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Detection of virulence associated genes, haemolysin and protease amongst Vibrio cholerae isolated in Malaysia

Iyer L, Vadivelu J, Puthucheary SD

Epidemiol Infect, 2000 Aug;125(1):27-34.
PMID: 11057956

Eighty-four strains of Vibrio cholerae O1, O139 and non-O1/non-O139 from clinical and environmental sources were investigated for the presence of the toxin co-regulated pilus gene, tcpA, the virulence cassette genes ctxA, zot, ace and cep and also for their ability to elaborate haemolysin and protease. The ctxA and zot genes were detected using DNA-DNA hybridization while the ace, cep and tcpA genes were detected using PCR. Production of haemolysin and protease was detected using mammalian erythrocytes and an agar diffusion assay respectively. Analysis of their virulence profiles showed six different groups designated Type I to Type VI and the major distinguishing factor among these profiles was in the in vitro production of haemolysin and/or protease. Clinical O1, O139 and environmental O1 strains were similar with regard to presence of the virulence cassette genes. All environmental O1 strains with the exception of one were found to possess ctxA, zot and ace giving rise to the probability that these strains may actually be of clinical origin. One strain which had only cep but none of the toxin genes may be a true environmental isolate. The virulence cassette and colonization factor genes were absent in all non-O1/non-O139 environmental strains but production of both the haemolysin and protease was present, indicating that these may be putative virulence factors. These findings suggest that with regard to its pathogenic potential, only strains of the O1 and O139 serogroup that possess the tcpA gene which encodes the phage receptor, have the potential to acquire the CTX genetic element and become choleragenic.
Detection of virulence genes in Malaysian Shigella species by multiplex PCR assay

Thong KL, Hoe SL, Puthucheary SD, Yasin R

BMC Infect Dis, 2005 Feb 14;5:8.
PMID: 15707504

In Malaysia, Shigella spp. was reported to be the third commonest bacterial agent responsible for childhood diarrhoea. Currently, isolation of the bacterium and confirmation of the disease by microbiological and biochemical methods remain as the "gold standard". This study aimed to detect the prevalence of four Shigella virulence genes present concurrently, in randomly selected Malaysian strains via a rapid multiplex PCR (mPCR) assay.
Development of a multiplex PCR assay for rapid identification of Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia mallei and Burkholderia cepacia complex

Koh SF, Tay ST, Sermswan R, Wongratanacheewin S, Chua KH, Puthucheary SD

J Microbiol Methods, 2012 Sep;90(3):305-8.
PMID: 22705921 DOI: 10.1016/j.mimet.2012.06.002

We have developed a multiplex PCR assay for rapid identification and differentiation of cultures for Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia mallei and Burkholderia cepacia complex. The assay is valuable for use in clinical and veterinary laboratories, and in a deployable laboratory during outbreaks.
Development of a species-specific PCR-RFLP targeting rpoD gene fragment for discrimination of Aeromonas species

Puah SM, Khor WC, Kee BP, Tan JAMA, Puthucheary SD, Chua KH

J Med Microbiol, 2018 Sep;67(9):1271-1278.
PMID: 30024365 DOI: 10.1099/jmm.0.000796

PURPOSE: The taxonomy of Aeromonas keeps expanding and their identification remains problematic due to their phenotypic and genotypic heterogeneity. In this study, we aimed to develop a rapid and reliable polymerase chain reaction-restriction fragment length polymorphism assay targeting the rpoD gene to enable the differentiation of aeromonads into 27 distinct species using microfluidic capillary electrophoresis.
METHODOLOGY: A pair of degenerate primers (Aero F: 5'-YGARATCGAYATCGCCAARCGB-3' and Aero R: 5'-GRCCDATGCTCATRCGRCGGTT-3') was designed that amplified the rpoD gene of 27 Aeromonas species. Subsequently, in silico analysis enabled the differentiation of 25 species using the single restriction endonuclease AluI, while 2 species, A. sanarelli and A. taiwanensis, required an additional restriction endonuclease, HpyCH4IV. Twelve type strains (A. hydrophila ATCC7966T, A. caviae ATCC15468T, A. veronii ATCC9071T, A. media DSM4881T, A. allosaccharophila DSM11576T, A. dhakensis DSM17689T, A. enteropelogens DSM7312T, A. jandaei DSM7311T, A. rivuli DSM22539T, A. salmonicida ATCC33658T, A. taiwanensis DSM24096T and A. sanarelli DSM24094T) were randomly selected from the 27 Aeromonas species for experimental validation.Results/key findings. The twelve type strains demonstrated distinctive RFLP patterns and supported the in silico digestion. Subsequently, 60 clinical and environmental strains from our collection, comprising nine Aeromonas species, were used for screening examinations, and the results were in agreement.
CONCLUSION: This method provides an alternative method for laboratory identification, surveillance and epidemiological investigations of clinical and environmental specimens.
Diagnostic and prognostic value of an immunofluorescent assay for melioidosis

Vadivelu J, Puthucheary SD

Am J Trop Med Hyg, 2000 Feb;62(2):297-300.
PMID: 10813488

Melioidosis caused by Burkholderia pseudomallei is endemic in southeast Asia. The clinical manifestations range from wound infections to acute septicemia. In some cases, recurrence can also occur following complete recovery. Case fatality rates are high and a major factor is the delay in the culture and identification of the bacterium. An immunofluorescent assay (IFAT) using whole-cell antigen for the detection of total antibodies to B. pseudomallei was tested with 650 sera. Using a cut-off value of 1:80, 66 sera from culture-confirmed cases were positive with titers > or = 320. In another 523 sera from patients in which no other etiology could be found, 149 (23.4%) were positive. To monitor disease activity, persistence of antibody levels was investigated on 61 serial sera samples collected from 14 other confirmed cases on follow-up visits while on oral maintenance therapy. The IFAT demonstrated a reduction in titers in cases of localized infections, suggesting that either the infection was being resolved or arrested while septicemic patients maintained high IFAT titers on follow-up, suggesting the possibility of continuous sequestration of antigen from an intracellular source.
Diagnostic use of Burkholderia pseudomallei selective media in a low prevalence setting

Roesnita B, Tay ST, Puthucheary SD, Sam IC

Trans R Soc Trop Med Hyg, 2012 Feb;106(2):131-3.
PMID: 22112687 DOI: 10.1016/j.trstmh.2011.10.007

Routine use of selective media improves diagnosis of Burkholderia pseudomallei, but resources may be limited in endemic developing countries. To maximise yield in the relatively low-prevalence setting of Kuala Lumpur, Malaysia, B. pseudomallei selective agar and broth were compared with routine media for 154 respiratory specimens from patients with community-acquired disease. Selective media detected three additional culture-positive specimens and one additional melioidosis patient, at a consumables cost of US$75. Burkholderia pseudomallei was not isolated from 74 diabetic foot ulcer samples. Following careful local evaluation, focused use of selective media may be cost-effective.
Distribution of group B streptococcal serotypes in Malaysia

Ngeow YF, Puthucheary SD

J Trop Med Hyg, 1987 Aug;90(4):193-6.
PMID: 3309355

A total of 350 strains of group B streptococci (308 from adults and 42 from neonates) were serotyped. The majority of the isolates from adult patients were from the genito-urinary tract and most of the neonatal isolates were from babies less than 10 days old. Serotype III was the predominant type among all groups of patients and accounted for 50.9% of all serotypes. The second and third most frequently occurring serotypes were II and Ic respectively. Together with type III they made up 87% of the isolates.
Distribution of immunoglobulin classes and IgG subclasses against a culture filtrate antigen of Burkholderia pseudomallei in melioidosis patients

Chenthamarakshan V, Kumutha MV, Vadivelu J, Puthucheary SD

J Med Microbiol, 2001 Jan;50(1):55-61.
PMID: 11192506 DOI: 10.1099/0022-1317-50-1-55

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.
Fulltext Draft genome sequence of an Aeromonas sp. strain 159 clinical isolate that shows quorum-sensing activity

Chan XY, Chua KH, Puthucheary SD, Yin WF, Chan KG

J Bacteriol, 2012 Nov;194(22):6350.
PMID: 23105081 DOI: 10.1128/JB.01642-12

Aeromonas is a pathogenic organism that is often found to infect humans. Here we report the draft genome of a clinical isolate in Malaysia, Aeromonas sp. strain 159, which shows N-acylhomoserine lactone production. In the draft genome of strain 159, luxI and luxR homologue genes were found to be located at contig 47, and these genes are believed to be important for the quorum-sensing system present in this pathogen.
Effect of soy protein on the small bowel mucosa of young infants recovering from acute gastroenteritis

Iyngkaran N, Yadav M, Looi LM, Boey CG, Lam KL, Balabaskaran S, et al.

J Pediatr Gastroenterol Nutr, 1988 Jan-Feb;7(1):68-75.
PMID: 3335989

The effect of soy protein on the small bowel mucosa of 18 infants with acute gastroenteritis was studied. The infants were maintained on a protein hydrolysate formula for 6-8 weeks, following which they were readmitted for soy protein challenge studies. Jejunal biopsy was performed before and 24 h after challenge. On the basis of the clinical and histological reaction to soy protein challenge, three groups were identified. Group 1 consisted of three infants who had clinical and histological reaction. There was associated depletion of mucosal enzymes, lactase, sucrase, malatase, alkaline phosphatase, and blood xylose levels. Group 2 consisted of seven infants who had histological reaction but no clinical symptoms. Two of these seven infants, however, developed clinical reaction when rechallenged with soy protein 2 and 90 days later. Following challenge, mucosal enzymes and blood xylose levels were depressed in five of the seven infants tested. Group 3 consisted of eight infants who did not have either a clinical or a histological reaction. The mucosal enzymes and blood xylose levels were not depressed in four infants tested. The present study shows that the small bowel mucosa of some young infants recovering from acute gastroenteritis remains sensitive to soy protein for a variable period of time. The feeding of soy protein to these infants may result in the persistence of mucosal damage and perpetuation of diarrhea.(ABSTRACT TRUNCATED AT 250 WORDS)
Eikenella corrodens from a brain abscess

Karunakaran R, Marret MJ, Hassan H, Puthucheary SD

Malays J Pathol, 2004 Jun;26(1):49-52.
PMID: 16190107

A 2-year-old boy with underlying congenital cyanotic heart disease presented with seizures and fever and was found to have bilateral parietal cerebral abscesses. Drainage of the pus from the abscesses was done in stages; on the day of admission, four days after admission and 3 weeks after admission. Although the pus from the first drainage did not grow any organisms, the pus from the second drainage on the fourth day of admission yielded a mixed growth of Eikenella corrodens and Streptococcus milleri. Following the second drainage of pus, the child was noted to have mild weakness (grade 3/5) and increased tone in the left upper limb. Three weeks after admission, due to recurring fever, further neurological signs and findings of an enlarging right cerebral abscess on a repeat CT scan, a third drainage was carried out. However no growth was obtained from this specimen. This patient was managed both surgically and with appropriate antibiotics. Over the next four months, serial CT scans revealed gradual resolution of the abscesses with disappearance of the surrounding oedema. The child showed gradual recovery of his left sided weakness with resolution of tone and reflexes to normal.
Enterotoxigenic Escherichia coli in the domestic environment of a Malaysian village

Vadivelu J, Feachem RG, Drasar BS, Harrison TJ, Parasakthi N, Thambypillai V, et al.

Epidemiol Infect, 1989 Dec;103(3):497-511.
PMID: 2691267

The membrane-filter assay, GM1-ELISA, and DNA-DNA hybridization assay, were used to detect enterotoxigenic Escherichia coli (ETEC) in samples of water, weaning food, food preparation surface swabs, fingerprints of mothers, and the fingerprints and stools of children under 5 years of age, in 20 households in a Malaysian village. Weaning food and environmental samples were frequently contaminated by faecal coliforms, including ETEC. The membrane-filter assay detected and enumerated faecal coliforms and LT-ETEC in all types of water and weaning food samples. Highest concentrations of faecal coliforms and LT-ETEC were found in weaning food, followed by well-water, stored water and stored drinking water. The GM1-ELISA detected LT-ETEC in weaning food, food preparation surfaces, fingerprints and stool samples. The DNA-DNA hybridization assay detected a larger proportion of STa2-ETEC than the other toxotypes, either singly or in combination. All the assays in combination detected the presence of ETEC in all types of samples on at least one occasion in each household. It was not possible to classify households as consistently more or less contaminated with ETEC. On individual occasions it was possible to show a significant association of the presence of LT-ETEC between the fingerprints of children and their stools, fingerprints of mothers and children, and weaning food and the stools of the child consuming the food.
Fulltext Enzymatic profiling of clinical and environmental isolates of Burkholderia pseudomallei

Liew SM, Tay ST, Wongratanacheewin S, Puthucheary SD

Trop Biomed, 2012 Mar;29(1):160-8.
PMID: 22543616 MyJurnal

Melioidosis has been recognized as an important cause of sepsis in the tropics. The disease caused by an environmental saprophyte Burkholderia pseudomallei, affects mostly adults with underlying immunocompromised conditions. In this study, the enzymatic profiles of 91 clinical and 9 environmental isolates of B. pseudomallei were evaluated using the APIZYM system, in addition to assessment of protease, phospholipase C and sialidase activities using agar plate methods and other assays. The activity of 10 enzymes - alkaline phosphatase, esterase, esterase lipase, lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and N-acetyl-β-glucosaminidase were detected in >75% of the clinical isolates. The majority of B. pseudomallei isolates in this study exhibited protease and phospholipase activities. No sialidase activity was detected. Five Burkholderia thailandensis isolates had similar APIZYM profiles as B. pseudomallei clinical isolates except for the lower detection rate for N-acetyl-β-glucosaminidase. The subtle differences in the number of enzymes secreted and the levels of enzymatic activities of phenotypically identical clinical and environmental strains of B. pseudomallei give weight to the fact that the causative agent of melioidodis originates from the environment.
Fulltext Exotoxin profiles of Campylobacters isolated in Malaysia

Tee TS, Devi S, Puthucheary SD, Kautner IM

Southeast Asian J. Trop. Med. Public Health, 1994 Sep;25(3):443-8.
PMID: 7777904

Approximately 57% of clinical and 33% of poultry isolates examined produced a cytotoxin. Cytotoxic activity was detected in 25 (50%) isolates of Campylobacter of which 12 were isolated from bloody diarrhea and 9 from watery stools. The cytotoxin titers were low, ranging from 2 to 16. The crude filtrates from 50 Campylobacter isolates showed no cytotoxic effect in Vero cells, no fluid accumulation in suckling mice and no hemolytic activity.
Exotoxin profiles of clinical isolates of Aeromonas hydrophila

Vadivelu J, Puthucheary SD, Navaratnam P

J Med Microbiol, 1991 Jun;34(6):363-7.
PMID: 2056519

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.
Extra-intestinal non-typhoidal Salmonella infections in children

Lee WS, Puthucheary SD, Parasakthi N

Ann Trop Paediatr, 2000 Jun;20(2):125-9.
PMID: 10945063

Extra-intestinal non-typhoidal Salmonella (NTS) infections are uncommon in developed countries but common in developing ones. The risk factors, clinical features and outcome of children admitted to the Department of Paediatrics, University of Malaya Medical Center, Kuala Lumpur from 1978 to 1998 with extra-intestinal NTS infections were reviewed. All positive cultures of NTS, blood, cerebrospinal fluid, urine, synovial, pericardial and other body secretions (except stools), were included. Of the 98 cases reviewed, 56 were boys and 42 girls. The mean age was 2.1 years (range: newborn to 14 years). Twenty-seven children were severely immunocompromised and 21 had underlying chronic medical disorders. Bacteraemia was the most commonly detected type of infection and meningitis the commonest focal infection. The overall mortality rate was 15%. An immunocompromised state or underlying chronic medical disorder was associated with increased mortality. The three serotypes most commonly isolated were S. enteritidis, S. paratyphi B and S. typhimurium. Most isolates were sensitive to antibiotics commonly used in salmonellosis.
Fulltext First Report of Extended-Spectrum β-Lactamases TEM-116 and OXA-10 in Clinical Isolates of Alcaligenes Species from Kuala Lumpur, Malaysia

Puah SM, Puthucheary SD, Chua KH

Jpn J Infect Dis, 2019 Jul 24;72(4):266-269.
PMID: 30918144 DOI: 10.7883/yoken.JJID.2018.031

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.
Flagellar motility mediates biofilm formation in Aeromonas dhakensis

Lau TV, Puah SM, Tan JMA, Merino S, Puthucheary SD, Chua KH

Microb Pathog, 2023 Apr;177:106059.
PMID: 36878334 DOI: 10.1016/j.micpath.2023.106059

Aeromonas dhakensis possesses dual flagellar systems for motility under different environments. Flagella-mediated motility is necessary for biofilm formation through an initial attachment of bacteria to the surface, but this has not been elucidated in A. dhakensis. This study investigates the role of polar (flaH, maf1) and lateral (lafB, lafK and lafS) flagellar genes in the biofilm formation of a clinical A. dhakensis strain WT187 isolated from burn wound infection. Five deletion mutants and corresponding complemented strains were constructed using pDM4 and pBAD33 vectors, respectively, and analyzed for motility and biofilm formation using crystal violet staining and real-time impedance-based assays. All mutants were significantly reduced in swimming (p
Focal non-typhoidal Salmonella infections from a single center in Malaysia

Lee WS, Hafeez A, Hassan H, Raja NS, Puthucheary SD

Southeast Asian J. Trop. Med. Public Health, 2005 May;36(3):678-82.
PMID: 16124437

A retrospective review of patients with focal non-typhoidal Salmonella (NTS) infection was performed to determine its features and outcome. All patients with focal NTS infection admitted to the University of Malaya Medical Center, Malaysia, from 1993 to 2002 were studied. More than half (58%) of the 35 cases (54% male, median age 39 years, range 1.5 months to 79 years) were immunocompromized or had chronic medical conditions. One-third of the patients (34%) had superficial infections (lymphadenitis or subcutaneous tissue infection) and all recovered with antimicrobial therapy alone. Deep infections (66%) noted were: meningitis (9%), osteomyelitis or arthritis (26%), abscesses of the gastrointestinal tract or adjacent organs (20%), and others (11%). Deep infections were more likely to occur in the extremes of age (<6 months or >60 years, p< 0.04), associated with adverse outcomes with an overall mortality rate of 9%, or required major surgery (15%).
Fulltext Fulminant hepatic failure and shigella bacteremia

Abidin Z, Iyngkaran N, Puthucheary SD

Med J Malaysia, 1983 Jun;38(2):112-3.
PMID: 6353185

A three and a half year old boy with shigellosis developed fulminant hepatic failure. The hepatic derangements rapidly improved over a period of two weeks after treatment of the shigellosis with parenteral gentamicin. We believe this is the first documented report of fulminant hepatic failure due to shigella sepsis.

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