Identification of Legionella spp. by 19 European reference laboratories: results of the European Working Group for Legionella Infections External Quality Assessment Scheme using DNA sequencing of the macrophage infectivity potentiator gene and dedicated online tools.

Identification of Legionella spp. can be achieved by DNA sequencing of the macrophage infectivity potentiator (mip) gene. The External Quality Assurance (EQA) scheme described in this report is the first to assess the proficiency of laboratories using this methodology. The results obtained from two EQA distributions sent to European reference laboratories involved in Legionella outbreak control and environmental monitoring are presented. Each distribution contained a panel of ten coded Legionella strains. All strains were from clinical and environmental sources and were considered to be wild-type strains. Participants used dedicated online tools to compare sequence text files against a database of known Legionella spp. The majority of centres (seven of ten, and 11 of 12) correctly identified all strains tested, in the first and second distributions, respectively. Typically, sequence similarity values of 98-100% were obtained when the test strains were compared with sequences contained in the database. In all but one case, lower values indicated a poor quality sequence. The exception was associated with the identification of a putative new species in the first panel. Genotypic identification of Legionella can be achieved by the use of standard protocols, dedicated identification libraries, and online tools. EQA schemes provide an independent measure of performance, and it is recommended that laboratories performing these techniques participate in such schemes, thereby allowing optimisation of and improvements in their performance.

Internationally distributed frozen oyster meat causing multiple outbreaks of norovirus infection in Australia.

BACKGROUND: Between November 2003 and January 2004, outbreaks of norovirus in 3 Australian jurisdictions involving 83 cases of illness were associated with imported oyster meat. METHODS: Cohort studies were conducted in 2 jurisdictions to identify relative risks of illness for the consumption of oysters. A case series was conducted in the third jurisdiction. RESULTS: The cohort studies conducted in the first 2 jurisdictions identified relative risks of illness of 17 (95% confidence interval, 5-51) and 35 (95% confidence interval, 5-243), respectively, for the consumption of oysters. Multiple strains of norovirus were detected in fecal specimens from 8 of 14 patients and in 1 of the 3 batches of implicated oyster meat using seminested reverse-transcriptase polymerase chain reaction methods. Traceback investigations revealed that all oyster meat was harvested from the same estuary system in Japan within the same month. CONCLUSIONS: These outbreaks demonstrate the potential of foodborne disease to spread internationally and the need for national and international collaboration to investigate such outbreaks. Foodborne illness related to norovirus is underestimated because of underreporting of human cases and challenges in laboratory detection of viruses in foods, both of which can delay public health action.

Molecular diversity of Campylobacter coli and C. jejuni isolated from pigs at slaughter by flaA-RFLP analysis and ribotyping.

A population of porcine isolates of Camplobacter jejuni (n = 11) and C. coli (n = 17) were examined for genotypic relatedness employing ribotyping, as well as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the flagellin (fla)A gene locus. PCR was employed to amplify a 533 bp fragment from the flaA gene, including the previously described short variable region (SVR), employing the novel primers, A2 and Al and successfully generated this amplicon for all wild-type strains examined (n = 28) of both C. jejuni and C. coli, as well as with both type strains, i.e. C. jejuni NCTC 11351 and C. coli NCTC 11366. Individual genotypes were assigned to each isolate typed employing the four typing methods (flaA-RFLP(Hae) III, flaA-RFLP(Pst) I ribotyping(Hae) III and ribotyping(Pst) I) and were assigned an arbitrary genotype code in ascending alphabetical order in comparison with a database of established genotypes for each of the methods employed. This study showed that several flaA-RFLP and ribopatterns existed within C. jejuni and C. coli, and demonstrated a heterogeneous diversity of strains occurring in the pigs examined. Ribotyping of strains with 16S and 23S rRNA with Pst I and Hae III digested chromosomal DNA allowed subdivision of strains into nine and eight groups, respectively. RFLP analyses with Pst I and Hae III digests probed with the flaA gene probe allowed subdivision of strains into eight and eleven subtypes, respectively. Employment of RFLP with the flaA nucleic acid probe and Hae III digests produced the greatest amount of variation of any genotyping scheme employed. Although there was a high degree of variability demonstrated by both typing methods, most isolates ( > 60%) clustered into four main genotypes, i.e. genotypes A-D. FlaA-PCR-RFLP typing demonstrated that the majority of isolates, 67.9 and 60.7%, were included in these four main genotypes for Pst I and Hae III restriction digests, respectively, although there was a high prevalence (7/11; 63.6%) of fla(Hae) III genotype A occurring within the C. jejuni isolates. Likewise, ribotyping studies demonstrated that most isolates were clustered into these four main genotypes, accounting for 81.5 and 60.7% of isolates for Pst I and Hae III restriction digests, respectively. This may indicate that the clonal population of campylobacters within this pig population is largely composed of persistent and dominant types, with a smaller number of hypervariable subtypes. Such data may useful in determining epidemiological routes of transmission of campylobacters from animal to animal, as well as helping to identify virulence determinants in persistent subtype populations.

Legionella drozanskii sp. nov., Legionella rowbothamii sp. nov. and Legionella fallonii sp. nov.: three unusual new Legionella species.

Seven strains of Legionella-like amoebal pathogens (LLAPs) were characterized on the basis of their cultural and staining characteristics, biochemical reactions, serology, cellular fatty acids (CFAs), isoprenoid quinone composition, total DNA relatedness, analysis of 16S rRNA and macrophage infectivity potentiator (mip) gene sequence analyses. All seven strains exhibited limited growth on buffered charcoal yeast extract alpha (BCYE) agar, required cysteine for growth and contained branched-chain CFAs and quinones typical of Legionella species. The bacilli were Gram-negative and catalase-positive. There were varying degrees of serological cross-reactions between these LLAP strains and other previously described Legionella species. Results from the various tests revealed that four LLAP strains represent three unusual new species of Legionella: Legionella drozanskii sp. nov., type strain LLAP-1T; Legionella rowbothamii sp. nov., type strain LLAP-6T; and Legionella fallonii sp. nov., type strain LLAP-10T. Three other LLAP strains, designated LLAP-7FL, LLAP-7NF and LLAP-9, were shown to be members of the species Legionella lytica. The deductions made from the phenetic characteristics of these bacteria were consistent with the phylogenetic relationships inferred from 16S rRNA and mip gene sequence analyses. This study is the first to speciate LLAP strains on the basis of data including quantitative DNA hybridization.

Hepatitis C genotyping by direct sequencing of the product from the Roche AMPLICOR test: methodology and application to a South Australian population.

The Roche AMPLICOR RT-PCR amplifies a 244 nucleotide sequence within the 5' non coding region (5'NCR) of the viral genome and is a widely used commercial test for the qualitative determination of hepatitis C RNA from sera. This paper describes a routine procedure for the purification of the PCR product, and its use in automated DNA sequencing, for determining the genotype of hepatitis C virus (HCV) isolates. Direct sequencing of the purified product was possible for 86% of samples, whilst 14% required additional amplification using a nested PCR method in order to read the resulting electropherogram. This method of genotyping is considerably less expensive than currently available commercial kits, and is convenient for the increasing number of laboratories that have access to automated DNA sequencers. The highly conserved nature of the 5'NCR limited differentiation of types and subtypes to an extent comparable to commercial HCV typing methods. Using this method on available laboratory samples and on patients about to commence interferon therapy, we found a predominance of genotype 1 (59%) and 3a (31%). Analysis of data on the interferon patients showed the median length of time from first exposure to diagnosis to be significantly longer for patients with genotype 1 than genotype 3a.

Sequence-based classification scheme for the genus Legionella targeting the mip gene.

The identification and speciation of strains of Legionella is often difficult, and even the more successful chromatographic classification techniques have struggled to discriminate newly described species. A sequence-based genotypic classification scheme is reported, targeting approximately 700 nucleotide bases of the mip gene and utilizing gene amplification and direct amplicon sequencing. With the exception of Legionella geestiana, for which an amplicon was not produced, the scheme clearly and unambiguously discriminated among the remaining 39 Legionella species and correctly grouped 26 additional serogroup and reference strains within those species. Additionally, the genotypic classification of approximately 150 wild strains from several continents was consistent with their phenotypic classification, with the exception of a few strains where serological cross-reactivity was complex, potentially confusing the latter classification. Strains thought to represent currently uncharacterized species were also found to be genotypically unique. The scheme is technically simple for a laboratory with even basic molecular capabilities and equipment, if access to a sequencing laboratory is available.

Interspecies sequence differences in the Mip protein from the genus Legionella: implications for function and evolutionary relatedness.

The nucleotide sequence of the mip genes and their inferred amino acid sequences were determined from 35 Legionella species and compared with the published sequences for L. pneumophila, L. micdadei and L. longbeachae. The sequences were 69-97% conserved at the nucleotide level and 82-99% at the amino acid level, with total conservation of amino acids determined to be associated with sites known to be involved in peptidyl prolyl cis-trans isomerase activity. No apparent difference could be determined in the arrangement of amino acids that would predict a functional difference in Mip from species associated with disease and Mip from species isolated only from the environment. Additionally, a phylogenetic comparison of the sequences with published 16S RNA sequences, using both genetic distance and maximum parsimony methods, was performed. Few relationships were apparent that were well supported by both data sets, the most robust being a clade comprising ([(cincinnatiensis, longbeachae, sainthelensi, santicrucis) gratiana] (moravica, quateirensis, shakespearei, worsleiensis) anisa, bozemanii, cherrii, dumoffii, gormanii, jordanis, parisiensis, pneumophila, steigerwaltii, tucsonensis, and wadsworthii).

Molecular microbiological investigation of an outbreak of hemolytic-uremic syndrome caused by dry fermented sausage contaminated with Shiga-like toxin-producing Escherichia coli.

Shiga-like toxin-producing Escherichia coli (SLTEC) strains are a diverse group of organisms which are known to cause diarrhea and hemorrhagic colitis in humans. This can lead to potentially fatal systemic sequelae, such as hemolytic-uremic syndrome (HUS). Strains belonging to more than 100 different O:H serotypes have been associated with severe SLTEC disease in humans, of which only O157 strains (which are uncommon in Australia) have a distinguishable cultural characteristic (sorbitol negative). During an outbreak of HUS in Adelaide, South Australia, a sensitive PCR assay specific for Shiga-like toxin genes (slt) was used to test cultures of feces and suspected foods. This enabled rapid confirmation of infection and identified a locally produced dry fermented sausage (mettwurst) as the source of infection. Cultures of feces from 19 of 21 HUS patients and 7 of 8 mettwurst samples collected from their homes were PCR positive for slt-I and slt-II genes. SLTEC isolates belonging to serotype O111:H- was subsequently isolated from 16 patients and 4 mettwurst samples. Subsequent restriction fragment length polymorphism analysis of chromosomal DNA from these isolates with slt-specific probes indicated that at least three different O111:H- genotypes were associated with the outbreak. Pulsed-field gel electrophoresis of genomic DNA restricted with XbaI showed that two of these restriction fragment length polymorphism types were closely related, but the third was quite distinct. However, SLTEC strains of other serotypes, including O157:H-, were also isolated from some of the HUS patients.

Use of gene amplification to detect Clostridium difficile in clinical specimens.

The combined use of an enrichment broth and gene amplification following simple DNA extraction to detect toxigenic strains of Clostridium difficile in feces was investigated by examining feces from 329 cases of suspected C. difficile infection. DNA was extracted by heating the washed centrifuged deposit from the broth in a microwave oven. For comparison, specimens were tested concurrently using standard methods for culture and cytotoxin testing. Amplified fragments were identified by molecular weight estimation, restriction enzyme digestion patterns and Southern blot hybridization. The combination of an enrichment broth followed by gene amplification was shown to be a sensitive, specific and rapid method for detecting toxigenic strains of C. difficile in feces. Use of the method in diagnostic laboratories may require the development of improved detection and verification systems for the amplified gene fragment.

Problems associated with identification of Legionella species from the environment and isolation of six possible new species.

Following investigation of an outbreak of legionellosis in South Australia, numerous Legionella-like organisms were isolated from water samples. Because of the limited number of commercially available direct fluorescent-antibody reagents and the cross-reactions found with some reagents, non-pneumophila legionellae proved to be difficult to identify and these isolates were stored at -70 degrees C for later study. Latex agglutination reagents for Legionella pneumophila and Legionella anisa developed by the Institute of Medical and Veterinary Science, Adelaide, Australia, were found to be useful as rapid screening aids. Autofluorescence was useful for placing isolates into broad groups. Cellular fatty acid analysis, ubiquinone analysis, and DNA hybridization techniques were necessary to provide definitive identification. The species which were isolated most frequently were L. pneumophila, followed by L. anisa, Legionella jamestowniensis, Legionella quinlivanii, Legionella rubrilucens, Legionella spiritensis, and a single isolate each of Legionella erythra, Legionella jordanis, Legionella birminghamensis, and Legionella cincinnatiensis. In addition, 10 isolates were found by DNA hybridization studies to be unrelated to any of the 26 currently known species, representing what we believe to be 6 possible new species.

The resistance of antimicrobial agents in salmonella from veterinary sources in Australia from 1975 to 1982.

A survey of antibiotic resistance in 1,287 strains of Salmonella from bovine, porcine and avian sources in Australia was carried out from 1975 to 1982. Isolates were tested against ampicillin, chloramphenicol, furazolidone, neomycin, streptomycin and tetracycline. Resistance was found to streptomycin in 286 isolates and to tetracycline in 282 isolates. Resistance to other antimicrobials was low and was unrelated to source. One hundred and seventy-three isolates showed multiple resistance to 2 or more antimicrobial agents with resistance to streptomycin and tetracycline being the most common. The overall level of resistance did not change over the period examined.

Protection of pregnant swine by vaccination against Leptospira infection.

The protection conferred on pregnant gilts by 2 commercially available leptospira interrogans serovars pomona and tarassovi bacterins was evaluated. Gilts vaccinated either 3, 6 or 12 months prior to natural challenge with L. interrogans serovar pomona had significantly lower abortion rates (2% vs 69%) and foetal mortality rates (14% vs 57%) than unvaccinated controls. One vaccine was significantly superior to the other and contained approximately twice the number of L. interrogans serovar pomona organisms per vaccine dose. Neither vaccine protected against renal colonisation but vaccination reduced urinary excretion of leptospires. Both vaccines reduced agglutinating antibody response to infection, as measured by the microscopic agglutination (MA) test. This may prevent the detection of a carrier animal by serology. Foetal pigs did not develop specific MA titres. Cultural methods were not reliable in making a diagnosis of foetal infection. Histopathology of foetal liver and kidneys helped in making a diagnosis of foetal infection.

Comparison of five methods for the assay of serum gentamicin.

The microbiological bioassay, the adenylation method, and the radiometric, enzyme, and fluorescent immunoassay methods for assaying serum gentamicin were compared. The precision, reproducibility, and specificity of each method was assessed and proved satisfactory, with the exception of the radioimmunoassay, which gave artificially high results. Good correlations between the other four methods was obtained in a comparison involving 103 patient sera. The other aspects of performance were also compared, namely, simplicity, speed, cost, ease of automation, and application to large and small workloads. The enzyme immunoassay performed best in this comparison, being accurate, specific, rapid, and very simple to perform. However, other laboratories might find that workload, staffing, and available equipment make other methods more attractive.

The importance of cephradine in hip surgery.

Effective concentrations of antibiotic in the fluid bathing implanted hip prostheses are essential to prevent infection by micro-organisms. Twenty patients undergoing total hip replacement were given one gram of Cephradine intramuscularly one hour before operation and one other received a single bolus of Cephradine intravenously before operation and one other received a single bolus of Cephradine intravenously before operation. The concentrations of antibiotic were greater and persisted longer in the tissue fluid than in the blood. The antibiotic was sufficient to inhibit most micro-organisms causing contamination. We recommend that Cephradine is given intramuscularly one hour before operation and at six-hourly intervals after operation until the drainage tubes and intravenous lines have been removed.

Evaluation of an enzyme immunoassay for serum gentamicin.

The EMIT (Enzyme Multiplied Immunoassay Technique) for serum gentamicin determination was evaluated by a standard procedure. The precision, accuracy, and specificity were assessed and proved satisfactory. Comparison with a bioassay was done, and results for patient samples showed a correlation coefficient of 0.95 between the two methods. Advantages of the enzyme immunoassay system were the provision of results within 15 min of receipt of blood in the laboratory, a requirement of minimal technical expertise, and an applicability to both large and small workloads. The EMIT proved during evaluation to be a practical alternative to current bioassays for the determination of serum gentamicin concentrations.