Distribution of Aeromonas species in waters with different levels of pollution

Strains of Aeromonas spp. (883) were isolated from 10 stations in the north-west of Spain. Biotyping of the strains gave: 55% Aeromonas caviae, 34% A. hydrophila, 6% A. sobria and 5% Aeromonas spp. Phenotypic characters that have been claimed to be related to virulence such as haemolysis and the Voges-Proskauer reaction were detected mostly in A. hydrophila and A. sobria. The distribution of the species was significantly related to levels of faecal pollution in waters. Aeromonas caviae predominated in sewage and waters with a high degree of faecal pollution. In less polluted waters, either fresh or marine, A. caviae and A. hydrophila were almost equally distributed. In waters with low or no faecal pollution, the proportion of A. sobria to other species increased considerably.     

Distribution of Aeromonas species in waters with different levels of pollution

Strains of Aeromonas spp. (883) were isolated from 10 stations in the north-west of Spain. Biotyping of the strains gave: 55% Aeromonas caviae , 34% A. hydrophila , 6% A. sobria and 5% Aeromonas spp. Phenotypic characters that have been claimed to be related to virulence such as haemolysis and the Voges-Proskauer reaction were detected mostly in A. hydrophila and A. sobria. The distribution of the species was significantly related to levels of faecal pollution in waters. Aeromonas caviae predominated in sewage and waters with a high degree of faecal pollution. In less polluted waters, either fresh or marine, A. caviae and A. hydrophila were almost equally distributed. In waters with low or no faecal pollution, the proportion of A. sobria to other species increased considerably.     

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.     

Significance of sinking particles in the distribution of motile Aeromonas during the winter season.

The role of sinking particles in the distribution of motile Aeromonas species was studied during the winter season. Various environmental parameters and microbial populations were investigated to elucidate the relationship with motile aeromonads. Motile Aeromonas species were enumerated by most probable number technique with alkaline peptone water as the enrichment medium and modified pril-xylose ampicillin agar as the plating medium. Aeromonas species were isolated in a water column in any one of the two procedures but sediment and plankton samples exhibited an irregular isolation pattern for these organisms. Aeoromonas species were continuously isolated in sinking particles with the highest counts during January. Of the 206 isolates identified, three known motile Aeromonas species were observed of which A. caviae accounted for 51.4% of the total. Toxin characterization showed that 20% of the strains produced haemolysin as well as cytotoxin, and A. hydrophila was highly toxigenic. Statistical analyses revealed that nutrients govern the distribution of Aeromonas. It may be that riverine discharge influences the distribution of motile aeromonads in this environment.     

Enterotoxigenic aeromonads on retail lamb meat and offal

Enrichment in alkaline peptone water was compared with the direct plating method for the isolation of Aeromonas spp. from lamb meat and offal samples. The enrichment method significantly increased the isolation rate of aeromonads. Motile Aeromonas species ( A. hydrophila, A. sobria and A. caviae ) were present in all kinds of samples investigated. Seventy-three Aeromonas strains isolated in this survey were characterized to species level and examined for their ability to produce virulence factors. Strains identified as A. sobria were the strongest producers of haemolysin and enterotoxin, whereas A. caviae strains were consistently non-haemolytic and non-enterotoxigenic. Thus it is likely that lamb meat and offal are potentially significant sources of virulent Aeromonas species and may play an important role in the aetiology of Aeromonas -associated gastro-enteritis.     

Prevalence, in-vitro secretory activity, and cytotoxicity of Aeromonas species associated with childhood gastroenteritis in Chennai (Madras), India

An investigation on the prevalence of Aeromonas in gastrointestinal illnesses of pediatric inpatients 1 month to 3 years of age was conducted from February 1997 through January 1998 in Madras. Sixteen Aeromonas spp. were isolated from 11 male and five female children among the 341 pediatric inpatients suffering from acute diarrhoea. A. caviae, which was isolated from nine cases, was found to be the most predominant isolate, followed by A. veronii biovar sobria, isolated from six cases, and A. hydrophila, isolated from one case. Shigella flexneri was recovered along with Aeromonas veronii biotype sobria serotype 035 from one 5-month-old female child. We did not notice any seasonal pattern in the association between Aeromonas and childhood gastroenteritis. None of the 147 stool samples obtained from age-matched non-diarrhoeic control children yielded Aeromonas spp. Isolation of Aeromonas spp. from patients suffering from gastroenteritis was found to be significant (chi 2 = 7.1312; P = 0.008, < 0.01). Among the 16 Aeromonas isolates, seven isolates of A. caviae and two isolates of A. veronii biovar sobria induced a secretory response in rabbit intestinal mucosa mounted in Ussing chambers as demonstrated by a significant increase in the short circuit current. Nine of the 16 Aeromonas isolates, including three isolates of A. caviae, five isolates of A. veronii biovar sobria, and the solitary isolate of A. hydrophila were also cytotoxic to CHO cells. Five of the six isolates of A. veronii biovar sobria and the A. hydrophila isolate produced hemolysin. The results of this study indicate that Aeromonas species are important causative agents of diarrhoea in childhood gastroenteritis and are prevalent throughout the year in Madras.     

Enterotoxigenic aeromonads on retail lamb meat and offal

Enrichment in alkaline peptone water was compared with the direct plating method for the isolation of Aeromonas spp. from lamb meat and offal samples. The enrichment method significantly increased the isolation rate of aeromonads. Motile Aeromonas species (A. hydrophila, A. sobria and A. caviae) were present in all kinds of samples investigated. Seventy-three Aeromonas strains isolated in this survey were characterized to species level and examined for their ability to produce virulence factors. Strains identified as A. sobria were the strongest producers of haemolysin and enterotoxin, whereas A. caviae strains were consistently non-haemolytic and non-enterotoxigenic. Thus it is likely that lamb meat and offal are potentially significant sources of virulent Aeromonas species and may play an important role in the aetiology of Aeromonas-associated gastro-enteritis.     

Comparative electrophoretic profiles of esterases, and of glutamate, lactate and malate dehydrogenases, from Aeromonas hydrophila, A. caviae and A. sobria

Esterases, and glutamate, lactate and malate dehydrogenases of 64 Aeromonas hydrophila, A. caviae and A. sobria strains, were analysed by polyacrylamide agarose gel electrophoresis and by thin layer isoelectrofocusing. On the basis of the isoelectric points of malate dehydrogenase from the three species and the mobility of lactate dehydrogenase from A. sobria, 8 species specific zymotypes were defined: three for A. hydrophila strains, three for A. caviae strains and two for A. sobria strains. These zymotypes correlated with previously established DNA hybridization groups. The other electrophoretic data were found to be less useful for distinction between A. hydrophila and A. sobria strains, but supported differentiation into zymotypes for A. caviae strains. The two-dimensional electrophoretic profile established by plotting isoelectric point against electrophoretic mobility of the major esterase illustrated the degree of enzyme polymorphism among the strains of the three species. Variation in electrophoretic patterns within A. hydrophila and A. caviae might provide useful epidemiological markers.     

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.     

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.     

Genetic diversity and antimicrobial susceptibility of motile aeromonads isolated from rainbow trout (Oncorhynchus mykiss, Walbaum) farms

A total of 22 motile Aeromonas strains were detected in 48 (18.53%) of 259 fish and 6 (10.71%) of 56 water samples obtained from seven commercial rainbow trout ( Oncorhynchus mykiss , Walbaum) farms in the province of Mersin, Turkey. These strains were identified by conventional microbiological techniques and by using an ID32GN system. Of these isolates 20 (91.3%) were identified as Aeromonas hydrophila and 2 (8.7%) as Aeromonas sobria . While 8 of the A. hydrophila strains were isolated from water samples, 12 isolates were from fish samples. Whereas A. hydrophila strains were found in all farms, A. sobria was detected in only two farms. Genetic diversity by arbitrarily primed polymerase chain reaction (AP-PCR) and antimicrobial sensitivity tests were carried out on eight A. hydrophila isolates obtained from water samples, and isolates from seven A. hydrophila and one A. sobria from fish samples. The AP-PCR band patterns of motile aeromonads demonstrated weak similarity to the A. hydrophila reference strain ATCC 7966. Five A. hydrophila strains in the water samples displayed genetic similarity, but three others were different. Aeromonas hydrophila isolates from fish samples possessed slight similarities, and A. sobria was genetically distant to all A. hydrophila strains. An antimicrobial sensitivity test of 16 isolates revealed that 100% were sensitive to gentamicin, 87.5% to sulphamethoxazole–trimethoprim, 62.5% to enrofloxacin, 43.8% to oxytetracycline, 37.5% to neomycin, 18.75% to streptomycin and 6.25% to erythromycin. All isolates were resistant to novobiocin.     

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.     

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.     

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.     

Occurrence, diversity and pathogenicity of mesophilic Aeromonas in estuarine waters of the Italian coast of the Adriatic Sea

A total of 208 strains of Aeromonas were isolated by monthly sampling from two estuaries (one provided with, and the other devoid of a waste-water treatment system) on the Italian coast of the Adriatic sea between September 1994 and August 1995. Biotyping at the species level allowed the identification of 96 strains (46%) as Aer. caviae , 46 (22%) as Aer. sobria , 33 (16%) as Aer. hydrophila and 25 (12%) as Aer. veronii . Eight strains (4%) were regarded as unnamed aeromonads. Aeromonas caviae was the most prevalent species in water with a high degree of pollution, while Aer. hydrophila strains were more commonly isolated from cleaner water. Aeromonas sobria and Aer. veronii were equally distributed in both estuaries. There was no correlation between temperature and numbers of aeromonads in either estuary. Using a biochemical fingerprinting method, strains were divided into similarity groups (PhP-types) based on their biochemical phenotypes. Several different PhP-types were found in each estuary, yielding a high diversity for these strains. However, some identical PhP-types were also found in both estuaries and at different times of the year, indicating that certain Aeromonas strains can survive more widely varying physico-chemical conditions. The production of toxins capable of causing cytoskeletal-dependent changes in the morphology of Chinese hamster ovary (CHO) cells was detected in 14 strains and appeared to be dependent on the season.     

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.     

Assessment of media used for selective isolation of Aeromonas spp.

A. GAVRIEL AND A.J. LAMB. 1995. A group of selective media were evaluated for their ability to support growth and recovery of type species of the mesophilic, motile group from the genus Aeromonas . With both low and high inoculum densities Aer. hydrophila and Aer. caviae grew well on ampicillin dextrin agar, glutamate starch penicillin agar and starch glutamate ampicillin penicillin agar whereas Aer. veronii only grew with a high level of inoculum. Aeromonas sobria and Aer. schubertii displayed little growth on any of these media regardless of the inoculum size. These results indicate that environmental studies attempting to evaluate the relative distribution and abundance of the members of the motile Aeromonas group would not recover all the strains with equal effectiveness using these particular media.     

Distribution and survival of motile Aeromonas spp. in brackish water receiving sewage treatment effluent.

The spatiotemporal distributions of Aeromonas spp. and fecal coliforms in a cove receiving sewage treatment effluent and draining into a brackish lagoon were studied for 34 months with sampling at six stations. A total of 452 strains of Aeromonas spp. were isolated and identified at the outflow of the treatment system and at stations in the cove. Hemolytic activity of 289 Aeromonas strains was determined. The Aeromonas spp. and fecal coliform distributions showed seasonal cycles in the pond effluent. These seasonal bacterial cycles were persistent in effluent, at the discharge point, and in the cove. However, the abundance levels of these bacterial distributions decreased gradually from the coast to the open lagoon. A dilution model showed that the Aeromonas spp. and fecal coliform distributions in the cove water were subject not only to dilution effect but also to other environmental factors, such as salinity. A. sobria is the most common species identified in the Aeromonas population present in the cove water. Survival studies confirmed that Aeromonas spp., especially A. sobria, were more sensitive to saline and/or marine stress than fecal coliforms. Among the Aeromonas hydrophila and A. sobria strains, 96 and 97%, respectively, produced hemolysin, whereas among the Aeromonas caviae strains, 95% were nonhemolytic.     

Distribution and survival of motile Aeromonas spp. in brackish water receiving sewage treatment effluent.

The spatiotemporal distributions of Aeromonas spp. and fecal coliforms in a cove receiving sewage treatment effluent and draining into a brackish lagoon were studied for 34 months with sampling at six stations. A total of 452 strains of Aeromonas spp. were isolated and identified at the outflow of the treatment system and at stations in the cove. Hemolytic activity of 289 Aeromonas strains was determined. The Aeromonas spp. and fecal coliform distributions showed seasonal cycles in the pond effluent. These seasonal bacterial cycles were persistent in effluent, at the discharge point, and in the cove. However, the abundance levels of these bacterial distributions decreased gradually from the coast to the open lagoon. A dilution model showed that the Aeromonas spp. and fecal coliform distributions in the cove water were subject not only to dilution effect but also to other environmental factors, such as salinity. A. sobria is the most common species identified in the Aeromonas population present in the cove water. Survival studies confirmed that Aeromonas spp., especially A. sobria, were more sensitive to saline and/or marine stress than fecal coliforms. Among the Aeromonas hydrophila and A. sobria strains, 96 and 97%, respectively, produced hemolysin, whereas among the Aeromonas caviae strains, 95% were nonhemolytic.