The occurrence of aerolysin-positive Aeromonas spp. and their cytotoxicity in Norwegian water sources

Aims:  The aim of the study was to investigate the occurrence of Aeromonas spp. and their aerolysin status in Norwegian natural water sources.
Methods and Results:  Seventy-one samples from 33 Norwegian water sources were examined for the presence of Aeromonas spp. From most of the sample sites, the strains were isolated on blood-ampicillin-agar and Difco Aeromonas agar simultaneously. The majority of the samples (73/77) contained Aeromonas spp., with an average of 35–100 cfu 100 ml^–1. The highest counts were found in faecally-contaminated water. Using PCR, 445 isolates were screened for the presence of aerolysin, and 79% of them were found to be carriers of the aerolysin gene. A selection of the isolated strains was tested on Vero cell cultures and 83% of them showed cytotoxicity.
Conclusions:  There is widespread occurrence of aerolysin-positive cytotoxic Aeromonas spp. in many different Norwegian natural waters, including drinking water sources.
Significance and Impact of the Study:  The widespread occurrence of potentially-pathogenic Aeromonas spp. in the environment demands that these bacteria should not be ignored in drinking water supplies and in the food industry.     

Occurrence of Staphylococcus and enteropathogens in soft cheese commercialized in the city of Rio de Janeiro,Brazil

AIMS: To investigate the presence of Staphylococcus aureus, enteropathogenic Escherichia coli (EPEC), Aeromonas spp. and Yersinia spp. in soft cheese commercialized in Rio de Janeiro, Brazil. METHODS AND RESULTS: A total of 45 samples of cheese from three different brands marketed in Rio de Janeiro city were analysed for faecal coliform levels using the Most Probable Number (MPN) technique. The samples were also analysed using conventional methodology for the investigation of food-borne pathogens.High levels of faecal contamination were detected in 95.5% of cheese samples. Staphylococcus aureus was isolated from 20% of samples, of which 17.7% were above the limits allowed by Brazilian legislation. Aeromonas hydrophila and Aer. caviae were detected in 17.7% of the samples. Yersinia spp. were not found in this study. EPEC was isolated from 21.1% of the samples and the most frequently found serogroups were O127, followed by O55 and O26. CONCLUSIONS: Our results showed that 95.5% of cheese samples had high levels of faecal coliforms. The isolation of Staph. aureus, serogroups of EPEC and Aeromonas spp. suggested that the soft cheese commercialized in the city of Rio de Janeiro may represent a health risk for the consumers. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that soft cheese may act as an important vehicle of transmission for well-established pathogens.     

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.     

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.     

Prevalence and virulence properties of Vibrio cholerae non-O1, Aeromonas spp. and Plesiomonas shigelloides isolated from Cambé Stream (State of Paraná, Brazil)

The incidence of Vibrio cholerae, Aeromonas spp. and Plesiomonas shigelloides was determined in water samples from Cambé Stream. The samples were collected from seven different sites. The serogroups, virulence markers and drug resistance profiles were also evaluated. Twelve Aer. hydrophila, 12Aer. caviae, eight Aer. sobria, seven Ple. shigelloides and two V. cholerae non-O1 were isolated. They belonged to different serogroups and all produced haemolysis in different assays. Five of the Aeromonas strains and one of V cholerae non-O1 were positive for enterotoxin activity. Haemagglutination and its inhibition, using erythrocytes of different origins, was variable for Aeromonas spp. and V. cholerae, while none of the Ple. shigelloides haemagglutinated in association with any type of erythrocyte. All isolates exhibited multiple drug resistance. These results indicate that the occurrence of V. cholerae non-O1, Aeromonas spp. and Ple. shigelloides, in water used for vegetable irrigation, human recreation and animal consumption, among others, represents a potential risk for humans.     

Comparative study of the prevalence and clinical profiles of diarrheas due to Aeromonas and other enteric pathogens

The prevalence of Aeromonas spp. and other enteric pathogens in stool specimens from diarrheic and non-diarrheic patients was studied over a 12 month period (January to December, 1986). Except for the absence of fever, all the clinical features in Aeromonas diarrhea were comparable to those associated with other diarrheagenic agents. These features included abdominal pain (30%), vomiting (24.5%), fever (31.5%), dehydration (9.5%) and hematochezia (19.5%). Aeromonas spp. were more frequently isolated from patients with gastroenteritis (2.5%) than from control patients (1.0%) (P less than 0.05). Isolates were recovered more often during the dry months (66.7%), than during the wet months (33.3%). Among the enteric pathogens isolated, Aeromonas spp. (2.5%) ranked next to Esch. coli (14.5%) and Shigella spp. (6.3%) in prevalence. Other bacterial isolates included Plesiomonas shigelloides (1.5%) Vibrio spp. (1.0%), Yersinia enterocolitica (1.0%) and Salmonella spp. (1.8%).     

从新疆哈萨克族2型糖尿病患者粪便中分离的气单胞菌对小鼠的作用研究

目的 从新疆哈萨克族2型糖尿病患者粪便中分离培养气单胞菌,并回接到小鼠体内,检测其对小鼠血糖、血脂等2型糖尿病相关中间表型的作用。方法 （1）收集新鲜哈萨克族２型糖尿病患者的粪便样本,用气单胞菌专属培养基进行分离培养,微量生化反应管进行初步鉴定;提取所得菌株的DNA,使用气单胞菌属引物对此DNA进行特异性扩增,结合全自动细菌鉴定仪对菌株进行鉴定;（2）将气单胞菌菌液回接小鼠,采用羰花青素染料（CM-Dil）标记法对菌定植力情况进行检测。（3）检测气单胞菌菌液对小鼠体重、随机血糖（GLU）、空腹血糖（FBG）、总胆固醇（TC）和甘油三酯（TG）的作用,进行葡萄糖耐量（OGTT）、胰岛素耐量（ITT）试验。结果 （1）所得到的菌株经全自动细菌鉴定仪确定为气单胞菌;（2）从小鼠粪便中分离培养后的细菌能检测到荧光标记;（3）与正常饮食组小鼠相比,气单胞菌稀释液、原液、浓缩液组小鼠体重均升高;气单胞菌原液组小鼠GLU升高。小鼠FBG、TC和TG在各组间差异均无统计学意义;气单胞菌菌液组小鼠口服葡萄糖耐量试验血糖曲线下面积增加;气单胞菌原液、浓缩液组小鼠胰岛素耐量试验血糖曲线下面积增加。结论 （1）从新疆哈萨克族2型糖尿病患者粪便中分离培养得到气单胞菌;（2）从糖尿病患者粪便中分离的气单胞菌可定植于小鼠体内,并使小鼠的体重增加,GLU升高,给予气单胞菌稀释液、菌原液、菌浓缩液后第8周,小鼠出现糖耐量受损,胰岛素敏感性下降。     

冷却猪肉中气单胞菌生长预测模型的建立和检验

为探讨冷却猪肉中气单胞菌的生长规律,将气单胞茵接种到经过80℃无菌水灭菌的冷却猪肉中,构建Gompertz和Baranyi初级模型来描述气单胞菌在不同温度下的生长状况。结果表明：修正的Gompertz模型对气单胞菌生长曲线的拟合效果优于Baranyi模型,R2均在0．97以上。应用平方根模型和Arrhenius模型对由修正的Gompertz模型得出的最大比生长速率进行拟合,所得平方根模型拟合效果略优于Arrhenius模型。实验所建二级模型能预测0-35℃贮藏条件下气单胞菌的生长。     

Incidence of Aeromonas species in influent and effluent of urban wastewater purification plants

Influent and effluent samples from urban wastewater plants were examined for presumptive Aeromonas. Removal efficiency was comparatively low for both presumptive Aeromonas (96.5%) and faecal coliforms (95.8%). Starch ampicillin agar (SA) medium was superior to mA medium for selectivity, with 32.3% of typical colonies confirmed as Aeromonas spp., and for the recovery of Aer. veronii biotype veronii.     

Isolation of Aeromonas spp. from an unchlorinated domestic water supply.

The recovery of Aeromonas spp. from the unchlorinated water supply for a Western Australian city of 21,000 people was monitored at several sampling points during a period of 1 year. Membrane filtration techniques were used to count colonies of Aeromonas spp., coliforms, and Escherichia coli in water sampled before entry to service reservoirs, during storage in service reservoirs, and in distribution systems. Aeromonas spp. were identified by subculture on blood agar with ampicillin, oxidase tests, and the use of Kaper medium and then were tested for production of enterotoxins and hemolysins. During the same period, two-thirds of all fecal specimens sent for microbiological examination were cultured on ampicillin-blood agar for Aeromonas spp. Recovery of Aeromonas spp. from water supplies at distribution points correlated with fecal isolations and continued during autumn and winter. Coliforms and E. coli were found most commonly in late summer to autumn. This pattern differs from the summer peak of Aeromonas isolations both from water and from patients with Aeromonas spp.-associated gastroenteritis in Perth, Western Australia, a city with a chlorinated domestic water supply. Of the Aeromonas strains from water, 61% were enterotoxigenic, and 64% produced hemolysins.     

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 of Aeromonas spp. as identified by 16S rDNA restriction fragment length polymorphism analysis in a trout farm

AIMS: This study used restriction fragment length polymorphism (RFLP) with Aeromonas-specific primers to identify species of Aeromonas and to investigate their distribution in a trout farm and stream. METHODS AND RESULTS: In January, May, August and November 2000, presumptive Aeromonas species were recovered from a farm and a sedimentation pond in a fish farm and stream, and identified by PCR-RFLP analysis with Aeromonas-specific primers. The specificity of Aeromonas-specific primers and the suitability of PCR-RFLP analysis for identifying Aeromonas spp. were confirmed with fatty acid methyl esters (FAMEs) and 16S rDNA sequencing analyses, respectively. Levels of Aeromonas spp. sampled in May and August were higher than in January and November at all sampling sites. Aeromonas salmonicida was the dominant species in January and November, and the proportion of pathogenic species (Aer. hydrophila, Aer. caviae and Aer. veronii) increased in May and August. CONCLUSIONS: PCR-RFLP analysis with Aeromonas-specific primers is a rapid and reliable method for identifying widely distributed Aeromonas spp. from environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: To minimize human health risk, monitoring the levels and species composition of Aeromonas in fish farm is advisable.     

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.     

SGAP-10C agar for the isolation and quantification of Aeromonas from water

Glutamate starch penicillin (GSP) medium was used for the simultaneous isolation of Pseudomonas and Aeromonas. Modifications to reduce the number of Pseudomonas and background flora and to improve the recovery of Aeromonas from water samples are described. The original medium was modified by adding glucose and ampicillin. The addition of 10 micrograms/l of C-glucose to the medium (SGAP-10C) permitted better recuperation of stressed cells of aeromonads and the ampicillin reduced the numbers of Pseudomonas. The best temperature for the recovery of aquatic aeromonads was 28 degrees C. The recovery of different species of Aeromonas on SGAP-10C was 93%. The selectivity of the medium was validated because 95.5% of 28 colonies tested with an Aeromonas-like morphology belonged to the genus Aeromonas. Moreover, when 45 strains of different genera were cultured on the medium, only Vibrio alginolyticus presented a confusing morphology. When the SGAP-10C was compared with GSP with 45 river samples, the new medium gave a significantly better recovery of Aeromonas spp., especially when large numbers of Pseudomonas spp. were present. SGAP-10C used at 28 degrees C and 48 h was an efficient selective medium for the isolation of Aeromonas from fresh waters.     

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.     

Production of acylated homoserine lactones by Aeromonas and Pseudomonas strains isolated from municipal activated sludge

Up to now, the production and role of N-acyl homoserine lactones (AHLs) in activated sludge have been poorly understood. In this study, cross-feeding assays with the reporter strains Agrobacterium tumefaciens NTL4 and Chromobacterium violaceum CV026 were used to investigate AHL signal production by municipal activated sludge samples. AHL signal production was consistently detected from municipal activated sludge when different samples were incubated on nutrient media. From one municipal activated sludge sample, 10 strains producing AHL-like auto inducers were isolated by an overlay technique. 16S rDNA-based phylogenetic analysis showed that eight of the isolates belonged to Aeromonas spp. and two to Pseudomonas spp. Box-PCR indicated that six of these Aeromonas isolates were different strains and the two Pseudomonas strains were identical. The production of AHL or AHL-like compounds by these strains was confirmed by thin layer chromatography and biosensor overlays. The six different Aeromonas strains were found to produce the same set of AHLs, including N-hexanoyl-L-homoserine lactone. These results may indicate the possible presence of AHLs in municipal activated sludge. The potential roles of AHL in this eco system are briefly discussed.     

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.     

SGAP-10C agar for the isolation and quantification of Aeromonas from water

Glutamate starch penicillin (GSP) medium was used for the simultaneous isolation of Pseudomonas and Aeromonas . Modifications to reduce the number of Pseudomonas and background flora and to improve the recovery of Aeromonas from water samples are described. The original medium was modified by adding glucose and ampicillin. The addition of 10 μg/l of C-glucose to the medium (SGAP-10C) permitted better recuperation of stressed cells of aeromonads and the ampicillin reduced the numbers of Pseudomonas . The best temperature for the recovery of aquatic aeromonads was 28°C. The recovery of different species of Aeromonas on SGAP-10C was 93%. The selectivity of the medium was validated because 95·5% of 28 colonies tested with an Aeromonas -like morphology belonged to the genus Aeromonas . Moreover, when 45 strains of different genera were cultured on the medium, only Vibrio alginolyticus presented a confusing morphology. When the SGAP-10C was compared with GSP with 45 river samples, the new medium gave a significantly better recovery of Aeromonas spp., especially when large numbers of Pseudomonas spp. were present. SGAP-10C used at 28°C and 48 h was an efficient selective medium for the isolation of Aeromonas from fresh waters.     

Age and population group related distribution of enteropathogens in the Eastern Cape, South Africa

The frequency of Salmonella spp., Shigella spp., Campylobacter spp., Yersinia enterocolitica, Aeromonas hydrophilia and Helminths in samples from various hospitals of the Eastern Cape, South Africa, were analysed for trends involving age distribution and population group. Significant differences in the distribution of these pathogens could be attributed to hygiene and environmental difference between the age and population groups which suggests that rapid urbanization and poor water supply remain major problems in the drought prone Eastern Cape.