Unique Resistance Genes Research Articles

A landscape of resistance gene analogs in sour cherry (Prunus cerasus L.)

ObjectiveThis research aims to analyze the presence and distribution of resistance genes in the avium and fruticosa subgenomes of Prunus cerasus through computational methods and bioinformatics tools.ResultsAnalysis of genome and transcriptome sequencing data revealed a total of 19,570 transcripts with at least one resistance gene domain in Prunus cerasus subgenome avium and 19,142 in Prunus cerasus subgenome fruticosa. Key findings include the identification of 804 “complete” resistance gene transcripts in Prunus cerasus subgenome avium and 817 in Prunus cerasus subgenome fruticosa, with distinct distributions of resistance gene classes observed between the subgenomes. Phylogenetic analysis showed clustering of resistance genes, and unique resistance proteins were identified in each subgenome. Functional annotation comparisons with Arabidopsis thaliana highlighted shared and unique resistance genes, emphasizing the complexity of disease resistance in cherry species. Additionally, a higher diversity of RLKs and RLPs was observed, with 504 transcripts identified and 18 showing similarity to known reference genes.

Unveiling the genomic diversity and ecological impact of phage communities in hospital wastewater

Phages are pivotal in shaping microbial communities and biogeochemical cycles, while our understanding of the diversity, functions potential, and resistance gene carriage of phages in hospital wastewater (HWW) remains limited. We collected influent and effluent samples from the 3 hospital wastewater treatment plants (HWTPs) to assess the diversity and fate of phages, the interactions between phages and hosts, and the presence of resistance genes and auxiliary metabolic genes (AMGs) encoded by phages. Compared to influent, effluent showed reduced phage abundance and altered composition, with decreases in Microviridae and Inoviridae. The gene-sharing network highlights that many phages in HWW are not classified in known viral genera, suggesting HWW as a rich source of new viruses. There was a significant association between phages and microorganisms, with approximately 32.57 % of phages expected to be capable of infecting microbial hosts, characterized primarily by lytic activity. A total of 8 unique antibiotic resistance genes, 13 unique metal resistance genes, and 5 mobile genetic elements were detected in 3 HWTPs phageomes. Phage AMGs have the potential to influence carbon, nitrogen, phosphorus, and sulfur metabolism, impacting biogeochemical cycles. This study reveals the genomic diversity and ecological role of phages in HWTPs, highlighting their environmental and ecosystem impact.

The effects of antibiotic exposures on the gut resistome during hematopoietic cell transplantation in children

ABSTRACT Antibiotic resistance is a global threat driven primarily by antibiotic use. We evaluated the effects of antibiotic exposures on the gut microbiomes and resistomes of children at high risk of colonization by antibiotic-resistant bacteria. We performed shotgun metagenomic sequencing of 691 serially collected fecal samples from 80 children (<18 years) undergoing hematopoietic cell transplantation. We evaluated the effects of aerobic (cefepime, vancomycin, fluoroquinolones, aminoglycosides, macrolides, and trimethoprim-sulfamethoxazole) and anaerobic (piperacillin-tazobactam, carbapenems, metronidazole, and clindamycin) antibiotic exposures on the diversity and composition of the gut microbiome and resistome. We identified 372 unique antibiotic resistance genes (ARGs); the most frequent ARGs identified encode resistance to tetracyclines (n = 88), beta-lactams (n = 84), and fluoroquinolones (n = 79). Both aerobic and anaerobic antibiotic exposures were associated with a decrease in the number of bacterial species (aerobic, β = 0.71, 95% CI: 0.64, 0.79; anaerobic, β = 0.66, 95% CI: 0.53, 0.82) and the number of unique ARGs (aerobic, β = 0.81, 95% CI: 0.74, 0.90; anaerobic, β = 0.73, 95% CI: 0.61, 0.88) within the gut metagenome. However, only antibiotic regimens that included anaerobic activity were associated with an increase in acquisition of new ARGs (anaerobic, β = 1.50; 95% CI: 1.12, 2.01) and an increase in the relative abundance of ARGs in the gut resistome (anaerobic, β = 1.62; 95% CI: 1.15, 2.27). Specific antibiotic exposures were associated with distinct changes in the number and abundance of ARGs for individual antibiotic classes. Our findings detail the impact of antibiotics on the gut microbiome and resistome and demonstrate that anaerobic antibiotics are particularly likely to promote acquisition and expansion of antibiotic-resistant bacteria.

Airway "Resistotypes" and Clinical Outcomes in Bronchiectasis.

Rationale: Chronic infection and inflammation shapes the airway microbiome in bronchiectasis. Utilizing whole-genome shotgun metagenomics to analyze the airway resistome provides insight into interplay between microbes, resistance genes, and clinical outcomes. Objectives: To apply whole-genome shotgun metagenomics to the airway microbiome in bronchiectasis to highlight a diverse pool of antimicrobial resistance genes: the "resistome," the clinical significance of which remains unclear. Methods: Individuals with bronchiectasis were prospectively recruited into cross-sectional and longitudinal cohorts (n = 280), including the international multicenter cross-sectional Cohort of Asian and Matched European Bronchiectasis 2 (CAMEB 2) study (n = 251) and two independent cohorts, one describing patients experiencing acute exacerbation and a further cohort of patients undergoing Pseudomonas aeruginosa eradication treatment. Sputum was subjected to metagenomic sequencing, and the bronchiectasis resistome was evaluated in association with clinical outcomes and underlying host microbiomes. Measurements and Main Results: The bronchiectasis resistome features a unique resistance gene profile and increased counts of aminoglycoside, bicyclomycin, phenicol, triclosan, and multidrug resistance genes. Longitudinally, it exhibits within-patient stability over time and during exacerbations despite between-patient heterogeneity. Proportional differences in baseline resistome profiles, including increased macrolide and multidrug resistance genes, associate with shorter intervals to the next exacerbation, whereas distinct resistome archetypes associate with frequent exacerbations, poorer lung function, geographic origin, and the host microbiome. Unsupervised analysis of resistome profiles identified two clinically relevant "resistotypes," RT1 and RT2, the latter characterized by poor clinical outcomes, increased multidrug resistance, and P. aeruginosa. Successful targeted eradication in P. aeruginosa-colonized individuals mediated reversion from RT2 to RT1, a more clinically favorable resistome profile demonstrating reduced resistance gene diversity. Conclusions: The bronchiectasis resistome associates with clinical outcomes, geographic origin, and the underlying host microbiome. Bronchiectasis resistotypes link to clinical disease and are modifiable through targeted antimicrobial therapy.

Identification and specificity validation of unique and antimicrobial resistance genes to trace suspected pathogenic AMR bacteria and to monitor the development of AMR in non-AMR strains in the environment and clinical settings

The detection of developing antimicrobial resistance (AMR) has become a global issue. The detection of developing antimicrobial resistance has become a global issue. The growing number of AMR bacteria poses a new threat to public health. Therefore, a less laborious and quick confirmatory test becomes important for further investigations into developing AMR in the environment and in clinical settings. This study aims to present a comprehensive analysis and validation of unique and antimicrobial-resistant strains from the WHO priority list of antimicrobial-resistant bacteria and previously reported AMR strains such as Acinetobacter baumannii, Aeromonas spp., Anaeromonas frigoriresistens, Anaeromonas gelatinfytica, Bacillus spp., Campylobacter jejuni subsp. jejuni, Enterococcus faecalis, Escherichia coli, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumonia subsp. pneumoniae, Pseudomonas aeruginosa, Salmonella enterica subsp. enterica serovar Typhimurium, Thermanaeromonas toyohensis, and Vibrio proteolyticus. Using in-house designed gene-specific primers, 18 different antibiotic resistance genes (algJ, alpB, AQU-1, CEPH-A3, ciaB, CMY-1-MOX-7, CMY-1-MOX-9, CMY-1/MOX, cphA2, cphA5, cphA7, ebpA, ECP_4655, fliC, OXA-51, RfbU, ThiU2, and tolB) from 46 strains were selected and validated. Hence, this study provides insight into the identification of strain-specific, unique antimicrobial resistance genes. Targeted amplification and verification using selected unique marker genes have been reported. Thus, the present detection and validation use a robust method for the entire experiment. Results also highlight the presence ofanother set of 18 antibiotic-resistant and unique genes (Aqu1, cphA2, cphA3, cphA5, cphA7, cmy1/mox7, cmy1/mox9, asaI, ascV, asoB, oxa-12, acr-2, pepA, uo65, pliI, dr0274, tapY2, and cpeT). Of these sets of genes, 15 were found to be suitable for the detection of pathogenic strains belonging to the genera Aeromonas, Pseudomonas, Helicobacter, Campylobacter, Enterococcus, Klebsiella, Acinetobacter, Salmonella, Haemophilus, and Bacillus. Thus, we have detected and verified sets of unique and antimicrobial resistance genes in bacteria on the WHO Priority List and from published reports on AMR bacteria. This study offers advantages for confirming antimicrobial resistance in all suspected AMR bacteria and monitoring the development of AMR in non-AMR bacteria, in the environment, and in clinical settings.

Prevalence and antimicrobial resistance profiles of Vibrio spp. and Enterococcus spp. in retail shrimp in Northern California.

Shrimp is one of the most consumed seafood products globally. Antimicrobial drugs play an integral role in disease mitigation in aquaculture settings, but their prevalent use raises public health concerns on the emergence and spread of antimicrobial resistant microorganisms. Vibrio spp., as the most common causative agents of seafood-borne infections in humans, and Enterococcus spp., as an indicator organism, are focal bacteria of interest for the monitoring of antimicrobial resistance (AMR) in seafood. In this study, 400 samples of retail shrimp were collected from randomly selected grocery stores in the Greater Sacramento, California, area between September 2019 and June 2020. The prevalence of Vibrio spp. and Enterococcus spp. was 60.25% (241/400) and 89.75% (359/400), respectively. Subsamples of Vibrio (n = 110) and Enterococcus (n = 110) isolates were subjected to antimicrobial susceptibility testing (AST). Vibrio isolates had high phenotypic resistance to ampicillin (52/110, 47.27%) and cefoxitin (39/110, 35.45%). Enterococcus were most frequently resistant to lincomycin (106/110, 96.36%), quinupristin-dalfopristin (96/110, 87.27%), ciprofloxacin (93/110, 84.55%), linezolid (86/110, 78.18%), and erythromycin (58/110, 52.73%). For both Vibrio and Enterococcus, no significant associations were observed between multidrug resistance (MDR, resistance to ≥3 drug classes) in isolates from farm raised and wild caught shrimp (p > 0.05) and in isolates of domestic and imported origin (p > 0.05). Whole genome sequencing (WGS) of a subset of Vibrio isolates (n = 42) speciated isolates as primarily V. metschnikovii (24/42; 57.14%) and V. parahaemolyticus (12/42; 28.57%), and detected 27 unique antimicrobial resistance genes (ARGs) across these isolates, most commonly qnrVC6 (19.05%, 8/42), dfrA31 (11.90%, 5/42), dfrA6 (9.5%, 4/42), qnrVC1 (9.5%, 4/42). Additionally, WGS predicted phenotypic resistance in Vibrio isolates with an overall sensitivity of 11.54% and specificity of 96.05%. This study provides insights on the prevalence and distribution of AMR in Vibrio spp. and Enterococcus spp. from retail shrimp in California which are important for food safety and public health and exemplifies the value of surveillance in monitoring the spread of AMR and its genetic determinants.

Aeromonas nosocomial cluster: Investigation review of possible modes of transmission

Background:Aeromonas is a gram-negative rod known to be present in water, sewage and soil which may cause infections especially in immunocompromised hosts. Cases of Aeromonas gastroenteritis have been associated with warmer weather. In total, 3 patients with extensively drug resistant (XDR) Aeromonas were identified at our facility between August and September 2022 on 2 intensive care units (ICUs). Our infection prevention, microbiology, and facility teams investigated these cases to determine whether a common source could be the mode of transmission. Methods: To first determine whether patients’ Aeromonas specimens were related, whole-genome sequencing (WGS) of the clinical isolates from 3 patients was performed using the Illumina DNA Prep Kit and Illumina MiSeq. Sequencing analysis was performed using CLC Genomics Workbench for de novo assembly, single-nucleotide polymorphisms (SNP) calling, and tree generation, Geneious Prime for reference-based assembly, annotation, and quality assessment, KmerFinder for reference identification, and the Comprehensive Antibiotic Resistance Database for resistance gene detection via protein homology. Chart review revealed that patients occupied multiple rooms between 2 ICUs (Fig. 1). Because water is a known source of Aeromonas, facility records were reviewed for water intrusion events. This analysis identified several cases in the affected patient and surrounding rooms. Sinks and faucets from 10 rooms were swabbed followed by direct plating on blood, MacConkey agar, and Aeromonas-selective cefsulodin-Irgasan-novobiocin (CIN) agar plates. Lastly, the city temperatures before and after positive cases were reviewed to identify whether any correlation could be shown between temperature and timing of infection. Results: WGS analysis revealed that the 3 Aeromonas isolates (all identified as A. hydrophila) were not directly related (minimum distance, 934 SNPs) and harbored between 4 and 19 unique antimicrobial resistance genes, including co-occurring carbapenemases VIM-2 and KPC-3 in 1 isolate and OXA-232 in another. Of the 20 environmental samples, few gram-negative nonfermenting bacteria and no Aeromonas isolates were detected (Fig. 1). Elevated city did loosely proceed patient cases of Aeromonas, suggesting a possible role of higher temperature, which may have promoted the growth of Aeromonas during the periods of the 3 cases and thus may contribute to the nosocomial infections (Fig. 2). Conclusions: Although our investigation did not reveal a definitive cause for the Aeromonas cases, it did show the importance prompt identification and investigation can have on mitigating the spread of a cluster. Our facility has not identified any additional nosocomial cases. Monitoring water intrusion events and plans for remediation continue to be a priority.Disclosures: None

A Core Genome Multilocus Sequence Typing Scheme for Proteus mirabilis

ObjectiveA core genome multilocus sequence typing (cgMLST) scheme to genotype and identify potential risk clonal groups (CGs) in Proteus mirabilis. MethodsIn this work, we propose a publicly available cgMLST scheme for P. mirabilis using chewBBACA. In total 72 complete P. mirabilis genomes, representing the diversity of this species, were used to set up a cgMLST scheme targeting 1,842 genes, 635 unfinished (contig, chromosome, and scaffold) genomes were used for its validation. ResultsWe identified a total of 205 CGs from 695 P. mirabilis strains with regional distribution characteristics. Of these, 159 unique CGs were distributed in 16 countries. CG20 and CG3 carried large numbers of shared and unique antibiotic resistance genes. Nine virulence genes (papC, papD, papE, papF, papG, papH, papI, papJ, and papK) related to the P fimbrial operon that cause severe urinary tract infections were only found in CG20. These CGs require attention due to potential risks. ConclusionThis research innovatively performs high-resolution molecular typing of P. mirabilis using whole-genome sequencing technology combined with a bioinformatics pipeline (chewBBACA). We found that the CGs of P. mirabilis showed regional distribution differences. We expect that our research will contribute to the establishment of cgMLST for P. mirabilis.

Prevalence and heterogeneity of antibiotic resistance genes in Orientia tsutsugamushi and other rickettsial genomes

Despite a million infections every year and an estimated one billion people at risk, scrub typhus is regarded as a neglected tropical disease. The causative bacterium Orientia tsutsugamushi, a member of rickettsiae, seems to be intrinsically resistant to several classes of antibiotics. The emergence of antibiotic-resistant scrub typhus is likely to become a global public health concern. Yet, it is unknown as to how common antibiotic resistance genes are in O. tsutsugamushi, and how variable these loci are among the genomes of rickettsiae. By using the comprehensive antibiotic resistance database, we explored 79 complete genomes from 24 species of rickettsiae for antibiotic resistance loci. There were 244 unique antibiotic resistance genes in rickettsiae. Both the total and unique antibiotic resistance genes in O. tsutsugamushi were significantly less compared to other members of rickettsiae. However, antibiotic resistance genes in O. tsutsugamushi genomes were more unique and highly variable. Many genes such as resistant variants of evgS, and vanS A/G were present in numerous copies. These results will have important implications in the context of antibiotic-resistant scrub typhus.

137. Comparison of genotypic resistance pattern between Enterococcus faecium and Enterococcus faecalis to better understand the occurrence of mutations.

Abstract Background Enterococcus is one of the major causes of nosocomial infections many with vancomycin resistance. About 50,000 infections are reported per year due to vancomycin-resistant Enterococci. Understanding the genotypic mutations can help recognize the potential burden for antibiotic resistance that exists in a population. In this study, using whole genome sequencing (WGS) we aim to analyze the prevalence of genetic resistance markers for commonly used antibiotics for Enterococcal treatment. Methods Whole genome sequencing was performed using the NextSeq (Illumina Inc., CA) on 60 isolates of E. faecium and 29 isolates of E. faecalis. The isolates were obtained from two different Detroit area hospitals from 2017-2019. The data from WGS was analyzed using EPISEQ CSTM (BIOMÉRIEUX, Marcy l ‘Etoile, France) bioinformatic database to obtain specific resistance genes which would correspond to commonly used antibiotics for treatment of Enterococcal infections. Results Among the 89 Enterococcus faecium and Enterococcus faecalis investigated, we identified a total of 33 unique resistance genes across 18 classes of antibiotics (Table 1). We detected genetic mutations efmA, pbp5, tet(L), liar, liaS unique to E. faecium and only emeA unique to E. faecalis. E. faecium and E. faecalis share 14 similar resistance genes. E. faecium and E. faecalis had 12 and 7 unique resistance genes, respectively but are of same drug classes. The most common antibiotic classes include aminoglycosides, tetracyclines, quinolones, beta-lactams, glycopeptides, macrolides, and pyrimidine analogs. Table 1:Enterococcus faecium and E. faecalis genetic mutations and drug class resistance Conclusion Enterococcus faecium had a greater number of resistance genes with glycopeptide, aminoglycoside, and beta lactam resistance when compared to Enterococcus faecalis. Whereas Enterococcus faecalis had more resistance genes associated with macrolides, tetracyclines, pyrimidine analogs, and Bactrim. In general, both appear to have many genes that encode for resistance to major classes of antibiotics which are used for treatment of Enterococcal infections. Further comparison with phenotypic susceptibility testing data would help better understand the practical implications of our resistance gene testing. Disclosures Piyali Chatterjee, PhD, AHRQ Grant # 1R03HS027667-01: Grant/Research Support|AHRQ Grant # 1R03HS027667-01: Central Texas Veterans Health Care System Keith S. Kaye, MD, MPH, Allecra: Advisor/Consultant|GlaxoSmithKline plc.: Receiving symposia honoraria|GlaxoSmithKline plc.: GlaxoSmithKline plc.-sponsored study 212502|Merck: Advisor/Consultant|qpex: Advisor/Consultant|Shionogi: Grant/Research Support|Spero: Advisor/Consultant Chetan Jinadatha, MD, MPH, AHRQ R01 Grant-5R01HS025598: Grant/Research Support|EOS Surfaces: Copper Coupons and materials for testing.

Antarctic Sphingomonas sp. So64.6b showed evolutive divergence within its genus, including new biosynthetic gene clusters.

The antibiotic crisis is a major human health problem. Bioprospecting screenings suggest that proteobacteria and other extremophile microorganisms have biosynthetic potential for the production novel antimicrobial compounds. An Antarctic Sphingomonas strain (So64.6b) previously showed interesting antibiotic activity and elicitation response, then a relationship between environmental adaptations and its biosynthetic potential was hypothesized. We aimed to determine the genomic characteristics in So64.6b strain related to evolutive traits for the adaptation to the Antarctic environment that could lead to its diversity of potentially novel antibiotic metabolites. The complete genome sequence of the Antarctic strain was obtained and mined for Biosynthetic Gene Clusters (BGCs) and other unique genes related to adaptation to extreme environments. Comparative genome analysis based on multi-locus phylogenomics, BGC phylogeny, and pangenomics were conducted within the closest genus, aiming to determine the taxonomic affiliation and differential characteristics of the Antarctic strain. The Antarctic strain So64.6b showed a closest identity with Sphingomonas alpina, however containing a significant genomic difference of ortholog cluster related to degradation multiple pollutants. Strain So64.6b had a total of six BGC, which were predicted with low to no similarity with other reported clusters; three were associated with potential novel antibiotic compounds using ARTS tool. Phylogenetic and synteny analysis of a common BGC showed great diversity between Sphingomonas genus but grouping in clades according to similar isolation environments, suggesting an evolution of BGCs that could be linked to the specific ecosystems. Comparative genomic analysis also showed that Sphingomonas species isolated from extreme environments had the greatest number of predicted BGCs and a higher percentage of genetic content devoted to BGCs than the isolates from mesophilic environments. In addition, some extreme-exclusive clusters were found related to oxidative and thermal stress adaptations, while pangenome analysis showed unique resistance genes on the Antarctic strain included in genetic islands. Altogether, our results showed the unique genetic content on Antarctic strain Sphingomonas sp. So64.6, -a probable new species of this genetically divergent genus-, which could have potentially novel antibiotic compounds acquired to cope with Antarctic poly-extreme conditions.

Uncovering the diversity and contents of gene cassettes in class 1 integrons from the endophytes of raw vegetables

Rapid spread of antibiotic resistance genes (ARGs) in pathogens is threatening human health. Integrons allow bacteria to integrate and express foreign genes, facilitating horizontal transfer of ARGs in environments. Consumption of raw vegetables represents a pathway for human exposure to environmental ARGs. However, few studies have focused on integron-associated ARGs in the endophytes of raw vegetables. Here, based on the approach of qPCR and clone library, we quantified the abundance of integrase genes and analyzed the diversity and contents of resistance gene cassettes in class 1 integrons from the endophytes of six common raw vegetables. The results revealed that integrase genes for class 1 integron were most prevalent compared with class 2 and class 3 integron integrase genes (1–2 order magnitude, P < 0.05). The cucumber endophytes harbored a higher absolute abundance of integrase genes than other vegetables, while the highest bacterial abundance was detected in cabbage and cucumber endophytes. Thirty-two unique resistance gene cassettes were detected, the majority of which were associated with the genes encoding resistance to beta-lactam and aminoglycoside. Antibiotic resistance gene cassettes accounted for 52.5 % of the functionally annotated gene cassettes, and blaTEM-157 and aadA2 were the most frequently detected resistance cassettes. Additionally, carrot endophytes harbored the highest proportion of antibiotic resistance gene cassettes in the class 1 integrons. Collectively, these results provide an in-depth view of acquired resistance genes by integrons in the raw vegetable endophytes and highlight the potential health risk of the transmission of ARGs via the food chain.

Integrated omics approach to unveil antifungal bacterial polyynes as acetyl-CoA acetyltransferase inhibitors

Bacterial polyynes are highly active natural products with a broad spectrum of antimicrobial activities. However, their detailed mechanism of action remains unclear. By integrating comparative genomics, transcriptomics, functional genetics, and metabolomics analysis, we identified a unique polyyne resistance gene, masL (encoding acetyl-CoA acetyltransferase), in the biosynthesis gene cluster of antifungal polyynes (massilin A 1, massilin B 2, collimonin C 3, and collimonin D 4) of Massilia sp. YMA4. Crystallographic analysis indicated that bacterial polyynes serve as covalent inhibitors of acetyl-CoA acetyltransferase. Moreover, we confirmed that the bacterial polyynes disrupted cell membrane integrity and inhibited the cell viability of Candida albicans by targeting ERG10, the homolog of MasL. Thus, this study demonstrated that acetyl-CoA acetyltransferase is a potential target for developing antifungal agents.

Comparative genomics reveals the organic acid biosynthesis metabolic pathways among five lactic acid bacterial species isolated from fermented vegetables

Lactic acid bacteria (LAB) comprise a widespread bacterial group, inhabiting the niches of fermented vegetables and capable of producing beneficial organic acids. In the present study, several bioinformatics approaches were used to perform whole-genome sequencing and comparative genomics of five LAB species, Lactobacillus plantarum PC1–1, Pediococcus pentosaceus PC2–1(F2), Weissella hellenica PC1A, Lactobacillus buchneri PC-C1, and Enterococcus sp. YC2–6, to enhance understanding of their different genetic functionalities and organic acid biosynthesis. The results revealed major carbohydrate-active enzymes, putative operons and unique mobile genetic elements, including plasmids, resistance genes, insertion sequences and composite transposons involved in organic acid biosynthesis. The metabolic pathways of organic acid biosynthesis emphasize the key genes encoding specific enzymes required for organic acid metabolism. The five genomes were found to contain various regions of secondary metabolite biosynthetic gene clusters, including the type III polyketide synthases (T3PKS) enriched with unique genes encoding a hydroxymethylglutaryl-CoA synthase, capable of exhibiting specific antimicrobial activity with biopreservative potential, and a cyclic AMP receptor protein (CRP) transcription factor acting as a glucose sensor in organic acid biosynthesis. This could enable the organisms to prevail in the fermentation process, suggesting potential industrial applications.

Impact of long-term dietary habits on the human gut resistome in the Dutch population

The human gut microbiome plays a central role in health and disease. Environmental factors, such as lifestyle and diet, are known to shape the gut microbiome as well as the reservoir of resistance genes that these microbes harbour; the resistome. In this study we assessed whether long-term dietary habits within a single geographical region (the Netherlands) impact the human gut resistome. Faecal samples from Dutch omnivores, pescatarians, vegetarians and vegans were analysed by metagenomic shotgun sequencing (MSS) (n = 149) and resistome capture sequencing approach (ResCap) (n = 64). Among all diet groups, 119 and 145 unique antibiotic resistance genes (ARGs) were detected by MSS or ResCap, respectively. Five or fifteen ARGs were shared between all diet groups, based on MSS and ResCap, respectively. The total number of detected ARGs by MSS or ResCap was not significantly different between the groups. MSS also revealed that vegans have a distinct microbiome composition, compared to other diet groups. Vegans had a lower abundance of Streptococcus thermophilus and Lactococcus lactis compared to pescatarians and a lower abundance of S. thermophilus when compared to omnivores. In summary, our study showed that long-term dietary habits are not associated with a specific resistome signature.

Comparing gut resistome composition among patients with acute Campylobacter infections and healthy family members

Campylobacter commonly causes foodborne infections and antibiotic resistance is an imminent concern. It is not clear, however, if the human gut ‘resistome’ is affected by Campylobacter during infection. Application of shotgun metagenomics on stools from 26 cases with Campylobacter infections and 44 healthy family members (controls) identified 406 unique antibiotic resistance genes (ARGs) representing 153 genes/operons, 40 mechanisms, and 18 classes. Cases had greater ARG richness (p < 0.0001) and Shannon diversity (p < 0.0001) than controls with distinct compositions (p = 0.000999; PERMANOVA). Cases were defined by multidrug resistance genes and were dominated by Proteobacteria (40.8%), specifically those representing Escherichia (20.9%). Tetracycline resistance genes were most abundant in controls, which were dominated by Bacteroidetes (45.3%) and Firmicutes (44.4%). Hierarchical clustering of cases identified three clusters with distinct resistomes. Case clusters 1 and 3 differed from controls containing more urban and hospitalized patients. Relative to family members of the same household, ARG composition among matched cases was mostly distinct, though some familial controls had similar profiles that could be explained by a shorter time since exposure to the case. Together, these data indicate that Campylobacter infection is associated with an altered resistome composition and increased ARG diversity, raising concerns about the role of infection in the spread of resistance determinants.

Enrofloxacin Alters Fecal Microbiota and Resistome Irrespective of Its Dose in Calves.

Enrofloxacin is a fluoroquinolone drug used to prevent and control bovine respiratory disease (BRD) complex in multiple or single doses, ranging from 7.5 to 12.5 mg/kg body weight. Here, we examined the effects of high and low doses of a single subcutaneously injected enrofloxacin on gut microbiota and resistome in calves. Thirty-five calves sourced for this study were divided into five groups: control (n = 7), two low dose groups (n = 14, 7.5 mg/kg), and two high dose groups (n = 14, 12.5 mg/kg). One group in the low and high dose groups was challenged with Mannheimia haemolytica to induce BRD. Both alpha and beta diversities were significantly different between pre- and post-treatment microbial communities (q < 0.05). The high dose caused a shift in a larger number of genera than the low dose. Using metagenomic ProxiMeta Hi-C, 32 unique antimicrobial resistance genes (ARGs) conferring resistance to six antibiotic classes were detected with their reservoirs, and the high dose favored clonal expansion of ARG-carrying bacterial hosts. In conclusion, enrofloxacin treatment can alter fecal microbiota and resistome irrespective of its dose. Hi-C sequencing provides significant benefits for unlocking new insights into the ARG ecology of complex samples; however, limitations in sample size and sequencing depth suggest that further work is required to validate the findings.

849. Whole Genome Sequencing Analysis of Klebsiella pneumoniae Isolates Reveals Diversity in Genetic Antibiotic Resistance Patterns

Background Klebsiella pneumoniae is among the leading causes of healthcare-associated infections (HAI). Multidrug-resistant (MDR) Klebsiella variants are difficult to treat and have been reported with increasing frequency in hospitals. Whole genome multi-locus sequence typing (wg-MLST) of K. pneumoniae HAI isolates was used to compare antibiotic resistance genetic patterns against epidemiologic typing.Figure 1. Prevalence of ABX resistance genes for each drug class. Figure 2. Prevalence of ABX resistance genes by hospital. MethodsForty-six clinical bacterial HAI isolates were collected from patients admitted to two disparate tertiary care hospitals in Detroit, Michigan between 2017 and 2019. Data output from wg-MLST was de novo assembled using SPAdes (version 3.7.1) assembler on the Bionumerics calculation engine. Minimum spanning tree (MST) analysis categorized isolates into unique MLST Pasteur serotypes (ST). Antimicrobial resistance genes and/or chromosomal mutations were identified using the ResFinder Database (version 3.2). K. pneumoniae isolates were compared for antibiotic-resistance patterns by hospital, unit, and ST.Figure 3. Hospital 1 clusters and strains that detected gene qnr1, arranged by unit for visual comparison. Figure 4. Sample of clinically significant genes and prevalence, excluding SHV family of genes for simplicity. ResultsThere was significant genetic diversity among the isolates, and no predominant strain was identified. MST analysis revealed 17 unique strains. Only six strains had genetically unique resistance genes detected in more than one isolate, and only three of six were hospital-specific; none were unit-specific. Out of the 75 unique resistance genes detected, only 8 genes had a prevalence >50%: oqxA (100%), oqxB (100%), fosA (89%), blaCTX-M-15 (76%), aac(6’)-Ib-cr (61%), blaTEM-1B (52%), blaOXA-1 (52%), and catB3 (52%). No colistin resistance genes were detected. Of the remaining 69 low-prevalence resistance genes, only 8 hospital-specific genes were detected in more than one isolate (qnrB1, blaSHV-1, blaSHV-110, ac(6’)-Ib3, blaCTX-M-3, blaSHV-36, blaSHV-80, blaSHV-178) with a prevalence range of 4%-22%.ConclusionOur genetic analysis of antibiotic resistance patterns and wg-MLST revealed significant heterogeneity among the isolates, indicating no common source of transmission for either hospital. Although K. pneumoniae is a very common nosocomial pathogen, etiologic analysis suggests diverse community strains (e.g., gut colonization) may actually be responsible for previously-designated HAI.Disclosures Chetan Jinadatha, MD, MPH, AHRQ (Research Grant or Support)Department of Veterans Affairs (Other Financial or Material Support, Owner: Department of Veterans Affairs. Licensed to: Xenex Disinfection System, San Antonio, TX)Inventor (Other Financial or Material Support, Methods for organizing the disinfection of one or more items contaminated with biological agents)NiH/NINR (Research Grant or Support)NSF (Research Grant or Support)Xenex Healthcare Services (Research Grant or Support) Mark Stibich, PhD MHS, Xenex Disinfection Services, Inc (Board Member, Employee)

Simulated Winter Incubation of Soil With Swine Manure Differentially Affects Multiple Antimicrobial Resistance Elements.

Gastrointestinal bacteria that harbor antibiotic resistance genes (ARG) become enriched with antibiotic use. Livestock manure application to cropland for soil fertility presents a concern that ARG and bacteria may proliferate and be transported in the environment. In the United States, manure applications typically occur during autumn with slow mineralization until spring planting season. A laboratory soil incubation study was conducted mimicking autumn swine manure application to soils with concentrations of selected ARG monitored during simulated 120-day winter incubation with multiple freeze-thaw events. Additionally, the effects of two soil moistures [10 and 30% water holding capacity (WHC)] and two manure treatments [raw versus hydrated lime alkaline stabilization (HLAS)] were assessed. Fourteen tetracycline resistance genes were evaluated; tet(D), tet(G), and tet(L) were detected in background soil while swine manure contained tet(A), tet(B), tet(C), tet(G), tet(M), tet(O), tet(Q), and tet(X). By day 120, the manure-borne tet(M) and tet(O) were still detected while tet(C), tet(D), tet(L), and tet(X) genes were detected less frequently. Other tet resistance genes were detected rarely, if at all. The sum of unique tet resistance genes among all treatments decreased during the incubation from an average of 8.9 to 3.8 unique tet resistance genes. Four resistance elements, intI1, blactx–m–32, sul(I), erm(B), and 16s rRNA genes were measured using quantitative PCR. ARG abundances relative to 16S abundance were initially greater in the raw manure compared to background soil (−1.53 to −3.92 log abundance in manure; −4.02 to <−6.7 log abundance in soil). In the mixed manure/soil, relative abundance of the four resistance elements decreased (0.87 to 1.94 log abundance) during the incubation largely because 16S rRNA genes increased by 1.21 log abundance. Throughout the incubation, the abundance of intI1, blactx–m–32, sul(I), and erm(B) per gram in soil amended with HLAS-treated manure was lower than in soil amended with raw manure. Under low initial soil moisture conditions, HLAS treatment reduced the abundance of intI1 and resulted in loss of blactx–m–32, sul(I), and erm(B)] compared to other treatment-moisture combinations. Although one might expect antibiotic resistance to be relatively unchanged after simulated winter manure application to soil, a variety of changes in diversity and relative abundance can be expected.

Genetic diversity of bacterial pathogens in children with acute gastroenteritis in Bangladesh during 2014-2019

Background: Acute gastroenteritis is still the health burden in developed and developing countries. From different etiological agents, bacteria and viruses are considered to be significant enteropathogens of acute gastroenteritis in children worldwide including Bangladesh. Among these diarrheal bacteria, Escherichia coli, Non Typhoidal Salmonella, Shigella spp. and Vibrio cholerae are some of the major enetropathogens. Antibiotics are mostly used for the treatment of bacterial pathogens. Antibiotic resistance property of the above mentioned bacterial pathogens is a true hindrance in treatment of diarrheal diseases. Methods and materials: The study was carried on 387 fecal specimens, collected from all children of acute gastroenteritis in four clinics encompassing three localities (Savar, Cattogram and Sirajganj) in Bangladesh from January 2014 to January 2019. For diarrheagenic bacterial pathogens, conventional bacterial identification and molecular characterization methods were used. Prevalence of antibiotic resistance bacterial pathogens were also determined. Sequence data were analyzed by Chromas 2.32, BioEdit and other software. Results: A total of 387 fecal specimens were tested for bacterial and viral pathogens. 39.27% (152 of 387) of the samples were positive for bacterial pathogens and 42% (164 of 387) for viral pathogens. Among diarrheagenic bacterial pathogens, E.coli was the most prevalent (17.3%, 67 of 387) followed by V. cholerae (13.5%, 52 of 387), Salmonella spp. (4.9%, 19 of 387) and Shigella spp. (3.6%, 14 of 387), respectively. We found 50% to 80% isolates of V. cholerae were resistance against the tested antibiotics. While 20% to 60% of E. coli isolates were resistance against those antibiotics. Molecular analysis of antibiotic resistance genes confirmed the presence of qnrA and sul4 resistance elements in diarrheal pathogenic bacteria. To far our knowledge, the report of sul4 in this study is first in Bangladesh. Conclusion: This study will help to better understand the overall bacterial pathogens involved in infants and children diarrheal diseases in Bangladesh. This research work also confirmed the presence of antibiotic resistance bacterial pathogens with unique resistance gene in diarrheal pediatric population in Bangladesh. This study will also provide information for the betterment of detection methods, prevention and treatment strategies for diarrheal diseases in Bangladesh.