Tatumella saanichensis sp. nov., isolated from a cystic fibrosis patient.

Polyphasic taxonomic analysis was performed on a clinical isolate (NML 06-3099T) from a cystic fibrosis patient, including whole-genome sequencing, proteomics, phenotypic testing, electron microscopy, chemotaxonomy and a clinical investigation. Comparative whole-genome sequence analysis and multilocus sequence analysis (MLSA) between Tatumella ptyseos ATCC 33301T and clinical isolate NML 06-3099T suggested that the clinical isolate was closely related to, but distinct from, the species T. ptyseos. By 16S rRNA gene sequencing, the clinical isolate shared 98.7 % sequence identity with T. ptyseos ATCC 33301T. A concatenate of six MLSA loci (totalling 4500 bp) revealed < 93.9 % identity between T. ptyseos ATCC 33301T, other members of the genus and the clinical isolate. A whole-genome sequence comparison between NML 06-3099T and ATCC 33301T determined that the average nucleotide identity was 76.24 %. The overall DNA G+C content of NML 06-3099T was 51.27 %, consistent with members of the genus Tatumella. By matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis, NML 06-3099T had a genus-level match, but not a species-level match, to T. ptyseos. By shotgun proteomics, T. ptyseos ATCC 33301T and NML 06-3099T were found to have unique proteomes. The two strains had similar morphologies and multiple fimbriae, as observed by transmission electron microscopy, but were distinguishable by phenotypic testing. Cellular fatty acids found were typical for members of the Enterobacteriaceae. NML 06-3099T was susceptible to commonly used antibiotics. Based on these data, NML 06-3099T represents a novel species in the genus Tatumella, for which the name Tatumella saanichensis sp. nov. is proposed (type strain NML 06-3099T = CCUG 55408T = DSM 19846T).

Evaluation of MALDI-TOF mass spectroscopy methods for determination of Escherichia coli pathotypes.

It is rapidly becoming apparent that many E. coli pathotypes cause a considerable burden of human disease. Surveillance of these organisms is difficult because there are few or no simple, rapid methods for detecting and differentiating the different pathotypes. MALDI-TOF mass spectroscopy has recently been rapidly and enthusiastically adopted by many clinical laboratories as a diagnostic method because of its high throughput, relatively low cost, and adaptability to the laboratory workflow. To determine whether the method could be adapted for E. coli pathotype differentiation the Bruker Biotyper methodology and a second methodology adapted from the scientific literature were tested on isolates representing eight distinct pathotypes and two other groups of E. coli. A total of 136 isolates was used for this study. Results confirmed that the Bruker Biotyper methodology that included extraction of proteins from bacterial cells was capable of identifying E. coli isolates from all pathotypes to the species level and, furthermore, that the Bruker extraction and MALDI-TOF MS with the evaluation criteria developed in this work was effective for differentiating most pathotypes.

Emergence of multidrug-resistant Salmonella enterica serotype 4,[5],12:i:- involving human cases in Canada: results from the Canadian Integrated Program on Antimicrobial Resistance Surveillance (CIPARS), 2003-10.

OBJECTIVES: Over the last decade, a marked increase in Salmonella enterica serotype 4,[5],12:i:- with a core resistance to ampicillin, streptomycin, sulphonamides and tetracycline (ASSuT) has been observed in Europe. This study describes the emergence and characterization of isolates of multidrug-resistant Salmonella 4,[5],12:i:- in Canada. METHODS: Human clinical isolates of Salmonella 4,[5],12:i:- were identified by provincial laboratories from 2003 to 2010. Serotyping and phage typing were performed by standardized methodologies. MIC values were determined using broth microdilution. PCR was used to determine the presence of resistance genes. Multilocus sequence typing was performed on a selected number of isolates. RESULTS: A total of 26 251 Salmonella were submitted as part of the Canadian Integrated Program on Antibiotic Resistance Surveillance (CIPARS). Of these, Salmonella 4,[5],12:i:- accounted for a total of 766 isolates (2.9%), and the number increased significantly from 42 (1.4%) in 2003 to 164 (4.8%) in 2010. The ASSuT+ phenotype was observed in 11.9% (n = 91) of Salmonella 4,[5],12:i:- isolates and increased from two isolates in 2003 to 35 isolates in 2010. Two sequence types (STs) were observed. ST34 was mainly associated with the ASSuT isolates (n = 24; 38%), which contained blaTEM, strA-strB, tet(B) and sul2. ST19 was more likely to be associated with the ACSSuT phenotype and contained blaTEM, floR, strA-strB, sul2 and tet(A) or blaPSE-1, floR, aadA2, sul1 and tet(G). CONCLUSIONS: The prevalence of Salmonella 4,[5],12:i:- has significantly increased from 2003 to 2010 and it is now the fifth most common serotype reported in Canada causing human disease. Similar antimicrobial resistance patterns, phage types and STs have been observed in Europe.

MS-H: a novel proteomic approach to isolate and type the E. coli H antigen using membrane filtration and liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Serotyping is the long-standing gold standard method to determine E. coli H antigens; however, this method requires a panel of H-antigen specific antibodies and often culture-based induction of the H-antigen flagellar motility. In this study, a rapid and accurate method to isolate and identify the Escherichia coli (E. coli) H flagellar antigen was developed using membrane filtration and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Flagella were isolated from pure culture, digested with trypsin, and then subjected to LC-MS/MS using one of two systems (Agilent-nano-LC-QSTAR XL or Proxeon-nano-LC-LTQ-Orbitrap XL). The resulting peptide sequence data were searched against a custom E. coli flagella/H antigen database. This approach was evaluated using flagella isolated from reference E. coli strains representing all 53 known H antigen types and 41 clinical E. coli strains. The resulting LC-MS/MS classifications of H antigen types (MS-H) were concordant with the known H serogroup for all 53 reference types, and of 41 clinical isolates tested, 38 (92.7%) were concordant with the known H serogroup. MS-H clearly also identified two clinical isolates (4.9%) that were untypeable by serotyping. Notably, successful detection and classification of flagellar antigens with MS-H did not generally require induction of motility, establishing this proteomic approach as more rapid and cost-effective than traditional methods, while providing equitable specificity for typing E. coli H antigens.

The O28 Antigen Gene Clusters of Salmonella enterica subsp. enterica Serovar Dakar and Serovar Pomona Are Different.

A 10 kb O-antigen gene cluster was sequenced from a Salmonella enterica subsp. enterica Dakar O28 reference strain and from two S. Pomona serogroup O28 isolates. The two S. Pomona O antigen gene clusters showed only moderate identity with the S. Dakar O28 gene cluster, suggesting that the O antigen oligosaccharides may contain one or more sugars conferring the O28 epitope but may otherwise be different. These novel findings are absolutely critical for the correct interpretation of molecular serotyping assays targeting genes within the O antigen gene clusters of these Salmonella serotypes and suggest the possibility that the O antigen gene clusters of other Salmonella serovars may also be heterogenous.

Detection of Campylobacter jejuni by culture and real-time PCR in a French cohort of patients with Guillain-Barre syndrome.

Bacteriological culture and real-time PCR (RT-PCR) were used to detect Campylobacter jejuni in fecal samples from a French cohort of 237 patients with Guillain-Barré syndrome (GBS). We provide evidence that diverse serotypes and genotypes of C. jejuni are a major trigger of GBS in France.

Comparative genotyping of Campylobacter jejuni strains from patients with Guillain-Barré syndrome in Bangladesh.

BACKGROUND: Campylobacter jejuni is a common cause of acute gastroenteritis and is associated with post-infectious neuropathies such as the Guillain-Barré syndrome (GBS) and the Miller Fisher syndrome (MFS). We here present comparative genotyping of 49 C. jejuni strains from Bangladesh that were recovered from patients with enteritis or GBS. All strains were serotyped and analyzed by lipo-oligosaccharide (LOS) genotyping, amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). METHODOLOGY/PRINCIPAL FINDINGS: C. jejuni HS:23 was a predominant serotype among GBS patients (50%), and no specific serotype was significantly associated with GBS compared to enteritis. PCR screening showed that 38/49 (78%) of strains could be assigned to LOS classes A, B, C, or E. The class A locus (4/7 vs 3/39; p<0.01) was significantly associated in the GBS-related strains as compared to enteritis strains. All GBS/oculomotor related strains contained the class B locus; which was also detected in 46% of control strains. Overlapping clonal groups were defined by MLST, AFLP and PFGE for strains from patients with gastroenteritis and GBS. MLST defined 22 sequence types (STs) and 7 clonal complexes including 7 STs not previously identified (ST-3742, ST-3741, ST-3743, ST-3748, ST-3968, ST-3969 and ST-3970). C. jejuni HS:23 strains from patients with GBS or enteritis were clonal and all strains belonged to ST-403 complex. Concordance between LOS class B and ST-403 complex was revealed. AFLP defined 25 different types at 90% similarity. The predominant AFLP type AF-20 coincided with the C. jejuni HS:23 and ST-403 complex. CONCLUSION/SIGNIFICANCE: LOS genotyping, MLST, AFLP and PFGE helped to identify the HS:23 strains from GBS or enteritis patients as clonal. Overall, genotypes exclusive for enteritis or for GBS-related strains were not obtained although LOS class A was significantly associated with GBS strains. Particularly, the presence of a clonal and putative neuropathogenic C. jejuni HS:23 serotype may contribute to the high prevalence of C. jejuni related GBS in Bangladesh.

Isolation and detection of Shiga toxin-producing Escherichia coli in clinical stool samples using conventional and molecular methods.

The isolation of Shiga toxin-producing Escherichia coli (STEC) other than serogroup O157 from clinical stool samples is problematic due to the lack of differential phenotypic characteristics from non-pathogenic E. coli. The development of molecular reagents capable of identifying both toxin and serogroup-specific genetic determinants holds promise for a more comprehensive characterization of stool samples and isolation of STEC strains. In this study, 876 stool samples from paediatric patients with gastroenteritis were screened for STEC using a cytotoxicity assay, commercial immunoassay and a conventional PCR targeting Shiga-toxin determinants. In addition, routine culture methods for isolating O157 STEC were also performed. The screening assays identified 45 stools presumptively containing STEC, and using non-differential culture techniques a total of 20 O157 and 22 non-O157 strains were isolated. These included STEC serotypes O157 : H7, O26 : H11, O121 : H19, O26 : NM, O103 : H2, O111 : NM, O115 : H18, O121 : NM, O145 : NM, O177 : NM and O5 : NM. Notably, multiple STEC serotypes were isolated from two clinical stool samples (yielding O157 : H7 and O26 : H11, or O157 : H7 and O103 : H2 isolates). These data were compared to molecular serogroup profiles determined directly from the stool enrichment cultures using a LUX real-time PCR assay targeting the O157 fimbrial gene lpfA, a microsphere suspension array targeting allelic variants of espZ and a gnd-based molecular O-antigen serogrouping method. The genetic profile of individual stool cultures indicated that the espZ microsphere array and lpfA real-time PCR assay could accurately predict the presence and provide preliminary typing for the STEC strains present in clinical samples. The gnd-based molecular serogrouping method provided additional corroborative evidence of serogroup identities. This toolbox of molecular methods provided robust detection capabilities for STEC in clinical stool samples, including co-infection of multiple serogroups.

Escherichia coli O123 O antigen genes and polysaccharide structure are conserved in some Salmonella enterica serogroups.

The serotyping of O and H antigens is an important first step in the characterization of Salmonella enterica. However, serotyping has become increasingly technically demanding and expensive to perform. We have therefore sequenced additional S. enterica O antigen gene clusters to provide information for the development of DNA-based serotyping methods. Three S. enterica isolates had O antigen gene clusters with homology to the Escherichia coli O123 O antigen region. O antigen clusters from two serogroup O58 S. enterica strains had approximately 85 % identity with the E. coli O123 O antigen region over their entire length, suggesting that these Salmonella and E. coli O antigen regions evolved from a common ancestor. The O antigen cluster of a Salmonella serogroup O41 isolate had a lower level of identity with E. coli O123 over only part of its O antigen DNA cluster sequence, suggesting a different and more complex evolution of this gene cluster than those in the O58 strains. A large part of the Salmonella O41 O antigen DNA cluster had very close identity with the O antigen cluster of an O62 strain. This region of DNA homology included the wzx and wzy genes. Therefore, molecular serotyping tests using only the O41 or O62 wzx and wzy genes would not differentiate between the two serogroups. The E. coli O123 O-antigenic polysaccharide and its repeating unit were characterized, and the chemical structure for E. coli O123 was entirely consistent with the O antigen gene cluster sequences of E. coli O123 and the Salmonella O58 isolates. An understanding of both the genetic and structural composition of Salmonella and E. coli O antigens is necessary for the development of novel molecular methods for serotyping these organisms.

Endemic and epidemic lineages of Escherichia coli that cause urinary tract infections.

Women with urinary tract infections (UTIs) in California, USA (1999-2001), were infected with closely related or indistinguishable strains of Escherichia coli (clonal groups), which suggests point source dissemination. We compared strains of UTI-causing E. coli in California with strains causing such infections in Montréal, Québec, Canada. Urine specimens from women with community-acquired UTIs in Montréal (2006) were cultured for E. coli. Isolates that caused 256 consecutive episodes of UTI were characterized by antimicrobial drug susceptibility profile, enterobacterial repetitive intergenic consensus 2 PCR, serotyping, XbaI and NotI pulsed-field gel electrophoresis, multilocus sequence typing, and phylogenetic typing. We confirmed the presence of drug-resistant, genetically related, and temporally clustered E. coli clonal groups that caused community-acquired UTIs in unrelated women in 2 locations and 2 different times. Two clonal groups were identified in both locations. Epidemic transmission followed by endemic transmission of UTI-causing clonal groups may explain these clusters of UTI cases.

Lineage specific recombination and positive selection in coding and intragenic regions contributed to evolution of the main Listeria monocytogenes virulence gene cluster.

The major virulence cluster of Listeria monocytogenes harbors six virulence genes that encode proteins critical for the intracellular life cycle of this human and animal pathogen. In this study, we determined the sequence (8709nt) of the virulence gene cluster (including the six main virulence genes) in 40 L. monocytogenes isolates from different source populations (human clinical cases, animal clinical cases, foods, and natural environments). An alignment of the full length cluster as well as individual gene alignments and alignments of intragenic regions were used for phylogenetic, recombination, and positive selection analyses. Initial phylogenetic analyses showed that the sequences represented two main clusters, consistent with previously defined L. monocytogenes phylogenetic lineages. The 40 sequences represented 25 distinct allelic types and the overall alignment included 592 polymorphic sites. Overall, our data show that (i) virulence genes in the main L. monocytogenes virulence gene cluster include highly conserved genes (i.e., hly and prfA) as well as diverse genes that appear to have evolved by positive selection (mpl, actA, and plcA), (ii) recombination has played an important role in the evolution of the virulence gene cluster, but is limited to lineage II isolates, and (iii) the promoter region driving the transcription of virulence genes transcribed early in intracellular infection (i.e., hly and plcA) has evolved by positive selection. The genes and intragenic regions in the L. monocytogenes virulence gene cluster thus have evolved independently, despite their close physical linkage, likely reflecting distinct selective pressures associated with expression and function of the proteins encoded in this region.

Isolation and genetic characterization of a coinfection of non-O157 Shiga toxin-producing Escherichia coli.

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.

Genetic determinants and polymorphisms specific for human-adapted serovars of Salmonella enterica that cause enteric fever.

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.

Identification and characterization of Shigella boydii 20 serovar nov., a new and emerging Shigella serotype.

Analysis of 163 putative Shigella isolates from Canada and the USA showed biochemical reactions consistent with Shigella species, although none of the isolates reacted with antiserum raised against any of the well-established or provisional Shigella serotypes. All these isolates, provisionally designated serotype SH108, were positive for the ipaH gene and the invasion-associated locus. All fermented mannitol, were serologically indistinguishable from each other and showed no reaction in antisera prepared against Escherichia coli serotypes O1 to O181. PCR-RFLP analysis of the genes involved in O-antigen synthesis revealed a common pattern among these isolates that was distinct from recognized Shigella serotypes and E. coli. Between 1999 and 2003, isolates from across Canada were submitted to the National Laboratory for Enteric Pathogens for antibiotic susceptibility testing, phage typing and PFGE. These assays revealed heterogeneity among the members of this serotype. Antimicrobial susceptibility testing with seven antibiotics identified six profiles, with 90 % (45/50) of the isolates resistant to four or more antibiotics and 72 % (36/50) resistant to five or more. All isolates were typable using a panel of 16 phages, with 11 different phage types (PTs) represented. The most common PTs found were PT 3 (64 %), PT 6 (10 %) and PT 16 (6 %). Analysis of XbaI-restricted genomic DNA revealed 16 highly related patterns that were not readily distinguishable from those obtained for some other Shigella serotypes. The World Health Organization Collaborating Center for Shigella has added serotype SH108 to the Shigella scheme as S. boydii serotype 20 (serovar nov.). Strain SH108 (isolate 99-4528) is the reference strain for this serotype.