The spread of pESI-mediated extended-spectrum cephalosporin resistance in Salmonella serovars-Infantis, Senftenberg, and Alachua isolated from food animal sources in the United States.

The goal of this study is to investigate the origin, prevalence, and evolution of the pESI megaplasmid in Salmonella isolated from animals, foods, and humans. We queried 510,097 Salmonella genomes under the National Center for Biotechnology Information (NCBI) Pathogen Detection (PD) database for the presence of potential sequences containing the pESI plasmid in animal, food, and environmental sources. The presence of the pESI megaplasmid was confirmed by using seven plasmid-specific markers (rdA, pilL, SogS, TrbA, ipf, ipr2 and IncFIB(pN55391)). The plasmid and chromosome phylogeny of these isolates was inferred from single nucleotide polymorphisms (SNPs). Our search resolved six Salmonella clusters carrying the pESI plasmid. Four were emergent Salmonella Infantis clusters, and one each belonged to serovar Senftenberg and Alachua. The Infantis cluster with a pESI plasmid carrying blaCTX-M-65 gene was the biggest of the four emergent Infantis clusters, with over 10,000 isolates. This cluster was first detected in South America and has since spread widely in United States. Over time the composition of pESI in United States has changed with the average number of resistance genes showing a decrease from 9 in 2014 to 5 in 2022, resulting from changes in gene content in two integrons present in the plasmid. A recent and emerging cluster of Senftenberg, which carries the blaCTX-M-65 gene and is primarily associated with turkey sources, was the second largest in the United States. SNP analysis showed that this cluster likely originated in North Carolina with the recent acquisition of the pESI plasmid. A single Alachua isolate from turkey was also found to carry the pESI plasmid containing blaCTX-M-65 gene. The study of the pESI plasmid, its evolution and mechanism of spread can help us in developing appropriate strategies for the prevention and further spread of this multi-drug resistant plasmid in Salmonella in poultry and humans.

Genomic analysis of azithromycin-resistant Salmonella from food animals at slaughter and processing, and retail meats, 2011-2021, United States.

IMPORTANCE: Macrolides of different ring sizes are critically important antimicrobials for human medicine and veterinary medicine, though the widely used 15-membered ring azithromycin in humans is not approved for use in veterinary medicine. We document here the emergence of azithromycin-resistant Salmonella among the NARMS culture collections between 2011 and 2021 in food animals and retail meats, some with co-resistance to ceftriaxone or decreased susceptibility to ciprofloxacin. We also provide insights into the underlying genetic mechanisms and genomic contexts, including the first report of a novel combination of azithromycin resistance determinants and the characterization of multidrug-resistant plasmids. Further, we highlight the emergence of a multidrug-resistant Salmonella Newport clone in food animals (mainly cattle) with both azithromycin resistance and decreased susceptibility to ciprofloxacin. These findings contribute to a better understating of azithromycin resistance mechanisms in Salmonella and warrant further investigations on the drivers behind the emergence of resistant clones.

A Multidrug-Resistant Salmonella Infantis Clone is Spreading and Recombining in the United States.

Recently, there have been reports worldwide of a multidrug-resistant, emergent Salmonella Infantis (ESI) clone with a large megaplasmid (pESI), often containing the extended-spectrum beta-lactamase gene blaCTX-M-65. This clone also has a gyrA mutation conferring fluoroquinolone resistance, further limiting treatment options. In the United States, this clone has also been found in poultry sources, indicating a likely source of human illnesses. We conducted short-read sequencing of Salmonella enterica isolated from retail meats as part of routine surveillance by the National Antimicrobial Resistance Monitoring System (NARMS). We analyzed the resulting data temporally and geographically to determine when and where the ESI clone has spread in the United States. We found the ESI clone was first found in retail meats in Tennessee in 2014, but by 2019 was throughout the United States and comprised 29% of all Salmonella isolated from retail chickens, and 7% from retail turkey. Of these isolates, 85.0% were within 20 single nucleotide polymorphisms (SNPs) of those causing human illnesses. Long-read sequencing data indicated substantial recombination in the pESI plasmid resulting in the presence of 0-10 resistance genes, despite all their chromosomes being within 31 SNPs of one another. This work demonstrates the rapid spread of this clone of Salmonella Infantis in poultry in the United States, with the potential for increased burden of human illness attributed to this multidrug-resistant pathogen.

Draft Genome Assemblies of Clinical Isolates of Klebsiella pneumoniae V9011662 and Enterobacter hormaechei Entb306.

Enterobacter hormaechei and Klebsiella pneumoniae are pathogenic Enterobacteriaceae that have been associated with the spread of antibiotic resistance. Here, we report draft genome assemblies of an Enterobacter hormaechei clinical isolate and a multidrug-resistant clinical isolate of Klebsiella pneumoniae.

lptG contributes to changes in membrane permeability and the emergence of multidrug hypersusceptibility in a cystic fibrosis isolate of Pseudomonas aeruginosa.

PURPOSE: In the lungs of cystic fibrosis patients, Pseudomonas aeruginosa is exposed to a myriad of antibiotics leading to alterations in antibiotic susceptibility. This study identifies mutations resulting in hypersusceptibility in isogenic mutants of a P. aeruginosa clinical isolate, PA34. METHODS: PA34 was exposed to subinhibitory concentrations of doripenem or meropenem during growth to mid-log phase. Antibiotic susceptibility of surviving colonies was determined by agar dilution. Two carbapenem-resistant colonies hypersusceptible to non-carbapenem antibiotics were selected for further analysis. Antibiotic resistance gene expression was evaluated by RT-rtPCR and OprD production by SDS-PAGE. PA34 and isogenic mutants were evaluated with whole genome sequencing. Sequence variants were confirmed by Sanger sequencing, and cognate genes in eight carbapenem-resistant clinical isolates hypersusceptible to non-carbapenem antibiotics were sequenced. Lipopolysaccharide preparations of PA34 and hypersusceptible mutants were evaluated with ProQ-Emerald stain. RESULTS: Isogenic mutants showed 4- to 8-fold MIC increase for imipenem, meropenem, and doripenem. However, they were hypersusceptible (≥4-fold MIC decrease) to aminoglycosides, fluoroquinolones, and non-carbapenem ß-lactams. Expression of ampC or mex-opr efflux pumps was unchanged, but OprD production was decreased. Mutations causing Q86H AlgU and G77C LptG amino acid substitutions and nonsense mutations within OprD were observed in both mutants. Lipopolysaccharide modifications were observed between isogenic mutants and PA34. Non-synonymous mutations in LptF or LptG were observed in 6/8 hypersusceptible clinical isolates resistant to carbapenem antibiotics. CONCLUSION: Evaluation of hypersusceptible mutants identified the association between lptG and a hypersusceptible phenotype. Modifications in lipopolysaccharide profiles suggests LptG modification interferes with lipopolysaccharide transport and contributes to hypersusceptibility.

Draft Genome Sequence of the Mucoid Pseudomonas aeruginosa Clinical Isolate PA34.

Pseudomonas aeruginosa is a serious threat to patients suffering from cystic fibrosis. These organisms are exposed to a unique set of selective pressures within the lung. Here, we report the draft genome sequence of a mucoid P. aeruginosa clinical isolate obtained from a cystic fibrosis patient colonized with P. aeruginosa.

High-Resolution Melting Analysis for Rapid Detection of Sequence Type 131 Escherichia coli.

Escherichia coli isolates belonging to the sequence type 131 (ST131) clonal complex have been associated with the global distribution of fluoroquinolone and ß-lactam resistance. Whole-genome sequencing and multilocus sequence typing identify sequence type but are expensive when evaluating large numbers of samples. This study was designed to develop a cost-effective screening tool using high-resolution melting (HRM) analysis to differentiate ST131 from non-ST131 E. coli in large sample populations in the absence of sequence analysis. The method was optimized using DNA from 12 E. coli isolates. Singleplex PCR was performed using 10 ng of DNA, Type-it HRM buffer, and multilocus sequence typing primers and was followed by multiplex PCR. The amplicon sizes ranged from 630 to 737 bp. Melt temperature peaks were determined by performing HRM analysis at 0.1°C resolution from 50 to 95°C on a Rotor-Gene Q 5-plex HRM system. Derivative melt curves were compared between sequence types and analyzed by principal component analysis. A blinded study of 191 E. coli isolates of ST131 and unknown sequence types validated this methodology. This methodology returned 99.2% specificity (124 true negatives and 1 false positive) and 100% sensitivity (66 true positives and 0 false negatives). This HRM methodology distinguishes ST131 from non-ST131 E. coli without sequence analysis. The analysis can be accomplished in about 3 h in any laboratory with an HRM-capable instrument and principal component analysis software. Therefore, this assay is a fast and cost-effective alternative to sequencing-based ST131 identification.