Distribution and characterization of hemolytic, and enteropathogenic motile Aeromonas in aquatic environment

A year-long survey on the distribution of motile Aeromonas species in the surface waters of riverine and marine environments was conducted. The filtered membranes were directly placed onto the modified Pril-xylose-ampicillin agar for the enumeration of Aeromonas species. High counts of motile aromonads were found in riverine stations and this bacterial population was also observed in significant quantities in polluted marine samples. In the identification of 2,444 isolates, three species of motile Aeromonas were observed. A. caviae (43%) was prevalent followed by A. sobria (35%) and A. hydrophila (20%). A. hydrophila was high in clean riverine samples, A. sobria was predominantly isolated from a stagnant water sampling area, and A. caviae was distributed more in marine samples. Statistical analyses suggested that the densities of Aeromonas were related to the cumulative effect of various physicochemical parameters rather than to a single factor. Among the species of Aeromonas, A. hydrophila, and A. sobria were highly hemolytic whereas only 11% of A. caviae were observed to lyse sheep erythrocytes. Suckling-mouse assay was performed to elucidate the enterotoxicity of motile aeromonads and 21% of the tested strains (one A. caviae strain) were found to produce enterotoxin.     

黄沙鳖白底板病病原菌的分离鉴定及6种毒力基因检测

用常规方法从患典型白底板病黄沙鳖的心脏、肝脏等处进行细菌的接种分离, 通过人工感染确定分离菌株的致病性, API 20NE、16S rRNA基因序列分析进行病原菌鉴定和确定其系统发育地位, K-B纸片扩散法进行药敏试验, PCR检测病原菌的6种毒力基因。试验结果, 共分离到13株病原菌, 其中嗜水气单胞菌9株, 温和气单胞菌4株。在9株嗜水气单胞菌中, 有5株与Aeromonas hydrophila ATCC 7966菌株亲源关系最近, 4株与Aeromonas hydrophila北京株QDC01的亲源关系最近; 而4株温和气单胞菌与Aeromonas sobria ATCC 43979的亲源关系最近。药敏试验结果, 仅头孢哌酮对13株病原菌都高度敏感, 来源于不同养殖区域的病原菌药敏结果相差较大。6种毒力基因的阳性率, Aer、Act和ahp均为100%, hly和Alt为92.31%, ahal为76.92%; 毒力基因型共有4种, 嗜水气单胞菌主要为hly+Aer+Alt+Act+ahal+ahp+基因型, 而温和气单胞菌主要为 hly+Aer+Alt－Act+ahal+ahp+基因型, 同时携带hly基因的菌株其致病力更强。       

Virulence markers in Aeromonas hydrophila and Aeromonas veronii biovar sobria isolates from freshwater fish and from a diarrhoea case

AIMS: To evaluate the public health significance of representative strains of two Aeromonas spp., mainly from freshwater fish, on the basis of production of virulence-associated factors and presence of the haemolytic genes aerA and hlyA. METHODS AND RESULTS: Eleven strains of Aer. hydrophila, three strains of Aer. veronii biovar sobria (all from freshwater fish) and one strain of Aer. hydrophila from human diarrhoea were tested for potential virulence traits and for the presence of the haemolytic genes aerA and hlyA. Ten Aer. hydrophila isolates were aerA(+)hlyA(+) and two aerA(+)hlyA(-). Aeromonas veronii biovar sobria isolates were aerA(-)hlyA(-). Strains from the three genotypes showed enterotoxic activity in the suckling mouse assay. At 28 degrees C, four Aer. hydrophila fish strains could be considered as potentially virulent (possessing at least two of these characteristics: haemolytic, cytotoxic and enterotoxic). One Aer. veronii biovar sobria strain and the clinical isolate were cytotoxic on Vero cells. When grown at 4 degrees C, these six isolates fulfilled virulence criterion, but at 37 degrees C, only one fish strain, an Aer. hydrophila, did. CONCLUSIONS: The potential health risk derived from the presence of Aer. hydrophila and Aer. veronii biovar sobria in ice-stored freshwater fish should not be underestimated. SIGNIFICANCE AND IMPACT OF THE STUDY: Expression of virulence factors is affected by temperature incubation and not always related to the presence of haemolytic genes.     

The activity of ceftiofur sodium for Aeromonas spp. isolated from ornamental fish.

Our objective was to determine the activity of ceftiofur sodium against Aeromonas hydrophila and A. sobria isolated from a variety of domestic and imported tropical fish. Twelve antimicrobial drugs were tested for effectiveness against these aeromonads using the Kirby-Bauer Disk Diffusion technique and minimum inhibitory concentration determinations. Ceftiofur sodium was highly effective in vitro against aeromonads isolated from ornamental fish. Of the 42 isolates of Aeromonas spp. tested, none were resistant to ceftiofur sodium; however, all isolates were resistant to ampicillin, and 71% were resistant to tetracycline.     

Pathogenicity of live bacteria and extracellular products of motile Aeromonas isolated from eels

The pathogenic activities in vitro and in vivo of live bacteria and extracellular products (ECP) of 24 motile Aeromonas strains were investigated. Most Aer. hydrophila and Aer. jandaei isolates were pathogenic for eels (LD₅₀ 10^5·4-10^7·6 cfu fish^-1) but no Aer. sobria , Aer. caviae and Aer. allosaccharophila caused mortality in eels at doses of > 10^8·4 cfu fish^-1. Of these Aeromonas strains, Aer. hydrophila and Aer. jandaei in particular produced elastases and haemolysins against fish erythrocytes. ECP from Aer. hydrophila and Aer. jandaei caused degenerative changes in fish cell lines and were strongly toxic for eels (LD⁵⁰ 1·0–3·2 μg (g fish)^-1) reproducing the symptoms associated with natural disease. ECP from non-pathogenic species were inactive on fish cell lines as well as being poorly lethal for eels (LD⁵⁰ > 9·2 μg (g fish)^-1). All these biological activities of Aeromonas ECP were lost after heat treatment. These findings indicate differences between pathogenic and non-pathogenic Aeromonas species with respect to the expression of virulence factors, and show that elastases, haemolysins and exotoxins play a leading role in the pathogenicity of motile Aeromonas for eels.     

Rapid detection of virulence factors of Aeromonas isolated from a trout farm by hexaplex-PCR

The detection of virulence factors of Aeromonas is a key component in determining potential pathogenicity because these factors act multifunctionally and multifactorially. In this study water samples were collected from a trout farm on a seasonal basis, and diseased fish and Aeromonas species were isolated and identified. For rapid detection of six virulence factors of isolated Aeromonas, a hexaplex-polymerase chain reaction (hexaplex-PCR) assay was used. The detected virulence factors include aerolysin (aer), GCAT (gcat), serine protease (ser), nuclease (nuc) lipase (lip) and lateral flagella (laf). The dominant strain found in our isolates was Aeromonas sobria, and the dominant virulence factors were aer and nuc for all seasons. We confirmed that A. sobria and two of the virulence genes (aer and nuc) are related. We proposed a method by which one can identify the major strains of Aeromonas: A. hydrophila, A. sobria, A. caviae, and A. veronii, using hexaplex-PCR.     

Incidence and identification of mesophilic Aeromonas spp. from retail foods

Sixty-eight food samples were examined for the presence of mesophilic Aeromonas species both qualitatively and quantitatively. Aeromonads were isolated from 26% of the vegetable samples, 70% of the meat and poultry samples and 72% of the fish and shrimps. Numbers of motile aeromonads present in the food samples varied from <10(2) cfu g(-1) to >10(5) cfu g(-1). GLC analysis of FAMEs was used to identify a selection of presumptive Aeromonas colonies to fenospecies or genomic species level. Aeromonas strains belonging to the Aer. caviae complex, which also includes the potentially pathogenic genospecies HG4, were mostly isolated from vegetables but were also found in meat, poultry and fish. In addition, three strains of the virulent taxon Aer. veronii biovar sobria HG8 were isolated from poultry and minced meat. All members of the Aer. hydrophila complex, predominant in the fish, meat and poultry samples, were classified in the non-virulent taxon HG3. Although the significance of Aeromonas in foods remains undefined, the isolation of Aeromonas HG4 and HG8 strains from a variety of retail foods may indicate that these products can act as possible vehicles for the dissemination of food-borne Aeromonas gastroenteritis.     

Distribution of Aeromonas Species in the Intestinal Tracts of River Fish

Aeromonas isolates were obtained from fish intestines, water, and sediments from an urban river and identified by the DNA-DNA microplate hybridization method. The isolates were Aeromonas veronii (22%), Aeromonas caviae (18%), Aeromonas hydrophila (13%), Aeromonas sobria (8%), Aeromonas jandaei (7%), and other Aeromonas spp. (33%). Aeromonas species occurred at high densities with high incidences, regardless of season. The results strongly suggest that aeromonads are indigenous in fish intestines, water, and sediments of rivers and have the potential to be predominant in aquatic environments.     

Comparative studies on Aeromonas strains isolated from Lakes Balaton (Hungary) and Fertó/Neusiedlersee (Hungary)

Ecological and comparative taxonomic investigations were carried out on 49 Aeromonas strains isolated from water samples of two moderately alkaline lakes of Hungary, Lake Balaton and Lake Fertó/Neusiedlersee together with 3 authentic strains of Aeromonas hydrophila. Five phena were created at greater than 92% similarity value using the UPGMA method with the Jaccard coefficient. Strains isolated from Lake Balaton were determined as A. hydrophila, while strains originated from Lake Fertó were identified as A. hydrophila and A. sobria. The Fertó isolates of A. hydrophila grew only at higher salt concentration (5% NaCl). This might be an adaptation to the higher salt contents in the water of Lake Fertó. However, no specific differences were detected in their behaviour against alkaline pH values. The wide range of their degradative enzymes indicate that aeromonads can play an important role in nutrient cycling.     

Survival ability of cytotoxic strains of motile Aeromonas spp. in different types of water

The ability of motile Aeromonas spp. to survive in drinking water (mineral and tap water) and in sea water was experimentally tested. Clinically isolated cytotoxic strains of A. hydrophila, A. caviae and A. sobria were selected for this study. After contamination of water samples, the survival of Aeromonas strains was studied for at least three months using viable counts. The results obtained show that the survival of the Aeromonas spp. varies considerably depending on species and water type. For all three species, the survival time was longest in mineral water, where viable bacteria of each strain were still detected after 100 d. Moreover, A hydrophila and A. caviae also re-grew on the first day. In tap water all strains showed marked survival, although to a lesser extent than in mineral water. Aeromonas cells showed a rapid decline in sea water (90% reduction in viable cells after about two d) and thus seem to be more sensitive to saline/marine stress than chlorination.     

Multilocus genetic relationships between clinical and environmental Aeromonas strains

Allelic variations in the chromosomal genome of 120 isolates of motile Aeromonas (A. hydrophila, A. sobria and A. caviae) and eight reference strains with one Plesiomonas shigelloides were assessed by analysis of electrophoretically demonstrable polymorphism in 16 genes encoding metabolic enzymes. The strains were collected from humans (n = 59) and from the aquatic environment (n = 61) in canton Tessin, Switzerland. Clustering of the electrophoretic types (ET) from a matrix of pairwise genetic distances, based on the 16 enzyme loci, confirmed the genetic distinctness of the three species. Furthermore, A. hydrophila and A. sobria were divided in three and two groups respectively. For each species clinical strains were well differentiated from those collected in the environment.     

Incidence of aeromonads in samples from an abattoir processing lambs

Two hundred and thirty three samples of lamb carcasses, livers, kidneys and faeces, collected at a local abattoir, were examined to determine the incidence of motile Aeromonas spp. Wash water from the abattoir was also tested. Direct plating on starch ampicillin agar and enrichment in alkaline peptone water were used. The incidence of aeromonads was low. They were detected only after enrichment in 5/47 faecal samples and 11/50 carcass samples. The 41 strains of Aeromonas isolated were identified to species level and 93% of them were able to grow at 5 degrees C. The ability to produce both haemolysin and enterotoxin was species-related and was more common in Aeromonas hydrophila and A. sobria strains than in A. caviae strains.     

A method for the enumeration of Aeromonas bacteriophage in aquatic environments

K.P. FLINT. 1996. Bacteriophage were isolated against type strains and environmental isolates of Aeromonas hydrophila, Aeromonas sobria and Aeromonas caviae . A most probable number method for estimating the number of bacteriophage in a water sample was devised and tested using some of these isolates. The maximum number of bacteriophage against all three type strains were found in water from below a sewage effluent outfall. This corresponds to the increased numbers of each species of bacterium also found in this water sample. High numbers of bacteriophage against Aer. hydrophila were also found in the lake sample examined. Bacteriophage against Aer. caviae were rare in water samples other than those contaminated with sewage effluent.     

Virulence properties of motile aeromonads isolated from farmed frogs Rana tigerina and R. rugulosa

Virulence factors were compared in Aeromonas species isolated from clinically normal and septicaemic farmed frogs from Thailand. Haemolysin activities against frog erythrocytes were significantly different within the collection of aeromonads. Groups of high haemolytic activity (unspeciated Aeromonas, Au), moderate haemolytic activity (A. hydrophila), and low haemolytic activity (A. veronii biovar sobria, A. veronii biovar veronii, A. caviae, A. schubertii) were noted. DNA colony hybridisation studies revealed that Au isolates possessed a haemolysin gene (ASH1) which was not present in any of the other Thai aeromonads or type strains tested. Elastinolytic activity was demonstrated in 90% of the Au isolates, 60% of the A. hydrophila isolates and in none of the other motile aeromonads. The cytotoxic activity of the Aeromonas isolates varied according to the source of cells used in the assays. Cells from rainbow trout were extremely sensitive to Au toxins but less so to toxins produced by other species. In contrast mammalian cells showed very little sensitivity to Au toxins but were more sensitive to toxins produced by A. hydrophila. Selection of suitable assay substrates is therefore important.     

Is Aeromonas hydrophila the dominant motile Aeromonas species that causes disease outbreaks in aquaculture production in the Zhejiang Province of China?

The significance of Aeromonas hydrophila in association with disease outbreaks in aquaculture production in the Zhejiang province of China was investigated. Bacteriological examination of moribund fish and crabs resulted in 95 bacterial isolates: 88 bacterial isolates from fish and 7 isolates from crabs. PCR and traditional biochemical methods were used for identification of A. hydrophila. Out of 69 motile aeromonads, 35 isolates were identified as A. hydrophila by biochemical tests. However, 6 of those were not identified as A. hydrophila by a species specific PCR method. Serotyping revealed 2 dominant serotypes (O9 and O97) among A. hydrophila isolates. The data presented show that approximately 42% of the motile aeromonads isolated from disease outbreaks among various fish species were A. hydrophila. It is noteworthy that A. hydrophila accounted for more than 50% of the isolated aeromonands isolated from crucian carp Carassius carassius and Wuchang bream Megalobrama amblycephala with haemorrhagic septicaemia. Although this species was the most frequently isolated organism from internal organs of diseased fish and crabs in the present study, other motile Aeromonas spp. were also found. The PCR assay was useful in preventing misidentification of A. hydrophila, which may occur when only phenotypic tests are employed.     

Distribution of two hemolytic toxin genes in clinical and environmental isolates of Aeromonas spp.: correlation with virulence in a suckling mouse model

Previous studies have shown that two hemolytic toxins, HlyA and AerA, contribute to the virulence of Aeromonas hydrophila. A survey was performed to gauge the distribution of hlyA and aerA genes in clinical and environmental Aeromonas isolates. For A. hydrophila, A. veronii biotype sobria and A caviae, 96%, 12% and 35% of strains, respectively, were hlyA positive, whereas, 78%, 97%, 41%, respectively, were aerA positive. All virulent A. hydrophila isolates were hlyA+ aerA+. This genotype was most common in A. hydrophila (75.4%) followed by A. caviae (29.4%) and A. veronii biotype sobria (9.6%). For A. hydrophila, a two-hemolytic toxin model of virulence provides the best prediction of virulence in an animal model.     

Application of ribotyping for differentiating aeromonads isolated from clinical and environmental sources.

We have investigated the usefulness of ribotyping for the differentiation of aeromonads isolated from five patients with gastroenteritis and from the source water, treatment plant, and distribution system of a small public water supply. Aeromonas hydrophila and Aeromonas caviae were isolated from fecal specimens preserved in Cary-Blair transport medium by using blood ampicillin agar or alkaline peptone water (pH 8.4) subcultured to blood ampicillin agar plates. A. hydrophila, Aeromonas sobria, and A. caviae were isolated from duplicate 100-ml water samples by the membrane filter technique by using ampicillin dextrin agar for quantitative determination of growth and alkaline peptone water enrichment for detection of the presence or absence of aeromonads below the detection limit of the membrane filter method. In addition, free chlorine residuals and pH values were determined for all water samples and heterotrophic plate counts and total and fecal coliform analyses were performed on them. Ribotyping patterns of aeromonads recovered from well 1, detention basin, sand filter, softener, and distribution samples were compared with those of the five clinical isolates. All patient strains were unique; however, identical ribotypes of A. hydrophila and A. sobria isolated from multiple sites in the water system indicated colonization of a well, sand filters, and the softener, with the potential for sporadic contamination of distribution water. Plant operational deficiencies were noted and corrected. Ribotyping can effectively differentiate otherwise indistinguishable strains of bacteria, thus providing a powerful tool for investigation of waterborne diseases and bacteriological problems within water treatment plants and distribution systems.     

Application of ribotyping for differentiating aeromonads isolated from clinical and environmental sources.

We have investigated the usefulness of ribotyping for the differentiation of aeromonads isolated from five patients with gastroenteritis and from the source water, treatment plant, and distribution system of a small public water supply. Aeromonas hydrophila and Aeromonas caviae were isolated from fecal specimens preserved in Cary-Blair transport medium by using blood ampicillin agar or alkaline peptone water (pH 8.4) subcultured to blood ampicillin agar plates. A. hydrophila, Aeromonas sobria, and A. caviae were isolated from duplicate 100-ml water samples by the membrane filter technique by using ampicillin dextrin agar for quantitative determination of growth and alkaline peptone water enrichment for detection of the presence or absence of aeromonads below the detection limit of the membrane filter method. In addition, free chlorine residuals and pH values were determined for all water samples and heterotrophic plate counts and total and fecal coliform analyses were performed on them. Ribotyping patterns of aeromonads recovered from well 1, detention basin, sand filter, softener, and distribution samples were compared with those of the five clinical isolates. All patient strains were unique; however, identical ribotypes of A. hydrophila and A. sobria isolated from multiple sites in the water system indicated colonization of a well, sand filters, and the softener, with the potential for sporadic contamination of distribution water. Plant operational deficiencies were noted and corrected. Ribotyping can effectively differentiate otherwise indistinguishable strains of bacteria, thus providing a powerful tool for investigation of waterborne diseases and bacteriological problems within water treatment plants and distribution systems.     

Rapid Aeromonas hydrophila identification by TaqMan PCR assay: comparison with a phenotypic method

Aims:  Aeromonas hydrophila is recognized as a human pathogen following wound exposure or ingestion of contaminated water and food. For rapid identification of this bacterium, a TaqMan-based real-time PCR assay has been developed.
Methods and Results:  Primers and probes that target specific sequences of the 16S rRNA gene and cytolytic enterotoxin gene ( aerA ) were combined in a duplex assay. Presence and size of PCR products were confirmed with microchannel fluidics electrophoresis analysis. After validation, using type strain CIP7614T DNA, the PCR assay was tested on 12 positive and negative controls. Twenty-one Aeromonas strains were isolated from environmental samples and were identified with biochemical tests as Aer. sobria , Aer. caviae and Aer. hydrophila . Only Aer. hydrophila strains tested positive by PCR assay.
Conclusions:  The PCR developed here was successfully applied for the identification of Aer. hydrophila from reference, clinical and environmental samples and showed a high discrimination between Aer. hydrophila and other Aeromonas species.
Significance and Impact of the Study:  This molecular method is convenient, rapid (2·5 h vs 24 h), specific to identify Aer. hydrophila and usable for diagnosis in medical and veterinary laboratories.     

Biochemical identification and numerical taxonomy of Aeromonas spp. isolated from environmental and clinical samples in Spain

AIMS: To study the phenotypic characteristics of Aeromonas spp. from environmental and clinical samples in Spain and to cluster these strains by numerical taxonomy. METHODS AND RESULTS: A collection of 202 Aeromonas strains isolated from bivalve molluscs, water and clinical samples was tested for 64 phenotypic properties; 91% of these isolates were identified at species level. Aeromonas caviae was predominant in bivalve molluscs and Aerom. bestiarum in freshwater samples. Cluster analyses revealed eight different phena: three containing more than one DNA-DNA hybridization group but including strains that belong to the same phenospecies complex (Aerom. hydrophila, Aerom. sobria and Aerom. caviae), Aerom. encheleia, Aerom. trota and three containing unidentified Aeromonas strains isolated from bivalve molluscs. CONCLUSIONS:Aeromonas spp. are widely distributed in environmental and clinical sources. A selection of 16 of the phenotypical tests chosen allowed the identification of most isolates (91%), although some strains remain unidentified, mainly isolates from bivalve molluscs, suggesting the presence of new Aeromonas species. Numerical taxonomy was not in total concordance with the identification of the studied strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Numerical taxonomy of Aeromonas strains isolated from different sources revealed the presence of potentially pathogenic Aeromonas spp., especially in bivalve molluscs, and phena with unidentified strains that suggest new Aeromonas species.