Genomic profiling of antimicrobial resistance genes in clinical isolates of Salmonella Typhi from patients infected with Typhoid fever in India

Background: Hospital wastewater is a major source of antimicrobial resistance (AMR) outflow into the environment. This study uses metagenomics to study how hospital clinical activity impacts antimicrobial resistance genes (ARGs) abundances in hospital wastewater.Methods: Sewage was collected over a 24-h period from multiple wastewater collection points (CPs) representing different specialties within a tertiary hospital site and simultaneously from community sewage works. High throughput shotgun sequencing was performed using Illumina HiSeq4000. ARG abundances were correlated to hospital antimicrobial usage (AMU), data on clinical activity and resistance prevalence in clinical isolates.Results: Microbiota and ARG composition varied between CPs and overall ARG abundance was higher in hospital wastewater than in community influent. ARG and microbiota compositions were correlated (Procrustes analysis, p=0.014). Total antimicrobial usage was not associated with higher ARG abundance in wastewater. However, there was a small positive association between resistance genes and antimicrobial usage matched to ARG phenotype (IRR 1.11, CI 1.06–1.16, p<0.001). Furthermore, analyzing carbapenem and vancomycin resistance separately indicated that counts of ARGs to these antimicrobials were positively associated with their increased usage [carbapenem rate ratio (RR) 1.91, 95% CI 1.01–3.72, p=0.07, and vancomycin RR 10.25, CI 2.32–49.10, p<0.01]. Overall, ARG abundance within hospital wastewater did not reflect resistance patterns in clinical isolates from concurrent hospital inpatients. However, for clinical isolates of the family Enterococcaceae and Staphylococcaceae, there was a positive relationship with wastewater ARG abundance [odds ratio (OR) 1.62, CI 1.33–2.00, p<0.001, and OR 1.65, CI 1.21–2.30, p=0.006 respectively].Conclusion: We found that the relationship between hospital wastewater ARGs and antimicrobial usage or clinical isolate resistance varies by specific antimicrobial and bacterial family studied. One explanation, we consider is that relationships observed from multiple departments within a single hospital site will be detectable only for ARGs against parenteral antimicrobials uniquely used in the hospital setting. Our work highlights that using metagenomics to identify the full range of ARGs in hospital wastewater is a useful surveillance tool to monitor hospital ARG carriage and outflow and guide environmental policy on AMR.

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Molecular epidemiology and antimicrobial resistance features of Acinetobacter baumannii clinical isolates from Pakistan

Annals of Clinical Microbiology and Antimicrobials ◽

10.1186/s12941-019-0344-7 ◽

2020 ◽

Vol 19 (1) ◽

Cited By ~ 2

Author(s):

Nabil Karah ◽

Fizza Khalid ◽

Sun Nyunt Wai ◽

Bernt Eric Uhlin ◽

Irfan Ahmad

Keyword(s):

Antimicrobial Resistance ◽

Molecular Epidemiology ◽

Acinetobacter Baumannii ◽

Resistance Genes ◽

Antimicrobial Agents ◽

Opportunistic Pathogen ◽

Clinical Isolates ◽

Diffusion Method ◽

Agar Disc ◽

Antimicrobial Resistance Genes

Abstract Background Acinetobacter baumannii is a Gram-negative opportunistic pathogen with a notorious reputation of being resistant to antimicrobial agents. The capability of A. baumannii to persist and disseminate between healthcare settings has raised a major concern worldwide. Methods Our study investigated the antibiotic resistance features and molecular epidemiology of 52 clinical isolates of A. baumannii collected in Pakistan between 2013 and 2015. Antimicrobial susceptibility patterns were determined by the agar disc diffusion method. Comparative sequence analyses of the ampC and blaOXA-51-like alleles were used to assign the isolates into clusters. The whole genomes of 25 representative isolates were sequenced using the MiSeq Desktop Sequencer. Free online applications were used to determine the phylogeny of genomic sequences, retrieve the multilocus sequence types (ST), and detect acquired antimicrobial resistance genes. Results Overall, the isolates were grouped into 7 clusters and 3 sporadic isolates. The largest cluster, Ab-Pak-cluster-1 (blaOXA-66 and ISAba1-ampC-19) included 24 isolates, belonged to ST2 and International clone (IC) II, and was distributed between two geographical far-off cities, Lahore and Peshawar. Ab-Pak-clusters-2 (blaOXA-66, ISAba1-ampC-2), and -3 (blaOXA-66, ISAba1-ampC-20) and the individual isolate Ab-Pak-Lah-01 (ISAba1-blaOXA-66, ISAba1-ampC-2) were also assigned to ST2 and IC II. On the other hand, Ab-Pak-clusters-4 (blaOXA-69, ampC-1), -5 (blaOXA-69, ISAba1-ampC-78), and -6A (blaOXA-371, ISAba1-ampC-3) belonged to ST1, while Ab-Pak-cluster-6B (blaOXA-371, ISAba1-ampC-8) belonged to ST1106, with both ST1 and ST1106 being members of IC I. Five isolates belonged to Ab-Pak-cluster-7 (blaOXA-65, ampC-43). This cluster corresponded to ST158, showed a well-delineated position on the genomic phylogenetic tree, and was equipped with several antimicrobial resistance genes including blaOXA-23 and blaGES-11. Conclusions Our study detected the occurrence of 7 clusters of A. baumannii in Pakistan. Altogether, 6/7 of the clusters and 45/52 (86.5%) of the isolates belonged to IC I (n = 9) or II (n = 36), making Pakistan no exception to the global domination of these two clones. The onset of ST158 in Pakistan marked a geographical dispersal of this clone beyond the Middle East and brought up the need for a detailed characterization.

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Comparison of Antimicrobial Resistance and Pan-Genome of Clinical and Non-Clinical Enterococcus cecorum from Poultry Using Whole-Genome Sequencing

Foods ◽

10.3390/foods9060686 ◽

2020 ◽

Vol 9 (6) ◽

pp. 686

Author(s):

Poonam Sharma ◽

Sushim K. Gupta ◽

John B. Barrett ◽

Lari M. Hiott ◽

Tiffanie A. Woodley ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Resistance Genes ◽

Gene Families ◽

Clinical Isolates ◽

Whole Genome ◽

Pan Genome ◽

Antimicrobial Resistance Genes ◽

Enterococcus Cecorum

Enterococcus cecorum is an emerging avian pathogen, particularly in chickens, but can be found in both diseased (clinical) and healthy (non-clinical) poultry. To better define differences between E. cecorum from the two groups, whole-genome sequencing (WGS) was used to identify and compare antimicrobial resistance genes as well as the pan-genome among the isolates. Eighteen strains selected from our previous study were subjected to WGS using Illumina MiSeq and comparatively analyzed. Assembled contigs were analyzed for resistance genes using ARG-ANNOT. Resistance to erythromycin was mediated by ermB, ermG, and mefA, in clinical isolates and ermB and mefA, in non-clinical isolates. Lincomycin resistance genes were identified as linB, lnuB, lnuC, and lnuD with lnuD found only in non-clinical E. cecorum; however, lnuB and linB were found in only one clinical isolate. For both groups of isolates, kanamycin resistance was mediated by aph3-III, while tetracycline resistance was conferred by tetM, tetO, and tetL. No mutations or known resistance genes were found for isolates resistant to either linezolid or chloramphenicol, suggesting possible new mechanisms of resistance to these drugs. A comparison of WGS results confirmed that non-clinical isolates contained more resistance genes than clinical isolates. The pan-genome of clinical and non-clinical isolates resulted in 3651 and 4950 gene families, respectively, whereas the core gene sets were comprised of 1559 and 1534 gene families in clinical and non-clinical isolates, respectively. Unique genes were found more frequently in non-clinical isolates than clinical. Phylogenetic analysis of the isolates and all the available complete and draft genomes showed no correlation between healthy and diseased poultry. Additional genomic comparison is required to elucidate genetic factors in E. cecorum that contribute to disease in poultry.

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Characterization of integrons and antimicrobial resistance genes in clinical isolates of Gram-negative bacteria from Palestinian hospitals

Microbiology and Immunology ◽

10.1111/j.1348-0421.2009.00168.x ◽

2009 ◽

Vol 53 (11) ◽

pp. 595-602 ◽

Cited By ~ 18

Author(s):

Amjad I. A. Hussein ◽

Ashraf M. Ahmed ◽

Maiko Sato ◽

Tadashi Shimamoto

Keyword(s):

Antimicrobial Resistance ◽

Resistance Genes ◽

Clinical Isolates ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Antimicrobial Resistance Genes

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Prevalence of antimicrobial resistance genes in Bacteroides spp. and Prevotella spp. Dutch clinical isolates

Clinical Microbiology and Infection ◽

10.1016/j.cmi.2019.02.017 ◽

2019 ◽

Vol 25 (9) ◽

pp. 1156.e9-1156.e13 ◽

Cited By ~ 13

Author(s):

A.C.M. Veloo ◽

W.H. Baas ◽

F.J. Haan ◽

J. Coco ◽

J.W. Rossen

Keyword(s):

Antimicrobial Resistance ◽

Resistance Genes ◽

Clinical Isolates ◽

Antimicrobial Resistance Genes

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230. Nontyphoidal Salmonella from Clinical and Retail Meat Sources Reveal Antimicrobial Resistance Genes for Ceftriaxone and Ciprofloxacin

Open Forum Infectious Diseases ◽

10.1093/ofid/ofab466.432 ◽

2021 ◽

Vol 8 (Supplement_1) ◽

pp. S225-S225

Author(s):

Nkuchia M M’ikanatha ◽

Xin Yin ◽

Yezhi Fu ◽

Sameera Sayeed ◽

Christopher Carr ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Resistance Genes ◽

Antimicrobial Agents ◽

The United States ◽

Clinical Isolates ◽

Food Sources ◽

Fluoroquinolone Resistance ◽

Third Generation ◽

Antimicrobial Resistance Genes ◽

Retail Meat

Abstract Background Pennsylvania participates in the National Antimicrobial Resistance Monitoring System (NARMS), which includes monitoring of Nontyphoidal Salmonella (NTS), a leading cause of bacterial foodborne illnesses in the United States. Methods Clinical NTS isolates submitted to the Pennsylvania Department of Health (2015-18) were tested for susceptibility to 15 antimicrobial agents and analyzed by whole-genome sequencing (WGS). Concurrently, we conducted a prospective microbiological survey of NTS in retail meat products (chicken breasts, ground turkey, and pork chops) with susceptibility testing and WGS. Results Of a sample of 426 clinical Salmonella isolates from humans analyzed for antimicrobial susceptibility, 65 (15.3%) had decreased susceptibility to ciprofloxacin (DSC). Ampicillin resistance was observed in 39 (9.2%) and 15 (3.5%) were ceftriaxone-resistant. Ten ceftriaxone-resistant isolates had genetic elements that confer resistance to third generation extended-spectrum cephalosporins (ESC) [blaCMY−2, n=8 and blaCTX-M-65, n=2]. The blaCTX-M-65- positive isolates had a mutation in gyrA that confers fluoroquinolone resistance. Thirteen clinical isolates carried plasmid-mediated fluoroquinolone resistance genes (PMQR) [qnrB19, qnrS1, qnrA1]. We detected NTS in 131 (3.8%) of 3480 meat samples tested. 7 (5.3%) had DSC, while 38 (29%) and 21 (16%) were resistant to ampicillin and ceftriaxone, respectively. Four S. Infantis isolates had DSC and a blaCTX-M-65 gene plus a mutation in gyrA. Thirteen meat isolates had the blaCMY-2 gene. One additional blaCTX-M-65-positive S. Infantis without gyrA from ground turkey (SRR6351119) differed from four clinical isolates by ≤10 single-nucleotide polymorphisms. Percent of isolates from patients and meat sources that demonstrated resistance to amoxicillin-clavulanate (AMC), ceftriaxone, and decreased susceptibility to ciprofloxacin (DSC) to nine antimicrobial classes tested. Among isolates from patients, resistance to ceftriaxone, a third-generation cephalosporin preferred for severe infections in children, increased from zero in 2015 to 5.8% in 2017. Overall, DSC increased in isolates from human sources while in strains from meat sources, DSC increased from zero in 2015 to over five percent in 2018. Conclusion NTS isolated from human and meat sources were multi-drug resistant. Demonstration of similar resistance genes in meat and in ill humans may be consistent with spread of antibiotic-resistant pathogens from food sources. Dissemination of genes that confer resistance to third generation cephalosporins and fluoroquinolones, including some on mobile plasmids, may undermine recommended treatment for severe NTS infections. These results underscore the need for antimicrobial stewardship efforts in both agriculture and human medicine. Disclosures All Authors: No reported disclosures

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Culture-Independent Genotyping, Virulence and Antimicrobial Resistance Gene Identification of Staphylococcus aureus from Orthopaedic Implant-Associated Infections

Microorganisms ◽

10.3390/microorganisms9040707 ◽

2021 ◽

Vol 9 (4) ◽

pp. 707

Author(s):

J. Christopher Noone ◽

Fabienne Antunes Ferreira ◽

Hege Vangstein Aamot

Keyword(s):

Staphylococcus Aureus ◽

Antimicrobial Resistance ◽

Resistance Genes ◽

Virulence Gene ◽

Orthopaedic Implant ◽

Metagenomic Sequencing ◽

Gene Detection ◽

Shotgun Metagenomic Sequencing ◽

Antimicrobial Resistance Genes ◽

Culture Independent

Our culture-independent nanopore shotgun metagenomic sequencing protocol on biopsies has the potential for same-day diagnostics of orthopaedic implant-associated infections (OIAI). As OIAI are frequently caused by Staphylococcus aureus, we included S. aureus genotyping and virulence gene detection to exploit the protocol to its fullest. The aim was to evaluate S. aureus genotyping, virulence and antimicrobial resistance genes detection using the shotgun metagenomic sequencing protocol. This proof of concept study included six patients with S. aureus-associated OIAI at Akershus University Hospital, Norway. Five tissue biopsies from each patient were divided in two: (1) conventional microbiological diagnostics and genotyping, and whole genome sequencing (WGS) of S. aureus isolates; (2) shotgun metagenomic sequencing of DNA from the biopsies. Consensus sequences were analysed using spaTyper, MLST, VirulenceFinder, and ResFinder from the Center for Genomic Epidemiology (CGE). MLST was also compared using krocus. All spa-types, one CGE and four krocus MLST results matched Sanger sequencing results. Virulence gene detection matched between WGS and shotgun metagenomic sequencing. ResFinder results corresponded to resistance phenotype. S. aureus spa-typing, and identification of virulence and antimicrobial resistance genes are possible using our shotgun metagenomics protocol. MLST requires further optimization. The protocol has potential application to other species and infection types.

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