Multiple-locus variable-number tandem-repeat analysis for discriminating within Salmonella enterica serovar Typhimurium definitive types and investigation of outbreaks.

The discriminatory power of multiple-locus variable-number tandem-repeat analysis (MLVA) needs to be evaluated for all Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium) phage types so that the power of this methodology is understood and results can be interpreted correctly during outbreak investigations. We evaluated the ability of MLVA to characterize four definitive phage types (DT) problematic in New Zealand. MLVA discriminated between DT104 isolates although there was very limited variation in the MLVA profiles for isolates with an RDNC phage type (reacts but does not conform to a recognized Typhimurium phage pattern) first observed in New Zealand's Enteric Reference Laboratory in May 2006. Most DT101 isolates had indistinguishable MLVA profiles or profiles that differed at one or two loci. This was also observed in DT160 isolates. MLVA may not identify all common-source outbreaks although it provided valuable data when applied to case isolates from two S. Typhimurium outbreaks.

Characterization of Escherichia coli O157:H7 in New Zealand using multiple-locus variable-number tandem-repeat analysis.

Recently, multiple-locus variable-number tandem-repeat analysis (MLVA) has been proposed as an alternative to pulsed-field gel electrophoresis (PFGE) for characterization of Escherichia coli O157:H7. In this study we characterized 118 E. coli O157:H7 isolates from cases of gastrointestinal disease in New Zealand using XbaI PFGE profiles and a MLVA scheme that assessed variability in eight polymorphic loci. The 118 isolates characterized included all 80 E. coli O157:H7 referred to New Zealand's Enteric Reference Laboratory in 2006 and 29 phage-type 2 isolates from 2005. When applied to these isolates the discriminatory power of PFGE and MLVA was not significantly different. However, MLVA data may be more epidemiologically relevant as isolates from family clusters of disease had identical MLVA profiles, even when the XbaI PFGE profiles differed slightly. Furthermore, most isolates with indistinguishable XbaI PFGE profiles that did not appear to be epidemiologically related had distinct MLVA profiles.

Clonal analysis of the serogroup B meningococci causing New Zealand's epidemic.

An epidemic of meningococcal disease caused by serogroup B meningococci expressing the P1.7-2,4 PorA protein began in New Zealand in 1991. The PorA type has remained stable. Different porB have been found in association with the P1.7-2,4 PorA, although type 4 has been most common. The clonal origins of B:P1.7-2,4 meningococci isolated from cases during 1990 to the end of 2003 were analysed. In 1990, the year immediately preceding the recognized increase in disease rates, all three subclones (ST-41, ST-42, and ST-154) of the ST-41/44 clonal complex occurred among the five isolates of B:P1.7-2,4. The two sequence types, ST-42 and ST-154, continued to cause most disease throughout New Zealand. Isolates belonging to subclone ST-41 were mostly identified early in the epidemic and in the South Island. 16S rRNA typing indicated that isolates belonging to the subclones ST-41 and ST-154 share a common ancestor, with those typing as ST-42 more distantly related with some genetically ambiguous. It is possible that ST-41 and ST-154 may have evolved one from the other but evolution to ST-42 is more difficult to explain. It is possible that one or more of the ST types could have been introduced into New Zealand prior to the first detection of clinical cases in 1990. Genetic diversity may have occurred during carriage in the community.

Stability of PorA during a meningococcal disease epidemic.

Meningococci causing New Zealand's epidemic, which began in 1991, are defined as group B, serosubtype P1.4 (subtype P1.7-2,4), belonging to the ST-41/ST-44 complex, lineage III. Of the 2,358 group B isolates obtained from disease cases from 1991 through 2003, 85.7% (2,021 of 2,358) were determined to be serosubtype P1.4. Of the remaining isolates, 156 (6.6%) were not serosubtypeable (NST). Molecular analysis of the porA gene from these B:NST meningococcal isolates was used to determine the reason. Most NST isolates (156, 88.5%) expressed a PorA that was distinct from P1.7-2,4 PorA. Fifteen isolates expressed variants of P1.7-2,4 PorA, and a further three expressed P1.7-2,4 PorA without any sequence variation. These three isolates expressed P1.7-2,4 PorA at very low levels, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, and showed variation in the porA promoter region. Among the 15 meningococcal isolates expressing variants of P1.7-2,4 PorA, 11 different sequence variations were found. Compared with the P1.7-2,4 PorA sequence, the sequences of these variants contained deletions, insertions, or single-nucleotide substitutions in the VR2 region of the protein. Multilocus restriction typing was used to assess the clonal derivations of B:NST case isolates. Meningococcal isolates expressing distinct PorA proteins belonged mostly to clonal types that were unrelated to the epidemic strain, whereas all meningococcal isolates expressing variants of P1.7-2,4 PorA belonged to the ST-41/ST-44 complex, lineage III. These results, together with those obtained serologically, demonstrate that the P1.7-2,4 PorA protein of meningococci responsible for New Zealand's epidemic has remained relatively stable over 13 years and support the use of a strain-specific outer membrane vesicle vaccine to control the epidemic.