A rapid monitoring assay for the detection of Salmonella spp. and Salmonella Senftenberg strain W775 in composts

AIMS: The composting process needs to be validated for its hygienic status in order to ensure that it is free of pathogens. Generally, this is evaluated through temperature monitoring, or additionally through active inoculation and monitoring of indicator organisms. We aimed to develop a monitoring method for the heat-resistant indicator organism Salmonella enterica ssp. enterica serovar Senftenberg strain W775 for detection in composting biowastes. METHODS AND RESULTS: The method development is comprised of: (i) optimization of molecular detection of bacteria belonging to the genus Salmonella; (ii) identification of a DNA marker for Salmonella strain W775; and (iii) development of a multiplex polymerase chain reaction (PCR)-based on both DNA markers. Subsequently, Salmonella strain W775 was inoculated and monitored during composting of biowastes in an industrial composting facility. CONCLUSIONS: A highly sensitive and specific detection of viable cells was obtained by enriching the compost sample prior to multiplex PCR analysis. Complete inactivation of Salmonella strain W775 was obtained within 4 days in an industrial composting facility at temperatures ranging between 41 and 57 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: We describe a monitoring method for the simultaneous detection of naturally occurring Salmonella strains and artificially introduced Salmonella strain W775 in composting biowastes that can be applied in routine analysis.     

High-resolution analysis of salmonellae from turtles within a headwater spring ecosystem

Sediments and water from the pristine headwaters of the San Marcos River, Texas, USA, as well as swabs from biofilms on the carapace and from the cloacae of 17 musk turtles (Sternotherus odoratus) and one snapping turtle (Chelydra serpentina serpentina) caught at the same site, were analysed for salmonellae by culture and molecular techniques. Whereas enrichment cultures from sediment and water samples were negative for salmonellae in PCR- and in situ hybridization-based analyses, both techniques detected salmonellae after enrichments from both carapace and cloacae of nine (i.e. of 53%) musk turtles. Further characterization of 10 isolates obtained from the enrichment cultures of four selected individuals and confirmed as salmonellae by PCR analysis was achieved by fingerprinting techniques (rep-PCR). The results show differences between individuals and, in one case, variation among isolates from a single individual. All isolates from two individuals displayed identical profiles. These profiles were different from those obtained from the isolates of the third individual, which were, themselves, also identical for all isolates. Salmonellae were much more diverse in samples from the carapace of the last individual with five different rep-PCR profiles retrieved. Serotyping of seven isolates representative for each rep-PCR profile identified all isolates as representing Salmonella enterica subspecies enterica serotype Rubislaw, which demonstrates the presence of different strains of potentially human pathogenic salmonellae naturally occurring on turtles even within pristine environments. The frequent detection of these organisms in biofilms on the carapace opens the door for speculations on the role of this habitat as a reservoir for salmonellae, and on potential implications for turtles acting as a dispersal vector.     

Detection of Campylobacter jejuni and Salmonella typhimurium in chicken using PCR for virulence factor hipO and invA genes (Saudi Arabia)

Campylobacter jejuni and Salmonella typhimurium are the leading causes of bacterial food contamination in chicken carcasses. Contamination is particularly associated with the slaughtering process. The present study isolated C. jejuni and S. typhimurim from fifty chicken carcass samples, all of which were acquired from different companies in Riyadh, Saudi Arabia. The identification of C. jejuni was performed phenotypically by using a hippurate test and genetically using a polymerase chain reaction with primers for 16S rRNA and hippurate hydrolase (hipO gene). For the dentification of S. typhimurim, a serological Widal test was carried out using serum anti-S. typhimurium antibodies. Strains were genetically detected using invA gene primers. The positive isolates for C. jejuni showed a specific molecular size of 1448 bp for 16S rRNA and 1148 bp for hipO genes. However, the positive isolates of the invA gene exhibited a specific molecular size at 244 bp using polymerase chain reaction (PCR). Comparing sequencing was performed with respect to the invA gene and the BLAST nucleotide isolates that were identified as Salmonella enterica subsp. enterica serovar typhimurium strain ST45, thereby producing a similarity of 100%. The testing identified C. jejuni for hippuricase, GenBank: {"type":"entrez-nucleotide","attrs":{"text":"Z36940.1","term_id":"535805"}}Z36940.1. While many isolates of Salmonella spp. that contained the invA gene were not necessarily identified as S. typhimurim, the limiting factor for the Widal test used antiS. typhimurum antibodies. The multidrug resistance (MDR) of C. jejuni isolates in chickens was compared with the standard C. jejuni strain ATCC 22931. Similarly, S. typhimurium isolates were compared with the standard S. typhimurium strain ATCC 14028.     

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.     

Rapid and simple detection of the invA gene in Salmonella spp. by isothermal target and probe amplification (iTPA)

Aims: Salmonella spp. are an important cause of food‐borne infections throughout world, and the availability of rapid and simple detection techniques is critical for the food industry. Salmonella enterica serovars Enteritidis and Typhimurium cause the majority of human gastroenteritis infections, and there are a reported 40 000 cases of salmonellosis in the United States each year. Methods and Results: A novel rapid and simple isothermal target and probe amplification (iTPA) assay that rapidly amplifies target DNA (Salmonella invA gene) using a FRET‐based signal probe in an isothermal environment was developed for detection Salmonella spp. in pre‐enriched food samples. The assay was able to specifically detect all of 10 Salmonella spp. strains without detecting 40 non‐Salmonella strains. The detection limit was 4 × 10¹ CFU per assay. The iTPA assay detected at an initial inoculum level of <10 CFU in the pre‐enriched food samples (egg yolk, chicken breast and peanut butter). Conclusions: This detection system requires only a water bath and a fluorometer and has great potential for use as a hand‐held device or point‐of‐care‐testing diagnostics. The iTPA assay is sensitive and specific and has potential for rapid screening of Salmonella spp. by food industry.     

Comparison of primers for the detection of Salmonella enterica serovars using real-time PCR

AIMS: To evaluate the specificity and sensitivity of PCR primers for the detection of Salmonella enterica in a real-time PCR assay using pure cultures. METHODS AND RESULTS: Unenriched whole cells in sterile water were used as template for each PCR. SYBR Green dye was used for the nonspecific detection of dsDNA. The real-time PCR detection limits of five previously published primer sets used in conventional PCR applications were not below 3 x 10(3) CFU per reaction (rxn). A new primer set, Sen, was designed, which detected Salm. enterica Newport down to 6 CFU rxn(-1) in one case, and gave an average detection limit of 35 CFU rxn(-1) over three separate runs. CONCLUSIONS: Primers originally designed for end-point PCR did not have adequate specificity or sensitivity compared with those specifically designed for real-time PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: This study emphasizes the importance of evaluating real-time PCR primer sets in pure cultures prior to testing in field samples. This study will benefit other researchers in selecting an appropriate primer set for real-time PCR detection of Salm. enterica.     

用环介导等温扩增技术快速检测粪便样本中的沙门菌

目的:建立快速检测粪便样本中的沙门菌的环介导等温扩增技术(LAMP),并着重在灵敏度和特异性方面对此方法进行评价。方法:利用LAMP针对沙门菌特定基因invA(靶基因)设计的6条特异引物,通过引物特异性识别特定基因invA上的8个独立区域来快速检测沙门菌;LAMP反应过程中会产生白色沉淀焦磷酸镁,故可以通过监测浊度来判定反应结果。结果:实时浊度仪监测反应结果表明,LAMP反应在60~65℃等温条件下50 min内完成;如果在反应前添加羟基萘酚兰,蓝色阳性结果很明显区别于紫色阴性结果;LAMP的最低检出限为6.97 pg/μL,PCR为69.7pg/μL,LAMP方法的检测灵敏度是PCR的10倍,且具有良好的特异性。结论:LAMP方法用于快速检测沙门菌,具有检测过程简单、实验装置简便、反应结果肉眼可辨、灵敏度高、特异性强的特点,对非沙门菌菌株的结果呈阴性,表明引物设计有很好的特异性。对粪便样本进行检测,发现具有同样的敏感性和特异性。这表明LAMP法是潜在的和有价值的在粪便样本中直接检测沙门菌的方法,具有快速、简便、低成本的特点。LAMP法适用于快速临床诊断。