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Fulltext Culture-positive thoracic empyema in adults

Liam CK, Pendek R, Navaratnam P, Hassan H, Puthucheary SD, Abdul Majid A, et al.

Med J Malaysia, 1990 Jun;45(2):169-76.
PMID: 2152022

Twenty-nine adult patients with culture-positive thoracic empyema were seen at the University Hospital Kuala Lumpur from 1984 to 1988. Cough, fever, chest pain, dyspnoea and weight loss were the common presenting symptoms. The empyema in 16 patients was associated with primary bronchopulmonary infections, nine occurred following thoracentesis of culture-sterile pleural effusions, two occurred as post-thoracic surgery complications, one following a subdiaphragmatic abscess and one as a result of a stab wound. The most common culture isolates were Streptococcus milleri, Pseudomonas aeruginosa and Klebsiella pneumoniae. Closed tube thoracostomy, the most common form of drainage procedure employed, was able to effect a cure or control of the empyema in 11 out of 19 patients in whom it was used.
Non-typhoid Salmonella gastroenteritis

Lee WS, Puthucheary SD, Boey CC

J Paediatr Child Health, 1998 Aug;34(4):387-90.
PMID: 9727185

OBJECTIVE: To study the clinical features of non-typhoid Salmonella gastroenteritis and the incidence, risk factors and outcome of invasive complications in urban Malaysian children. To describe the serotypes of Salmonella species isolated and the pattern of antibiotic susceptibility.
METHODOLOGY: Retrospective review of a group of 131 children with non-typhoid Salmonella gastroenteritis seen at the University Hospital, Kuala Lumpur, Malaysia from January 1994 to December 1996.
RESULTS: Sixty-seven percent were infants below one year of age. Fever and vomiting were seen in nearly half of children. Seven children (5.3%) had invasive complications: 5 bacteraemia and 2 meningitis. Age below 6 months, fever > 38.0 degrees C, and dehydration on admission were significantly associated with invasive complications. The commonest serotypes isolated were S. enteritidis, S. paratyphi B, and S. bovis-morbificans. A total of 94-100% of isolates were susceptible to commonly prescribed antibiotics.
CONCLUSIONS: Children with Salmonella gastroenteritis below 6 months of age who are febrile and dehydrated should be treated empirically with antibiotics until the result of blood culture is available.
Fulltext Whole-Genome Analysis of Aeromonas hydrophila Strain 187, Exhibiting Quorum-Sensing Activity

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

Genome Announc, 2014;2(6).
PMID: 25540357 DOI: 10.1128/genomeA.01360-14

Aeromonas hydrophila is a quorum-sensing (QS) bacterium that causes diarrhea in humans upon infection. Here, we report the genome of pathogenic Aeromonas hydrophila strain 187, which possesses a QS gene responsible for signaling molecule N-acyl homoserine lactone (AHL) synthesis and has been found to be located at contig 36.
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.
In-vitro and in-vivo studies of a cytotoxin from Campylobacter jejuni

Pang T, Wong PY, Puthucheary SD, Sihotang K, Chang WK

J Med Microbiol, 1987 May;23(3):193-8.
PMID: 3585956

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.
Possible virulence factors involved in bacteraemia caused by Aeromonas hydrophila

Vadivelu J, Puthucheary SD, Phipps M, Chee YW

J Med Microbiol, 1995 Mar;42(3):171-4.
PMID: 7884797

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.
Fulltext Campylobacter enteritis in children: clinical and laboratory findings in 137 cases

Puthucheary SD, Parasakthi N, Liew ST, Chee YW

Singapore Med J, 1994 Oct;35(5):453-6.
PMID: 7701360

One hundred and thirty-seven children with Campylobacter diarrhoea were reviewed. The predominant species was C. jejuni. Ninety-five percent of the children were below 5 years of age with 61% of these being 2-12 months old. A slight male preponderance was noted. About half the cases presented with fever and bloody diarrhoea; vomiting was seen in 28% and abdominal colic in only 8%. Moderate to severe diarrhoea was present in 48% of the children. Thirty-seven percent had a history of recent or concurrent illness. Other bacterial enteropathogens together with Campylobacter were isolated in 15% of the children. Erythromycin, the most useful drug, when indicated for Campylobacter infections, had an MIC90 of 2 mg/l with 96.2% of the strains being sensitive.
Antimicrobial susceptibility and distribution of non-typhoidal Salmonella serovars isolated in Malaysian children

Lee WS, Puthucheary SD, Parasakthi N, Choo KE

J Trop Pediatr, 2003 Feb;49(1):37-41.
PMID: 12630719

There is widespread resistance of Salmonella species to commonly prescribed antimicrobials the world over. We aimed to determine the antimicrobial susceptibility and serovar distribution of non-typhoidal Salmonella (NTS) isolated from blood cultures of Malaysian children. Positive isolates of NTS from blood cultures obtained from children admitted to the pediatric wards of University of Malaya Medical Center (UMMC), a large urban hospital from Kuala Lumpur (1991-2001), and Hospital Kota Bharu (HKB), from the predominantly rural state of Kelantan (1991-1999), Malaysia, were reviewed retrospectively. Serovar distribution and antimicrobial susceptibility were ascertained. A total of 64 and 55 isolates of NTS were obtained from blood cultures of children admitted to UMMC and HKB, respectively. The commonest serovar isolated was Salmonella enteritidis in both centers. The NTS isolated were highly sensitive to the antimicrobials tested: ampicillin 98 per cent, chloramphenicol 98 per cent, gentamicin 97 per cent, trimethoprim-sulfamethoxazole (TMP-SMX) 98 per cent, and ceftriaxone 100 per cent in UMMC; ampicillin 100 per cent, chloramphenicol 87 per cent, kanamycin 100 per cent, streptomycin 96 per cent, TMP-SMX 93 per cent, and tetracycline 89 per cent in HKB. There were only one and five multi-resistant isolates in UMMC and HKB, respectively. In conclusion, NTS isolated from blood cultures of Malaysian children from Kuala Lumpur and Kota Bharu were highly sensitive to commonly prescribed antibiotics. We speculate that this is due to the restriction of sales of antimicrobials in Malaysia except by prescription. Continuing vigilance and frequent antmicrobial surveillance is necessary.
Fulltext Phenotypic and Genetic Diversity of Aeromonas Species Isolated from Fresh Water Lakes in Malaysia

Khor WC, Puah SM, Tan JA, Puthucheary SD, Chua KH

PLoS One, 2015;10(12):e0145933.
PMID: 26710336 DOI: 10.1371/journal.pone.0145933

Gram-negative bacilli of the genus Aeromonas are primarily inhabitants of the aquatic environment. Humans acquire this organism from a wide range of food and water sources as well as during aquatic recreational activities. In the present study, the diversity and distribution of Aeromonas species from freshwater lakes in Malaysia was investigated using glycerophospholipid-cholesterol acyltransferase (GCAT) and RNA polymerase sigma-factor (rpoD) genes for speciation. A total of 122 possible Aeromonas strains were isolated and confirmed to genus level using the API20E system. The clonality of the isolates was investigated using ERIC-PCR and 20 duplicate isolates were excluded from the study. The specific GCAT-PCR identified all isolates as belonging to the genus Aeromonas, in agreement with the biochemical identification. A phylogenetic tree was constructed using the rpoD gene sequence and all 102 isolates were identified as: A. veronii 43%, A. jandaei 37%, A. hydrophila 6%, A. caviae 4%, A. salmonicida 2%, A. media 2%, A. allosaccharophila 1%, A. dhakensis 1% and Aeromonas spp. 4%. Twelve virulence genes were present in the following proportions--exu 96%, ser 93%, aer 87%, fla 83%, enolase 70%, ela 62%, act 54%, aexT 33%, lip 16%, dam 16%, alt 8% and ast 4%, and at least 2 of these genes were present in all 102 strains. The ascV, aexU and hlyA genes were not detected among the isolates. A. hydrophila was the main species containing virulence genes alt and ast either present alone or in combination. It is possible that different mechanisms may be used by each genospecies to demonstrate virulence. In summary, with the use of GCAT and rpoD genes, unambiguous identification of Aeromonas species is possible and provides valuable data on the phylogenetic diversity of the organism.
Fulltext Molecular characterization of putative virulence determinants in Burkholderia pseudomallei

Puah SM, Puthucheary SD, Wang JT, Pan YJ, Chua KH

ScientificWorldJournal, 2014;2014:590803.
PMID: 25215325 DOI: 10.1155/2014/590803

The Gram-negative saprophyte Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease which is endemic in Southeast Asia and northern Australia. This bacterium possesses many virulence factors which are thought to contribute to its survival and pathogenicity. Using a virulent clinical isolate of B. pseudomallei and an attenuated strain of the same B. pseudomallei isolate, 6 genes BPSL2033, BP1026B_I2784, BP1026B_I2780, BURPS1106A_A0094, BURPS1106A_1131, and BURPS1710A_1419 were identified earlier by PCR-based subtractive hybridization. These genes were extensively characterized at the molecular level, together with an additional gene BPSL3147 that had been identified by other investigators. Through a reverse genetic approach, single-gene knockout mutants were successfully constructed by using site-specific insertion mutagenesis and were confirmed by PCR. BPSL2033::Km and BURPS1710A_1419::Km mutants showed reduced rates of survival inside macrophage RAW 264.7 cells and also low levels of virulence in the nematode infection model. BPSL2033::Km demonstrated weak statistical significance (P = 0.049) at 8 hours after infection in macrophage infection study but this was not seen in BURPS1710A_1419::Km. Nevertheless, complemented strains of both genes were able to partially restore the gene defects in both in vitro and in vivo studies, thus suggesting that they individually play a minor role in the virulence of B. pseudomallei.
Fulltext Potential immunogenic polypeptides of Burkholderia pseudomallei identified by shotgun expression library and evaluation of their efficacy for serodiagnosis of melioidosis

Puah SM, Puthucheary SD, Chua KH

Int J Med Sci, 2013;10(5):539-47.
PMID: 23532805 DOI: 10.7150/ijms.5516

The search for novel immunogenic polypeptides to improve the accuracy and reliability of serologic diagnostic methods for Burkholderia pseudomallei infection is ongoing. We employed a rapid and efficient approach to identify such polypeptides with sera from melioidosis patients using a small insert genomic expression library created from clinically confirmed local virulent isolates of B. pseudomallei. After 2 rounds of immunoscreening, 6 sero-positive clones expressing immunogenic peptides were sequenced and their identities were: benzoate 1,2-dioxygenase beta subunit, a putative 200 kDa antigen p200, phosphotransferase enzyme family protein, short chain dehydrogenase and 2 hypothetical proteins. These immunogens were then transferred to an ELISA platform for further large scale screening. By combining shotgun expression library and ELISA assays, we identified 2 polypeptides BPSS1904 (benzoate 1,2-dioxygenase beta subunit) and BPSL3130 (hypothetical protein), which had sensitivities of 78.9% and 79.4% and specificities of 88.1% and 94.8%, respectively in ELISA test, thus suggesting that both are potential candidate antigens for the serodiagnosis of infections caused by B. pseudomallei.
Molecular investigation of virulence determinants between a virulent clinical strain and an attenuated strain of Burkholderia pseudomallei

Puthucheary SD, Puah SM, Chai HC, Thong KL, Chua KH

J. Mol. Microbiol. Biotechnol., 2012;22(3):198-204.
PMID: 22846664 DOI: 10.1159/000338985

Burkholderia pseudomallei is the causative agent of melioidosis. We initiated this investigation with a virulent and an attenuated strain of B. pseudomallei. Pulsed-field gel electrophoresis was carried out initially for macrogenomic comparison of both strains of B. pseudomallei. However, the pulsotypes obtained were identical and therefore we applied a subtractive hybridization technique to distinguish and determine the possible differences between the two strains. Six virulence strain-specific DNA fragments were obtained and the encoding homolog proteins were identified as a xenobiotic-responsive element family of transcriptional regulator, a hypothetical protein, an unknown protein, a plasmid recombination enzyme, a regulatory protein and a putative hemolysin activator protein. A combination of at least three of these determinants was identified in 45 clinical isolates when screening was carried out with self-designed multiplex PCR targeting the six putative virulent determinants. Our data demonstrated that different combinations of the six putative virulence genes were present in the clinical isolates indicating their probable role in the pathogenesis of B. pseudomallei infections.
Fulltext Molecular characterization of clinical isolates of Aeromonas species from Malaysia

Puthucheary SD, Puah SM, Chua KH

PLoS One, 2012;7(2):e30205.
PMID: 22383958 DOI: 10.1371/journal.pone.0030205

BACKGROUND: Aeromonas species are common inhabitants of aquatic environments giving rise to infections in both fish and humans. Identification of aeromonads to the species level is problematic and complex due to their phenotypic and genotypic heterogeneity.
METHODOLOGY/PRINCIPAL FINDINGS: Aeromonas hydrophila or Aeromonas sp were genetically re-identified using a combination of previously published methods targeting GCAT, 16S rDNA and rpoD genes. Characterization based on the genus specific GCAT-PCR showed that 94 (96%) of the 98 strains belonged to the genus Aeromonas. Considering the patterns obtained for the 94 isolates with the 16S rDNA-RFLP identification method, 3 clusters were recognised, i.e. A. caviae (61%), A. hydrophila (17%) and an unknown group (22%) with atypical RFLP restriction patterns. However, the phylogenetic tree constructed with the obtained rpoD sequences showed that 47 strains (50%) clustered with the sequence of the type strain of A. aquariorum, 18 (19%) with A. caviae, 16 (17%) with A. hydrophila, 12 (13%) with A. veronii and one strain (1%) with the type strain of A. trota. PCR investigation revealed the presence of 10 virulence genes in the 94 isolates as: lip (91%), exu (87%), ela (86%), alt (79%), ser (77%), fla (74%), aer (72%), act (43%), aexT (24%) and ast (23%).
CONCLUSIONS/SIGNIFICANCE: This study emphasizes the importance of using more than one method for the correct identification of Aeromonas strains. The sequences of the rpoD gene enabled the unambiguous identication of the 94 Aeromonas isolates in accordance with results of other recent studies. Aeromonas aquariorum showed to be the most prevalent species (50%) containing an important subset of virulence genes lip/alt/ser/fla/aer. Different combinations of the virulence genes present in the isolates indicate their probable role in the pathogenesis of Aeromonas infections.
Quorum sensing in Aeromonas species isolated from patients in Malaysia

Chan KG, Puthucheary SD, Chan XY, Yin WF, Wong CS, Too WS, et al.

Curr Microbiol, 2011 Jan;62(1):167-72.
PMID: 20544198 DOI: 10.1007/s00284-010-9689-z

Bacterial quorum sensing signal molecules called N-acylhomoserine lactone (AHL) controls the expression of virulence determinants in many Gram-negative bacteria. We determined AHL production in 22 Aeromonas strains isolated from various infected sites from patients (bile, blood, peritoneal fluid, pus, stool and urine). All isolates produced the two principal AHLs, N-butanoylhomoserine lactone (C4-HSL) and N-hexanoyl homoserine lactone (C6-HSL). Ten isolates also produced additional AHLs. This report is the first documentation of Aeromonas sobria producing C6-HSL and two additional AHLs with N-acyl side chain longer than C(6). Our data provides a better understanding of the mechanism(s) of this environmental bacterium emerging as a human pathogen.
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.
Proteomic profiling of clinical and environmental strains of Pseudomonas aeruginosa

Liew SM, Puthucheary SD, Rajasekaram G, Chai HC, Chua KH

Mol Biol Rep, 2021 Mar;48(3):2325-2333.
PMID: 33728559 DOI: 10.1007/s11033-021-06262-8

Pseudomonas aeruginosa is a ubiquitous bacterium, which is able to change its physiological characteristics in response to different habitats. Environmental strains are presumably less pathogenic than clinical strains and whether or not the clinical strains originate from the environment or through inter-host transmission remains poorly understood. To minimize the risk of infection, a better understanding of proteomic profiling of P. aeruginosa is necessary for elucidating the correlation between environmental and clinical strains. Based on antimicrobial susceptibility and patterns of virulence, we selected 12 clinical and environmental strains: (i) environmental, (ii) multidrug resistant (MDR) clinical and (iii) susceptible clinical strains. Whole-cell protein was extracted from each strain and subjected to two-dimensional differential gel electrophoresis (2-D DIGE) and liquid chromatography tandem mass spectrometry quadrupole time-of-flight (LC-MS QTOF). All 12 strains were clustered into 3 distinct groups based on their variance in protein expression. A total of 526 matched spots were detected and four differentially expressed protein spots (p < 0.05) were identified and all differential spots were downregulated in MDR strain J3. Upregulation of chitin binding and BON domain proteins was present in the environmental and some MDR strains, whereas the clinical strains exhibited distinct proteomic profiles with increased expression of serine protein kinase and arginine/ornithine transport ATP-binding proteins. Significant difference in expression was observed between susceptible clinical and MDR strains, as well as susceptible clinical and environmental strains. Transition from an environmental saprophyte to a clinical strain could alter its physiological characteristics to further increase its adaptation.
Aeromonas aquariorum clinical isolates: antimicrobial profiles, plasmids and genetic determinants

Puah SM, Puthucheary SD, Liew FY, Chua KH

Int J Antimicrob Agents, 2013 Mar;41(3):281-4.
PMID: 23312608 DOI: 10.1016/j.ijantimicag.2012.11.012

The objective of this study was to investigate the antimicrobial resistance patterns of 47 clinical isolates of Aeromonas aquariorum and to identify the presence of plasmids and the relevant antibiotic resistance genes (ARGs). Antibiotic susceptibilities were determined by the standard disc diffusion method. The presence of plasmids and ARGs was detected by gel electrophoresis and monoplex PCR. Resistance to amoxicillin/clavulanic acid (98%), amoxicillin (91%), gentamicin (13%), trimethoprim/sulfamethoxazole (11%) and kanamycin (6%) was observed, whilst no ciprofloxacin- or amikacin-resistant strains were detected. All isolates harboured plasmids with sizes ranging from ca. 2 kb to 10 kb. PCR revealed that A. aquariorum carried three β-lactam resistance genes (bla(TEM), bla(MOX) and bla(PSE)) and two sulphonamide resistance genes (sul1 and sul2). This study provides further understanding of the phenotypic and genotypic characteristics of multiresistant A. aquariorum clinical isolates.
Fulltext Genetic relatedness and novel sequence types of clinical Aeromonas dhakensis from Malaysia

Lau TTV, Tan JMA, Puthucheary SD, Puah SM, Chua KH

Braz J Microbiol, 2020 Sep;51(3):909-918.
PMID: 32067209 DOI: 10.1007/s42770-020-00239-8

Aeromonas dhakensis is an emergent human pathogen with medical importance. This study was aimed to determine the sequence types (STs), genetic diversity, and phylogenetic relationships of different clinical sources of 47 A. dhakensis from Malaysia using multilocus sequence typing (MLST), goeBURST, and phylogenetic analyses. The analysis of a concatenated six-gene tree with a nucleotide length of 2994 bp based on six housekeeping genes (gyrB, groL, gltA, metG, ppsA, and recA) and independent analyses of single gene fragments was performed. MLST was able to group 47 A. dhakensis from our collection into 36 STs in which 34 STs are novel STs. The most abundant ST521 consisted of five strains from peritoneal fluid and two strains from stools. Comparison of 62 global A. dhakensis was carried out via goeBURST; 94.4% (34/36) of the identified STs are novel and unique in Malaysia. Two STs (111 and 541) were grouped into clonal complexes among our strains and 32 STs occurred as singletons. Single-gene phylogenetic trees showed varying topologies; groL and rpoD grouped all A. dhakensis into a tight-cluster with bootstrap values of 100% and 99%, respectively. A poor phylogenetic resolution encountered in single-gene analyses was buffered by the multilocus phylogenetic tree that offered high discriminatory power (bootstrap value = 100%) in resolving all A. dhakensis from A. hydrophila and delineating the relationship among other taxa. Genetic diversity analysis showed groL as the most conserved gene and ppsA as the most variable gene. This study revealed novel STs and high genetic diversity among clinical A. dhakensis from Malaysia.
Detection of VIM-2-, IMP-1- and NDM-1-producing multidrug-resistant Pseudomonas aeruginosa in Malaysia

Liew SM, Rajasekaram G, Puthucheary SD, Chua KH

J Glob Antimicrob Resist, 2018 06;13:271-273.
PMID: 29432937 DOI: 10.1016/j.jgar.2018.01.026

OBJECTIVES: The increasing incidence of carbapenem-resistant Pseudomonas aeruginosa along with the discovery of novel metallo-β-lactamases (MBLs) is of concern. In this study, the isolation of MBL-producing P. aeruginosa clinical strains in Malaysia was investigated.
METHODS: A total of 53 P. aeruginosa clinical strains were isolated from different patients in Sultanah Aminah Hospital (Johor Bahru, Malaysia) in 2015. Antimicrobial susceptibility testing was performed, and minimum inhibitory concentrations (MICs) of imipenem and meropenem were determined by Etest. Carbapenem-resistant strains were screened for MBL production by the imipenem-ethylene diamine tetra-acetic acid (IMP-EDTA) double-disk synergy test, MBL imipenem/imipenem-inhibitor (IP/IPI) Etest and PCR. Multilocus sequence typing (MLST) analysis was performed for genotyping of the isolates.
RESULTS: Among the 53 clinical strains, 3 (5.7%) were identified as MBL-producers. Multidrug resistance was observed in all three strains, and two were resistant to all of the antimicrobials tested. Sequencing analysis confirmed that the three strains harboured carbapenemase genes (blaIMP-1, blaVIM-2 and blaNDM-1 in one isolate each). These multidrug-resistant strains were identified as sequence type 235 (ST235) and ST308.
CONCLUSIONS: The blaIMP-1 and blaNDM-1 genes have not previously been reported in Malaysian P. aeruginosa isolates. The emergence of imipenemase 1 (IMP-1)- and New Delhi metallo-β-lactamase 1 (NDM-1)-producing P. aeruginosa in Malaysia maybe travel-associated.
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.

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