Molecular characterization of clinical isolates of Aeromonas species from Malaysia

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.     

High frequency of hemolytic and cytotoxic activity in Aeromonas spp. isolated from clinical, food and environmental in Rio de Janeiro, Brazil

Molecular study of aerolysin and cytotonic enterotoxin genes by PCR and colony blot hybridization was performed in 117 strains of Aeromonas spp. isolated from different sources. Homogeneous distribution of these genes in A. hydrophila complex strains was observed. For A. caviae and A. sobria complex strains, aerolysin genes were more frequent than cytotonic enterotoxins genes. Of 64 A. caviae complex strains, only one (1.5%) amplified the 451 bp product for the aer gene, however, the same primers detected a 400 bp product in 50 (78%) strains. This product was sequenced and had two short regions with homology to several hemolysin genes. The genotype aer ⁺/aerA⁺/hly ⁺/ast ⁺/alt ⁺ was detected in six A. hydrophila strains from food and environmental source. The most common genotype found in A. hydrophila strains was hly ⁺ (85%) and aerA⁺ (78.7%), while in A. caviae complex strains was aerA⁺ (32.8%). All A. veronii complex sobria strains were aer ⁺/aerA⁺. All A. caviae and A. hydrophila were positive when tested with aer probe using the colony blot test. Thirty-seven percent of A. hydrophila and 53% of A. caviae tested were positive for ast probe. Eighty-nine percent of samples were cytotoxic in Vero cells. Our data demonstrated that Aeromonas spp. can harbor and express virulence genes and reinforce the potential of Aeromonas as a human pathogen.     

A molecular study on the prevalence and virulence potential of Aeromonas spp. recovered from patients suffering from diarrhea in Israel

Background

Species of the genus Aeromonas are native inhabitants of aquatic environments and have recently been considered emerging human pathogens. Although the gastrointestinal tract is by far the most common anatomic site from which aeromonads are recovered, their role as etiologic agents of bacterial diarrhea is still disputed. Aeromonas-associated diarrhea is a phenomenon occurring worldwide; however, the exact prevalence of Aeromonas infections on a global scale is unknown.

Methodology/Principal Findings

The prevalence and virulence potential of Aeromonas in patients suffering from diarrhea in Israel was studied using molecular methods. 1,033 diarrheal stools were sampled between April and September 2010 and Aeromonas species were identified in 17 (∼2%) patients by sequencing the rpoD gene. Aeromonas species identity and abundance was: A. caviae (65%), A. veronii (29%) and Aeromonas taiwanensis (6%). This is the first clinical record of A. taiwanensis as a diarrheal causative since its recent discovery from a wound infection in a patient in Taiwan. Most of the patients (77%) from which Aeromonas species were isolated were negative for any other pathogens. The patients ranged from 1 to 92 years in age. Aeromonas isolates were found to possess different virulence-associated genes: ahpB (88%), pla/lip/lipH3/apl-1 (71%), act/hlyA/aerA (35%), alt (18%), ast (6%), fla (65%), lafA (41%), TTSS ascV (12%), TTSS ascF-ascG (12%), TTSS-dependent ADP-ribosylating toxins aexU (41%) and aexT (6%) in various combinations. Most of the identified strains were resistant to beta-lactam antibiotics but susceptible to third-generation cephalosporin antibiotics.

Conclusions

Aeromonas may be a causative agent of diarrhea in patients in Israel and therefore should be included in routine bacteriological screenings.     

Aeromonas hydrophila and Aeromonas veronii Predominate among Potentially Pathogenic Ciprofloxacin- and Tetracycline-Resistant Aeromonas Isolates from Lake Erie

Members of the genus Aeromonas are ubiquitous in nature and have increasingly been implicated in numerous diseases of humans and other animal taxa. Although some species of aeromonads are human pathogens, their presence, density, and relative abundance are rarely considered in assessing water quality. The objectives of this study were to identify Aeromonas species within Lake Erie, determine their antibiotic resistance patterns, and assess their potential pathogenicity. Aeromonas strains were isolated from Lake Erie water by use of Aeromonas selective agar with and without tetracycline and ciprofloxacin. All isolates were analyzed for hemolytic ability and cytotoxicity against human epithelial cells and were identified to the species level by using 16S rRNA gene restriction fragment length polymorphisms and phylogenetic analysis based on gyrB gene sequences. A molecular virulence profile was identified for each isolate, using multiplex PCR analysis of six virulence genes. We demonstrated that Aeromonas comprised 16% of all culturable bacteria from Lake Erie. Among 119 Aeromonas isolates, six species were identified, though only two species (Aeromonas hydrophila and A. veronii) predominated among tetracycline- and ciprofloxacin-resistant isolates. Additionally, both of these species demonstrated pathogenic phenotypes in vitro. Virulence gene profiles demonstrated a high prevalence of aerolysin and serine protease genes among A. hydrophila and A. veronii isolates, a genetic profile which corresponded with pathogenic phenotypes. Together, our findings demonstrate increased antibiotic resistance among potentially pathogenic strains of aeromonads, illustrating an emerging potential health concern.     

Characterization of Aeromonas strains isolated from Indian foods using rpoD gene sequencing and whole cell protein analysis

Aeromonas are responsible for causing gastroenteritis and extra-intestinal infections in humans. Twenty-two Aeromonas strains isolated from different food sources were re-identified up to species level using rpoD gene sequence analysis. Biochemical tests and 16S rRNA gene sequencing were insufficient to identify Aeromonas till species level. However, incorporation of additional biochemical tests lead to correct identification of 95.5 % strains up to species level. The 16S rRNA gene sequencing was useful to identify Aeromonas isolates at the genus level only. Sequences of the rpoD gene showed greater discriminatory power than 16S rRNA gene and provided conclusive discrimination of the strains for which the phenotypic species identification was uncertain. All these 22 strains were accurately identified up to species level by rpoD gene as A. salmonicida (6), A. veronii bv. veronii (4), A. caviae (3), A. hydrophila (2), A. veronii bv. sobria (2), A. jandaei (1), A. trota (1), A. sobria (1), A. allosaccharophila (1) and A. bivalvium (1). All these strains were also characterized using whole cell protein (WCP) analysis by gradient SDS-PAGE and showed different whole cell protein (WCP) profile [22–28 polypeptide bands (~10 to >97 kDa)], indicating high genetic diversity. The present work emphasizes the use of molecular methods such as rpoD gene sequencing along with comprehensive biochemical tests for the rapid and accurate identification of Aeromonas isolates till species level. The WCP profile can be subsequently used to characterize Aeromonas isolates below species level.     

Interaction of Aeromonas Strains with Lactic Acid Bacteria via Caco-2 Cells

The genus Aeromonas includes some species that have now been identified as human pathogens of significant medical importance. We investigated the ability of 13 selected Aeromonas strains belonging to nine species isolated from clinical cases (n = 5), environmental waters (n = 5), and fish (n = 3) to adhere to and translocate Caco-2 cells in the absence and presence of two lactic acid bacteria (LAB), i.e., Lactobacillus acidophilus and Bifidobacterium breve. Aeromonas isolates were also assessed for their cytotoxicity, the presence of virulence genes, and hemolysin production. Among the clinical isolates, one strain of Aeromonas veronii biovar veronii and two strains of Aeromonas hydrophila carried cytotoxin (act), heat-labile toxin (alt), hemolysin (hlyA), and aerolysin (aerA) genes, were cytotoxic to Vero cells, produced hemolysin, and showed higher adherence to Caco-2 cells. In contrast, this was seen in only one environmental strain, a strain of A. veronii biovar sobria. When Aeromonas strains were coinoculated with LAB onto Caco-2 cells, their level of adhesion was reduced. However, their rate of translocation in the presence of LAB increased and was significantly (P < 0.05) higher among fish strains. We suggest that either the interaction between Aeromonas and LAB strains could have a detrimental effect on the Caco-2 cells, allowing the Aeromonas to translocate more readily, or the presence of the LAB stimulated the Aeromonas strains to produce more toxins and/or increase their translocation rate.     

Identity and virulence properties of Aeromonas isolates from diseased fish, healthy controls and water environment in China

Aims: To investigate the species distribution in Aeromonas isolates from diseased fish, healthy controls and water environment in China; to evaluate the frequency of the aerolysin (aer), cytotonic enterotoxin (alt), cytotoxic enterotoxin (act), temperature‐sensitive protease (eprCAI) and serine protease (ahp) genes in Aeromonas isolates; and to determine the potential pathogenicity of these isolates. Methods and Results: Two hundred and two Aeromonas isolates from diseased fish (n = 42), healthy fish (n = 120) and water environment (n = 40) in China were identified to species levels based on sequencing of the housekeeping gene gyrB, while the distribution of five virulence factors, including aer, alt, act, eprCAI and ahp, was investigated by PCR. Aeromonas veronii (25/42; 60%) and Aeromonas hydrophila (14/42; 33%) were the species most commonly isolated from diseased fish, while Aer. veronii was the most common species in healthy fish (90/120; 75%) and water samples (25/40; 62·5%). All the five virulence genes were present in 9% (19/202), among which 10 strains were from diseased fish and nine were identified as Aer. hydrophila. For the strains carrying five virulence genes, the average 50% lethal doses (LD_50s) of strains from diseased fish were lower when compared with the strains from healthy fish and water environment. Conclusions: Aeromonas veronii is the most common species, but no significant difference exists in the isolates obtained from diseased fish and from healthy fish. However, Aer. hydrophila isolates were significantly more frequent from diseased fish than from healthy fish. aer⁺alt⁺act⁺eprCAI⁺ahp⁺ was more frequent virulence genotype in Aeromonas isolates from diseased fish than from healthy fish and water environment, and the aer⁺alt⁺act⁺eprCAI⁺ahp⁺ isolates were more virulent to zebrafish comparing to the other genetic profiles. Significant and Impact of the Study: Aeromonas species in aquatic environments are various and have considerable virulence potential, and therefore, there is a need for more careful and intensive epidemiology studies.     

Comparison of phenotypical and genetic identification of Aeromonas strains isolated from diseased fish

Phenotypicaly identified Aeromonas strains (n=119) recovered mainly from diseased fish were genetically re-identified and the concordance between the results was analysed. Molecular characterization based on the GCAT genus specific gene showed that only 90 (75.6%) strains belonged to the genus Aeromonas. The 16S rDNA-RFLP method identified correctly most of the strains with the exception of a few that belonged to A. bestiarum, A. salmonicida or A. piscicola. Separation of these 3 species was correctly assessed with the rpoD gene sequences, which revealed that 5 strains with the RFLP pattern of A. salmonicida belonged to A. piscicola, as did 1 strain with the pattern of A. bestiarum. Correct phenotypic identification occurred in only 32 (35.5%) of the 90 strains. Only 14 (21.8%) of the 64 phenotypically identified A. hydrophila strains belonged to this species. However, coincident results were obtained in 88% (15/17) of the genetically identified A. salmonicida strains. Phenotypic tests were re-evaluated on the 90 genetically characterized Aeromonas strains and there were contradictions in the species A. sobria for a number of previously published species-specific traits. After genetic identification, the prevailing species were A. sobria, A. salmonicida, A. bestiarum, A. hydrophila, A. piscicola and A. media but we could also identify a new isolate of the recently described species A. tecta. This work emphasizes the need to rely on the 16S rDNA-RFLP method and sequencing of housekeeping genes such as rpoD for the correct identification of Aeromonas strains.     

嗜水气单胞菌菌落多重PCR方法的建立及应用

[背景]嗜水气单胞菌(Aeromonas hydrophila)对水产动物、畜禽和人类均有致病性.基因表达的溶血素、气溶素和肠毒素是重要毒力因子,在致病性嗜水气单胞菌早期检测及防治中尤为重要.目前采用菌落直接提取DNA用于多重PCR研究的相关报道较少.[目的]基于菌落PCR方法建立针对嗜水气单胞菌溶血性基因、肠毒素基因...     

Virulence Diversity among Bacteremic Aeromonas Isolates: Ex Vivo,Animal, and Clinical Evidences

Background

The objective of this study was to compare virulence among different Aeromonas species causing bloodstream infections.

Methodology/Principal Findings

Nine of four species of Aeromonas blood isolates, including A. dhakensis, A. hydrophila, A. veronii and A. caviae were randomly selected for analysis. The species was identified by the DNA sequence matching of rpoD. Clinically, the patients with A. dhakensis bacteremia had a higher sepsis-related mortality rate than those with other species (37.5% vs. 0%, P = 0.028). Virulence of different Aeromonas species were tested in C. elegans, mouse fibroblast C2C12 cell line and BALB/c mice models. C. elegans fed with A. dhakensis and A. caviae had the lowest and highest survival rates compared with other species, respectively (all P values <0.0001). A. dhakensis isolates also exhibited more cytotoxicity in C2C12 cell line (all P values <0.0001). Fourteen-day survival rate of mice intramuscularly inoculated with A. dhakensis was lower than that of other species (all P values <0.0001). Hemolytic activity and several virulence factor genes were rarely detected in the A. caviae isolates.

Conclusions/Significance

Clinical data, ex vivo experiments, and animal studies suggest there is virulence variation among clinically important Aeromonas species.     

Complete Type III Secretion System of a Mesophilic Aeromonas hydrophila Strain

We have investigated the existence and genetic organization of a functional type III secretion system (TTSS) in a mesophilic Aeromonas strain by initially using the Aeromonas hydrophila strain AH-3. We report for the first time the complete TTSS DNA sequence of an Aeromonas strain that comprises 35 genes organized in a similar disposition as that in Pseudomonas aeruginosa. Using several gene probes, we also determined the presence of a TTSS in clinical or environmental strains of different Aeromonas species: A. hydrophila, A. veronii, and A. caviae. By using one of the TTSS genes (ascV), we were able to obtain a defined insertion mutant in strain AH-3 (AH-3AscV), which showed reduced toxicity and virulence in comparison with the wild-type strain. Complementation of the mutant strain with a plasmid vector carrying ascV was fully able to restore the wild-type toxicity and virulence.     

Nonselectivity of Rimler-Shotts Medium for Aeromonas hydrophila in Estuarine Environments

Thirty presumptive Aeromonas hydrophila isolates were collected from estuarine sources using Rimler-Shotts media. Many of the isolates, especially the strains from a low-salinity site, were also identified as A. hydrophila using the API 20E system and two differential techniques devised for the identification of A. hydrophila. All of the isolates, however, had deoxyribonucleic acid base compositions with guanine-plus-cytosine ratios between 40 and 49 mol%, which excludes these strains from the genus Aeromonas.     

Prevalence of virulence and extended-spectrum β-lactamase (ESBL) genes harbouring Vibrio spp. isolated from cockles (Tegillarca granosa) marketed in Korea

This study aimed to examine incidence, virulence and antimicrobial properties in Aeromonas spp. isolated from cockles (Tegillarca granosa) in Korea. Firstly, genomic DNA was extracted from 32 Aeromonas spp. isolates, and PCR screening for virulence, antimicrobial resistance genes was carried out. The disk diffusion assay was used to examine antimicrobial susceptibility. Aeromonas spp. isolates comprised, A. hydrophila (n = 8), A. veronii (n = 15), A. media (n = 2), A. salmonicida (n = 2), A. allosaccharophila (n = 1), A. bestiarum (n = 1), A. culicicola (n = 1), A. enteropelogenes (n = 1) and A. rivipollensis (n = 1). High prevalence of virulence-related genes reported as; act (69%), alt (47%), ast (41%), aerA (56%), lip (50%), ahyB (47%), ser (28%), fla (66%), gcat (44%), ascV (50%) and hlyA (72%). All isolates were multidrug resistant, while highest resistance level observed for ampicillin (100%), followed by imipenem (81%), rifampicin (78%), cephalothin (72%), piperacillin (47%) and Colistin sulfate (31%). The presence of bla_SHV, bla_CTX, tetE, aac(6’)-Ib, strA-strB, qnrS, qnrB and IntI1 genes were reported in varying combinations. Nevertheless, bla_TEM, bla_IMP, tetA, tetB, qnrA, qnrB and aphAI-IAB genes and the class1 integron were not detected. The high occurrence of virulence and antimicrobial resistance genes in cockles reveals that it can be a potential health risk source for consumers.     

Characterization and virulence potential of phenotypically diverse Aeromonas veronii isolates recovered from moribund freshwater ornamental fishes of Kerala, India

In the present study, we investigated the involvement of Aeromonas spp. in eliciting disease outbreaks in freshwater ornamental fishes across the state of Kerala, India. We investigated three incidences of disease, in which the moribund fishes exhibited clinical signs such as haemorrhagic septicemia (in gouramy, Trichogaster sp.), dropsy (in Oscar, Astronotus ocellatus) and tail rot/fin rot (in gold fish, Carassius carassius). Pure cultures (n = 20 from each fish; 60 in total) of Aeromonas spp. were recovered from the abdominal fluid as well as from internal organs of affected fishes, although they could not be identified to species level because of the variations in their phenotypic characters. The molecular fingerprinting of the isolates using Enterobacterial Repetitive Intergenic Consensus PCR proved the genetic diversity of the isolates from the three sites. The phylogenetic trees constructed using concatenated sequences (using 16S rRNA, gyrA, gyrB and rpoD genes) indicated that they were related to Aeromonas veronii. They exhibited marked cytotoxic and haemolytic activity, which were responsible for the pathogenic potential of the isolates. The isolates possessed multiple virulence genes such as enterotoxins (act and alt), haemolytic toxins (aerA and hlyA), genes involved in type III secretion system (ascV, aexT and ascF–ascG), glycerophospholipid-cholesterol acyltransferase (gcat) and a type IV pilus (tapA) gene, as determined by PCR. Virulence of representative isolates to goldfish was also tested, and we found LD₅₀ values of 10^4.07–10^5.35 cfu/fish. Furthermore, the organisms could be recovered as pure cultures from the lesions as well as from the internal organs.     

Detection of three virulence genes alt, ahp and aerA in Aeromonas hydrophila and their relationship with actual virulence to zebrafish

Aims: To evaluate the frequency of the aerolysin (aerA), cytotoxic enterotoxin (alt) and serine protease (ahp) genes in Aeromonas hydrophila isolates from different sources, and to determine the relationship between the presence of these genes and virulence of A. hydrophila in zebrafish. Methods and Results: Aeromonas hydrophila isolates from clinical cases (n = 40), from healthy fish (n = 22) and from water environment (n = 21) were analysed with respect to the prevalence of aerA, alt and ahp genes by PCR assay. These virulence factors occur among clinical isolates as well as among isolates from healthy fish and water environment. The majority (97·6%) of the strains examined carried one or more virulence genes. The isolates were divided into seven genetic profiles on the basis of PCR result: aerA⁺alt⁺ahp⁺ (62·7%), aerA⁺alt⁺ahp^? (13·3%), aerA⁺alt^?ahp⁺ (10·8%), aerA^?alt⁺ahp⁺ (4·8%), aerA^?alt^?ahp⁺ (3·6%), aerA⁺alt^?ahp^? (2·4%) and aerA^?alt^?ahp^? (2·4%). A higher frequency of genetic group aerA⁺alt⁺ahp⁺ was determined in the isolates from diseased animals compared to those from healthy fish or water environments. Virulence properties of 26 representative strains belonging to the seven genetic profiles were further characterized. Results demonstrated that as the present of virulence genes increased, the proteolytic, haemolytic and cytotoxic activities of extracellular products also increased. And the 50% lethal doses (LD₅₀s) of aerA⁺alt⁺ahp⁺ isolates (<10⁵) in zebrafish were lower when compared with the strains expressing one or combinations of two virulence genes (>10⁶). Conclusions: Virulence properties of A. hydrophila correlated well with the presence of virulence genes tested. aerA⁺alt⁺ahp⁺ was more frequent virulence genotype in A. hydrophila isolates from clinical diseases than from healthy fish and water environment, and the aerA⁺alt⁺ahp⁺ isolates were more virulent to zebrafish compared to the other six genetic profiles. Significant and Impact of the Study: The detection for aerA, alt and ahp can be used for virulence typing of A. hydrophila isolates.     

<Emphasis Type="Italic">Aeromonas</Emphasis> and <Emphasis Type="Italic">Plesiomonas</Emphasis> species from scarlet ibis (<Emphasis Type="Italic">Eudocimus ruber</Emphasis>) and their environment: monitoring antimicrobial susceptibility and virulence

The present study aimed at evaluating the role of captive scarlet ibises (Eudocimus ruber) and their environment as reservoirs of Aeromonas spp. and Plesiomonas spp., and analyzing the in vitro antimicrobial susceptibility and virulence of the recovered bacterial isolates. Thus, non-lactose and weak-lactose fermenting, oxidase positive Gram-negative bacilli were recovered from cloacal samples (n = 30) of scarlet ibises kept in a conservational facility and from water samples (n = 30) from their environment. Then, the antimicrobial susceptibility, hemolytic activity and biofilm production of the recovered Aeromonas spp. and Plesiomonas shigelloides strains were assessed. In addition, the virulence-associated genes of Aeromonas spp. were detected. Ten Aeromonas veronii bv. sobria, 2 Aeromonas hydrophila complex and 10 P. shigelloides were recovered. Intermediate susceptibility to piperacillin-tazobactam and cefepime was observed in 2 Aeromonas spp. and 1 P. shigelloides, respectively, and resistance to gentamicin was observed in 4 P. shigelloides. The automated susceptibility analysis revealed resistance to piperacillin-tazobactam and meropenem among Aeromonas spp. and intermediate susceptibility to gentamicin among P. shigelloides. All Aeromonas isolates presented hemolytic activity, while 3 P. shigelloides were non-hemolytic. All Aeromonas spp. and 3/10 P. shigelloides were biofilm-producers, at 28 °C, while 10 Aeromonas spp. and 6/10 P. shigelloides produced biofilms, at 37 °C. The most prevalent virulence genes of Aeromonas spp. were asa1 and ascV. Scarlet ibises and their environment harbour potentially pathogenic bacteria, thus requiring monitoring and measures to prevent contamination of humans and other animals.     

The prevalence of antibiotic resistance genes among Aeromonas species in aquatic environments

The global rise in antimicrobial resistance (AMR) among bacteria causing infectious diseases is well documented, and the associated risks for human health are well known. There is much less research on AMR with regard to environmental strains, both opportunistic and pathogenic ones. The genus Aeromonas is widely distributed in the environment and causes many variable diseases in fish and humans. Infections in humans are predominantly caused by Aeromonas veronii, A. hydrophila and A. caviae (A. punctata) in a form of bacteremia, gastroenteritis or even septicaemia in immunocompetent and immunocompromised individuals. Different groups of antibiotics are used in the treatment, but studies indicate that fluoroquinolones and cefotaxime are the most efficient. A disturbing consequence of antibiotic overuse is an increasing number of detection of various antibiotic resistance genes (ARG) within this genus. The water environment is one of the major modes of transmission of resistant bacteria from animals to humans, and, thus, the dissemination of antibiotic resistance genes, particularly those located in mobile genetic elements (MGE) occurs in such as plasmids and transposons. This review summarizes recently published information on the type, distribution, and transmission of ARG by MGE, widespread in Aeromonas strains living in various aquatic environments, including wastewater, natural water, aquaculture and urban drinking water. The data available indicate that the opportunistic pathogens like Aeromonas spp. might serve as important vectors of ARG for clinically relevant pathogens present in such bodies of water .     

Identification and Characterization of Pathogenic Aeromonas veronii Biovar Sobria Associated with Epizootic Ulcerative Syndrome in Fish in Bangladesh

Sparse information is available on the virulence factors of Aeromonas strains isolated from diseased fish, from the environment, and from humans. In the present study, 52 Aeromonas isolates obtained from epizootic ulcerative syndrome (EUS) lesions in fish, from the aquatic environment, and from children with diarrhea in Bangladesh were identified by biochemical phenotyping (i.e., PhenePlate [PhP] typing) and DNA fingerprinting and then characterized with respect to certain putative virulence factors. The isolates from the fish exhibiting EUS symptoms were identified to be Aeromonas veronii biovar sobria by fatty acid methyl ester analysis and amplified fragment length polymorphism fingerprinting. Biochemical phenotyping revealed that all EUS-associated isolates belonged to a unique phenotype which was not identified among more than 1,600 environmental and diarrheal isolates in a previously collected database of PhP types of Bangladeshi Aeromonas isolates. The 52 Aeromonas isolates were investigated for the production of hemolysin and cytotoxin; for hemagglutination with erythrocytes from fish, human, and rabbit sources; for the presence of a cytolytic enterotoxin gene; and for adhesion to and invasion into fish cell lines. All of the EUS isolates produced all of the virulence factors investigated, as did also some of the environmental isolates, but the isolates from EUS were unique in their ability to agglutinate fish erythrocytes. Our results suggest that a clonal group of A. veronii biovar sobria is associated with, and may be a causative agent of, EUS in fish in Bangladesh.     

Multilocus phylogenetic analysis of the genus Aeromonas

A broad multilocus phylogenetic analysis (MLPA) of the representative diversity of a genus offers the opportunity to incorporate concatenated inter-species phylogenies into bacterial systematics. Recent analyses based on single housekeeping genes have provided coherent phylogenies of Aeromonas. However, to date, a multi-gene phylogenetic analysis has never been tackled. In the present study, the intra- and inter-species phylogenetic relationships of 115 strains representing all Aeromonas species described to date were investigated by MLPA. The study included the independent analysis of seven single gene fragments (gyrB, rpoD, recA, dnaJ, gyrA, dnaX, and atpD), and the tree resulting from the concatenated 4705 bp sequence. The phylogenies obtained were consistent with each other, and clustering agreed with the Aeromonas taxonomy recognized to date. The highest clustering robustness was found for the concatenated tree (i.e. all Aeromonas species split into 100% bootstrap clusters). Both possible chronometric distortions and poor resolution encountered when using single-gene analysis were buffered in the concatenated MLPA tree. However, reliable phylogenetic species delineation required an MLPA including several “bona fide” strains representing all described species.