenical transport, but no additional antibiotic resistance genes. This isolate and the pSB89A/IMI-2 was compared to three human clinical isolates positive for bla (IMI-2) available from the Swedish antibiotic monitoring program Swedres. It was shown that one of the human isolates carried a plasmid similar with regards to gene content to the pSB89A/IMI-2 except for the plasmid transfer system, but that the order of genes was different. The pSB89A/IMI-2 did however share the same transfer system as the bla (IMI-2) carrying plasmids from the other two human isolates. The pSB89A/IMI-2 was also compared to previously published plasmids carrying bla (IMI-2), but no identical plasmids could be identified. However, most shared part of the plasmid transfer system and DNA replication genes, and the bla (IMI-2) gene was located next the transcription regulator imiR. The IS3-family insertion element downstream of imiR in the pSB89A was also related to the IS elements in other bla (IMI)-carrying plasmids.202236338068
1566150.9970Accumulation of Antibiotic Resistance Genes in Carbapenem-Resistant Acinetobacter baumannii Isolates Belonging to Lineage 2, Global Clone 1, from Outbreaks in 2012-2013 at a Tehran Burns Hospital. The worldwide distribution of carbapenem-resistant Acinetobacter baumannii (CRAB) has become a global concern, particularly in countries where antibiotic prescription is not tightly regulated. However, knowledge of the genomic aspects of CRAB from many parts of the world is still limited. Here, 50 carbapenem-resistant A. baumannii isolates recovered at a single hospital in Tehran, Iran, during several outbreaks in 2012 and 2013 were found to be resistant to multiple antibiotics. They were examined using PCR mapping and multilocus sequence typing (MLST). All Iranian strains belonged to sequence type 328 in the Institut Pasteur MLST scheme (ST328(IP)), a single-locus variant of ST81(IP,) and all Iranian strains contained two carbapenem resistance genes, oxa23 and oxa24. The oxa23 gene is in the transposon Tn2006 in AbaR4, which interrupts the chromosomal comM gene. Phylogenetic analysis using whole-genome sequence (WGS) data for 9 isolates showed that they belonged to the same clade, designated the ST81/ST328 clade, within lineage 2 of global clone 1 (GC1). However, there were two groups that included either KL13 or KL18 at the K locus (KL) for capsular polysaccharide synthesis and either a tet39 or an aadB resistance gene, respectively. The genetic context of the resistance genes was determined, and the oxa24 (OXA-72 variant) and tet39 (tetracycline resistance) genes were each in a pdif module in different plasmids. The aadB gene cassette (which encodes gentamicin, kanamycin, and tobramycin resistance) was harbored by pRAY*, and the aphA6 gene (which encodes amikacin resistance) and sul2 gene (which encodes sulfamethoxazole resistance) were each harbored by a different plasmid. The sequences obtained here will underpin future studies of GC1 CRAB strains from the Middle East region.IMPORTANCE Carbapenem-resistant Acinetobacter baumannii strains are among the most critical antibiotic-resistant bacteria causing hospital-acquired infections and treatment failures. The global spread of two clones has been responsible for the bulk of the resistance, in particular, carbapenem resistance. However, there is a substantial gap in our knowledge of which clones and which specific lineages within each clone are circulating in many parts of the world, including Africa and the Middle East region. This is the first genomic analysis of carbapenem-resistant A. baumannii strains from Iran. All the isolates, from a single hospital, belonged to lineage 2 of global clone 1 (GC1) but fell into two groups distinguished by genes in the locus for capsule biosynthesis. The analysis suggests a potential origin of multiply antibiotic-resistant lineage 2 in the Middle East region and highlights the ongoing evolution of carbapenem-resistant GC1 A. baumannii strains. It will enhance future studies on the local and global GC1 population structure.202032269158
3016160.9970Complete nucleotide sequence of the conjugative tetracycline resistance plasmid pFBAOT6, a member of a group of IncU plasmids with global ubiquity. This study presents the first complete sequence of an IncU plasmid, pFBAOT6. This plasmid was originally isolated from a strain of Aeromonas caviae from hospital effluent (Westmorland General Hospital, Kendal, United Kingdom) in September 1997 (G. Rhodes, G. Huys, J. Swings, P. McGann, M. Hiney, P. Smith, and R. W. Pickup, Appl. Environ. Microbiol. 66:3883-3890, 2000) and belongs to a group of related plasmids with global ubiquity. pFBAOT6 is 84,748 bp long and has 94 predicted coding sequences, only 12 of which do not have a possible function that has been attributed. Putative replication, maintenance, and transfer functions have been identified and are located in a region in the first 31 kb of the plasmid. The replication region is poorly understood but exhibits some identity at the protein level with replication proteins from the gram-positive bacteria Bacillus and Clostridium. The mating pair formation system is a virB homologue, type IV secretory pathway that is similar in its structural organization to the mating pair formation systems of the related broad-host-range (BHR) environmental plasmids pIPO2, pXF51, and pSB102 from plant-associated bacteria. Partitioning and maintenance genes are homologues of genes in IncP plasmids. The DNA transfer genes and the putative oriT site also exhibit high levels of similarity with those of plasmids pIPO2, pXF51, and pSB102. The genetic load region encompasses 54 kb, comprises the resistance genes, and includes a class I integron, an IS630 relative, and other transposable elements in a 43-kb region that may be a novel Tn1721-flanked composite transposon. This region also contains 24 genes that exhibit the highest levels of identity to chromosomal genes of several plant-associated bacteria. The features of the backbone of pFBAOT6 that are shared with this newly defined group of environmental BHR plasmids suggest that pFBAOT6 may be a relative of this group, but a relative that was isolated from a clinical bacterial environment rather than a plant-associated bacterial environment.200415574953
9887170.9970PCR-Based Analysis of ColE1 Plasmids in Clinical Isolates and Metagenomic Samples Reveals Their Importance as Gene Capture Platforms. ColE1 plasmids are important vehicles for the spread of antibiotic resistance in the Enterobacteriaceae and Pasteurellaceae families of bacteria. Their monitoring is essential, as they harbor important resistant determinants in humans, animals and the environment. In this work, we have analyzed ColE1 replicons using bioinformatic and experimental approaches. First, we carried out a computational study examining the structure of different ColE1 plasmids deposited in databases. Bioinformatic analysis of these ColE1 replicons revealed a mosaic genetic structure consisting of a host-adapted conserved region responsible for the housekeeping functions of the plasmid, and a variable region encoding a wide variety of genes, including multiple antibiotic resistance determinants. From this exhaustive computational analysis we developed a new PCR-based technique, targeting a specific sequence in the conserved region, for the screening, capture and sequencing of these small plasmids, either specific for Enterobacteriaceae or specific for Pasteurellaceae. To validate this PCR-based system, we tested various collections of isolates from both bacterial families, finding that ColE1 replicons were not only highly prevalent in antibiotic-resistant isolates, but also present in susceptible bacteria. In Pasteurellaceae, ColE1 plasmids carried almost exclusively antibiotic resistance genes. In Enterobacteriaceae, these plasmids encoded a large range of traits, including not only antibiotic resistance determinants, but also a wide variety of genes, showing the huge genetic plasticity of these small replicons. Finally, we also used a metagenomic approach in order to validate this technique, performing this PCR system using total DNA extractions from fecal samples from poultry, turkeys, pigs and humans. Using Illumina sequencing of the PCR products we identified a great diversity of genes encoded by ColE1 replicons, including different antibiotic resistance determinants, supporting the previous results achieved with the collections of bacterial isolates. In addition, we detected cryptic ColE1 plasmids in both families with no known genes in their variable region, which we have named sentinel plasmids. In conclusion, in this work we present a useful genetic tool for the detection and analysis of ColE1 plasmids, and confirm their important role in the dissemination of antibiotic resistance, especially in the Pasteurellaceae family of bacteria.201829615998
2082180.9969Rapid screening technique for class 1 integrons in Enterobacteriaceae and nonfermenting gram-negative bacteria and its use in molecular epidemiology. A screening technique for integrons in members of the family Enterobacteriaceae and nonfermenting gram-negative bacteria by real-time PCR is reported. A total of 226 isolates of gram-negative bacteria obtained from a variety of clinical specimens were screened for class 1 integrons by real-time PCR performed on a LightCycler instrument. This technique used a primer pair specific for a 300-bp conserved region at the 5' ends of class 1 integrons. The screening assay was evaluated by comparison with results obtained by the conventional, thermal-block PCR (long PCR) by using established conditions and primers for the detection of class 1 integrons, and the real-time PCR technique was thus shown to be both sensitive and specific. DNA from 50 of 226 (22%) isolates screened was identified as containing an integron by the screening PCR, and sequence data were obtained across the integron for 34 of 50 (68%) of these isolates. In an attempt to study the molecular epidemiology of antimicrobial resistance genes carried within integrons, a comparison of the types of gene cassettes carried by isolates from different patients was made. Adenyltransferase genes conferring resistance to streptomycin and spectinomycin were the predominant gene cassettes amplified in the study. Resistance to trimethoprim was also frequently found to be encoded within integrons. Furthermore, multiple bacterial isolates obtained from one patient over a 5-month period were all shown to carry an integron containing the same single adenyltransferase gene cassette, suggesting that these elements were relatively stable in this case.200111257011
9966190.9969The A to Z of A/C plasmids. Plasmids belonging to incompatibility groups A and C (now A/C) were among the earliest to be associated with antibiotic resistance in Gram-negative bacteria. A/C plasmids are large, conjugative plasmids with a broad host range. The prevalence of A/C plasmids in collections of clinical isolates has revealed their importance in the dissemination of extended-spectrum β-lactamases and carbapenemases. They also mobilize SGI1-type resistance islands. Revived interest in the family has yielded many complete A/C plasmid sequences, revealing that RA1, designated A/C1, is different from the remainder, designated A/C2. There are two distinct A/C2 lineages. Backbones of 128-130 kb include over 120 genes or ORFs encoding proteins of at least 100 amino acids, but very few have been characterized. Genes potentially required for replication, stability and transfer have been identified, but only the replication system of RA1 and the regulation of transfer have been studied. There is enormous variety in the antibiotic resistance genes carried by A/C2 plasmids but they are usually clustered in larger regions at various locations in the backbone. The ARI-A and ARI-B resistance islands are always at a specific location but have variable content. ARI-A is only found in type 1 A/C2 plasmids, which disseminate blaCMY-2 and blaNDM-1 genes, whereas ARI-B, carrying the sul2 gene, is found in both type 1 and type 2. This review summarizes current knowledge of A/C plasmids, and highlights areas of research to be considered in the future.201525910948