Mesophilic Aeromonas Species Research Articles

Reassessment of the Enteropathogenicity of Mesophilic Aeromonas Species.

Cases of Aeromonas diarrhea have been described all over the world. The genus Aeromonas includes ca. 30 species, of which 10 have been isolated in association with gastroenteritis. The dominating species that account for ca. 96% of the identified strains are Aeromonas caviae, A. veronii, A. dhakensis, and A. hydrophila. However, the role of Aeromonas as a true enteropathogen has been questioned on the basis of the lack of outbreaks, the non-fulfillment of Koch’s postulates and the low numbers of acute illnesses in the only existing human challenge study. In the present study we reassess the enteropathogenicity of Aeromonas using dose response models for microbial infection and acute illness. The analysis uses the data from the human challenge study and additional data from selected outbreak investigations where the numbers exposed and the dose were reported, allowing their inclusion as “natural experiments”. In the challenge study several cases of asymptomatic shedding were found (26.3%, 15/57), however, only 3.5% (2/57) of those challenged with Aeromonas developed acute enteric symptoms (i.e., diarrhea). The “natural experiments” showed a much higher risk of illness associated with exposure to Aeromonas, even at moderate to low doses. The median dose required for 1% illness risk, was ~1.4 × 104 times higher in the challenge study (1.24 × 104 cfu) compared to natural exposure events (0.9 cfu). The dose response assessment presented in this study shows that the combined challenge and outbreak data are consistent with high infectivity of Aeromonas, and a wide range of susceptibility to acute enteric illness. To illustrate the outcomes, we simulate the risk associated with concentrations of Aeromonas found in different water and food matrices, indicating the disease burden potentially associated with these bacteria. In conclusion this study showed that Aeromonas is highly infectious, and that human susceptibility to illness may be high, similar to undisputed enteropathogens like Campylobacter or Salmonella.

Multiresistant waterborne pathogens isolated from water reservoirs and cooling systems

To determine the incidence of multiple antibiotic-resistant strains of the emergent human pathogens Legionella pneumophila, Pseudomonas aeruginosa and mesophilic Aeromonas species among those isolated from water reservoirs and industrial cooling systems. Water from four natural water reservoirs and four industrial cooling towers was sampled for 1 year period. The total heterotrophs, mesophilic Aeromonas, Pseudomonas spp. and Legionella spp. counts were performed as recommended by standard procedures, and the sensitivity of the isolates to 27 antibiotics was tested. A total of 117 Aeromonas, 60 P. aeruginosa and 15 L. pneumophila strains were isolated and identified by means of biochemical tests and DNA probes. 46.4% of Aeromonas, and 100% of P. aeruginosa isolates presented multiple resistance. Legionella pneumophila strains were generally sensitive to the drugs used. Antibiotic-resistant pathogenic bacteria belonging to P. aeruginosa and mesophilic Aeromonas species are common in natural aquatic environments. Thus, the 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. These data confirm the emergence of bacteria resistant to antibiotics in aquatic environments.

Aeromonas bivalvium sp. nov., isolated from bivalve molluscs

A polyphasic study was performed to determine the taxonomic position of two Aeromonas strains, 665N and 868E(T), isolated from bivalve molluscs, that could not be identified at the species level in a previous numerical taxonomy study. The DNA G+C content of these isolates was 62.3 and 62.6 mol%, respectively. Sequence analysis of the 16S rRNA gene showed that the two new strains were closely related to members of the genus Aeromonas. Fluorescence amplified fragment length polymorphism fingerprinting revealed that strains 665N and 868E(T) clustered together with a similarity of 78 % but did not cluster with any of the Aeromonas genomospecies. DNA-DNA hybridization experiments revealed a high level of relatedness between the two new isolates (76 %) but low levels of relatedness between these and phylogenetically most closely related Aeromonas genomospecies (30-44 %). Useful tests for the phenotypic differentiation of strains 665N and 868E(T) from other mesophilic Aeromonas species included those for gas from glucose, lysine decarboxylase, Voges-Proskauer reaction, acid from L-arabinose, hydrolysis of aesculin and utilization of L-lactate. On the basis of genotypic and phenotypic evidence, strains 665N and 868E(T) are considered to represent a novel species of the genus Aeromonas, for which the name Aeromonas bivalvium sp. nov. is proposed. The type strain is 868E(T) (=CECT 7113(T)=LMG 23376(T)).

Comparative study of cytotoxicity of Aeromonas spp. on four different cell lines

In vitro cytotoxicity is an important virulence property of motile mesophilic Aeromonas species. Cell-free supernatant prepared from 55 Aeromonas isolates including one A. hydrophila type strain (MTCC 646) were examined for their cytotoxic potential on four different cell lines (Vero, BHK-21, MDBK, B 95a). Results of the study revealed cytotoxic potential in 92.72% of the isolates. Analysis of data exposed significant variation among isolates in respect of their cytotoxicity. Vero cells proved to be most sensitive to aeromonal toxins and B 95a cells showed significantly (P<0.01) lower response compared to other cell lines. Sensitivities of BHK-21 and MDBK cell lines were in between Vero and B 95a.

Aeromonas simiae sp. nov., isolated from monkey faeces.

Two Aeromonas strains, IBS S6874(T) and IBS S6652, were isolated from the faeces of two healthy monkeys (Macaca fascicularis) from Mauritius that were kept in quarantine in the Centre for Primatology, Strasbourg, France. Phylogenetic analysis based on 16S rRNA gene sequences showed that the two isolates formed an unknown genetic lineage within the genus Aeromonas. The two isolates had nearly identical sequences (0.1 % nucleotide substitution) that were related closely to those of recognized Aeromonas species (1.7-3.5 % nucleotide substitution). DNA-DNA hybridization showed that strains IBS S6874(T) and IBS S6652 had high DNA-DNA similarity (89 %) to each other and a low level of DNA-DNA similarity to closely related taxa (18 % relatedness to Aeromonas trota and 16 % relatedness to Aeromonas schubertii). Phenotypically, the two monkey isolates differed from most previously described mesophilic Aeromonas species by their lack of haemolysis on sheep-blood agar and inability to produce indole, gas from glucose or acid from mannitol. They differed from the most closely related species, A. schubertii, by their ability to produce acid from D-cellobiose and D-sucrose and by their pyrazinamidase activity. The name Aeromonas simiae sp. nov. is proposed for these isolates; strain IBS S6874(T) (=CIP 107798(T)=CCUG 47378(T)) is the type strain.

Aeromonas Causes Severe Skin Lesions in Rainbow Trout (Oncorhynchus mykiss): Clinical Pathology, Haematology, and Biochemistry

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Role of flm locus in mesophilic Aeromonas species adherence.

The adherence mechanism of Aeromonas caviae Sch3N to HEp-2 cells was initially investigated through four mini-Tn5 mutants that showed a 10-fold decrease in adherence. These mutants lost motility, flagella, and their lipopolysaccharide (LPS) O antigen (O-Ag). Three genes, flmB-neuA-flmD, were found to be interrupted by the transposon insertions; additionally, two other genes, one lying upstream (flmA) and one downstream (neuB), were found to be clustered in the same operon. While the flmA and flmB genes were present in all mesophilic Aeromonas spp. (A. hydrophila, A. caviae, A. veronii bv. veronii, and A. veronii bv. sobria) tested, this was not the case for the neuA-flmD-neuB genes. Construction and characterization of flmB insertion mutants in five other mesophilic Aeromonas strains revealed the loss of motility, flagella, and adherence but did not alter the LPS composition of these strains. Taking the above findings into consideration, we conclude (i) that flagella and possibly the LPS O-Ag are involved in the adherence of the mesophilic Aeromonas to human epithelial cells; (ii) flmA and flmB are genes widely distributed in the mesophilic Aeromonas and are involved in flagella assembly, and thus adherence; and (iii) in A. caviae Sch3N the flmA and flmB genes are found in a putative operon together with neuA, flmD, and neuB and are involved in LPS O-Ag biosynthesis and probably have a role in flagellum assembly.

Incidence and identification of mesophilic Aeromonas spp. from retail foods.

Sixty-eight food samples were examined for the presence of mesophilic Aeromonas species both qualitatively and quantitatively. Aeromonads were isolated from 26% of the vegetable samples, 70% of the meat and poultry samples and 72% of the fish and shrimps. Numbers of motile aeromonads present in the food samples varied from <10(2) cfu g(-1) to >10(5) cfu g(-1). GLC analysis of FAMEs was used to identify a selection of presumptive Aeromonas colonies to fenospecies or genomic species level. Aeromonas strains belonging to the Aer. caviae complex, which also includes the potentially pathogenic genospecies HG4, were mostly isolated from vegetables but were also found in meat, poultry and fish. In addition, three strains of the virulent taxon Aer. veronii biovar sobria HG8 were isolated from poultry and minced meat. All members of the Aer. hydrophila complex, predominant in the fish, meat and poultry samples, were classified in the non-virulent taxon HG3. Although the significance of Aeromonas in foods remains undefined, the isolation of Aeromonas HG4 and HG8 strains from a variety of retail foods may indicate that these products can act as possible vehicles for the dissemination of food-borne Aeromonas gastroenteritis.

Evaluation of the Synergistic Haemolysis (CAMP-like) test in the Identification of Motile, Mesophilic Aeromonas Species

Usefulness of the synergistic haemolysis (CAMP-like) test in the identification of Aeromonas hydrophila, Aeromonas sobria, and Aeromonas caviae was evaluated, using beta-toxin producing Staphylococcus aureus ATCC 25923 and alpha-toxin producing Clostridium perfringens ATCC 13124 as sphingomyelinase producing indicator strains. The tests were done on Columbia Blood Agar Base supplemented with 5% sheep blood (CBA; Difco Laboratories, Detroit, MI, USA). When cultured aerobically, 77% of A. hydrophila isolates, 75% of A. sobria isolates, and 23% of A. caviae isolates elicited synergistic haemolysis of sheep erythrocytes with the beta-toxin producing strain of S. aureus. Anaerobically, using the same indicator strain, synergistic haemolysis was given by 91%, 51% and 82% of A. hydrophila, A. sobria, and A. caviae, respectively. When alpha-toxin producing C. perfringens was used in the test instead of beta-toxin producing S. aureus, 100%, 86%, and 93% of A. hydrophila, A. sobria, and A. caviae isolates, respectively, showed synergistic haemolysis of sheep erythrocytes. Due to the high number of the tested isolates of A. hydrophila, A. sobria, and A. caviae giving a positive synergistic haemolysis reaction in both atmospheric conditions of culture, this test cannot be used to identify any of the three Aeromonas spp. The use of alpha-toxin producing C. perfringens did not improve discriminatory power of the test. Because the nature of the product of Aeromonas spp. responsible for the lytic phenomenon is not known, the use of Aeromonas factor and synergistic haemolysis terms was proposed rather than CAMP (reaction, test) or CAMP (-like) factor.

Pril-ampicillin-dextrin-ethanol agar for the isolation and quantification of Aeromonas spp. from polluted environmental waters

Several selective media were evaluated for their suitability for the isolation and quantification of mesophilic Aeromonas species from naturally polluted samples. Satisfactory recoveries were obtained with most of them but only when densities of background microflora were low. When analysed samples were from highly polluted waters, results were inconsistent because they did not give quantitative recovery of mesophilic aeromonads or they did not permit ready differentiation of Aeromonas species from the competitive bacteria. A new medium was developed on the basis of the combination of some positive aspects of several published media, pril‐ampicillin‐dextrin‐ethanol (PADE) agar. The medium employs dextrin (Merck 3006) as a fermentable carbohydrate and pril, ampicillin and ethanol as inhibitory substances. Recovery on PADE agar from suspensions of 15 tested strains of Aeromonas prepared from pure cultures was excellent. The confirmation rate of typical colonies designated Aeromonas spp. isolated from polluted samples exceeded 90%. Recoveries of stressed aeromonad strains on both PADE agar and a non‐selective medium (TSA) did not show any significant difference (P 0.05). PADE agar was more reliable for quantitative recovery of mesophilic aeromonads than the other selective media because of its characteristics: (i) inhibition of the swarming of Proteus, (ii) good reduction of the background, (iii) inhibition of the over growth of Klebsiella spp., (iv) absence of NaCl makes it unfavourable for the growth of halophilic vibrios, (v) combination of two pH indicators permitted a very easy differentiation between Aeromonas colonies and the competitive microflora. The medium can also be used for isolation of aeromonads from various sources by membrane filtration.

Pril-ampicillin-dextrin-ethanol agar for the isolation and quantification ofAeromonasspp. from polluted environmental waters

Several selective media were evaluated for their suitability for the isolation and quantification of mesophilic Aeromonas species from naturally polluted samples. Satisfactory recoveries were obtained with most of them but only when densities of background microflora were low. When analysed samples were from highly polluted waters, results were inconsistent because they did not give quantitative recovery of mesophilic aeromonads or they did not permit ready differentiation of Aeromonas species from the competitive bacteria. A new medium was developed on the basis of the combination of some positive aspects of several published media, pril-ampicillin-dextrin-ethanol (PADE) agar. The medium employs dextrin (Merck 3006) as a fermentable carbohydrate and pril, ampicillin and ethanol as inhibitory substances. Recovery on PADE agar from suspensions of 15 tested strains of Aeromonas prepared from pure cultures was excellent. The confirmation rate of typical colonies designated Aeromonas spp. isolated from polluted samples exceeded 90%. Recoveries of stressed aeromonad strains on both PADE agar and a non-selective medium (TSA) did not show any significant difference (P > 0.05). PADE agar was more reliable for quantitative recovery of mesophilic aeromonads than the other selective media because of its characteristics: (i) inhibition of the swarming of Proteus, (ii) good reduction of the background, (iii) inhibition of the over growth of Klebsiella spp., (iv) absence of NaCl makes it unfavourable for the growth of halophilic vibrios, (v) combination of two pH indicators permitted a very easy differentiation between Aeromonas colonies and the competitive microflora. The medium can also be used for isolation of aeromonads from various sources by membrane filtration.

Comparison of seven selective media for the isolation of mesophilic Aeromonas species in fish and meat

Seven selective agar media and two enrichment broths were evaluated for their suitability for the isolation of mesophilic Aeromonas species from meat, fish, and shellfish samples. In a first trial, aeromonads were inoculated in fish and meat samples and reisolated using all selected media. For qualitative isolation, enrichment in alkaline peptone water (pH 8.7 ± 0.1) at 28 °C and subsequent plating onto sheep blood agar supplemented with 30 mg L ampicillin (ASBA 30) and bile salts-irgasan-brilliant green agar (BIBG) at 35 °C led to the best results. For quantitative assays, direct plating on the same agar media is recommended. In a second trial, 829 meat, fish, and shellfish samples were investigated with the same methods. The results show that BIBG is the most selective medium and that presumptive identification of aeromonads on ASBA 30 is very easy. Finally, we could confirm the opinion of other workers that optimal recovery of mesophilic Aeromonas spp. requires the use of more than one agar medium.

Distribution of mesophilic Aeromonas species in raw and ready-to-eat fish and meat products in Switzerland

A total of 829 poultry, meat, shellfish and fish products commonly consumed in Switzerland were qualitatively and quantitatively examined for the presence of mesophilic Aeromonas spp. Overall, aeromonads occurred in 24.1% of the samples. Raw food products were frequently contaminated (e.g. 94.1% in minced meat), with colony counts up to 6.0·10 6/g. Some ready-to-eat products had a relatively high percentage of positive samples as well, such as cooked ham in slices (38.2%), mortadella (12.9%), smoked cooked sausage (15.6%), hot and cold smoked fish (10.9–14.3%) and gravad salmon (10.5%). Colony counts, however, were somewhat lower (up to 1.7·10 3/g). The high contamination rate of cooked or hot smoked foods suggests recontamination after cooking or smoking, e.g., at the slicing and packaging stage. 61.2% of the identified strains were Aeromonas hydrophila, followed by 22.5% Aeromonas caviae and 16.3% Aeromonas sobria.

Acquisition of iron from host sources by mesophilic Aeromonas species

The mesophilic Aeromonas species are opportunistic pathogens that produce either of the siderophores amonabactin or enterobactin. Acquisition of iron for growth from Fe-transferrin in serum was dependent on the siderophore amonabactin; 50 of 54 amonabactin-producing isolates grew in heat-inactivated serum, whereas none of 30 enterobactin-producing strains were able to grow. Most isolates (regardless of siderophore produced) used haem as a sole source of iron for growth; all of 33 isolates grew with either haematin or haemoglobin and 30 of these used haemoglobin when complexed to human haptoglobin. Mutants unable to synthesize a siderophore used iron from haem, suggesting that this capacity was unrelated to siderophore production. Some members of the mesophilic Aeromonas species have evolved both siderophore-dependent and -independent mechanisms for acquisition of iron from a host.

Extended serogrouping scheme for motile, mesophilic Aeromonas species

A total of 1,255 strains of motile, mesophilic Aeromonas species isolated from clinical and environmental specimens in the United Kingdom and 258 strains isolated in Australia, Brazil, Peru, and the United States were examined by using antisera for serogroups O1 to O44 (R. Sakazaki and T. Shimada, Jpn. J. Med. Sci. 37:247-255, 1984) and for unpublished serogroup O45 (R. Sakazaki). The typeability rate for strains isolated in the United Kingdom was 35%; the strains isolated in other countries had typeability rates of between 14 and 43%. A total of 52 provisional new serogroups were identified, and the strains with unidentified O groups were examined by using antisera for these provisional new serogroups. The typeability rate for strains isolated in the United Kingdom was increased to 66% (70% of smooth strains). The typeability rates were 76% for A. hydrophila and 63% for both A. caviae and A. sobria. The 52 antisera for the provisional new serogroups increased the typeability rate for strains isolated outside the United Kingdom to between 43 and 68%. This extended serogrouping scheme would be of value in determining the importance of Aeromonas strains as human intestinal pathogens and in investigating the pathogenic mechanisms that may be involved in the production of diarrheal disease.