We performed genome size estimation of 17 recent human isolates of Salmonella typhi from geographically diverse regions using pulsed-field gel electrophoresis (PFGE) after digestion of chromosomal DNA with restriction endonucleases XbaI (5'-TCTAGA-3'), AvrII (5'-CCTAGG-3') and SpeI (5'-ACTAGT-3'), and summation of the sizes of restriction fragments obtained. All 17 isolates had circular chromosomes, and genome sizes differed by as much as 959 kb, ranging from 3,964 to 4,923 kb (mean genome size = 4,528 kb). The data obtained confirm the usefulness of PFGE in studies of bacterial genome size and are in agreement with recent results indicating considerable genetic diversity and genomic plasticity of S. typhi. The variation in genome sizes noted may be relevant to the observed biological properties of this important human pathogen, including its virulence.
We examined human tissues infected by Burkholderia (Pseudomonas) pseudomallei which is endemic in Malaysia to study the types of inflammation invoked, and to look for histopathological clues to its diagnosis. The lesions which varied from acute to chronic granulomatous inflammation were not tissue-specific. In five autopsy cases, the inflammation was usually a focal or diffuse, acute necrotising inflammation with varying numbers of neutrophils, macrophages, lymphocytes and 'giant cells'. The 'giant cells' probably represent giant macrophages with phagocytosed leukocytes. There were numerous gram-negative, non-acid-fast, intra- and extracellular bacilli, occurring either singly or in chains. Intracellular bacteria within macrophages and 'giant cells' were so numerous as to resemble globi. This feature has not been previously reported and may be a useful diagnostic clue in melioidosis. In 14 surgical cases biopsies showed acute inflammatory lesions that appeared no different from acute inflammation due to other causes. In many biopsies, however, the inflammation was either an acute-on-chronic inflammation with a focal granulomatous component, or was purely granulomatous in character. Bacilli were difficult to demonstrate in surgical biopsies even with the gram strain.
Fifty cases of septicaemic melioidosis were reviewed. There was a preponderance of disease among males (male:female ratio 3.2:1) and those aged over 30 years. The presenting clinical features were very varied and not pathognomonic, ranging from fever, cough and septicaemia to fulminant septicaemia and shock. Pulmonary involvement was recorded in 58% of the patients. Skin and soft tissue sepsis was seen in 24%, but many had signs and symptoms of multiorgan involvement. Associated underlying illness was identified in 76% of patients, diabetes mellitus being the commonest (38%), while 34% had more than one predisposing factor. The mortality of 65% in our series is a reflection of the less than satisfactory status of the diagnosis and therapy of septicaemic melioidosis. Only 24% of our patients received appropriate empirical antibiotic therapy. A high index of suspicion of melioidosis in endemic areas and the use of appropriate empirical antimicrobial therapy would be a step towards reducing the high mortality rate.
A report is presented of seven patients with acute septicaemic melioidosis seen at the University Hospital, Kuala Lumpur, Malaysia, during 1976-1979. All had associated disorders which rendered them more susceptible to infection. As prognosis depends on early diagnosis it is important that this disease be considered in the differential diagnosis of a septicaemic illness in such patients from endemic areas. The treatment of choice is a combination of tetracyclines and chloramphenicol, initially used in massive doses, and continued for at least six month to prevent relapses.
170 clinical isolates of Pseudomonas aeruginosa were tested for in vitro susceptibility to gentamicin, amikacin, tobramycin, netilmicin, kanamycin, streptomycin, cefotaxime, ceftriaxone, cefoperazone, ceftazidime, moxalactam, azlocillin, piperacillin and ticarcillin. Against 93 gentamicin-sensitive strains, the most active antibiotics were in descending order, ceftazidime, tobramycin, gentamicin, amikacin, and the ureidopenicillins. Against 77 gentamicin-resistant strains, only ceftazidime, amikacin and moxalactam had mode minimum inhibitory concentrations within achievable peak serum levels after standard therapeutic dosage. There was no correlation between cephalosporin resistance and aminoglycoside resistance except for cefoperazone, which, together with the ureidopenicillins and ticarcillin, showed marked decrease in activity against gentamicin-resistant strains.
A 13-year old boy with acute lymphoblastic leukemia on chemotherapy developed neutropenia and acute cellulitis progressing to fulminating septicemia due to Pseudomonas pseudomallei. Septicemic melioidosis should be considered in the differential diagnosis of a febrile illness in children who are susceptible to infections.
There is an alarming increase in the prevalence of extended-spectrum β-lactamases (ESBLs) present mainly in Enterobacteriaceae and other nonfermenting gram-negative bacteria, such as Alcaligenes faecalis, which is the only species in that genus that is clinically relevant. We investigated Alcaligenes species from 7 cases (6 inpatients and one outpatient) at our tertiary-care hospital. Four patients had urinary tract infections, and one each had systemic lupus erythematosus, pulmonary stenosis, and diabetic ulcer. All 7 isolates were identified as Alcaligenes spp. based on their 16S rRNA gene sequences, and antibiotic susceptibility was determined using a Vitek 2 system with AST-GN87 cards. All the strains were resistant to cefazolin; 6 were resistant to trimethoprim/sulfamethoxazole; 5 manifested resistance to ampicillin/sulbactam, cefepime, tobramycin, ciprofloxacin, and nitrofurantoin; whereas 5 had multidrug resistance profiles. All the strains (7/7) expressed ESBL activity; PCR screening and sequencing showed evidence of genes blaTEM-116 (7/7) and blaOXA-10 (4/7), and we believe that this is the first report on the presence of TEM-116 and OXA-10 in an Alcaligenes spp. A combination of the 2 genes was present in 4 strains. All 7 strains were found to harbor at least one ESBL gene probably contributing to the drug resistance.
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.
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.
Burkholderia pseudomallei the causative agent of melioidosis, is being increasingly recognized as an important cause of morbidity and mortality in South East Asia. Biofilm formation of B. pseudomallei may be responsible for dormancy, latency and relapse of melioidosis. Based on the colonial morphology of the bacteria on B. pseudomallei selective agar medium, seven distinct morphotypes were identified. This study was conducted to assess the in vitro biofilm produced by B. pseudomallei and to investigate possible correlation between B. pseudomallei morphotypes with biofilm forming abilities of the isolates. Using a standard biofilm crystal violet staining assay, comparison was made between the biofilm forming ability of 76 isolates of B. pseudomallei and Burkholderia thailandensis ATCC 700388. Amongst the blood isolates, 30.2% were considered as high biofilm producers and 27.9% were low producers, 33.3% of the pus isolates were considered as high and 16% low biofilm producers. Most of the isolates were identified as morphotype group 1 which displayed a rough centre with irregular circumference on the agar medium. However, we did not find any correlation of B. pseudomallei morphotypes with biofilm forming abilities (p > 0.05). Additional studies are needed to identify internal and external factors which contribute to the high and low biofilm formation of B. pseudomallei.
Ciprofloxacin, a quinolone with good intracellular penetration may possibly be used for treatment of melioidosis caused by Burkholderia pseudomallei, but problems with resistance may be encountered. Amino acid substitutions in gyrA/gyrB have given rise to fluoroquinolone resistance in various microorganisms. Using published primers for gyrA and gyrB, PCR was performed on 11 isolates of B. pseudomallei with varying degrees of sensitivity to ciprofloxacin, followed by DNA sequencing to detect possible mutations. Results showed an absence of any point mutation in either gene. Local isolates have yet to develop full resistance to ciprofloxacin and probably other mechanisms of resistance may have been involved in the decreased sensitivity to ciprofloxacin.
Current diagnosis of melioidosis is based on bacterial culture and/or serology which is becoming increasingly useful. An IgM-ELISA using heat-killed whole cells of Pseudomonas pseudomallei was developed and compared with an indirect haemagglutination technique (IHAT) and an indirect immunofluorescent technique(IFAT). The IgM-ELISA using a P:N ratio of > or = 2 had a sensitivity of 91% and a specificity of 96%. All 3 assays were further used in a seroepidemiological survey amongst different groups of patients and healthy individuals. It was found that the IFAT performed better than the IHAT, detecting antibodies to P. pseudomallei in 6% of diabetics, 5% of pyrexics, 8% of pregnant women and 3% of farmers. For the same groups the IgM-ELISA detected antibodies in 1% of pyrexics, 8% of pregnant women and a further 14% of farmers. The IgM-ELISA was found to be sensitive and useful for the serological diagnosis of acute melioidosis.
Eight-five clinical and 15 poultry isolates of Campylobacter species were characterised by biotyping, serotyping and by using a radiolabelled DNA probe. A total of 80% of the isolates from both sources were identified as C. jejuni. Also amongst the clinical strains were 5 c. jejuni subsp. doylei, 7 C. coli, 3 C. lari and 8 were untypable. The similarity in the distribution of C. jejuni in the clinical and poultry isolates adds credibility to published reports of chickens being the most common source of Campylobacter infections. Although the gold standard for identification of C. jejuni is the DNA probe, serotyping is more discriminating while biotyping is the most feasible method in most laboratories.
A set of twins born to a 24-year-old primigravida had evidence of sepsis 24 to 60 hours after birth and were treated empirically with penicillin and gentamicin. A non-encapsulated H. influenzae biotype IV strain was isolated from the blood cultures of both and from the CSF of twin II. The isolates were beta-lactamase positive and hence showed resistance to ampicillin and therapy was changed to chloramphenicol only. Twin II recovered but Twin I developed a brain abscess in the left occipital region which resolved with extended antibiotic treatment. Although ampicillin-resistant H. influenzae have been reported in Malaysia, invasive disease by such strains are rare.
A nosocomial outbreak of bacteraemia, caused by Enterobacter gergoviae infected 11 babies, nine of whom were premature, and was investigated in the neonatal intensive care unit (NICU) of a general hospital in Johor Bahru, Malaysia. The strain that was isolated from the babies was also isolated from the dextrose saline used for the dilution of parenteral antibiotics and from the hands of a healthcare worker on duty in the nursery. Pulsed-field gel electrophoresis (PFGE) of Xba I-digested chromosomal DNA confirmed a possible cross-contamination of parenteral dextrose saline and the healthcare worker. Prompt and effective control measures were initiated within NICU and the nosocomial infection of E. gergoviae was brought to an abrupt end. To the best of our knowledge, this is the first documented outbreak of E. gergoviae in the NICU in a hospital in the state of Johor, Malaysia.
Pulsed field gel electrophoresis (PFGE), the gold standard of molecular typing methods, has a major disadvantage of an unusually long electrophoretic time. From the original protocol of 6 days, it was modified to 3 days and subsequently to a single day. We describe the procedure of stacking five to six gels one on top of another in order to increase and maximize the output in a shorter time without compromising the resolution and reproducibility. All the variables that affect pulsed field gels during electrophoresis were taken into consideration. We firstly optimized the parameters to be used and secondly determined whether stacking of five to six gels had any effect on the molecular separation during electrophoresis in comparison with a single gel run. DNA preparation, restriction, electrophoresis, staining and gel documentation was carried out based on previously published methods. Gels were analysed using BioNumerics and dice coefficient and unweighted pair group methods were used to generate dendrograms based on 1.5% tolerance values. Identical band profiles and band resolution-separation were seen in the PFGE patterns with single gel and multiple stacking gels. Cluster analysis further strengthened the fact that results from stacking gels were reproducible and comparable with a single gel run. This method of stacking gels saves time and maximizes the output at the same time. The run time for a single gel was about 28 hours, but with six stacked gels the run time was 54 hours compared with 28 x 6 = 168 hours if they were run separately as single gels thus saving time of 67.86%. Beside the big factor of saving time, stacking gels save resources (electricity, reagents, water, chemicals and working time) by increasing the sample throughput in a shorter time without compromising on quality of data. But optimization of working parameters is vital depending on the PFGE system used.
Four flagellin allelic types (I to IV) of Burkholderia pseudomallei were identified based on their sequence variation and restriction fragment length polymorphism (RFLP) analysis of the amplified flagellin gene. Flagellin allelic type I was the most predominantly (75.0%) found among the 100 clinical isolates of B. pseudomallei investigated in this study.