Dobryan M. Tracz

Limited genetic diversity in Salmonella enterica Serovar Enteritidis PT13

BackgroundSalmonella enterica serovar Enteritidis has emerged as a significant foodborne pathogen throughout the world and is commonly characterized by phage typing. In Canada phage types (PT) 4, 8 and 13 predominate and in 2005 a large foodborne PT13 outbreak occurred in the province of Ontario. The ability to link strains during this outbreak was difficult due to the apparent clonality of PT13 isolates in Canada, as there was a single dominant pulsed-field gel electrophoresis (PFGE) profile amongst epidemiologically linked human and food isolates as well as concurrent sporadic strains. The aim of this study was to perform comparative genomic hybridization (CGH), DNA sequence-based typing (SBT) genomic analyses, plasmid analyses, and automated repetitive sequence-based PCR (rep-PCR) to identify epidemiologically significant traits capable of subtyping S. Enteritidis PT13.ResultsCGH using an oligonucleotide array based upon chromosomal coding sequences of S. enterica serovar Typhimurium strain LT2 and the Salmonella genomic island 1 successfully determined major genetic differences between S. Typhimurium and S. Enteritidis PT13, but no significant strain-to-strain differences were observed between S. Enteritidis PT13 isolates. Individual loci (safA and fliC) that were identified as potentially divergent in the CGH data set were sequenced in a panel of S. Enteritidis strains, and no differences were detected between the PT13 strains. Additional sequence-based typing was performed at the fimA, mdh, manB, cyaA, citT, caiC, dmsA, ratA and STM0660 loci. Similarly, no diversity was observed amongst PT13 strains. Variation in plasmid content between PT13 strains was observed, but macrorestriction with Bgl II did not identify further differences. Automated rep-PCR patterns were variable between serovars, but S. Enteritidis PT13 strains could not be differentiated.ConclusionNone of the methods identified any significant variation between PT13 strains. Greater than 11,300 base pairs of sequence for each of seven S. Enteritidis PT13 strains were analyzed without detecting a single polymorphic site, although diversity between different phage types of S. Enteritidis was observed. These data suggest that Canadian S. Enteritidis PT13 strains are highly related genetically.

Genetic characterization of clinical and agri-food isolates of multi drug resistant Salmonella enterica serovar Heidelberg from Canada

BackgroundSalmonella enterica serovar Heidelberg ranks amongst the most prevalent causes of human salmonellosis in Canada and an increase in resistance to extended spectrum cephalosporins (ESC) has been observed by the Canadian Integrated Program for Antimicrobial Resistance Surveillance. This study examined the genetic relationship between S. Heidelberg isolates from livestock, abattoir, retail meat, and clinical human specimens to determine whether there was a link between the emergence of MDR S. Heidelberg in chicken agri-food sources and the simultaneous increase of MDR S. Heidelberg in human clinical samples.ResultsChromosomal genetic homogeneity was observed by pulsed-field gel electrophoresis (PFGE), DNA sequence-based typing (SBT) and DNA microarray-based comparative genomic hybridization (CGH). Sixty one percent of isolates were indistinguishable by PFGE conducted using Xba I and Bln I restriction enzymes. An additional 15% of isolates had PFGE patterns that were closely related to the main cluster. SBT did not identify DNA polymorphisms and CGH revealed only genetic differences between the reference S. Typhimurium strain and S. Heidelberg isolates. Genetic variation observed by CGH between S. Heidelberg isolates could be attributed to experimental variation. Alternatively, plasmid content was responsible for differences in antimicrobial susceptibility, and restriction fragment length polymorphism (RFLP) analyses followed by replicon typing identified two divergent plasmid types responsible for ESC resistance.ConclusionDue to the overall limited genetic diversity among the isolates, it was not possible to identify variable traits that would be suitable for source tracking between human and agri-food isolates of S. Heidelberg in Canada.

Genetic Determinants and Polymorphisms Specific for Human-Adapted Serovars of Salmonella enterica That Cause Enteric Fever

ABSTRACT Salmonella enterica serovars Typhi, Paratyphi A, and Sendai are human-adapted pathogens that cause typhoid (enteric) fever. The acute prevalence in some global regions and the disease severity of typhoidal Salmonella have necessitated the development of rapid and specific detection tests. Most of the methodologies currently used to detect serovar Typhi do not identify serovars Paratyphi A or Sendai. To assist in this aim, comparative sequence analyses were performed at the loci of core bacterial genetic determinants and Salmonella pathogenicity island 2 genes encoded by clinically significant S. enterica serovars. Genetic polymorphisms specific for serovar Typhi (at trpS), as well as polymorphisms unique to human-adapted typhoidal serovars (at sseC and sseF), were observed. Furthermore, entire coding sequences unique to human-adapted typhoidal Salmonella strains (i.e., serovar-specific genetic loci rather than polymorphisms) were observed in publicly available comparative genomic DNA microarray data sets. These polymorphisms and loci were developed into real-time PCR, standard PCR, and liquid microsphere suspension array-based molecular protocols and tested for with a panel of clinical and reference subspecies I S. enterica strains. A proportion of the nontyphoidal Salmonella strains hybridized with the allele-specific oligonucleotide probes for sseC and sseF; but the trpS allele was unique to serovar Typhi (with a singular serovar Paratyphi B strain as an exception), and the coding sequences STY4220 and STY4221 were unique among serovars Typhi, Paratyphi A, and Sendai. These determinants provided phylogenetic data on the genetic relatedness of serovars Typhi, Paratyphi A, and Sendai; and the protocols developed might allow the rapid identification of these Salmonella serovars that cause enteric fever.

Use of the espZ Gene Encoded in the Locus of Enterocyte Effacement for Molecular Typing of Shiga Toxin-Producing Escherichia coli

ABSTRACT Infections with Shiga toxin-producing Escherichia coli (STEC) result in frequent cases of sporadic and outbreak-associated enteric bacterial disease in humans. Classification of STEC is by stx genotype (encoding the Shiga toxins), O and H antigen serotype, and seropathotype (subgroupings based upon the clinical relevance and virulence-related genotypes of individual serotypes). The espZ gene is encoded in the locus of enterocyte effacement (LEE) pathogenicity island responsible for the attaching and effacing (A/E) lesions caused by various E. coli pathogens (but not limited to STEC), and this individual gene (∼300 bp) has previously been identified as hypervariable among these A/E pathogens. Sequence analysis of the espZ locus encoded by additional STEC serotypes and strains (including O26:H11, O121:H19, O111:NM, O145:NM, O165:H25, O121:NM, O157:NM, O157:H7, and O5:NM) indicated that distinct sequence variants exist which correlate to subgroups among these serotypes. Allelic discrimination at the espZ locus was achieved using Light Upon eXtension real-time PCR and by liquid microsphere suspension arrays. The allele subtype of espZ did not correlate with STEC seropathotype classification; however, a correlation with the allele type of the LEE-encoded intimin (eae) gene was supported, and these sequence variations were conserved among individual serotypes. The study focused on the characterization of three clinically significant seropathotypes of LEE-positive STEC, and we have used the observed genetic variation at a pathogen-specific locus for detection and subtyping of STEC.

Isolation and Genetic Characterization of a Coinfection of Non-O157 Shiga Toxin-Producing Escherichia coli

ABSTRACT A coinfection of O177:NM and O55:H7 Shiga toxin-producing Escherichia coli (STEC) was identified for a child with acute bloody diarrhea and hemolytic uremic syndrome by using culture and serotype-specific molecular reagents. The profile of O157-related genetic islands revealed that the O55:H7 isolate was highly similar to O157 STEC whereas the O177:NM isolate lacked several fimbrial O islands and non-locus-of-enterocyte-effacement effector determinants. However, both STEC serotypes are known to cause serious disease, and the significant repertoire of virulence determinants in both strains made it impossible to determine their individual contributions to the clinical symptoms.

Multilocus Sequence Typing of Escherichia coli O26:H11 Isolates Carrying stx in Canada Does Not Identify Genetic Diversity

ABSTRACT Multilocus sequence typing of 31 stx-carrying Escherichia coli O26:H11 strains isolated in Canada between 1999 and 2003 revealed a high degree of genetic relatedness at 10 loci, suggesting either that this is a clonal serotype (similar to O157:H7) or that additional genetic loci need to be examined.

Effect of gamma radiation on the identification of bacterial pathogens by MALDI-TOF MS.

MALDI-TOF MS is a well-established method for rapid identification of bacteria; however there are no reports to date on its performance with gamma-irradiated samples typically used in BSL-3 laboratories for sample inactivation. In this report we demonstrate that gamma-irradiated bacteria can be accurately identified by MALDI-TOF MS in most cases, but a decrease in identification scores is observed.

A simple shotgun proteomics method for rapid bacterial identification

Bacterial pathogens were rapidly identified by shotgun proteomics using a novel, easy to implement database search strategy. Peptide sequence data from nano-LC-MS/MS was searched against a database represented by concatenated proteomes of completed genome sequences. Select bacterial species, including BSL-3 select agents, were used to demonstrate this method.

Development and characterization of neutralizing monoclonal antibodies against the pandemic H1N1 virus (2009)

The 2009 H1N1 influenza pandemic was a major international public health crisis which caused considerable morbidity and mortality worldwide. The goal of this study was to produce anti-H1 monoclonal antibodies (MAbs) for improving diagnostic immunological assays and to develop potential immunotherapeutics. Nine MAbs were produced after immunizing mice with recombinant hemagglutinin (HA) protein from A/California/06/09. Two spleenocyte myeloma fusions yielded 1588 hybridoma cultures. After screening the hybridoma culture supernatants for antibody reactivity to rHA, nine clones were selected for further characterization. Cross-reactivity studies of the anti-rHA antibodies against a panel of influenza viruses (H1-H16) revealed eight out of nine MAbs were specific to the pandemic H1 subtype, except for MAb F256G2sc1 which also cross-reacted with H5 subtype virus. All MAbs were of the IgG1κ isotype, except F256G2sc1 which was IgG2aκ. The anti-rHA MAbs had binding affinities to rHA that ranged from a K(D) (disassociation constant) of 1.34×10(-9)M (F255G7sc1) to the weakest affinity of 4.60×10(-8)M (F255G4sc1). Interestingly, in a plaque reduction neutralization assay, all MAbs except F255G3sc1 demonstrated neutralizing ability. Furthermore, all MAbs except F255G3sc1 and F255G9sc1 exhibited anti-hemagglutinin activity against pandemic H1N1 viruses, but not against classical North American swine influenza viruses of the same subtype. Immunofluorescence assay (IFA) demonstrated that all MAbs except F255G1sc1 and F255G3sc1 were able to detect 2009 pandemic H1N1 (2009) virus- infected MDCK cells. The MAbs were also evaluated for potential use in competitive ELISA (cELISA), and with the exception of F255G3sc1, all MAbs showed competitive activity with serum collected from pigs infected with pandemic H1N1 virus (2009). The developed MAbs have demonstrated utility as immunodiagnostic and research reagents, and their neutralizing capabilities also hold potential for designing antiviral drugs against pandemic influenza.

Effects of the Campylobacter jejuni CJIE1 prophage homologs on adherence and invasion in culture, patient symptoms, and source of infection

BackgroundProphages of enteric bacteria are frequently of key importance for the biology, virulence, or host adaptation of their host. Some C. jejuni isolates carry homologs of the CJIE1 (CMLP 1) prophage that carry cargo genes potentially involved in virulence. Possible role(s) of CJIE1 homologs in the biology and virulence of C. jejuni were therefore investigated by using in vitro cell culture assays and by assessing the association of C. jejuni isolates with and without these prophages with patients’ symptoms, with source, and with clonal lineages within the C. jejuni population.ResultsFour C. jejuni isolates, three carrying the CJIE1-like prophage and one without, were tested in cell culture assays for adherence and invasion. Both adherence and invasion of C. jejuni to cells in culture were increased by the presence of the CJIE1-family prophage. Differences in motility and growth rate did not appear to be responsible. The CJIE1 prophage was present in 23% of isolates from human and non-human sources combined that were obtained through sentinel-site surveillance, and the distribution of CJIE1 in this population showed modest clonal associations. There was no correlation between the presence of the CJIE1 prophage in C. jejuni and patient symptoms, although there was some statistical support for lower rates of abdominal pain and fever when the prophage was present. Little evidence was found for a role of the prophage in host adaptation or host specificity.ConclusionThese biological effects suggest that the presence of the prophage may be a marker for differential virulence of some C. jejuni isolates. Ongoing research into the effects of the prophage on protein expression may provide additional insights into the roles the prophage may play in the biology of its host bacterium.