Primers for Salmonella serovar detection by polymerase chain reaction.

Salmonella serovar detection was studied by polymerase chain reaction (PCR). The primers were designed from Salmonella specific clone, A18:2 which was previously constructed and studied for genus specificity through colony hybridization. The primers were subsequently tested for specificity and sensitivity and showed that they amplified DNA fragment of all Salmonellae tested but did not amplify all isolates of non-Salmonellae tested. The amplified fragment was confirmed and increased sensitivity by nested PCR. Salmonella isolates amplified by the primers in the first round PCR were all positive in the second round. The sensitivity in the first and second round were 7 pg and 80 fg, respectively. The result indicated that the primers can be used as molecular tool for future field survey of Salmonella both in food and in clinical specimens.

Construction of specific DNA probe for the detection of Salmonella in food.

The Salmonella specific DNA fragment from genomic DNA of S. typhimurium ATCC 23566 was cloned in E. coli and successfully used as a digoxigenin labeled probe for detecting the presence of Salmonella serotypes in both artificially contaminated food and natural contaminated food samples.

Salmonella krefeld in Thailand: I. Epidemiology, infection and drug resistance.

Information from the National Salmonella Shigella Center (NSSC), Thailand indicated that the most frequently isolated Salmonella serotype from humans during 1974-1975 was Salmonella typhi (33.1%), during 1976-1982 was S. krefeld (26.6%) and during 1983-1987 was S. derby (12.6%). Antimicrobial susceptibility study of various Salmonella serotypes indicated that S. krefeld was the serotype with multiple drug resistance persisting for the longest period of time. Human salmonellosis due to S. krefeld is very rare. During 1976-1978, a large outbreak of S. krefeld gastroenteritis occurred in Thailand, mainly in children. The outbreak spread countrywide and is currently endemic. Gastrointestinal symptoms are severe in young infants. Systemic invasion with bacteremia, meningitis and pneumonitis were reported. The antimicrobial susceptibility pattern of isolates varied from sensitive to multiply drug resistant. The common antibiotic resistances were to ampicillin (75-92%), chloramphenicol (33-75%), kanamycin (67-90%) and sulfamethoxazole-trimethoprim (15-52%). Resistance to gentamicin and sulfamethoxazole-trimethoprim declined after the period of the epidemic. Antimicrobial resistance patterns of 150 S. krefeld strains isolated in Thailand during 1978-1987 showed multiple drug resistance with up to seven drugs. The most common patterns were ApCmKmSuTp and ApCmKmSmSuTc.

Salmonella krefeld in Thailand: II. Molecular biology of drug resistance.

Human salmonellosis due to Salmonella krefeld is very rare. During 1976-1978, a large outbreak of S. krefeld gastroenteritis occurred in Thailand, mainly in children. The majority of strains were multiply drug resistant with high minimum inhibitory concentration (MIC). The MIC for these drugs were ampicillin (Ap) 256-4096 mg/l, chloramphenicol (Cm) 256-512 mg/l, kanamycin (Km) 512- greater than 4096 mg/l, streptomycin (Sm) greater than 1024 mg/l, sulfamethoxazole (Su) 4096- greater than 8192 mg/l, tetracycline (Tc) 64-128 mg/l and trimethoprim (Tp) 64-256 mg/l. Resistance to Su and Tp declined after the period of the epidemic. The resistance genes were found to be highly transferable at a rate of 10(-2) to 10(-4). All strains with more than five resistance markers had large molecular weight plasmids of 120-140 megadaltons. The restriction profile analysis of plasmids from isolates collected from various regions of the country showed similarity of DNA fragment pattern. These isolates were resistant to Ap, Cm, Km, Sm, Su and Tc.

Epidemiological study of sulfonamide and trimethoprim resistance genes in Enterobacteriaceae.

Sulfonamide (Su) and trimethoprim (Tp) resistance are known to caused by the production of drug resistant dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR), respectively. Sulfonamide and trimethoprim are often used in combination under the name cotrimoxazole. Cotrimoxazole resistance in various enteric bacteria isolated at Ramathibodi Hospital was studied. The rate of resistance from 1984-1989 of many genera was rather constant at 40%-60% except in Shigella spp in which the rate increased rapidly in 1987 till 1989. Seventy-five percent of Su-Tp resistant (Sur-Tpr) bacteria were also found to be resistant to other drugs such as ampicillin, aminoglycosides, tetracycline and chloramphenicol in addition to cotrimoxazole. Two hundred and forty Su-Tp resistant strains were analysed for the presence of type I and II dihydropteroate synthase as well as type I and V dihydrofolate reductase genes by hybridization with the corresponding gene probes. Type I DHPS gene predominated in Su-Tp resistant bacteria at 60.8% whereas type II DHPS was found in only 25%. Some strains (11.7%) had both genotypes but 2.5% did not have any. In the trimethoprim resistance study, the DHFR type I gene was also found more frequently (30%) whereas type V DHFR was only 19%. The remaining of Tp resistance (51%) was unclassified. The coexistence of Su and Tp resistance genes of each type was investigated among 118 Su and Tp resistant strains. It was found that type I DHPS gene was found together with either type I or V DHFR gene and type II DHPS was found with type I DHFR gene at about the same rate (28.9%, 27.1% and 26.3%, respectively). However, the presence of type II DHPS together with type V DHFR was rather low, only 5.9% of isolates were found to have both types of genes.

Biotinylated probes for epidemiological studies of drug resistance in Salmonella krefeld.

A gene probe for ampicillin resistance and one for sulphonamide resistance were prepared to study the origin and the relation of multiple drug resistances in Salmonella krefeld. The resistance genes were cloned into the pACYC184 vector of Escherichia coli from a common plasmid of S. krefeld that encoded for resistance to ampicillin, chloramphenicol, kanamycin, streptomycin, sulphonamide and tetracycline resistance. Restriction map analysis and deletion analysis of a recombinant plasmid (pACSS1) showed that the gene determining ampicillin resistance was located on a 1.34 and 1.12 kb PstI fragment, and that the gene for sulphonamide resistance was located on a 0.85 kb PstI fragment. These fragments were used as probes. Their specificity was tested by colony hybridization with various bacterial species, including sensitive and resistance S. krefeld isolates. Further study indicated that the ampicillin resistance gene probe reacted with the gene for TEM-1 beta-lactamase and that the gene probe for sulphonamide resistance reacted with the gene for type II dihydropteroate synthase. The two probes were sufficiently specific to allow study of the epidemiology of resistance in S. krefeld and other enteric bacteria.