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81900.9946Trimethoprim resistance transposon Tn4003 from Staphylococcus aureus encodes genes for a dihydrofolate reductase and thymidylate synthetase flanked by three copies of IS257. Trimethoprim resistance mediated by the Staphylococcus aureus multi-resistance plasmid pSK1 is encoded by a structure with characteristics of a composite transposon which we have designated Tn4003. Nucleotide sequence analysis of Tn4003 revealed it to be 4717 bp in length and to contain three copies of the insertion element IS257 (789-790 bp), the outside two of which are flanked by directly repeated 8-bp target sequences. IS257 has imperfect terminal inverted repeats of 27-28 bp and encodes for a putative transposase with two potential alpha-helix-turn-alpha-helix DNA recognition motifs. IS257 shares sequence similarities with members of the IS15 family of insertion sequences from Gram-negative bacteria and with ISS1 from Streptococcus lactis. The central region of the transposon contains the dfrA gene that specifies the S1 dihydrofolate reductase (DHFR) responsible for trimethoprim resistance. The S1 enzyme shows sequence homology with type I and V trimethoprim-resistant DHFRs from Gram-negative bacteria and with chromosomally encoded DHFRs from Gram-positive and Gram-negative bacteria. 5' to dfrA is a thymidylate synthetase gene, designated thyE.19892548057
300810.9940Sequence 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
244620.9939Low selection of topoisomerase mutants from strains of Escherichia coli harbouring plasmid-borne qnr genes. OBJECTIVES: To investigate mutations in the type II topoisomerase genes in quinolone-resistant mutants selected from bacteria harbouring plasmid-borne qnr genes. METHODS: Mutants were selected by nalidixic acid, ciprofloxacin and moxifloxacin from two Escherichia coli reference strains and corresponding transconjugants harbouring qnrA1, qnrA3, qnrB2 or qnrS1 genes. RESULTS: The proportion of resistant mutants selected by the three quinolones was, respectively, in the same range for qnr-positive transconjugants and reference strains. Only 20% (65/329) of the mutants selected from the transconjugants showed a gyrase mutation, whereas 79% (94/119) of those from the reference strains without a qnr gene did (P < 0.0001). At four times the MIC of the selector quinolone, gyrA mutants represented 49% and 95% of the mutants selected with nalidixic acid, 4% and 94% with ciprofloxacin and 0% and 54% with moxifloxacin for qnr-positive transconjugants and reference strains, respectively. Mutations within gyrA were distributed at codon 87 (D87G, H, N or Y) and at codon 83 (S83L) with three novel mutations (gyrA Ser83stop, gyrA Asp82Asn and gyrB insertion of Glu at 465) and three rare mutations (gyrA Gly81Asp, gyrA Asp82Gly and gyrA Ser431Pro), mainly obtained from reference strains after moxifloxacin selection. Strikingly, none of the mutants selected by moxifloxacin from qnr-positive transconjugants harboured a mutation in the topoisomerase genes. CONCLUSIONS: Topoisomerase mutants are rarely selected by ciprofloxacin and moxifloxacin from strains harbouring qnr. This suggests that the quinolone resistance-determining region domains are protected from quinolones by the Qnr protein and consequently other mechanisms are developed to acquire a further step of fluoroquinolone resistance.200818325893
200730.9938Novel ISCR1-linked resistance genes found in multidrug-resistant Gram-negative bacteria in southern China. Non-duplicate multidrug-resistant (MDR) Gram-negative bacteria (n=1329) isolated from southern China between January 2008 and December 2009 were investigated for the presence of ISCR1 as well as characterisation of ISCR1-linked resistance genes. Of 433 ISCR1-positive strains, 151 appeared to carry ISCR1-linked resistance genes. Seven different ISCR1-linked resistance gene arrays were identified by restriction fragment length polymorphism (RFLP) and DNA sequencing analysis. Many of these arrays are reported in some species for the first time. A total of 12 genes, including a novel ABC transporter (GenBank accession no. GU944725), qnrA1, qnrB2, qnrB6, bla(DHA-1), ampR, bla(CTX-M-9), bla(PER-1), insB, sapA-like peptide transport periplasmic protein, putative glutathione S-transferase and short-chain dehydrogenase/reductase, were detected. This study was the first to employ PCR-RFLP using HinfI and RsaI to analyse ISCR1-linked genes. ISCR1 was widely disseminated among MDR Gram-negative bacteria and was in close association with quinolone resistance and β-lactamase genes (class A and class C) in southern China.201222890194
40640.9938Naturally occurring macrolide-lincosamide-streptogramin B resistance in Bacillus licheniformis. Resistance to the macrolide-lincosamide-streptogramin B (MLS) group of antibiotics is widespread and of clinical importance. B. Weisblum and his coworkers have demonstrated that this resistance is associated with methylation of the 23S ribosomal ribonucleic acid of the large ribosomal subunit which results in a diminished affinity of this organelle for these antibiotics (Lai et al, J. Mol. Biol. 74:67-72, 1973). We report that 10 of 15 natural isolates of Bacillus licheniformis, a common soil organism, are resistant to the MLS antibiotics. The properties of this resistance (high level of tolerance for erythromycin, broad cross-resistance spectrum, and inducibility) suggest that resistance is conferred as described above. The resistance determinant from one of these strains was cloned onto a B. subtilis plasmid vector, and the resulting hybrid plasmid (pBD90) was used to prepare radioactive probe deoxyribonucleic acid for hybridization studies. All of the resistance B. licheniformis strains studied exhibited homology with the pBD90 insert. Plasmid pBD90 showed no homology to the following staphylococcal and streptococcal MLS-resistance plasmids: pE194, pE5, pAM77, pI258. Plasmids pE194 and pE5, on the other hand, carry homologous MLS genes but showed no detectable homology to one another in their replication genes. pBD90 specified a 35,000-dalton erythromycin-inducible protein, detectable in minicells, which therefore appears different from the 29,000-dalton inducible resistance protein specified by pE194. We conclude that there are at least three distinct MLS resistance determinants to be found among gram-positive bacteria.19816780509
522150.9936Molecular cloning of the DNA gyrase genes from Methylovorus sp. strain SS1 and the mechanism of intrinsic quinolone resistance in methylotrophic bacteria. The genes encoding the DNA gyrase A (GyrA) and B subunits (GyrB) of Methylovorus sp. strain SS1 were cloned and sequenced. gyrA and gyrB coded for proteins of 846 and 799 amino acids with calculated molecular weights of 94,328 and 88,714, respectively, and complemented Escherichia coli gyrA and gyrB temperature sensitive (ts) mutants. To analyze the role of type II topoisomerases in the intrinsic quinolone resistance of methylotrophic bacteria, the sequences of the quinolone resistance-determining regions (QRDRs) in the A subunit of DNA gyrase and the C subunit (ParC) of topoisomerase IV (Topo IV) of Methylovorus sp. strain SS1, Methylobacterium extorquens AM1 NCIB 9133, Methylobacillus sp, strain SK1 DSM 8269, and Methylophilus methylotrophus NCIB 10515 were determined. The deduced amino acid sequences of the QRDRs of the ParCs in the four methylotrophic bacteria were identical to that of E. coli ParC. The sequences of the QRDR in GyrA were also identical to those in E. coli GyrA except for the amino acids at positions 83, 87, or 95. The Ser83 to Thr substitution in Methylovorus sp. strain SS1, and the Ser83 to Leu and Asp87 to Asn substitutions in the three other methylotrophs, agreed well with the minimal inhibitory concentrations of quinolones in the four bacteria, suggesting that these residues play a role in the intrinsic susceptibility of methylotrophic bacteria to quinolones.200516404155
200960.9936Aminoglycoside resistance genes aph(2")-Ib and aac(6')-Im detected together in strains of both Escherichia coli and Enterococcus faecium. Escherichia coli SCH92111602 expresses an aminoglycoside resistance profile similar to that conferred by the aac(6')-Ie-aph(2")-Ia gene found in gram-positive cocci and was found to contain the aminoglycoside resistance genes aph(2")-Ib and aac(6')-Im (only 44 nucleotides apart). aph(2")-Ib had been reported previously in Enterococcus faecium SF11770. aac(6')-Im had not been detected previously in enterococci and was found to be present also 44 nucleotides downstream from aph(2")-Ib in E. faecium SF11770. aph(2")-Ib and aac(6')-Im are separate open reading frames, each with its own putative ribosome binding site, whereas aac(6')-Ie-aph(2")-Ia appears to be a fusion of two genes with just one start and one stop codon. The deduced AAC(6')-Im protein exhibits 56% identity and 80% similarity to the AAC(6')-Ie domain of the bifunctional enzyme AAC(6')-APH(2"). Our results document the existence of a member of the aph(2") family of genes in gram-negative bacteria and provide evidence suggesting the horizontal transfer of aph(2")-Ib and aac(6')-Im as a unit between gram-positive and gram-negative bacteria.200111557456
587570.9935Detection of the staphylococcal multiresistance gene cfr in Macrococcus caseolyticus and Jeotgalicoccus pinnipedialis. OBJECTIVES: To investigate the presence and the genetic environment of the multiresistance gene cfr in Jeotgalicoccus pinnipedialis and Macrococcus caseolyticus from pigs. METHODS: A total of 391 bacterial isolates with florfenicol MICs ≥16 mg/L were obtained from nasal swabs of 557 individual pigs; of these, 75 Gram-positive isolates other than staphylococci and enterococci were screened by PCR for the presence of known florfenicol resistance genes. Species assignments of the cfr-carrying isolates were based on the results of biochemical profiling and 16S rDNA sequencing. The locations of the cfr gene were determined by Southern blotting. Regions flanking each cfr gene were sequenced by a modified random primer walking strategy, and the transferability of cfr was assessed by electrotransformation. RESULTS: Two M. caseolyticus isolates and one J. pinnipedialis isolate were cfr positive. The cfr gene was located either on a 7057 bp plasmid, pSS-03, which was widely distributed among staphylococci of pig origin, or on the ∼53 kb plasmid pJP1. The region of pJP1 that included the cfr gene and the adjacent IS21-558, showed 99.7% identity to the corresponding region of plasmid pSCFS3. In addition, the genes aadD + aacA-aphD, ble and erm(C), coding for aminoglycoside, bleomycin and macrolide-lincosamide-streptogramin B resistance, respectively, were also identified on plasmid pJP1. CONCLUSIONS: This study showed that plasmids carrying the multidrug resistance gene cfr are present in two new genera of commensal and environmental bacteria, Macrococcus and Jeotgalicoccus. This observation underlines the role of commensal and environmental flora in the dissemination of clinically important resistance genes, such as cfr.201222577104
40580.9935Characterization of a small plasmid (pMBCP) from bovine Pseudomonas pickettii that confers cadmium resistance. This is the first report of isolation of Pseudomonas pickettii from a normal adult bovine duodenum. This organism was one of several bacteria isolated as part of a study to examine cadmium resistance genes (cad(r)) for use in generating transgenic plants to reclaim cadmium-contaminated soils in Kansas. P. pickettii containing a plasmid of 2.2kb (designated pMBCP) grew in Luria-Bertani broth and agar containing up to 800 microM of cadmium chloride and was resistant to 16 antibiotics. Curing the organism of plasmid revealed that antibiotic resistances were not plasmid-mediated. Low-level cadmium resistance was conferred by the plasmid because uncured organism grew significantly better (P<0.05) at 55 microM compared to cured organism. Both plasmid and chromosomal DNA were probed by DNA-DNA hybridization for the presence of known cadmium resistance genes (cadA, cadC, and cadD from Gram-positive (Staphylococcus aureus), but none were detected. The plasmid had one restriction site each for BamHI, PstI, SmaI, and XhoI; two sites each for HincII, SacI, and SphI; and multiple sites for AluI and XcmI. DNA sequence analyses of the cloned and original plasmids showed a GC content of greater than 60% and no homology to any published sequences in the GenBank, European Bioinformatics Institute, or Japanese Genome Net databases. The DNA sequence is contained in GenBank accession number AF144733. Thus, pMBCP offers low-level cadmium resistance to P. picketttii.200312651180
585090.9935Gram-positive merA gene in gram-negative oral and urine bacteria. Clinical mercury resistant (Hg(r)) Gram-negative bacteria carrying Gram-positive mercury reductase (merA)-like genes were characterized using DNA-DNA hybridization, PCR and sequencing. A PCR assay was developed which discriminated between the merA genes related to Staphylococcus and those related to the Bacillus/Streptococcus merA genes by the difference in size of the PCR product. DNA sequence analysis correlated with the PCR assay. The merA genes from Acinetobacter junii, Enterobacter cloacae and Escherichia coli were sequenced and shared 98-99% identical nucleotide (nt) and 99.6-100% amino acid identity with the Staphylococcus aureus MerA protein. A fourth merA gene, from Pantoeae agglomerans, was partially sequenced (60%) and had 99% identical nt and 100% amino acid identity with the Streptococcus oralis MerA protein. All the Hg(r) Gram-negative bacteria transferred their Gram-positive merA genes to a Gram-positive Enterococcus faecalis recipient with the resulting transconjugants expressing mercury resistance. These Gram-positive merA genes join Gram-positive tetracycline resistance and Gram-positive macrolide resistance genes in their association with mobile elements which are able to transfer and express in Gram-negative bacteria.200415358427
820100.9934Nucleotide sequence analysis of a transposon (Tn5393) carrying streptomycin resistance genes in Erwinia amylovora and other gram-negative bacteria. A class II Tn3-type transposable element, designated Tn5393 and located on plasmid pEa34 from streptomycin-resistant strain CA11 of Erwinia amylovora, was identified by its ability to move from pEa34 to different sites in plasmids pGEM3Zf(+) and pUCD800. Nucleotide sequence analysis reveals that Tn5393 consists of 6,705 bp with 81-bp terminal inverted repeats and generates 5-bp duplications of the target DNA following insertion. Tn5393 contains open reading frames that encode a putative transposase (tnpA) and resolvase (tnpR) of 961 and 181 amino acids, respectively. The two open reading frames are separated by a putative recombination site (res) consisting of 194 bp. Two streptomycin resistance genes, strA and strB, were identified on the basis of their DNA sequence homology to streptomycin resistance genes in plasmid RSF1010. StrA is separated from tnpR by a 1.2-kb insertion element designated IS1133. The tnpA-res-tnpR region of Tn5393 was detected in Pseudomonas syringae pv. papulans Psp36 and in many other gram-negative bacteria harboring strA and strB. Except for some strains of Erwinia herbicola, these other gram-negative bacteria lacked insertion sequence IS1133. The prevalence of strA and strB could be accounted for by transposition of Tn5393 to conjugative plasmids that are then disseminated widely among gram-negative bacteria.19938380801
2453110.9934Prevalence and molecular determinants of colistin resistance among commensal Enterobacteriaceae isolated from poultry in northwest of Iran. BACKGROUND: The emergence of colistin-resistant Enterobacteriaceae from human and animal sources is a public health concern as this antibiotic is considered to be the last line therapeutic option for infections caused by multidrug-resistant Gram-negative bacteria. Here we aimed to determine the prevalence of colistin resistance, among enterobacteria isolated from poultry and the possible underlying colistin resistance mechanisms. METHODS: A collection of 944 cloacal samples were obtained from poultry and screened for colistin resistance. To uncover the molecular mechanism behind colistin resistance, the presence of plasmid encoded colistin resistance genes mcr-1, mcr-2, mcr-3 and mcr-4 was examined by PCR. The nucleotide sequences of the mgrB, pmrA, pmrB, phoP, phoQ, crrA and crrB genes were determined. The genetic relatedness of the colistin resistant (ColR) isolates was evaluated by Multilocus sequence typing. Three ColR mutants were generated in vitro by repetitive drug exposure. RESULTS: Overall from 931 enteric bacteria isolated from poultry samples obtained from 131 farms, nine ColR bacteria (0.96%) with high level colistin resistance (MICs ≥ 64 mg/L) were detected all being identified as K. pneumoniae. The 9 ColR bacteria originated from different farms and belonged to 7 distinct Sequence types including ST11 (22.2%) and ST726 (22.2%) being the most prevalent STs followed by ST37, ST74, ST485, ST525 and novel sequence type 3380 (11.1% each). mcr-type genes were not detected in any isolate. In 88.8% of the isolates (n = 8), MgrB was inactivated by Insertion of IS elements (IS1-like, IS3-like, IS5-like families, positions + 75, + 113, + 117, + 135) and nonsense mutations at codons 8, 16, 30. All ColR isolates harboured wild type PmrA, PhoP, PhoQ or polymorphic variants of PmrB. Sequence analysis of the CrrB revealed a familiar S195N and 4 novel I27V, T150R, F303S and K325R substitutions. PmrB T93N substitution and mgrB locus deletion were identified in two laboratory induced ColR mutants and one mutant lacked alteration in the studied loci. In one ColR isolate with wild type MgrB an A83V substitution was detected in CrrA. CONCLUSION: It is concluded from our results that colistin resistance in the studied avian K. pneumoniae isolates was mostly linked to alterations identified within the mgrB gene.201930728861
818120.9933Characterization of a staphylococcal plasmid related to pUB110 and carrying two novel genes, vatC and vgbB, encoding resistance to streptogramins A and B and similar antibiotics. We isolated and sequenced a plasmid, named pIP1714 (4,978 bp), which specifies resistance to streptogramins A and B and the mixture of these compounds. pIP1714 was isolated from a Staphylococcus cohnii subsp. cohnii strain found in the environment of a hospital where pristinamycin was extensively used. Resistance to both compounds and related antibiotics is encoded by two novel, probably cotranscribed genes, (i) vatC, encoding a 212-amino-acid (aa) acetyltransferase that inactivates streptogramin A and that exhibits 58.2 to 69.8% aa identity with the Vat, VatB, and SatA proteins, and (ii) vgbB, encoding a 295-aa lactonase that inactivates streptogramin B and that shows 67% aa identity with the Vgb lactonase. pIP1714 includes a 2,985-bp fragment also found in two rolling-circle replication and mobilizable plasmids, pUB110 and pBC16, from gram-positive bacteria. In all three plasmids, the common fragment was delimited by two direct repeats of four nucleotides (GGGC) and included (i) putative genes closely related to repB, which encodes a replication protein, and to pre(mob), which encodes a protein required for conjugative mobilization and site-specific recombination, and (ii) sequences very similar to the double- and single-strand origins (dso, ssoU) and the recombination site, RSA. The antibiotic resistance genes repB and pre(mob) carried by each of these plasmids were found in the same transcriptional orientation.19989661023
831130.9933RmtC and RmtF 16S rRNA Methyltransferase in NDM-1-Producing Pseudomonas aeruginosa. We investigated 16S rRNA methyltransferases in 38 blaNDM-1-positive Pseudomonas aeruginosa isolates and found RmtC in 3 isolates, 1 of which also harbored RmtF. The isolates were clonally unrelated; rmtC and rmtF genes were located on a chromosome with the blaNDM-1 gene. Strategies are needed to limit the spread of such isolates.201526488937
1403140.9932Evaluation of the AusDiagnostics MT CRE EU assay for the detection of carbapenemase genes and transferable colistin resistance determinants mcr-1/-2 in MDR Gram-negative bacteria. OBJECTIVES: To evaluate the AusDiagnostics MT CRE EU assay for the detection of carbapenemase and acquired colistin resistance genes in Gram-negative bacteria. METHODS: The assay allows the detection of blaKPC, blaOXA-48-like, blaNDM, blaVIM, blaIMP, blaSIM, blaGIM, blaSPM, blaFRI, blaIMI, blaGES (differentiating ESBL and carbapenemase variants), blaSME and mcr-1/-2. It was evaluated against a panel of isolates including Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp. retrospectively (n = 210) and prospectively (n = 182). RESULTS: The CRE EU assay was able to detect 268/268 carbapenemase genes, with 239 belonging to the 'big five' families (KPC, OXA-48-like, NDM, VIM and IMP) and 29 carbapenemase genes of the SIM, GIM, SPM, FRI, IMI, SME and GES families. It could distinguish between ESBL and carbapenemase variants of GES. It also allowed detection of mcr-1/-2 colistin resistance genes on their own or in isolates co-producing a carbapenemase. CONCLUSIONS: The AusDiagnostics MT CRE EU assay offered wide coverage for detection of acquired carbapenemase genes. It required minimal hands-on time and delivered results in less than 4 h from bacterial culture.201830189011
1439150.9932Molecular characteristics of carbapenem-resistant gram-negative bacteria in southern China. A total of 368 nonreplicate gram-negative bacteria with resistance to imipenem or meropenem were collected to search for carbapenemase genes, class 1 integrons, and insertion sequence with common region 1 (ISCR1). The carbapenemase genes blaIMP-4, blaKPC-2, and blaNDM-1 were found in two Enterobacteriaceae and seven Pseudomonas aeruginosa isolates, nine Klebsiella pneumoniae isolates, and seven Enterobacteriaceae and two Acinetobacter spp. isolates. The class D OXA-type carbapenemase genes blaOXA-23-like, blaOXA-24-like, blaOXA-58, and blaOXA-51-like were detected in 59 (34.9%), 2 (1.2%), 16 (9.5%), and 126 (74.6%) Acinetobacter strains. This is the first description of blaNDM-1 in Enterobacter hormaechei and Acinetobacter genomic species 13TU. Of the integrase-positive strains, 135 (90.0%) Acinetobacter spp., 22 (61.1%) P. aeruginosa, and 14 (100%) Enterobacteriaceae isolates were identified by five, ten, and four different gene cassette arrays, respectively. Three novel gene cassette arrays aadB-aadA1, dfrA25, and dfrA16-aadA2 were reported for the first time in some species. Of the ISCR1-positive strains, the nonfermentative strains (102 Acinetobacter spp. and 13 P. aeruginosa. isolates) contained the same arrangement blaPER-1-putative glutathione-S-transferase-novel type ABC transporter, and three Enterobacteriaceae isolates harbored three different arrangements. Four distinct complex class 1 integron structures were observed. The complex class 1 integron detected in New Delhi, metallo-β-lactamase (NDM-1)-producing E. hormaechei, was found to coexist in the NDM-1-carrying plasmid. Our results suggested that we should pay more attention to the strict implementation of infection control measures and active antibiotic resistance surveillance to avoid the rapid spread or outbreak of carbapenemase-producing gram-negative bacteria.201525469995
2454160.9932Colistin resistance in Gram-negative bacteria analysed by five phenotypic assays and inference of the underlying genomic mechanisms. BACKGROUND: Colistin is used against multi-drug resistant pathogens, yet resistance emerges through dissemination of plasmid-mediated genes (mcr) or chromosomal mutation of genes involved in lipopolysaccharide synthesis (i.e. mgrB, phoPQ, pmrCAB). Phenotypic susceptibility testing is challenging due to poor diffusion of colistin in agar media, leading to an underestimation of resistance. Performance of five phenotypic approaches was compared in the context of different molecular mechanisms of resistance. We evaluated Vitek 2® (bioMérieux, AST N242), Colistin MIC Test Strip (Liofilchem Diagnostici), UMIC (Biocentric), and Rapid Polymyxin™ NP test (ELITechGroup) against the standard broth microdilution (BMD) method. We used whole genome sequencing (WGS) to infer molecular resistance mechanisms. We analysed 97 Enterobacterales and non-fermenting bacterial isolates, largely clinical isolates collected up to 2018. Data was analysed by comparing susceptibility categories (susceptible or resistant) and minimal inhibitory concentrations (MIC). Susceptibility category concordance is the percentage of test results sharing the same category to BMD. MIC concordance was calculated similarly but considering ±1 MIC titre error range. We determined genomic diversity by core genome multi locus sequencing typing (cgMLST) and identified putative antimicrobial resistance genes using NCBI and CARD databases, and manual annotation. RESULTS: Of 97 isolates, 54 (56%) were resistant with standard BMD. Highest susceptibility category concordance was achieved by Rapid Polymyxin™ NP (98.8%) followed by UMIC (97.9%), Colistin E-test MIC strip (96.9%) and Vitek 2® (95.6%). Highest MIC concordance was achieved by UMIC (80.4%), followed by Vitek 2® (72.5%) and Colistin E-test MIC strip (62.9%). Among resistant isolates, 23/54 (43%) were intrinsically resistant to colistin, whereas 31/54 (57%) isolates had acquired colistin resistance. Of these, mcr-1 was detected in four isolates and mcr-2 in one isolate. Non-synonymous mutations in mgrB, phoQ, pmrA, pmrB, and pmrC genes were encountered in Klebsiella pneumoniae, Escherichia coli, and Acinetobacter bereziniae resistant isolates. Mutations found in mgrB and pmrB were only identified in isolates exhibiting MICs of ≥16 mg/L. CONCLUSIONS: The Rapid Polymyxin™ NP test showed highest categorical concordance and the UMIC test provided MIC values with high concordance to BMD. We found colistin resistance in diverse species occurred predominantly through spontaneous chromosomal mutation rather than plasmid-mediated resistance.202134798825
829170.9932Loop-mediated isothermal amplification assay for 16S rRNA methylase genes in Gram-negative bacteria. Using the loop-mediated isothermal amplification (LAMP) method, we developed a rapid assay for detection of 16S rRNA methylase genes (rmtA, rmtB, and armA), and investigated 16S rRNA methylase-producing strains among clinical isolates. Primer Explorer V3 software was used to design the LAMP primers. LAMP primers were prepared for each gene, including two outer primers (F3 and B3), two inner primers (FIP and BIP), and two loop primers (LF and LB). Detection was performed with the Loopamp DNA amplification kit. For all three genes (rmtA, rmtB, and armA), 10(2) copies/tube could be detected with a reaction time of 60 min. When nine bacterial species (65 strains saved in National Institute of Infectious Diseases) were tested, which had been confirmed to possess rmtA, rmtB, or armA by PCR and DNA sequencing, the genes were detected correctly in these bacteria with no false negative or false positive results. Among 8447 clinical isolates isolated at 36 medical institutions, the LAMP method was conducted for 191 strains that were resistant to aminoglycosides based on the results of antimicrobial susceptibility tests. Eight strains were found to produce 16S rRNA methylase (0.09%), with rmtB being identified in three strains (0.06%) of 4929 isolates of Enterobacteriaceae, rmtA in three strains (0.10%) of 3284 isolates of Pseudomonas aeruginosa, and armA in two strains (0.85%) of 234 isolates of Acinetobacter spp. At present, the incidence of strains possessing 16S rRNA methylase genes is very low in Japan. However, when Gram-negative bacteria showing high resistance to aminoglycosides are isolated by clinical laboratories, it seems very important to investigate the status of 16S rRNA methylase gene-harboring bacilli and monitor their trends among Japanese clinical settings.201425179393
1477180.9932Multicenter Evaluation of the BIOFIRE Blood Culture Identification 2 Panel for Detection of Bacteria, Yeasts, and Antimicrobial Resistance Genes in Positive Blood Culture Samples. Diagnostic tools that can rapidly identify and characterize microbes growing in blood cultures are important components of clinical microbiology practice because they help to provide timely information that can be used to optimize patient management. This publication describes the bioMérieux BIOFIRE Blood Culture Identification 2 (BCID2) Panel clinical study that was submitted to the U.S. Food & Drug Administration. Results obtained with the BIOFIRE BCID2 Panel were compared to standard-of-care (SoC) results, sequencing results, PCR results, and reference laboratory antimicrobial susceptibility testing results to evaluate the accuracy of its performance. Results for 1,093 retrospectively and prospectively collected positive blood culture samples were initially enrolled, and 1,074 samples met the study criteria and were included in the final analyses. The BIOFIRE BCID2 Panel demonstrated an overall sensitivity of 98.9% (1,712/1,731) and an overall specificity of 99.6% (33,592/33,711) for Gram-positive bacteria, Gram-negative bacteria and yeast targets which the panel is designed to detect. One hundred eighteen off-panel organisms, which the BIOFIRE BCID2 Panel is not designed to detect, were identified by SoC in 10.6% (114/1,074) of samples. The BIOFIRE BCID2 Panel also demonstrated an overall positive percent agreement (PPA) of 97.9% (325/332) and an overall negative percent agreement (NPA) of 99.9% (2,465/2,767) for antimicrobial resistance determinants which the panel is designed to detect. The presence or absence of resistance markers in Enterobacterales correlated closely with phenotypic susceptibility and resistance. We conclude that the BIOFIRE BCID2 Panel produced accurate results in this clinical trial.202337227281
5846190.9932Distribution of tetracycline resistance genes and transposons among phylloplane bacteria in Michigan apple orchards. The extent and nature of tetracycline resistance in bacterial populations of two apple orchards with no or a limited history of oxytetracycline usage were assessed. Tetracycline-resistant (Tc(r)) bacteria were mostly gram negative and represented from 0 to 47% of the total bacterial population on blossoms and leaves (versus 26 to 84% for streptomycin-resistant bacteria). A total of 87 isolates were screened for the presence of specific Tc(r) determinants. Tc(r) was determined to be due to the presence of Tet B in Pantoea agglomerans and other members of the family Enterobacteriacae and Tet A, Tet C, or Tet G in most Pseudomonas isolates. The cause of Tc(r) was not identified in 16% of the isolates studied. The Tc(r) genes were almost always found on large plasmids which also carried the streptomycin resistance transposon Tn5393. Transposable elements with Tc(r) determinants were detected by entrapment following introduction into Escherichia coli. Tet B was found within Tn10. Two of eighteen Tet B-containing isolates had an insertion sequence within Tn10; one had IS911 located within IS10-R and one had Tn1000 located upstream of Tet B. Tet A was found within a novel variant of Tn1721, named Tn1720, which lacks the left-end orfI of Tn1721. Tet C was located within a 19-kb transposon, Tn1404, with transposition genes similar to those of Tn501, streptomycin (aadA2) and sulfonamide (sulI) resistance genes within an integron, Tet C flanked by direct repeats of IS26, and four open reading frames, one of which may encode a sulfate permease. Two variants of Tet G with 92% sequence identity were detected.199910543801