Genetic Determinants Of Resistance Research Articles

Determination of molecular types and resistance to macrolides in <i>Treponema pallidum</i> isolates isolated in the Russian Federation

Background. The number of syphilis cases in the Russian Federation increased significantly in 2022. Control of heterogeneity of Treponema pallidum subtypes is important to monitor the emergence and spread of antibiotic-resistant strains Aims. To determine molecular subtypes and resistance to macrolides in T. pallidum isolates isolated in the Russian Federation in 2022. Methods. We analyzed DNA isolated from 49 samples of clinical material obtained from patients from dermatovenerological treatment and prevention facilities in three federal districts (CFD, SFD, SCFD) of the Russian Federation in 2022 with diagnoses of primary syphilis and secondary syphilis. T. pallidum DNA isolation and confirmation of the presence of genetic material were performed according to the existing algorithms. To search for genetic determinants of resistance to macrolides, a fragment of the 23S rRNA gene was analyzed. Primary decoding of nucleotide sequences was performed in Sequencing Analysis 5.3.1. Mega 11 program was used to align the analyzed fragments of target genes to T. pallidum reference sequences. Results. In 2022, three subtypes of T. pallidum were identified in the territory of the represented federal districts of the Russian Federation: 14d/f, 14d/g, 14d/d with continued dominance of subtype 14d/f. The macrolide-resistant subtype 14d/d was identified in two federal districts, which is new for the Russian Federation. Conclusions. The population of T. pallidum continues to expand in the Russian Federation, including the emergence of azithromycin-resistant strains. The data obtained confirm the need for continuous monitoring of circulating strains and may facilitate understanding of their geographic distribution.

Assessment of spillover of antimicrobial resistance to untreated children 7 to 12 years old after mass drug administration of azithromycin for child survival in Niger: a secondary analysis of the MORDOR cluster-randomized trial.

The risk of antibiotic resistance is complicated by the potential for spillover effects from one treated population to another. Azithromycin mass drug administration programs report higher rates of antibiotic resistance among treatment arms in targeted groups. This study aims to understand the risk of spillover of antibiotic resistance to non-target groups in these programs. Data was used from a cluster-randomized trial comparing the effect of biannual azithromycin and placebo distribution to children 1-59 months on child mortality. Nasopharyngeal samples from untreated children 7-12 years old were tested for genetic determinants of macrolide resistance (primary outcome) and resistance to other antibiotic classes (secondary outcomes). Linear regression was used to compare the community-level mean difference in prevalence by arm at the 24-month timepoint adjusting for baseline prevalence. 1,103 children 7-12 years old in 30 communities were included in the analysis (15 azithromycin, 15 placebo). Adjusted mean differences in prevalence of resistance determinants for macrolides, beta-lactams and tetracyclines were 3.4% (95% CI -4.1% to 10.8%, P-value 0.37), -1.2% (95% CI -7.9% to 5.5%, P-value 0.72), and -3.3% (95% CI -9.5% to 2.8%, P-value 0.61), respectively. We were unable to demonstrate a statistically significant increase in macrolide resistance determinants in untreated groups in an azithromycin mass drug administration program. While the result might be consistent with a small spillover effect, this study was not powered to detect such a small difference. Larger studies are warranted to better understand the potential for spillover effects within these programs.

Bacterial enrichment prior to third-generation metagenomic sequencing improves detection of BRD pathogens and genetic determinants of antimicrobial resistance in feedlot cattle.

Bovine respiratory disease (BRD) is one of the most important animal health problems in the beef industry. While bacterial culture and antimicrobial susceptibility testing have been used for diagnostic testing, the common practice of examining one isolate per species does not fully reflect the bacterial population in the sample. In contrast, a recent study with metagenomic sequencing of nasal swabs from feedlot cattle is promising in terms of bacterial pathogen identification and detection of antimicrobial resistance genes (ARGs). However, the sensitivity of metagenomic sequencing was impeded by the high proportion of host biomass in the nasal swab samples. This pilot study employed a non-selective bacterial enrichment step before nucleic acid extraction to increase the relative proportion of bacterial DNA for sequencing. Non-selective bacterial enrichment increased the proportion of bacteria relative to host sequence data, allowing increased detection of BRD pathogens compared with unenriched samples. This process also allowed for enhanced detection of ARGs with species-level resolution, including detection of ARGs for bacterial species of interest that were not targeted for culture and susceptibility testing. The long-read sequencing approach enabled ARG detection on individual bacterial reads without the need for assembly. Metagenomics following non-selective bacterial enrichment resulted in substantial agreement for four of six comparisons with culture for respiratory bacteria and substantial or better correlation with qPCR. Comparison between isolate susceptibility results and detection of ARGs was best for macrolide ARGs in Mannheimia haemolytica reads but was also substantial for sulfonamide ARGs within M. haemolytica and Pasteurella multocida reads and tetracycline ARGs in Histophilus somni reads. By increasing the proportion of bacterial DNA relative to host DNA through non-selective enrichment, we demonstrated a corresponding increase in the proportion of sequencing data identifying BRD-associated pathogens and ARGs in deep nasopharyngeal swabs from feedlot cattle using long-read metagenomic sequencing. This method shows promise as a detection strategy for BRD pathogens and ARGs and strikes a balance between processing time, input costs, and generation of on-target data. This approach could serve as a valuable tool to inform antimicrobial management for BRD and support antimicrobial stewardship.

Prolonged mass azithromycin distributions and macrolide resistance determinants among preschool children in Niger: A sub-study of a cluster-randomized trial (MORDOR).

Randomized controlled trials found that twice-yearly mass azithromycin administration (MDA) reduces childhood mortality, presumably by reducing infection burden. World Health Organization (WHO) issued conditional guidelines for mass azithromycin administration in high-mortality settings in sub-Saharan Africa given concerns for antibiotic resistance. While prolonged twice-yearly MDA has been shown to increase antibiotic resistance in small randomized controlled trials, the objective of this study was to determine if macrolide and non-macrolide resistance in the gut increases with the duration of azithromycin MDA in a larger setting. The Macrolide Oraux pour Réduire les Décès avec un Oeil sur la Résistance (MORDOR) study was conducted in Niger from December 2014 to June 2020. It was a cluster-randomized trial of azithromycin (A) versus placebo (P) aimed at evaluating childhood mortality. This is a sub-study in the MORDOR trial to track changes in antibiotic resistance after prolonged azithromycin MDA. A total of 594 communities were eligible. Children 1 to 59 months in 163 randomly chosen communities were eligible to receive treatment and included in resistance monitoring. Participants, staff, and investigators were masked to treatment allocation. At the conclusion of MORDOR Phase I, by design, all communities received an additional year of twice-yearly azithromycin treatments (Phase II). Thus, at the conclusion of Phase II, the treatment history (1 letter per 6-month period) for the participating communities was either (PP-PP-AA) or (AA-AA-AA). In Phase III, participating communities were then re-randomized to receive either another 3 rounds of azithromycin or placebo, thus resulting in 4 treatment histories: Group 1 (AA-AA-AA-AA-A, N = 51), Group 2 (PP-PP-AA-AA-A, N = 40), Group 3 (AA-AA-AA-PP-P, N = 27), and Group 4 (PP-PP-AA-PP-P, N = 32). Rectal swabs from each child (N = 5,340) were obtained 6 months after the last treatment. Each child contributed 1 rectal swab and these were pooled at the community level, processed for DNA-seq, and analyzed for genetic resistance determinants. The primary prespecified outcome was macrolide resistance determinants in the gut. Secondary outcomes were resistance to beta-lactams and other antibiotic classes. Communities recently randomized to azithromycin (groups 1 and 2) had significantly more macrolide resistance determinants than those recently randomized to placebo (groups 3 and 4) (fold change 2.18, 95% CI 1.5 to 3.51, Punadj < 0.001). However, there was no significant increase in macrolide resistance in communities treated 4.5 years (group 1) compared to just the most recent 2.5 years (group 2) (fold change 0.80, 95% CI 0.50 to 1.00, Padj = 0.010), or between communities that had been treated for 3 years in the past (group 3) versus just 1 year in the past (group 4) (fold change 1.00, 95% CI 0.78 to 2.35, Padj = 0.52). We also found no significant differences for beta-lactams or other antibiotic classes. The main limitations of our study were the absence of phenotypic characterization of resistance, no complete placebo arm, and no monitoring outside of Niger limiting generalizability. In this study, we observed that mass azithromycin distribution for childhood mortality among preschool children in Niger increased macrolide resistance determinants in the gut but that resistance may plateau after 2 to 3 years of treatment. Co-selection to other classes needs to be monitored. NCT02047981 https://classic.clinicaltrials.gov/ct2/show/NCT02047981.

Resistome, Virulome, and Clonal Variation in Methicillin-Resistant Staphylococcus aureus (MRSA) in Healthy Swine Populations: A Cross-Sectional Study.

This cross-sectional study investigates the methicillin-resistant Staphylococcus aureus (MRSA): its prevalence, antimicrobial resistance, and molecular characteristics in healthy swine populations in central Portugal. A total of 213 samples were collected from pigs on twelve farms, and MRSA prevalence was assessed using selective agar plates and confirmed via molecular methods. Antimicrobial susceptibility testing and whole genome sequencing (WGS) were performed to characterize resistance profiles and genetic determinants. Among the 107 MRSA-positive samples (83.1% prevalence), fattening pigs and breeding sows exhibited notably high carriage rates. The genome of 20 isolates revealed the predominance of the ST398 clonal complex, with diverse spa types identified. Antimicrobial susceptibility testing demonstrated resistance to multiple antimicrobial agents, including penicillin, cefoxitin, and tetracycline. WGS analysis identified a diverse array of resistance genes, highlighting the genetic basis of antimicrobial resistance. Moreover, virulence gene profiling revealed the presence of genes associated with pathogenicity. These findings underscore the significant prevalence of MRSA in swine populations and emphasize the need for enhanced surveillance and control measures to mitigate zoonotic transmission risks. Implementation of prudent antimicrobial use practices and targeted intervention strategies is essential to reducing MRSA prevalence and safeguarding public health. Continued research efforts are warranted to elucidate transmission dynamics and virulence potential, ultimately ensuring food safety and public health protection.

Genetic determinants of antimicrobial resistance in polymyxin B resistant Pseudomonas aeruginosa isolated from airways of patients with cystic fibrosis.

Pseudomonas aeruginosa is the main pathogen associated with pulmonary exacerbation in patients with cystic fibrosis (CF). CF is a multisystemic genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene, which mainly affects pulmonary function. P. aeruginosa isolated from individuals with CF in Brazil is not commonly associated with multidrug resistance (MDR), especially when compared to global occurrence, where the presence of epidemic clones, capable of expressing resistance to several drugs, is often reported. Due to the recent observations of MDR isolates of P. aeruginosa in our centers, combined with these characteristics, whole-genome sequencing was employed for analyses related to antimicrobial resistance, plasmid identification, search for phages, and characterization of CF clones. All isolates in this study were polymyxin B resistant, exhibiting diverse mutations and reduced susceptibility to carbapenems. Alterations in mexZ can result in the overexpression of the MexXY efflux pump. Mutations in oprD, pmrB, parS, gyrA and parC may confer reduced susceptibility to antimicrobials by affecting permeability, as observed in phenotypic tests. The phage findings led to the assumption of horizontal genetic transfer, implicating dissemination between P. aeruginosa isolates. New sequence types were described, and none of the isolates showed an association with epidemic CF clones. Analysis of the genetic context of P. aeruginosa resistance to polymyxin B allowed us to understand the different mechanisms of resistance to antimicrobials, in addition to subsidizing the understanding of possible relationships with epidemic strains that circulate among individuals with CF observed in other countries.

Genetic determinants of resistance to antimicrobial therapeutics are rare in publicly available Clostridioides difficile genome sequences.

To determine the frequencies and clonal distributions of putative genetic determinants of resistance to antimicrobials applied for treatment of Clostridioides difficile infection (CDI), as documented in the genomic record. We scanned 26 557 C. difficile genome sequences publicly available from the EnteroBase platform for plasmids, point mutations and gene truncations previously reported to reduce susceptibility to vancomycin, fidaxomicin or metronidazole, respectively. We measured the antimicrobial susceptibility of 143 selected C. difficile isolates. The frequency of mutations causing reduced susceptibility to vancomycin and metronidazole, respectively, increased strongly after 2000, peaking at up to 52% of all sequenced C. difficile genomes. However, both mutations declined sharply more recently, reflecting major changes in CDI epidemiology. We detected mutations associated with fidaxomicin resistance in several major genotypes, but found no evidence of international spread of resistant clones. The pCD-METRO plasmid, conferring metronidazole resistance, was detected in a single previously unreported C. difficile isolate, recovered from a hospital patient in Germany in 2008. The pX18-498 plasmid, putatively associated with decreased vancomycin susceptibility, was confined to related, recent isolates from the USA. Phenotype measurements confirmed that most of those genetic features were useful predictors of antibiotic susceptibility, even though ranges of MICs typically overlapped among isolates with and without specific mutations. Genomic data suggested that resistance to therapeutic antimicrobial drugs is rare in C. difficile. Public antimicrobial resistance marker databases were not equipped to detect most of the genetic determinants relevant to antibiotic therapy of CDI.

The fly route of extended-spectrum-β-lactamase-producing Enterobacteriaceae dissemination in a cattle farm: from the ecosystem to the molecular scale

IntroductionThis study aimed to understand the origin and to explain the maintenance of extended-spectrum β-lactamase (ESBL) Enterobacteriaceae isolated from food-producing animals in a third-generation cephalosporin (3GC)-free farm.MethodsCulture and molecular approaches were used to test molecules other than 3GC such as antibiotics (tetracycline and oxytetracycline), antiparasitics (ivermectin, flumethrin, fenbendazol, and amitraz), heavy metal [arsenic, HNO3, aluminum, HNO3, cadmium (CdSO4), zinc (ZnCl2), copper (CuSO4), iron (FeCl3), and aluminum (Al2SO4)], and antioxidant (butylated hydroxytoluene) as sources of selective pressure. Whole-genome sequencing using short read (Illumina™) and long read (Nanopore™) technologies was performed on 34 genomes. In silico gene screening and comparative analyses were used to characterize the genetic determinants of resistance, their mobility, and the genomic relatedness among isolates.ResultsOur analysis unveiled a low diversity among the animal ESBL-producing strains. Notably, E. coli ST3268 was recurrently isolated from both flies (n = 9) and cattle (n = 5). These E. coli ST3268/blaCTX-M-15/blaTEM-1B have accumulated multiple plasmids and genes, thereby representing a reservoir of resistance and virulence factors. Our findings suggest that flies could act as effective mechanical vectors for antimicrobial gene transfer and are capable of transporting resistant bacteria across different environments and to multiple hosts, facilitating the spread of pathogenic traits. A significantly higher mean minimum inhibitory concentration of oxytetracycline (841.4 ± 323.5 mg/L vs. 36.0 ± 52.6 mg/L, p = 0.0022) in ESBL E. coli than in non-ESBL E. coli and blaCTX-M-15 gene overexpression in oxytetracycline-treated vs. untreated ESBL E. coli (RQOxy = 3.593, p = 0.024) confirmed oxytetracycline as a source of selective pressure in ESBL E. coli.DiscussionThe occurrence of ESBL E. coli in a farm without 3GC use is probably due to an as yet undefined human origin of Enterobacteriaceae blaCTX-M-15 gene transmission to animals in close contact with cattle farm workers and the maintenance of the local ESBL E. coli reservoir by a high fly diversity and oxytetracycline selective pressure. These findings highlight the critical need for stringent vector control to mitigate antimicrobial resistance spread for preserving public health. Addressing this issue necessitates a multifaceted approach combining microbial genetics, vector ecology, and farm management practices.

Antimicrobial resistance genetic determinants and susceptibility profile of Pseudomonas aeruginosa isolated from clinical samples in a tertiary hospital in Ogun State, Nigeria.

Genetic determinants are known to promote antibiotic resistance through horizontal gene transfer. We molecularly characterized integrons, plasmid replicon types and metallo-β-lactamase-encoding genes of 38 Pseudomonas aeruginosa strains isolated from clinical samples. The P. aeruginosa isolates displayed high resistance (97.4%) to β-lactams. Seventeen (44.74%) of them possessed plasmids. Of the 17 isolates that possessed plasmids, 11 (64.7%) of them harboured IncFIA plasmid replicon type, while 6 (35.3%), 5 (29.4%) and 5 (29.4%) were of the IncFIB, IncF and IncW types, respectively. The intI1 gene was detected in 19 (50%) of the isolates. The blaNDM-A, blaNDM-B and blaVIM genes were detected in 14 (35.9%), 4 (10.3%) and 5 (12.8%) of the isolates, respectively. High resistance to β-lactams was observed among P. aeruginosa strains of clinical origin in this study. They possessed transmissible genetic elements indicating the potential for continuous dissemination, thus continuous surveillance is advocated.

Characteristics of &lt;i&gt;Streptococcus pneumoniae&lt;/i&gt; carriage in the pediatric population

Objective: to investigate the regional peculiarities of Streptococcus pneumoniae carriage in the pediatric population and characterize the dominant serotypes of the pathogen.&#x0D; Materials and methods. The clinical study group consisted of 509 healthy children attending preschool institutions. Examination of nasopharyngeal samples for the detection of S. pneumonae was carried out by classical bacteriological and molecular biological methods. The serotype was determined by real-time PCR. Genome-wide sequencing of the serogroups 15 and 11 isolates and bioinformatic analysis were performed.&#x0D; Results. The S. pneumoniae bacterial carriers in the group of healthy children was detected in 207 children (40.7%), while the frequency of detection of S. pneumoniae in urban children living in Kazan was significantly higher than in children living in rural area and amounted to 53.4 and 31.1%, respectively (p 0.05). Among children vaccinated with the 13-valent pneumococcal conjugate vaccine (PCV-13), S. pneumoniae carriers were not detected in 57.5% of cases. There were no significant differences in the degree of nasopharyngeal contamination depending on the vaccination status. Analysis of the serotype composition indicates the predominance of vaccine serotypes (57.7%), while the share of serotypes included in the PСV-13 vaccine accounts for only 24.7%, the share of non-vaccine serotypes was 32.1%, untyped — 10.2%. In unvaccinated children, vaccine serotypes that are part of the PCV-13 and 23-valent polysaccharide pneumococcal vaccine prevailed (PPSV-23): 6ABCD (21%), 11 AD (15%), 14 (13%). In vaccinated children, serotypes not included in the active vaccines dominated: 15AF (17.4%), 23A (19.2%), as well as 11AD (19.6%) (11А is included in PPSV-23). The 27 Kz isolate (serotype 15C) belonged to one of the most common sequence types ST1025. The 105_Kz isolate (serotype 11D) belonged to another common sequence type ST 62.&#x0D; Conclusion. In order to improve epidemiological surveillance of pneumococcal infection, it is necessary to introduce the monitoring of circulating clonal complexes of dominant S. pneumoniae serogroups and analyze the genetic determinants of antibiotic resistance and virulence depending on the sequence type.

Genetic determinants of thermal resistance in foodborne bacterial pathogens

AbstractThermal food processing stands as the conventional and prevalent intervention utilized by food industries to uphold food safety standards. Some bacteria are known to withstand thermal treatments up to definite extents. This ability primarily stems from genetically determined traits that can be influenced by environmental conditions. This review focuses on the genetic determinants of thermal resistance in foodborne pathogens including the locus of heat resistance (LHR). Genes contributing to the expression of proteins that might indirectly contribute to thermal resistance have also been covered. The emergence of thermal tolerance in these pathogens raises significant food safety concerns, given global worries about bacterial resistance. The review consolidates existing research on genetic components and loci of heat resistance and highlights potential areas of gaps that warrant further research. Furthermore, acquired cross‐protection leading to resistance across multiple interventions has also been highlighted as an emerging concern. Acquiring insights into the environmental conditions that promote the selection of the LHR will empower the adoption of more efficient strategies to obstruct the pathways by which these pathogens persist within processing chains.

Phenotypic and genetic characterization of antimicrobial resistance in Salmonella enterica serovar Choleraesuis isolates from humans and animals in Spain from 2006 to 2021.

While an increase in the levels of MDR in Salmonella enterica sevorar Choleraesuis has been reported in Europe, little is known about the situation in Spain. Therefore, we first aimed to assess the phenotypic resistance profile and to determine the presence of genetic determinants of resistance of S. Choleraesuis isolates collected in animal and human. Our second objective was to identify and characterize clusters of highly related isolates. We analysed 50 human and 45 animal isolates retrieved from 2006 to 2021 using the disc diffusion method and performed WGS followed by analyses of genetic determinants and phylogenetic analysis. All isolates were of ST145 and corresponded to the variant Kunzendorf. Swine isolates harboured a significantly higher number of antimicrobial resistance genes than human isolates, and often carried plasmid replicons of the IncHI2/IncHI2A type (42% of all animal isolates). In addition, we identified several MDR S. Choleraesuis strains circulating in humans and swine between 2006 and 2021. The phylogenetic analyses identified four clades associated with specific patterns of resistance genes and plasmid replicons. The clades also included isolates that differed in terms of year and region of isolation as well as host of origin. This One Health approach highlights that reducing human MDR S. Choleraesuis infections may require the adoption of strategies that not only seek to prevent cases in humans but also to characterize and reduce the infection burden in swine.

Anthropogenic contamination sources drive differences in antimicrobial-resistant Escherichia coli in three urban lakes.

Antimicrobial resistance (AMR) is an ever-present threat to the treatment of infectious diseases. However, the potential relevance of this phenomenon in environmental reservoirs still raises many questions. Detection of antimicrobial-resistant bacteria in the environment is a critical aspect for understanding the prevalence of resistance outside of clinical settings, as detection in the environment indicates that resistance is likely already widespread. We isolated antimicrobial-resistant Escherichia coli from three urban waterbodies over a 15-month time series, determined their antimicrobial susceptibilities, investigated their population structure, and identified genetic determinants of resistance. We found that E. coli populations at each site were composed of different dominant phylotypes and showed distinct patterns of antimicrobial and multidrug resistance, despite close geographic proximity. Many strains that were genome-sequenced belonged to sequence types of international concern, particularly the ST131 clonal complex. We found widespread resistance to clinically important antimicrobials such as amoxicillin, cefotaxime, and ciprofloxacin, but found that all strains were susceptible to amikacin and the last-line antimicrobials meropenem and fosfomycin. Resistance was most often due to acquirable antimicrobial resistance genes, while chromosomal mutations in gyrA, parC, and parE conferred resistance to quinolones. Whole-genome analysis of a subset of strains further revealed the diversity of the population of E. coli present, with a wide array of AMR and virulence genes identified, many of which were present on the chromosome, including blaCTX-M. Finally, we determined that environmental persistence, transmission between sites, most likely mediated by wild birds, and transfer of mobile genetic elements likely contributed significantly to the patterns observed.IMPORTANCEA One Health perspective is crucial to understand the extent of antimicrobial resistance (AMR) globally, and investigation of AMR in the environment has been increasing in recent years. However, most studies have focused on waterways that are directly polluted by sewage, industrial manufacturing, or agricultural activities. Therefore, there remains a lack of knowledge about more natural, less overtly impacted environments. Through phenotypic and genotypic investigation of AMR in Escherichia coli, this study adds to our understanding of the extent and patterns of resistance in these types of environments, including over a time series, and showed that complex biotic and abiotic factors contribute to the patterns observed. Our study further emphasizes the importance of incorporating the surveillance of microbes in freshwater environments in order to better comprehend potential risks for both human and animal health and how the environment may serve as a sentinel for potential future clinical infections.

Identification of genetic determinants of bedaquiline resistance in Mycobacterium tuberculosis in Ural region, Russia.

Bedaquiline (BDQ) is a new anti-tuberculosis drug. The phenotypic and genotypic data describing the mechanism of drug resistance are critical for the design of rapid and accurate diagnostic tests. We consider that our work, which describes genotypic and phenotypic resistance to BDQ, can contribute to the standardization of drug susceptibility testing.

Microbial associations for pneumonia causative agents and level of their resistance to antimicrobial drugs during a new coronavirus infection pandemic

Introduction. Bacterial coinfection and secondary bacterial infection are considered critical risk factors for the severity and mortality of SARS-CoV-2-caused pneumonia. The aim of the study was to analyze a pattern of microbial associations between K. pneumoniae and A. baumannii isolated from the lower respiratory tract discharge and sectional material (lung tissue) of patients diagnosed with pneumonia, and to compare resistance level in monoculture and associations during new coronavirus infection pandemic. Materials and methods. A bacteriological study of 2689 sputum and bronchial washing samples from patients at infectious diseases hospitals, and 1411 lung pathological material samples was carried out. Bacterial isolates were identified by mass spectrometry. Antibiotic sensitivity for isolates was determined by the disk diffusion method. Genetic determinants of resistance to beta-lactam antibiotics were detected by PCR. Statistical data processing was performed using SPSS version 22 software. Results. K. pneumoniae and A. baumannii isolates were predominantly found in two- and three-pathogen associations. It was established that the resistance level of K. pneumoniae isolates in association with A. baumannii is significantly higher compared to that in monoculture for all antimicrobial drugs studied. At the same time, K. pneumoniae in combination with Candida spp. vs monoculture showed significantly lower level of resistance to ciprofloxacin, amikacin, cefotaxime, ceftazidime and amoxicillin/clavulanic acid. K. pneumoniae isolates carried resistance determinants to extended-spectrum beta-lactamases: OXA-48 — (22.5%), OXA-51 — (5.6%), OXA-23 — (4.2%), KPC — 70.9%, NDM — 7%. Of these, 14.1% of strains had the ability to co-produce serine carbapenemases OXA-48 and KPC. Sputum and lung tissue A. baumannii isolates exhibited extremely high multiple resistance regardless of their associations with other microorganisms. Microbiome species similarity in the lower respiratory tract and lung tissue discharge was revealed. The proportion of lung tissue vs sputum resistant strains of K. pneumoniae and A. baumannii was significantly higher. Conclusion. The detection of of multiple drug resistant K. pneumoniae and A. baumannii isolates as well as their associations may indicate aggravated pneumonia severity.

Whole-genome long-read sequencing to unveil Enterococcus antimicrobial resistance in dairy cattle farms exposed a widespread occurrence of Enterococcus lactis.

Enterococcus faecalis (Efs) and Enterococcus faecium (Efm) are major causes of multiresistant healthcare-associated or nosocomial infections. Efm has been traditionally divided into clades A (healthcare associated) and B (community associated) but clade B has been recently reassigned to Enterococcus lactis (Elc). However, identification techniques do not routinely differentiate Elc from Efm. As part of a longitudinal study to investigate the antimicrobial resistance of Enterococcus in dairy cattle, isolates initially identified as Efm were confirmed as Elc after Oxford-Nanopore long-fragment whole-genome sequencing and genome comparisons. An Efm-specific PCR assay was developed and used to identify isolates recovered from animal feces on five farms, resulting in 44 Efs, 23 Efm, and 59 Elc. Resistance, determined by broth microdilution, was more frequent in Efs than in Efm and Elc but all isolates were susceptible to ampicillin, daptomycin, teicoplanin, tigecycline, and vancomycin. Genome sequencing analysis of 32 isolates identified 23 antimicrobial resistance genes (ARGs, mostly plasmid-located) and 2 single nucleotide polymorphisms associated with resistance to 10 antimicrobial classes, showing high concordance with phenotypic resistance. Notably, linezolid resistance in Efm was encoded by the optrA gene, located in plasmids downstream of the fexA gene. Although most Elc lacked virulence factors and genetic determinants of resistance, one isolate carried a plasmid with eight ARGs. This study showed that Elc is more prevalent than Efm in dairy cattle but carries fewer ARGs and virulence genes. However, Elc can carry multi-drug-resistant plasmids like those harbored by Efm and could act as a donor of ARGs for other pathogenic enterococcal species.IMPORTANCEEnterococcus species identification is crucial due to differences in pathogenicity and antibiotic resistance profiles. The failure of traditional methods or whole-genome sequencing-based taxonomic classifiers to distinguish Enterococcus lactis (Elc) from Enterococcus faecium (Efm) results in a biased interpretation of Efm epidemiology. The Efm species-specific real-time PCR assay developed here will help to properly identify Efm (only the formerly known clade A) in future studies. Here, we showed that Elc is prevalent in dairy cattle, and although this species carries fewer genetic determinants of resistance (GDRs) than Enterococcus faecalis (Efs) and Efm, it can carry multi-drug-resistant (MDR) plasmids and could act as a donor of resistance genes for other pathogenic enterococcal species. Although all isolates (Efs, Efm, and Elc) were susceptible to critically or highly important antibiotics like daptomycin, teicoplanin, tigecycline, and vancomycin, the presence of GDRs in MDR-plasmids is a concern since antimicrobials commonly used in livestock could co-select and confer resistance to critically important antimicrobials not used in food-producing animals.

Whole Genome Sequences, De Novo Assembly, and Annotation of Antibiotic Resistant Campylobacter jejuni Strains S27, S33, and S36 Newly Isolated from Chicken Meat.

Campylobacter is a leading bacterial cause of gastrointestinal infections in humans and has imposed substantial medical and public health burdens worldwide. Among a total of 39 species in the Campylobacter genus, C. jejuni is the most important species responsible for approx. 90% of human Campylobacter illness. Most cases of the infection were acquired by ingesting undercooked poultry meat due to the high prevalence of Campylobacter in the products. Here, we reported the dataset of raw sequences, de novo assembled and annotated genomes of C. jejuni strains S27, S33, and S36 recently isolated from retail chicken by using PacBio highly accurate long-read sequencing technology combined with bioinformatics tools. Our data revealed several virulence and antibiotic resistance genes in each of the chromosomes, a type IV secretion system in the plasmid (pCjS33) of C. jejuni S33, and a type VI secretion system and a phage in the plasmid (pCjS36) of C. jejuni S36. This study not only provides new sequence data but also extends the knowledge pertaining to the genomic and functional aspects of this important foodborne pathogen, including the genetic determinants of virulence and antibiotic resistance.

Quantitative measurement of antibiotic resistance in Mycobacterium tuberculosis reveals genetic determinants of resistance and susceptibility in a target gene approach

The World Health Organization has a goal of universal drug susceptibility testing for patients with tuberculosis. However, molecular diagnostics to date have focused largely on first-line drugs and predicting susceptibilities in a binary manner (classifying strains as either susceptible or resistant). Here, we used a multivariable linear mixed model alongside whole genome sequencing and a quantitative microtiter plate assay to relate genomic mutations to minimum inhibitory concentration (MIC) in 15,211 Mycobacterium tuberculosis clinical isolates from 23 countries across five continents. We identified 492 unique MIC-elevating variants across 13 drugs, as well as 91 mutations likely linked to hypersensitivity. Our results advance genetics-based diagnostics for tuberculosis and serve as a curated training/testing dataset for development of drug resistance prediction algorithms.

Antibiotic resistance determination using Enterococcus faecium whole-genome sequences: a diagnostic accuracy study using genotypic and phenotypic data

DNA sequencing could become an alternative to invitro antibiotic susceptibility testing (AST) methods for determining antibiotic resistance by detecting genetic determinants associated with decreased antibiotic susceptibility. Here, we aimed to assess and improve the accuracy of antibiotic resistance determination from Enterococcus faecium genomes for diagnosis and surveillance purposes. In this retrospective diagnostic accuracy study, we first conducted a literature search in PubMed on Jan14,2021, to compile a catalogue of genes and mutations predictive of antibiotic resistance in E faecium. We then evaluated the diagnostic accuracy of this database to determine susceptibility to 12 different, clinically relevant antibiotics using a diverse population of 4382 E faecium isolates with available whole-genome sequences and invitro culture-based AST phenotypes. Isolates were obtained from various sources in 11 countries worldwide between 2000 and 2018. We included isolates tested with broth microdilution, Vitek 2, and disc diffusion, and antibiotics with at least 50 susceptible and 50 resistant isolates. Phenotypic resistance was derived from raw minimum inhibitory concentrations and measured inhibition diameters, and harmonised primarily using the breakpoints set by the European Committee on Antimicrobial Susceptibility Testing. A bioinformatics pipeline was developed to process raw sequencing reads, identify antibiotic resistance genetic determinants, and report genotypic resistance. We used our curated database, as well as ResFinder, AMRFinderPlus, and LRE-Finder, to assess the accuracy of genotypic predictions against phenotypic resistance. We curated a catalogue of 228 genetic markers involved in resistance to 12 antibiotics in E faecium. Very accurate genotypic predictions were obtained for ampicillin (sensitivity 99·7% [95% CI 99·5-99·9] and specificity 97·9% [95·8-99·0]), ciprofloxacin (98·0% [96·4-98·9] and 98·8% [95·9-99·7]), vancomycin (98·8% [98·3-99·2] and 98·8% [98·0-99·3]), andlinezolid resistance (after re-testing false negatives: 100·0% [90·8-100·0] and 98·3% [97·8-98·7]). High sensitivity was obtained for tetracycline (99·5% [99·1-99·7]), teicoplanin (98·9% [98·4-99·3]), and high-level resistance to aminoglycosides (97·7% [96·6-98·4] for streptomycin and 96·8% [95·8-97·5] for gentamicin), although at lower specificity (60-90%). Sensitivity was expectedly low for daptomycin (73·6% [65·1-80·6]) and tigecycline (38·3% [27·1-51·0]), for which the genetic basis of resistance is not fully characterised. Compared with other antibiotic resistance databases and bioinformatic tools, our curated database was similarly accurate at detecting resistance to ciprofloxacin and linezolid and high-level resistance to streptomycin and gentamicin, but had better sensitivity for detecting resistance to ampicillin, tigecycline, daptomycin, and quinupristin-dalfopristin, and better specificity for ampicillin, vancomycin, teicoplanin, and tetracycline resistance. In a validation dataset of 382 isolates, similar or improved diagnostic accuracies were also achieved. To our knowledge, this work represents the largest published evaluation to date of the accuracy of antibiotic susceptibility predictions from E faecium genomes. The results and resources will facilitate the adoption of whole-genome sequencing as a tool for the diagnosis and surveillance of antimicrobial resistance in E faecium. A complete characterisation of the genetic basis of resistance to last-line antibiotics, and the mechanisms mediating antibiotic resistance silencing, are needed to close the remaining sensitivity and specificity gaps in genotypic predictions. Wellcome Trust, UK Department of Health, British Society for Antimicrobial Chemotherapy, Academy of Medical Sciences and the Health Foundation, Medical Research Council Newton Fund, Vietnamese Ministry of Science and Technology, and European Society of Clinical Microbiology and Infectious Disease.

Genomic investigation unveils high-risk ESBL producing Enterobacteriaceae within a rural environmental water body

Antimicrobial resistance is regarded as a global threat to public health, animals, and the environment, emerging in response to extensive utilization of antimicrobials. The determinants of antimicrobial resistance are transported to susceptible bacterial populations through genetic recombination or through gene transfer, mediated by bacteriophages, plasmids, transposons, and insertion sequences. To determine the penetration of antimicrobial resistance into the bacterial population of the Thiruvandarkoil Lake, a water body located in the rural settings of Puducherry, India, culture-based microbiological and genomic approaches were used. Resistant bacterial isolates obtained from microbiological screening were subjected to whole genome sequencing and the genetic determinants of antimicrobial resistance were identified using in silico genomic tools. Cephalosporin-resistant isolates were found to produce extended spectrum beta lactamases, encoded by blaVEB-6 (in Proteus mirabilis PS01), blaSHV-12 and ompK36 mutation (in Klebsiella quasipneumoniae PS02) and blaSHV-12, blaACT-16, blaCTX-M and blaNDM-1 in (Enterobacter hormaechei PS03). Genes encoding heavy metal resistance, virulence and resistance to detergents were also detected in these resistant isolates. Among ESBL-producing organisms, one mcr-9-positive Enterobacter hormaechei was also identified in this study. To our knowledge, this is the first report of mcr-9 carrying bacterium in the environment in India. This study seeks the immediate attention of policy makers, researchers, government officials and environmental activists in India, to develop surveillance programs to monitor the dissemination of antimicrobial resistance in the environment.