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.     

Enumeration and characterization of Aeromonas hydrophila and Aeromonas caviae isolated from grocery store produce

Starch-ampicillin agar was used to quantitatively isolate Aeromonas sp. from retail grocery store produce. All produce sampled, including parsley, spinach, celery, alfalfa sprouts, broccoli, and lettuce, contained Aeromonas sp. In most instances, the count of Aeromonas sp. increased 10- to 1,000-fold during 2 weeks of storage at 5 degrees C. Eleven (92%) of 12 kinds of produce yielded cytotoxic Aeromonas sp. Identification as Aeromonas hydrophila was the strongest indicator of cytotoxicity, and all 29 (100%) A. hydrophila isolates and 1 (6%) of 16 A. caviae isolates were cytotoxic. Twenty-seven (90%) of 30 cytotoxic Aeromonas sp. strains produced hemolysins. Strong correlations were also noted between ability to produce cytotoxin and positive Voges-Proskauer, lysine decarboxylase, and sorbitol fermentation reactions. It appears that grocery store produce is a potentially significant source of cytotoxic Aeromonas sp. and should be considered in the epidemiology of A. hydrophila gastroenteritis.     

Enumeration and characterization of Aeromonas hydrophila and Aeromonas caviae isolated from grocery store produce.

Starch-ampicillin agar was used to quantitatively isolate Aeromonas sp. from retail grocery store produce. All produce sampled, including parsley, spinach, celery, alfalfa sprouts, broccoli, and lettuce, contained Aeromonas sp. In most instances, the count of Aeromonas sp. increased 10- to 1,000-fold during 2 weeks of storage at 5 degrees C. Eleven (92%) of 12 kinds of produce yielded cytotoxic Aeromonas sp. Identification as Aeromonas hydrophila was the strongest indicator of cytotoxicity, and all 29 (100%) A. hydrophila isolates and 1 (6%) of 16 A. caviae isolates were cytotoxic. Twenty-seven (90%) of 30 cytotoxic Aeromonas sp. strains produced hemolysins. Strong correlations were also noted between ability to produce cytotoxin and positive Voges-Proskauer, lysine decarboxylase, and sorbitol fermentation reactions. It appears that grocery store produce is a potentially significant source of cytotoxic Aeromonas sp. and should be considered in the epidemiology of A. hydrophila gastroenteritis.     

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.     

Peritrichous flagella in mesophilic strains of Aeromonas

A total of 186 mesophilic strains of Aeromonas, comprising 151 A. hydrophila and 35 A. caviae, were tested for peritrichous flagella by Leifson's staining method. When incubated on solid medium for 18 hr at 22 C, peritrichous flagella were demonstrated in 28 (18.5%) of the 151 strains of A. hydrophila and in 6 (17.1%) of the 35 strains of A. caviae. The peritrichous flagella in these 34 mesophilic strains of Aeromonas were also observed by electron microscopy.     

Virulence factors in Vibrios and Aeromonads isolated from seafood

Thirty-one isolates from seafood, identified as Aeromonas hydrophila (7), Aeromonas caviae (11), Vibrio parahaemolyticus (3), Vibrio fluvialis (5), Vibrio alginolytictus (3), Vibrio metschnikovii (1) and Vibrio damsela (1), were tested for possible virulence factors including extracellular hydrolytic enzymes, haemolysins, cytotoxins (VERO and HEp-2 cells) and adherence ability (HEp-2 cells). All the A. hydrophila strains were beta-haemolytic and produced cytotoxins as well as one strain of V. fluvialis. A. hydrophila and A. caviae strains, frequently adhesive, showed both aggregative and diffusive patterns, while five Vibrio strains only (three V. fluvialis, one V. parahaemolyticus and one V. alginolyticus) were adhesive with an aggregative pattern.     

Production of exotoxins by Aeromonas spp. at 5 degrees C

The ability of 60 strains of Aeromonas to produce enterotoxin and haemolysin after cultivation at 5 degrees C for 7-10 d was investigated. The strains were isolated from lamb meat, offal, carcasses and faeces, and had previously been tested for their ability to produce these exotoxins at 37 degrees C. The results showed that some strains of Aeromonas hydrophila and A. sobria were capable of producing enterotoxin and haemolysin at 5 degrees C, but none of the A. caviae strains tested produced these two factors. Of the 30 A. hydrophila strains investigated 25 and 27 were enterotoxigenic and haemolytic respectively. Likewise, of the 24 A. sobria strains investigated 16 and 18 were enterotoxigenic and haemolytic respectively. The results indicate that certain strains of Aeromonas species, in particular A. hydrophila and A. sobria, are of potential public health significance in meats stored at refrigeration temperature.     

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.     

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.     

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.     

Interactions of clinical and environmental Aeromonas isolates with Caco-2 and HT29 intestinal epithelial cells

AIM: Evaluation of adherence and invasion of Aeromonas spp. to human colon carcinoma cell lines Caco-2 and HT29 and assessment of cytotoxic activity. METHODS AND RESULTS: A number of 27 strains of Aeromonas caviae and 23 strains of Aeromonas hydrophila was analysed. All strains were capable to adhere to sub-confluent monolayers of Caco-2 and HT29 cell types, presenting aggregative and diffuse adherence patterns cells, respectively. In the cytotoxic assays all strains showed cytopathic and/or cytotoxic activities to Vero cells. The evaluation of the tetrazolium salt (MTT test) reduction capability was carried out in Vero, Caco-2, and HT29 cells. MTT test showed that Vero cell line was the most sensitive cell type. In the invasion test, 13 strains were analysed on Caco-2 and HT29 monolayers. Only two (15%) of the 13 strains, A. hydrophila and A. caviae species, both isolated from vegetables were invasive to Caco-2 cells. No strains were able to invade the HT29 cells. CONCLUSIONS: A. hydrophila and A. caviae isolated from human diarrhoeic faeces, vegetables, and water, were able to adhere to and produce cytotoxic/cytopathic effects in intestinal epithelial cell lines. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of Aeromonas spp. in food and water samples expressing virulence factors suggest that these sources may act as dissemination vehicles of human pathogen with implication in the public health.     

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.     

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.     

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.     

Production of exotoxins by Aeromonas spp. at 5°C

The ability of 60 strains of Aeromonas to produce enterotoxin and haemolysin after cultivation at 5°C for 7–10 d was investigated. The strains were isolated from lamb meat, offal, carcasses and faeces, and had previously been tested for their ability to produce these exotoxins at 37°C. The results showed that some strains of Aeromonas hydrophila and A. sobria were capable of producing enterotoxin and haemolysin at 5°C, but none of the A. caviae strains tested produced these two factors. Of the 30 A. hydrophila strains investigated 25 and 27 were enterotoxigenic and haemolytic respectively. Likewise, of the 24 A. sobria strains investigated 16 and 18 were enterotoxigenic and haemolytic respectively. The results indicate that certain strains of Aeromonas species, in particular A. hydrophila and A. sobria , are of potential public health significance in meats stored at refrigeration temperature.     

Production of cholera-like enterotoxin by Aeromonas hydrophila

A total of 249 strains of mesophilic Aeromonas including 179 A. hydrophila and 70 A. caviae were tested for production of cholera-like enterotoxin by reversed passive latex agglutination (RPLA) assay. A cholera-like enterotoxin neutralized with cholera antitoxin was demonstrated in the culture filtrates from eight (4.5%) of the 179 A. hydrophila strains, while none of A. caviae strains revealed the enterotoxin production in the test. Production of the cholera-like enterotoxin in the eight strains of A. hydrophila was also confirmed by enzyme-linked immunosorbent assay (ELISA).     

Occurrence of Vibrionaceae in crude and mechanically purified urban sewage; evaluation of their sensitivity to selected drugs

The aim of this study was to ascertain whether bacteria from Vibrionaceae family are present in crude and mechanically cleaned urban sewage and what is their profile of resistance to selected chemotherapeutics. The presence in sewage of bacteria of Vibrionaceae family was proved. They constitute only small percentage of total number of bacteria. No influence of mechanical purification process of sewage on the reduction of total number of bacteria and bacteria from Vibrionaceae family was seen. The number of bacteria from Vibrionaceae family amounted to 6-9 cells per 100 ml of crude sewage and 2-16 cells per 100 ml of mechanically cleaned sewage. From samples tested the following species were isolated: non 01 V. cholerae, V. fluvialis, V. parahaemolyticus, A. hydrophila, A. caviae, A. sobria. All strains were sensitive to neomycin and nalidixic acid and with few exceptions of A. caviae strains to streptomycin, gentamicin, doxycycline and chloramphenicol .     

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.