Overcoming Microbial Inhibition of S. Sonnei Through the Exploitation of Genomically Predicted Antibiotic Resistance Profiles for the Development of Food Enrichment Media.

Linking outbreaks of Shigella spp. to specific foods is challenging due to poor selectivity of current enrichment media. We have previously shown that enrichment media, tailored to the genomically-predicted antimicrobial resistance (AMR) of Shiga toxigenic E. coli strains, enhances their isolation from foods. This study investigates the application of this approach for Shigella isolation. The AMR gene profiles of 21,908 published S. sonnei genomes indicated a high prevalence of genes conferring resistance to streptomycin (aadA, aph(3″)-Ib, aph(6)-Id, 92.8%), sulfonamides (sul1, sul2, 74.8%), and/or trimethoprim (dfrA, 96.2%). Genomic analysis and antibiotic susceptibility testing conducted with a panel of 17 outbreak-associated S. sonnei strains confirmed the correlation of AMR gene detection with resistance phenotypes. Supplementation of Shigella Broth (SB) with up to 400 µg/mL of trimethoprim or sulfadiazine did not suppress the growth of sensitive strains, whereas 100 µg/mL of streptomycin increased the selectivity of this broth. All three antibiotics increased the selectivity of modified Tryptone Soya Broth (mTSB). Based on these results, supplemented media formulations were developed and assessed by measuring the relative growth of S. sonnei in cultures coinoculated with a strain of bacteriocin-producing E. coli that is inhibitory to Shigella growth. S. sonnei was not recovered from cocultures grown in SB or mTSB without antibiotics. In contrast, media supplemented with streptomycin at 50 and 100 µg/mL, trimethoprim at 25 and 50 µg/mL, and sulfadiazine at 100 µg/mL increased the relative proportion of S. sonnei in postenrichment cultures. The enhanced recovery of resistant S. sonnei strains achieved in this study indicates that, in cases where genomic data are available for clinical S. sonnei isolates, customization of selective enrichment media based on AMR gene detection could be a valuable tool for supporting the investigation of foodborne shigellosis outbreaks.

Strain-specific Recovery of S. sonnei from Artificially Contaminated Baby Carrots: Enhancing Food-safety Investigations with a Customized Shigella Detection Method Based on Genomically predicted Antibiotic Resistance Traits.

Shigella spp. are Gram-negative gastrointestinal bacterial pathogens that cause bacillary dysentery or shigellosis in humans. Isolation of Shigella from outbreak-associated foods is often problematic due to the lack of selectivity of cultural enrichment broths. To facilitate Shigella recovery from foods, we have developed strain-specific enrichment media based on the genomically-predicted antimicrobial resistance (AMR) features of an outbreak-associated Shigella sonnei strain harboring resistance genes for streptomycin (STR) and trimethoprim (TMP). To assess performance of the method, baby carrots were artificially contaminated with the S. sonnei strain at low (2.4 CFU), medium (23.5 CFU), and high levels (235 CFU) along with 10-fold higher levels of a Shigella-inhibiting Escherichia coli strain. The target S. sonnei strain was successfully recovered from artificially-contaminated baby carrots when enriched in modified Tryptone Soya Broth (mTSB) supplemented with TMP, whereas Shigella was not recovered from Shigella broth (SB) or SB supplemented with STR. Quantitative PCR analysis indicated that supplementation of the enrichment broths with TMP or STR increased the relative proportion of S. sonnei in enrichment cultures, except at the lowest inoculation level for STR. Microbiome profiling of the baby carrot enrichment cultures conducted by 16S rRNA gene sequencing indicated that both SB-STR and mTSB-TMP repressed the growth of competing Enterobacteriaceae in the enrichment cultures, relative to SB without supplementation. Overall, improved Shigella recovery was achieved with the addition of the appropriate custom selective agent during cultural enrichments demonstrating that genomically informed custom selective enrichment of Shigella could be a valuable tool for supporting future foodborne shigellosis outbreak investigations.

Analysis of Antimicrobial Resistance in Bacterial Pathogens Recovered from Food and Human Sources: Insights from 639,087 Bacterial Whole-Genome Sequences in the NCBI Pathogen Detection Database.

Understanding the role of foods in the emergence and spread of antimicrobial resistance necessitates the initial documentation of antibiotic resistance genes within bacterial species found in foods. Here, the NCBI Pathogen Detection database was used to query antimicrobial resistance gene prevalence in foodborne and human clinical bacterial isolates. Of the 1,843,630 sequence entries, 639,087 (34.7%) were assigned to foodborne or human clinical sources with 147,788 (23.14%) from food and 427,614 (76.88%) from humans. The majority of foodborne isolates were either Salmonella (47.88%), Campylobacter (23.03%), Escherichia (11.79%), or Listeria (11.3%), and the remaining 6% belonged to 20 other genera. Most foodborne isolates were from meat/poultry (95,251 or 64.45%), followed by multi-product mixed food sources (29,892 or 20.23%) and fish/seafood (6503 or 4.4%); however, the most prominent isolation source varied depending on the genus/species. Resistance gene carriage also varied depending on isolation source and genus/species. Of note, Klebsiella pneumoniae and Enterobacter spp. carried larger proportions of the quinolone resistance gene qnrS and some clinically relevant beta-lactam resistance genes in comparison to Salmonella and Escherichia coli. The prevalence of mec in S. aureus did not significantly differ between meat/poultry and multi-product sources relative to clinical sources, whereas this resistance was rare in isolates from dairy sources. The proportion of biocide resistance in Bacillus and Escherichia was significantly higher in clinical isolates compared to many foodborne sources but significantly lower in clinical Listeria compared to foodborne Listeria. This work exposes the gaps in current publicly available sequence data repositories, which are largely composed of clinical isolates and are biased towards specific highly abundant pathogenic species. We also highlight the importance of requiring and curating metadata on sequence submission to not only ensure correct information and data interpretation but also foster efficient analysis, sharing, and collaboration. To effectively monitor resistance carriage in food production, additional work on sequencing and characterizing AMR carriage in common commensal foodborne bacteria is critical.

Modeling the limits of detection for antimicrobial resistance genes in agri-food samples: a comparative analysis of bioinformatics tools.

BACKGROUND: Although the spread of antimicrobial resistance (AMR) through food and its production poses a significant concern, there is limited research on the prevalence of AMR bacteria in various agri-food products. Sequencing technologies are increasingly being used to track the spread of AMR genes (ARGs) in bacteria, and metagenomics has the potential to bypass some of the limitations of single isolate characterization by allowing simultaneous analysis of the agri-food product microbiome and associated resistome. However, metagenomics may still be hindered by methodological biases, presence of eukaryotic DNA, and difficulties in detecting low abundance targets within an attainable sequence coverage. The goal of this study was to assess whether limits of detection of ARGs in agri-food metagenomes were influenced by sample type and bioinformatic approaches. RESULTS: We simulated metagenomes containing different proportions of AMR pathogens and analysed them for taxonomic composition and ARGs using several common bioinformatic tools. Kraken2/Bracken estimates of species abundance were closest to expected values. However, analysis by both Kraken2/Bracken indicated presence of organisms not included in the synthetic metagenomes. Metaphlan3/Metaphlan4 analysis of community composition was more specific but with lower sensitivity than the Kraken2/Bracken analysis. Accurate detection of ARGs dropped drastically below 5X isolate genome coverage. However, it was sometimes possible to detect ARGs and closely related alleles at lower coverage levels if using a lower ARG-target coverage cutoff (< 80%). While KMA and CARD-RGI only predicted presence of expected ARG-targets or closely related gene-alleles, SRST2 (which allows read to map to multiple targets) falsely reported presence of distantly related ARGs at all isolate genome coverage levels. The presence of background microbiota in metagenomes influenced the accuracy of ARG detection by KMA, resulting in mcr-1 detection at 0.1X isolate coverage in the lettuce but not in the beef metagenome. CONCLUSIONS: This study demonstrates accurate detection of ARGs in synthetic metagenomes using various bioinformatic methods, provided that reads from the ARG-encoding organism exceed approximately 5X isolate coverage (i.e. 0.4% of a 40 million read metagenome). While lowering thresholds for target gene detection improved sensitivity, this led to the identification of alternative ARG-alleles, potentially confounding the identification of critical ARGs in the resistome. Further advancements in sequencing technologies providing increased coverage depth or extended read lengths may improve ARG detection in agri-food metagenomic samples, enabling use of this approach for tracking clinically important ARGs in agri-food samples.

Development and delivery of a higher diploma in emergency medicine and critical care for clinical officers in Kenya.

The provision of emergency medicine and critical care in a cost-efficient manner has the potential to address many preventable deaths in low- and middle-income countries. Here, utilising Kern's framework for curriculum development, we describe the origins, development and implementation of the Emergency Medicine and Critical Care Clinical Officer training program; Kenya's first training programme for clinical officers in emergency medicine and critical care. Graduates are scattered across the country in diverse settings, ranging from national referral hospitals in the capital, Nairobi, to rural hospitals in northern Kenya. In these locations, they provide clinical care, leadership, and teaching. Similar programmes could be replicated in other locations to help plug the gap in critical care provision in Sub-Saharan Africa.

Global Prevalence of Severe Neonatal Jaundice among Hospital Admissions: A Systematic Review and Meta-Analysis.

Evidence regarding the adverse burden of severe neonatal jaundice (SNJ) in hospitalized neonates in resource-constrained settings is sparse. We attempted to determine the prevalence of SNJ, described using clinical outcome markers, in all World Health Organization (WHO) regions in the world. Data were sourced from Ovid Medline, Ovid Embase, Cochrane Library, African Journals Online, and Global Index Medicus. Hospital-based studies, including the total number of neonatal admissions with at least one clinical outcome marker of SNJ, defined as acute bilirubin encephalopathy (ABE), exchange blood transfusions (EBT), jaundice-related death, or abnormal brainstem audio-evoked response (aBAER), were independently reviewed for inclusion in this meta-analysis. Of 84 articles, 64 (76.19%) were from low- and lower-middle-income countries (LMICs), and 14.26% of the represented neonates with jaundice in these studies had SNJ. The prevelance of SNJ among all admitted neonates varied across WHO regions, ranging from 0.73 to 3.34%. Among all neonatal admissions, SNJ clinical outcome markers for EBT ranged from 0.74 to 3.81%, with the highest percentage observed in the African and South-East Asian regions; ABE ranged from 0.16 to 2.75%, with the highest percentages observed in the African and Eastern Mediterranean regions; and jaundice-related deaths ranged from 0 to 1.49%, with the highest percentage observed in the African and Eastern Mediterranean regions. Among the cohort of neonates with jaundice, the prevalence of SNJ ranged from 8.31 to 31.49%, with the highest percentage observed in the African region; EBT ranged from 9.76 to 28.97%, with the highest percentages reported for the African region; ABE was highest in the Eastern Mediterranean (22.73%) and African regions (14.51%). Jaundice-related deaths were 13.02%, 7.52%, 2.01% and 0.07%, respectively, in the Eastern Mediterranean, African, South-East Asian and European regions, with none reported in the Americas. aBAER numbers were too small, and the Western Pacific region was represented by only one study, limiting the ability to make regional comparisons. The global burden of SNJ in hospitalized neonates remains high, causing substantial, preventable morbidity and mortality especially in LMICs.

Exploiting a targeted resistome sequencing approach in assessing antimicrobial resistance in retail foods.

BACKGROUND: With the escalating risk of antimicrobial resistance (AMR), there are limited analytical options available that can comprehensively assess the burden of AMR carried by clinical/environmental samples. Food can be a potential source of AMR bacteria for humans, but its significance in driving the clinical spread of AMR remains unclear, largely due to the lack of holistic-yet-sensitive tools for surveillance and evaluation. Metagenomics is a culture-independent approach well suited for uncovering genetic determinants of defined microbial traits, such as AMR, present within unknown bacterial communities. Despite its popularity, the conventional approach of non-selectively sequencing a sample's metagenome (namely, shotgun-metagenomics) has several technical drawbacks that lead to uncertainty about its effectiveness for AMR assessment; for instance, the low discovery rate of resistance-associated genes due to their naturally small genomic footprint within the vast metagenome. Here, we describe the development of a targeted resistome sequencing method and demonstrate its application in the characterization of the AMR gene profile of bacteria associated with several retail foods. RESULT: A targeted-metagenomic sequencing workflow using a customized bait-capture system targeting over 4,000 referenced AMR genes and 263 plasmid replicon sequences was validated against both mock and sample-derived bacterial community preparations. Compared to shotgun-metagenomics, the targeted method consistently provided for improved recovery of resistance gene targets with a much-improved target detection efficiency (> 300-fold). Targeted resistome analyses conducted on 36 retail-acquired food samples (fresh sprouts, n = 10; ground meat, n = 26) and their corresponding bacterial enrichment cultures (n = 36) reveals in-depth features regarding the identity and diversity of AMR genes, most of which were otherwise undetected by the whole-metagenome shotgun sequencing method. Furthermore, our findings suggest that foodborne Gammaproteobacteria could be the major reservoir of food-associated AMR genetic determinants, and that the resistome structure of the selected high-risk food commodities are, to a large extent, dictated by microbiome composition. CONCLUSIONS: For metagenomic sequencing-based surveillance of AMR, the target-capture method presented herein represents a more sensitive and efficient approach to evaluate the resistome profile of complex food or environmental samples. This study also further implicates retail foods as carriers of diverse resistance-conferring genes indicating a potential impact on the dissemination of AMR.

Development and delivery of a higher diploma in emergency medicine and critical care for clinical officers in Kenya

The provision of emergency medicine and critical care in a cost-efficient manner has the potential to address many preventable deaths in low- and middle-income countries. Here, utilising Kern's framework for curriculum development, we describe the origins, development and implementation of the Emergency Medicine and Critical Care Clinical Officer training program; Kenya's first training programme for clinical officers in emergency medicine and critical care. Graduates are scattered across the country in diverse settings, ranging from national referral hospitals in the capital, Nairobi, to rural hospitals in northern Kenya. In these locations, they provide clinical care, leadership, and teaching. Similar programmes could be replicated in other locations to help plug the gap in critical care provision in Sub-Saharan Africa.

Impact of a Mechanical Ventilation Curriculum on Respiratory Therapist Recognition of Patient-Ventilator Asynchrony.

BACKGROUND: Respiratory therapists (RTs) play a crucial role in managing mechanically ventilated patients, such as addressing patient-ventilator asynchronies that may contribute to patient harm. Waveform analysis is integral to the evaluation of patient-ventilator asynchronies; despite this, no published studies have assessed the ability of practicing RTs to interpret ventilator waveform abnormalities. METHODS: The study took place between June 2017-February 2019. Eighty-six RTs from 2 academic medical centers enrolled in a one-day mechanical ventilation course. The scores of 79 first-time attendees were included in the analysis. Prior to and following the course, RTs were asked to identify abnormalities on a 5-question, multiple-choice ventilator waveform exam. They were also asked to provide a self-assessment of their ventilator management skills on a 1 (complete novice) to 5 (expert) scale. RESULTS: Initial scores were low but improved after one day of ventilator instruction (19.4 ± 17.1 vs 29.6 ± 19.0, P < .001). No significant difference was noted in mean confidence levels between the pre- and post-course assessments (3.8 ± 0.9 vs 3.8 ± 1.0, P = .56). RTs with fewer years of clinical experience (0-10 y) had a statistically significant improvement in their post-course test scores relative to their pre-course scores (0-5 y: 12.5 ± 10.1 to 46.0 ± 10.8, P < .001; 6-10 y: 18.7 ± 15.8 to 32.1 ± 16.7, P = .02), whereas those with > 11 y of clinical experience did not (11-20 y: 22.4 ± 15.5 to 27.4 ± 19.0, P = .44; 21+ y: 19.6 ± 22.1 to 15.3 ± 13.8, P = .50). CONCLUSIONS: RTs may benefit from additional training in ventilator waveform interpretation, especially early in their clinical training. More work is needed to determine the optimal length and content of a mechanical ventilation curriculum for RTs.

Microbial Antagonism in Food-Enrichment Culture: Inhibition of Shiga Toxin-Producing Escherichia coli and Shigella Species.

Bacterial pathogens, such as Shiga toxin-producing Escherichia coli (STEC) and Shigella spp., are important causes of foodborne illness internationally. Recovery of these organisms from foods is critical for food safety investigations to support attribution of illnesses to specific food commodities; however, isolation of bacterial cultures can be challenging. Methods for the isolation of STEC and Shigella spp. from foods typically require enrichment to amplify target organisms to detectable levels. Yet, during enrichment, target organisms can be outcompeted by other bacteria in food matrices due to faster growth rates, or through production of antimicrobial agents such as bacteriocins or bacteriophages. The purpose of this study was to evaluate the occurrence of Shigella and STEC inhibitors produced by food microbiota. The production of antimicrobial compounds in cell-free extracts from 200 bacterial strains and 332 food-enrichment broths was assessed. Cell-free extracts produced by 23 (11.5%) of the strains tested inhibited growth of at least one of the five Shigella and seven STEC indicator strains used in this study. Of the 332 enrichment broths tested, cell-free extracts from 25 (7.5%) samples inhibited growth of at least one of the indicator strains tested. Inhibition was most commonly associated with E. coli recovered from meat products. Most of the inhibiting compounds were determined to be proteinaceous (34 of the 48 positive samples, 71%; including 17 strains, 17 foods) based on inactivation by proteolytic enzymes, indicating presence of bacteriocins. The cell-free extracts from 13 samples (27%, eight strains, five foods) were determined to contain bacteriophages based on the observation of plaques in diluted extracts and/or resistance to proteolytic enzymes. These results indicate that the production of inhibitors by food microbiota may be an important challenge for the recovery of foodborne pathogens, particularly for Shigella sonnei. The performance of enrichment media for recovery of Shigella and STEC could be improved by mitigating the impact of inhibitors produced by food microbiota during the enrichment process.

How I Teach Auto-PEEP: Applying the Physiology of Expiration.

Teaching complex topics in mechanical ventilation can prove challenging for clinical educators, both at the bedside and in the classroom setting. Some of these topics, such as the topic of auto-positive end-expiratory pressure (auto-PEEP), consist of complicated physiological principles that can be difficult to convey in an organized and intuitive manner. In this entry of "How I Teach," we provide an approach to teaching the concept of auto-PEEP to senior residents and fellows working in the intensive care unit. We offer a framework for educators to effectively present the concepts of auto-PEEP to learners, either at the bedside or in the classroom setting, by summarizing key concepts and including concrete examples of the educational techniques we use. This framework includes specific content we emphasize, how to present this content using a variety of educational resources, assessing learner understanding, and how to modify the topic on the basis of location, time, or resource constraints.

Systematic Evaluation of Whole-Genome Sequencing Based Prediction of Antimicrobial Resistance in Campylobacter jejuni and C. coli.

The increasing prevalence of antimicrobial resistance (AMR) in Campylobacter spp. is a global concern. This study evaluated the use of whole-genome sequencing (WGS) to predict AMR in Campylobacter jejuni and C. coli. A panel of 271 isolates recovered from Canadian poultry was used to compare AMR genotype to antimicrobial susceptibility testing (AST) results (azithromycin, ciprofloxacin, erythromycin, gentamicin, tetracycline, florfenicol, nalidixic acid, telithromycin, and clindamycin). The presence of antibiotic resistance genes (ARGs) was determined for each isolate using five computational approaches to evaluate the effect of: ARG screening software, input data (i.e., raw reads, draft genome assemblies), genome coverage and genome assembly software. Overall, concordance between the genotype and phenotype was influenced by the computational pipelines, level of genome coverage and the type of ARG but not by input data. For example, three of the pipelines showed a 99% agreement between detection of a tet(O) gene and tetracycline resistance, whereas agreement between the detection of tet(O) and TET resistance was 98 and 93% for two pipelines. Overall, higher levels of genome coverage were needed to reliably detect some ARGs; for example, at 15X coverage a tet(O) gene was detected in >70% of the genomes, compared to <60% of the genomes for bla(OXA). No genes associated with florfenicol or gentamicin resistance were found in the set of strains included in this study, consistent with AST results. Macrolide and fluoroquinolone resistance was associated 100% with mutations in the 23S rRNA (A2075G) and gyrA (T86I) genes, respectively. A lower association between a A2075G 23S rRNA gene mutation and resistance to clindamycin and telithromycin (92.8 and 78.6%, respectively) was found. While WGS is an effective approach to predicting AMR in Campylobacter, this study demonstrated the impact that computational pipelines, genome coverage and the genes can have on the reliable identification of an AMR genotype.

Genomic Markers for Quaternary Ammonium Compound Resistance as a Persistence Indicator for Listeria monocytogenes Contamination in Food Manufacturing Environments.

ABSTRACT: Persistent contamination of food manufacturing environments by Listeria monocytogenes is an important public health risk, because such contamination events defy standard sanitization protocols, for example, the application of quaternary ammonium compounds such as benzalkonium chloride (BC), providing a source for prolonged dissemination of the bacteria in food products. We performed whole genome sequencing analyses of 1,279 well-characterized L. monocytogenes isolates from various foods and food manufacturing environments and identified the bcrABC gene cassette associated with BC resistance in 531 (41.5%) isolates. The bcrABC cassette was significantly associated with L. monocytogenes isolates belonging to clonal complex (CC) 321, CC155, CC204, and CC199, which are among the 10 most prevalent genotypes recovered from foods and food production environments. All but 1 of the 177 CC321 isolates harbored the bcrABC cassette. In addition, 384 (38.6%) of the 994 isolates recovered from foods representing 67 different CCs and 119 (59.2%) of isolates from food manufacturing environmental samples representing 26 different CCs were found to harbor the intact bcrABC cassette. A representative set of 69 isolates with and without bcrABC was assayed for the ability to grow in the presence of BC, and 34 of 35 isolates harboring the bcrABC cassette exhibited MICs of ≥10 µg/mL BC. Determination of bcrABC in isolates could be achieved using both PCR and whole genome sequencing techniques, providing food testing laboratories with options for the characterization of isolates. The ability to determine markers of quaternary ammonium compound resistance such as bcrABC and epidemiologic lineage may provide risk managers with a tool to assess the potential for persistent contamination of the food manufacturing environment and the need for more targeted surveillance to ensure the efficacy of mitigation actions.

Pragmatic Recommendations for the Management of COVID-19 Patients with Shock in Low- and Middle-Income Countries.

As some patients infected with the novel coronavirus progress to critical illness, a subset will eventually develop shock. High-quality data on management of these patients are scarce, and further investigation will provide valuable information in the context of the pandemic. A group of experts identify a set of pragmatic recommendations for the care of patients with SARS-CoV-2 and shock in resource-limited environments. We define shock as life-threatening circulatory failure that results in inadequate tissue perfusion and cellular dysoxia/hypoxia, and suggest that it can be operationalized via clinical observations. We suggest a thorough evaluation for other potential causes of shock and suggest against indiscriminate testing for coinfections. We suggest the use of the quick Sequential Organ Failure Assessment (qSOFA) as a simple bedside prognostic score for COVID-19 patients and point-of-care ultrasound (POCUS) to evaluate the etiology of shock. Regarding fluid therapy for the treatment of COVID-19 patients with shock in low-middle-income countries, we favor balanced crystalloids and recommend using a conservative fluid strategy for resuscitation. Where available and not prohibited by cost, we recommend using norepinephrine, given its safety profile. We favor avoiding the routine use of central venous or arterial catheters, where availability and costs are strong considerations. We also recommend using low-dose corticosteroids in patients with refractory shock. In addressing targets of resuscitation, we recommend the use of simple bedside parameters such as capillary refill time and suggest that POCUS be used to assess the need for further fluid resuscitation, if available.

Pragmatic Recommendations for Therapeutics of Hospitalized COVID-19 Patients in Low- and Middle-Income Countries.

The therapeutic options for COVID-19 patients are currently limited, but numerous randomized controlled trials are being completed, and many are on the way. For COVID-19 patients in low- and middle-income countries (LMICs), we recommend against using remdesivir outside of a clinical trial. We recommend against using hydroxychloroquine ± azithromycin or lopinavir-ritonavir. We suggest empiric antimicrobial treatment for likely coinfecting pathogens if an alternative infectious cause is likely. We suggest close monitoring without additional empiric antimicrobials if there are no clinical or laboratory signs of other infections. We recommend using oral or intravenous low-dose dexamethasone in adults with COVID-19 disease who require oxygen or mechanical ventilation. We recommend against using dexamethasone in patients with COVID-19 who do not require supplemental oxygen. We recommend using alternate equivalent doses of steroids in the event that dexamethasone is unavailable. We also recommend using low-dose corticosteroids in patients with refractory shock requiring vasopressor support. We recommend against the use of convalescent plasma and interleukin-6 inhibitors, such as tocilizumab, for the treatment of COVID-19 in LMICs outside of clinical trials.

Centrifugal microfluidic lab-on-a-chip system with automated sample lysis, DNA amplification and microarray hybridization for identification of enterohemorrhagic Escherichia coli culture isolates.

The development of technology for the rapid, automated identification of bacterial culture isolates can help regulatory agencies to shorten response times in food safety surveillance, compliance, and enforcement as well as outbreak investigations. While molecular methods such as polymerase chain reaction (PCR) enable the identification of microbial organisms with high sensitivity and specificity, they generally rely on sophisticated instrumentation and elaborate workflows for sample preparation with an undesirably high level of hands-on engagement. Herein, we describe the design, operation and performance of a lab-on-a-chip system integrating thermal lysis, PCR amplification and microarray hybridization on the same cartridge. The assay is performed on a centrifugal microfluidic platform that allows for pneumatic actuation of liquids during rotation, making it possible to perform all fluidic operations in a fully-automated fashion without the need for integrating active control elements on the microfluidic cartridge. The cartridge, which is fabricated from hard and soft thermoplastic polymers, is compatible with high-volume manufacturing (e.g., injection molding). Chip design and thermal interface were both optimized to ensure efficient heat transfer and allow for fast thermal cycling during the PCR process. The integrated workflow comprises 14 steps and takes less than 2 h to complete. The only manual steps are related to loading of the sample and reagents on the cartridge as well as fluorescence imaging of the microarray. On-chip lysis and PCR amplification both provided results comparable to those obtained by bench-top instrumentation. The microarray, incorporating a panel of oligonucleotide probes for multiplexed detection of seven enterohemorrhagic E. coli priority serotypes, was implemented on a cyclic olefin copolymer substrate using a novel activation scheme that involves the conversion of hydroxyl groups (derived from oxygen plasma treatment) into reactive cyanate ester using cyanogen bromide. On-chip hybridization was demonstrated in a non-quantitative fashion using fluorescently-labelled gene markers for E. coli O157:H7 (rfbO157, eae, vt1, and vt2) obtained through a multiplexed PCR amplification step.

Systematic Evaluation of Whole Genome Sequence-Based Predictions of Salmonella Serotype and Antimicrobial Resistance.

Whole-genome sequencing (WGS) is used increasingly in public-health laboratories for typing and characterizing foodborne pathogens. To evaluate the performance of existing bioinformatic tools for in silico prediction of antimicrobial resistance (AMR) and serotypes of Salmonella enterica, WGS-based genotype predictions were compared with the results of traditional phenotyping assays. A total of 111 S. enterica isolates recovered from a Canadian baseline study on broiler chicken conducted in 2012-2013 were selected based on phenotypic resistance to 15 different antibiotics and isolates were subjected to WGS. Both SeqSero2 and SISTR accurately determined S. enterica serotypes, with full matches to laboratory results for 87.4 and 89.2% of isolates, respectively, and partial matches for the remaining isolates. Antimicrobial resistance genes (ARGs) were identified using several bioinformatics tools including the Comprehensive Antibiotic Resistance Database - Resistance Gene Identifier (CARD-RGI), Center for Genomic Epidemiology (CGE) ResFinder web tool, Short Read Sequence Typing for Bacterial Pathogens (SRST2 v 0.2.0), and k-mer alignment method (KMA v 1.17). All ARG identification tools had ≥ 99% accuracy for predicting resistance to all antibiotics tested except streptomycin (accuracy 94.6%). Evaluation of ARG detection in assembled versus raw-read WGS data found minimal observable differences that were gene- and coverage- dependent. Where initial phenotypic results indicated isolates were sensitive, yet ARGs were detected, repeat AMR testing corrected discrepancies. All tools failed to find resistance-determining genes for one gentamicin- and two streptomycin-resistant isolates. Further investigation found a single nucleotide polymorphism (SNP) in the nuoF coding region of one of the isolates which may be responsible for the observed streptomycin-resistant phenotype. Overall, WGS-based predictions of AMR and serotype were highly concordant with phenotype determination regardless of computational approach used.

Polymer Micropillar Arrays for Colorimetric DNA Detection.

We describe the use of periodic micropillar arrays, produced from cyclic olefin copolymer using high-fidelity microfabrication, as templates for colorimetric DNA detection. The assay involves PCR-amplified gene markers for E. coli O157:H7 (rfbO157, eae, vt1, and vt2) incorporating a detectable digoxigenin label, which is revealed through an immunoenzymatic process following hybridization with target-specific oligonucleotide capture probes. The capacity of micropillar arrays to induce wicking is used to distribute and confine capture probes with spatial control, making it possible to achieve a uniform signal while allowing multiple, independent probes to be arranged in close proximity on the same substrate. The kinetic profile of color pigment formation on the surface was followed using absorbance measurements, showing maximum signal increase between 20 and 60 min of reaction time. The relationship between microstructure and colorimetric signal was investigated through variation of geometric parameters, such as pitch (10-50 µm), pillar diameter (5-40 µm), and height (16-48 µm). Our findings suggest that signal intensity is largely influenced by the edges of the pillars and less by their height such that it deviates from a linear relationship when both aspect ratio and pillar density become very high. A theoretical model used to simulate the changes in surface composition at the molecular level suggests that differences in the temporal and spatial accumulation of assay components account for this observation.