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Isolation and molecular identification of Aeromonas species from the tank water of ornamental fishes

Cheok YY, Puah SM, Chua KH, Tan JAMA

Acta Vet Hung, 2020 10 13;68(2):130-139.
PMID: 33055305 DOI: 10.1556/004.2020.00029

Aeromonads are recognised as important pathogens of fishes. In this study, ten water samples were randomly collected from pet shops' fish tanks and home aquaria inhabited by several fish species (silver arowana, koi, goldfish, catfish, pictus fish, silver shark and silver dollar fish). Altogether 298 colonies were isolated using Aeromonas selective agar. A total of 154 isolates were then confirmed as belonging to the genus Aeromonas using the GCAT gene. Using ERIC-PCR, a total of 40 duplicate isolates were excluded from the study and 114 isolates were subjected to PCR-RFLP targeting the RNA polymerase sigma factor (rpoD) gene using lab-on-chip. A total of 13 different Aeromonas species were identified. The most prevalent species were A. veronii (27%, 31/114), followed by A. dhakensis (17%, 19/114), A. finlandiensis (9%, 10/114), A. caviae (8%, 9/114), A. hydrophila (4%, 4/114), A. jandaei (4%, 4/114), A. rivuli (3%, 3/114), A. enteropelogens (2%, 2/114), A. tecta (2%, 2/114), A. allosaccharophila (1%, 1/114), A. eucrenophila (1%, 1/114), A. media (1%, 1/114) and A. diversa (1%, 1/114). Twenty-six isolates (23%) were unidentifiable at species level. The present study demonstrates that Aeromonas species are highly diverse in freshwater fish tanks, and suggests the potential risks posed by the isolated bacteria to the health of ornamental fish species.

Matched MeSH terms: Aeromonas/isolation & purification*
Isolation and identification of bacterial populations of zoonotic importance from captive non-venomous snakes in Malaysia

Abba Y, Ilyasu YM, Noordin MM

Microb Pathog, 2017 Jul;108:49-54.
PMID: 28478198 DOI: 10.1016/j.micpath.2017.04.038

AIM: Captivity of non-venomous snakes such as python and boa are common in zoos, aquariums and as pets in households. Poor captivity conditions expose these reptiles to numerous pathogens which may result in disease conditions. The purpose of this study was to investigate the common bacteria isolated from necropsied captive snakes in Malaysia over a five year period.
MATERIALS AND METHODS: A total of 27 snake carcasses presented for necropsy at the Universiti Putra Malaysia (UPM) were used in this survey. Samples were aseptically obtained at necropsy from different organs/tissues (lung, liver, heart, kindey, oesophagus, lymph node, stomach, spinal cord, spleen, intestine) and cultured onto 5% blood and McConkey agar, respectively. Gram staining, morphological evaluation and biochemical test such as oxidase, catalase and coagulase were used to tentatively identify the presumptive bacterial isolates.
RESULTS: Pythons had the highest number of cases (81.3%) followed by anaconda (14.8%) and boa (3.7%). Mixed infection accounted for 81.5% in all snakes and was highest in pythons (63%). However, single infection was only observed in pythons (18.5%). A total of 82.7%, 95.4% and 100% of the bacterial isolates from python, anaconda and boa, respectively were gram negative. Aeromonas spp was the most frequently isolated bacteria in pythons and anaconda with incidences of 25 (18%) and 8 (36.6%) with no difference (p > 0.05) in incidence, respectively, while Salmonella spp was the most frequently isolated in boa and significantly higher (p

Matched MeSH terms: Aeromonas/isolation & purification
Detection of Tilapia Lake Virus in Egyptian fish farms experiencing high mortalities in 2015

Nicholson P, Fathi MA, Fischer A, Mohan C, Schieck E, Mishra N, et al.

J Fish Dis, 2017 Dec;40(12):1925-1928.
PMID: 28590067 DOI: 10.1111/jfd.12650
Matched MeSH terms: Aeromonas/isolation & purification
Necrotising fasciitis caused by aeromonas sobria: Not just a simple catfish sting

Ng BW, Ong KC, Ahmad-Azraf A, Abdul-Muttalib AW

Med J Malaysia, 2019 12;74(6):543-544.
PMID: 31929484

Necrotising fasciitis is a life-threatening infection of the soft tissue which can be caused by different microorganisms, but infection caused by Aeromonas spp. or Vibrio spp. is frequently associated with higher mortality rate. Necrotising fasciitis progresses rapidly and often need aggressive surgical intervention. We present a rare case of necrotising fasciitis cause by Aeromonas sobria which mortality was successfully prevented by swift diagnosis and aggressive surgery.

Matched MeSH terms: Aeromonas/isolation & purification*
Characterization of the relationship between polar and lateral flagellar genes in clinical Aeromonas dhakensis: phenotypic, genetic and biochemical analyses

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

Braz J Microbiol, 2021 Jun;52(2):517-529.
PMID: 33768508 DOI: 10.1007/s42770-021-00457-8

Flagellar-mediated motility is a crucial virulence factor in many bacterial species. A dual flagellar system has been described in aeromonads; however, there is no flagella-related study in the emergent human pathogen Aeromonas dhakensis. Using 46 clinical A. dhakensis, phenotypic motility, genotypic characteristics (flagellar genes and sequence types), biochemical properties and their relationship were investigated in this study. All 46 strains showed swimming motility at 30 °C in 0.3% Bacto agar and carried the most prevalent 6 polar flagellar genes cheA, flgE, flgG, flgH, flgL, and flgN. On the contrary, only 18 strains (39%) demonstrated swarming motility on 0.5% Eiken agar at 30 °C and they harbored 11 lateral flagellar genes lafB, lafK, lafS, lafT, lafU, flgCL, flgGL, flgNL, fliEL, fliFL, and fliGL. No association was found between biochemical properties and motility phenotypes. Interestingly, a significant association between swarming and strains isolated from pus was observed (p = 0.0171). Three strains 187, 277, and 289 isolated from pus belonged to novel sequence types (ST522 and ST524) exhibited fast swimming and swarming profiles, and they harbored > 90% of the flagellar genes tested. Our findings provide a fundamental understanding of flagellar-mediated motility in A. dhakensis.

Matched MeSH terms: Aeromonas/isolation & purification
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.

Matched MeSH terms: Aeromonas/isolation & purification*
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.

Matched MeSH terms: Aeromonas/isolation & purification
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.

Matched MeSH terms: Aeromonas/isolation & purification
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.

Matched MeSH terms: Aeromonas/isolation & purification
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.

Matched MeSH terms: Aeromonas/isolation & purification*
Population dynamics, antibiotics resistance and biofilm formation of Aeromonas and Vibrio species isolated from aquatic sources in Northern Malaysia

Odeyemi OA, Ahmad A

Microb Pathog, 2017 Feb;103:178-185.
PMID: 28062284 DOI: 10.1016/j.micpath.2017.01.007

This study aimed to compare population dynamics, antibiotic resistance and biofilm formation of Aeromonas and Vibrio species from seawater and sediment collected from Northern Malaysia. Isolates with different colony morphology were characterized using both biochemical and molecular methods before testing for antibiotic resistance and biofilm formation. Results obtained from this study showed that in Kedah, the population of Aeromonas isolated from sediment was highest in Pantai Merdeka (8.22 log CFU/ml), Pulau Bunting recorded the highest population of Aeromonas from sediment (8.43 log CFU/g). It was observed that Vibrio species isolated from seawater and sediment were highest in Kuala Sanglang (9.21 log CFU/ml). In Kuala Perlis, the population of Aeromonas isolated from seawater was highest in Jeti (7.94 log CFU/ml). Highest population of Aeromonas from sediment was recorded in Kampong Tanah Baru (7.99 log CFU/g). It was observed that Vibrio species isolated from seawater was highest in Padang Benta (8.42 log CFU/g) while Jeti Kuala Perlis had highest population of Vibrio isolated from sediment. It was observed that location does not influence population of Aeromonas. The results of the independent t - test revealed that there was no significant relationship between location and population of Vibrio (df = 10, t = 1.144, p > 0.05). The occurrence of biofilm formation and prevalence of antibiotic resistant Aeromonas and Vibrio species in seawater and sediment pose danger to human and aquatic animals' health.

Matched MeSH terms: Aeromonas/isolation & purification
Comparison of Clinical Isolates of Aeromonas from Singapore and Malaysia with Regard to Molecular Identification, Virulence, and Antimicrobial Profiles

Khor WC, Puah SM, Koh TH, Tan JAMA, Puthucheary SD, Chua KH

Microb Drug Resist, 2018 May;24(4):469-478.
PMID: 29461928 DOI: 10.1089/mdr.2017.0083

OBJECTIVE: The objective of this study was to examine the species distribution, genetic relatedness, virulence gene profiles, antimicrobial sensitivities, and resistance gene distribution of clinical Aeromonas strains from Singapore and Malaysia.
METHODS: A total of 210 Aeromonas clinical isolates were investigated: 116 from Singapore General Hospital and 94 archived clinical isolates from University of Malaya Medical Center, Malaysia. The isolates were genetically identified based on the gcat gene screening and the partial sequences of the rpoD housekeeping gene. Genetic relatedness, distribution of 15 virulence genes and 4 beta-lactamase resistance genes, and susceptibility patterns to 11 antimicrobial agents were compared.
RESULTS: Of the 210 Aeromonas isolates, A. dhakensis-94 (45%) was the dominant species in Singapore and Malaysia. Species composition was similar and enterobacterial repetitive intergenic consensus-PCR did not show genetic relatedness between strains from the two countries. Of the 15 virulence genes, A. dhakensis and A. hydrophila harbored the most compared with other species. Different combinations of 9 virulence genes (exu, fla, lip, eno, alt, dam, hlyA, aexU, and ascV) were present in A. dhakensis, A. hydrophila, and A. veronii from both the countries. Distribution of virulence genes was species and anatomic site related. Majority (>80%) of the strains were susceptible to all antimicrobial agents tested, except amoxicillin and cephalothin. A. dhakensis strains from Malaysia significantly harbored the cphA gene compared with A. dhakensis from Singapore. Multidrug resistance was mostly detected in strains from peritoneal fluids of dialysis patients.
CONCLUSION: This study revealed A. dhakensis as the dominant species isolated in both geographic regions, and that it carried a high number of virulence genes. It also highlights the geographic-related differences of virulence gene distribution and antimicrobial resistance profiles of clinical Aeromonas strains from Singapore and Malaysia.

Matched MeSH terms: Aeromonas/isolation & purification*
Isolations of enteric pathogens from synanthropic flies trapped in downtown Kuala Lumpur

Sulaiman S, Othman MZ, Aziz AH

J Vector Ecol, 2000 Jun;25(1):90-3.
PMID: 10925800

Four species of synanthropic flies were trapped in downtown Kuala Lumpur: Chrysomya megacephala, Chrysomya rufifacies, Musca domestica, and Musca sorbens. Burkholderia pseudomallei, the organism causing melioidosis, was the dominant bacteria isolated from Chrysomya megacephala. Klebsiella oxytoca, commonly associated with nosocomial infections, was commonly isolated from Chrysomya megacephala, Musca domestica, and Musca sorbens. Aeromonas hydrophila, the bacteria causing gastroenteritis, was predominantly isolated from Chrysomya megacephala and also from Musca domestica and Musca sorbens. A total of 18 bacterial species was isolated from the synanthropic flies trapped. Burkholderia pseudomallei had been reported for the first time.

Matched MeSH terms: Aeromonas/isolation & purification
White spot syndrome virus isolates of tiger shrimp Penaeus monodon (Fabricious) in India are similar to exotic isolates as revealed by polymerase chain reaction and electron microscopy

Mishra SS, Shekhar MS

Indian J Exp Biol, 2005 Jul;43(7):654-61.
PMID: 16053274

Microbiological analysis of samples collected from cases of white spot disease outbreaks in cultured shrimp in different farms located in three regions along East Coast of India viz. Chidambram (Tamil Nadu), Nellore (Andhra Pradesh) and Balasore (Orissa), revealed presence of Vibrio alginolyticus, Vibrio parahaemolyticus, and Aeromonas spp. but experimental infection trials in Penaeus monodon with these isolates did not induce any acute mortality or formation of white spots on carapace. Infection trials using filtered tissue extracts by oral and injection method induced mortality in healthy P. monodon with all samples and 100% mortality was noted by the end of 7 day post-inoculation. Histopathological analysis demonstrated degenerated cells characterized by hypertrophied nuclei in gills, hepatopancreas and lymphoid organ with presence of intranuclear basophilic or eosino-basophilic bodies in tubular cells and intercellular spaces. Analysis of samples using 3 different primer sets as used by other for detection of white spot syndrome virus (WSSV) generated 643, 1447 and 520bp amplified DNA products in all samples except in one instance. Variable size virions with mean size in the range of 110 x 320 +/- 20 nm were observed under electron microscope. It could be concluded that the viral isolates in India involved with white spot syndrome in cultured shrimp are similar to RV-PJ and SEMBV in Japan, WSBV in Taiwan and WSSV in Malaysia, Indonesia, Thailand, China and Japan.

Matched MeSH terms: Aeromonas/isolation & purification

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