Mobile Genetic Elements Drive Antimicrobial Resistance Gene Spread in Pasteurellaceae Species

Mobile genetic elements (MGEs) and antimicrobial resistance (AMR) drive important ecological relationships in microbial communities and pathogen-host interaction. In this study, we investigated the resistome-associated mobilome in 345 publicly available Pasteurellaceae genomes, a large family of Gram-negative bacteria including major human and animal pathogens. We generated a comprehensive dataset of the mobilome integrated into genomes, including 10,820 insertion sequences, 2,939 prophages, and 43 integrative and conjugative elements. Also, we assessed plasmid sequences of Pasteurellaceae. Our findings greatly expand the diversity of MGEs for the family, including a description of novel elements. We discovered that MGEs are comparable and dispersed across species and that they also co-occur in genomes, contributing to the family’s ecology via gene transfer. In addition, we investigated the impact of these elements in the dissemination and shaping of AMR genes. A total of 55 different AMR genes were mapped to 721 locations in the dataset. MGEs are linked with 77.6% of AMR genes discovered, indicating their important involvement in the acquisition and transmission of such genes. This study provides an uncharted view of the Pasteurellaceae by demonstrating the global distribution of resistance genes linked with MGEs.

One Health Genomic Study of Human and Animal Klebsiella pneumoniae Isolated at Diagnostic Laboratories on a Small Caribbean Island

Klebsiella pneumoniae causes a variety of infections in both humans and animals. In this study, we characterised the genomes of human and animal isolates from two diagnostic laboratories on St. Kitts, a small Caribbean island inhabited by a large population of vervet monkeys. In view of the increased chances of direct or indirect contact with humans and other animal species, we used the One Health approach to assess transmission of K. pneumoniae across host species by sequencing 82 presumptive K. pneumoniae clinical isolates from humans (n = 51), vervets (n = 21), horses (n = 5), dogs (n = 4) and a cat (n = 1). Whole genome sequencing (WGS) was carried out using Illumina technology. De novo assembly was performed in CLC Genomics Workbench v.11.0. Single nucleotide polymorphisms were detected using NASP followed by phylogenetic analysis using IQ-TREE. Virulence and antimicrobial resistance gene contents were analysed using the Kleborate and CGE pipelines. WGS-based analysis showed that 72 isolates were K. pneumoniae sensu stricto and five K. quasipneumoniae and five K. variicola. K. pneumoniae isolates belonged to 35 sequence types (ST), three of which were occasionally shared between humans and animals: ST23, ST37 and ST307. The ST23 strains from vervets formed a separate cluster amongst publicly available sequenced ST23 strains, indicating the presence of a specific vervet sublineage. Animal strains harbored fewer resistance genes and displayed distinct virulence traits that appeared to be host-specific in vervet isolates. Our results show that K. pneumoniae infections on this Caribbean island are usually caused by host-specific lineages.

Genomic epidemiology and longitudinal sampling of ward wastewater environments and patients reveals complexity of the transmission dynamics of blaKPC-carbapenemase-producing Enterobacterales in a hospital setting

Background Healthcare-associated wastewater reservoirs and asymptomatic gastrointestinal patient colonisation by carbapenemase-producing Enterobacterales (CPE) can be important in nosocomial CPE dissemination and infection. We characterised these niches and within-niche diversity in a blaKPC-associated CPE (KPC-E) endemic healthcare setting, to better understand transmission potential. Methods We systematically sampled wastewater sites and patients across three units (six wards) over 6-12 months in 2016 in a KPC-E endemic hospital. We used Illumina sequencing to characterise up to five isolates per sample. Recombination-adjusted phylogenies were used to define genetically related strains; assembly and mapping-based typing approaches were used to characterise antimicrobial resistance gene and insertion sequence profiles, and Tn4401 types/target site sequences. The wider accessory genome was evaluated in a subset of the largest clusters, and those crossing niches. Findings Wastewater site KPC-E-positivity was substantial (101/349 sites [28.9%] positive, 319/4,488 [7.1%] sampling events positive); 183/4,425 (4.1%) of patients were CPE culture-positive over the same timeframe. 13 species and 109 KPC-E strains were observed across niches, and 24% of wastewater and 26% of patient KPC-E-positive samples harboured ≥1 strain. Most diversity was explained by the individual niche, suggesting highly localised factors are important in selection and spread. Tn4401+target site sequence (TSS) diversity was greater in wastewater sites (p<0.001), suggesting these might favour Tn4401-associated transposition/evolution and dissemination. Shower/bath and sluice/mop-associated sites were more likely to be KPC-E-positive (Adjusted Odds Ratio [95% CI]: 2.69 [1.44-5.01], p=0.0019 and 2.60 [1.04-6.52], p=0.0410, respectively). Different strains had different transmission and blaKPC dissemination dynamics. Interpretation There may be substantial KPC-E colonisation of wastewater sites and patients in KPC-E-endemic healthcare settings. Niche-specific factors, and different strains with different transmission dynamics influence carbapenemase gene dissemination. New transmission models incorporating complex, multi-level dynamics are needed to better quantify CPE dissemination to inform interventions and reduce transmission. Funding This study was supported by the National Institute for Health Research, UK.

Characterization of Aminoglycoside Modifying Enzymes Producing E. coli and Klebsiella pneumoniae Clinical Isolates

Antimicrobial resistance gene profile characterization and dissemination offer useful detail on the possible challenge in treating bacteria. The development of aminoglycoside modifying enzymes (AMEs) is considered as the primary mechanism of resistance to aminoglycosides, in addition to the 16S rRNA methylases. This study aimed at isolation and characterization of aminoglycosides resistant clinical isolates of enterobacteriaceae family from different clinical samples. Over a period of 24 months, thirty samples were collected and 49 clinical isolates of E. coli [n=25], Klebsiella [n=13], Enterobacter species (n=7) and Proteus species (n=4) were isolated from Egyptian clinical laboratories. The identities of the cultures were confirmed following standard microbiological procedures. Resistance of the isolates to aminoglycosides was determined by the disc diffusion method and isolates with highest resistance (n=9) were selected and investigated for 16S rRNA methylase and AMES encoding genes by polymerase chain reaction (PCR) and sequencing. In general, aminoglycoside resistance was found in 95% of the isolates; the isolates displayed the highest rate of resistance to netilmicin (75%) and kanamycin (55%), while resistance to gentamycin (18%) and tobramycin (16%) was low. A total of 9 isolates have the highest aminoglycoside resistant rate, showed the highest appearance for aac(6′)-Ib as well as ant (3″)-Ia resistant genes, with aac (3)-II (44%) and ant (4′)-IIb (34%) following closely. The high prevalence of AMEs observed among resistant isolates in this study suggests the urgent need for more efficient treatment designs to mitigate the selection burden as well as improved care of patients who have been infected with these drug-resistant organisms.

Comparative analysis of Bacillus cereus group isolates' resistance using disk diffusion and broth microdilution and the correlation between resistance phenotypes and genotypes

A collection of 85 Bacillus cereus group isolates were screened for phenotypic resistance to nine antibiotics using disk diffusion and broth microdilution. The broth microdilution antimicrobial results were interpreted using the CLSI M45 breakpoints for Bacillus spp. Due to the lack of Bacillus spp. disk diffusion breakpoints, the results obtained with the disk diffusion assay were interpreted using the CLSI M100 breakpoints for Staphylococcus spp. We identified significant (p < 0.05) discrepancies in resistance interpretation between the two methods for ampicillin, gentamicin, rifampicin, tetracycline, and trimethoprim/sulfamethoxazole. Antimicrobial resistance genes were detected using unassembled and assembled whole-genome sequences with Ariba and Abricate, respectively, to assess the sensitivity and specificity for predicting phenotypic resistance based on the presence of antimicrobial resistance genes. We found antimicrobial resistance gene presence to be a poor indicator for phenotypic resistance, calling for further investigation of mechanisms underlying antimicrobial resistance in the B. cereusgroup. Genes with poor sensitivity and/or specificity, as determined based on broth microdilution results included rph(rifampicin, 0%, 95%), mphgenes (erythromycin, 0%, 96%), and all vangenes (vancomycin, 100%, 35%). However, Bc(ampicillin, 64%, 100%) andtet genes (tetracycline, 67%, 100%) were highly specific, albeit moderately sensitive indicators of phenotypic resistance based on broth microdilution results. Only beta-lactam resistance genes (Bc, BcII, and blaTEM) were highly sensitive (94%) and specific (100%) markers of resistance to ceftriaxone based on the disk diffusion results, providing further evidence of these beta-lactams' role in nonsusceptibility of Bacillus cereus group isolates to ceftriaxone.

792. Outbreak of Multidrug-resistant Pseudomonas aeruginosa in a Tertiary Healthcare System in Detroit

Background Pseudomonas aeruginosa is one of the most common causes of healthcare-associated infections in critically ill patients and those with suboptimal immunity. However, the development of multidrug resistant Pseudomonas aeruginosa (MDR Pa) creates an even great disease burden and threat to both the hospital and local community health. The purpose of this study is to illustrate a descriptive analysis of a cluster of MDR Pseudomonas, during a local surge of SARS-CoV-2 (COVID 19) pandemic. The goal is to shed more light on the troublesome parallel during outbreaks, such as COVID-19 and consequential secondary outcomes. Methods From November 2020 through February 2021, 16 patients exposed to the intensive care units of a tertiary healthcare system were infected or colonized with a multidrug-resistant strain of P. aeruginosa (Figure 1). Outbreak investigation was conducted via retrospective chart review of the first eight cases and prospective analysis of the latter eight cases. The isolates collected prospectively were analyzed for taxonomic identification, antimicrobial resistance profile, and phylogenetic analysis. Clinical characteristics of all patients were collected, and epidemiological investigation was carried out. MDR is defined as resistance to at least four classes of antibiotics: third-generation cephalosporins, fluoroquinolones, aminoglycosides, and carbapenems. Figure 1. Epidemiological Curve of Cases of Multidrug-resistant Pseudomonas aeruginosa in the Detroit Medical Center from November 2020-February 2021 Results Of the 16 cases of MDR Pa infections, seven died within five months (Table 1). Antimicrobial resistance gene profiling detected blaOXA and blaPAO betalactamase genes in all the samples. One sample contained an additional blaVIM resistance gene, although this patient was colonized and not actively infected. The analysis suggests existence of two clusters demonstrating relatedness and possible horizontal transmission. Timing of this cluster of cases coincides with surge of COVID-19 cases. This highlights the importance of infection control measures and antimicrobial stewardship. Table 1. Characteristics of patients infected with multidrug-resistant Pseudomonas aeruginosa (MDR-Pa) at Detroit Medical Center, November 2020 to February 2021 Conclusion Since early 2017 studies show there is a growing prevalence worldwide in transferable resistance, particularly for β-lactamases and carbapenemases, MDR Pseudomonas. This study emphasizes an irony paralleled during a pandemic, the needed efforts to prevent unintentional lapses in patient safety. Disclosures All Authors: No reported disclosures

Profile variation of bla genes among non-lactose fermenting Gram-negative bacilli between clinical and environmental isolates of Dr. Soetomo Hospital, Surabaya, Indonesia

Endraputra PN, Kuntaman K, Wasito EB, Shirakawa T, Raharjo D, Setyarini W. 2021. Profile variation of bla genes among non-lactose fermenting Gram negative bacilli between clinical and environmental isolates of Dr. Soetomo Hospital, Surabaya, Indonesia. Biodiversitas 22: 5047-5054. Carbapenem-resistant non-fermenter Gram-negative bacilli are notorious opportunistic pathogens in hospitalized patients and hospital environments. This study explored the carbapenemase gene among non-fermenter Gram-negative bacilli from hospital wastewater and clinical isolates in Dr. Soetomo Hospital, Surabaya, Indonesia. All samples were screened on MacConkey agar with meropenem 2 µg/ml and gene detected by Multiplex PCR. All samples were screened on MacConkey agar with meropenem 2 µg/ml and gene detected by Multiplex PCR. A total of 121 isolates consisted of 76 clinical (41 carbapenem-resistant Acinetobacter baumannii and 35 carbapenem-resistant Pseudomonas aeruginosa), 45 environmental isolates (6 carbapenem-resistant Pseudomonas aeruginosa and 32 carbapenem-resistant Pseudomonas spp.), and 7 screening samples (all CRPAs). Clinical isolates carbapenemase genes were identified, blaOXA-23-like 21 (28%), blaOXA-24-like 30 (39%), blaNDM-1 1 (1%), and blaIMP-1 6 (8%) while environmental isolates were blaOXA-23-like 5 (13%), blaOXA-24-like 4 (11%), blaOXA-48-like 2 (5%), blaNDM-1 13 (34%), blaVIM 12 (32%), and blaIMP-1 4 (11%). Rectal swab screening specimens presented blaOXA-23-like 3 (43%), blaOXA-24-like 3 (43%), and blaNDM-1 1 (14%). The carbapenemase gene pattern was different between clinical and environmental isolates. The blaOXA-23-like and blaOXA-24-like were most prevalent among in both clinical and wastewater, while blaVIM was mostly in wastewater. The presence of carbapenem-resistant non-fermenter Gram-negative bacilli carrying carbapenemase genes in hospital effluents indicated that the community river was seeded with an antimicrobial resistance gene.

Investigating the Effect of an Oxytetracycline Treatment on the Gut Microbiome and Antimicrobial Resistance Gene Dynamics in Nile Tilapia (Oreochromis niloticus)

Antibiotics play a vital role in aquaculture where they are commonly used to treat bacterial diseases. However, the impact of antibiotic treatment on the gut microbiome and the development of antimicrobial resistance in Nile tilapia (Oreochromis niloticus) over time remains to be fully understood. In this study, fish were fed a single treatment of oxytetracycline (100 mg/kg/day) for eight days, followed by a 14-day withdrawal period. Changes in the distal gut microbiome were measured using 16S rRNA sequencing. In addition, the abundance of antimicrobial resistance genes was quantified using real-time qPCR methods. Overall, the gut microbiome community diversity and structure of Nile tilapia was resilient to oxytetracycline treatment. However, antibiotic treatment was associated with an enrichment in Plesiomonas, accompanied by a decline in other bacteria taxa. Oxytetracycline treatment increased the proportion of tetA in the distal gut of fish and tank biofilms of the treated group. Furthermore, the abundance of tetA along with other tetracycline resistance genes was strongly correlated with a number of microbiome members, including Plesiomonas. The findings from this study demonstrate that antibiotic treatment can exert selective pressures on the gut microbiome of fish in favour of resistant populations, which may have long-term impacts on fish health.