High prevalence of plasmid-mediated quinolone resistance (PMQR) among E. coli from aquatic environments in Bangladesh

Fluro(quinolones) is an important class of antibiotic used widely in both human and veterinary medicine. Resistance to fluro(quinolones) can be acquired by either chromosomal point mutations or plasmid-mediated quinolone resistance (PMQR). There is a lack of studies on the prevalence of PMQR in organisms from environmental sources in Bangladesh. In this study, we investigated the occurrence of PMQR genes in E. coli from various water sources and analysed associations between multi-drug resistance (MDR) and resistance to extended spectrum β-lactam antibiotics. We analysed 300 E. coli isolates from wastewaters of urban live-bird markets (n = 74) and rural households (n = 80), rural ponds (n = 71) and river water samples (n = 75) during 2017–2018. We isolated E. coli by filtering 100 ml of water samples through a 0.2μm cellulose membrane and incubating on mTEC agar media followed by identification of isolated colonies using biochemical tests. We selected one isolate per sample for detection of PMQR genes by multiplex PCR and tested for antibiotic susceptibility by disc diffusion. Clonal relatedness of PMQR-positive isolates was evaluated by enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). About 66% (n = 199) of E. coli isolates harbored PMQR-genes, predominantly qnrS (82%, n = 164) followed by aac(6’)-lb-cr (9%, n = 17), oqxAB (7%, n = 13), qnrB (6%, n = 11) and qepA (4%, n = 8). Around 68% (n = 135) of PMQR-positive isolates were MDR and 92% (n = 183) were extended spectrum β-lactamase (ESBL)-producing of which the proportion of positive samples was 87% (n = 159) for blaCTX-M-1’ 34% (n = 62) for blaTEM, 9% (n = 16) for blaOXA-1, blaOXA-47 and blaCMY-2, and 2% (n = 4) for blaSHV. Further, 16% (n = 32) of PMQR-positive isolates were resistant to carbapenems of which 20 isolates carried blaNDM-1. Class 1 integron (int1) was found in 36% (n = 72) of PMQR-positive E. coli isolates. PMQR genes were significantly associated with ESBL phenotypes (p≤0.001). The presence of several PMQR genes were positively associated with ESBL and carbapenemase encoding genes such as qnrS with blaCTXM-1 (p<0.001), qnrB with blaTEM (p<0.001) and blaOXA-1 (p = 0.005), oqxAB and aac(6’)-lb-cr with blaSHV and blaOXA-1 (p<0.001), qnrB with blaNDM-1 (p<0.001), aac(6’)-lb-cr with blaOXA-47 (p<0.001) and blaNDM-1 (p = 0.002). Further, int1 was found to correlate with qnrB (p<0.001) and qepA (p = 0.011). ERIC-PCR profiles allowed identification of 84 of 199 isolates with 85% matching profiles which were further grouped into 33 clusters. Only 5 clusters had isolates (n = 11) with identical ERIC-PCR profiles suggesting that PMQR-positive E. coli isolates are genetically heterogeneous. Overall, PMQR-positive MDR E. coli were widely distributed in aquatic environments of Bangladesh indicating poor wastewater treatment and highlighting the risk of transmission to humans and animals.

Undetectable Production of the VIM-1 Carbapenemase in an Atlantibacter hermannii Clinical Isolate

The differential expression of VIM-1 in Atlantibacter hermannii WEB-2 and Enterobacter hormaechei ssp. hoffmannii WEB-1 clinical isolates from a rectal swab of a hospitalized patient in France was investigated. A. hermannii WEB-2 was resistant to all β-lactams except carbapenems. It produced ESBL SHV-12, but the Carba NP test failed to detect any carbapenemase activity despite the production of VIM-1. Conversely, E. hormaechei WEB-1, previously recovered from the same patient, was positive for the detection of carbapenemase activity. The blaVIM–1 gene was located on a plasmid and embedded within class 1 integron. Both plasmids were of the same IncA incompatibility group and conferred the same resistance pattern when electroporated in Escherichia coli TOP10 or Enterobacter cloacae CIP7933. Quantitative RT-PCR experiments indicated a weaker replication of pWEB-2 in A. hermannii as compared to E. hormaechei. An isogenic mutant of A. hermannii WEB-2 selected after sequential passages with increased concentrations of imipenem possessed higher MICs for carbapenems and cephalosporins including cefiderocol, higher levels of the blaVIM–1 gene transcripts, and detectable carbapenemase activity using the Carba NP test. Assessment of read coverage demonstrated that a duplication of the region surrounding blaVIM–1 gene occurred in the A. hermannii mutant with detectable carbapenemase activity. The lack of detection of the VIM-1 carbapenemase activity in A. hermannii WEB-2 isolate was likely due to a weak replication of the IncA plasmid harboring the blaVIM–1 gene. Imipenem as selective pressure led to a duplication of this gene on the plasmid and to the restoration of a significant carbapenem-hydrolyzing phenotype.

Complete Genome Sequence of Schaalia turicensis Strain CT001, Isolated from a Patient with Gonococcal Urethritis in Thailand

Schaalia turicensis , a Gram-positive bacillus, is a potential pathogen in genital infections. Here, we report the complete genome sequence of S. turicensis strain CT001, which was coisolated with Neisseria gonorrhoeae . Comprehensive analysis revealed the presence of a composite transposon carrying an imperfect class 1 integron in S. turicensis .

Quorum Sensing Mediated Pathogenicity, Virulence Genes and Class 1 Integron in Carbapenem-Resistant Clinical Pseudomonas Aeruginosa Isolates

Carbapenems are the most effective agents for treating carbapenem-resistant clinical P. aeruginosa (CRPsA) infections. During an infection, a quorum-sensing (QS) system and its regulating virulence genes have a great role. The aim of the study was to detect the presence of a las and rhl QS system and related virulence genes and a class 1 (Cls1) integron. A total of 52 CRPsA isolates obtained from Kastamonu, Turkey was analyzed. A conventional culture method, an oprL gene-based molecular assay for P. aeruginosa isolates, and an automated VITEK-2 compact system were applied for the isolation and identification of CRPsA isolates. The oprL gene was detected in all of the isolates tested. At least one of the las or rhl system genes was detected in 98.07% of the isolates, and the percentage of las system genes (98.07%) were higher than that of rhl system genes (90.38%). algD, lasB, toxA and aprA genes were detected in between 46.15% and 88.46% of the isolates, and co-existence of four genes were detected in 40.38% of the isolates. Cls1 integron and slime production using Congo Red Agar (CRA) were present in 51.92% and 67.30%, respectively, of the isolates. There was a significant positive correlation (p <.10) between the las system and the rhl system and a strongly positive correlation (p<.01 or p <.05) between the rhl system-four virulence genes and slime production-and among some virulence genes. In conclusion, the CRPsA isolates tested in the study are highly virulent and QS systems have a significant role in pathogenesis.

Virulence genotype and phenotype of multiple Antimicrobial Resistant Escherichia coli isolates from Broilers assessed in a “One-Health” Perspective

Extraintestinal pathogenic Escherichia coli (ExPEC) include several serotypes which have been associated with colibacillosis in poultry,while urinary tract infections (UTI) and newborn meningitis in humans. In this study, 57 antimicrobial resistant E. coli from apparently healthy broiler chickens were characterized for their health and safety risks. These isolates belonged to 12 serotypes and isolates of the same serotype were determined to be clonal based on Single Nucleotide Variant (SNV) analysis. Most of the isolates harbored plasmids, with Inc C and Inc FIA being frequently detected. The majority of the resistant isolates harboured plasmid-mediated resistance genes including aph (3'')-Ib , aph(6)-Id , bla CMY-2 , floR , sul (1 and 2) and tet (A and B) in agreement with their resistant phenotypes. The Class 1 integron was detected in all serotypes except O124:H25 and O7:H6. Of the 57 broilers E. coli , 27 were APEC among which 18 were also defined as uropathogenic E. coli (UPEC) and the remainder as other ExPEC. Two isolates of serotype O161:H4 (ST117) were genetically related to the control APEC and a clinical isolate associated with UTI . A strain of serotype O159:H45 (ST101) was also found to be closely related to an UTI case isolate. The detected virulence factors included adhesins, invasions, siderophores, type III secretion systems and toxins in combination with other virulence genes. A broiler isolate of serotype O7:H18 (ST 38) carried the ibe A gene encoding a protein involved in invasion of brain endothelium on a 102 kbp genetic island. This isolate demonstrated a moderate level of Caco-2 cells invasion and induced mortality (42.5 %) in a day-old chick infection model.

Molecular Characteristics and Antimicrobial Resistance of Salmonella enterica Serovar Schwarzengrund from Chicken Meat in Japan

Our previous study revealed that Salmonella enterica serovar Schwarzengrund-contaminated areas of broiler chickens have expanded from West Japan to East Japan. The present study investigated the antimicrobial resistance and molecular characteristics of 124 S. Schwarzengrund isolates obtained from chicken meat produced in East and West Japan from 2008 to 2019. Comparing the isolates obtained in 2008 and 2015–2019, an increase in the proportion of those resistant to kanamycin [51.4–89.7% (p < 0.001)] was observed. In contrast, the proportion of isolates resistant to both streptomycin and tetracycline and those that harbored a 1.0-kb class 1 integron, aadA1, and tetA, significantly decreased from 100% in 2008 to 47.1% in 2015–2019 (p < 0.001). A 1.0-kb class 1 integron containing aadA1, harbored by 78 isolates, was different from that reported in globally distributed S. Schwarzengrund strains (1.9 kb, containing the dfrA12-aadA2 gene cassette). Twenty-five isolates from different product districts and years of isolation were typed as sequence type (ST) 241 with multilocus sequence typing. Our results suggest that S. Schwarzengrund, which contaminates chicken meat in Japan, shares a common ancestor regardless of the product district from 2008 to recent years. Moreover, S. Schwarzengrund ST241 may have spread from western to eastern Japan.

Carriage of antibiotic resistant bacteria in endangered and declining Australian pinniped pups

The rapid emergence of antimicrobial resistance (AMR) is a major concern for wildlife and ecosystem health globally. Genetic determinants of AMR have become indicators of anthropogenic pollution due to their greater association with humans and rarer presence in environments less affected by humans. The objective of this study was to determine the distribution and frequency of the class 1 integron, a genetic determinant of AMR, in both the faecal microbiome and in Escherichia coli isolated from neonates of three pinniped species. Australian sea lion ( Neophoca cinerea ), Australian fur seal ( Arctocephalus pusillus doriferus ) and long-nosed fur seal ( Arctocephalus forsteri ) pups from eight breeding colonies along the Southern Australian coast were sampled between 2016-2019. DNA from faecal samples ( n =309) and from E. coli ( n =795) isolated from 884 faecal samples were analysed for class 1 integrons using PCRs targeting the conserved integrase gene ( intI ) and the gene cassette array. Class 1 integrons were detected in A. p. doriferus and N. cinerea pups sampled at seven of the eight breeding colonies investigated in 4.85% of faecal samples ( n =15) and 4.52% of E. coli isolates ( n =36). Integrons were not detected in any A. forsteri samples. DNA sequencing of the class 1 integron gene cassette array identified diverse genes conferring resistance to four antibiotic classes. The relationship between class 1 integron carriage and the concentration of five trace elements and heavy metals was also investigated, finding no significant association. The results of this study add to the growing evidence of the extent to which antimicrobial resistant bacteria are polluting the marine environment. As AMR determinants are frequently associated with bacterial pathogens, their occurrence suggests that these pinniped species are vulnerable to potential health risks. The implications for individual and population health as a consequence of AMR carriage is a critical component of ongoing health investigations.

Emergence of rare carbapenemases (FRI, GES-5, IMI, SFC, and SFH-1) in Enterobacterales isolated from surface waters in Japan

ABSTRACTObjectivesCarbapenemase-producing Enterobacterales (CPE) pose serious threats to public health. Compared with clinical CPE, the genetic characteristics of environmental CPE are not well understood. This study aimed to characterize the genetic determinants of carbapenem resistance in CPE isolated from environmental waters in Japan.MethodsEighty-five water samples were collected from rivers and a lake in Japan. CPE were identified using selective media, and genome sequencing was performed for the obtained isolates (n = 21).ResultsVarious rare/novel carbapenemases were identified: GES-5 in Raoultella planticola (n = 1), FRI-8 and FRI-11 in Enterobacter spp. (n = 8), IMI-22 and IMI-23 in Serratia ureilytica (n = 3), and SFC-1, SFC-2 and SFH-1 in Serratia fonticola (n = 9). Genomes of 11 isolates could be closed, allowing the elucidation of the genetic contexts of the carbapenemase genes. The blaGES-5 gene was located within a class 1 integron, In2071 (cassette array, blaGES-5-aacA3-aadA16), on a 33 kb IncP6 plasmid. The blaFRI-8 genes were carried on IncFII(Yp) plasmids ranging in size from 191 kb to 244 kb, and the blaFRI-11 genes were carried on 70 kb and 74 kb IncFII(pECLA)/IncR plasmids. The blaIMI-22 and blaIMI-23 genes were colocated on a 107 kb plasmid. The blaSFC and blaSFH-1 genes were found on putative genomic islands inserted at tRNA-Phe genes in chromosomes.ConclusionsThis study revealed the presence of rare/novel carbapenemases among CPE in aquatic environments, suggesting that the environment may act as a potential reservoir of these minor carbapenemases.