Human campylobacteriosis exhibits a distinctive seasonality in temperate regions. This paper aims to identify the origins of this seasonality. Clinical isolates [typed by multi-locus sequence typing (MLST)] and epidemiological data were collected from Scotland. Young rural children were found to have an increased burden of disease in the late spring due to strains of non-chicken origin (e.g. ruminant and wild bird strains from environmental sources). In contrast the adult population had an extended summer peak associated with chicken strains. Travel abroad and UK mainland travel were associated with up to 17% and 18% of cases, respectively. International strains were associated with chicken, had a higher diversity than indigenous strains and a different spectrum of MLST types representative of these countries. Integrating empirical epidemiology and molecular subtyping can successfully elucidate the seasonal components of human campylobacteriosis. The findings will enable public health officials to focus strategies to reduce the disease burden.

There is concern that current procedures for the heat inactivation of Mycobacterium tuberculosis may not be adequate. This raises serious safety issues for laboratory staff performing molecular investigations such as IS6110 restriction fragment length polymorphism typing. This paper confirms that the protocol of van Embden et al, as performed routinely in this laboratory, is safe and effective for the heat inactivation of M tuberculosis. This procedure involves complete immersion of a tube containing a suspension of one loopfull of growth in a water bath at 80°C for 20 minutes. Seventy four isolates were included in this investigation. Despite prolonged incubation for 20 weeks, none of the heat killed M tuberculosis suspensions produced visible colonies or gave a positive growth signal from liquid culture. This method did not affect the integrity of the DNA for subsequent molecular investigations. I S6110 restriction fragment length polymorphism analysis is considered to be the "gold standard" typing method for DNA fingerprinting of Mycobacterium tuberculosis.1 Before DNA extraction, M tuberculosis must be heat inactivated to render it safe for manipulation outwith a containment level 3 facility. Two reports have raised concerns that some heat killing procedures used for the inactivation of M tuberculosis are not reliably effective. This may put laboratory workers using molecular techniques at risk of laboratory acquired infection (P Bemer-Melchior et al. Transmission of Mycobacterium tuberculosis in a mycobacteriology laboratory. Presented at the 5th International Conference on the Prevention of Infection,1998). Zwadyk and colleagues 2 first suggested that temperatures below 100°C do not consistently kill M tuberculosis. They showed survival of 50% and 25% of the organisms after heat inactivation at 95°C in a dry heat block for 20 and 30 minutes, respectively. These findings were confirmed by Bemer-Melchior and Drugeon, 3 who investigated several different inactivation protocols involving heat killing at either 80°C for 20 minutes or 100°C for five minutes, followed by either lysozyme (0.5 mg/ml) or a combined proteinase K (0.4 mg/ml) and lysozyme (0.5 mg/ml) digestion. They reported the growth of M tuberculosis in 80% of subcultures on Löwenstein-Jensen (L-J) medium after heat inactivation at 80°C for 20 minutes and treatment with lysozyme and in 10% after heat inactivation at 80ºC for 20 minutes and treatment with both lysozyme and proteinase K. In addition, Zwadyk and colleagues 2 showed that increasing exposure time did not always correlate with a decrease in viability. In view of these findings, we investigated the suitability of the heat killing procedure currently used in our laboratory. This assessment involved detailed attention to procedures used during heat inactivation and included extended viability checks before and after heat inactivation."Two reports have raised concerns that some heat killing procedures used for the inactivation of Mycobacterium tuberculosis are not reliably effective" METHODSA...

Transposable elements can affect an organism's fitness through the insertional inactivation of genes and can therefore be used to identify genes that are nonessential for growth in vitro or in animal models. However, these models may not adequately represent the genetic requirements during chains of human infection. We have therefore conducted a genome-wide survey of transposon mutations in Mycobacterium tuberculosis isolates from cases of human infection, identifying the precise, base-specific insertion sites of the naturally occurring transposable element IS6110. Of 294 distinct insertions mapped to the strain H37Rv genome, 180 were intragenic, affecting 100 open reading frames. The number of genes carrying IS6110 in clinical isolates, and hence apparently not essential for infection and transmission, is very much lower than the estimates of nonessential genes derived from in vitro studies. This suggests that most genes in M. tuberculosis play a significant role in human infection chains. IS6110 insertions were underrepresented in genes associated with virulence, information pathways, lipid metabolism, and membrane proteins but overrepresented in multicopy genes of the PPE family, genes of unknown function, and intergenic sequences. Population genomic analysis of isolates recovered from an organism's natural habitat is an important tool for determining the significance of genes or classes of genes in the natural biology of an organism.

g Closely related bacterial isolates can display divergent phenotypes. This can limit the usefulness of phylogenetic studies for understanding bacterial ecology and evolution. Here, we compare phenotyping based on Raman spectrometric analysis of cellular composition to phylogenetic classification by ribosomal multilocus sequence typing (rMLST) in 108 isolates of the zoonotic pathogens Campylobacter jejuni and C. coli. Automatic relevance determination (ARD) was used to identify informative peaks in the Raman spectra that could be used to distinguish strains in taxonomic and host source groups (species, clade, clonal complex, and isolate source/host). Phenotypic characterization based on Raman spectra showed a degree of agreement with genotypic classification using rMLST, with segregation accuracy between species (83.95%), clade (in C. coli, 98.41%), and, to some extent, clonal complex (86.89% C. jejuni ST-21 and ST-45 complexes) being achieved. This confirmed the utility of Raman spectroscopy for lineage classification and the correlation between genotypic and phenotypic classification. In parallel analysis, relatively distantly related isolates (different clonal complexes) were assigned the correct host origin irrespective of the clonal origin (74.07 to 96.97% accuracy) based upon different Raman peaks. This suggests that the phenotypic characteristics, from which the phenotypic signal is derived, are not fixed by clonal descent but are influenced by the host environment and change as strains move between hosts.

Butyrivibrio fibrisolvens strains resistant to tetracycline were isolated from the bovine rumen. Two of three Tc r B. fibrisolvens tested were able to donate tetracycline resistance at frequencies ranging from 10 ؊7 to 10 ؊1 per donor cell in anaerobic filter matings to a rifampin-resistant mutant of the type strain of B. fibrisolvens, 2221 R. The recipient strain 2221 R exhibited rapid autoaggregation, which might be a factor in the high transfer rates observed. Tc r transconjugants of B. fibrisolvens 2221 R were also capable of further transferring tetracycline resistance to a fusidic acid-resistant mutant, 2221 F. Comparison of genomic DNAs by pulsed-field gel electrophoresis demonstrated altered band profiles in transconjugants, consistent with the acquisition of a large mobile chromosomal element. The transferable elements from the two B. fibrisolvens donors 1.23 and 1.230 (TnB123 and TnB1230, respectively) showed the same preferred insertion site in the B. fibrisolvens 2221 R chromosome and are likely to be similar, or identical, elements. Hybridization experiments showed no close relationship between TnB1230 and int-xis regions from Tn916 or Tn5253. Although DNA from the B. fibrisolvens donor strains hybridized with probes carrying tet(M) or tet(O) sequences, transconjugants were found to have acquired a distinct band that hybridized only weakly with these probes, suggesting that a second, distantly related Tc r determinant had been transferred.