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519900.9944Whole genome sequencing uncovers a novel IND-16 metallo-β-lactamase from an extensively drug-resistant Chryseobacterium indologenes strain J31. BACKGROUND: Chryseobacterium indologenes is an emerging opportunistic pathogen in hospital-acquired infection, which is intrinsically resistant to most antimicrobial agents against gram-negative bacteria. In the purpose of extending our understanding of the resistance mechanism of C. indologenes, we sequenced and analyzed the genome of an extensively antibiotic resistant C. indologenes strain, isolated from a Chinese prostate cancer patient. We also investigated the presence of antibiotic resistance genes, particularly metallo-β-lactamase (MBL) genes, and performed a comparative genomic analysis with other Chryseobacterium species. RESULTS: 16s rRNA sequencing indicated the isolate belongs to C. indologenes. We assembled a total of 1095M bp clean-filtered reads into 171 contigs by de novo assembly. The draft genome of C. indologenes J31 consisted of 5,830,795 bp with a GC content of 36.9 %. RAST analysis revealed the genome contained 5196 coding sequences (CDSs), 28 rRNAs, 81 tRNAs and 114 pseudogenes. We detected 90 antibiotic resistance genes from different drug classes in the whole genome. Notably, a novel bla(IND) allele bla(IND-16) was identified, which shared 99 % identity with bla(IND-8) and bla(IND-10). By comparing strain J31 genome to the closely four related neighbors in the genus Chryseobacterium, we identified 2634 conserved genes, and 1449 unique genes. CONCLUSIONS: In this study, we described the whole genome sequence of C. indologenes strain J31. Numerous resistance determinants were detected in the genome and might be responsible for the extensively antibiotic resistance of this strain. Comparative genomic analysis revealed the presence of considerable strain-specific genes which would contribute to the distinctive characteristics of strain J31. Our study provides the insight of the multidrug resistance mechanism in genus Chryseobacterium.201627785154
520010.9941Whole genome sequencing of the multidrug-resistant Chryseobacterium indologenes isolated from a patient in Brazil. Chryseobacterium indologenes is a non-glucose-fermenting Gram-negative bacillus. This emerging multidrug resistant opportunistic nosocomial pathogen can cause severe infections in neonates and immunocompromised patients. This study aimed to present the first detailed draft genome sequence of a multidrug-resistant C. indologenes strain isolated from the cerebrospinal fluid of an infant hospitalized at the Neonatal Intensive Care Unit of Brazilian Tertiary Hospital. We first analyzed the susceptibility of C. indologenes strain to different antibiotics using the VITEK 2 system. The strain demonstrated an outstanding resistance to all the antibiotic classes tested, including β-lactams, aminoglycosides, glycylcycline, and polymyxin. Next, C. indologenes was whole-genome-sequenced, annotated using Prokka and Rapid Annotation using Subsystems Technology (RAST), and screened for orthologous groups (EggNOG), gene ontology (GO), resistance genes, virulence genes, and mobile genetic elements using different software tools. The draft genome contained one circular chromosome of 4,836,765 bp with 37.32% GC content. The genomic features of the chromosome present numerous genes related to cellular processes that are essential to bacteria. The MDR C. indologenes revealed the presence of genes that corresponded to the resistance phenotypes, including genes to β-lactamases (bla (IND-13), bla (CIA-3), bla (TEM-116), bla (OXA-209), bla (VEB-15)), quinolone (mcbG), tigecycline (tet(X6)), and genes encoding efflux pumps which confer resistance to aminoglycosides (RanA/RanB), and colistin (HlyD/TolC). Amino acid substitutions related to quinolone resistance were observed in GyrA (S83Y) and GyrB (L425I and K473R). A mutation that may play a role in the development of colistin resistance was detected in lpxA (G68D). Chryseobacterium indologenes isolate harbored 19 virulence factors, most of which were involved in infection pathways. We identified 13 Genomic Islands (GIs) and some elements associated with one integrative and conjugative element (ICEs). Other elements linked to mobile genetic elements (MGEs), such as insertion sequence (ISEIsp1), transposon (Tn5393), and integron (In31), were also present in the C. indologenes genome. Although plasmids were not detected, a ColRNAI replicon type and the most resistance genes detected in singletons were identified in unaligned scaffolds. We provided a wide range of information toward the understanding of the genomic diversity of C. indologenes, which can contribute to controlling the evolution and dissemination of this pathogen in healthcare settings.202235966843
540520.9941Characterization of florfenicol resistance genes in the coagulase-negative Staphylococcus (CoNS) isolates and genomic features of a multidrug-resistant Staphylococcus lentus strain H29. BACKGROUND: With the wide use of florfenicol to prevent and treat the bacterial infection of domestic animals, the emergence of the florfenicol resistance bacteria is increasingly serious. It is very important to elucidate the molecular mechanism of the bacteria's resistance to florfenicol. METHODS: The minimum inhibitory concentration (MIC) levels were determined by the agar dilution method, and polymerase chain reaction was conducted to analyze the distribution of florfenicol resistance genes in 39 CoNS strains isolated from poultry and livestock animals and seafood. The whole genome sequence of one multidrug resistant strain, Staphylococcus lentus H29, was characterized, and comparative genomics analysis of the resistance gene-related sequences was also performed. RESULTS: As a result, the isolates from the animals showed a higher resistance rate (23/28, 82.1%) and much higher MIC levels to florfenicol than those from seafood. Twenty-seven animal isolates carried 37 florfenicol resistance genes (including 26 fexA, 6 cfr and 5 fexB genes) with one carrying a cfr gene, 16 each harboring a fexA gene, 5 with both a fexA gene and a fexB gene and the other 5 with both a fexA gene and a cfr gene. On the other hand, all 11 isolates from seafood were sensitive to florfenicol, and only 3 carried a fexA gene each. The whole genome sequence of S. lentus H29 was composed of a chromosome and two plasmids (pH29-46, pH29-26) and harbored 11 resistance genes, including 6 genes [cfr, fexA, ant(6)-Ia, aacA-aphD, mecA and mph(C)] encoded on the chromosome, 4 genes [cfr, fexA, aacA-aphD and tcaA] on pH29-46 and 1 gene (fosD) on pH29-26. We found that the S. lentus H29 genome carried two identical copies of the gene arrays of radC-tnpABC-hp-fexA (5671 bp) and IS256-cfr (2690 bp), of which one copy of the two gene arrays was encoded on plasmid pH29-46, while the other was encoded on the chromosome. CONCLUSIONS: The current study revealed the wide distribution of florfenicol resistance genes (cfr, fexA and fexB) in animal bacteria, and to the best of our knowledge, this is the first report that one S. lentus strain carried two identical copies of florfenicol resistance-related gene arrays.202133413633
245330.9940Prevalence 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
245440.9939Colistin 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
200750.9938Novel 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
514360.9937Genomic Insights Into the Pathogenicity of a Novel Biofilm-Forming Enterococcus sp. Bacteria (Enterococcus lacertideformus) Identified in Reptiles. Whole genome analysis of a novel species of enterococci, Enterococcus lacertideformus, causing multi-systemic and invariably fatal disease in critically endangered Christmas Island reptiles was undertaken to determine the genetic elements and potential mechanisms conferring its pathogenic nature, biofilm-forming capabilities, immune recognition avoidance, and inability to grow in vitro. Comparative genomic analyses with related and clinically significant enterococci were further undertaken to infer the evolutionary history of the bacterium and identify genes both novel and absent. The genome had a G + C content of 35.1%, consisted of a circular chromosome, no plasmids, and was 2,419,934 bp in length (2,321 genes, 47 tRNAs, and 13 rRNAs). Multi-locus sequence typing (MLST), and single nucleotide polymorphism (SNP) analysis of multiple E. lacertideformus samples revealed they were effectively indistinguishable from one another and highly clonal. E. lacertideformus was found to be located within the Enterococcus faecium species clade and was closely related to Enterococcus villorum F1129D based on 16S rDNA and MLST house-keeping gene analysis. Antimicrobial resistance (DfreE, EfrB, tetM, bcrRABD, and sat4) and virulence genes (Fss3 and ClpP), and genes conferring tolerance to metals and biocides (n = 9) were identified. The detection of relatively few genes encoding antimicrobial resistance and virulence indicates that this bacterium may have had no exposure to recently developed and clinically significant antibiotics. Genes potentially imparting beneficial functional properties were identified, including prophages, insertion elements, integrative conjugative elements, and genomic islands. Functional CRISPR-Cas arrays, and a defective prophage region were identified in the genome. The study also revealed many genomic loci unique to E. lacertideformus which contained genes enriched in cell wall/membrane/envelop biogenesis, and carbohydrate metabolism and transport functionality. This finding and the detection of putative enterococcal biofilm determinants (EfaAfs, srtC, and scm) may underpin the novel biofilm phenotype observed for this bacterium. Comparative analysis of E. lacertideformus with phylogenetically related and clinically significant enterococci (E. villorum F1129D, Enterococcus hirae R17, E. faecium AUS0085, and Enterococcus faecalis OG1RF) revealed an absence of genes (n = 54) in E. lacertideformus, that encode metabolic functionality, which potentially hinders nutrient acquisition and/or utilization by the bacterium and precludes growth in vitro. These data provide genetic insights into the previously determined phenotype and pathogenic nature of the bacterium.202133737921
541670.9936Limited predictive power of known resistance genes for phenotypic drug resistance in clinical Mycobacterium abscessus complex from Beijing in China. Mycobacterium abscessus complex (MABC) is an emerging pathogen with intrinsic multidrug resistance. Genomic sequencing technology has been widely applied to predict bacterial resistance in other bacteria, but the catalog of known resistance-determining genes to explain phenotypic resistance in the MABC is incomplete for many antibiotics. Eighty-one MABC strains were isolated from sputum samples of patients with pulmonary disease in the Beijing Chest Hospital. All isolates were tested for minimum inhibitory concentrations (MICs) to eight antibiotics and underwent whole-genome sequencing (WGS). Of the total 81 MABC isolates, six strains exhibited clarithromycin (CLM) resistance by day 3 in culture, but only one (16.7%, 1/6) contained a mutation in the rrl gene. All M. abscessus strains contained the erm (41)28T (100.0%, 49/49) polymorphism and exhibited CLM-induced resistance after 14 days in culture. Of the 61 imipenem-resistant strains, 12 (19.7%, 12/61) had mutations in the bla gene. Although there were four (4.9%) amikacin-resistant, nine (11.1%) linezolid-resistant, eight (9.9%) clofazimine-resistant, 23 (28.4%) bedaquiline-resistant, and 27 (33.3%) cefoxitin-resistant strains, no known mutations associated with resistance to these antibiotics were found. These results suggest that the explanatory power of known resistance genes for clinical MABC resistance is limited and that other unidentified genes or novel resistance mechanisms may be involved.202540422286
199080.9936Genomic Analysis of Aeromonas veronii C198, a Novel Mcr-3.41-Harboring Isolate from a Patient with Septicemia in Thailand. The resistance of Gram-negative bacteria to colistin, mediated by plasmid-borne mcr genes, is an emerging public health concern. The complete genome sequence (4.55 Mb) of a clinical isolate of Aeromonas veronii biovar veronii obtained from a patient with septicemia was determined using short-read and long-read platforms. This isolate (C198) was found to harbor a novel mcr-3 gene, designated mcr-3.41. Isolate C198 revealed adjacent mcr-3.41 and mcr-3-like genes. It contained one chromosome and two plasmids, both of which encoded a RepB replication protein. Other antimicrobial resistance genes, including bla(cphA3), bla(OXA-12), tetA, rsmA, and adeF, were also present. Isolate C198 was resistant to amoxicillin-clavulanate, ampicillin-sulbactam and tetracycline, and showed intermediate resistance to trimethoprim-sulfamethoxazole. The isolate was susceptible to piperacillin-tazobactam, carbapenem, third-generation cephalosporins, fluoroquinolones, chloramphenicol, and aminoglycosides. Putative virulence genes in the C198 genome encoded type II, III, and VI secretion systems; type IV Aeromonas pili; and type I fimbria, flagella, hemagglutinin, aerolysin, and hemolysins. Multilocus sequence typing revealed a novel sequence type (ST), ST720 for C198. Phylogenetic analysis of the single nucleotide polymorphisms in C198 demonstrated that the strain was closely related to A. veronii 17ISAe. The present study provides insights into the genomic characteristics of human A. veronii isolates.202033317051
121290.9936Virulence Factors and Antimicrobial Resistance of Uropathogenic Escherichia coli EQ101 UPEC Isolated from UTI Patient in Quetta, Balochistan, Pakistan. Infectious diseases have been tremendously increasing as the organisms of even normal flora become opportunistic and cause an infection, and Escherichia coli (E. coli EQ101) is one of them. Urinary tract infections are caused by various microorganisms, but Escherichia coli is the primary cause of almost 70%-90% of all UTIs. It has multiple strains, possessing diverse virulence factors, contributing to its pathogenicity. Furthermore, these virulent strains also can cause overlapping pathogenesis by sharing resistance and virulence factors among each other. The current study is aimed at analyzing the genetic variants associated with multi-drug-resistant (MDR) E. coli using the whole genome sequencing platform. The study includes 100 uropathogenic Escherichia coli (UPEC) microorganisms obtained from urine samples out of which 44% were multi-drug-resistant (MDR) E. coli. Bacteria have been isolated and antimicrobial susceptibility test (AST) was determined by disk diffusion method on the Mueller-Hinton agar plate as recommended by the Clinical and Laboratory Standards Institute (CLSI) 2020, and one isolate has been selected which shows resistance to most of the antibiotics, and that isolate has been analyzed by whole genome sequencing (WGS), accompanied by data and phylogenetic analysis, respectively. Organisms were showing resistance against ampicillin (10 μg), cefixime (5 μg), ceftriaxone (30 μg), nalidixic acid (30 μg), ciprofloxacin (5 μg), and ofloxacin (5 μg) on antimicrobial susceptibility test. WGS were done on selected isolate which identified 25 virulence genes (air, astA, chuA, fyuA, gad, hra, iha, irp2, iss, iucC, iutA, kpsE, kpsMII_K1, lpfA, mchF, ompT, papA_F43, sat, senB, sitA, terC, traT, usp, vat, and yfcV) and seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA). Among resistance genes, seven genes (TolC, emrR, evgA, qacEdelta1, H-NS, cpxA, and mdtM) were identified to be involved in antibiotic efflux, three AMR genes (aadA5, mphA, and CTX-M-15) were involved in antibiotic inactivation, and two genes (sul1 and dfrA14) were found to be involved in antibiotic drug replacement. Our data identified antibiotic resistance and virulence genes of the isolate. We suggest further research work to establish region-based resistance profile in comparison with the global resistance pattern.202337727279
5872100.9936Characterization of the plasmids harbouring the florfenicol resistance gene floR in Glaesserella parasuis and Actinobacillus indolicus. OBJECTIVES: The aim of this study was to characterize the floR-carrying plasmids originating from Glaesserella parasuis and Actinobacillus indolicus isolated from pigs with respiratory disease in China. METHODS: A total of 125 G. parasuis and 28 A. indolicus strains collected between 2009 and 2022 were screened for florfenicol resistance. Characterization of floR-positive isolates and plasmids were determined by antimicrobial susceptibility testing, serotyping, multilocus sequence typing (MLST), conjugation and transformation assays, whole-genome sequencing (WGS), and phylogenetic analysis. RESULTS: One A. indolicus and six G. parasuis were identified as positive for floR. The six G. parasuis were assigned to four different serovars, including serovars 6, 7, 9, and unknown. In addition to strain XP11, six floR genes were located on plasmids. The six floR-bearing plasmids could be transformed into Pasteurella multocida and divided into two different types, including ∼5000 bp and ∼6000 bp plasmids. The ∼5000 bp plasmids consisting of rep, lysR, mobB, and floR genes, exhibited high similarity among Pasteurellaceae bacteria. Furthermore, the ∼6000 bp plasmids, consisting of rep, lysR, mobC, mobA/L, and floR genes, showed high similarity between G. parasuis and Actinobacillus Spp. Notably, WGS results showed that the floR modules of the two types of plasmids could be transferred and integrated into the diverse Pasteurellaceae- origined plasmids. CONCLUSION: This study firstly reported the characterization of floR-carrying plasmids from A. indolicus and a non-virulent serovar of G. parasuis in pigs in China and elucidated the transmission mechanism of the floR resistance gene among the Pasteurellaceae family.202337726088
5214110.9936Comparative genomic analysis of a new tellurite-resistant Psychrobacter strain isolated from the Antarctic Peninsula. The Psychrobacter genus is a cosmopolitan and diverse group of aerobic, cold-adapted, Gram-negative bacteria exhibiting biotechnological potential for low-temperature applications including bioremediation. Here, we present the draft genome sequence of a bacterium from the Psychrobacter genus isolated from a sediment sample from King George Island, Antarctica (3,490,622 bp; 18 scaffolds; G + C = 42.76%). Using phylogenetic analysis, biochemical properties and scanning electron microscopy the bacterium was identified as Psychrobacter glacincola BNF20, making it the first genome sequence reported for this species. P. glacincola BNF20 showed high tellurite (MIC 2.3 mM) and chromate (MIC 6.0 mM) resistance, respectively. Genome-wide nucleotide identity comparisons revealed that P. glacincola BNF20 is highly similar (>90%) to other uncharacterized Psychrobacter spp. such as JCM18903, JCM18902, and P11F6. Bayesian multi-locus phylogenetic analysis showed that P. glacincola BNF20 belongs to a polyphyletic clade with other bacteria isolated from polar regions. A high number of genes related to metal(loid) resistance were found, including tellurite resistance genetic determinants located in two contigs: Contig LIQB01000002.1 exhibited five ter genes, each showing putative promoter sequences (terACDEZ), whereas contig LIQB1000003.2 showed a variant of the terZ gene. Finally, investigating the presence and taxonomic distribution of ter genes in the NCBI's RefSeq bacterial database (5,398 genomes, as January 2017), revealed that 2,623 (48.59%) genomes showed at least one ter gene. At the family level, most (68.7%) genomes harbored one ter gene and 15.6% exhibited five (including P. glacincola BNF20). Overall, our results highlight the diverse nature (genetic and geographic diversity) of the Psychrobacter genus, provide insights into potential mechanisms of metal resistance, and exemplify the benefits of sampling remote locations for prospecting new molecular determinants.201829479501
1211120.9935Molecular characterization of multidrug-resistant Escherichia coli of the phylogroups A and C in dairy calves with meningitis and septicemia. Escherichia coli is an important cause of septicemia (SEPEC) and neonatal meningitis (NMEC) in dairy calves. However, the diversity of virulence profiles, phylogroups, antimicrobial resistance patterns, carriage of integron structures, and fluoroquinolone (FQ) resistance mechanisms have not been fully investigated. Also, there is a paucity of knowledge about the virulence profiles and frequency of potential SEPEC in feces from calves with or without diarrhea. This study aimed to characterize the virulence potential, phylogroups, antimicrobial susceptibility, integron content, and FQ-resistance mechanisms in Escherichia coli isolated from calves with meningitis and septicemia. Additionally, the virulence genes (VGs) and profiles of E. coli isolated from diarrheic and non-diarrheic calves were compared between them and together with NMEC and SEPEC in order to identify shared profiles. Tissue and fluid samples from eight dairy calves with septicemia, four of which had concurrent meningitis, were processed for bacteriology and histopathology. Typing of VGs was assessed in 166 isolates from diverse samples of each calf. Selected isolates were evaluated for antimicrobial susceptibility by the disk diffusion test. Phylogroups, integron gene cassettes cartography, and FQ-resistance determinants were analyzed by PCR, sequencing, and bioinformatic tools. Furthermore, 109 fecal samples and 700 fecal isolates from dairy calves with or without diarrhea were evaluated to detect 19 VGs by uniplex PCR. Highly diverse VG profiles were characterized among NMEC and SEPEC isolates, but iucD was the predominant virulence marker. Histologic lesions in all calves supported their pathogenicity. Selected isolates mainly belonged to phylogroups A and C and showed multidrug resistance. Classic (dfrA17 and arr3-dfrA27) and complex (dfrA17-aadA5::ISCR1::bla(CTX-M-2)) class 1 integrons were identified. Target-site mutations in GyrA (S83L and D87N) and ParC (S80I) encoding genes were associated with FQ resistance. The VGs detected more frequently in fecal samples included f17G (50%), papC (30%), iucD (20%), clpG (19%), eae (16%), and afaE-8 (13%). Fecal isolates displaying the profiles of f17 or potential SEPEC were found in 25% of calves with and without diarrhea. The frequency of E. coli VGs and profiles did not differ between both groups (p > 0.05) and were identical or similar to those found in NMEC and SEPEC. Overall, multidrug-resistant E. coli isolates with diverse VG profiles and belonging to phylogroups A and C can be implicated in natural cases of meningitis and septicemia. Their resistance phenotypes can be partially explained by class 1 integron gene cassettes and target-site mutations in gyrA and parC. These results highlight the value of antimicrobial resistance surveillance in pathogenic bacteria isolated from food-producing animals. Besides, calves frequently shed potential SEPEC in their feces as commensals ("Trojan horse"). Thus, these bacteria may be disseminated in the farm environment, causing septicemia and meningitis under predisposing factors.202234982979
1506130.9935Detection of Five mcr-9-Carrying Enterobacterales Isolates in Four Czech Hospitals. The aim of this study was to report the characterization of the first mcr-positive Enterobacterales isolated from Czech hospitals. In 2019, one Citrobacter freundii and four Enterobacter isolates were recovered from Czech hospitals. The production of carbapenemases was examined by a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) imipenem hydrolysis assay. Additionally, bacteria were screened for the presence of carbapenemase-encoding genes and plasmid-mediated colistin resistance genes by PCR. To define the genetic units carrying mcr genes, the genomic DNAs of mcr-carrying clinical isolates were sequenced on the PacBio Sequel I platform. Results showed that all isolates carried bla(VIM)- and mcr-like genes. Analysis of whole-genome sequencing (WGS) data revealed that all isolates carried mcr-9-like alleles. Furthermore, the three sequence type 106 (ST106) Enterobacter hormaechei isolates harbored the bla(VIM-1) gene, while the ST764 E. hormaechei and ST95 C. freundii included bla(VIM-4) Analysis of plasmid sequences showed that, in all isolates, mcr-9 was carried on IncHI2 plasmids. Additionally, at least one multidrug resistance (MDR) region was identified in each mcr-9-carrying IncHI2 plasmid. The bla(VIM-4) gene was found in the MDR regions of p48880_MCR_VIM and p51929_MCR_VIM. In the three remaining isolates, bla(VIM-1) was localized on plasmids (∼55 kb) exhibiting repA-like sequences 99% identical to the respective gene of pKPC-CAV1193. In conclusion, to the best of our knowledge, these 5 isolates were the first mcr-9-positive bacteria of clinical origin identified in the Czech Republic. Additionally, the carriage of the bla(VIM-1) on pKPC-CAV1193-like plasmids is described for the first time. Thus, our findings underline the ongoing evolution of mobile elements implicated in the dissemination of clinically important resistance determinants.IMPORTANCE Infections caused by carbapenemase-producing bacteria have led to the revival of polymyxins as the "last-resort" antibiotic. Since 2016, several reports describing the presence of plasmid-mediated colistin resistance genes, mcr, in different host species and geographic areas were published. Here, we report the first detection of Enterobacterales carrying mcr-9-like alleles isolated from Czech hospitals in 2019. Furthermore, the three ST106 Enterobacter hormaechei isolates harbored bla(VIM-1), while the ST764 E. hormaechei and ST95 Citrobacter freundii isolates included bla(VIM-4) Analysis of WGS data showed that, in all isolates, mcr-9 was carried on IncHI2 plasmids. bla(VIM-4) was found in the MDR regions of IncHI2 plasmids, while bla(VIM-1) was localized on pKPC-CAV1193-like plasmids, described here for the first time. These findings underline the ongoing evolution of mobile elements implicated in dissemination of clinically important resistance determinants. Thus, WGS characterization of MDR bacteria is crucial to unravel the mechanisms involved in dissemination of resistance mechanisms.202033298573
1640140.9935First Identification of bla (NDM-1) Producing Escherichia coli ST 9499 Isolated from Musca domestica in the Urban Center of Rio de Janeiro, Brazil. The present study presents phenotypic and molecular characterization of a multidrug-resistant strain of Escherichia coli (Lemef26), belonging to sequence type ST9499 carrying a bla(NDM-1) carbapenem resistance gene. The bacterium was isolated from a specimen of Musca domestica, collected in proximity to a hospital in Rio de Janeiro City, Brazil. The strain was identified as E. coli by matrix-assisted laser desorption-ionization time of flight mass spectrometry (Maldi-TOF-MS) and via genotypic analysis (Whole-Genome Sequencing-WGS), followed by phylogenetic analysis, antibiotic resistance profiling (using phenotypic and genotypic methods) and virulence genotyping. Interestingly, the bla(NDM-1) was the only resistance determinant detected using a panel of common resistance genes, as evaluated by PCR. In contrast, WGS detected genes conferring resistance to aminoglycosides, fluoroquinolones, quinolones, trimethoprim, beta-lactams, chloramphenicol, macrolides, sulfonamide, tetracycline, lincosamide and streptogramin B. Conjugation experiments demonstrated the transfer of carbapenem resistance, via acquisition of the bla(NDM-1) sequence, to a sensitive receptor strain of E. coli, indicating that bla(NDM-1) is located on a conjugative plasmid (most likely of the IncA/C incompatibility group, in association with the transposon Tn3000). Phylogenetic analyses placed Lemef26 within a clade of strains exhibiting allelic and environment diversity, with the greatest level of relatedness recorded with a strain isolated from a human source suggesting a possible anthropogenic origin. Analysis of the virulome revealed the presence of fimbrial and pilus genes, including a CFA/I fimbriae (cfaABCDE), common pilus (ecpABCDER), laminin-bind fimbrae (elfADG), hemorrhagic pilus (hcpABC) and fimbrial adherence determinants (stjC) indicates the ability of strain Lemef26 to colonize animal hosts. To the best of our knowledge, this study represents the first report of bla(NDM-1) carbapenemase gene in an E. coli strain isolated from M. domestica. In concordance with the findings of previous studies on the carriage of MDR bacteria by flies, the data presented herein provide support to the idea that flies may represent a convenient means (as sentinel animals) for the monitoring of environmental contamination with multidrug-resistant bacteria.202337436443
5196150.9934Phenomics and genomic features of Enterococcus avium IRMC1622a isolated from a clinical sample of hospitalized patient. BACKGROUND: Enterococcus avium (E. avium) is a Gram-positive nosocomial pathogen that is commonly isolated from the alimentary tract. The objective of this functional genomics study was to identify the resistant genes by analyzing the genome of E. avium IRMC1622a, a type of bacteria found in feces collected from a patient at a Saudi Arabian tertiary hospital. METHODS: The bacterial strain IRMC1622a was identified by 16 S rRNA sequencing as Enterococcus sp. The resistance phenomics were performed using VITEK® 2, and morphological analysis was achieved using a scanning electron microscope (SEM). Finally, the whole bacterial genome of the bacterial strain IRMC1622a was subjected to sequencing during October 2023 using Oxford Nanopore long-read sequencing technology, and mining for resistant genes. RESULTS: The results of antimicrobial resistant phenomics indicated that the IRMC1622a strain was sensitive to all tested antimicrobial agents except for erythromycin, and the same result was confirmed by genomic analysis in addition to other classes of antibiotics. SEM showed E. avium IRMC1622a is ovoid shape, in single cells (L 1.2797 ± 0.1490 µm), in pairs (L 1.7333 ± 0.1054 µm), and in chains (L 2.44033 ± 0.1978 µm). The E. avium IRMC1622a genome has 14 (in CARD) antimicrobial resistance genes that were identified with several mechanisms of antimicrobial resistance, such as the efflux pump and conferring antibiotic resistance. The present study revealed that the E. avium IRMC1622a genome contains a high number of genes associated with virulence factors, and 14 matched pathogenic protein families and predicted as human pathogen (probability score 0.855). We report two (ISEnfa4 and ISEfa5) mobile genetic elements for the first time in the E. avium genome. CONCLUSIONS: The study concludes that E. avium IRMC1622a is susceptible to all tested antibacterials except erythromycin. The IRMC1622a has 14 genes encoding antimicrobial resistance mechanisms, including the efflux pump and conferring antibiotic resistance. This could indicate a potential rise in E. avium resistance in healthcare facilities. These observations may raise concerns regarding E. avium resistance in healthcare. We need more research to understand the pathophysiology of E. avium, which leads to hospital-acquired infections.202438833914
1213160.9934Fosfomycin resistance determinants in Escherichia coli isolates of human and animal origin from Iran. BACKGROUND: Fosfomycin has regained clinical interest over the last years due to its superior activity against multidrug-resistant bacterial pathogens. In the current study we aimed to characterize genotypic and phenotypic features of fosfomycin resistant (FosR) Escherichia coli isolates originating from human and animal. METHODS AND RESULTS: Five FosR bacteria were selected from a population of 150 E. coli isolates of human and broiler chickens. The sequence types of isolates were determined by multi-locus sequencing typing. Fosfomycin MICs were determined by agar dilution and gradient diffusion methods. Molecular detection of plasmid encoded fosfomycin resistance genes, fosA, fosA3, fosA4, fosA5 and fosC2 was performed by PCR. The modifications of fosfomycin target (MurA), transporters (GlpT, UhpT), and transporter regulator (PtsI) were investigated by gene sequencing. The MICs of fosfomycin were found to be ≥ 128 mg/L according to agar dilution and > 1024 mg/L according to gradient diffusion method. FosR isolates belonged to sequence types ST10 (n = 2), ST361, ST209 and ST1158 (n = 1). While all FosR isolates carried fos genes (fosA3 (n = 2), fosA5 (n = 2) and fosA4(n = 1)), only three isolates revealed amino acid substations in MurA, PtsI and GlpT with MurA P99S change being predicted to have deleterious impact on the function of protein. CONCLUSIONS: Emergence of fosfomycin resistance among studied isolates was mainly attributed to plasmid genes coding for fosfomycin modifying enzymes. The similarity in fosfomycin resistance determinants among clonally diverse E. coli isolates of human and animals indicates a possible cross-sectoral dissemination of fos genes by epidemic plasmids between bacterial isolates of clinical settings and those from animals.202540465084
1997170.9934Genetic Characterization of bla (CTX-M-55) -Bearing Plasmids Harbored by Food-Borne Cephalosporin-Resistant Vibrio parahaemolyticus Strains in China. This study aims to investigate and compare the complete nucleotide sequences of the multidrug resistance plasmids pVb0267 and pVb0499, which were recovered from foodborne Vibrio parahaemolyticus isolates, and analyze the genetic environment of bla(CTX-M-55) to provide insight into the dissemination mechanisms of this resistance element. Analysis of the sequences of plasmids pVb0267 (166,467 bp) and pVb0499 (192,739 bp) revealed that the backbones of these two plasmids exhibited a high degree of similarity with pR148, a recognized type 1a IncC plasmid recovered from Aeromonas hydrophila (99% identity). The resistance genes, found in both plasmids, included qacH, aadB, arr2, bla (OXA-10) , aadA1, sul1, tet(A), and bla (CTX-M-55), which were mostly arranged in a specific region designated ARI-A. Plasmid pVb0499 was found to possess a larger size of ARI-A than pVb0267, which lacked a mer determination region, a qnr A segment, an aacC3 gene and several mobility-encoding genes. Comparative analysis of resistance island (RI) of these plasmids and others revealed the potential evolution route of these RI sequences. In conclusion, plasmids harboring the bla (CTX-M-55) gene has been recovered in Vibrio parahaemolyticus strains of food origin. It is alarming to find that IncC plasmids harboring resistance islands are disseminating in aquatic bacterial strains. The continuous evolution of resistance genes in conjugative plasmid in aquatic bacteria could be due to bacterial adaption to aquaculture environment, where antibiotics were increasingly used.201931275270
848180.9934Molecular Characterization of Escherichia coli Causing Urinary Tract Infections Through Next-Generation Sequencing: A Comprehensive Analysis of Serotypes, Sequence Types, and Antimicrobial and Virulence Genes. Introduction An enormous increase in antimicrobial resistance (AMR) among bacteria isolated from human clinical specimens contributed to treatment failures. Increased surveillance through next-generation sequencing (NGS) or whole genome sequencing (WGS) could facilitate the study of the epidemiology of drug-resistant bacterial strains, resistance genes, and other virulence determinants they are potentially carrying. Methods This study included 30 Escherichia coli (E. coli) isolates obtained from patients suffering from urinary tract infections (UTIs) attending Prathima Institute of Medical Sciences, Karimnagar, India. All bacterial isolates were identified, and antimicrobial susceptibility patterns were determined through conventional microbiological techniques and confirmed by automated systems. All the isolates were investigated using NGS to identify genes coding for resistance, such as extended-spectrum beta-lactamases (ESBLs), metallo-beta-lactamases, and virulence genes. Multilocus sequence typing (MLST) was used to understand the prevalent strain types, and serotyping was carried out to evaluate the type of O (cell wall antigen) and H (flagellar antigen) serotypes carried by the isolates. Results The conventional antimicrobial susceptibility testing revealed that 15 (50%) isolates were resistant to imipenem (IPM), 10 (33.33%) were resistant to amikacin (AK), 13 (43.33%) were resistant to piperacillin-tazobactam (PTZ), 17 (56.66%) were resistant to cephalosporins, and 14 (46.66%) were resistant to nitrofurantoin (NIT). Among the isolates, 26 (86.66%) had revealed the presence of multiple antibiotic-resistant genes with evidence of at least one gene coding for beta-lactamase resistance. There was a high prevalence of bla(CTX-M )(19/30, 63.33%) genes, followed by bla(TEM) and bla(OXA-1). The bla(NDM-5) gene was found in three isolates (3/30, 10%). The virulence genes identified in the present study were iutA, sat, iss, and papC, among others. The E. coli serotype found predominantly belonged to O25:H4 (5, 16.66%), followed by O102:H6 (4, 13.33%). A total of 16 MLST variants were identified among the examined samples. Of the MLST-based sequence types (STs) identified, ST-131 (7, 23.33%) was the predominant one, followed by ST-167 (3, 10%) and ST-12 (3, 10%). Conclusions The study results demonstrated that the E. coli strains isolated from patients suffering from UTIs potentially carried antimicrobial resistance and virulence genes and belonged to different strain types based on MLST. Careful evaluation of bacterial strains using molecular analyses such as NGS could facilitate an improved understanding of bacterial antibiotic resistance and its virulence potential. This could enable physicians to choose appropriate antimicrobial agents and contribute to better patient management, thereby preventing the emergence and spread of drug-resistant bacteria.202438576671
5438190.9934Genomic Insights into Staphylococcus aureus Isolates Exhibiting Diminished Daptomycin Susceptibility. Daptomycin is one of the last therapeutic resources for multidrug-resistant gram-positive bacteria. Despite its structural similarities with glycopeptides, its mechanisms of action and resistance are different and in some respects are not completely understood. Mutations in several genes have been associated with daptomycin resistance, especially in mprF, walkR, rpoB and rpoC, but their role and importance remain to be elucidated. We have studied mutations in 11 genes, which have been previously associated with daptomycin non-susceptibility, in nine daptomycin-non-susceptible Staphylococcus aureus clinical isolates (daptomycin MIC: >1 mg/L). Susceptibility to daptomycin, vancomycin, linezolid, oxacillin, telavancin and dalbavancin was studied. walkR, agrA, cls1, cls2, fakA, pnpA, clpP, prs, rpoB, rpoC and mprF were amplified by PCR and sequenced. The sequences were compared with the S. aureus ATCC 25923 complete genome (GenBank gi: 685631213) by using BLAST(®) software. We did not find any changes in walkR, pnpA, prs and clpP. All isolates excepting isolate MSa5 showed a high number of significant mutations (between 13 and 25 amino acid changes) in mprF. Most isolates also showed mutations in the rpo genes, the cls genes and fakA. Daptomycin non-susceptibility in S. aureus clinical isolates seems to be reached through different mutation combinations when compared to S. aureus ATCC 25293. Especially mprF and cls1 showed very high polymorphism in most isolates. Meanwhile, one isolate, MSa5, showed only single mutation in mprF (P314T).202438535549