Identification by subtractive hybridization of sequences specific for Salmonella enterica serovar enteritidis.

Salmonella enterica serovar Enteritidis, a major cause of food poisoning, can be transmitted to humans through intact chicken eggs when the contents have not been thoroughly cooked. Infection in chickens is asymptomatic; therefore, simple, sensitive, and specific detection methods are crucial for efforts to limit human exposure. Suppression subtractive hybridization was used to isolate DNA restriction fragments present in Salmonella serovar Enteritidis but absent in other bacteria found in poultry environments. Oligonucleotide primers to candidate regions were used in polymerase chain reactions to test 73 non-Enteritidis S. enterica isolates comprising 34 different serovars, including Dublin and Pullorum, two very close relatives of Enteritidis. A primer pair to one Salmonella difference fragment (termed Sdf I) clearly distinguished serovar Enteritidis from all other serovars tested, while two other primer pairs only identified a few non-Enteritidis strains. These primer pairs were also useful for the detection of a diverse collection of clinical and environmental Salmonella serovar Enteritidis isolates. In addition, five bacterial genera commonly found with Salmonella serovar Enteritidis were not detected. By treating total DNA with an exonuclease that degrades sheared chromosomal DNA but not intact circular plasmid DNA, it was shown that Sdf I is located on the chromosome. The Sdf I primers were used to screen a Salmonella serovar Enteritidis genomic library and a unique 4,060-bp region was defined. These results provide a basis for developing a rapid, sensitive, and highly specific detection system for Salmonella serovar Enteritidis and provide sequence information that may be relevant to the unique characteristics of this serovar.     

Detection of hilA gene sequences in serovars of Salmonella enterica subspecies enterica

hilA gene promoter, component of the Salmonella Pathogenicity Island 1, has been found in Salmonella serovar Typhimurium, being important for the regulation of type III secretion apparatus genes. We detected hilA gene sequences in Salmonella serovars Typhi, Enteritidis, Choleraesuis, Paratyphi A and B, and Pullorum, by polymerase chain reaction (PCR) and hybridization techniques. The primers to carry out PCR were designed according to hilA sequence. A low stringency hybridization with the probe pVV441 (hilA open-reading-frame plasmid) was carried out. To find hilA gene sequences in other Salmonella sp. suggest that these serovars could have similar sequences of this kind of virulence genes.     

New O-acetyltransferase-deficient Ames Salmonella strains generated by specific gene disruption

CoASAc-dependent N-hydroxyarylamine O-acetyltransferase (OAT) is an enzyme involved in the intracellular metabolic activation of N-hydroxyarylamines derived from mutagenic nitroarenes and aromatic amines. The oat gene encoding the enzyme of S. typhimurium TA98 and TA100 was specifically disrupted and the sensitivities of the resulting strains, i.e., YG7130 and YG7126, to mutagens were compared with those of the conventional oat-deficient strains, i.e., TA98/1,8DNP6 and TA100/1,8DNP, respectively. The new oat-deficient strains and the conventional strains exhibited similar sensitivity against most of the chemicals tested: both strains YG7130 and strain TA98/1,8-DNP6 were resistant to mutagenicity by 1,8-dinitropyrene (1, 8-DNP), 1-nitropyrene, 2-amino-6-methyldipyrido[1,2-alpha:3', 2'-d]imidazole (Glu-P-1) and 2-amino-3-methyl-3H-imidazo[4, 5-f]quinoline (IQ); neither strain YG7130 nor strain TA98/1,8-DNP6 was resistant to the mutagenicity of 3-amino-1-methyl-5H-pyrido[4, 3-b]indole (Trp-P-2); strain YG7126 and strain TA100/1,8-DNP were refractory to the mutagenicity of 1,8-DNP. However, the order of the sensitivity against 2-nitrofluorene (2-NF) was TA98>YG7130>TA98/1, 8-DNP6 and TA100>YG7126>TA100/1,8-DNP. Since the strains YG7130 and YG7126 have chloramphenicol resistance (Cmr) gene in place of the chromosomal oat gene for gene disruption, the possible involvement of chloramphenicol acetyltransferase (CAT) encoded by the Cmr gene in the activation of 2-NF was examined. Strikingly, introduction of plasmid pACYC184 carrying the Cmr gene alone substantially enhanced the sensitivity of the conventional oat-deficient strains to 2-NF. These results suggest that the new strains as well as the conventional strains are useful to assess the roles of OAT in the metabolic activation of nitroaromatics and aromatic amines in S. typhimurium, and also that CAT has the ability to activate N-hydroxy aromatic amines to mutagens.     

Low expression of AcrB in the deoxycholate-sensitive strains of Salmonella enterica subspecies enterica serovar Pullorum

We investigated the mechanism responsible for bile susceptibility in three deoxycholate-sensitive (DCs) strains of Salmonella enterica subspecies enterica serovar Pullorum isolated in 1958 in Japan. Of the genes encoding the AcrAB-TolC efflux system, the expression of acrB mRNA was 10-fold lower in the DCs strains than in a deoxycholate-resistant (DCr) strain, whereas those of the acrA and tolC genes were two-fold lower. These results suggested that low expression of acrB was strongly correlated with bile susceptibility in the DCs strains. In addition, the increase in tolC expression levels was not detected in the DCr mutants derived from the DCs strains, suggesting that difference in the expression levels of tolC is not associated with bile susceptibility.     

Salmonella enterotoxin (stn) gene is prevalent among strains of Salmonella enterica, but not among Salmonella bongori and other Enterobacteriaceae

Abstract All strains and serovars of Salmonella enterica such as serovar Typhimurium, Enteritidis, Dublin, Typhi, etc. were found to carry the Salmonella enterotoxin determinant stn as far as examined in PCR and hybridization studies. However, using MDCK cells for testing the toxicity of the strains under investigation, only a limited number of stn positive strains revealed phenotypically the Salmonella enterotoxin Stn. In contrast to S. enterica , other Enterobacteriaceae including Salmonella bongori were found neither genotypically nor phenotypically Stn toxin positive.     

Identification of Salmonella enterica subspecies I, Salmonella enterica serovars Typhimurium, Enteritidis and Typhi using multiplex PCR

This study was designed to develop a multiplex PCR method with five specific primer pairs for the detection of Salmonella spp., Salmonella subspecies I, Salmonella enterica serovars Typhimurium, Typhi and Enteritidis. A multiplex PCR was constructed with five primer pairs for the detection of Salmonella and pathogenic Salmonella serovars, including a specific primer pair for Salmonella Typhi, based on the sequence comparison between genomic DNA sequences of 12 Salmonella strains. Each primer pair was specifically targeted to Salmonella spp., Salmonella subspecies I, Salmonella Typhimurium, Typhi and Enteritidis. This multiplex PCR was evaluated with various DNAs of Salmonella serovars that yielded high specificity for amplifying the expected PCR products of Salmonella serovars. Using this primer pair, a set of multiplex PCR was performed for the rapid identification of salmonellae and major pathogenic Salmonella serovars. Although this multiplex PCR method will need to be evaluated for a wide range of Salmonella serovars among multilaboratories, it should be useful for identifying clinically significant strains of Salmonella serovars rapidly and accurately without the need for serological testing.     

Variation of the rfb gene clusters in Salmonella enterica.

In order to explore the genetic variation of O antigens of Salmonella enterica, we surveyed 164 strains (132 serovars) belonging to 45 serogroups, using 25 mostly single-gene rfb DNA probes for colony hybridization. The results revealed that strains within a serogroup have very similar or identical rfb genes. At least three of the four rhamnose genes were detected in all 17 serogroups reported to contain rhamnose, and one or more were detected in three others. The likelihood of being detected decreased in the order rfbB, rfbC, rfbA, and rfbD, which is the map order, suggesting a gradient of divergence. Mannose pathway genes were much less conserved, and of 27 groups reported to contain mannose or mannose derivatives colitose or fucose, only 9 hybridized to the rfbM and rfbK probes. Dideoxyhexose genes were found only in groups reported to contain dideoxyhexoses. Group D2, which had not been studied previously, appears to resemble group D1, with the substitution of one gene from group E1 to give a change in one linkage. In contrast to sugar pathway genes, sugar transferase genes did not in general hybridize to strains of other groups outside the closely related groups A, B, and D, with the exception of the galactose transferase gene also shared by groups C2, C3, and all E groups.     

The occurrence and characterisation of oxy-imino-beta-lactams resistant strains among Salmonella enterica subsp. enterica isolated in Poland

Extended-spectrum beta-lactamases (ESBLs) are enzymes manifesting considerable hydrolyzing activity on a wide variety of beta-lactam antibiotics including oxyiminocephalosporins and aztreonam. In the study reported here we investigated the types of ESBL produced by Salmonella enterica subsp. enterica strains isolated from clinical samples in the microbiological laboratories of sanitary-epidemiological units in Poland from 1999 to 2004. Among 239 ampicillin-resistant Salmonella enterica subsp. enterica strains isolated from clinical samples in the microbiological laboratories of sanitary-epidemiological units in Poland, 68 isolates of oximino-beta-lactams resistant of 6 serovars were found. There were 16 epidemiological unrelated strains (6 isolates of S. Enteritidis, 5 isolates of S. Thompson, 3 isolates of S. Typhimurium, one-fold isolate of S. Muenster and S. enterica 1,9,12:-:-) coming from different areas of country and 52 epidemiologically related isolates of S. Oranienburg, coming from a prolonged outbreak in an orphanage in Lód?. All the strains were identified as the ESBLs producers. The molecular analysis revealed that most of them expressed CTX-M-3 ESBL which is widely observed in Poland and additional enzyme TEM-1. All tested isolates of S. Thompson and one of three S. Typhimurium isolates were found to produce SHV-5 ESBL. This is the first report regarding the presence of SHV-5 in the genus Salmonella in Poland.     

Identification of host-specific colonization factors of Salmonella enterica serovar Typhimurium

The severity of infections caused by Salmonella enterica serovar Typhimurium varies depending on the host species. Numerous virulence genes have been identified in S. Typhimurium, largely from studies in mice, but their roles in infections of other species remain unclear. In the most comprehensive survey of its kind, through the use of signature-tagged mutagenesis of S. Typhimurium we have identified mutants that were unable to colonize calf intestines, mutants unable to colonize chick intestines and mutants unable to colonize both species. The type three secretion systems encoded on Salmonella pathogenicity islands (SPIs) 1 and 2 were required for efficient colonization of cattle. However, disruption of these secretion systems only caused a minor defect in S. Typhimurium colonization of chicks. Transposon insertions in SPI-4 compromised S. Typhimurium colonization of cattle, but not chicks. This is the first data confirming a role for SPI-4 in pathogenesis. We have also been able to ascribe a role in colonization for cell surface polysaccharides, cell envelope proteins, and many 'housekeeping' genes and genes of unknown function. We conclude that S. Typhimurium uses different strategies to colonize calves and chicks. This has major implications for vaccine design.     

Genome sequences of Salmonella enterica serovar typhimurium, Choleraesuis, Dublin, and Gallinarum strains of well- defined virulence in food-producing animals

Salmonella enterica is an animal and zoonotic pathogen of worldwide importance and may be classified into serovars differing in virulence and host range. We sequenced and annotated the genomes of serovar Typhimurium, Choleraesuis, Dublin, and Gallinarum strains of defined virulence in each of three food-producing animal hosts. This provides valuable measures of intraserovar diversity and opportunities to formally link genotypes to phenotypes in target animals.     

Characterization of lysine decarboxylase-negative strains of Salmonella enterica serovar Enteritidis disseminated in Japan

Salmonella enterica serovar Enteritidis is one of the leading causes of food-borne diseases in Japan. Typically, Salmonella spp. test positive for lysine-decarboxylase. However, the number of isolates of serovar Enteritidis without lysine-decarboxylase activity increased in Japan in 2003. Among 109 strains from distinct outbreaks, 10 lacked lysine-decarboxylase activity. Nine of the ten lysine-decarboxylase-negative strains showed quite similar pulsed-field gel electrophoresis profiles. Their lysine-decarboxylase phenotype was recovered by introduction of the cadBA locus from an lysine-decarboxylase-positive strain. Although the cad loci of the lysine-decarboxylase-negative strains seemed to be intact without any insertion sequences, cadC, a positive regulator of cadBA, had a single-base deletion at the same position, the 973rd base (cytosine), in all the nine lysine-decarboxylase-negative strains, whereas the wild-type cadC gene has a 1542 bp coding region (514 amino acids). This deletion was expected to produce a truncated (338 amino acids) form of CadC due to a frameshift. Because CadC senses environmental cues such as external pH and lysine through its putative C-terminal periplasmic domain, it is likely that the truncated CadC is not sensitive enough to external signaling to activate the cadBA operon, resulting in loss of the lysine-decarboxylase activity. Our results suggest that dissemination of these genetically closely related strains of serovar Enteritidis accounts for the unusual increase in the isolation of lysine-decarboxylase-negative strains.     

SMM-system: A mining tool to identify specific markers in Salmonella enterica

This report presents SMM-system, a software package that implements various personalized pre- and post-BLASTN tasks for mining specific markers of microbial pathogens. The main functionalities of SMM-system are summarized as follows: (i) converting multi-FASTA file, (ii) cutting interesting genomic sequence, (iii) automatic high-throughput BLASTN searches, and (iv) screening target sequences. The utility of SMM-system was demonstrated by using it to identify 214 Salmonella enterica-specific protein-coding sequences (CDSs). Eighteen primer pairs were designed based on eighteen S. enterica-specific CDSs, respectively. Seven of these primer pairs were validated with PCR assay, which showed 100% inclusivity for the 101 S. enterica genomes and 100% exclusivity of 30 non-S. enterica genomes. Three specific primer pairs were chosen to develop a multiplex PCR assay, which generated specific amplicons with a size of 180 bp (SC1286), 238 bp (SC1598) and 405 bp (SC4361), respectively. This study demonstrates that SMM-system is a high-throughput specific marker generation tool that can be used to identify genus-, species-, serogroup- and even serovar-specific DNA sequences of microbial pathogens, which has a potential to be applied in food industries, diagnostics and taxonomic studies. SMM-system is freely available and can be downloaded from http://foodsafety.sjtu.edu.cn/SMM-system.html.