Antimicrobial Resistance Determinants Research Articles

Carriage of three plasmids in a single human clinical isolate of Clostridioides difficile

A subset of clinical isolates of Clostridioides difficile contains one or more plasmids and these plasmids can harbor virulence and antimicrobial resistance determinants. Despite their potential importance, C. difficile plasmids remain poorly characterized. Here, we provide the complete genome sequence of a human clinical isolate that carries three high-copy number plasmids from three different plasmid families that are therefore compatible. For two of these, we identify a region capable of sustaining plasmid replication in C. difficile that is also compatible with the plasmid pCD630 that is found in many laboratory strains. Together, our data advance our understanding of C. difficile plasmid biology.

151. Molecular diversity and resistance mechanisms of Klebsiella pneumoniae bloodstream infections in Peru

Abstract Background Klebsiella pneumoniae, a leading pathogen for mortality associated with antimicrobial resistance (AMR), was responsible for > 250,000 deaths in 2019. Genomic surveillance can guide the development of vaccines and new antibiotics against this pathogen. While AMR disproportionally affects low-and middle-income countries, limited genomic data are available from these countries. To close this gap, this study provides genomic characterization of K. pneumoniae blood isolates recovered in Peru. Methods Consecutive non-duplicate K. pneumoniae blood culture isolates were collected during an AMR surveillance study (VIRAPERU) from Jul 2017 to Oct 2019, from 15 tertiary hospitals from 13 regions of Peru. DNA extraction (GeneJET, Thermo Fisher Scientific), DNA library (Nextera XT, Illumina) and genome sequencing (MiSeq 500bp-V2, Illumina) were conducted. De novo assembling (SPAdes v3.13.1), quality assessment and annotation (Nullarbor v2.0), and identification of species, ST group, K/O loci, AMR (Kleborate v2.0.1) were conducted. Phylogenetic trees were built with Microreact v.192. Results From 119 K. pneumoniae isolates, 114 were recovered and confirmed to belong to the Klebsiella taxon. Six species were identified, the most prevalent being K. pneumoniae (106), followed by K. quasipneumoniae (3). Among carbapenem-resistant isolates (n=13), 10 (77%) carried the blaNDM-1 gene, while other three carried blaKPC-2 (1), blaIMP-16 (1) or no carbapenemase (1). Phenotypic co-resistance to colistin was present in 2 isolates, both negative for mcr-1 gene. The most common mechanisms of resistance to 3rd generation cephalosporins, quinolones and to aminoglycosides were CTX-M-15 (74.4%), qnrB (63.6%), aac(6')-Ib-cr (87.5%), respectively. Many other AMR genetic determinants were identified (Fig. 1). Sixty ST groups were identified, but only 6 ST groups carried a carbapenemase gene (Fig. 2). A wide diversity of K and O locus was also identified (54 distinct K-loci and 14 distinct O-loci). Figure 1.Genetic determinants of antimicrobial resistance and virulence found in Klebsiella pneumoniae blood isolates of PeruFigure 2.Distribution of ST groups according to specific antimicrobial resistance profiles Conclusion Bloodstream infections in Peru are caused by a wide diversity of K. pneumoniae strains, carrying multiple AMR genes. Carbapenem resistance is principally a result of blaNDM-1 carriage, found across 6 specific ST groups. Disclosures Omai Garner, PhD, Beckman Coulter, Inc.: Clinical trial data collection funded by Beckman Coulter, Inc. Victor Nizet, MD, Cellics Therapeutics: Advisor/Consultant|Clarametyx Biosciences: Advisor/Consultant|Vaxcyte, Inc.: Advisor/Consultant|Vaxcyte, Inc.: Grant/Research Support.

Australian Group on Antimicrobial Resistance (AGAR) Australian Staphylococcus aureus Surveillance Outcome Program (ASSOP).

From 1 January to 31 December 2021, forty-eight institutions around Australia participated in the Australian Staphylococcus aureus Surveillance Outcome Programme (ASSOP). The aim of ASSOP 2021 was to determine the proportion of Staphylococcus aureus bacteraemia (SAB) isolates in Australia that were antimicrobial resistant, with particular emphasis on susceptibility to methicillin and on characterisation of the molecular epidemiology of the methicillin-resistant isolates. A total of 2,928 SAB episodes were reported, of which 78.4% were community-onset. Overall, 16.9% of S. aureus isolates were methicillin resistant. The 30-day all-cause mortality associated with methicillin-resistant SAB was 15.0%, which was not significantly different from the 14.4% all-cause mortality associated with methicillin-susceptible SAB (p = 0.7). With the exception of the β-lactams and erythromycin, antimicrobial resistance in methicillin-susceptible S. aureus was rare. However, in addition to the β-lactams, approximately 36% of methicillin-resistant S. aureus (MRSA) were resistant to ciprofloxacin; 30% to erythromycin; 15% to tetracycline; 16% to gentamicin; and 3% to co-trimoxazole. When applying the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints, teicoplanin resistance was detected in three S. aureus isolates. Resistance to vancomycin or linezolid was not detected. Resistance to non-β-lactam antimicrobials was largely attributable to the healthcare-associated MRSA (HA-MRSA) clone ST22-IV [2B] (EMRSA-15), and the community-associated MRSA (CA-MRSA) clone ST45-V [5C2&5] which has acquired multiple antimicrobial resistance determinants including ciprofloxacin, erythromycin, clindamycin, gentamicin and tetracycline. The ST22-IV [2B] (EMRSA-15) clone is the predominant HA-MRSA clone in Australia. Nonetheless, 85% of methicillin-resistant SAB episodes were due to CA-MRSA clones. Although polyclonal, approximately 68% of CA-MRSA clones were characterised as ST93-IV [2B] (Queensland clone); ST45-V [5C2&5]; ST5-IV [2B]; ST1-IV [2B]; ST30-IV [2B]; and ST97-IV [2B]. As CA-MRSA is well established in the Australian community, it is important to monitor antimicrobial resistance patterns in community- and healthcare-associated SAB as this information will guide therapeutic practices in treating S. aureus bacteraemia.

Advancing antimicrobial resistance monitoring in surface waters with metagenomic and quasimetagenomic methods

The National Antimicrobial Resistance Monitoring System (NARMS) has monitored antimicrobial resistance (AMR) associated with pathogens of humans and animals since 1996. In alignment with One Health strategic planning, NARMS is currently exploring the inclusion of surface waters as an environmental modality for monitoring AMR. From a One Health perspective, surface waters function as key environmental integrators between humans, animals, agriculture, and the environment. Surface waters however, due to their dilute nature present a unique challenge for monitoring critically important antimicrobial resistance. Selective enrichments from water paired with genomic sequencing effectively describe AMR for single genomes but do not provide data to describe a broader environmental resistome. Metagenomic data effectively describe a broad range of AMR from certain matrices however, depth of coverage is usually insufficient to describe clinically significant AMR from aquatic matrices. Thus, the coupling of biological enrichments of surface water with shotgun NGS sequencing has been shown to greatly enhance the capacity to report an expansive profile of clinically significant antimicrobial resistance genes. Here we demonstrate, using water samples from distinct sites (a creek in close proximity to a hospital and a reservoir used for recreation and municipal water), that the AMR portfolio provided by enriched (quasimetagenomic) data is capable of describing almost 30% of NARMS surveillance targets contrasted to only 1% by metagenomic data. Additionally, the quasimetagenomic data supported reporting of statistically significant (P< 0.05) differential abundance of specific AMR genes between sites. A single time-point for two sites is a small pilot, but the robust results describing critically important AMR determinants from each water source, provide proof of concept that quasimetagenomics can be applied to aquatic AMR surveillance efforts for local, national, and global monitoring.

Plasmid-Coded Linezolid Resistance in Methicillin-Resistant Staphylococcus aureus from Food and Livestock in Germany

Resistance of methicillin-resistant Staphylococcus aureus (MRSA) from food and livestock to last resort antibiotics such as linezolid is highly concerning, since treatment options for infections in humans might be diminished. Known mechanisms of linezolid resistance include point mutations in the 23S rRNA gene and in the ribosomal proteins L3, L4 and L22 as well as an acquisition of the cfr, optrA or poxtA gene. The objective of our study was to characterize antimicrobial resistance (AMR) determinants and phylogenetic relationships among linezolid-resistant (LR-) MRSA from food and livestock. In total, from more than 4000 incoming isolates in the years 2012 to 2021, only two strains from 2015 originating from pig samples exhibited linezolid resistance in the antimicrobial susceptibility testing with MICs of ≥8 mg/L. These LR-MRSA were characterized in detail by whole-genome sequencing and phylogenetic analyses using cgMLST. The LR-MRSA strains showed resistances to ten and eight different antibiotics, respectively. Both strains harbored plasmid-coded cfr genes mediating the linezolid resistance. The cfr genes showed identical sequences in both strains. In addition to the cfr gene, genes for phenicol and clindamycin resistance were detected on the respective plasmids, opening the possibility for a co-selection. The LR-MRSA differed distantly in the phylogenetic analyses and also to other MRSA from pig samples in the year 2015. In conclusion, the occurrence of LR-MRSA in food and livestock seems to be very rare in Germany. However, carriage of plasmids with linezolid resistance determinants could lead to further linezolid-resistant strains by horizontal gene transfer.

Genomic epidemiology of Escherichia coli: antimicrobial resistance through a One Health lens in sympatric humans, livestock and peri-domestic wildlife in Nairobi, Kenya.

Livestock systems have been proposed as a reservoir for antimicrobial-resistant (AMR) bacteria and AMR genetic determinants that may infect or colonise humans, yet quantitative evidence regarding their epidemiological role remains lacking. Here, we used a combination of genomics, epidemiology and ecology to investigate patterns of AMR gene carriage in Escherichia coli, regarded as a sentinel organism. We conducted a structured epidemiological survey of 99 households across Nairobi, Kenya, and whole genome sequenced E. coli isolates from 311 human, 606 livestock and 399 wildlife faecal samples. We used statistical models to investigate the prevalence of AMR carriage and characterise AMR gene diversity and structure of AMR genes in different host populations across the city. We also investigated household-level risk factors for the exchange of AMR genes between sympatric humans and livestock. We detected 56 unique acquired genes along with 13 point mutations present in variable proportions in human and animal isolates, known to confer resistance to nine antibiotic classes. We find that AMR gene community composition is not associated with host species, but AMR genes were frequently co-located, potentially enabling the acquisition and dispersal of multi-drug resistance in a single step. We find that whilst keeping livestock had no influence on human AMR gene carriage, the potential for AMR transmission across human-livestock interfaces is greatest when manure is poorly disposed of and in larger households. Findings of widespread carriage of AMR bacteria in human and animal populations, including in long-distance wildlife species, in community settings highlight the value of evidence-based surveillance to address antimicrobial resistance on a global scale. Our genomic analysis provided an in-depth understanding of AMR determinants at the interfaces of One Health sectors that will inform AMR prevention and control.

Genetic diversity and variation in antimicrobial-resistance determinants of non-serotype 2 Streptococcus suis isolates from healthy pigs.

Streptococcus suis is a leading cause of bacterial meningitis in South-East Asia, with frequent zoonotic transfer to humans associated with close contact with pigs. A small number of invasive lineages are responsible for endemic infection in the swine industry, causing considerable global economic losses. A lack of surveillance and a rising trend in clinical treatment failure has raised concerns of growing antimicrobial resistance (AMR) among invasive S. suis . Gene flow between healthy and disease isolates is poorly understood and, in this study, we sample and sequence a collection of isolates predominantly from healthy pigs in Chiang Mai province, Northern Thailand. Pangenome characterization identified extensive genetic diversity and frequent AMR carriage in isolates from healthy pigs. Multiple AMR genes were identified, conferring resistance to aminoglycosides, lincosamides, tetracycline and macrolides. All isolates were non-susceptible to three or more different antimicrobial classes, and 75 % of non-serotype 2 isolates were non-susceptible to six or more classes (compared to 37.5 % of serotype 2 isolates). AMR genes were found on integrative and conjugative elements previously observed in other species, suggesting a mobile gene pool that can be accessed by invasive disease isolates. This article contains data hosted by Microreact.

High rate of fluoroquinolone resistant Neisseria gonorrhoeae detected by molecular surveillance of antimicrobial resistance determinants in Bulgaria

Antimicrobial-resistant Neisseria gonorrhoeae is a major public health concern. The surveillance of antimicrobial resistance benefits from rapid and accurate molecular techniques in molecular diagnostics to facilitate individualised medicine and antimicrobial stewardship. To support the recommendations for empirical treatment of gonococcal infections in Bulgaria, we investigated N. gonorrhoeae-positive clinical specimens from 2018 to 2021 for the presence of genetic determinants associated with antimicrobial resistance. N. gonorrhoeae-positive samples stored at the National Center of Infectious and Parasitic Diseases during the four-year study period were retrospectively analysed by polymerase chain reaction and DNA sequencing assays for resistance determinants to fluoroquinolones, third-generation cephalosporins and macrolides. The detected determinants indicated a high rate of fluoroquinolone resistance (59%), very low level of decreased susceptibility to third-generation cephalosporins (3%) but no macrolide resistance (0%). These findings validate the utilisation of the international guidelines’ recommendations for empirical dual therapy with ceftriaxone/cefixime and azithromycin in Bulgaria. Because of the high fluoroquinolone resistance rate, ciprofloxacin should only be considered as treatment if phenotypic or molecular antimicrobial susceptibility data indicate susceptibility to ciprofloxacin. For the purposes of surveillance and individualised medicine, molecular assays for resistance determinants could complement culture-based phenotypic gonococcal antimicrobial resistance testing.

MEGARes and AMR++, v3.0: an updated comprehensive database of antimicrobial resistance determinants and an improved software pipeline for classification using high-throughput sequencing.

Antimicrobial resistance (AMR) is considered a critical threat to public health, and genomic/metagenomic investigations featuring high-throughput analysis of sequence data are increasingly common and important. We previously introduced MEGARes, a comprehensive AMR database with an acyclic hierarchical annotation structure that facilitates high-throughput computational analysis, as well as AMR++, a customized bioinformatic pipeline specifically designed to use MEGARes in high-throughput analysis for characterizing AMR genes (ARGs) in metagenomic sequence data. Here, we present MEGARes v3.0, a comprehensive database of published ARG sequences for antimicrobial drugs, biocides, and metals, and AMR++ v3.0, an update to our customized bioinformatic pipeline for high-throughput analysis of metagenomic data (available at MEGLab.org). Database annotations have been expanded to include information regarding specific genomic locations for single-nucleotide polymorphisms (SNPs) and insertions and/or deletions (indels) when required by specific ARGs for resistance expression, and the updated AMR++ pipeline uses this information to check for presence of resistance-conferring genetic variants in metagenomic sequenced reads. This new information encompasses 337 ARGs, whose resistance-conferring variants could not previously be confirmed in such a manner. In MEGARes 3.0, the nodes of the acyclic hierarchical ontology include 4 antimicrobial compound types, 59 resistance classes, 233 mechanismsand 1448 gene groups that classify the 8733 accessions.

Genomic Insights into the Mobilome and Resistome of Sentinel Microorganisms Originating from Farms of Two Different Swine Production Systems.

Antimicrobial resistance (AMR) is a threat to public health due to long-term antimicrobial use (AMU), which promotes the bacterial acquisition of antimicrobial resistance determinants (ARDs). Within food-producing animals, organic and extensive Iberian swine production is based on sustainable and eco-friendly management systems, providing an excellent opportunity to evaluate how sustained differences in AMU impact the development and spread of AMR. Here, through a whole-genome sequencing approach, we provide an in-depth characterization of the resistome and mobilome and their interaction in 466 sentinel bacteria, namely, Escherichia coli, Enterococcus spp., Campylobacter coli, and Staphylococcus spp., recovered from 37 intensive and organic-extensive pig farms. Both ARDs and mobile genetic elements (MGEs) were primarily taxon-associated, with higher similarities among bacteria which were closely phylogenetically related. E. coli exhibited the most diverse resistome and mobilome, with 85.4% mobilizable ARDs, 50.3% of which were plasmid-associated. Staphylococcus spp. exhibited a broad repertoire of ARDs and MGEs, with 52.3% of its resistome being mobilizable. Although Enterococcus spp. carried the highest number of ARDs per isolate and its plasmidome was similar in size to that of E. coli, 43.7% of its resistome was mobilizable. A narrow spectrum of ARDs constituted the C. coli resistome, with point mutations as its main AMR driver. A constrained AMU, as observed in organic-extensive herds, determined a reduction in the quantitative composition of the resistome and the complexity of the resistome-mobilome interaction. These results demonstrate taxon-associated AMR-MGE interactions and evidence that responsible AMU can contribute to reducing AMR pressure in the food chain. IMPORTANCE This study provides the first integral genomic characterization of the resistome and mobilome of sentinel microorganisms for antimicrobial resistance (AMR) surveillance from two different swine production systems. Relevant differences were observed among taxa in the resistomes and mobilomes they harbored, revealing their distinctive risk in AMR dissemination and spread. Thus, Escherichia coli and, to a lesser extent, Staphylococcus spp. constituted the main reservoirs of mobilizable antimicrobial resistance genes, which were predominantly plasmid-associated; in contrast to Campylobacter coli, whose resistome was mainly determined by point mutations. The reduced complexity of mobilome-resistome interaction in Enterococcus spp. suggested its limited role in AMR dissemination from swine farms. The significant differences in antimicrobial use among the studied farms allowed us to assess the suitability of whole-genome sequencing as a rapid and efficient technique for the assessment of mid- to long-term on-farm interventions for the reduction of antimicrobial use and the evaluation of AMR status.

Determination of Enterococcus faecalis and Enterococcus faecium Antimicrobial Resistance and Virulence Factors and Their Association with Clinical and Demographic Factors in Kenya.

Enterococci are clinically significant because of their increasing antibiotic resistance and their ability to cause severe infections due to an arsenal of virulence genes. Few studies in the developing world have examined virulence factors that may significantly impact patient outcomes. This study describes the antimicrobial resistance profiles and prevalence of five key Enterococcal virulence genes gelE, asa, cylA, esp, and hyl in forty-four clinical Enterococcus faecalis and E. faecium isolates in Kenya and their association with patients' demographic and clinical characteristics. All E. faecium isolates were obtained from hospital-acquired skin and soft tissue infections. While E. faecalis was associated with community-acquired urinary tract infections. All isolates were resistant to erythromycin, whereas 11/44 (27.5%), 25/44 (56.8%), 28/44 (63.6%), 37/44 (84.1%), 40/44 (90.0%), and 43/44 (97.5%) were susceptible to tetracycline, levofloxacin, gentamicin, ampicillin, nitrofurantoin, and teicoplanin, respectively. All isolates were susceptible to tigecycline, vancomycin, and linezolid. There was little difference in the antibiotic resistance profiles between E. faecalis and E. faecium. The prevalence of the virulence genes among the 44 isolates were 27 (61.4%) for gelE, 26 (59.1%) for asa1, 16 (36.3%) for esp, 11 (25.0%) for cylA, and 1 (2.3%) for hyl. 72.9% of E. faecalis isolates had multiple virulence genes compared to 57% of E. faecium isolates with no virulence genes. The hyl gene was only detected in E. faecium, while cylA and asa1 were only detected in E. faecalis. A significant correlation was observed between the presence of asa1 and esp virulence genes and tetracycline resistance (P=0.0305 and 0.0363, respectively). A significant correlation was also observed between the presence of virulence genes gelE and asa1 and nitrofurantoin resistance (P=0.0175 and 0.0225, respectively) and ampicillin resistance (P=0.0005 and 0.0008, respectively). The study highlights the high levels of erythromycin resistance in E. faecalis and E. faecium, the demographic factors influencing the species distribution among patients, and the accumulation of multiple virulence genes in E. faecalis. The significant association of gelE, asa1, and esp virulence genes with drug resistance could explain the pathogenic success of E. faecalis and provides a guide for future studies.

Replacement of KPC-producing pandemic lineages and dissemination of plasmids associated with antimicrobial resistance determinants during inpatient’s hospitalization

The emergence of blaKPC-2 within nosocomial settings has become a major public health crisis worldwide. Our aim was to perform whole-genome sequencing (WGS) of three KPC-producing Gram-negative bacilli (KPC-GNB) strains isolated from a hospitalized patient to identify acquired antimicrobial resistance genes (ARGs). WGS was performed using Illumina MiSeq-I, and de novo assembly was achieved using SPAdes. Bioinformatics analysis was done using Resfinder, AMRFinder, ISFinder, plasmidSPAdes, PlasmidFinder, MOB-suite, PLSDB database, and IntegronFinder. Conjugation assays were performed to assess the ability of blaKPC-2 to transfer via a plasmid-related mobilization mechanism. High-risk clone KPC-producing Klebsiella pneumoniae sequence type (ST) 258 (HA3) was colonizing an inpatient who later was infected by KPC-producing Escherichia coli ST730 (HA4) and subsequently by KPC-producing K. pneumoniae ST11 (HA15) during hospitalization. Although belonging to different species, both strains causing infections harbored the same gene configuration for dissemination of blaKPC-2 in related IncM1 plasmids recently found in other KPC-GNB isolated from Hospital Alemán at Ciudad Autónoma de Buenos Aires. Conjugation assays revealed that only pDCVEA4-KPC from E. coli HA4 was successfully transferred with a conjugation frequency of 3.66×101. Interchange of multidrug-resistant K. pneumoniae lineages ST258 replaced by ST11 in the framework of colonization and infection by KPC-GNB of an inpatient from our institution was found. In addition, the transfer of the gene configuration of blaKPC-2 between infecting strains may have occurred in the nosocomial environment, but we cannot rule out that the event took place in vivo, within the patient, during hospitalization.

Incidence of antibiotic resistance genotypes of Vibrio species recovered from selected freshwaters in Southwest Nigeria

Vibrio species are classified as potent hazards because of their tendency to effect serious diseases like cholera and other gastrointestinal ailments in humans, as well as vibriosis in fish. A total of 144 freshwater samples were aseptically collected monthly across four rivers (Asejire, Ona, Dandaru and Erinle rivers) over a 12-month period from which Vibrio spp. were isolated using culture procedures, confirmed by means of biochemical test as well as Polymerase Chain Reaction (PCR) assay and further characterized for their phenotypic antibiotic susceptibilities and relevant antimicrobial resistant determinants by PCR. Three hundred and fifteen (58%) isolates confirmed across the sampled sites (Asejire = 75, Dandaru = 87, Eleyele = 72, Erinle = 81) showed high resistance against erythromycin—95%, Sulphamethoxazole—94%, rifampicin—92%, doxycycline—82%, tetracycline—75%, amoxicillin—45%, cephalothin—43% and varied susceptibilities to other antibiotics. The multiple antibiotic resistance indices of 97% of the Vibrio isolates were above the 0.2 threshold limit with MAR phenotype pattern E-SUL-RF-TET-DOX (0.38) found to be the most prevalent pattern among the isolates. The distributions of resistance determinant of the tested antibiotics were revealed as follows: sulII 33%, sulI 19% (sulfonamides); blaOXA 27%, ampC 39%, blapse 11% (beta-lactams); tetA 28%, tetE 20%, tet39 8%, (tetracyclines) and strA 39%. aacC2 24%, aphA1 14% (aminoglycosides). Strong positive associations were observed among tetA, sulI, tetE and sulII. This study raises concerns as these selected rivers may contribute to the environmental spread of waterborne diseases and antibiotic resistance genes. Therefore, we recommend environmental context-tailored strategies for monitoring and surveillance of resistance genes so as to safeguard the environment from becoming reservoirs of virulent and infectious Vibrio species.

Genomic Analysis and Molecular Characteristics in Carbapenem-Resistant Klebsiella pneumoniae Strains.

Klebsiella pneumoniae is an important bacterium and responsible for both infections acquired in hospital and community because of its multidrug resistance and the virulence. The aim of this research was to investigate clonal lineages, antibiotic resistance profiles, and virulence factors of the hospital isolated carbapenem-resistant strains. Fifty carbapenem-resistant isolates were phenotypically confirmed extended-spectrum beta-lactamases ESBLs producers. MLST analysis revealed 94% sequence type 11. These isolates mainly belonged to three clones according to the PFGE DNA patterns. PFGE patterns have good discrimination than ST profiles. One isolate, K. pneumoniae KPX, undergoing whole-genome sequencing comprised one circular chromosome and four circular plasmids. This isolate harbored a variety of antimicrobial resistance and virulence determinants. The closest relative of K. pneumoniae KPX was another ST11 clinical isolate recovered Sichuan. In addition, KPC-2 (98.0%), SHV-11 (98.0%), TEM-1 (76.0%), CTX-M (76.0%), oqxB1(66%), qnrS (70%), Int1 (42.0%), sul1 (82.0%), sul2 (96.0%), iutA (88%), iucC(10%), and rmpA2 (100%) genes were presented in multiple drug-resistant isolates. The dataset presented in this study provided the genomic and epidemiological analysis of carbapenem-resistant K. pneumoniae in hospital settings. Antimicrobial-resistant profiles suggested the presence of significant selective antibiotic pressure. Appropriate surveillance is essential to the development of effective control strategies in the prevention of nosocomial infection.

Comparative Analysis of the Molecular Characteristics of Group B Streptococcus Isolates Collected from Pregnant Korean Women Using Whole-genome Sequencing.

The incidence of early- and late-onset sepsis and meningitis in neonates due to maternal rectovaginal group B Streptococcus (GBS) colonization may differ with serotype distribution and clonal complex (CC). CC17 strains are associated with hypervirulence and poor disease outcomes. GBS serotypes are distinguished based on the polysaccharide capsule, the most important virulence factor. We determined the sequence type distribution of GBS isolates from pregnant women in Korea and validated whole-genome sequencing (WGS)-based prediction of antimicrobial susceptibility and capsular serotypes in GBS isolates. Seventy-five GBS isolates collected from pregnant Korean women visiting Wonju Severance Christian Hospital, Wonju, Korea between 2017 and 2019 were subjected to WGS using the NovaSeq 6000 system (Illumina, San Diego, CA, USA). Multilocus sequence types, serotypes, antimicrobial resistance genes, and hemolysin operon mutations were determined by WGS, and the latter three were compared with the results of conventional phenotypic methods. The predominant lineage was CC1 (37.3%), followed by CC19 (32.0%), CC12 (17.3%), and CC17 (4.0%). All isolates were cps typeable (100%, (75/75), and 89.3% of cps genotypes (67/75) were concordant with serotypes obtained using latex agglutination. The cps genotypes of the 75 isolates were serotypes III (24.0%), V (22.7%), and VIII (17.3%). All isolates harboring intact ermB and tet were non-susceptible to erythromycin and tetracycline, respectively. Three non-hemolytic strains had 1-bp frameshift insertions in cylE. The low prevalence of CC17 GBS colonization may explain the low frequency of neonatal GBS infections. WGS is a useful tool for simultaneous genotyping and antimicrobial resistance determination.

The Impact of Chlorine Disinfection of Hospital Wastewater on Clonal Similarity and ESBL-Production in Selected Bacteria of the Family Enterobacteriaceae.

Hospitals are regarded as ecological niches of antibiotic-resistant bacteria (ARB). ARB can spread outside the hospital environment via hospital wastewater (HWW). Therefore, HWW is often disinfected in local stations to minimize that risk. Chlorine-based treatment is the most popular method of HWW disinfection around the world, however, recent research has suggested that it can contribute to the spread of antimicrobial resistance (AMR). The aim of this study is to determine the impact of HWW disinfection on the clonal similarity of Enterobacteriaceae species and their ability to produce extended-spectrum beta-lactamases (ESBLs). The study was conducted in a hospital with a local chlorine-based disinfection station. Samples of wastewater before disinfection and samples of disinfected wastewater, collected in four research seasons, were analyzed. Bacteria potentially belonging to the Enterobacteriaceae family were isolated from HWW. The Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) method was used to generate DNA fingerprints of all bacterial isolates. The isolates were phenotypically tested for the production of ESBLs. Antibiotic resistance genes (blaSHV, blaTEM, and blaOXA, blaCTX-M-1-group, blaCTX-M-2-group, blaCTX-9-group and blaCTX-M-8/25-group) were detected by PCR in strains with confirmed phenotypic ability to produce ESBLs. The ESBL+ isolates were identified by the sequencing of 16S rDNA. In the present study, the same bacterial clones were isolated from HWW before and after disinfection and HWW was sampled in different seasons. Genetic and phenotypic variations were observed in bacterial clones. ESBL+ strains were isolated significantly more often from disinfected than from non-disinfected HWW. The blaOXA gene was significantly more prevalent in isolates from disinfected than non-disinfected HWW. Enterobacter hormaechei and Klebsiella pneumoniae were the dominant species in ESBL+ strains isolated from both sampling sites. The results of this study indicate that chlorine-based disinfection promotes the survival of ESBL-producing bacteria and/or the transmission of genetic determinants of antimicrobial resistance. As a result, chlorination increases the proportion of ESBL-producing Enterobacteriaceae in disinfected wastewater. Consequently, chlorine-based disinfection practices may pose a risk to the environment and public health by accelerating the spread of antimicrobial resistance.

Emergence and Genomic Characterization of the First Reported optrA-Carrying Linezolid-Resistant Enterococci Isolated from Retail Broiler Meat in the United Arab Emirates

The foodborne transfer of resistant genes from enterococci to humans and their tolerance to several commonly used antimicrobials are of growing concern worldwide. Linezolid is a last-line drug for managing complicated illnesses resulting from multidrug-resistant Gram-positive bacteria. The optrA gene has been reported in enterococci as one of the acquired linezolid resistance mechanisms. The present study uses whole-genome sequencing analysis to characterize the first reported isolates of linezolid-resistant E. faecium (n = 6) and E. faecalis (n = 10) harboring the optrA gene isolated from samples of supermarket broiler meat (n = 165) in the United Arab Emirates (UAE). The sequenced genomes were used to appraise the study isolates’ genetic relatedness, antimicrobial resistance determinants, and virulence traits. All 16 isolates carrying the optrA gene demonstrated multidrug-resistance profiles. Genome-based relatedness classified the isolates into five clusters that were independent of the isolate sources. The most frequently known genotype among the isolates was the sequence type ST476 among E. faecalis (50% (5/10)). The study isolates revealed five novel sequence types. Antimicrobial resistance genes (ranging from 5 to 13) were found among all isolates that conferred resistance against 6 to 11 different classes of antimicrobials. Sixteen different virulence genes were found distributed across the optrA-carrying E. faecalis isolates. The virulence genes in E. faecalis included genes encoding invasion, cell adhesion, sex pheromones, aggregation, toxins production, the formation of biofilms, immunity, antiphagocytic activity, proteases, and the production of cytolysin. This study presented the first description and in-depth genomic characterization of the optrA-gene-carrying linezolid-resistant enterococci from retail broiler meat in the UAE and the Middle East. Our results call for further monitoring of the emergence of linezolid resistance at the retail and farm levels. These findings elaborate on the importance of adopting a One Health surveillance approach involving enterococci as a prospective bacterial indicator for antimicrobial resistance spread at the human–food interface.

Determination and quantification of microbial communities and antimicrobial resistance on food through host DNA-depleted metagenomics

Food products carry bacteria unless specifically sterilised. These bacteria can be pathogenic, commensal or associated with food spoilage, and may also be resistant to antimicrobials. Current methods for detecting bacteria on food rely on culturing for specific bacteria, a time-consuming process, or 16S rRNA metabarcoding that can identify different taxa but not their genetic content. Directly sequencing metagenomes of food is inefficient as its own DNA vastly outnumbers the bacterial DNA present. We optimised host DNA depletion enabling efficient sequencing of food microbiota, thereby increasing the proportion of non-host DNA sequenced 13-fold (mean; range: 1.3–40-fold) compared to untreated samples. The method performed best on chicken, pork and leafy green samples which had high mean prokaryotic read proportions post-depletion (0.64, 0.74 and 0.74, respectively), with lower mean prokaryotic read proportions in salmon (0.50) and prawn samples (0.19). We show that bacterial compositions and concentrations of antimicrobial resistance (AMR) genes differed by food type, and that salmon metagenomes were influenced by the production/harvesting method. The approach described in this study is an efficient and effective method of identifying and quantifying the predominant bacteria and AMR genes on food.

Characterization of Resistance and Virulence of Pasteurella multocida Isolated from Pet Cats in South China

Pasteurella multocida, one of the major zoonotic pathogens, may cause localized cellulitis, bacteremia, septic shock, and other symptoms in people. Accidental bites or scratches in close contact between humans and cats are one of the causes of P. multocida infection in humans. The prevalence of diseases due to P. multocida in humans may be rising as a result of the popularity of cat cafes in China. However, the epidemiology of P. multocida in cat-themed cafes in South China is yet to be reported. P. multocida in 92 healthy cats from four cafes with pet-cat themes was examined in their tonsils and oral cavities. The antibiotic susceptibility of each isolate was evaluated by using the disk-susceptibility testing method. The 19 P. multocida were selected for whole-genome sequencing (WGS), together with the genome data of eight previously described clinical strains isolated from humans, and the analysis of antimicrobial resistance (AMR) determinants, capsular genotyping, MLST genotyping, and virulence gene profiles was carried out. The results showed that 48.91% of cats (45/90) were positive for P. multocida. All isolated P. multocida stains were highly resistant to erythromycin (15 μg) (100%) and nonsensitive to fluoroquinolones (5 μg) (37.38%), while they were all susceptible to penicillin (10 untis), tetracycline (30 μg), doxycycline (30 μg), and chloramphenicol (30 μg). According to WGS analysis, those with the gyrA resistance gene were all insensitive to fluoroquinolone antibiotics. Virulence gene profiles showed that the genes pfhA, nanH, and plpB were practically all present in cats compared to humans. To conclude, increased antibiotic resistance, along with virulence factors, complicates the treatment of P. multocida infection. Thus, clinical treatment for P. multocida infection should be performed more cautiously.

Oral and Rectal Colonization by Antimicrobial-Resistant Gram-Negative Bacteria and Their Association with Death among Residents of Long-Term Care Facilities: A Prospective, Multicenter, Observational, Cohort Study

Introduction: The prevalence of antimicrobial-resistant bacteria (ARB) in long-term care facilities (LTCFs) remains unclear. Furthermore, the effect of ARB colonization on the clinical outcomes of LTCF residents has not been explored. Methods: We conducted a prospective multicenter cohort study and investigated the residents (N = 178) of six Japanese LTCFs (three Welfare Facilities for the Elderly Requiring Long-term Care and three Geriatric Health Service Facilities) for oral and rectal carriage of ARB. The clinical outcomes of the residents were evaluated based on isolating bacterial strains and subjecting them to whole-genome sequencing. Results: Of the 178 participants, 32 belonging to Geriatric Health Service Facilities with no information on their clinical outcome were excluded, and the remaining 146 were followed up for at most 21 months. Extended-spectrum β-lactamases (ESBL)-producing Enterobacterales and Pseudomonas aeruginosa were detected in 42.7% (n = 76) and 2.8% (n = 5) of the rectal swabs and 5.6% (n = 10) and 3.4% (n = 6) of the oral swabs, respectively. Detection of ARB in the oral and rectal cavities showed remarkable association with enteral nutrition. Further, P. aeruginosa was significantly associated with an increase in mortality of the residents, but there were not significant association between ESBL-producing Enterobacterales and mortality. Core-genome phylogeny of P. aeruginosa revealed a wide-spread distribution of the isolated strains across the phylogeny, which included a cluster of ST235 strains with substantially higher biofilm formation ability than the other isolated P. aeruginosa strains. Discussion/Conclusion: This study is the first to investigate the carriage of both oral and rectal ARB, genomic relatedness and determinants of antimicrobial resistance in isolated strains, and clinical outcomes of LTCF residents. Our study provides the first direct evidence for the burden of antimicrobial resistance in LTCFs.