Evaluation of different target genes for the detection of Salmonella sp. by loop-mediated isothermal amplification.

The loop-mediated isothermal amplification (LAMP) technique was used to investigate six salmonella-specific sequences for their suitability to serve as targets for the pathogen identification. Sequences selected for designing LAMP primers were genes invA, bcfD, phoP, siiA, gene62181533 and a region within the ttrRSBCA locus. Primers including single nucleotide polymorphisms were configured as degenerate primers. Specificity of the designed primer sets was determined by means of 46 salmonella and 32 other food- and waterborne bacterial reference species and strains. Primers targeting the ttrRSBCA locus showed 100 % inclusivity of target and exclusivity of other test species and strains. Other primer sets revealed deficiencies, especially regarding Salmonella enterica subsp. II-IV and Salmonella bongori. Additionally, primers targeting the siiA gene failed to detect S. enterica subsp. enterica serotypes Newport and Stanley, whereas bcfD primers did not amplify DNA of S. enterica subsp. enterica serotype Schleissheim. TtrRSBCA primers, providing short detection times and constant melting temperatures of amplification products, achieved best overall performance.

An outbreak of Salmonella Newport associated with mung bean sprouts in Germany and the Netherlands, October to November 2011.

The largest Salmonella enterica serovar Newport outbreak (n=106) ever reported in Germany occurred in October and November 2011. Twenty associated cases were reported in the Netherlands. The outbreak investigation included an analytical epidemiological study, molecular typing of human and food isolates and food traceback investigations. Unspecified Salmonella had been detected in samples of mung bean sprouts at a sprout producer (producer A) in the Netherlands and mung bean sprouts contaminated with S. Newport had been found during routine sampling at a sprout distributor in Germany. Therefore, we tested the hypothesis of sprouts being the infection vehicle. In a case-control study, we compared 50 notified adult S. Newport cases with 45 Salmonella enterica serovar Enteritidis cases regarding their food consumption in the three days before illness. In multivariable logistic regression analysis, only sprout consumption was significantly associated with S. Newport infection (odds ratio: 18.4; 95% confidence interval: 2.2-150.2). Molecular typing patterns of human isolates were indistinguishable from a mung bean sprouts isolate. Traceback of sprouts led to distributors and producer A in the Netherlands. Since sprouts are frequently contaminated with microorganisms, consumers need to be aware that consumption of raw or insufficiently cooked sprouts may pose a health risk.

Use of multilocus variable-number tandem repeat analysis (MLVA) in eight European countries, 2012.

Genotyping of important medical or veterinary prokaryotes has become a very important tool during the last decades. Rapid development of fragment-separation and sequencing technologies has made many new genotyping strategies possible. Among these new methods is multilocus variable-number tandem repeat analysis (MLVA). Here we present an update on the use of MLVA in eight European countries (Denmark, France, Germany, Ireland, Italy, the Netherlands, Norway and Sweden). Researchers in Europe have been active in developing and implementing a large array of different assays. MLVA has been used as a typing tool in several contexts, from aiding in resolving outbreaks of foodborne bacteria to typing organisms that may pose a bioterrorist threat, as well as in scientific studies.

Different mutations in the oafA gene lead to loss of O5-antigen expression in Salmonella enterica serovar Typhimurium.

AIMS: To analyse genetic changes in the oafA gene explaining the loss of O5-antigen expression in Salmonella Typhimurium and Salm. 4,[5],12:i:-. METHODS AND RESULTS: The oafA gene in 52 O5-antigen-negative and 77 O5-antigen-positive Salm. Typhimurium (N = 47) and Salm. 4,[5],12:i:- (monophasic Salm. Typhimurium strains, N = 82) was investigated by a combination of PCR screening and DNA sequencing to identify mutations leading to the suppression of the O5-antigen. Various DNA sequence changes within the open reading frame (ORF) of oafA in O5-antigen-negative strains could be identified. In 77% of the O5-antigen-negative strains, a 7-bp deletion of a duplicated sequence within the functional oafA gene led to a frameshift in the ORF. In four strains, an IS4 element and in two, an IS1 element was inserted at different positions. Four other strains carried at different positions single base pair substitutions causing a premature stop codon. Finally, in two strains, a deletion of the oafA 3'end of undetermined size was responsible for the lack of O5-antigen expression. In none of the strains investigated, the complete ORF of oafA was deleted. Primers were designed and used to detect the most prominent variants. CONCLUSIONS: O5-antigen-negative Salm. Typhimurium and Salm. 4,[5],12:i:- strains carry an oafA pseudogene caused by different genetic events indicating that there is a selection for oafA mutations leading to the loss of O5-antigen expression. SIGNIFICANCE AND IMPACT OF THE STUDY: The loss of O5-antigen expression may be an example of a common evolutionary mechanism to escape host defence or to adapt to environmental changes. The data are the basis for the development of diagnostic PCR assays for the differentiation of O5-antigen-positive and O5-antigen-negative Salm. Typhimurium and its monophasic (Salm. 4,[5],12:i-) strains.

[Protracted nosocomial outbreak of Salmonella Enteritidis LT 8/7]. / Protrahiert verlaufender nosokomialer Ausbruch von Salmonella Enteritidis LT 8/7.

INTRODUCTION: In spring 2007a cluster of nosocomial salmonellosis cases, culture confirmed for SALMONELLA Enteritidis lysotype (LT) 8/7, occurred in a Wolfsburg hospital. An outbreak investigation was initiated to determine the epidemiology of the outbreak and to identify and control the possible sources. METHODS: A multidisciplinary outbreak team was formed including members from hospital hygiene and local, state and national health and veterinarian authorities. Active surveillance was set up in the hospital to find new cases. A retrospective case control study was conducted to identify possible risk factors for disease. Hospital workers and food samples were tested for the pathogen and positive isolates were typed. RESULTS: Between calendar weeks 4 and 24 of the year 2007, a total of 28 patients with a median age 66 years had nosocomial salmonellosis culture confirmed for SALMONELLA Enteritidis LT 8/7. In food samples from February, the same Salmonella lysotype was isolated in 4 different food samples. SALMONELLA Enteritidis LT 8/7 was also identified in stool samples from 5 kitchen personnel. The case control study indicated antacida therapy (odds ratio: 5.5, 95 % CI 1.2 - 26.0) as a risk factor for nosocomial salmonellosis among patients. No particular diet was associated with an increased risk of disease. DISCUSSION: This nosocomial salmonellosis outbreak was characterised by prolonged duration and a low infection rate among patients. The epidemiological investigation suggests that the origin of the outbreak was food that was probably associated with a contamination in the hospital kitchen. Furthermore, kitchen staff could, as carriers, have contributed to a low-level contamination of various foods for a long period. The intermittent occurrence of the cases is best explained by a low level of contaminated food which primarily led to clinical symptoms among especially vulnerable persons (older patients with antacida therapy). Considering the unusual progression of this outbreak, hospitals should initiate an intensive epidemiological and microbiological investigation, even if only few nosocomial salmonellosis cases occur.

Incidence of quinolone resistance in strains of Salmonella isolated from poultry, cattle and pigs in Germany between 1998 and 2001.

This paper reports the susceptibility to the quinolone nalidixic acid and the fluoroquinolone ciprofloxacin of 14,514 strains of Salmonella isolated in Germany from poultry, cattle and pigs between 1998 and 2001. Quinolone-resistant salmonellae were most frequently isolated from poultry, with a prevalence of 10.2 to 16.8 per cent. Poultry-associated serotypes, such as Salmonella Paratyphi B (d-tartrate positive), Salmonella Hadar and Salmonella Virchow, had the highest prevalence of quinolone resistance, ranging between 35 and 74 per cent. All the nalidixic acid-resistant strains also had a reduced susceptibility to ciprofloxacin, with minimum inhibitory concentrations (MICS) of 0.125 to 2 microg/ml. A comparison of the MICS for ciprofloxacin of the strains of these poultry-associated serotypes and Salmonella Enteritidis phage type 4 isolated in 1998/99 and 2000/01 indicated that there had been a shift towards higher MIC values of up to 2 microg/ml. The quinolone resistance-determining region (QRDR) of the gyrA gene and the homologue region of the parC gene of 31 selected strains were sequenced. Several different amino acid changes were observed in gyrA of the quinolone-resistant isolates at positions 83 and 87, but no substitutions were observed in parC.

Evaluation of molecular typing methods for Salmonella enterica serovar Typhimurium DT104 isolated in Germany from healthy pigs.

The discriminatory power of four different DNA based typing methods was tested for the molecular subtyping of Salmonella Typhimurium phage type DT104 isolates. German DT104 strains (n = 133) originating from slaughter pigs were analysed by plasmid profiling, and 32 of them by pulsed-field gel electrophoresis (PFGE) using the restriction enzymes XbaI, SpeI or BlnI, random amplification of polymorphic DNA (RAPD) using 13 different primers and IS200 typing. A resulting subtyping scheme was obtained which is based on the most discriminatory power of the individual methods i.e. plasmid profiling and PFGE with all three enzymes. The index of discrimination obtained by the subtyping scheme was 0.909 closely approaching the maximum value of one. Although minor differences occurred in the molecular DNA pattern of single DT104 strains, a dominating subtyping pattern was observed confirming other studies which showed, that S. Typhimurium DT104 isolates are highly clonal.

Incidence of quinolone resistance over the period 1986 to 1998 in veterinary Salmonella isolates from Germany.

A total of 24,591 nonhuman salmonella strains isolated in Germany between 1986 and 1998 were examined for their resistance to nalidixic acid by an agar diffusion method. The rate of resistance (inhibition zone,

[Investigation of foodborne outbreak due to Salmonella infantis using epidemiological and microbiological methods]. / Untersuchung einer lebensmittelbedingten Salmonella infantis-Gruppenerkrankung mit Methoden der Epidemiologie und Mikrobiologie.

In foodborne outbreaks, direct microbiological diagnosis is often not possible due to lack of remaining food samples. Therefore, in this investigation of an outbreak of Salmonella infantis at a fair, we chose an epidemiological approach in addition to microbiological testing. In a case control study, fair participants with symptoms of acute gastroenteritis as well as participants showing no signs of disease were interviewed by telephone. Questions concerning what food had been eaten at the fair and the course of disease had priority. Data analysis showed a significantly elevated odds ratio of 144 (p < 0.00001) for the consumption of potato salad. Salmonella infantis was cultured in faeces of symptomatic individuals as well as from left-over potato salad in high concentration. In conclusion, our data show that the cause of a foodborne outbreak can be detected through the application of epidemiologic methods with a high degree of certainty. In order to eliminate memory bias, a structured interview should be carried out as soon as possible after the initial outbreak.

Sequence diversity, predicted two-dimensional protein structure, and epitope mapping of neisserial Opa proteins.

The sequence diversity of 45 Opa outer membrane proteins from Neisseria meningitidis, Neisseria gonorrhoeae, Neisseria sicca, and Neisseria flava indicates that horizontal genetic exchange of opa alleles has been rare between these species. A two-dimensional structural model containing four surface-exposed loops was constructed based on rules derived from porin crystal structure and on conservation of sequence homology within transmembrane beta-strands. The minimal continuous epitopes recognized by 23 monoclonal antibodies were mapped to loops 2 and 3. Some of these epitopes are localized on the bacterial cell surface, in support of the model.

Clonal descent and microevolution of Neisseria meningitidis during 30 years of epidemic spread.

Serogroup A meningococci of subgroups III, IV-1 and IV-2 are probably descended from a common ancestor that existed in the nineteenth century. The 10.5kb sequences spanning five distinct chromosomal loci, encoding cell-surface antigens, a secreted protease or housekeeping genes and intergenic regions, were almost identical in strains of those subgroups isolated in 1966, 1966 and 1917 respectively. During the subsequent two to three decades, all of these loci varied as a result of mutation, translocation or import of DNA from unrelated neisseriae. Thus, microevolution occurs frequently in naturally transformable bacteria. Many variants were isolated only once or within a single geographical location and disappeared thereafter. Other variants achieved genetic fixation within months or a few years. The speed with which sequence variation is either eliminated or fixed may reflect sequential bottlenecks associated with epidemic spread and contrasts with the results of phylogenetic analyses from bacteria that do not cause epidemics.

The porA alleles are identical in subgroup III serogroup A Neisseria meningitidis strains isolated in China in the 1960s and 1980s.

The porA genes from serogroup A, subgroup III strains isolated in the People's Republic of China in 1966 and in 1984 and 1985 were amplified by PCR at an annealing temperature of 75 degrees C. The DNA sequences (5 strains) and the restriction patterns generated by MspI (14 strains) were identical, unlike the results reported by Peixuan et al. (Z. Peixuan, H. Xujing, and X. Li, J. Clin. Microbiol. 33:458-462, 1995). Furthermore, PCR products which were amplified at an annealing temperature of 60 degrees C, as described by Peixuan et al., were heterogeneous in our study.