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.     

Identification of a new hemolysin from diarrheal isolate SSU of Aeromonas hydrophila

A clinical strain SSU of Aeromonas hydrophila produces a potent cytotoxic enterotoxin (Act) with cytotoxic, enterotoxic, and hemolytic activities. A new gene, which encoded a hemolysin of 439-amino acid residues with a molecular mass of 49 kDa, was identified. This hemolysin (HlyA) was detected based on the observation that the act gene minus mutant of A. hydrophila SSU still had residual hemolytic activity. The new hemolysin gene (hlyA) was cloned, sequenced, and overexpressed in Escherichia coli. The hlyA gene exhibited 96% identity with its homolog found in a recently annotated genome sequence of an environmental isolate, namely the type strain ATCC 7966 of A. hydrophila subspecies hydrophila. The hlyA gene did not exhibit any homology with other known hemolysins and aerolysin genes detected in Aeromonas isolates. However, this hemolysin exhibited significant homology with hemolysin of Vibrio vulnificus as well as with the cystathionine beta synthase domain protein of Shewanella oneidensis. The HlyA protein was activated only after treatment with trypsin and the resulting hemolytic activity was not neutralizable with antibodies to Act. The presence of the hlyA gene in clinical and water Aeromonas isolates was investigated and DNA fingerprint analysis was performed to demonstrate its possible role in Aeromonas virulence.     

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.     

Biochemical and molecular characterization of tetracycline-resistant Aeromonas veronii isolates from catfish

Eighty-one tetracycline-resistant Aeromonas sp. strains were isolated from farm-raised catfish. Morphological and biochemical characteristics indicated that 23 of the 81 aeromonads were Aeromonas hydrophila, 7 isolates were Aeromonas trota, 6 isolates were Aeromonas caviae, 42 isolates were Aeromonas veronii, and 3 isolates were Aeromonas jandaei. However, the AluI and MboI restriction fragment length polymorphism (RFLP) patterns of the PCR-amplified 1.4-kb 16S rRNA gene from all 81 tetracycline-resistant aeromonads from catfish were identical to the RFLP banding patterns of A. veronii ATCC 35626, indicating that all 81 isolates were strains of A. veronii. A multiplex PCR assay successfully amplified the 5 tetracycline-resistant genes (tetA to E) from the genomic DNA of all 81 isolates. The assay determined that tetE was the dominant gene occurring in 73/81 (90.0%) of the aeromonads. Plasmids (2.0 to 20 kb) were isolated from 33 of the 81 isolates. Dendrogram analysis of the SpeI pulsed-field gel electrophoresis identified 15 distinct macrorestriction patterns among the isolates. Our results indicate the need for use of 16S rRNA in the identification of Aeromonas spp. and the prevalence of catfish as a reservoir of tet genes.     

Virulence potential and genetic diversity of Aeromonas caviae, Aeromonas veronii, and Aeromonas hydrophila clinical isolates from Mexico and Spain: a comparative study

A comparative study of 109 Aeromonas clinical isolates belonging to the 3 species most frequently isolated from patients with diarrhea in Mexico and Spain was performed to investigate the distribution of 3 prominent toxin genes and the gene encoding flagellin of lateral flagella; 4 well-established virulence factors in the genus Aeromonas. The aerolysin-hemolysin toxin genes were the most prevalent, being present in 89% of the total isolates. The ast toxin gene was conspicuously absent from the Aeromonas caviae and Aeromonas veronii groups but was present in 91% of the Aeromonas hydrophila isolates. Both the alt toxin gene and the lafA flagellin gene also had a low incidence in A. caviae and A. veronii. Differences in the prevalence of alt and lafA were observed between isolates from Mexico and Spain, confirming genus heterogeneity according to geographic location. Carriage of multiple toxin genes was primarily restricted to A. hydrophila isolates, suggesting that A. caviae and A. veronii isolates circulating in Mexico and Spain possess a limited array of virulence genes. Enterobacterial repetitive intergenetic consensus - polymerase chain reaction showed that the Aeromonas populations sampled lack dominant clones and were genetically heterogeneous, with A. caviae being the most diverse species. Further surveys of virulence determinants in genetically heterogeneous populations of Aeromonas isolates circulating worldwide are required to enhance the understanding of their capacity to cause disease.     

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.     

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

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

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.     

DNA probes specific for Aeromonas hydrophila (HG1)

Aeromonas hydrophila (HG1)-specific RAPD-PCR fragments were investigated for their potential as DNA probes. From 20 RAPD-PCR fragment bands, it was found that two were specific to all isolates of Aeromonas hydrophila (HG1) tested. Cloning and nucleotide sequence determination of one of these bands showed that co-migration of similar sized amplicons had occurred and that this band (designated '7e') contained at least four fragments of different sequences. Three of these individual amplicons had a sequence specific to Aer. hydrophila (HG1) isolates. The sequence of one of these amplicons ('7e5') was used to design primers for a specific polymerase chain reaction (PCR). The specificity of the PCR was achieved using a modified hot-start procedure. The identity of the PCR amplicons was confirmed by high stringency hybridization with a digoxygenin-labelled 7e5 probe.     

嗜水气单胞菌感染现状及耐药分析

目的调查湖州市中心医院嗜水气单胞菌感染现状和耐药情况。方法采用常规方法分离,用VITEK-32全自动微生物分析仪进行菌种鉴定为嗜水气单胞菌或豚鼠气单胞菌,依据葡萄糖产气反应鉴定为嗜水气单胞菌。并根据配套药敏卡进行药敏试验。结果共分离到34株嗜水气单胞菌,主要来自痰液、胆汁、腹腔引流液或腹水。嗜水气单胞菌对哌拉西林、替卡西林、阿莫西彬克拉维酸、妥布霉素、头孢呋辛、头孢噻肟、头孢他啶、环丙沙星、复方新诺明耐药率为52．9％～73．5％。结论目前嗜水气单胞菌也呈现多重耐药现象,临床上应予以重视。     

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.     

Isolation of enterotoxigenic Aeromonas from fish.

This paper reports two different enterotoxigenic classes of the genus Aeromonas isolated from fishes. Strains of A. sobria were isolated from healthy fishes only wheras strains of A. hydrophila were isolated from both healthy and moribund fishes. Aeromonas sobria and hydrophilia strains produced a cytotoxic factor and were highly proteolytic. Seventy-five percent (75%) of the A. sobria strains produced only one type of hemolysis on 5% blood agar, but 92% of the A. hydrophilia produced two types of hemolysis. Enterotoxigenicity of all strains was tested by the rabbit gut loop technique and the suckling mouse test. Most of those Aeromonads were enterotoxigenic. Enterotoxigenic strains appeared to produce two different enterotoxins which were antigenically related, but they seemed to have different mechanisms of action. Enterotoxins of Aeromonas were shown to be antigenically different from those of E. coli.     

Detection of metallo-β-lactamases-encoding genes in environmental isolates of Aeromonas hydrophila and Aeromonas jandaei

Aims:  To determine the prevalence and expression of metallo-β-lactamases (MBL)-encoding genes in Aeromonas species recovered from natural water reservoirs in southeastern Brazil.
Methods and Results:  Eighty - seven Aeromonas isolates belonging to Aeromonas hydrophila ( n  =   41) and Aer. jandaei ( n  =   46) species were tested for MBL production by the combined disk test using imipenem and meropenem disks as substrates and EDTA or thioglycolic acid as inhibitors. The presence of MBL genes was investigated by PCR and sequencing using new consensus primer pairs designed in this study. The cphA gene was found in 97·6% and 100% of Aer. hydrophila and Aer. jandaei isolates, respectively, whereas the acquired MBL genes bla _IMP , bla _VIM and bla _SPM-1 were not detected. On the other hand, production of MBL activity was detectable in 87·8% and 10·9% of the cphA -positive Aer. hydrophila and Aer. jandaei isolates respectively.
Conclusions:  Our results indicate that cphA seems to be intrinsic in the environmental isolates of Aer. hydrophila and Aer. jandaei in southeastern Brazil, although, based on the combined disk test, not all of them are apparently able to express the enzymatic activity.
Significance and Impact of the Study:  These data confirm the presence of MBL-producing Aeromonas species in natural water reservoirs. Risk of waterborne diseases owing to domestic and industrial uses of freshwater should be re-examined from the increase of bacterial resistance point of view.     

Cloning and expression of an outer membrane protein OmpW of Aeromonas hydrophila and study of its distribution in Aeromonas spp.

Aims:  The main aims of this study were to clone and express an outer membrane protein (OMP), OmpW, of Aeromonas hydrophila and to study its distribution in Aeromonas spp.
Methods and Results:  The gene encoding OmpW in A. hydrophila has been cloned and expressed in Escherichia coli . Primers were designed for amplification of full-length ompW gene and used for identification of this gene in different Aeromonas spp. Of the 42 Aeromonas strains tested, all the isolates were positive by polymerase chain reaction (PCR) except one strain of Aeromonas veronii biovar veronii (VTE338). None of the other gram-negative bacteria were positive by PCR with primers specific to ompW gene of A. hydrophila . Polyclonal antibodies were raised in rabbit against the purified recombinant protein and the reaction of these antibodies was confirmed by western blotting using the purified recombinant protein and 42 Aeromonas cultures grown at various salt concentrations.
Conclusions:  The ompW -based PCR method developed in this study was found to be 100% specific and 97% sensitive. Expression of OmpW protein of Aeromonas was found to be salt-dependant. Recombinant OmpW protein was found to be highly immunogenic in fish.
Significance and Impact of the Study:  To our knowledge, this is the first report on cloning and expression of OmpW protein of A. hydrophila . Full-length ompW gene amplification by PCR can be used for the detection of Aeromonas . Recombinant OmpW protein can be useful for vaccination of fish against Aeromonas spp.     

Molecular typing of Aeromonas isolates in natural mineral waters

A total of 103 isolates of Aeromonas spp. were obtained over a 3-year period from a natural mineral water and from surface streams located within the boundaries of the watershed of the natural mineral water wells and were typed by macrorestriction analysis of genomic DNA with XbaI and by pulsed-field gel electrophoresis. All Aeromonas caviae isolates from the natural mineral water belonged to the same clone, and an analogous clonal identity was found among Aeromonas hydrophila isolates. These two clones expressed no hemolytic or cytotoxic activities. Aeromonas isolates from surface waters showed high molecular heterogeneity and were not related to the clones found in the natural mineral water. The presence of aeromonads chronically found in the natural mineral water was a likely consequence of a localized development of a biofilm, with no exogenous contamination of the aquifer. Molecular fingerprinting of drinking water isolates is a useful tool in explaining possible reasons for bacterial occurrences.     

Aeromonas hydrophila: Ecology and Toxigenicity of Isolates from an Estuary

A microbiological survey of Aeromonas hydrophila in Chesapeake Bay and its tributaries showed that this species is ubiquitous, occurring in numbers ranging from <0.3/l to 5 × 10³/ml in the water column and ca. 4.6 × 10²/g in sediment. It was recovered from water samples collected at several locations in Chesapeake Bay representing various salinity regimes, but the numbers of A. hydrophila in higher salinity water, i.e. 15^O/OO, were low. Results of stepwise multiple linear regression analysis showed that concentrations of A. hydrophila were correlated with total, aerobic, viable, heterotrophic, bacterial counts, and, in addition, were inversely related to salinity and to concentration of dissolved oxygen. Seasonal occurrence was recorded, with fewer strains of A. hydrophila encountered during the winter months. The potential pathogenicity of A. hydrophila strains isolated from Chesapeake Bay was estimated by testing selected isolates for toxigenicity, using the Y-1 adrenal cell assay. Of 116 isolates tested, 83 (71%) produced a cytotoxic response, a characteristic found to be correlated with the lysine decarboxylase and Voges-Proskauer reactions. Eight of 11 strains tested, which elicited fluid accumulation in the rabbit ligated ileal loop assay, also provoked a cytotoxic reaction in the Y-l adrenal cell assay. Results of the study indicate that large numbers of toxigenic A. hydrophila can be found in an estuary and such strains may be pathogenic for man and/or animals.     

An attenuated plasmid-cured strain of Aeromonas hydrophila elicits protective immunity in Clarias batrachus L

Wild type Aeromonas hydrophila (Strain AO1) isolated from the lesions of ulcerative disease syndrome (UDS) affected fish bears a 21 kb virulence plasmid. With plasmid curing the isolates became attenuated and failed to induce fatal haemorrhagic ulcers in fish. The objective of the present work was to check the immunogenicity of these plasmid-cured derivatives and determine whether such strains could be used as candidate antigens for eliciting protective immunity to A. hydrophila infections in the Indian catfish Clarias batrachus L. It was observed that the plasmid-cured strains were immunogenic since infection with live plasmid-cured AO1 isolates generated effective T cell responses and led to increase in serum antibacterial agglutinin titres in C. batrachus. Plasmid-cured AO1 strains injected into C. batrachus could disseminate into head kidney (HK) and spleen but never attained the same bacterial loads obtained with wild type AO1 and were cleared rapidly from the host. Immunisation with plasmid-cured bacteria prevented systemic spread and conferred protection against lethal challenge (10 x LD(50)) with wild type A. hydrophila as well as other pathogenic strains of Aeromonas sp. These results demonstrate the potentials of plasmid-cured A. hydrophila derivatives as candidate antigens for eliciting protective immunity in fish and the possibility of using such isolates as shuttle vectors in aquaculture.     

Amonabactin, a novel tryptophan- or phenylalanine-containing phenolate siderophore in Aeromonas hydrophila.

Aeromonas hydrophila 495A2 excreted two forms of amonabactin, a new phenolate siderophore composed of 2,3-dihydroxybenzoic acid, lysine, glycine, and either tryptophan (amonabactin T) or phenylalanine (amonabactin P). Supplementing cultures with L-tryptophan (0.3 mM) caused exclusive synthesis of amonabactin T, whereas supplements of L-phenylalanine (0.3 to 30 mM) gave predominant production of amonabactin P. The two forms of amonabactin were separately purified by a combination of production and polyamide column chromatographic methods. Both forms were biologically active, stimulating growth in iron-deficient medium of an amonabactin-negative mutant. Of 43 additional siderophore-producing isolates of the Aeromonas species that were tested, 76% (19 of 25) of the A. hydrophila isolates were amonabactin positive, whereas only 19% (3 of 16) of the A. sobria isolates and all (3 of 3) of the A. caviae isolates produced amonabactin, suggesting a predominant synthesis of amonabactin in certain Aeromonas species.     

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.     

Occurrence of conjugated R-plasmids in bacteria of the Aeromonas genus isolated from purified urban sewage water]

The aim of the study was to determine a participation of Aeromonas sp., having conjugation R plasmids, in a population of the bacteria present in purified urban sewage . In 1 ml of sewage 1.8 x 10(3) - 50 x 10(3) of Aeromonas sp., were found. The isolated strains belonged to the following genera: A. hydrophila, A. caviae, A. sorbia. All tested strains were sensitive to gentamicin, tetracycline, kanamycin and nalidixic acid. Among 186 strains of Aeromonas sp., two belonging to A. caviae genus transferred resistance to streptomycin to A. hydrophila and E. coli recipients on the other hand two strains of A. caviae transferred resistance to streptomycin exclusively to A. hydrophila recipient.