Salmonella detection in foods and feeds in 27 hours by an enzyme immunoassay

A heterogeneous enzyme immunoassay (EIA), which could be completed within 27 h, was developed for the detection of salmonellae in foods. Samples were subjected to the usual non-selective enrichment for 16–18 at 35°C and to a short (6 h) post-enrichment in a moderately selective broth. The EIA was carried out on polystyrene microtitration plates. A pooled polyvalent Salmonella flagellar antiserum and a protein A-alkaline phosphatase conjugate were used. The sensitivity of the enzyme immunoassay compared favorably with that of the conventional cultural technique for detection of Salmonella in 40 naturally contaminated food and feed samples. No sample was positive only by the cultural technique; samples positive only by the enzyme immunoassay were observed for feeds. Some specimens yielded high background values indicating possible interference from food proteins.     

Molecular phylogenetic typing of pandemic isolates of Salmonella enteritidis

Salmonella enteritidis is now the most common Salmonella serovar in many countries. We have used cloned DNA probes to analyze genome interrelationships between strains chosen to represent the current S. enteritidis pandemic, and included designated type strains of the seven subspecies of Salmonella in order to compare the levels of discrimination of probes. DNA sequence divergence and rearrangements were analyzed in and around the rfa, fim and umuDC loci, and around insertion sites of the Salmonella-specific DNA insertion element, IS200. The S. enteritidis isolates showed a high degree of genome homogeneity. Chromosomal genetic loci exhibited characteristic DNA sequence divergence between subspecies of Salmonella, but no intraserovar divergence or difference with the subspecies I type strain was observed for S. enteritidis. The locus umuDC was not found in S. enteritidis. S. enteritidis contains a conserved and a variable site of insertion of insertion sequence IS200 and the analysis of DNA rearrangements around the second of these sites showed that three distinct evolutionary lines or races exist within pandemic isolates associated with human gasteroenteritis. IS200 profiles of a range of U.K. isolates of the epidemic phage type PT4 showed that all belonged to a single clonal line.     

Susceptibility of Salmonella typhimurium and Salmonella typhi to oxygen metabolites

Abstract The susceptibility of Salmonella typhimurium LT2 and of S. typhi 1079 to oxygen metabolites were compared. S. typhimurium LT2 and S. typhi 1079 were killed to an equal extent (about 40%) by the xanthine-xanthine oxidase (200 mU/ml) system. Among the various scavengers of oxygen metabolites, catalase alone inhibited the killing of S. typhimurium LT2 and S. typhi 1079 by the xanthine-xanthine oxidase system, indicating that hydrogen peroxide contributed to the killing of Salmonellae . The respiratory burst of murine macrophages was efficiently triggered by the ingestion of S. typhimurium LT2, S. typhimurium SL1102, and S. typhi 1079 and all to the same extent. However, in the range of the concentration of hydrogen peroxide produced by murine macrophages, neither S. typhimurium LT2 nor S. typhi 1079 were killed. Only S. typhimurium SL1102, a rough mutant of S. typhimurium LT2, was markedly susceptible under these conditions. The findings suggest that both S. typhimurium LT2 and S. typhi 1079 are resistant to oxygen-dependent killing mechanisms.     

Serotyping, PCR, phage-typing and antibiotic sensitivity testing of Salmonella serovars isolated from urban drinking water supply systems of Nepal

AIMS: To study the occurrence and diversity of Salmonella serovars in urban water supply systems of Nepal. METHODS AND RESULTS: Occurrence of Salmonella was detected in 42 out of 300 water samples by enrichment culture technique in selenite F broth followed by plating on Salmonella Shigella agar. A total of 54 isolates identified to genus level by standard tests were subsequently confirmed by serotyping, phage typing and PCR detection of virulence genes (inv A and spv C). The predominant serotype was Salmonella Typhimurium, followed by Salm. Typhi, Salm. Paratyphi A and Salmonella Enteritidis. Most of the Salm. Typhi isolates were E1 phage type followed by UVS4, A and UVS1. All isolates of Salm. Paratyphi A and Salm. Enteritidis were an untypable (UT) phage type. The majority of isolates were multi-drug resistant as revealed by Kirby-Bauer disc diffusion technique. Ceftriaxone resistant isolates of Salm. Enteritidis indicated the presence of one of the ESBL genes, blaSHV, whereas the genes blaTEM and blaCTX were absent. CONCLUSIONS: The microbiological quality of the urban water supply is poor and indicates possibility of fatal outbreaks of enteric fever and related infections in Nepal. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study will be useful in water borne disease control and prevention strategy formulation in Nepal and in the global context.     

Towards more virulent and antibiotic-resistant Salmonella?

Salmonella are well-known pathogens. Virulence determinants can be present on the chromosome, usually encoded on pathogenicity islands, or on plasmids and bacteriophages. Antibiotic resistance determinants usually are encoded on plasmids, but can also be present on the multidrug resistance region of Salmonella Genomic Island 1 (SGI1). Virulence plasmids show a remarkable diversity in the combination of virulence factors they encode, which appears to adapt them to specific hosts and the ability to cause gastroenteritidis or systemic disease. The appearance of plasmids with two replicons may help to extend the host range of these plasmids and thereby increase the virulence of previously non- or low pathogenic serovars. Antibiotic resistance among Salmonella is also increasing. This increase is not only in the percentage isolates resistant to a particular antibiotic, but also the development of resistance against newer antibiotics. The increased occurrence of integrons is particularly worrying. Integrons can harbour a varying set of antibiotic resistance encoding gene cassettes. Gene cassettes can be exchanged between integrons. Although the gene cassettes currently present in Salmonella integrons encode for older antibiotics (however, some still frequently used) gene cassettes encoding resistance against the newest antibiotics has been documented in Enterobacteriaceae. Furthermore, beta-lactamases with activity against broad-spectrum cephalosporins, which are often used in empiric therapy, have been found associated with integrons. So, empiric treatment of Salmonella infections becomes increasingly more difficult. The most worrisome finding is that virulence and resistance plasmids form cointegrates. These newly formed plasmids can be selected by antibiotic pressure and thereby for virulence factors. Taken together these trends may lead to more virulent and antibiotic-resistant Salmonella.     

2005-2013年肠道致病菌中志贺菌和沙门菌的分布及检测方法经验总结

目的分析杭州市第三人民医院2005-2013年肠道致病菌中志贺菌和沙门菌的分布并对检测方法进行经验总结。方法采用定位显色培养基和营养琼脂初筛疑似志贺菌和沙门菌,并参考该院历年菌种分离情况进行血清凝集。采用EXCEL统计处理数据,χ2检验比较采用定位显色培养基和营养琼脂平板之前和之后,粪便培养志贺菌和沙门菌的分离率之间差异的显著性。结果采用定位显色培养基和营养琼脂平板初筛疑似菌株后,志贺菌和沙门菌的分离率差异存在统计学意义。结论采用定位显色培养基和营养琼脂初筛疑似菌株,并对本院历年菌种分离情况进行总结,对改善志贺菌和沙门菌的分离率,提升检验科微生物室的工作效率有较大帮助。     

The sialic acid-containing lipopolysaccharides of Salmonella djakarta and Salmonella isaszeg (serogroup O: 48): Chemical characterization and reactivity with a sialic acid-binding lectin from Cepaea hortensis

Abstract Lipopolysaccharides (LPS) of Salmonella djakarta and Salmonella isaszeg , as well as of a spontaneous R-mutant of S. djakarta were investigated as to their content in neuraminic acid (Neu) and its individual linkage. The two Salmonella serovars both belong to the O:48 serogroup of Salmonella , but to two different subgroups. LPS of both S-forms contained high amounts of Neu, although in different quantities, whereas the R-form was completely devoid of it. Methylation analysis indicated that Neu is exclusively terminally linked in S. djakarta whereas both terminal and 4-linked Neu were recognized in S. isaszeg . Although terminally linked, a sialidase from Arthrobacter ureafaciens was unable to split Neu even after prolonged incubation from both S-type LPSs. When LPS was first treated by mild alkali, however, the total amount of Neu from S. djakarta LPS and about 50% from that of LPS of S. isaszeg could be removed. In contrast, alkali-treated LPS, but also the non-treated one, proved to be effective inhibitors for a sialic acid-binding lectin from Cepaea hortensis . The resistance of terminal Neu towards sialidase may be due to the presence of an O-acetyl group which would be removed during the methylation analysis but would, especially when linked to C-4, not interfere with the reactivity of the lectin.     

Real-time PCR method for Salmonella spp. targeting the stn gene

AIM: To develop a real-time PCR assay for Salmonella spp. targeting the stn gene. METHODS AND RESULTS: The presence of stn in the Salmonella bongori genome was found by a BLAST with Salmonella enterica stn sequence. Manual alignment of stn sequences showed that Salm. bongori had 88% sequence identity with Salm. enterica. Two primers (stnL-433 and stnR-561) and a probe (stnP-452) were designed to target conserved regions in stn and meet the requirements of a 5'-nuclease assay. The primers and probe were evaluated against 353 isolates, including 255 Salm. enterica representing 158 serotypes, 14 Salm. bongori representing 12 serotypes and 84 non-Salmonella representing 56 species from 31 genera. All isolates were correctly identified, with the exception of three isolates of Citrobacter amalonaticus, which gave false positives. The limit of detection with cultured Salmonella was 3 CFU per reaction. CONCLUSIONS: The stn real-time PCR method had 100% inclusivity, 96.4% exclusivity and a level of detection of 3 CFU per reaction for cultured Salmonella spp. SIGNIFICANCE AND IMPACT OF THE STUDY: The study showed that stn is present in Salm. bongori and is a valid target for both species of Salmonella. The Salmonella s tn real-time PCR is a useful method for identifying Salmonella spp.     

Salmonella enteritidis temperature-sensitive mutants protect mice against challenge with virulent Salmonella strains of different serotypes

The protection conferred by temperature-sensitive mutants of Salmonella enteritidis against different wild-type Salmonella serotypes was investigated. Oral immunization with the single temperature-sensitive mutant E/1/3 or with a temperature-sensitive thymine-requiring double mutant (E/1/3T) conferred: (i) significant protection against the homologous wild-type Salmonella strains; (ii) significant cross-protection toward high challenge doses of S. typhimurium. Significant antibody levels against homologous lipopolysaccharide and against homologous and heterologous protein antigens were detected in sera from immunized mice. Moreover, a wide range of protein antigens from different Salmonella O serotypes were recognized by sera from immunized animals. Besides, primed lymphocytes from E/1/3 immunized mice recognized Salmonella antigens from different serotypes. Taken together, these results indicate that temperature-sensitive mutants of S. enteritidis are good candidates for the construction of live vaccines against Salmonella.     

Flagellin expressed by live Salmonella vaccine strains induces distinct antibody responses following delivery via systemic or mucosal immunization routes

Salmonella flagellin, expressed as flagella in live attenuated vaccine strains, elicits distinct systemic (IgG) and secreted (IgA) antibody responses in mice following delivery via mucosal (nasal/oral) or parenteral (intraperitoneal (i.p.)) immunization routes. Reduced flagellin-specific antibodies were detected either systemically or locally following delivery of flagellated derivatives of aroA Salmonella enterica serovar Dublin SL1438 via the nasal route, the most effective mucosal site for activation of immune responses in mice. In contrast, flagellin represents the most potent Salmonella antigen for the generation of specific serum antibody (IgG) responses following i.p. inoculations. The distinct immunogenic properties of Salmonella flagellin could not be ascribed to deficient colonization, reduced invasive ability or loss of the flagellin expression by the flagellated vaccine strains.     

沙门氏菌Ⅲb的一个新血清型

1989年8月从蛇肠内容物中检出一株沙门氏菌,编号为S.3337经鉴定为新血清型,其生化特性符合沙门氏菌属的定义.根据该菌株不发酵卫矛醇,能利用丙二酸盐,迅速发酵乳糖,ONPG为阳性,具有双相H抗原,应归属于沙门氏菌Ⅲb.经抗原分析该菌株的抗原式为65:z:z_(55).     

伤寒杆菌和甲型副伤寒杆菌的稳定L型外膜蛋白和染色体DNA酶切研究

非高渗培养基传代培养的伤寒杆菌和甲型副伤寒杆菌的稳定L型丧失了主要外膜蛋白、特异性表面抗原和染色体DNA部分片段,保留了沙门氏菌共同的内部抗原和形成L型独特的表面抗原。提示伤寒杆菌和甲型副伤寒杆菌的稳定L型多种特性的丧失同其基因或主要外膜蛋白的缺失有关。     

Antimicrobial drug resistance in Salmonella: problems and perspectives in food- and water-borne infections

Strains of Salmonella spp. with resistance to antimicrobial drugs are now widespread in both developed and developing countries. In developed countries it is now increasingly accepted that for the most part such strains are zoonotic in origin and acquire their resistance in the food-animal host before onward transmission to humans through the food chain. Of particular importance since the early 1990s has been a multiresistant strain of Salmonella typhimurium definitive phage type (DT) 104, displaying resistance to up to six commonly used antimicrobials, with about 15% of isolates also exhibiting decreased susceptibility to ciprofloxacin. Mutations in the gyrA gene in such isolates have been characterised by a PCR LightCycler-based gyrA mutation assay, and at least four different mutations have been identified. Multiple resistance (to four or more antimicrobials) is also common in the poultry-associated pathogens Salmonella virchow and Salmonella hadar, with an increasing number of strains of these serotypes exhibiting decreased susceptibility to ciprofloxacin. Multiple resistance is also being found in other serotypes in several other European countries, and has been associated with treatment failures. For Salmonella typhi, multiple drug resistance is now the norm in strains originating in the Indian subcontinent and south-east Asia. Such multiresistant strains have been responsible for several epidemics and some of these have been associated with contaminated water supplies. Furthermore, an increasing number of multiresistant strains of S. typhi are now exhibiting decreased susceptibility to ciprofloxacin, with concomitant treatment failures. In developed countries antimicrobial resistance in zoonotic salmonellas has been attributed to the injudicious use of antimicrobials in food-producing animals. It is hoped that the application of Codes of Practice for the use of such agents, which have been prepared by the pharmaceutical industry in response to widespread international concern about the development of drug resistance in bacterial pathogens, will now result in a widespread reduction in the incidence of drug-resistant salmonellas in food production animals and humans on an international scale.     

Distribution of "classic" virulence factors among Salmonella spp

Whether an infection with Salmonella spp. leads to a disease largely depends on the virulence of the strain and the constitution of the host. The virulence of the strain is determined by so-called virulence factors. Whereas a number of virulence factors of Salmonella have been identified only recently, others have been studied for decades. These latter virulence factors i.e., virulence-plasmids, toxins, fimbriae and flagella are therefore referred to as "classic" virulence factors. Here we present an overview on the distribution of (genes coding for) these virulence factors among Salmonella spp. The pathogenicity islands of Salmonella are also reviewed, all be it briefly, since they contain a major part of the virulence genes.     

PagR mediates the precise regulation of Salmonella pathogenicity island 2 gene expression in response to magnesium and phosphate signals in Salmonella Typhimurium

To establish systemic infections, Salmonella enterica serovar Typhimurium (S. Typhimurium) requires Salmonella pathogenicity island 2 (SPI‐2) to survive and replicate within macrophages. High expression of many SPI‐2 genes during the entire intracellular growth period within macrophages is essential, as it contributes to the formation of Salmonella‐containing vacuole and bacterial replication. However, the regulatory mechanisms underlying the sustained induction of SPI‐2 within macrophages are not fully understood. Here, we revealed a time‐dependent regulation of SPI‐2 expression mediated by a novel regulator PagR (STM2345) in response to the low Mg²⁺ and low phosphate (P_i) signals, which ensured the high induction of SPI‐2 during the entire intramacrophage growth period. Deletion of pagR results in reduced bacterial replication in macrophages and attenuation of systemic virulence in mice. The effects of pagR on virulence are dependent on upregulating the expression of slyA, a regulator of SPI‐2. At the early (0–4 hr) and later (after 4 hr) stage post‐infection of macrophages, pagR is induced by the low P_i via PhoB/R two‐component systems and low Mg²⁺ via PhoP/Q systems, respectively. Collectively, our findings revealed that the PagR‐mediated regulatory mechanism contributes to the precise and sustained activation of SPI‐2 genes within macrophages, which is essential for S. Typhimurium systemic virulence.     

Detection of peptidoglycan and β-glucan with silkworm larvae plasma test

Abstract Discrimination of 70 Salmonella strains previously studied by ribotyping was realized by RAPD and ERIC-PCR analysis. RAPD results on the 56 S. typhimurium isolates did not closely match those of ribotyping. With ERIC-PCR, two fingerprints only were obtained. For the 14 S. enteritidis strains, a helpful discrimination was obtained with RAPD analysis, while ERIC-PCR resulted in a single fingerprint.