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Resistance to beta-lactam antimicrobials caused by extended-spectrum beta-lactamase (ESBL)-producing organisms is a global health concern. The objectives of this study were to (1) summarise the prevalence of potential ESBL-producing Escherichia coli (ESBL-EC) and Salmonella spp. (ESBL-SA) isolates from agrifood and human sources in Canada from 2012 to 2017, and (2) describe the distribution of ESBL genotypes among these isolates. All data were obtained from the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS). CIPARS analysed samples for the presence of ESBLs through phenotypic classification and identified beta-lactamase genes (blaTEM, blaSHV, blaCTX, blaOXA, blaCMY−2) using polymerase chain reaction (PCR) and whole genome sequencing (WGS). The prevalence of PCR-confirmed ESBL-EC in agrifood samples ranged from 0.5% to 3% across the surveillance years, and was detected most frequently in samples from broiler chicken farms. The overall prevalence of PCR-confirmed ESBL-SA varied between 1% and 4% between 2012 and 2017, and was most frequently detected in clinical isolates from domestic cattle. The TEM-CMY2 gene combination was the most frequently detected genotype for both ESBL-EC and ESBL-SA. The data suggest that the prevalence of ESBL-EC and ESBL-SA in Canada was low (i.e.

Antimicrobial resistance (AMR) has become a critical threat to public health worldwide. The use of antimicrobials in food and livestock agriculture, including the production of poultry, is thought to contribute to the dissemination of antibiotic resistant bacteria (ARB) and the genes and plasmids that confer the resistant phenotype (ARG). However, the relative contribution of each of these processes to the emergence of resistant pathogens in poultry production and their potential role in the transmission of resistant pathogens in human infections, requires a deeper understanding of the dynamics of ARB and ARG in food production and the factors involved in the increased risk of transmission.

We investigated whether gentamicin resistance (Gen(r)) in Escherichia coli isolates from human infections was related to Gen(r) E. coli in chicken and whether resistance may be due to coselection from use of lincomycin-spectinomycin in chickens on farms. Whole-genome sequencing was performed on 483 Gen(r) E. coli isolates isolated between 2014 and 2017. These included 205 human-source isolates collected by the Canadian Ward (CANWARD) program and 278 chicken-source isolates: 167 from live/recently slaughtered chickens (animals) and 111 from retail chicken meat collected by the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS). The predominant Gen(r) gene was different in human and chicken sources; however, both sources carried aac(3)-IId, aac(3)-VIa, and aac(3)-IVa. Forty-one percent of human clinical isolates of Gen(r) E. coli contained a bla(CTX-M) extended-spectrum beta-lactamase (ESBL) gene (84/205), and 53% of these were sequence type 131 (ST131). Phylogenomic analysis revealed a high diversity of Gen(r) isolates; however, there were three small clusters of closely related isolates from human and chicken sources. Gen(r) and spectinomycin resistance (Spec(r)) genes were colocated in 148/167 (89%) chicken animal isolates, 94/111 (85%) chicken retail meat isolates, and 137/205 (67%) human-source isolates. Long-read sequencing of 23 isolates showed linkage of the Gen(r) and Spec(r) genes on the same plasmid in 14/15 (93%) isolates from chicken(s) and 6/8 (75%) isolates from humans. The use of lincomycin-spectinomycin on farms may be coselecting for gentamicin-resistant plasmids in E. coli in broiler chickens; however, Gen(r) isolates and plasmids were mostly different in chickens and humans.

Non-typhoidal

Whole genome sequencing (WGS) of

We investigated whether the increased prevalence of gentamicin resistance in Salmonella from human infections was related to a similar increased prevalence in isolates from broiler chickens and whether this increase may have been due to coselection from use of lincomycin-spectinomycin in chickens on farms. Whole-genome sequencing was performed on gentamicin-resistant (Gen(r)) Salmonella isolates from human and chicken sources collected from 2014 to 2017 by the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS). We determined the genomic relatedness of strains and characterized resistance genes and plasmids. From 2014 to 2017, 247 isolates of Gen(r) Salmonella were identified by CIPARS: 188 were from humans, and 59 were from chicken sources (26 from live animals on farm and 33 from retail meat). The five most common Gen(r) serovars were Salmonella enterica serovars Heidelberg (n = 93; 31.5%), 4,[5],12:i:− (n = 42; 14.2%), Kentucky (n = 37; 12.5%), Infantis (n = 33; 11.2%), and Typhimurium (n = 23; 7.8%). Phylogenomic analysis revealed that for S. Heidelberg and S. Infantis, there were closely related isolates from human and chicken sources. In both sources, resistance to gentamicin and spectinomycin was most frequently conferred by aac(3)-VIa and ant(3′′)-Ia, respectively. Plasmid closure confirmed linkages of gentamicin and spectinomycin resistance genes and revealed instances of similar plasmids from both sources. Gentamicin and spectinomycin resistance genes were linked on the same plasmids, and some plasmids and isolates from humans and chickens were genetically similar, suggesting that the use of lincomycin-spectinomycin in chickens may be selecting for gentamicin-resistant Salmonella in broiler chickens and that these resistant strains may be acquired by humans.

Candida auris is an emerging yeast that is associated with antifungal resistance and healthcare-associated outbreaks. From 2012 to 2019, there were 24 known cases of C. auris colonization or infection in Canada. Isolates were from axilla/groin (n = 6), ear (n = 5), blood (n = 4), toe (n = 2), and a variety of other sites (n = 7). Canadian isolates belonged to the four main genomic clades: Clade I (formerly called South Asian clade, n = 12), Clade II (East Asian, n = 3), Clade III (African, n = 4), and Clade IV (South American, n = 5). Isolates within each clade were clonal; however, whole genome sequencing may be helpful in identifying clusters within healthcare facilities. Lay summary The fungal pathogen Candida auris has caused many hospital outbreaks and is often multidrug resistant. All four major strains of C. auris were identified in Canada from 2012 to 2019. Genomic epidemiology may be useful for identifying and reducing transmission of C. auris within hospitals.

Staphylococcus aureus chronic airway infection in patients with cystic fibrosis (CF) allows this pathogen to adapt over time in response to different selection pressures. We have previously shown that the main sequence types related to community-acquired methicillin-resistant S. aureus (MRSA) infections in Argentina – ST5 and ST30 – are also frequently isolated from the sputum of patients with CF, but in these patients they usually display multi-drug antimicrobial resistance. In this study, we sequenced the genomes of MRSA from four paediatric CF patients with the goal of identifying mutations among sequential isolates, especially those possibly related to antimicrobial resistance and virulence, which might contribute to the adaptation of the pathogen in the airways of patients with CF. Our results revealed genetic differences in sequential MRSA strains isolated from patients with CF in both their core and accessory genomes. Although the genetic adaptation of S. aureus was distinct in different hosts, we detected independent mutations in thyA, htrA, rpsJ and gyrA – which are known to have crucial roles in S. aureus virulence and antimicrobial resistance – in isolates recovered from multiple patients. Moreover, we identified allelic variants that were detected in all of the isolates recovered after a certain time point; these non-synonymous mutations were in genes associated with antimicrobial resistance, virulence, iron scavenging and oxidative stress resistance. In conclusion, our results provide evidence of genetic variability among sequential MRSA isolates that could be implicated in the adaptation of these strains during chronic CF airway infection.

Background Candida auris was first described in Japan in 2009 and has since been detected in over 40 countries. The yeast is concerning for multiple reasons, primarily: (1) challenges with accurate identification; (2) reported multidrug resistance; (3) published mortality rates of 30%-60%; and (4) persistence in the environment associated with human transmission. We report the emergence of a healthcare-associated cluster in the Greater Vancouver area in 2018 and describe the measures implemented to contain its transmission. Methods Cases were identified through passive and ring surveillance of affected wards. Positive isolates were sent to provincial and national reference laboratories for confirmation and genomic characterization. Extensive infection control measures were implemented immediately after the initial case was identified. Results Four cases were identified during the outbreak. In a 4-month period, over 700 swabs were collected in order to screen 180 contacts. Whole genome sequencing concluded that all isolates clustered together and belonged to the South Asian clade. No isolates harbored FKS gene mutations associated with resistance to echinocandins. Infection control measures, including surveillance, education, cleaning and/or disinfection, patient cohorting, isolation, and hand hygiene, effectively contained the outbreak; it was declared over within 2 months. Conclusions The spread of C auris in healthcare facilities has not spared Canadian institutions. Our experience demonstrates that strict infection control measures combined with microbiological screening can effectively halt transmission in healthcare centers. The necessity of active prospective screening remains unclear.

In less than a decade, C. auris has emerged in health care settings worldwide; this species is capable of colonizing skin and causing outbreaks of invasive candidiasis. In contrast to other Candida species, C. auris is unique in its ability to spread via nosocomial transmission and its high rates of drug resistance. As part of the public health response, whole-genome sequencing has played a major role in characterizing transmission dynamics and detecting new C. auris introductions. Through a global collaboration, we assessed genome evolution of isolates of C. auris from 19 countries. Here, we described estimated timing of the expansion of each C. auris clade and of fluconazole resistance, characterized discrete phylogeographic population structure of each clade, and compared genome data to sensitivity measurements to describe how antifungal resistance mechanisms vary across the population. These efforts are critical for a sustained, robust public health response that effectively utilizes molecular epidemiology., Candida auris has emerged globally as a multidrug-resistant yeast that can spread via nosocomial transmission. An initial phylogenetic study of isolates from Japan, India, Pakistan, South Africa, and Venezuela revealed four populations (clades I, II, III, and IV) corresponding to these geographic regions. Since this description, C. auris has been reported in more than 30 additional countries. To trace this global emergence, we compared the genomes of 304 C. auris isolates from 19 countries on six continents. We found that four predominant clades persist across wide geographic locations. We observed phylogeographic mixing in most clades; clade IV, with isolates mainly from South America, demonstrated the strongest phylogeographic substructure. C. auris isolates from two clades with opposite mating types were detected contemporaneously in a single health care facility in Kenya. We estimated a Bayesian molecular clock phylogeny and dated the origin of each clade within the last 360 years; outbreak-causing clusters from clades I, III, and IV originated 36 to 38 years ago. We observed high rates of antifungal resistance in clade I, including four isolates resistant to all three major classes of antifungals. Mutations that contribute to resistance varied between the clades, with Y132F in ERG11 as the most widespread mutation associated with azole resistance and S639P in FKS1 for echinocandin resistance. Copy number variants in ERG11 predominantly appeared in clade III and were associated with fluconazole resistance. These results provide a global context for the phylogeography, population structure, and mechanisms associated with antifungal resistance in C. auris.

We surveyed Canadian Nosocomial Infection Surveillance Program hospitals to evaluate infection prevention and microbiology laboratory preparedness for Candida auris. We identified significant gaps: most hospitals were not prepared to screen patients for colonization, and only one-half of laboratories reported identifying all clinically significant Candida isolates to the species level.

To better understand the contribution of wildlife to the dissemination of Salmonella and antimicrobial resistance in Salmonella and Escherichia coli, we examined whole-genome sequence data from Salmonella and E. coli isolates collected from raccoons (Procyon lotor) and environmental sources on farms in southern Ontario. All Salmonella and phenotypically resistant E. coli collected from raccoons, soil, and manure pits on five swine farms as part of a previous study were included. We assessed for evidence of potential transmission of these organisms between different sources and farms utilizing a combination of population structure assessments (using core-genome multi-locus sequence typing), direct comparisons of multi-drug resistant isolates, and epidemiological modeling of antimicrobial resistance (AMR) genes and plasmid incompatibility (Inc) types. Univariable logistic regression models were fit to assess the impact of source type, farm location, and sampling year on the occurrence of select resistance genes and Inc types. A total of 159 Salmonella and 96 resistant E. coli isolates were included. A diversity of Salmonella serovars and sequence types were identified, and, in some cases, we found similar or identical Salmonella isolates and resistance genes between raccoons, soil, and swine manure pits. Certain Inc types and resistance genes associated with source type were consistently more likely to be identified in isolates from raccoons than swine manure pits, suggesting that manure pits are not likely a primary source of those particular resistance determinants for raccoons. Overall, our data suggest that transmission of Salmonella and AMR determinants between raccoons and swine manure pits is uncommon, but soil-raccoon transmission appears to be occurring frequently. More comprehensive sampling of farms, and assessment of farms with other livestock species, as well as additional environmental sources (e.g., rivers) may help to further elucidate the movement of resistance genes between these various sources.

We have identified a Salmonella enterica serotype 4,[5],12:i:- containing a mcr-3.2 in a patient who travelled to Thailand 1 month prior to the identification of it in Canada. The isolate was multidrug resistant, but remained susceptible to the carbapenems, amikacin and piperacillin/tazobactam. The mcr-3.2 was carried on a 261 Kb variant of the IncHI2 pWJ1. This report provides further evidence of the emergence of a ST34 colistin-resistant clone.

A strain of extensively drug-resistant (XDR) Salmonella enterica serovar Typhi has caused a large ongoing outbreak in Pakistan since 2016. In Ontario, Canada, 10 cases of mainly bloodstream infections ( n = 9) were identified in patients who traveled to Pakistan. Whole-genome sequencing showed that Canadian cases were genetically related to the Pakistan outbreak strain. The appearance of XDR typhoid cases in Ontario prompted a provincial wide alert to physicians to recommend treatment with carbapenems or azithromycin in suspected typhoid cases with travel history to Pakistan.

We report three cases of hospital-acquired mucormycosis in heart and lung transplant patients over a 6-month period. Traditional epidemiological investigation tools were used to look for a common link between patients to explain the outbreak. Genome sequencing of each fungal strain was used to supplement the investigation. By disproving a close genetic link between infecting strains of mucormycosis, we were able to conclude the outbreak investigation. Genome sequencing is a novel tool that can be used in addition to traditional epidemiologic investigations to help determine linkage of patients during outbreak investigations.

Salmonella enterica subsp. enterica serovar Dublin is a zoonotic pathogen that often leads to invasive bloodstream infections in humans that are multidrug resistant. Described here are the results of Canadian national surveillance of S. Dublin from 2003 to 2015 in humans and bovines, principally collected through the Canadian Integrated Program for Antibiotic Resistance Surveillance (CIPARS). An increase in human infections due to multidrug-resistant (MDR) S. Dublin was observed in 2010, many of which were bloodstream infections. Phylogenomic analysis of human and bovine isolates revealed a closely related network that differed by only 0 to 17 single nucleotide variants (SNVs), suggesting some potential transmission between humans and bovines. Phylogenomic comparison of global publicly available sequences of S. Dublin showed that Canadian isolates clustered closely with those from the United States. A high correlation between phenotypic and genotypic antimicrobial susceptibility was observed in Canadian isolates. IS26 replication was widespread among U.S. and Canadian isolates and caused the truncation and inactivation of the resistance genes strA and bla(TEM-1B). A hybrid virulence and MDR plasmid (pN13-01125) isolated from a Canadian S. Dublin isolate was searched against NCBI SRA data of bacteria. The pN13-01125 coding sequences were found in 13 Salmonella serovars, but S. Dublin appears to be a specific reservoir. In summary, we have observed the rise of invasive MDR S. Dublin in humans in Canada and found that they are closely related to bovine isolates and to American isolates in their mobile and chromosomal contents.

Purpose Antimicrobial resistance (AMR), especially multidrug resistance, is one of the most serious global threats facing public health. The authors proof‐of‐concept study assessing the suitability of shotgun proteomics as an additional approach to whole‐genome sequencing (WGS) for detecting AMR determinants. Experimental Design Previously published shotgun proteomics and WGS data on four isolates of Campylobacter jejuni are used to perform AMR detection by searching the Comprehensive Antibiotic Resistance Database, and their detection ability relative to genomics screening and traditional phenotypic testing measured by minimum inhibitory concentration is assessed. Results Both genomic and proteomic approaches identify the wild‐type and variant molecular determinants responsible for resistance to tetracycline and ciprofloxacin, in agreement with phenotypic testing. In contrast, the genomic method identifies the presence of the β‐lactamase gene, bla OXA ‐ 61, in three isolates. However, its corresponding protein product is detected in only a single isolate, consistent with results obtained from phenotypic testing.

Pan-aminoglycoside (amikacin, gentamicin, tobramycin, plazomicin)–resistant Escherichia coli and Klebsiella pneumoniae clinical isolates from patients in Canadian Hospitals (2013–2017) were evaluated by whole genome sequencing for 16S ribosomal RNA methyltransferase genes. The rmtB gene was detected in 2 isolates (1 of 3094 E. coli [0.03%], 1 of 1039 K. pneumoniae [0.1%]).

To characterize the relationship between penicillin-binding protein 2 (PBP2/ penA ) and susceptibility to extended-spectrum cephalosporins (ESCs) and carbapenem antibiotics, we compared 17 PBP2 variants in Neisseria gonorrhoeae . Nonmosaic and mosaic variants of PBP2 caused decreased susceptibility to ESCs and, to a lesser extent, to carbapenems. An A501P substitution in mosaic XXXIV_A501P conferred decreased susceptibility to ESCs but restored carbapenem susceptibility to wild-type levels. These results could aid the molecular surveillance of antimicrobial resistance to these agents.

First description of rpsJ and mepA mutations associated with tigecycline resistance in Staphylococcus aureus isolated from a cystic fibrosis patient during antibiotic therapy. Fil: Haim, Maria Sol. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina Fil: Di Gregorio, Sabrina Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina Fil: Galanternik, L.. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina Fil: Lubovich, S.. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina Fil: Vázquez, M.. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina Fil: Zaheer, A.. Public Health Agency; Canadá Fil: Golding, G. R.. Public Health Agency; Canadá Fil: Graham, M.. Public Health Agency; Canadá Fil: Van Domselaar, G.. Public Health Agency; Canadá Fil: Cardona, S. T.. Public Health Agency; Canadá Fil: Mollerach, Marta Eugenia. Public Health Agency; Canadá. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Bharat, R.. Public Health Agency; Canadá

A curated Web-based user-friendly sequence typing tool based on antimicrobial resistance determinants in Neisseria gonorrhoeae was developed and is publicly accessible ( https://ngstar.canada.ca ). The N. gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR) molecular typing scheme uses the DNA sequences of 7 genes ( penA , mtrR , porB , ponA , gyrA , parC , and 23S rRNA) associated with resistance to β-lactam antimicrobials, macrolides, or fluoroquinolones. NG-STAR uses the entire penA sequence, combining the historical nomenclature for penA types I to XXXVIII with novel nucleotide sequence designations; the full mtrR sequence and a portion of its promoter region; portions of ponA , porB , gyrA , and parC ; and 23S rRNA sequences. NG-STAR grouped 768 isolates into 139 sequence types (STs) ( n = 660) consisting of 29 clonal complexes (CCs) having a maximum of a single-locus variation, and 76 NG-STAR STs ( n = 109) were identified as unrelated singletons. NG-STAR had a high Simpson's diversity index value of 96.5% (95% confidence interval [CI] = 0.959 to 0.969). The most common STs were NG-STAR ST-90 ( n = 100; 13.0%), ST-42 and ST-91 ( n = 45; 5.9%), ST-64 ( n = 44; 5.72%), and ST-139 ( n = 42; 5.5%). Decreased susceptibility to azithromycin was associated with NG-STAR ST-58, ST-61, ST-64, ST-79, ST-91, and ST-139 ( n = 156; 92.3%); decreased susceptibility to cephalosporins was associated with NG-STAR ST-90, ST-91, and ST-97 ( n = 162; 94.2%); and ciprofloxacin resistance was associated with NG-STAR ST-26, ST-90, ST-91, ST-97, ST-150, and ST-158 ( n = 196; 98.0%). All isolates of NG-STAR ST-42, ST-43, ST-63, ST-81, and ST-160 ( n = 106) were susceptible to all four antimicrobials. The standardization of nomenclature associated with antimicrobial resistance determinants through an internationally available database will facilitate the monitoring of the global dissemination of antimicrobial-resistant N. gonorrhoeae strains.

High-throughput screening (HTS) of chemical and microbial strain collections is an indispensable tool for modern chemical and systems biology; however, HTS data sets have inherent systematic and random error, which may lead to false-positive or false-negative results. Several methods of normalization of data exist; nevertheless, due to the limitations of each, no single method has been universally adopted. Here, we present a method of data visualization and normalization that is effective, intuitive, and easy to implement in a spreadsheet program. For each plate, the data are ordered by ascending values and a plot thereof yields a curve that is a signature of the plate data. Curve shape characteristics provide intuitive visualization of the frequency and strength of inhibitors, activators, and noise on the plate, allowing potentially problematic plates to be flagged. To reduce plate-to-plate variation, the data can be normalized by the mean of the middle 50% of ordered values, also called the interquartile mean (IQM) or the 50% trimmed mean of the plate. Positional effects due to bias in columns, rows, or wells can be corrected using the interquartile mean of each well position across all plates (IQM W ) as a second level of normalization. We illustrate the utility of this method using data sets from biochemical and phenotypic screens.

The EngA protein is a conserved and essential bacterial GTPase of largely enigmatic function. While most investigations of EngA have suggested a role in ribosome assembly, the protein has also been implicated in diverse elements of physiology including chromosome segregation, cell division and cell cycle control. Here, we have probed additional phenotypes related to ribosome biogenesis on depletion of EngA in Escherichia coli to better understand its role in the cell. Depletion of EngA resulted in cold sensitive growth and stimulation of a ribosomal rRNA promoter, both phenotypes associated with the disruption of ribosome biogenesis in bacteria. Amongst antibiotics that inhibit translation, depletion of EngA resulted in sensitization to the aminoglycoside class of antibiotics. EngA bound the alarmone ppGpp with equally high affinity as it bound GDP. These data offer additional support for a role in ribosome biogenesis for EngA, possibly in maturation of the A-site of the 50S subunit.

A case of an IMP-27-positive Morganella morganii isolate is reported, where the carbapenemase enzyme was demonstrated by whole genome sequencing. Carbapenemase detection using a multiplex PCR assay was negative due to mutations in the primer binding site. This case serves to illustrate the limitations of multiplex PCR for carbapenemase detection.

We developed a real-time PCR assay to detect single nucleotide polymorphisms associated with ciprofloxacin resistance in specimens submitted for nucleic acid amplification testing (NAAT). All three single nucleotide polymorphism (SNP) targets produced high sensitivity and specificity values. The presence of ≥2 SNPs was sufficient to predict ciprofloxacin resistance in an organism.

Published in the Journal of Biomolecular Screening, doi: 10.1177/1087057113486001 The synthesis of ribosomes is an essential process, which is aided by a variety of transacting factors in bacteria. Among these is a group of GTPases essential for bacterial viability and emerging as promising targets for new antibacterial agents. Herein, we describe a robust high-throughput screening process for inhibitors of one such GTPase, the Escherichia coli EngA protein. The primary screen employed an assay of phosphate production in 384-well density.Reaction conditions were chosen to maximize sensitivity for the discovery of competitive inhibitors while maintaining a strong signal amplitude and low noise. In a pilot screen of 31,800 chemical compounds, 44 active compounds were identified. Further, we describe the elimination of non-specific inhibitors that were detergent-sensitive or reactive as well as those that interfered with the high-throughput phosphate assay. Four inhibitors survived these common counterscreens for non-specificity but these chemicals were also inhibitors of the unrelated enzyme dihydrofolate reductase, suggesting that they too were promiscuously active. The highthroughput screen of the EngA protein described here provides a meticulous pilot study in the search for specific inhibitors of GTPases involved in ribosome biogenesis.

Conventional efforts to describe essential genes in bacteria have typically emphasized nutrient-rich growth conditions. Of note, however, are the set of genes that become essential when bacteria are grown under nutrient stress. For example, more than 100 genes become indispensable when the model bacterium Escherichia coli is grown on nutrient-limited media, and many of these nutrient stress genes have also been shown to be important for the growth of various bacterial pathogens in vivo. To better understand the genetic network that underpins nutrient stress in E. coli, we performed a genome-scale cross of strains harboring deletions in some 82 nutrient stress genes with the entire E. coli gene deletion collection (Keio) to create 315,400 double deletion mutants. An analysis of the growth of the resulting strains on rich microbiological media revealed an average of 23 synthetic sick or lethal genetic interactions for each nutrient stress gene, suggesting that the network defining nutrient stress is surprisingly complex. A vast majority of these interactions involved genes of unknown function or genes of unrelated pathways. The most profound synthetic lethal interactions were between nutrient acquisition and biosynthesis. Further, the interaction map reveals remarkable metabolic robustness in E. coli through pathway redundancies. In all, the genetic interaction network provides a powerful tool to mine and identify missing links in nutrient synthesis and to further characterize genes of unknown function in E. coli. Moreover, understanding of bacterial growth under nutrient stress could aid in the development of novel antibiotic discovery platforms., IMPORTANCE With the rise of antibiotic drug resistance, there is an urgent need for new antibacterial drugs. Here, we studied a group of genes that are essential for the growth of Escherichia coli under nutrient limitation, culture conditions that arguably better represent nutrient availability during an infection than rich microbiological media. Indeed, many such nutrient stress genes are essential for infection in a variety of pathogens. Thus, the respective proteins represent a pool of potential new targets for antibacterial drugs that have been largely unexplored. We have created all possible double deletion mutants through a genetic cross of nutrient stress genes and the E. coli deletion collection. An analysis of the growth of the resulting clones on rich media revealed a robust, dense, and complex network for nutrient acquisition and biosynthesis. Importantly, our data reveal new genetic connections to guide innovative approaches for the development of new antibacterial compounds targeting bacteria under nutrient stress.

Neisseria gonorrhoeae strains with coresistance to the first-line antimicrobial treatments azithromycin and ceftriaxone are an emerging public health threat. Here, we present the complete genome sequences of three strains of N. gonorrhoeae , including one susceptible strain and two strains with coresistance to ceftriaxone and azithromycin.

The emergence of Neisseria gonorrhoeae strains with decreased susceptibility to cephalosporins and azithromycin (AZM) resistance (AZM r ) represents a public health threat of untreatable gonorrhea infections. Genomic epidemiology through whole-genome sequencing was used to describe the emergence, dissemination, and spread of AZM r strains. The genomes of 213 AZM r and 23 AZM-susceptible N. gonorrhoeae isolates collected in Canada from 1989 to 2014 were sequenced. Core single nucleotide polymorphism (SNP) phylogenomic analysis resolved 246 isolates into 13 lineages. High-level AZM r (MICs ≥ 256 μg/ml) was found in 5 phylogenetically diverse isolates, all of which possessed the A2059G mutation ( Escherichia coli numbering) in all four 23S rRNA alleles. One isolate with high-level AZM r collected in 2009 concurrently had decreased susceptibility to ceftriaxone (MIC = 0.125 μg/ml). An increase in the number of 23S rRNA alleles with the C2611T mutations ( E. coli numbering) conferred low to moderate levels of AZM r (MICs = 2 to 4 and 8 to 32 μg/ml, respectively). Low-level AZM r was also associated with mtrR promoter mutations, including the −35A deletion and the presence of Neisseria meningitidis -like sequences. Geographic and temporal phylogenetic clustering indicates that emergent AZM r strains arise independently and can then rapidly expand clonally in a region through local sexual networks.

Background C. auris is a rapidly emerging pathogen which is potentially multidrug resistant, has caused large hospital outbreaks, and is difficult to identify in the routine microbiology laboratory. We surveyed CNISP sites to evaluate infection prevention and control (IPAC) and microbiology laboratory (MICRO) preparedness. Methods An electronic survey with five IPAC and 12 MICRO questions was sent out to IPAC and MICRO leads for all CNISP sites in January 2018. Data were entered and analyzed in Excel. Results We received 32 IPAC surveys representing 58/66 (88%) CNISP hospitals, and 27 MICRO surveys representing 27/32 (84%) CNISP labs. Four of 58 (7%) hospitals have a written policy for C. auris screening of patients; and 22 (38%) recommend screening; most commonly: roommates of any patient colonized/infected with any C. auris (n = 7), room/wardmates (RWM) of patients colonized/infected with any C. auris (n = 7) or RWM of patients with MDR C. auris (n = 3). Without resource limitations, 50 (86%) hospitals would screen RWM of C. auris patients and 34 (59%) would screen patients previously hospitalized in the Indian subcontinent. Overall, 13/27 (48%) labs identify all clinically significant Candida spp. to the species level and 13 identify sterile site (SS) isolates. Twenty-two (81%) labs use MALDI-TOF for identification: 10 Bruker Biotyper and 12 VitekMS. 26 (96%) labs refer non-identified species and commonly misidentified yeast from SS for definitive identification. Twenty-three (85%) labs perform antifungal susceptibility testing for all Candida from blood and CSF. Twenty-two (81%) labs are confident that their current laboratory protocol would identify C. auris if the isolate is from an SS, 17 (63%) if identified as being resistant to at least 1 antifungal and 20 (74%) if the isolate is from a non-SS culture and is identified to the species level. Four (15%) labs have a protocol for C. auriscolonization detection. Four labs have identified six C. auris isolates: two reported retrospective identification of three fluconazole susceptible C. auris; and two reported one resistant and two MDR isolates identified prospectively in 2017/2018. Conclusion MDR C. auris have been identified in Canada. Gaps remain in ensuring reliable identification of C. auris, particularly from non-SS, and most IPAC CNISP teams and MICRO do not yet have protocols for identification of C. auriscolonization. Disclosures All authors: No reported disclosures.

A robust and sensitive platform was developed for chemical-genomics in bacteria. Kinetic acquisitions of colony growth enable calculation of growth rates alongside conventional endpoint volume measurements, generating a wealth of chemical-genetic interactions. This kinetic platform is highly amenable to prokaryotic or eukaryotic strain collections., While genetic perturbation has been the conventional route to probing bacterial systems, small molecules are showing great promise as probes for cellular complexity. Indeed, systematic investigations of chemical-genetic interactions can provide new insights into cell networks and are often starting points for understanding the mechanism of action of novel chemical probes. We have developed a robust and sensitive platform for chemical-genomic investigations in bacteria. The approach monitors colony volume kinetically using transmissive scanning measurements, enabling acquisition of growth rates and conventional endpoint measurements. We found that chemical-genomic profiles were highly sensitive to concentration, necessitating careful selection of compound concentrations. Roughly 20,000,000 data points were collected for 15 different antibiotics. While 1052 chemical-genetic interactions were identified using the conventional endpoint biomass approach, adding interactions in growth rate resulted in 1564 interactions, a 50–200% increase depending on the drug, with many genes uncharacterized or poorly annotated. The chemical-genetic interaction maps generated from these data reveal common genes likely involved in multidrug resistance. Additionally, the maps identified deletion backgrounds exhibiting class-specific potentiation, revealing conceivable targets for combination approaches to drug discovery. This open platform is highly amenable to kinetic screening of any arrayable strain collection, be it prokaryotic or eukaryotic.

Antimicrobial resistant Neisseria gonorrhoeae is a major concern to public health due to decreased susceptibility to frontline antimicrobials. To find agents that are active against N. gonorrhoeae, we tested antimicrobials alone or in combination by Etest gradient strips. The potencies (as assessed by minimum inhibitory concentrations) of twenty-five antimicrobials were evaluated against nine reference strains of N. gonorrhoeae (WHO F, G, K, L, M, N, O, P and ATCC 49226). Potency was greatest for netilmicin, quinupristin-dalfopristin, ceftriaxone, ertapenem and piperacillin-tazobactam. Combinations of azithromycin, moxifloxacin, or gentamicin with ceftriaxone, doripenem, or aztreonam were tested against reference isolates and the fractional inhibitory concentration index (FICI) was calculated. All nine combinations resulted in indifference (>0.5 FICI ≤ 4). Combinations with FICI

The incidence of antimicrobial-resistant Neisseria gonorrhoeae continues to rise in Canada; however, antimicrobial resistance data are lacking for approximately 70% of gonorrhea infections that are diagnosed directly from clinical specimens by nucleic acid amplification tests (NAATs). We developed a molecular assay for surveillance use to detect mutations in genes associated with decreased susceptibility to cephalosporins that can be applied to both culture isolates and clinical samples. Real-time PCR assays were developed to detect single nucleotide polymorphisms (SNPs) in ponA , mtrR , penA , porB , and one N. gonorrhoeae -specific marker ( porA ). We tested the real-time PCR assay with 252 gonococcal isolates, 50 nongonococcal isolates, 24 N. gonorrhoeae -negative NAAT specimens, and 34 N. gonorrhoeae -positive NAAT specimens. Twenty-four of the N. gonorrhoeae -positive NAAT specimens had matched culture isolates. Assay results were confirmed by comparison with whole-genome sequencing data. For 252 N. gonorrhoeae strains, the agreement between the DNA sequence and real-time PCR was 100% for porA , ponA , and penA , 99.6% for mtrR , and 95.2% for porB . The presence of ≥2 SNPs correlated with decreased susceptibility to ceftriaxone (sensitivities of >98%) and cefixime (sensitivities of >96%). Of 24 NAAT specimens with matched cultures, the agreement between the DNA sequence and real-time PCR was 100% for porB , 95.8% for ponA and mtrR , and 91.7% for penA . We demonstrated the utility of a real-time PCR assay for sensitive detection of known markers for the decreased susceptibility to cephalosporins in N. gonorrhoeae . Preliminary results with clinical NAAT specimens were also promising, as they correlated well with bacterial culture results.

To probe the cellular phenotype and biochemical function associated with the G domains of Escherichia coli EngA (YfgK, Der), mutations were created in the phosphate binding loop of each. Neither an S16A nor an S217A variant of G domain 1 or 2, respectively, was able to support growth of an engA conditional null. Polysome profiles of EngA-depleted cells were significantly altered, and His 6 -EngA was found to cofractionate with the 50S ribosomal subunit. The variants were unable to complement the abnormal polysome profile and were furthermore significantly impacted with respect to in vitro GTPase activity. Together, these observations suggest that the G domains have a cooperative function in ribosome stability and/or biogenesis.