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 enteritidis outbreak in Thailand: study by random amplified polymorphic DNA (RAPD) analysis.

An outbreak of Salmonella enteritidis in Thailand was reported in 1990. The majority of isolates were found in chicken and human throughout the country. The continuation of a high rate of spreading which is presently continuing prompted us to investigate possible clonal involvement in the outbreak. One hundred and twenty five isolates of S. enteritidis which were isolated between 1990-1993 were clonally identified by the technique of Random Amplified Polymorphic DNA (RAPD) analysis. Eight profiles were found indicating the presence of 8 clones, designated no. 1-8. The predominant clone was profile no. 4 which was encountered in 93.6% of tested isolates while the rest of the profile comprised only 0.8-1.6%. The predominant clone was distributed mainly in isolates from chickens and humans which is suggestive that the profile no. 4 is the major clone involved in this outbreak and that chickens were the source of S. enteritidis infection. The information from the Microbiology Laboratory at Ramathibodi Hospital revealed that nearly 40% of S. enteritidis were isolated from blood specimens. This may reflect the invasiveness of S. enteritidis in Thailand. We concluded that the outbreak involved the single clone, RAPD profile no. 4 which may disperse dominantly during the epidemic.

Morphological variation in pathogenic strains of Penicillium marneffei.

Penicillium marneffei is a dimorphic fungus known to be pathogenic to animals and man. The natural reservoir of this organism was known to be bamboo rats found in South Vietnam, Thailand and China. The first two human infections were reported in 1959 and 1973 from the United States. Up to 1984, five new cases of human penicillosis were reported from Thailand. Since then several more cases have been reported from different parts of the world mainly from the southern part of China. However, there are very limited mycological descriptions of this fungi. In this report, five Thai strains were studied for colonial morphology in comparison with Reference strain PLM 689. Variation in mycelial pigment was observed ranging from yellowish-green to orange with water soluble red pigment produced in every strain which can be seen early from the reverse side. Ultrastructural study by both scanning electron microscopy (SEM) and transmission electron microscopy (TEM) was compared with that of the reference strain PLM 689. PLM 689 strain had only biverticillate penicilli, but all five strains from Thailand had both monoverticillate and biverticillate penicilli which occasionally appeared on the same branch. The conidia of the Thai isolates were oval in shape and 1.3-2 x 0.7-1.6 microns in size smaller than those of PLM 689 which were 2.5-4 x 2-3 microns. Phialides were also smaller and a little shorter but the number of phialides was similar to those of PLM 689 ranging 4-10 except for one strain which had 3-16 phialides. All Thai strains have stipes smaller and somewhat longer than those of PLM 689.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Haemophilus influenzae and Streptococcus pneumoniae in children with acute respiratory infection.

The incidence of Haemophilus influenzae and Streptococcus pneumoniae in children with acute respiratory infection (ARI) under 5 years was carried out by throat swab culture, blood culture, body fluid or tissue culture in 688 patients from a community, 744 patients from a teaching hospital in Bangkok, 766 normal children from the community and 303 children from a hospital well baby clinic. H. influenzae was found in the throats of 15-20% of patients and in the throats of 4-6% of normal children (p less than 0.001 for both hospital and community patients). Only 12/332 strains (3.6%) of H. influenzae were type b. The rest of H. influenzae were non type b. The most common biotype of H. influenzae non type b was biotype II. S. pneumoniae was found in hospital patients in highly significant numbers compared to the controls (12% vs 4%). No significant difference was observed in strains from the community patients.

Drug resistance of Haemophilus ducreyi.

Chancroid, the disease caused by H. ducreyi is one of the common sexually transmitted diseases (STD) in Thailand and other tropical countries. In Thailand, the diagnosis of chancroid is still based on clinical appearance which may be confused with other STD manifested by genital ulcers. In recent years the increasing resistance strains of H. ducreyi to these antimicrobial agents has been reported so that cultivation and antimicrobial susceptibility tests of this organism have become more important. This study showed that MBV is the best medium for isolation with a success rate of 48%. All strains tested from isolates of this study were resistant to ampicillin, due to production of beta-lactamase. Approximately 99% of the strains were resistant to tetracycline 92% of strains were resistant to sulfamethoxazole and 32% were resistant to trimethoprim. All isolates were susceptible to chloramphenicol, ceftriaxone, erythromycin and the fluorinated quinolones ciprofloxacin, norfloxacin, ofloxacin and pefloxacin. Beta-lactamase enzymes produced by 37 strains of H. ducreyi were determined for their isoelectric point (pI). All had pI of 5.4, indicative of plasmid-mediated beta-lactamase type TEM-1.

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.

Aetiologic agents of septic sore throat in Thai children.

A bacteriological study of children with respiratory infections in Bangkok during January to November 1976 revealed that 37% of the patients had symptoms and sign of bacterial pharyngotonsillitis. Twenty-six per cent of these children harboured Streptococcus pyogenes in their throats. The numbers of streptococci other than group A and Staphylococcus aureus were increased in the children with respiratory infections. However, Staph. aureus was found as the sole organism in children with exudate more often than in the children with only URI. The possible role of Staph. aureus in bacterial pharyngitis should not be ignored. Penicillin remains a drug of choice for the treatment of streptococcal pharyngitis. If penicillin is contraindicated, erythromycin should be preferred over lincomycin as a second choice of drug in order to avoid treatment failure if lincomycin resistant streptococci are present.

Pseudomonas pseudomallei: II. Laboratory and experimental studies in animals.

The cultural, biochemical characteristics and antibiotic sensitivity of strains of Pseudomonas pseudomallei isolated from four cases of melioidosis admitted to Ramathibodi Hospital are described. The organisms were gram-negative bacilli often with bipolar staining. The colonies were wrinkled when incubated for long periods. The characteristic non-specific uptake of dye from media into the colonies and their musty or earthy odour rendered them easily distinguishable from other organisms. All strains were sensitive to chloramphenicol and all but one were sensitive to tetracycline. All strains were resistant to colimycin and gentamicin. The pathogenicity of the strain isolated from a fatal case of peritonitis was studied in guinea pigs. The findings showed that following a large inoculation intraperitoneally, the animal developed acute septicaemia and died shortly afterwards. Only a few micro-abscesses were found on the surface of the liver. Chronic infection of longer duration occurred when a small number of organisms were introduced through a cutaneous abrasion. The lesions included pneumonitis and multiple abscesses of various organs including subcutaneous tissue, liver, spleen and mediastinum.