Studies were performed on a cytotoxin (CT) from human strains of Campylobacter jejuni isolated in Malaysia. CT was detected by cytopathic effect (CPE) on HeLa cells at titres from 8 to 32, in culture filtrates from 14 (48%) of 29 human isolates. The CPE correlated well with a quantitative 51Cr-release assay where a specific release of 54-68% was noted. CT production was lost after 5-7 subcultures. CT activity was also detected in 5 (26%) of 19 faecal filtrates from which CT-producing isolates were subsequently obtained. The mol. wt of CT was estimated by Sephadex G-50 chromatography to be greater than 30,000. In a suckling-mouse assay, CT consistently failed to demonstrate fluid accumulation after intragastric inoculation of culture filtrate. The Removable Intestinal Tie Adult Rabbit Diarrhoea (RITARD) assay was also used. Rabbits given CT-producing strains of C. jejuni developed bacteraemia and severe watery mucus-containing diarrhoea for the duration of the experiment with death of some animals. Rabbits given CT non-producing strains had less severe disease and none died. Rabbits given partially-purified CT had diarrhoea for 3 days but none died.
A simple adherence test to detect IgM antibodies in patients with typhoid is described. The test utilises the IgM-"capture" approach, in which the test serum is applied to microtitration plate wells previously coated with anti-human IgM, followed by application of a stained Salmonella typhi antigen suspension which shows adherence in positive cases. By this test, 58 (95%) of 61 sera from confirmed cases of typhoid possessed IgM antibodies to the H or O or both antigens of S. typhi. In patients for whom a diagnosis of typhoid was based only on a significant Widal-test titre, 31 (41%) of 76 sera had IgM antibodies to the H or O or both antigens of S. typhi. Some cross-reactivity of the IgM antibodies was detected, especially with the O antigens of S. paratyphi A and B. A total of 82 sera from non-typhoidal fevers (leptospirosis, typhus, dengue fever) showed no reactivity in this test. In normal sera there was no detectable IgM to the O antigen of S. typhi and only a small number (3.9%) had low levels of IgM to the H antigen. The significance and potential importance of this simple, sensitive, specific and economical test is discussed.
Eighty-six clinical isolates of Aeromonas hydrophila were studied for their ability to produce four exotoxins: a haemolysin active against rabbit erythrocytes, cytotoxin and enterotoxin detectable with Vero cell cultures, and the cholera toxin-like factor detected by an enzyme-linked immunosorbent assay. At least one exotoxin was produced by 80% of enteric and 96% of non-enteric isolates. The exotoxin profiles of non-enteric isolates were more restricted than those of enteric isolates, with haemolysin and cytotoxin producers preponderant. Although haemolysin and cytotoxin were produced by isolates from all sources, the enterotoxin and cholera toxin-like factor were more common amongst enteric isolates. The production of haemolysin and cytotoxin were closely related but the association between the enterotoxin and the cholera toxin-like factor was not significant.
There are several methods for the detection of haemolytic activity in campylobacters. However, we found the haemolytic effect of campylobacters on conventional blood agar plates to be variable, inconsistent and difficult to interpret. Blood agarose plates showed campylobacter haemolytic activity more clearly. The incubation conditions (temperature and gaseous) appear to be important for the expression of this activity. Ninety four percent of the Campylobacter isolates examined were found to be haemolytic by the microplate assay with minimal haemolytic units that ranged from 1 to 64. Haemolytic activity was detected only from live bacterial cultures and not from any of the 50 bacterial culture supernates, which suggests that campylobacters may possess a cell-associated haemolysin. The identification of such haemolytic activity in a large number of campylobacters (94%) suggests its potential role as a virulence factor in campylobacter gastroenteritis.
Eighteen strains of Aeromonas hydrophila from patients with bacteraemia were investigated for possible virulence factors. Cytotoxin and haemolysin were produced by all strains, whereas cholera toxin-like factor was produced by 33% of strains only. Enterotoxin production was not detected. Haemagglutination of guinea-pig, fowl and rabbit erythrocytes was demonstrated by 83%, 67% and 61% of strains, respectively. Fucose- and mannose-sensitive haemagglutinins were predominant. None of the strains agglutinated sheep erythrocytes. Extrachromosomal DNA was detected in 17 strains, 16 of which had a plasmid (3.6-5.1 MDa), the majority being between 4.6 and 5.1 MDa.
Aeromonas hydrophila strains obtained from diarrhoeal samples of human patients (19 isolates) and freshwater ponds (11 isolates) were analysed for siderophore production. Both clinical and environmental isolates showed significantly increased siderophore production under iron-limiting conditions both at 28 degrees C and at 37 degrees C. Clinical isolates consistently produced higher levels of siderophores than did the environmental isolates. The role of plasmids in moderating siderophore production was studied after curing with acridine orange. Treatment with acridine orange for 24 h removed the larger plasmids but the smaller plasmids (< 5 MDa), more common in the environmental isolates, were resistant to curing. As found in the untreated isolates, the cured clinical isolates produced higher mean levels of siderophores than the cured environmental isolates. Siderophore production in A. hydrophila was significantly influenced by iron-limiting cultural conditions and the source of isolates, but plasmid content and growth temperature at 28 degrees C or 37 degrees C had little effect on production. The basis for the greater production of siderophores in clinical isolates than in environmental isolates needs further study.
PCR and restriction fragment length polymorphism (RFLP) typing of flagellin genes (fliC) from 57 clinical isolates of Burkholderia cepacia indicated that only type 11 flagellins were present. Twenty-two isolates previously identified as the epidemic UK cystic fibrosis strain were indistinguishable by this method, as were 11 isolates from a pseudo-outbreak in Senegal. Other clinical isolates, including 19 from disparate sources in Malaysia, were separated into nine fliC RFLP groups, exhibiting a large degree of divergence. When isolates were indistinguishable by fliC genotyping, their similarity was confirmed by whole genome macro-restriction analysis with pulsed-field gel electrophoresis following XbaI digestion. The variation in fliC sequences of B. cepacia was far greater than that with B. pseudomallei, supporting the view that 'B. cepacia', as currently defined, may comprise several different genomic species.
The class and subclass distribution of antibody response to the culture filtrate antigen (CFA) of Burkholderia pseudomallei was examined in the sera of 45 septicaemic and 17 localised melioidosis cases and 40 cases clinically suspected of melioidosis and the results were compared with those from high-risk and healthy control groups. The geometric mean titre index (GMTI) values for all classes and subclasses of immunoglobulins examined were higher for sera from the proven and clinically suspected melioidosis cases than for the control groups. However, the highest response in the three patient groups was that of IgG with GMTIs ranging from 219.4 to 291.6 and the lowest was for IgM with GMTIs of 22.5, 24.3 and 28.7. The IgA response was intermediate with GMTIs ranging from 119.2 to 170. The GMTIs were highest for IgG in septicaemic and localised infections and for IgA and IgM in localised infections. As regards IgG subclass distribution, IgG1 and IgG2 were the predominant subclasses produced against the CFA in contrast to IgG3 and IgG4, which were produced in low amounts. None of the sera from the control groups had any significant titres of antibodies.
The aim of this study was to determine the role of macrophages in the Actinobacillus actinomycetemcomitans-induced murine immune response. BALB/c mice were given carrageenan solution by intraperitoneal injection before immunisation with heat-killed A. actinomycetemcomitans. Mice immunised with antigens and phosphate-buffered saline served as positive and negative controls, respectively. One week after the last immunisation, the delayed-type hypersensitivity (DTH) response was assessed by measurement of footpad swelling. Serum IgG and IgM anti-A. actinomycetemcomitans antibody levels and culture supernate levels of interferon (IFN)-gamma were determined by ELISA. The diameter of abscess formation was determined every 5 days. Sham-immunised spleen cells were transferred to carrageenan-untreated recipients (groups A and B) and to carrageenan-treated recipients (group D). Antigen-immunised spleen cells were transferred to carrageenan-untreated (group C) and carrageenan-treated (group E) recipients. The carrageenan-treated recipients in groups F and G received macrophages from antigen- and sham-immunised mice respectively. All mice except those in group A were immunised with antigen 24 h after cell transfer. After 1 week, a partial suppression of DTH response, reduced levels of IFN-gamma, serum IgG and IgM anti-A. actinomycetemcomitans antibodies and delayed healing were seen in carrageenan-treated mice when compared with the positive control. The immune response to A. actinomycetemcomitans in groups A, B and D was lower than that in groups C and E. Healing of the lesion in the former groups was also delayed when compared with the latter groups. The immune response and the healing of the lesion could be partially restored in carrageenan-treated mice that received antigen-pulsed macrophages (group F) but not in those that received naive macrophages (group G). These results suggest that macrophages play a partial role in the induction of the murine immune response to A. actinomycetemcomitans.
The emergence and spread of multiresistant methicillin-resistant Staphylococcus aureus (MRSA) strains, especially those resistant to fusidic acid and rifampicin, in Malaysian hospitals is of concern. In this study DNA fingerprinting by PFGE was performed on fusidic acid- and rifampicin-resistant isolates from Malaysian hospitals to determine the genetic relatedness of these isolates and their relationship with the endemic MRSA strains. In all, 32 of 640 MRSA isolates from 9 Malaysian hospitals were resistant to fusidic acid and rifampicin. Seven PFGE types (A, ZC, ZI, ZJ, ZK, ZL and ZM) were observed. The commonest type was type ZC, seen in 72% of isolates followed by type A, seen in 13%. Each of the other types (ZI, ZJ, ZK, ZL and ZM) was observed in a single isolate. Each type, even the commonest, was found in only one hospital. This suggests that the resistant strains had arisen from individual MRSA strains in each hospital and not as a result of the transmission of a common clone.
The aims of this study were to compare the genetic relatedness of: (i) sequential and single isolates of Candida strains from women with recurrent vaginal candidiasis (RVC); and (ii) Candida strains from women who had only one episode of infection within a 1-year period. In total, 87 isolates from 71 patients were cultured, speciated and genotyped by random amplification of polymorphic DNA (RAPD) analysis. Patients were categorized into three groups, namely those with: (i) a history of RVC from whom two or more yeast isolates were obtained (group A); (ii) a history of RVC from whom only a single isolate was obtained (group B); and (iii) a single episode of vaginal candidiasis within a 1-year period (group C). Six yeast species were detected: Candida albicans, Candida glabrata, Candida lusitaniae, Candida famata, Candida krusei and Candida parapsilosis. Interestingly, the prevalence of non-albicans species was higher in group A patients (50 %) than in patients in groups B (36 %) or C (18.9 %). Eighty RAPD profiles were observed, with a total of 61 polymorphic PCR fragments of distinct sizes. Clustering analysis showed that, overall, the majority of patients in group A had recurrent infections caused by highly similar, but not identical, sequential strains [mean pairwise similarity coefficient (S(AB)) = 0.721 +/- 0.308]. The range of mean S(AB) values for intergroup comparisons for C. albicans isolates alone was 0.50-0.56, suggesting that there was no significant relatedness between strains from different groups. Genetic similarity of C. albicans isolates from patients in group A was lower than that of C. albicans isolates from patients in group C (mean S(AB) = 0.532 +/- 0.249 and 0.636 +/- 0.206, respectively); this difference was statistically significant (P = 0.036). These results demonstrate that the cause of recurrent infections varies among individuals and ranges between strain maintenance, strain microevolution and strain replacement; the major scenario is strain maintenance with microevolution. They also show that C. albicans strains that cause recurrent infections are less similar to each other than strains that cause one-off infections, suggesting that the former may represent more virulent subtypes.
The suitability of a PCR procedure using a pair of primers targeting the hilA gene was evaluated as a means of detecting Salmonella species. A total of 33 Salmonella strains from 27 serovars and 15 non-Salmonella strains from eight different genera were included. PCR with all the Salmonella strains produced a 784 bp DNA fragment that was absent from all the non-Salmonella strains tested. The detection limit of the PCR was 100 pg with genomic DNA and 3 x 10(4) c.f.u. ml(-1) with serial dilutions of bacterial culture. An enrichment-PCR method was further developed to test the sensitivity of the hilA primers for the detection of Salmonella in faecal samples spiked with different concentrations of Salmonella choleraesuis subsp. choleraesuis serovar Typhimurium. The method described allowed the detection of Salmonella Typhimurium in faecal samples at a concentration of 3 x 10(2) c.f.u. ml(-1). In conclusion, the hilA primers are specific for Salmonella species and the PCR method presented may be suitable for the detection of Salmonella in faeces.
Streptococcus agalactiae or group B streptococci (GBS) often colonize the gastrointestinal and urogenital tracts of women, who may transmit these organisms to their offspring during the birth process. Using PFGE analysis, the genetic diversity of GBS was studied for strains isolated from pregnant women and their newborn infants in a teaching hospital. A total of 48 different PFGE profiles were obtained from 123 strains, with one profile (S1) appearing to be predominant among both groups studied. There was good overall correlation between the profiles obtained for strains from mother-infant pairs and for strains isolated from different body sites in the same individual. Occasional discrepancies seen in related body sites and among mother-infant pairs suggest concurrent carriage of different strains in the same individual as well as the possibility of an environmental source of organism for the neonate. The overall results demonstrated that many variants of GBS strains occur in Malaysia.
Cellulitis of the orbit is a common cause of proptosis in children, and also frequently arises in the elderly and the immunocompromised. The condition is characterized by infection and swelling of the soft tissues lining the eye socket, pushing the eye ball outwards and causing severe pain, redness, discharge of pus and some degree of blurred vision. There is a small risk of infection spreading to the meninges of the brain and causing meningitis. This paper reports the case of an adult in whom polymicrobial bilateral orbital cellulitis had developed due to Staphylococcus aureus and Neisseria gonorrhoeae infection. N. gonorrhoeae infections are acquired by sexual contact. Although the infection may disseminate to a variety of tissues, it usually affects the mucous membranes of the urethra in males and the endocervix and urethra in females. To the authors' knowledge this is the first report of polymicrobial bilateral orbital cellulitis due to S. aureus and N. gonorrhoeae in medical literature.
Vancomycin-resistant enterococci (VRE) are formidable organisms renowned for their ability to cause infections with limited treatment options and their potential for transferring resistance genes to other Gram-positive bacteria. Usually associated with nosocomial infections, VRE are rarely reported as a cause of community-acquired infection. Presented here is a case of community-acquired infection due to vancomycin-resistant Enterococcus faecium. The patient had been applying herbal leaves topically to his cheek to treat a buccal space abscess, resulting in a burn of the overlying skin. From pus aspirated via the skin a pure culture of E. faecium was grown that was resistant to vancomycin with a MIC of >256 microg ml-1 by the E test and resistant to teicoplanin by disc diffusion, consistent with the VanA phenotype. The organism was suspected of contaminating the leaf and infecting the patient via the burnt skin. This case highlights the need for further studies on the community prevalence of VRE among humans and animals to define unrecognized silent reservoirs for VRE, which may pose a threat to public health.
Helicobacter pylori CagA modifies the signalling of host cells and causes gastric diseases. Although CagA is injected into gastric epithelial cells through the type IV secretion machinery, it remains unclear how CagA is transported towards the machinery in the bacterial cytoplasm. In this study, it was determined that the proton-dependent intracytoplasmic transport system correlates with the priming of CagA secretion from H. pylori. The cytotoxicity of neutral-pH- and acidic-pH-treated H. pylori was examined in the AGS cell line. The amount of phosphorylated CagA in AGS cells incubated with acidic-pH- and neutral-pH-treated H. pylori was determined by enzyme immunoassay and Western blot. The production of CagA and adherence of the treated bacteria were examined by enzyme immunoassay and light microscopy, respectively. To clarify how CagA is transported towards the inner membrane of the treated bacteria, the localization of CagA was analysed by immunoelectron microscopy. The proportion of hummingbird cells in the AGS cell line rapidly increased following the inoculation of acidic-pH-treated H. pylori but increased more slowly with neutral-pH-treated H. pylori, and the phenomenon correlated with the amount of phosphorylated CagA in AGS cells. CagA was densely localized near the inner membrane in the acidic-pH-treated bacterial cytoplasm, but this localization was not observed in the neutral-pH-treated bacterial cytoplasm, suggesting that CagA shifts from the centre to the peripheral portion of the cytoplasm as a result of an extracellular decrease in pH. This phenomenon depended on the presence of UreI, a proton-dependent urea channel, but not on the presence of urea. The pH treatments did not enhance CagA production or the adherence of the bacterium to AGS cells. The authors propose that H. pylori possesses a proton-dependent intracytoplasmic transport system that probably accelerates priming for CagA injection.
The incidence of candidaemia among immunocompromised patients in Malaysia is increasing at an alarming rate. Isolation of clinical strains that are resistant to fluconazole has also risen markedly. We report here the repeated isolation of Candida tropicalis from the blood of a neonatal patient with Hirschsprung's disease. In vitro fluconazole susceptibility tests of the eight isolates obtained at different time points showed that seven of the isolates were resistant and one isolate was scored as susceptible dose-dependent. Random amplification of polymorphic DNA fingerprinting of the isolates using three primers and subsequent phylogenetic analysis revealed that these isolates were highly similar strains having minor genetic divergence, with a mean pairwise similarity coefficient of 0.893+/-0.041. The source of the infectious agent was thought to be the central venous catheter, as culture of its tip produced fluconazole-resistant C. tropicalis. This study demonstrates the utility of applying molecular epidemiology techniques to complement traditional mycological culture and drug susceptibility tests for accurate and appropriate management of recurrent candidaemia and highlights the need for newer antifungals that can combat the emergence of fluconazole-resistant C. tropicalis strains.