Reciprocal regulation of enterococcal cephalosporin resistance by products of the autoregulated yvcJ-glmR-yvcL operon enhances fitness during cephalosporin exposure.

Enterococci are commensal members of the gastrointestinal tract and also major nosocomial pathogens. They possess both intrinsic and acquired resistance to many antibiotics, including intrinsic resistance to cephalosporins that target bacterial cell wall synthesis. These antimicrobial resistance traits make enterococcal infections challenging to treat. Moreover, prior therapy with antibiotics, including broad-spectrum cephalosporins, promotes enterococcal proliferation in the gut, resulting in dissemination to other sites of the body and subsequent infection. As a result, a better understanding of mechanisms of cephalosporin resistance is needed to enable development of new therapies to treat or prevent enterococcal infections. We previously reported that flow of metabolites through the peptidoglycan biosynthesis pathway is one determinant of enterococcal cephalosporin resistance. One factor that has been implicated in regulating flow of metabolites into cell wall biosynthesis pathways of other Gram-positive bacteria is GlmR. In enterococci, GlmR is encoded as the middle gene of a predicted 3-gene operon along with YvcJ and YvcL, whose functions are poorly understood. Here we use genetics and biochemistry to investigate the function of the enterococcal yvcJ-glmR-yvcL gene cluster. Our results reveal that YvcL is a DNA-binding protein that regulates expression of the yvcJ-glmR-yvcL operon in response to cell wall stress. YvcJ and GlmR bind UDP-GlcNAc and reciprocally regulate cephalosporin resistance in E. faecalis, and binding of UDP-GlcNAc by YvcJ appears essential for its activity. Reciprocal regulation by YvcJ/GlmR is essential for fitness during exposure to cephalosporin stress. Additionally, our results indicate that enterococcal GlmR likely acts by a different mechanism than the previously studied GlmR of Bacillus subtilis, suggesting that the YvcJ/GlmR regulatory module has evolved unique targets in different species of bacteria.

PASTA-kinase-mediated signaling drives accumulation of the peptidoglycan synthesis protein MurAA to promote cephalosporin resistance in Enterococcus faecalis.

The bacterial PASTA kinase, IreK, is required for intrinsic cephalosporin resistance in the Gram-positive opportunistic pathogen, Enterococcus faecalis. IreK activity is enhanced in response to cell wall stress, such as cephalosporin exposure. The downstream consequences of IreK activation are not well understood in E. faecalis, but recent work in other low-GC Gram-positive bacteria demonstrated PASTA kinase-dependent regulation of MurAA, an enzyme that performs the first committed step in the peptidoglycan synthesis pathway. Here, we used genetic suppressor selections to identify MurAA as a downstream target of IreK signaling in E. faecalis. Using complementary genetic and biochemical approaches, we demonstrated that MurAA abundance is regulated by IreK signaling in response to physiologically relevant cell wall stress to modulate substrate flux through the peptidoglycan synthesis pathway. Specifically, the IreK substrate, IreB, promotes proteolysis of MurAA through a direct physical interaction in a manner responsive to phosphorylation by IreK. MurAB, a homolog of MurAA, also promotes MurAA proteolysis and interacts directly with IreB. Our results therefore establish a connection between the cell wall stress sensor IreK and one critical physiological output to modulate peptidoglycan synthesis and drive cephalosporin resistance.

Multidrug resistance in Salmonella isolates of swine origin: mobile genetic elements and plasmids associated with cephalosporin resistance with potential transmission to humans.

The emergence of foodborne Salmonella strains carrying antimicrobial resistance (AMR) in mobile genetic elements (MGE) is a significant public health threat in a One Health context requiring continuous surveillance. Resistance to ciprofloxacin and cephalosporins is of particular concern. Since pigs are a relevant source of foodborne Salmonella for human beings, we studied transmissible AMR genes and MGE in a collection of 83 strains showing 9 different serovars and 15 patterns of multidrug resistant (MDR) previously isolated from pigs raised in the conventional breeding system of Northern Spain. All isolates were susceptible to ciprofloxacin and three isolates carried blaCMY-2 or blaCTX-M-9 genes responsible for cefotaxime resistance. Filter mating experiments showed that the two plasmids carrying blaCTX-M-9 were conjugative while that carrying blaCMY-2 was self-transmissible by transformation. Whole-genome sequencing and comparative analyses were performed on the isolates and plasmids. The IncC plasmid pSB109, carrying blaCMY-2, was similar to one found in S. Reading from cattle, indicating potential horizontal transfer between serovars and animal sources. The IncHI2 plasmids pSH102 in S. Heidelberg and pSTM45 in S. Typhimurium ST34, carrying blaCTX-M-9, shared similar backbones and two novel "complex class 1 integrons" containing different AMR and heavy metal genes. Our findings emphasize the importance of sequencing techniques to identify emerging AMR regions in conjugative and stable plasmids from livestock production. The presence of MGE carrying clinically relevant AMR genes raises public health concerns, requiring monitoring to mitigate the emergence of bacteria carrying AMR genes and subsequent spread through animals and food.IMPORTANCEThe emergence of foodborne Salmonella strains carrying antimicrobial resistance (AMR) in mobile genetic elements (MGE) is a significant public health threat in a One Health context. Since pigs are a relevant source of foodborne Salmonella for humans, in this study, we investigate different aspects of AMR in a collection of 83 Salmonella showing nine different serovars and 15 patterns of multidrug resistant (MDR) isolated from pigs raised in the conventional breeding system. Our findings emphasize the importance of sequencing techniques to identify emerging AMR regions in conjugative and stable plasmids from livestock production. The presence of MGE carrying clinically relevant AMR genes raises public health concerns, requiring monitoring to mitigate the emergence of bacteria carrying AMR genes and subsequent spread through animals and food.

Use of an Interspecies Chimeric Receptor for Inducible Gene Expression Reveals that Metabolic Flux through the Peptidoglycan Biosynthesis Pathway is an Important Driver of Cephalosporin Resistance in Enterococcus faecalis.

Cephalosporins are commonly prescribed antibiotics that impair cross-linking of the bacterial cell wall. The Gram-positive opportunistic pathogen, Enterococcus faecalis, is intrinsically resistant to these antibiotics and proliferates substantially during cephalosporin therapy. As a result, the usage of cephalosporins has the potential to lead to life-threatening enterococcal infections. Yet, the molecular mechanisms that drive cephalosporin resistance (CR) are incompletely understood. Previously, we demonstrated that MurAA, an enzyme that catalyzes the first committed step in peptidoglycan (PG) synthesis, is required for CR. However, the mechanism by which MurAA contributes to CR remained unknown. Here, we tested the hypothesis that MurAA drives CR by controlling metabolic flux through the PG synthesis pathway. To do so, we developed and exploited an inducible gene expression system for E. faecalis based on an interspecies chimeric receptor that responds to exogenous nitrate for control of expression from a NisR-regulated promoter (PnisA). We used this tool to demonstrate synthetic lethality of MurAA with its homolog MurAB, to titrate expression of MurAA, and to conditionally deplete multiple PG synthesis enzymes downstream of MurAA that are predicted to be essential. These genetic manipulations, in addition to pharmacological inhibition of the PG synthesis pathway, all led to reductions in PG synthesis that correlated with reductions in CR. Our findings are consistent with a model in which control of metabolic flux through the PG synthesis pathway is a major driver of CR. IMPORTANCE Enterococci are dangerous opportunistic pathogens with the potential to cause life-threatening infections due in part to their intrinsic resistance to cephalosporin antibiotics. Elucidating the molecular mechanisms that provide this resistance is critical for the development of strategies to both prevent and treat enterococcal infections. Here, we report that the cell wall synthesis enzyme, MurAA, drives cephalosporin resistance at least in part by controlling metabolic flux through the peptidoglycan synthesis pathway. To demonstrate this, we designed and validated an inducible gene expression system based on a chimeric receptor that is functional in multiple lineages of E. faecalis. In doing so, we provided a new tool for inducible gene expression with broad applications beyond our studies, including studies of essential genes.

Extended Spectrum ß-Lactamase Activity and Cephalosporin Resistance in Escherichia coli from U.S. Mid-Atlantic Surface and Reclaimed Water.

Phylogenetic distribution and extended spectrum ß-lactamase (ESBL) activity of Escherichia coli recovered from surface and reclaimed water in the mid-Atlantic U.S. were evaluated. Among 488 isolates, phylogroups B1 and A were the most and least prevalent, respectively. Water type, but not season, affected phylogroup distribution. The likelihood of detecting group A isolates was higher in reclaimed than pond (P < 0.01), freshwater river (P < 0.01) or brackish river (P < 0.05) water. Homogeneity in group distribution was lowest in pond water, where group B1 comprised 50% of isolates. Only 16 (3.3%) isolates exhibited phenotypic resistance to one or more cephalosporins tested and only four had ESBL activity, representing groups B1, B2 isolates, and D. Phylogroup was a factor in antimicrobial resistance (P < 0.05), with group A (8.7%) and D (1.6%) exhibiting the highest and lowest rates. Resistance to cefoxitin was the most prevalent. Multi- versus single drug resistance was affected by phylogroup (P < 0.05) and more likely in groups D and B1 than A which carried resistance to cefoxitin only. The most detected ß-lactam resistance genes were blaCMY-2 and blaTEM. Water type was a factor for blaCTX-M gene detection (P < 0.05). Phenotypic resistance to cefotaxime, ceftriaxone, cefuroxime and ceftazidime, and genetic determinants for ESBL-mediated resistance were found predominantly in B2 and D isolates from rivers and reclaimed water. Overall, ESBL activity and cephalosporin resistance in reclaimed and surface water isolates were low. Integrating data on ESBL activity and ß-lactam resistance among E. coli populations can inform decisions on safety of irrigation water sources and One Health. IMPORTANCE Extended spectrum ß-lactamase (ESBL) producing bacteria, that are resistant to a broad range of antimicrobial agents, are spreading in the environment but data remain scarce. ESBL-producing Escherichia coli infections in the community are on the rise. This work was conducted to assess presence of ESBL-producing E. coli in water that could be used for irrigation of fresh produce. The study provides the most extensive evaluation of ESBL-producing E. coli in surface and reclaimed water in the mid-Atlantic United States. The prevalence of ESBL producers was low and phenotypic resistance to cephalosporins (types of ß-lactam antibiotics) was affected by season but not water type. Data on antimicrobial resistance among E. coli populations in water can inform decisions on safety of irrigation water sources and One Health.

Antimicrobial Resistance of Shigella flexneri in Pakistani Pediatric Population Reveals an Increased Trend of Third-Generation Cephalosporin Resistance.

The rapid emergence of resistance to third-generation cephalosporins in Shigella flexneri is crucial in pediatric shigellosis management. Limited studies have been conducted on molecular pattern of antibiotic resistance of S. flexneri in diarrhea endemic areas of Pakistan. The aim of the study was to analyze the antimicrobial resistance of S. flexneri isolated from pediatric diarrheal patients in Peshawar, Pakistan. A total of 199 S. flexneri isolates (clinical, n = 1 55 and non-clinical, n = 44) were investigated for drug resistance and mutational analysis of selected drug resistance genes. All isolates were found to be highly resistant to amoxicillin/clavulanic acid (88%), followed by trimethoprim-sulfamethoxazole (77%), chloramphenicol (43%), and quinolones (41.6%). About 34.5% S. flexneri isolates were found to be resistant to third-generation cephalosporin. None of the isolates was resistant to imipenem, piperacillin-tazobactam, and amikacin. Interestingly high frequency of third-generation cephalosporin resistance was observed in S. flexneri isolated from non-clinical samples (49%) when compared to clinical samples (30.5%). Furthermore, the most prevalent phenotypic-resistant patterns among third-generation cephalosporin-resistant isolates were AMC,CAZ,CPD,CFM,CRO,SXT (13%) followed by OFX,AMC,CAZ,CPD,CFM,CRO,SXT,NA,CIP (10%). The most frequently detected resistance genes were trimethoprim-sulfamethoxazole (sul2 = 84%), beta-lactamase genes (blaOXA = 87%), quinolones (qnrS = 77%), and chloramphenicol (cat = 64%). No mutation was detected in any drug-resistant genes. We are reporting for the first time the sequence of the blaTEM gene in S. flexneri. Furthermore, high third-generation cephalosporin resistance was observed in the patients who practiced self-medication as compared to those who took medication according to physician prescription. This study shows the high emergence of third-generation cephalosporin-resistant S. flexneri isolates, which is a potential threat to the community in the country. This finding will be helpful to develop a suitable antibiotic prescription regime to treat shigellosis.

Salmonella Typhi acquires diverse plasmids from other Enterobacteriaceae to develop cephalosporin resistance.

BACKGROUND: Recent reports have established the emergence and dissemination of extensively drug resistant (XDR) H58 Salmonella Typhi clone in Pakistan. In India where typhoid fever is endemic, only sporadic cases of ceftriaxone resistant S. Typhi are reported. This study aimed at elucidating the phylogenetic evolutionary framework of ceftriaxone resistant S. Typhi isolates from India to predict their potential dissemination. METHODS: Five ceftriaxone resistant S. Typhi isolates from three tertiary care hospitals in India were sequenced on an Ion Torrent Personal Genome Machine (PGM). A core genome single-nucleotide-polymorphism (SNP) based phylogeny of the isolates in comparison to the global collection of MDR and XDR S. Typhi isolates was built. Two of five isolates were additionally sequenced using Oxford Nanopore MinION to completely characterize the plasmid and understand its transmission dynamics within Enterobacteriaceae. RESULTS: Comparative genomic analysis and detailed plasmid characterization indicate that while in Pakistan (4.3.1 lineage I) the XDR trait is associated with blaCTX-M-15 gene on IncY plasmid, in India (4.3.1 lineage II), the ceftriaxone resistance is due to short term persistence of resistance plasmids such as IncX3 (blaSHV-12) or IncN (blaTEM-1B + blaDHA-1). CONCLUSION: Considering the selection pressure exerted by the extensive use of ceftriaxone in India, there are potential risks for the occurrence of plasmid transmission events in the predominant H58 lineages. Therefore, continuous monitoring of S. Typhi lineages carrying plasmid-mediated cephalosporin resistant genes is vital not just for India but also globally.

Population structure and uropathogenic potential of extended-spectrum cephalosporin-resistant Escherichia coli from retail chicken meat.

BACKGROUND: Food-producing animals and their products are considered a source for human acquisition of antimicrobial resistant (AMR) bacteria, and poultry are suggested to be a reservoir for Escherichia coli resistant to extended-spectrum cephalosporins (ESC), a group of antimicrobials used to treat community-onset urinary tract infections in humans. However, the zoonotic potential of ESC-resistant E. coli from poultry and their role as extraintestinal pathogens, including uropathogens, have been debated. The aim of this study was to characterize ESC-resistant E. coli isolated from domestically produced retail chicken meat regarding their population genetic structure, the presence of virulence-associated geno- and phenotypes as well as their carriage of antimicrobial resistance genes, in order to evaluate their uropathogenic potential. RESULTS: A collection of 141 ESC-resistant E. coli isolates from retail chicken in the Norwegian monitoring program for antimicrobial resistance in bacteria from food, feed and animals (NORM-VET) in 2012, 2014 and 2016 (n = 141) were whole genome sequenced and analyzed. The 141 isolates, all containing the beta-lactamase encoding gene blaCMY-2, were genetically diverse, grouping into 19 different sequence types (STs), and temporal variations in the distribution of STs were observed. Generally, a limited number of virulence-associated genes were identified in the isolates. Eighteen isolates were selected for further analysis of uropathogen-associated virulence traits including expression of type 1 fimbriae, motility, ability to form biofilm, serum resistance, adhesion- and invasion of eukaryotic cells and colicin production. These isolates demonstrated a high diversity in virulence-associated phenotypes suggesting that the uropathogenicity of ESC-resistant E. coli from chicken meat is correspondingly highly variable. For some isolates, there was a discrepancy between the presence of virulence-associated genes and corresponding expected phenotype, suggesting that mutations or regulatory mechanisms could influence their pathogenic potential. CONCLUSION: Our results indicate that the ESC-resistant E. coli from chicken meat have a low uropathogenic potential to humans, which is important knowledge for improvement of future risk assessments of AMR in the food chains.

Genomic surveillance of antimicrobial resistant bacterial colonisation and infection in intensive care patients.

BACKGROUND: Third-generation cephalosporin-resistant Gram-negatives (3GCR-GN) and vancomycin-resistant enterococci (VRE) are common causes of multi-drug resistant healthcare-associated infections, for which gut colonisation is considered a prerequisite. However, there remains a key knowledge gap about colonisation and infection dynamics in high-risk settings such as the intensive care unit (ICU), thus hampering infection prevention efforts. METHODS: We performed a three-month prospective genomic survey of infecting and gut-colonising 3GCR-GN and VRE among patients admitted to an Australian ICU. Bacteria were isolated from rectal swabs (n = 287 and n = 103 patients ≤2 and > 2 days from admission, respectively) and diagnostic clinical specimens between Dec 2013 and March 2014. Isolates were subjected to Illumina whole-genome sequencing (n = 127 3GCR-GN, n = 41 VRE). Multi-locus sequence types (STs) and antimicrobial resistance determinants were identified from de novo assemblies. Twenty-three isolates were selected for sequencing on the Oxford Nanopore MinION device to generate completed reference genomes (one for each ST isolated from ≥2 patients). Single nucleotide variants (SNVs) were identified by read mapping and variant calling against these references. RESULTS: Among 287 patients screened on admission, 17.4 and 8.4% were colonised by 3GCR-GN and VRE, respectively. Escherichia coli was the most common species (n = 36 episodes, 58.1%) and the most common cause of 3GCR-GN infection. Only two VRE infections were identified. The rate of infection among patients colonised with E. coli was low, but higher than those who were not colonised on admission (n = 2/33, 6% vs n = 4/254, 2%, respectively, p = 0.3). While few patients were colonised with 3GCR- Klebsiella pneumoniae or Pseudomonas aeruginosa on admission (n = 4), all such patients developed infections with the colonising strain. Genomic analyses revealed 10 putative nosocomial transmission clusters (≤20 SNVs for 3GCR-GN, ≤3 SNVs for VRE): four VRE, six 3GCR-GN, with epidemiologically linked clusters accounting for 21 and 6% of episodes, respectively (OR 4.3, p = 0.02). CONCLUSIONS: 3GCR-E. coli and VRE were the most common gut colonisers. E. coli was the most common cause of 3GCR-GN infection, but other 3GCR-GN species showed greater risk for infection in colonised patients. Larger studies are warranted to elucidate the relative risks of different colonisers and guide the use of screening in ICU infection control.

Molecular detection of blaCTX-M gene to predict phenotypic cephalosporin resistance and clinical outcome of Escherichia coli bloodstream infections in Vietnam.

BACKGROUND: Blood stream infections (BSI) caused by Extended Spectrum Beta-Lactamases (ESBLs) producing Enterobacteriaceae is a clinical challenge leading to high mortality, especially in developing countries. In this study, we sought to describe the epidemiology of ESBL-producing Escherichia coli strains isolated from Vietnamese individuals with BSI, to investigate the concordance of genotypic-phenotypic resistance, and clinical outcome of ESBL E. coli BSI. METHODS: A total of 459 hospitalized patients with BSI were screened between October 2014 and May 2016. 115 E. coli strains from 115 BSI patients were isolated and tested for antibiotic resistance using the VITEK®2 system. The ESBL phenotype was determined by double disk diffusion method following the guideline of Clinical and Laboratory Standards Institute. Screening for beta-lactamase (ESBL and carbapenemase) genes was performed using a multiplex-PCR assay. RESULTS: 58% (67/115) of the E. coli strains were ESBL-producers and all were susceptible to both imipenem and meropenem. Resistance to third-generation cephalosporin was common, 70% (81/115) were cefotaxime-resistant and 45% (52/115) were ceftazidime-resistant. blaCTX-M was the most common ESBL gene detected (70%; 80/115) The sensitivity and specificity of blaCTX-M-detection to predict the ESBL phenotype was 87% (76-93% 95% CI) and 54% (39-48% 95% CI), respectively. 28%% (22/80) of blaCTX-M were classified as non-ESBL producers by phenotypic testing for ESBL production. The detection of blaCTX-M in ESBL-negative E. coli BSI was associated with fatal clinical outcome (27%; 6/22 versus 8%; 2/26, p = 0.07). CONCLUSION: A high prevalence of ESBL-producing E. coli isolates harbouring blaCTX-M was observed in BSI patients in Vietnam. The genotypic detection of blaCTX-M may have added benefit in optimizing and guiding empirical antibiotic therapy of E. coli BSI to improve clinical outcome.

Use of critically important antimicrobial classes early in life may adversely impact bacterial resistance profiles during adult years: potential co-selection for plasmid-borne fluoroquinolone and macrolide resistance via extended-spectrum beta-lactam use in dairy cattle.

The transfer of antimicrobial resistance genes commonly occurs via vertical and horizontal gene transfer, as such genes are often found on the same mobile genetic element. This occurrence can lead to the co-selection of resistance to antimicrobials without their application. Dairy cattle located in the south-western United States were enrolled in a matched-pair longitudinal study to evaluate the effects of a two-dose ceftiofur treatment for metritis on levels of third-generation cephalosporin resistance among faecal Escherichia coli temporally. Escherichia coli chosen for further investigation were isolated on selective media, harboured extended-spectrum beta-lactam, fluoroquinolone and macrolide resistance genes. This combination has previously been unreported; importantly, it included genes encoding for resistance to antibiotics that can only be used in dairy cattle less than 20 months of age. Fluoroquinolones, macrolides and third and higher generation cephalosporins are considered critically important and highest priority for human medicine by the World Health Organization.

Molecular Characterization of Cephalosporin-Resistant Salmonella Enteritidis ST11 Isolates Carrying blaCTX-M from Children with Diarrhea.

Salmonella Enteritidis is an important foodborne pathogen with high prevalence of resistance to cephalosporins, imposing a serious threat to public health. Therefore, a total of 162 Salmonella Enteritidis isolates collected from child patients in China from 2007 to 2017 were characterized for their resistance to cephalosporins and investigated the transmission characteristics of cephalosporin resistance gene. We found that 15 (9.26%) isolates were all resistant to cefalotin (minimum inhibitory concentration [MIC] ≥512 µg/mL), ceftazidime (MIC 16-128 µg/mL), ceftriaxone (MIC 64 to ≥512 µg/mL), ceftiofur (MIC 64-256 µg/mL), and cefotaxime (MIC 64 to ≥512 µg/mL) with the possession of cephalosporin resistance genes blaCTX-M-55 (n = 13), blaCTX-M-101 (n = 1), and blaCTX-M-153 (n = 1). Molecular typing further revealed that these 15 isolates belonged to sequence type ST11 and shared close pulsed-field gel electrophoresis patterns, suggesting the possibility of clonal spread in Salmonella Enteritidis interspecies. Furthermore, conjugation experiments were successfully performed in 13 of 15 isolates, and blaCTX-M-55 was present on conjugative plasmids with sizes ranging from 54.7 to 173.4 kb. Compared with recipient Escherichia coli C600, transconjugants conferred elevated MICs for cephalosporins ranging from 2- to 2048-fold. The genetic structure surrounding of blaCTX-M-55 gene in transconjugants were ΔISEcp1-blaCTX-M-55-orf477 (n = 8) and ISEcp1-blaCTX-M-55-orf477 (n = 3), respectively. Taken together, blaCTX-M on the plasmids might contribute to cephalosporin resistance in Salmonella Enteritidis, and conjugative transfer of blaCTX-M-55 might facilitate the spread of cephalosporin resistance in Salmonella Enteritidis. Hence, effective mitigation measurements are needed to reduce the threat caused by cephalosporin-resistant Salmonella Enteritidis to public health.

Prior exposure of agriculture cephalosporin ceftiofur impaired conjugation of blaCTX-M-65 gene-bearing plasmid in Salmonella Saintpaul.

AIMS: Although a link between agricultural cephalosporin use and resistance in Salmonella has been demonstrated with the drug ceftiofur, the underlying mechanism of the correlation is unclear. This study investigated the impact of ceftiofur exposure in S. Saintpaul on ceftriaxone resistance, the gene expression and the conjugative transfer of the blaCTX-M-65 gene. METHODS AND RESULTS: Prior ceftiofur exposure caused a twofold increase in MIC from 1024 to 2048 µg ml-1 towards ceftriaxone and increased the enzymatic activity of BlaCTX-M-65 2·2 folds from 3·46 to 7·67 nmol nitrocefin hydrolysed min-1 . A threefold upregulation in gene expression of the blaCTX-M-65 gene was also observed. Donors exposed to ceftiofur subsequently demonstrated a 2·5-fold decrease in transfer efficiency. CONCLUSIONS: Prior exposure of S. Saintpaul to ceftiofur led to increased phenotypic resistance towards ceftriaxone while its ability to spread the cephalosporin resistance through conjugation, conversely, was impaired. SIGNIFICANCE AND IMPACT OF THE STUDY: Findings from this study shed light on one possible mechanism in which agricultural cephalosporin exposure in Salmonella may subsequently impact clinical treatment. The finding that cephalosporin exposure in donors may hinder the subsequent spread of resistance instead of aiding it up was counter-intuitive.

Detection of New Delhi Metallo-ß-lactamase 1 and Cephalosporin Resistance Genes Among Carbapenem-Resistant Enterobacteriaceae in Water Bodies Adjacent to Hospitals in India.

The prevalence of carbapenem resistance among bacterial isolates from selected water bodies receiving hospital effluents and adjoining aquaculture farms in Kerala, India, was studied. Klebsiella pneumoniae followed by Escherichia coli, Klebsiella oxytoca, Enterobacter aerogenes and Acinetobacter baumannii were the predominant isolates. Antibiotic sensitivity of these isolates was determined by Kirby-Bauer disc diffusion method. Nearly 60% of the Enterobacteriaceae isolates screened were multidrug resistant of which 16.6% were carbapenem resistant. The carbapenem-resistant Enterobacteriaceae were further screened for the presence of New Delhi metallo ß-lactamase-1 and cephalosporin resistance encoding genes. All NDM-1 isolates were highly resistant to carbapenem, cephalosporin, aminoglycosides, quinolones, tetracycline, and sulphonamides. K. pneumoniae harboring blaNDM-1 gene and E. coli isolates with blaCTX-M-15 and blaSHV-11 genes were detected in hospital discharge points. In aquaculture farms too, carbapenem-resistant K. pneumoniae with blaNDM-1 gene and E. coli isolates with blaCTX-M-15 were observed, although there was no use of antibiotics in these farms. However, other carbapenemase genes such as blaTEM, blaVIM, blaIMP and blaGIM were not detected in any of these isolates. The results suggest the increased prevalence of carbapenem-resistant Enterobacteriaceae in the water bodies receiving hospital effluent and its dissemination to adjacent aquaculture farms, posing a serious threat to public health.

Synergistic effects of functionally distinct substitutions in ß-lactamase variants shed light on the evolution of bacterial drug resistance.

The CTX-M ß-lactamases have emerged as the most widespread extended-spectrum ß-lactamases (ESBLs) in Gram-negative bacteria. These enzymes rapidly hydrolyze cefotaxime, but not the related cephalosporin, ceftazidime. ESBL variants have evolved, however, that provide enhanced ceftazidime resistance. We show here that a natural variant at a nonactive site, i.e. second-shell residue N106S, enhances enzyme stability but reduces catalytic efficiency for cefotaxime and ceftazidime and decreases resistance levels. However, when the N106S variant was combined with an active-site variant, D240G, that enhances enzyme catalytic efficiency, but decreases stability, the resultant double mutant exhibited higher resistance levels than predicted on the basis of the phenotypes of each variant. We found that this epistasis is due to compensatory effects, whereby increased stability provided by N106S overrides its cost of decreased catalytic activity. X-ray structures of the variant enzymes in complex with cefotaxime revealed conformational changes in the active-site loop spanning residues 103-106 that were caused by the N106S substitution and relieve steric strain to stabilize the enzyme, but also alter contacts with cefotaxime and thereby reduce catalytic activity. We noted that the 103-106 loop conformation in the N106S-containing variants is different from that of WT CTX-M but nearly identical to that of the non-ESBL, TEM-1 ß-lactamase, having a serine at the 106 position. Therefore, residue 106 may serve as a "switch" that toggles the conformations of the 103-106 loop. When it is serine, the loop is in the non-ESBL, TEM-like conformation, and when it is asparagine, the loop is in a CTX-M-like, cefotaximase-favorable conformation.

Genetic Characterization and Enhanced Surveillance of Ceftriaxone-Resistant Neisseria gonorrhoeae Strain, Alberta, Canada, 2018.

In July 2018, a case of Neisseria gonorrhoeae associated with ceftriaxone treatment failure was identified in Alberta, Canada. We identified the isolate and nucleic acid amplification testing (NAAT) specimen as the ceftriaxone-resistant strain multilocus sequence type 1903/NG-MAST 3435/NG-STAR 233, originally identified in Japan (FC428), with the same penA 60.001 mosaic allele and genetic resistance determinants. Core single-nucleotide variant (SNV) analysis identified 13 SNVs between this isolate and FC428. Culture-independent surveillance by PCR for the A311V mutation in the penA allele and N. gonorrhoeae multiantigen sequence typing directly from NAAT transport media positive for N. gonorrhoeae by NAAT did not detect spread of the strain. We identified multiple sequence types not previously detected in Alberta by routine surveillance. This case demonstrates the benefit of using culture-independent methods to enhance detection, public health investigations, and surveillance to address this global threat.

First Clinical Case of In Vivo Acquisition of DHA-1 Plasmid-Mediated AmpC in a Salmonella enterica subsp. enterica Isolate.

A pan-susceptible Salmonella enterica serovar Worthington isolate was detected in the stool of a man returning from Sri Lanka. Under ceftriaxone treatment, a third-generation cephalosporin (3GC)-resistant Salmonella Worthington was isolated after 8 days. Molecular analyses indicated that the two isolates were identical. However, the latter strain acquired a blaDHA-1-carrying IncFII plasmid probably from a Citrobacter amalonaticus isolate colonizing the gut. This is the first report of in vivo acquisition of plasmid-mediated resistance to 3GCs in S. enterica.

Circulation of Plasmids Harboring Resistance Genes to Quinolones and/or Extended-Spectrum Cephalosporins in Multiple Salmonella enterica Serotypes from Swine in the United States.

Nontyphoidal Salmonella enterica (NTS) poses a major public health risk worldwide that is amplified by the existence of antimicrobial-resistant strains, especially those resistant to quinolones and extended-spectrum cephalosporins (ESC). Little is known on the dissemination of plasmids harboring the acquired genetic determinants that confer resistance to these antimicrobials across NTS serotypes from livestock in the United States. NTS isolates (n = 183) from U.S. swine clinical cases retrieved during 2014 to 2016 were selected for sequencing based on their phenotypic resistance to enrofloxacin (quinolone) or ceftiofur (3rd-generation cephalosporin). De novo assemblies were used to identify chromosomal mutations and acquired antimicrobial resistance genes (AARGs). In addition, plasmids harboring AARGs were identified using short-read assemblies and characterized using a multistep approach that was validated by long-read sequencing. AARGs to quinolones [qnrB15, qnrB19, qnrB2, qnrD, qnrS1, qnrS2, and aac(6')Ib-cr] and ESC (blaCMY-2, blaCTX-M-1, blaCTX-M-27, and blaSHV-12) were distributed across serotypes and were harbored by several plasmids. In addition, chromosomal mutations associated with resistance to quinolones were identified in the target enzyme and efflux pump regulation genes. The predominant plasmid harboring the prevalent qnrB19 gene was distributed across serotypes. It was identical to a plasmid previously reported in S. enterica serovar Anatum from swine in the United States (GenBank accession number KY991369.1) and similar to Escherichia coli plasmids from humans in South America (GenBank accession numbers GQ374157.1 and JN979787.1). Our findings suggest that plasmids harboring AARGs encoding mechanisms of resistance to critically important antimicrobials are present in multiple NTS serotypes circulating in swine in the United States and can contribute to resistance expansion through horizontal transmission.

Characterization of CMY-2-type beta-lactamase-producing Escherichia coli isolated from chicken carcasses and human infection in a city of South Brazil.

BACKGROUND: Food-producing animals, mainly poultry, have been associated with the maintenance and dissemination of antibiotic-resistant bacteria, such as plasmid-mediated AmpC (pAmpC)-producing Enterobacteriaceae, to humans, thus impacting food safety. Many studies have shown that Escherichia coli strains isolated from poultry and humans infections share identical cephalosporin resistance, suggesting that transmission of resistance from poultry meat to humans may occur. The aim of this study was to characterize pAmpC-producing E. coli strains isolated from chicken carcasses and human infection in a restrict area and to determine their antimicrobial resistance profiles, and molecular type by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). RESULTS: A total of 14 pAmpC-producing E. coli strains were isolated, including eight strains from chicken carcasses and six strains from human infections (from urine, tissue and secretion). The blaCMY-2 gene was identified in all pAmpC-producing E. coli strains by polymerase chain reaction (PCR) and DNA sequencing. High percentages of strains resistant to tetracycline, nalidixic acid and sulfamethoxazole-trimethoprim (78-92%) were detected, all of which were considered multidrug-resistant. Among the non-beta-lactam resistance genes, the majority of the strains showed tetA, tetB, sulI and sulII. No strain was considered an extended-spectrum beta-lactamases (ESBL) producer, and the blaTEM-1 gene was found in 2 strains isolated from human infection. Six strains from chicken carcasses and four strains from humans infections were linked to an ISEcp1-like element. Through MLST, 11 sequence types were found. Three strains isolated from human infection and one strain isolated from chicken carcasses belonged to the same sequence type (ST354). However, considerable heterogeneity between the strains from chicken carcasses and humans was confirmed by PFGE analysis. CONCLUSION: This study showed the prevalence of E. coli strains producing blaCMY-2 linked to ISEcp1 that were present in both chickens and humans in a restricted area. Our results also suggest the presence of a highly diverse strains that harbor pAmpC, indicating no clonal dissemination. Therefore, continuous monitoring and comparative analyses of resistant bacteria from humans and food-producing animals are needed.

The role of extended-spectrum cephalosporin-resistance in recurrent community-onset Enterobacteriaceae urinary tract infections: a retrospective cohort study.

BACKGROUND: Bacterial resistance to first line antibiotics used to treat community-onset urinary tract infections (UTIs) continues to emerge. We sought to determine the association between extended-spectrum cephalosporin resistance (ESC-R) and recurrence among Enterobacteriaceae (EB) UTIs. METHODS: A retrospective cohort study was performed. All patients presenting to the Emergency Departments (EDs) or outpatient practices in a large health system with EB UTIs between 2010 and 2013 were included. Exposed patients had ESC-R EB UTIs. Unexposed patients had ESC-susceptible EB UTIs and were matched to exposed patients 1:1 on study year. Multivariable Cox proportional hazards regression analyses were performed to evaluate the association between ESC-R EB UTI and time to recurrent UTI within 12 months. RESULTS: A total of 302 patients with an index community-onset EB UTI were included, with 151 exposed and 151 unexposed. Overall, 163 (54%) patients experienced a recurrent UTI with a median time to recurrence of 69 days (interquartile range 25-183). On multivariable analyses, ESC-resistance was associated with an increased hazard of recurrent UTI (hazard ratio [HR] 1.39, 95% confidence interval [CI] 1.01-1.91, P = 0.04). Other variables that were independently associated with recurrence included a history of UTI prior to the index UTI and presence of a urinary catheter at the time of the index UTI. Secondarily, we found that when the treatment for the index UTI was adjusted for, there was no longer a significant association between ESC-R status and time to recurrent UTI (aHR 1.26, 95% CI 0.91-1.76, P = 0.17). CONCLUSIONS: Community-onset UTI due to EB demonstrating ESC-resistance is associated with a significantly increased hazard of recurrent UTI within 12 months compared to ESC-susceptible EB, even after adjusting for baseline factors that predispose patients to UTI recurrence. This association appears to be driven primarily by delayed or inappropriate treatment for the index ESC-R EB UTI.