CPHLN recommendations for the laboratory detection of Shiga toxin-producing Escherichia coli (O157 and non-O157).

Shiga toxin-producing Escherichia coli (STEC) are important enteric pathogens responsible for sporadic cases and outbreaks of gastroenteritis. E.coli O157:H7/NM (STEC O157) are the most commonly known STEC serotypes but it is now increasingly apparent that non-O157 STEC serotypes have been underreported in the past because they were not part of routine screening in many front-line laboratories. The Canadian Public Health Laboratory Network (CPHLN) has identified the need for improved detection and surveillance of non-O157 STEC and has developed the following recommendations to assist in the decision-making process for clinical and reference microbiology laboratories. These recommendations should be followed to the best of a laboratory's abilities based on the availability of technology and resources. The CPHLN recommends that when screening for the agents of bacterial gastroenteritis from a stool sample, front-line laboratories use either a chromogenic agar culture or a culture-independent diagnostic test (CIDT). CIDT options include nucleic acid amplification tests (NAATs) to detect Shiga toxin genes or enzyme immunoassays (EIAs) to detect Shiga toxins. If either CIDT method is positive for possible STEC, laboratories must have a mechanism to culture and isolate STEC in order to support both provincial and national surveillance as well as outbreak investigations and response. These CPHLN recommendations should result in improved detection of STEC in patients presenting with diarrhea, especially when due to the non-O157 serotypes. These measures should enhance the overall quality of healthcare and food safety, and provide better protection of the public via improved surveillance and outbreak detection and response.

Current trends of human infections and antibiotic resistance of the genus Shewanella.

Shewanella spp. are commonly known as environmental bacteria and are most frequently isolated from aquatic areas. Currently, diseases syndromes and multidrug resistance have increasingly been reported in the genus Shewanella. Some species are associated with various infections, such as skin and soft tissue infections, as well as bacteremia. Generally, these bacteria are opportunistic and mostly affect people with an impaired immune system. This genus is also a probable vehicle and progenitor of antibiotic resistance genes. In fact, several resistance genes and mobile genetic elements have been identified in some resistant species isolated from environmental or clinical settings. These genes confer resistance to different antibiotic classes, including those used in therapies such as ß-lactams and quinolones, and are generally located on the chromosome. Recently, a multidrug-resistant (MDR) plasmid harboring several drug resistance genes associated with transposons and integrons has been identified in Shewanella xiamenensis. These antibiotic resistance genes can circulate in the environment and contribute to the emergence of antibiotic resistance. This review describes different aspects of Shewanella, focusing on the infections caused by this genus, as well as their role in the propagation of antibiotic resistance via mobile genetic elements.

International outbreak of multiple Salmonella serotype infections linked to sprouted chia seed powder - USA and Canada, 2013-2014.

Salmonella is a leading cause of bacterial foodborne illness. We report the collaborative investigative efforts of US and Canadian public health officials during the 2013-2014 international outbreak of multiple Salmonella serotype infections linked to sprouted chia seed powder. The investigation included open-ended interviews of ill persons, traceback, product testing, facility inspections, and trace forward. Ninety-four persons infected with outbreak strains from 16 states and four provinces were identified; 21% were hospitalized and none died. Fifty-four (96%) of 56 persons who consumed chia seed powder, reported 13 different brands that traced back to a single Canadian firm, distributed by four US and eight Canadian companies. Laboratory testing yielded outbreak strains from leftover and intact product. Contaminated product was recalled. Although chia seed powder is a novel outbreak vehicle, sprouted seeds are recognized as an important cause of foodborne illness; firms should follow available guidance to reduce the risk of bacterial contamination during sprouting.

Salmonella Thompson outbreak associated with consumption of chicken shawarma and the usefulness of genome sequencing in the investigation.

BACKGROUND: A sudden increase in Salmonella Thompson (S. Thompson) cases distributed throughout three border regions in the province of Quebec in November 2016 triggered a provincial investigation to identify a common source of contamination and to put the appropriate control measures into place. OBJECTIVE: To report on the outbreak and to describe the use of genomic sequencing to identify the salmonella serotype responsible. METHODS: A descriptive survey of all reported cases of Salmonella serogroup C1 that had occurred between October 1, 2016 and February 15, 2017 was conducted. A case definition was developed. Pulsed field gel electrophoresis supplemented by analyses of genome sequences using the single nucleotide variant phylogenomics method were used to demarcate and manage the outbreak. RESULTS: Eighteen cases of S. Thompson were identified through whole genome sequencing. The onset dates of symptoms for the 16 cases that presented enteric symptoms were November 21-December 2, 2016. Two cases that presented with atypical symptoms were not reported until February 2017. Among the 18 cases, 16 had eaten or probably eaten chicken shawarma at the same restaurant chain and nine of these cases ate it at the same restaurant. In total, five restaurants from this chain, spread throughout three border regions of Quebec, were identified. CONCLUSION: Outbreaks associated with chicken shawarma have been identified in the past. Efforts must be made to ensure that the owners of this type of restaurant know the contamination risk associated with this type of cooking and take the necessary steps to reduce this risk. The use of the genome sequencing method was very useful in defining the outbreak.

Usefulness of High-Quality Core Genome Single-Nucleotide Variant Analysis for Subtyping the Highly Clonal and the Most Prevalent Salmonella enterica Serovar Heidelberg Clone in the Context of Outbreak Investigations.

Salmonella enterica serovar Heidelberg is the second most frequently occurring serovar in Quebec and the third-most prevalent in Canada. Given that conventional pulsed-field gel electrophoresis (PFGE) subtyping for common Salmonella serovars, such as S. Heidelberg, yields identical subtypes for the majority of isolates recovered, public health laboratories are desperate for new subtyping tools to resolve highly clonal S. Heidelberg strains involved in outbreak events. As PFGE was unable to discriminate isolates from three epidemiologically distinct outbreaks in Quebec, this study was conducted to evaluate whole-genome sequencing (WGS) and phylogenetic analysis as an alternative to conventional subtyping tools. Genomes of 46 isolates from 3 Quebec outbreaks (2012, 2013, and 2014) supported by strong epidemiological evidence were sequenced and analyzed using a high-quality core genome single-nucleotide variant (hqSNV) bioinformatics approach (SNV phylogenomics [SNVphyl] pipeline). Outbreaks were indistinguishable by conventional PFGE subtyping, exhibiting the same PFGE pattern (SHEXAI.0001/SHEBNI.0001). Phylogenetic analysis based on hqSNVs extracted from WGS separated the outbreak isolates into three distinct groups, 100% concordant with the epidemiological data. The minimum and maximum number of hqSNVs between isolates from the same outbreak was 0 and 4, respectively, while >59 hqSNVs were measured between 2 previously indistinguishable outbreaks having the same PFGE and phage type, thus corroborating their distinction as separate unrelated outbreaks. This study demonstrates that despite the previously reported high clonality of this serovar, the WGS-based hqSNV approach is a superior typing method, capable of resolving events that were previously indistinguishable using classic subtyping tools.

Outbreak of Shigella sonnei in Montréal's ultra-Orthodox Jewish community, 2015.

An outbreak of Shigella sonnei that occurred in the ultra-Orthodox Jewish community (UOJC) was the subject of an investigation and response by the Montréal Regional Public Health Department (DRSP), who collaborated with several health and community partners. A total of 27 confirmed cases were reported in this outbreak, which lasted from February to June 2015. The epidemic curve was compatible with a point source with secondary person-to-person transmission. In 11 of the 27 cases, pulsed-field gel electrophoresis (PFGE) analysis of strains found a single PFGE pattern newly identified in Quebec. Almost all strains tested showed resistance to ampicillin and trimethoprim-sulfamethoxazole (TMP/SMX). All the cases resided in Montréal Centre-West. Most of the cases were under 5 years old and attended a daycare centre, an environment recognized to be conducive to the transmission of enteric diseases. DRSP sent timely information to families, daycare and school stakeholders, community partners and synagogues in the UOJC, which helped reduce the transmission of shigellosis in the community.

Multiple-locus variable-number tandem-repeat analysis of Francisella tularensis from Quebec, Canada.

UNLABELLED: Francisella tularensis is ubiquitous in the Northern Hemisphere. Yet, little is known about the disease and its ecology within Canada as few serological studies have shown exposure to the disease and fewer case studies have been reported. This report is the first to describe the molecular subtyping of F. tularensis isolates within eastern Canada using multiple-locus variable-number tandem-repeat analysis. From 1998 to 2011, a total of 73 specimens were isolated from unique human and animal sources. As expected, F. tularensis subsp. tularensis AI and F. tularensis subsp. holarctica subtypes were observed, corresponding to the known geographical division within this species. The majority of human isolates (78%) and all animal (hare) isolates were of the more virulent, AI type. Half of the B isolates were isolated from patients living in a region of Quebec where muskrat densities are known to be high. A relatively high level of marker diversity was found, suggestive of multiple introductions of the organism to the region, or more likely ongoing endemicity. There was no evidence of ongoing outbreaks or transmission, and the bulk of cases were likely due to interaction between human activity and the environment (e.g. hunting/trapping activities). SIGNIFICANCE AND IMPACT OF THE STUDY: This study reveals the diversity of Francisella tularensis in eastern Canada using multiple-locus variable-number tandem-repeat analysis. It was initiated to further the understanding of the species within North America as previous studies elucidating the diversity and phylogeography of the species have consisted mostly of specimens from the United States. Type A tularaemia, the most life-threatening subtype of the species and a Category A biothreat agent, is restricted to North America, and this study serves to broaden the knowledge of the epidemiology and diversity of the organism.

A genetic linkage map of the soybean cyst nematode Heterodera glycines.

A genetic linkage map of the soybean cyst nematode (SCN) Heterodera glycines was constructed using a population of F2 individuals obtained from matings between two highly inbred SCN lines, TN16 and TN20. The AFLP fingerprinting technique was used to genotype 63 F2 progeny with two restriction enzyme combinations (EcoRI/MseI and PstI/TaqI) and 38 primer combinations. The same F2 population was also genotyped for Hg-cm-1 (H. glycines chorismate mutase-1), a putative virulence gene, using real-time quantitative PCR. Some of the markers were found to be distributed non-randomly. Even so, of the 230 markers analyzed, 131 could be mapped onto ten linkage groups at a minimum LOD of 3.0, for a total map distance of 539 cM. The Hg-cm-1 locus mapped to linkage group III together with 16 other markers. The size of the H. glycines genome was estimated to be in the range of 630-743 cM, indicating that the current map represents 73-86% of the genome, with a marker density of one per 4.5 cM, and a physical/genetic distance ratio of between 124 kb/cM and 147 kb/cM. This genetic map will be of great assistance in mapping H. glycines markers to genes of interest, such as nematode virulence genes and genes that control aspects of nematode parasitism.

'Candidatus pasteuria usgae' sp. nov., an obligate endoparasite of the phytoparasitic nematode Belonolaimus longicaudatus.

Taxonomically relevant characteristics of a fastidiously Gram-positive, obligately endoparasitic prokaryote (strain S-1) that uses the phytoparasitic sting nematode Belonolaimus longicaudatus as its host are reviewed. 16S rDNA sequence similarity (> or = 93%) confirms its congeneric ranking with other Pasteuria species and strains from nematodes and cladocerans and corroborates morphological, morphometric and host range evidence suggesting a novel taxon. The 16S rDNA sequence of strain S-1 has greatest similarity (96%) to the 16S rDNA sequences of both Pasteuria penetrans from root-knot nematodes (Meloidogyne species) and the recently reported strain of Pasteuria isolated from the soybean cyst nematode Heterodera glycines. Because the obligately endoparasitic nature of prokaryotes in the genus Pasteuria prevents isolation of definitive type strains, strain S-1 is proposed as 'Candidatus Pasteuria usgae' sp. nov.

Ultrastructure and Development of Pasteuria sp. (S-1 strain), an Obligate Endoparasite of Belonolaimus longicaudatus (Nemata: Tylenchida).

Pasteuria sp., strain S-1, is a gram-positive, obligate endoparasitic bacterium that uses the phytoparasitic sting nematode, Belonolaimus longicaudatus, as its host in Florida. The host attachment of S-1 appears to be specific to the genus Belonolaimus with development occurring only in juveniles and adults of B. longicaudatus. This bacterium is characterized from other described species of Pasteuria using ultrastructure of the mature endospore. Penetration, development, and sporogenesis were elucidated with TEM, LTSEM, and SEM and are similar to other nematode-specific Pasteuria. Recent analysis of 16S rDNA sequence homology confirms its congeneric ranking with other Pasteuria species and strains from nematodes and cladocerans, and corroborates ultrastructural, morphological, morphometric, and host-range evidence suggesting separate species status.

Phenotypic and molecular analysis of a pasteuria strain parasitic to the sting nematode.

Pasteuria strain S-1 was found to parasitize the sting nematode Belonolaimus longicaudatus. S-1 spores attached to several strains of B. longicaudatus from different geographical locations within the United States. However, they did not adhere to any of the following species: Heterodera schachtii, Longidorus africanus, Meloidogyne hapla, M. incognita, M. javanica, Pratylenchus brachyurus, P. scribneri, P. neglectus, P. penetrans, P. thornei, P. vulnus, and Xiphinema spp. The 16S rRNA genes from Pasteuria strain S-1 and P. penetrans strain Pp from Senegal were obtained by PCR amplification. A DNA sequence analysis showed that the S-1 16S rRNA had 96% or less similarity to the 16S rRNA genes from all previously reported Pasteuria species. Diverse phylogenetic methods all provided robust support for an association of Pasteuria strain S-1, Pasteuria strain NA parasitic to H. glycines, and P. penetrans strain Pp, to the exclusion of P. ramosa. In addition, our study showed intraspecific variation within P. penetrans as inferred by its 98% similarity to P. penetrans strain Pp.

Purification of Leuconostoc mesenteroides citrate lyase and cloning and characterization of the citCDEFG gene cluster.

A citrate lyase (EC 4.1.3.6) was purified 25-fold from Leuconostoc mesenteroides and was shown to contain three subunits. The first 42 amino acids of the beta subunit were identified, as well as an internal peptide sequence spanning some 20 amino acids into the alpha subunit. Using degenerated primers from these sequences, we amplified a 1.2-kb DNA fragment by PCR from Leuconostoc mesenteroides subsp. cremoris. This fragment was used as a probe for screening a Leuconostoc genomic bank to identify the structural genes. The 2.7-kb gene cluster encoding citrate lyase of L. mesenteroides is organized in three open reading frames, citD, citE, and citF, encoding, respectively, the three citrate lyase subunits gamma (acyl carrier protein [ACP]), beta (citryl-S-ACP lyase; EC 4.1.3.34), and alpha (citrate:acetyl-ACP transferase; EC 2.8.3.10). The gene (citC) encoding the citrate lyase ligase (EC 6.2.1.22) was localized in the region upstream of citD. Protein comparisons show similarities with the citrate lyase ligase and citrate lyase of Klebsiella pneumoniae and Haemophilus influenzae. Downstream of the citrate lyase cluster, a 1.4-kb open reading frame encoding a 52-kDa protein was found. The deduced protein is similar to CitG of the other bacteria, and its function remains unknown. Expression of the citCDEFG gene cluster in Escherichia coli led to the detection of a citrate lyase activity only in the presence of acetyl coenzyme A, which is a structural analog of the prosthetic group. This shows that the acetyl-ACP group of the citrate lyase form in E. coli is not complete or not linked to the protein.

Genetic diversity among a complex of cereal cyst nematodes inferred from RFLP analysis of the ribosomal internal transcribed spacer region.

This study examined the restriction polymorphism (RFLP) of the nuclear ribosomal DNA in Heterodera avenae, H. filipjevi, H. mani, H. latipons, and the taxonomically unclear Gotland strain in order to establish a molecular characterization and phylogenetic relationships in the complex of cereal cyst nematodes (CCN). The internal transcribed spacer (ITS) and 5.8S rDNA were amplified by PCR from a single female or a cyst of 27 different geographic isolates of the CCN complex and one population of H. schachtii, used as outgroup. The amplified product was 1.2 kb long and 14 of 15 enzymes produced restriction fragments for each isolate. Relationships between populations were determined from UPGMA analysis based on distance values calculated from RFLP data. Digestions with TaqI clearly differentiated H. avenae, H. latipons, and a group composed of H. filipjevi and the Gotland strain. Six endonucleases (HaeIII, HinfI, ItaI, PstI, TaqI, and Tru9I) produced the same restriction pattern with H. filipjevi and the Gotland strain, and both were clearly separated from H. avenae with PstI. Restriction sites have revealed a mixture of the species H. latipons and H. avenae, and possible infraspecific variation in H. avenae. The inferred phylogenetic relationships of species in the CCN complex are in agreement with their morphological characterization.