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 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.     

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.     

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.     

Dynamics of Aeromonas hydrophila, Aeromonas sobria, and Aeromonas caviae in a sewage treatment pond.

The spatiotemporal dynamics of Aeromonas spp. and fecal coliforms in the sewage treatment ponds of an urban wastewater center were studied after 20 months of sampling from five stations in these ponds. Isolation and identification of 247 Aeromonas strains were undertaken over four seasons at the inflow and outflow of this pond system. The hemolytic activity of these strains was determined. The Aeromonas spp. and the fecal coliform distributions showed seasonal cycles, the amplitude of which increased at distances further from the wastewater source, so that in the last pond there was an inversion of the Aeromonas spp. cycle in comparison with that of fecal coliforms. The main patterns in these cycles occurred simultaneously at all stations, indicating control of these bacterial populations by seasonal factors (temperature, solar radiation, phytoplankton), the effects of which were different on each bacterial group. The analysis of the Aeromonas spp. population structure showed that, regardless of the season, Aeromonas caviae was the dominant species at the pond system inflow. However at the outflow the Aeromonas spp. population was dominated by A. caviae in winter, whereas Aeromonas sobria was the dominant species in the treated effluent from spring to fall. Among the Aeromonas hydrophila and A. sobria strains, 100% produced hemolysin; whereas among the A. caviae strains, 96% were nonhemolytic.     

Bacteriocin-like substance (BLS) production in Aeromonas hydrophila water isolates

30 Aeromonas hydrophila water isolates were tested for bacteriocin-like substance (BLS) production using a target panel of closely related microorganisms and other Gram-positive and Gram-negative bacteria, including food-borne pathogens. A. hydrophila showed antibacterial activity against one or more indicator microorganisms, but the activity emerged only with non-phylogenetically related genera or species. In particular all A. hydrophila showed antibacterial activity against one or more of the tested Staphylococcus strains, five against Listeria spp. (Listeria seeligeri, Listeria welshimeri and Listeria ivanovii), and eight presented a weak antagonistic activity towards Streptococcus agalactiae and Lactobacillus spp. Inhibitory activity was not observed against the other Gram-positive (Listeria monocytogenes, Listeria innocua and Enterococcus spp.) and Gram-negative tested strains, including Aeromonas sobria, Aeromonas caviae and the same A. hydrophila, when used as indicator. Anti-staphylococcal activity was observed with a gradual increase of the inhibition zone during incubation and seemed to be influenced by A. hydrophila hemolytic expression. Extrachromosomal analysis showed the presence, in 70% of the strains, of one to five plasmids with molecular masses ranging from 2.1 to 41.5 MDa, but it was not possible to relate this result with BLS production.     

Dynamics of Aeromonas hydrophila, Aeromonas sobria, and Aeromonas caviae in a sewage treatment pond

The spatiotemporal dynamics of Aeromonas spp. and fecal coliforms in the sewage treatment ponds of an urban wastewater center were studied after 20 months of sampling from five stations in these ponds. Isolation and identification of 247 Aeromonas strains were undertaken over four seasons at the inflow and outflow of this pond system. The hemolytic activity of these strains was determined. The Aeromonas spp. and the fecal coliform distributions showed seasonal cycles, the amplitude of which increased at distances further from the wastewater source, so that in the last pond there was an inversion of the Aeromonas spp. cycle in comparison with that of fecal coliforms. The main patterns in these cycles occurred simultaneously at all stations, indicating control of these bacterial populations by seasonal factors (temperature, solar radiation, phytoplankton), the effects of which were different on each bacterial group. The analysis of the Aeromonas spp. population structure showed that, regardless of the season, Aeromonas caviae was the dominant species at the pond system inflow. However at the outflow the Aeromonas spp. population was dominated by A. caviae in winter, whereas Aeromonas sobria was the dominant species in the treated effluent from spring to fall. Among the Aeromonas hydrophila and A. sobria strains, 100% produced hemolysin; whereas among the A. caviae strains, 96% were nonhemolytic.     

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.     

The occurrence of Aeromonas species in drinking water supplies of an area of the Dolomite Mountains, Italy

A study was made of the occurrence of Aeromonas spp. in drinking water supplies in a mountain area in northeast Italy (the Dolomites). On account of its location, the water in question is exposed to a low level of pollution and systematic chemical disinfection is not necessary. Out of 7395 water samples analysed over a 3 year period, 1623 (21·95%) were found to be positive for Aeromonas , with levels ranging from 1 to 240 cfu 100 ml⁻¹ ; 72·4% of the strains were identified as Aer. hydrophila , 14·7% as Aer. caviae and 12·9% as Aer. sobria. The percentage of recovery from surface water (approximately 40%) was found to be higher than that of ground water (springs : 24·9% ; wells : 28·6%). Aeromonas spp. were isolated from 21·7% of samples from the distribution network and showed no significant variations compared with water from reservoirs. There was no evidence, therefore, of after-growth in the distribution system. No correlation was found between the concentrations of Aeromonas spp. and faecal indicator organisms. As the distribution of Aeromonas spp. was unrelated to anthropic pollution, it is believed that the search for these micro-organisms should be adopted as a further indicator of drinking water quality, especially in waters such as those in the present investigation not undergoing systematic purification treatment.     

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.     

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.     

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.     

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.     

Survival of Aeromonas hydrophila, Aeromonas caviae and Aeromonas sobria in soil

The survival of mesophilic Aeromonas spp. in soil in the presence or absence of indigenous microflora was evaluated in a laboratory study. Two cytotoxic ( Aer. hydrophila and Aer. caviae ) and one invasive ( Aer. sobria ) clinical isolate strains were selected for this study. After contamination of sterile or unsterilized soil with the three strains of Aeromonas , the number of living cells was determined over at least 5 months. For all strains the survival curves were characterized by an initial re-growth followed by a slow inactivation of bacteria, with significant differences due to the presence of indigenous microflora. The times necessary to achieve a 95% reduction of the initial population were > 140, 113 and 62 d in sterilized soil respectively for Aer. caviae, Aer. hydrophila and Aer. sobria , while the corresponding times in unsterilized soil were 42, 38 and 11 d. All strains preserved the virulence factors for the entire period of the study. These results suggest that the soil may be an important reservoir for Aeromonas spp. and, thus, may play an important role in the epidemiology of Aeromonas -associated human infections.     

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.     

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.     

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.     

Comparison of different media for the isolation and enumeration of Aeromonas spp. in foods

Starch ampicillin agar (SA), starch glutamate ampicillin penicillin agar (SGAP) and Aeromonas medium (AM) were evaluated for enumeration of Aeromonas spp. from foods. Recovery from pure cultures of Aer. hydrophila and Aer. caviae was excellent on all media. Recovery of Aer. sobria was best on AM agar, where 95.5% were recovered, compared with 31.9% on SA agar and 33.3% on SGAP medium.