Invasive infections caused by Corynebacterium diphtheriae in vaccinated and non-vaccinated individuals have been reported increasingly. In this study we used multilocus sequence typing (MLST) to study genetic relationships between six invasive strains of this bacterium isolated solely in the urban area of Rio de Janeiro, Brazil, during a 10-year period. Of note, all the strains rendered negative results in PCR reactions for the tox gene, and four strains presented an atypical sucrose-fermenting ability. Five strains represented new sequence types. MLST results did not support the hypothesis that invasive (sucrose-positive) strains of C. diphtheriae are part of a single clonal complex. Instead, one of the main findings of the study was that such strains can be normally found in clonal complexes with strains related to non-invasive disease. Comparative analyses with C. diphtheriae isolated in different countries provided further information on the geographical circulation of some sequence types.

ABSTRACT A single multilocus sequence typing (MLST) scheme was developed for precise characterization of the opportunistic pathogens of Burkholderia cepacia complex (BCC), a group composed of at least nine closely related species. Seven conserved housekeeping genes were selected after a comparison of five Burkholderia species, and a collection of strains was subjected to nucleotide sequence analysis using a nested PCR amplification approach for each gene. MLST differentiated all nine current BCC species and identified 114 sequence types within a collection of 119 strains. No differentiation was found between strains recovered from environmental or clinical sources. The improved resolution in strain identification offered by MLST was able to identify previously characterized epidemic strain lineages and also demonstrated the presence of four novel potential species groups within the complex. There was also evidence for recombination having an important role in the recent evolution of individual BCC species. This highly transferable, validated, MLST scheme provides a new means to assist in species identification as well as unambiguous strain discrimination of the BCC by a single approach. It is also the first MLST scheme designed at the outset to incorporate multiple species and should facilitate global epidemiological investigations of the BCC.

Objective: The tcdC gene of C. difficile negatively regulates the production of toxins A and B. Mutations within this gene may result in loss of the negative regulatory role, potentially leading to hyperproduction of toxins and increased virulence. This hypervirulent strain caused an epidemic of C. difficile-associated disease in Quebec in 2002 and has been seen in increasing numbers in the western provinces, including BC. The objective of this study was to produce a baseline of the molecular and phenotypic profile of strains from participating centres to complement future infection control surveillance programs for C. difficile infection in BC. Methods: Over a one month period in March 2008, all stool specimens positive for C. difficile testing were cultured at participating laboratories or forwarded to the reference laboratory (BCCDC) for culture. Methods were standardized across participating laboratories. C. difficile was identified by colony morphology, Gram-stain, aerotolerance and C. difficile latex agglutination kit. The presence of the tcdC gene deletion was determined for each isolate using fragment analysis PCR. A fingerprint pattern was generated using pulsed-field gel electrophoresis (PFGE) and assigned PFGE pattern numbers and NAP type using the Canadian C. difficile database. Results: The total number of specimens received over the month of March 2008 from all participating sites was 414. Of these, 368 were culture confirmed cases of C. difficile. The tcdC gene mutation was detected in 189 (51.4%) isolates and the remaining 179 (48.6%) were wildtype strains with no mutation. Of those detecting a mutation, 145 (76.7%) displayed a mutation at ( 18)( 1) in the tcdC gene, associated with NAP1 PFGE designation and the remaining 29 (15.3%) showed a mutation other than at the ( 18)( 1) region. 156 (42.4%) of the total cases matched the NAP1 PFGE designation. Less common were PFGE patterns with NAP2 designation (9.5%) and NAP4 designation (7.8%). Conclusions: The prevalence of the NAP1 strain of C. difficile in the province of BC was 42% for March 2008. Of the C. difficile isolates recovered, the majority contained mutation at ( 18)( 1), with 15.3% exhibiting mutations at other sites within PaLoc. This study provides a baseline for infection control and public health functions in BC. O30 The use of a multi locus sequence typing scheme to characterize Canadian isolates of Corynebacterium diphtheriae K. Bernard1 *, S. Schindle2, T. Burdz1, D. Wiebe1, A. Reimer, C. Dowson3, F. Bolt3, A. Baldwin3. 1National Microbiology Laboratory, Winnipeg, Canada, 2University of Manitoba, Winnipeg, Canada, 3University of Warwick, Warwick, United Kingdom

Interaction with plants around their roots and foliage forms the natural habitat for a wide range of gram-negative bacteria such as Burkholderia, Pseudomonas and Ralstonia. During these interactions many of these bacteria facilitate highly beneficial processes such as the breakdown of pollutants or enhancement of crop growth. All these bacterial species are also capable of causing opportunistic infections in vulnerable individuals, especially people with cystic fibrosis (CF). Here we will review the current understanding of the Burkholderia cepacia complex (Bcc) as a group of model opportunistic pathogens, contrasting their clinical epidemiology with their ecological importance. Currently, the B. cepacia complex is composed of nine formally named species groups which are all difficult to identify using phenotypic methods. Genetic methods such as 16S rRNA and recA gene sequence analysis have proven useful for Bcc species identification. Multilocus sequence typing (MLST) is also emerging as a very useful tool for both Bcc strain and species identification. Historically, Burkholderia cenocepacia was the most dominant Bcc pathogen in CF, however, probably as a result of strict infection control practices introduced to control the spread of this species, its prevalence has been reduced. Burkholderia multivorans is the now the most dominant Bcc infection encountered in the UK CF population, a changing epidemiology that also appears to be occurring in the US CF population. The distribution of Bcc species residing in the natural environment may vary considerably with the type of environment examined. Clonally identical Bcc strains have been found to occur in the natural environment and cause infection. The contamination of medical devices, disinfectants and pharmaceutical formulations has also been directly linked to several outbreaks of infection. In the last 10 years considerable progress has been made in understanding the natural biology and clinical infections caused by this fascinating group of bacteria.

Aims:The use of cough swabs (CS)which entrap respiratory secretions produce false negatives and are poorly tolerated.Using cough plates (CP) was proposed as an alternative sampling method [1].This study compares CS with CP in nonexpectorating CF pts.Methods: 48 pts, 4 16 yrs were enrolled into the study over 4 months.The pts had to be able to cough voluntarily and be unable to expectorate a sputum sample.Each pt had a CS inserted into the back of the oropharynx, following a cough, it was withdrawn.The pt then coughed deeply twice over 4 CP at 5 cm from their mouth.The CP were blood agar,chocolate agar, Sabourand's medium and Bukholderia cepacia sensative agar (all Oxoid).The CS and CP were incubated and processed.Results: The variety and frequency of micro-organisms was greater in CS compared to CR 6 of the 48 samples had concurrence between CS and CR These were 4 staphylococcus aureus, 2 Aspergillus spp.The following numbers of CS grew organisms for which the CP did not: 9 Staph aureus, including 2 MRSA; 14 Pseudomonas aeruginosa; 1 Burkholderia cepacia; Only 1 CP grew Staph aureus while the CS grew yeast.Conclusion: Our results do not confirm those of Maiya et al, who concluded that CP are more sensative than CS.However their pt group differed from ours in that their pts were able to expectorate, while ours were excluded from the study if they were able to expectorate sputum.There may have been a potential source of bias in that there was no randomisation of CS and CR but this alone could not explain the difference between the results.Our results suggest CS are far superior to CP in the non-expectorating CF pt.

A self-bioluminescent construct of Salmonella enterica serovar Typhimurium DT104 was used to evaluate dry and wet surface pasteurisation of a wide range of meat and vegetable surfaces in a test rig. Bioluminescence was shown to be a sensitive measure of metabolic activity in real time and spatial position. Recovery of bioluminescence showed that limited dry heating with holding times of 3min at both 60°C and 90°C, was relatively poor at reducing bacterial contamination. The type of food influenced the rates of bacterial recovery, with skinless chicken allowing the most rapid recovery. Whilst wet heat was shown to be very effective at reducing survivors to below detection levels, in some instances bioluminescence was detected after a 24h recovery period, not in the area of initial inoculation, but in protected areas between the edge of the sample and the sample holder. This finding may explain anomalous results reported by other workers using sample destructive viable counts and also shows that steaming can dislodge and redistribute surface contaminants without killing.

ABSTRACT Several transmissible Burkholderia cenocepacia strains that infect multiple cystic fibrosis patients contain a genomic island designated as the cenocepacia island (cci). The cci contains a predicted N-acylhomoserine lactone (AHL) synthase gene, cciI, and a predicted response regulator gene, cciR. AHL production profiles indicated that CciI catalyzes the synthesis of N-hexanoyl-l-homoserine lactone and minor amounts of N-octanoyl-l-homoserine lactone. The cciI and cciR genes were found to be cotranscribed by reverse transcription-PCR analysis, and the expression of a cciIR::luxCDABE fusion in a cciR mutant suggested that the cciIR system negatively regulates its own expression. B. cenocepacia strains also have a cepIR quorum-sensing system. Expression of cepI::luxCDABE or cepR::luxCDABE fusions in a cciR mutant showed that CciR negatively regulates cepI but does not regulate cepR. Expression of the cciIR::luxCDABE fusion in a cepR mutant indicated that functional CepR is required for cciIR expression. Phylogenetic analysis suggested that the cciIR system was acquired by horizontal gene transfer from a distantly related organism and subsequently incorporated into the ancestral cepIR regulatory network. Mutations in cciI, cciR, cepI cciI, and cepR cciR were constructed in B. cenocepacia K56-2. The cciI mutant had greater protease activity and less swarming motility than the parent strain. The cciR mutant had less protease activity than the parent strain. The phenotypes of the cepI cciI and cepR cciR mutants were similar to cepI or cepR mutants, with less protease activity and swarming motility than the parent strain.

ABSTRACTTheBurkholderia cepaciaepidemic strain marker (BCESM) is a useful epidemiological marker for virulentB. cenocepaciastrains that infect patients with cystic fibrosis. However, there was no evidence that the original marker, identified by random amplified polymorphic DNA fingerprinting, contributed to pathogenicity. Here we demonstrate that the BCESM is part of a novel genomic island encoding genes linked to both virulence and metabolism. The BCESM was present on a 31.7-kb low-GC-content island that encoded 35 predicted coding sequences (CDSs): anN-acyl homoserine lactone (AHL) synthase gene (cciI) and corresponding transcriptional regulator (cciR), representing the first time cell signaling genes have been found on a genomic island; fatty acid biosynthesis genes; an IS66family transposase; transcriptional regulator CDSs; amino acid metabolism genes; and a group of hypothetical genes. Mutagenesis of the AHL synthase, amidase (amiI), and porin (opcI) genes on the island was carried out. Testing of the isogenic mutants in a rat model of chronic lung infection demonstrated that the amidase played a role in persistence, while the AHL synthase and porin were both involved in virulence. The island, designated theB. cenocepaciaisland (cci), is the first genomic island to be defined in theB. cepaciacomplex and its discovery validates the original epidemiological correlation of the BCESM with virulent CF strains. The features of the cci, which overlap both pathogenicity and metabolism, expand the concept of bacterial pathogenicity islands and illustrate the diversity of accessory functions that can be acquired by lateral gene transfer in bacteria.