- Most prominent representative: Aeromonas hydrophila
- Ubiquitous in aquatic environments
- A. hydrophila, A. caviae and A. veronii bv. Sobria are considered opportunistic human pathogens while A. salmonicida and A. bestiarum are considered fish pathogens
- Role of Aeromonas in causing human infection through contaminated drinking water is still being debated
- Easily disinfected by chlorine and chloramines, but occurs in drinking water through regrowth in biofilms
- A number of detection methods available
- Poor agreement between phenotypic and genotypic classification systems
Members of the genus Aeromonas
are autochthonous in aquatic environments. They are gram negative, oxidase positive, ampicillin resistant (an exception is A. trota
, Albert et al. 2000
), non-spore forming, facultative anaerobic rods ( Carnahan and Joseph 1991
; Percival et al. 2004
) with a length between 1.0-3.5 μm and diameter between 0.3-1 μm. Aeromonas
spp. often interfere with tests for coliforms ( Landre et al. 1998
). Many strains mimic coliforms in displaying ß-galactosidase activity. However, coliforms are oxidase negative ( Leclerc et al. 2001
). Like coliforms, Aeromonas
spp. are Gamma-proteobacteria but they are taxonomically distinct from the Enterobacteriaceae in which coliform genera have been placed.
Aeromonas speciation based on phenotypic characteristics is difficult. The main reason for this is that current Aeromonas taxonomy is based on the DNA relatedness of the species within the genus ( Popoff et al. 1981; Carnahan et al. 1991) and phenotypic identification of the genomic species has not been completely possible by these methods. There have been reports of mistyping Aeromonas isolates as Vibrio cholera ( Abbott et al. 1998). It has been useful to divide the genus into non-motile psychrophiles that are commonly fish pathogens (e.g., A. salmonicida) and motile mesophiles (especially A. hydrophila, A. caviae, and A. sobria (now classified as A. veronii bv. sobria)) that are considered opportunistic human pathogens and the causative agents in gastroenteritis and wound infections ( Janda and Abbott 1998; Gavriel et al. 1998). Aeromonas hydrophila was on the U.S. EPA's Contaminant Candidate List-2 (CCL) primarily because of its potential to grow in water distribution system biofilms ( Edberg et al. 2007). No point-source disease outbreaks have been attributed to Aeromonas spp. ( U.S. EPA Office of Water 2006). Bernagozzi et al. 1995 suggested that Aeromonas spp. might be indicators of nutrient loading in surface waters rather than fecal contamination per se. However, they are present in feces of healthy animals and humans ( U.S. EPA Office of Water 2006). Percival et al. 2004 stated that Aeromonas spp. may be normal fecal microflora and concluded that "it is possible that Aeromonas may not even be a true enteric pathogen". Messi et al. 2002 thought A. hydrophila should "not be considered a normal inhabitant of the human gastrointestinal tract". Edberg et al. 2007 considered Aeromonas hydrophila a "putatively emerging enteric pathogen" but stated that "the role of drinking water consumption in Aeromonas infections is unclear". While some evidence for waterborne transmission has been reported from drinking untreated water, only one case of human infection (a 3 month old infant suffering from Kwashiorkor) has been reported from drinking treated water (Moyer and Larew 1988). The infectious dose is high enough that waterborne transmission is unlikely except in persons with fragile gastrointestinal tracts, e.g. small children. However, two recent studies, where clinical and water isolates have been compared for their genetic profile, suggest clearly a waterborne infection route for Aeromonas. In one study conducted in Leon, Spain, one A. caviae diarrheal isolate had the same genetic pattern as two drinking water isolates ( Pablos et al. 2010). In the other study which included isolates mostly from the US, it was noted that most of the strains belonged to three groups: A. hydrophila, A. caviae-A. media and A. veronii-A. sobria. Genetic characterization demonstrated identical profiles between diarrheal and water isolates for some of the A. caviae-A. media strains, providing evidence not only for a water-to- human transmission route for these strains, but also that they were capable of colonization and infection ( Khajanchi et al. 2010). Other circumstantial evidence for Aeromonas spp. as causative agents in diarrheal infections include the observations that counts are higher in the feces of symptomatic versus asymptomatic individuals, that Aeromonas spp. have been isolated in the absence of other identified pathogens, that Aeromonas enterotoxins have been identified in symptomatic patients, and that treatment with antiobiotics against Aeromonas spp. has been shown to improve symptoms ( Edberg et al. 2007). Several studies have identified toxins or other virulence factors in Aeromonas strains ( Albert et al. 2000). The virulence factors include hemolysins, cytotoxins, enterotoxins, proteases, elastase, lipases, DNases, and adhesins (type IV pili, lateral and polar flagella) (Agarwal 1998; Cascon et al. 2000; Rabaan et al. 2001; Sen and Lye 2007). Chopra and Houston 1999 provided a detailed review of Aeromonas toxins. Additional virulence factors and regulatory genes have been reported recently, and they include enolase, glucose-inhibited divison A (gidA), virulence-associated protein B (vacB), DNA adenine methyltransferase (dam), T3SS and T6SS effectors, and ToxR regulated lipprotein (tagA ) ( Khajanchi et al. 2010). It is likely that pathogenicity of Aeromonas strains is multifactorial, but the presence of a particular array of virulence genes probably distinguishes pathogenic from non-pathogenic strains ( Percival et al. 2004; von Graevenitz 2007). Several of the virulence factors have been identified in strains isolated from water ( Kuhn et al. 1997a; Handfield et al. 1996; Fernandez et al. 2000; Sen and Rodgers 2004; Pablos et al. 2009; Bhowmik et al. 2009). A high degree of genetic diversity within the species A. hydrophila, based on the distribution of virulence factors in environmental and clinical isolates, had been reported earlier by Aguilera-Arreola et al. 2005.
A U.S. EPA-approved protocol (Method 1605) for culturing Aeromonas spp. from finished water involves membrane filtration and plating on ampicillin dextrin agar containing vancomycin (ADA-V). Presumptive Aeromonas spp. are colonies that ferment dextrin with acid production (yellow colonies). Colonies are confirmed with assays including one for oxidase (U.S.EPA 2001). Handfield et al. 1996 found ampicillin dextrin agar superior to nine other media in the recovery of Aeromonas spp. from drinking water. Another common medium for recovering Aeromonas spp. from water is Aeromonas agar ( Bernagozzi et al. 1994). Culturing of Aeromonas spp. is still the primary means of detection. To date, molecular methods are mostly used for typing isolates.
spp. are ubiquitous in aquatic environments, which is evidence of their adaptability to a range of conditions (Vanderkooij 1991; Messi et al. 2002
). Percival et al. 2004
and Theron and Cloete 2002
summarized data on the occurrence of Aeromonas
spp. in different waters. Reported densities were in the range of 106
CFU/mL for raw sewage, 103
CFU/mL for sewage effluents, 102
CFU/mL for river water, and 1-100 CFU/mL for groundwater ( Theron and Cloete 2002
Selected publications on prevalence and abundance in water are summarized in the following:
- The occurrence of Aeromonas spp. in a municipal drinking water system was monitored for 1 year in Leon, Spain ( Pablos et al. 2009). While coliforms were found only in 3.8 % of samples (n=132), Aeromonas was found in 26 %. Aeromonas was recovered in the winter months when the temperature was below 14°C, and the residual chlorine was between 0.21-0.72 mg/L. A direct correlation was seen between rainfall and occurrence of Aeromonas. Selected isolates were identified either as A. caviae or A. media (n= 35). The aerA gene was found only in 6 isolates. laf and alt genes were found in all isolates.
- During 2000-2001 the U.S. EPA examined 16 drinking water utilities in four states in the U.S. and determined the frequency of occurrence of Aeromonas in drinking water. Altogether 205 isolates were obtained, that encompassed the species A. hydrophila, A. salmonicida, A. bestiarum, A. caviae, A. enchelia and A. veronii bv. sobria, HG11 ( Sen 2005) . Sen and Rodgers 2004 used PCR to determine the distribution of six virulence genes in these isolates. There was a variety of combinations of the genes among different strains of the same species. In agreement with results from Kuhn et al., they found that although multiple Aeromonas strains were isolated from a single source, one strain type was dominant ( Kuhn et al. 1997a).
- Aeromonas spp. were isolated from ~50 untreated well water samples (n ~1000) in Libya. Isolation frequency in this warm climate was higher in winter and lower in summer. The species distribution of 382 strains was 59 % A. hydrophila, 27 % A. caviae, and 11 % A. sobria. About 50 % of 171 strains were hemolytic against human erythrocytes ( Ghenghesh et al. 2001).
- Based on epidemiological evidence of significant Aeromonas-related gastrointestinal infections in a region of Scotland, Gavriel et al. 1998 investigated the incidence of Aeromonas spp. in a major public distribution system in the area. Culturable counts and chlorine residual data for 31 reservoirs were obtained weekly for one year. No Aeromonas spp. were isolated from the plant itself and a central reservoir. However, 21 of the 31 downstream reservoirs were positive for Aeromonas spp. on at least one occasion (maximum value ~600 CFU/300 mL). Although there was an inverse relationship between chlorine residual and culturable counts, there were cases of Aeromonas spp. recovery in the presence of a chlorine residual and failure to recover Aeromonas spp. in the absence of a chlorine residual. The authors concluded that maintenance of a chlorine residual may not be sufficient to inactivate Aeromonas spp.
- Havelaar et al. 1990 tracked Aeromonas spp. counts in source, treated, and distribution water at 20 treatment plants in the Netherlands. River water counts were in the range of 105 CFU/100 mL. Treatment reduced counts to <10 CFU/100 mL, but regrowth was observed in 16 of 20 water distribution systems (maximum ~3 x 103 CFU/mL).
- Knochel and Jeppsen 1990 cultured and typed Aeromonas spp. from distribution systems in Copenhagen, Denmark. Total chlorine residuals were typically <0.01 mg/L and temperatures were 8 - 16°C. Presumptive colonies were typed, and A. hydrophila isolates were tested for hemolysin activity. Approximately 30 % of drinking water samples (n=130) were positive for Aeromonas spp. (1-40 CFU/100 mL). Of 30 random isolates, 29 were A. hydrophila. Hemolysin activity was found in four A. hydrophila isolates assayed (n=11).
- Burke et al. detected Aeromonas spp. in an Australian metropolitan water supply that met all international drinking water standards, including absence of detectable E. coli. The authors suggested that the presence of Aeromonas spp. in drinking water merited evaluation as a potential health risk. A. hydrophila was cultured in ~20 % of chlorinated tap water samples (n=286), and in ~70 % of samples in which E. coli was also cultured ( Millership and Chattopadhyay 1985).
- Studies in the Netherlands have shown that densities of Aeromonas spp. in distribution systems can increase with residence time and with depletion of chlorine. This behavior is reflected in drinking water standards that specify a limit of 20 CFU/100 mL at the plant and 200 CFU/mL in distribution systems ( Vanderkooij 1988).
- Kuhn et al. found less diversity among Aeromonas isolates in distribution systems than in the associated source waters, suggesting preferential inactivation of some strains during treatment ( Kuhn et al. 1997a). Aeromonas counts were > 100 CFU/mL in 15 % of treated water samples (n=48) and >10 CFU/100 mL in 48 % of distribution samples (n=122).
- Kuhn et al. found evidence of both resident and transient Aeromonas strains during a four-year study of Swedish well water (not disinfected). A. hydrophila was cultured in 28 of 40 samples, was a dominant member of the culturable microflora in ~50 % of the samples, and accounted for 39 % of all isolates typed ( Kuhn et al. 1997b).
- A. hydrophila strains were isolated from 16 source waters in Kolkata India, where diarrheal diseases are endemic. Of the 21 environmental strains isolated from these waters that were regularly used for domestic purposes, 81% showed cytotoxicity, 71 % produced hemolysin, 90 % demonstrated human serum resistance and all were multiple drug resistant. Some of the isolates were able to induce fluid accumulation (enterotoxic) and colonize mouse intestines ( Bhowmik et al. 2009).
- Aeromonas spp. were cultured from 73 of 77 samples of Norwegian source waters (35-100 CFU/100 mL). Higher counts were related to fecal contamination. A PCR assay detected the aer gene (a marker for aerolysin) in ~80 % of the isolates (n=445) ( Ormen and Ostensvik 2001).
- Aeromonas spp. were cultured from ~30 % of environmental samples (n=2120) in Bangladesh, including surface waters, sediments, and aquatic plants. Colony blots from isolates were hybridized with probes for three toxin genes (act, alt, ast). The assortment of these genes among isolates of different species was very diverse. Only two A. hydrophila isolates (n=18) were positive for all three genes. Two of the genes, alt and ast, were considered reliable markers for strains responsible for diarrheal infections in children ( Albert et al. 2000).
- Pettibone found correlations between Aeromonas spp., fecal indicators, and total suspended solids in summer but not winter in the Buffalo River (NY) and some of its tributaries ( Pettibone 1998).
- Waters that have not been impacted by human contamination but which may have been exposed to naturally-occurring Aeromonas spp. are often distributed and consumed without disinfection. For example, non-disinfected mountain waters of Italy were not free of Aeromonas spp. ( Legnani et al. 1998). Aeromonas spp. were cultured from 40 % of surface waters, 25 % of spring waters, 29 % of well waters, and 22 % of distribution systems. As might be expected, occurrence of Aeromonas spp. was not correlated with fecal indicators in this study.
- Bernagozzi et al. 1994 cultured Aeromonas spp. and A. hydrophila from all samples from two rivers (n=21), two estuaries (n=16), and one reservoir supplying a drinking water treatment facility (n=8) in Italy, and from Adriatic seawater (n=17). Mean A.hydrophila counts (CFU/100 mL) were approximately 2.7 x 104, 2.6 x 104, 88, and 5.5 x 102 in river, estuarian, reservoir, and ocean water samples, respectively. Mean counts of Aeromonas spp. and A. hydrophila were consistently higher than (but not correlated with) fecal coliforms.
- Araujo et al. 1991 reported the distribution of 883 Aeromonas isolates from 10 surface water sites in Spain as 55 % A. caviae, 34 % A. hydrophila, 6 % A. sobria, and 5 % untyped. A. caviae and A. sobria were associated with higher and lower levels of pollution, respectively.
- Mesophilic Aeromonas spp., primarily A. hydrophila, A. caviae, and A. sobria were found in all seasons in high densities in all environments sampled in some freshwaters of Japan. Seasonal variations were insignificant and there were no correlations with fecal indicators. Prevalence decreased with increasing salinity ( Chowdhury et al. 1990).
Drinking water treatment strategies such as rapid or slow sand filtration, direct filtration and use of granular activated carbon (GAC) and hyperchlorination are effective in reducing Aeromonads to low levels.
Examples of studies investigating susceptibility to disinfection are summarized in the following:
- Meheus and Peeters 1989 reported mean reductions for the different stages of treatment as 30-60 % for flocculation/sedimentation, 70-90 % for rapid sand filtration, 80-90 % for filtration through GAC and 98-100 % for slow sand filtration. Hyperchlorination combined with filtration was successful in removing 99-100 % of Aeromonads.
- Chlorine levels that are maintained in distribution systems are enough to inactivate free Aeromonas cells. The response of environmental isolates of Aeromonas to disinfection by chlorine at 0.5 mg/L is similar to E. coli although there are strain differences. Thus some strains survived treatment at 0.3 mg/L ( Cattabani 1986; U.S. EPA Office of Water 2006) and others at 0.45 and 0.5 mg/L (Gavriel et al.1998; Massa et al. 1999). While strains from untreated (environmental) water were inactivated within one minute, those isolated from chlorinated waters were moderately resistant.
- The effect of chlorine is pH dependent and the rate of inactivation was greater at pH 6 than at pH 8 ( Massa et al. 1999).
- Failure to maintain a chlorine residual, however, can result in regrowth of Aeromonads ( Burke et al. 1984; van der Kooij 1988). The growth of Aeromonas spp. in chlorinated water is related to water temperature and content of free chlorine and the interaction between the two ( Burke et al. 1984). Available nutrients, contributing to the organic content of the water, also player a role in regrowth.
- A. hydrophila can readily establish a biofilm along with other heterotrophic bacteria. Mackerness et al. 1991 showed that Aeromonas in biofilms survived 0.6 ml/L of monochloramine, concentrations that were enough to kill E. coli-associated biofilms. Holmes et al. (1996) reported that even after disinfection with 1 mg/L of chlorine, Aeromonads were still detected in 10 % of the pipe lengths.
spp. tolerate a wide range of pH (5 -10) and can utilize a variety of organic substrates (amino acids, carbohydrates, peptides, caroxylic acids, etc.), which may account for their ability to persist in distribution system biofilms ( Percival et al. 2004
). Vanderkooij 1991 attributed their multiplication in distribution systems to their ability to use biofilm biomass substrates, since they compete poorly with autochthonous bacteria for the low levels of dissolved organic carbon (DOC) in the water (usually <10 µg C/L).
A selection of relevant publications is summarized below:
- When a test strain of A. hydrophila was seeded into drinking water in which indigenous bacteria had been heat inactivated, maximum attainable densities were <5 x 104 CFU/mL, which indicated that available substrate was < 10 µg C/L (Vanderkooij 1991). There is also evidence of tolerance to refrigeration, which has implications for both food and drink products (Edberg et al. 2007).
- Massa et al. 1999 found that two A. hydrophila strains isolated untreated water were immediately inactivated by chlorine, whereas two from tap water exhibited some chlorine resistance. However, the small sample size does not permit generalization. Strains of A. hydrophila, A. caviae, and A. sobria were more chlorine resistant than other Aeromonas strains but still less resistant than E. coli in general.
- Bomo et al. 2004 documented survival of Aeromonas spp. in simulated distribution system biofilms. Various model biofilm devices representing different surface materials were naturally inoculated from potable or recycled water. Aeromonas spp. were isolated from the recycled water systems only, and were not typed as belonging to species that present a health risk. When an existing biofilm was challenged with a slug dose of an ATTC strain of A. hydrophila, the organism was able to establish itself in the biofilm at an initial density of 103 -104 cells/cm2 and to persist at some lower level indefinitely. However, other Aeromonas spp. isolated during the study were not capable of establishing themselves in biofilms in the same way.
- Chauret et al. 2001 cultured A. hydrophila from ~8 % of distribution system biofilm samples (n= 26) but failed to culture cells from bulk water samples, which the authors attributed to resistance of biofilm A. hydrophila to disinfection.
- An A. hydrophila strain seeded into filtered-autoclaved water survived without decline for 10 days. However, in unfiltered water, the A. hydrophila counts decreased rapidly, presumably due to interactions with other microflora ( Kersters et al. 1996). A six-log reduction in culturable A. hydrophila in tap water (106 CFU/mL inoculum) was observed during a 9-hour incubation at pH 7 and 9 ( Silvestry-Rodriguez et al. 2007).
- Bottled uncarbonated mineral waters may be considered safer than tap water by many consumers and their market is increasing. They are generally marketed without any treatment. Aeromonas spp. may be a minor constituent of the microflora at the source but may grow heterotrophically during storage ( Croci et al. 2001). In one study, naturally occurring A. hydrophila proliferated in bottled mineral waters from different sources and varying in mineral content. Typical initial counts of ~101-102 CFU/100mL were measured within 48 h of bottling. Counts generally reached 102-103 CFU/mL by 14-21 days and remained at those levels before declining to near initial levels around day 55 ( Croci et al. 2001).
- Messi et al. 2002 detected survival of a strain of A. hydrophila for 150 days after it was inoculated into filtered and autoclaved uncarbonated mineral water at 106 CFU/mL. When it was co-inoculated with an E. coli strain, its survival was shortened by 30 days. When it was co-inoculated with Pseudomonas fluorescens and Pseudomonas putida, its survival actually increased by 30 and 60 days, respectively. These results suggested that survival may depend on both antagonistic and commensal relationships with other aquatic organisms.
- A. hydrophila seeded into five brands of mineral waters at 104 CFU/mL was cultured up to 180 days later in 3 of 20 samples ( Korzeniewaska et al. 2005).
- Brandi et al. 1999 seeded isolates of A. hydrophila, A. caviae, and A. sobria into tap water, bottled mineral water, and sea water, and measured survival over time. A. hydrophila and A. caviae counts actually increased about 10 % initially, suggesting they were able to grow on substrates present in the water. The time required for a one log reduction in culturable counts was similar for mineral and tap water samples (from ~10-22 days), compared to ~3 days for the sea water samples. There were no great differences in survival among the different species. Starting with an initial dose of ~106 CFU/mL, viable cells were still cultured for at least 100 days for all three species.
- Motile Aeromonas spp. were isolated and typed in Japanese freshwaters and their sediments. All 132 water samples and ~60 % of the sediment samples (n=514) were Aeromonas positive. Culturable counts were ~103 CFU/L and 106 CFU/g in water and sediments, respectively. Approximately 70 % of the isolates were typed as A. hydrophila, A. caviae, or A. sobria ( Fukuyama et al. 1989).
Data on infectious dose of Aeromonas
spp. are extremely limited. In the most detailed study we are aware of, five A. hydrophila
strains isolated from humans and found to possess various cytotoxin, hemolysin, and enterotoxin activities were administered to healthy adults. Challenge doses ranged from ~104
CFU, depending on the strain. Volunteers were monitored for symptoms of diarrhea. Only two volunteers (n=57) developed diarrhea, and from different strains ( Morgan et al. 1985
). In one volunteer, the infective dose was ~109
CFU, but seven other volunteers receiving 109
CFU were free of symptoms. In the other volunteer, the infective dose was ~107
CFU, but 15 other volunteers receiving from 107
CFU did not experience diarrhea. The authors concluded that the Aeromonas
virulence properties known at the time did not explain virulence in humans. The European Union has established a drinking water standard of no more than 20 CFU/100 mL for Aeromonas
spp. in water leaving the treatment plant and of no more than 200 CFU/100 mL in distribution system water.
It is generally easy to identify Aeromonas
spp. at the genus level in primary cultures as described under "General information". Presumptive colonies on plates are further confirmed by an oxidase test on media without carbohydrates, by indole production from tryptophan or the ability to ferment trehalose. Other biochemical tests include reduction of nitrate to nitrite, sensitivity to ampicillin, NaCl, ONPG, and resistance to vibriostatic reagent 0/129, their main purpose being able to distinguish Aeromonads from Pleisomonads or Vibrio
spp. ( Millership 1996
Identification to species level by phenotypic methods is more complicated. This is because classification of Aeromonas species or placement in Hybridization Groups is based on DNA-DNA hybridization. Thus, there is often poor agreement between phenotypic and genotypic classification systems ( Ormen et al. 2005). Phenotypic methods for identification of Aeromonas species include biotyping, which comprises specific biochemical reactions such as esculin hydrolysis, gas production from sucrose, and the Voges Proskauer test, among others ( Abbott et al. 2003), phage typing, where as many as 25 different phages have been used to compare strains, serotyping, protein analysis, resistotyping (resistance to various antibiotics), multilocus enzyme analysis, and cellular fatty acid composition. Details of these phenotyping procedures and their ability to identify genomospecies correctly have been described by Altwegg 1994.
Among the various rapid test systems that have been commercially developed the API systems (BioMerieux, France), such as the API 20 NE system, API 20 E or ID 32 GN, along with some additional biochemical tests, are most commonly used for speciation. A comparison of the API kits to identify Aeromonas species has been performed by Awan et al. 2005.
Most of the molecular methods developed for Aeromonas spp. have been used for genotyping and characterizing isolates, rather than for direct detection from environmental samples. Genotyping has been used to identify the presence of potentially pathogenic isolates.
Selected non-cultivation based detection methods are summarized in the following:
The classical method of identifying the different species of Aeromonas is by Hybridization Groups (HG), which is based on DNA-DNA hybridization analysis and determines relatedness or homology of a species within the genus ( Carnahan and Joseph 1991). A laborious method, the process includes isolation of genomic DNA of an isolate, labeling the DNA and subjecting it to hybridization to DNA from reference and type strains at stringent and optimal reassociation temperatures, and finally determining the relative binding ratios and the divergence in the melting temperature ( Carnahan and Joseph 1991). Fourteen HG were originally identified, with 7 new species being added since 2002 (Janda and Abbott 2010). The 21 different species are: A. hydrophila, A. bestiarum, A. salmonicida, A. caviae, A. media, A. eucrenophila, A. sobria, A. veronii, (biovars sobria and veronii), A. jandaei, A. schubertii, A. trota, A. allosaccharophila, A. enchelia (HG11), A. popoffii, A. simiae, A. culicicola, A. molluscorum, A. bivalvium, A. aquarirorium, A. sharmana, A. tecta. HGs of special public health interest include HG1 (A. hydrophila), HG4 (A. caviae), and HG7 (A. sobria). Edberg et al. listed identifying phenotypic characteristics of the different HG.
Sequencing of the 16S rRNA gene has played an important role in the identification of the species and it demonstrated that the some of the species are extremely similar, such as A. caviae and A. trota (single nucleotide difference), A. hydrophila and A. media, A. hydrophila and A. enchelia, or A. salmonicida and A. bestiarum (Martinez-Murcia 1992; Sen K 2005). In general, relative to other bacteria, the 16S rRNA gene is extremely conserved among Aeromonas spp. (98-100 % similar) (Martinez-Murcia 1992). Sequencing of two other housekeeping genes, rpoD and gyrB from different Aeromonas species demonstrated that these genes can give better resolution among some species (Soler et al 2004). Thus gyrB showed better resolution between Aeromonas spp. HG 11- A. enchelia, A. veronii and A. culicicola/A. allosaccharophila compared to 16S rRNA gene, while rpoD differentiated clearly between A. salmonicida and A. bestiarum.
Pulse Field Gel Electrophoresis:
PFGE patterns produced by restriction digestion of chromosomal DNA of strains are compared to establish epidemiological links between them.
- Pablos et al. 2009 demonstrated that one A. caviae diarrheal isolate had the same PFGE pattern as two drinking water isolates obtained during the same study period, from the Leon municipal drinking water system. The three isolates carried the same virulence genes that they were tested for.
- Khajanchi et al. 2010 were able to demonstrate a water-to-human transmission of A. caviae - A.media species isolated from different regions in the U.S., by studying the PFGE pattern as well as their virulence factor gene profile. They obtained three sets of isolates (n=7), comprised of both diarrheal and environmental isolates, that had identical PFGE patterns and the same virulence gene profile, which demonstrated that these strains were able to colonize and infect the human gut.
- Borchardt et al. 2003 compared Aeromonas isolates from diarrhea patients with those obtained from their drinking water by PFGE, in Wisconsin. No genetic relationship was obtained between the two.
Useful for establishing an epidemiological link as well as a taxonomic tool, the method involves the 16S rRNA gene. Labeled fragments made from the 16S rRNA gene, or from the entire operon coding for 16S, 5S and 23S rRNA, of E. coli are hybridized to an Aeromonas isolate's genomic DNA that has been digested by a suitable restriction enzyme, to obtain a characteristic pattern for an Aeromonas strain or species ( Martinetti and Altwegg 1992).
- Moyer et al. 2010 used ribotyping to demonstrate that clinical strains of Aeromonas had a different pattern than ones recovered from a water distribution system, the associated treatment plant, and source water.
- Demarta et al. 2000 used ribotyping to study the epidemiology of Aeromonas associated gastro-enteritis in young children. Ribo-patterns of 104 strains isolated from the stools of sick children and their household environment were compared. Three strains isolated from patients had the same riboprofile as their environment. The pattern was also found in the asymptomatic family members of these children (2 of 11 cases) suggesting a relationship between predisposing factors of host and Aeromonas infection.
- This method has been useful in distinguishing strains of HG2 from HG3 which are not easy to distinguish by phenotypic methods. On the other hand patterns of A. jandaei (HG 9) and A. veronii (HG 8/10) are very similar and thus they cannot be distinguished ( Altwegg 1999).
Most of the PCR based analysis is for one or a few species with a couple that detect most of the species. One such is based on RFLP and is described later. Since A. hydrophila strains may be the most likely strains to cause disease in humans, several PCR and probe based molecular detection methods have been developed in the last two decades to detect and identify exclusively A. hydrophila.
- A PCR assay targeting the lip gene was specific for A. hydrophila from HG1. The PCR assay had a sensitivity of ~1 pg of isolated DNA for the gene target which is also also called hydrolipase ( Cascon et al. 1996).
- Baloda et al. 1995 developed a PCR method to screen A. hydrophila and A. sobria isolates for the aer gene (encodes the ß-subunit of aerolysin). PCR products (~210 bp) were amplified only from A. hydrophila strains with a hemolytic phenotype. Hemolytic A. sobria strains were not detected. These results essentially confirmed earlier research by Lior and Johnson 1991 in which aer PCR products were obtained from hemolytic, cytotoxic, and enterotoxic A. hydrophila strains, but not from hemolytic A. sobria.
- Dorsch et al. 1994 developed species specific probes for A. hydrophila and A. veronii, targeted to their 16S rRNA gene, and were able to distinguish these species from 67 environmental Aeromonas isolates obtained from sewage effluents and surface waters around Sydney.
- Ash et al. 1993 used PCR to amplify the 16S rRNA gene and used two highly species specific oligonucleotides to identify A. schubertii and A. jandaei.
- Several PCR based detection schemes use virulence factors to differentiate between the different species. Chu and Lu 2005 developed a multiplex PCR (mPCR) method that targeted a gene associated with virulence (aero1) and a region of the 16S rDNA that is species-specific. There was ~95% agreement between mPCR and an ELISA method when both were used to test 36 clinical isolates. The mPCR was primarily used to identify pathogenic isolates. There was no discussion of use of this method for environmental DNA, where the two target genes, if present, could arise from different strains.
- PCR primers were developed for a phylogenetic study of the gyrB gene within Aeromonas spp. ( Yanez et al. 2003). An ~1100 bp amplicon was generated from all 53 Aeromonas isolates studied. No non-Aeromonas were tested and there was no discussion of the application of these primers for environmental detection of Aeromonas spp.
- Both genera- and species-specific Aeromonas PCR primers were developed to target the 16S - 23S rDNA intergenic spacer region ( Kong et al. 1999).
- Sen created a multiplex PCR method for typing Aeromonas isolates. It targeted gyrB (two primer pairs), two lipase genes, an elastase gene, and 16S rDNA sequences (several primer pairs; Sen 2005). Because of the large number of primer sets, three separate multiplex reactions were required. The mPCR provided good species discrimination and correctly identified 59 of 63 isolates from water that were earlier identified by biochemical tests.
- Nam et al. developed a multiplex PCR for typing Aeromonas isolates from fish farm waters. It targeted six known genes for virulence factors (aer, ser, nuc, lip, laf, and gcat). Those Aeromonas strains most relevant to infectious disease could be identified by the assortment of target genes they contained.
- Borrell et al. 1997 developed a species identification scheme based on the RFLP analysis of the 16S rRNA gene of the different species. The 16S rRNA gene was amplified by PCR before being subjected to restriction analysis by the enzyme pairs AluI and MboI. When 76 previously characterized clinical isolates were compared with RFLP analysis, one strain identified as A. veronii by biochemical tests showed the RFLP pattern of A. hydrophila. Conversely, three strains of A. hydrophila showed RFLP patterns of A. veronii. However, the groups A. salmonicida - A. bestiarum and Aeromonas sp. HG 11- A. enchelia- A.popoffii cannot be distinguished by this analysis. Thus a second and third round of RFLP analysis is necessary, involving digestion with different sets of enzymes, to correctly distinguish between these groups ( Figueras et al. 2000).
- Kingombe et al. 1999 used the cytolytic enterotoxin (achytoen) from A. hydrophila and the homologus aerolysin and hemolysin genes from different Aeromonas spp. to develop PCR primers that were used in RFLP and/or PCR amplicon sequence analysis (PSA) to characterize the distribution of these genes in different environmental, food , clinical and reference strains (n=350).
- Trakhna et al. 2009 developed a duplex TaqMan PCR with primers and probes targeted to the 16S rRNA gene and the aerA enterotoxin gene to rapidly identify environmental A. hydrophila strains that were previously characterized by biochemical means. A. sobria and A. caviae also isolated from these samples were negative by this assay.
- Recently, a method was developed to detect Aeromonas spp. in the environment without culturing. An adhesin gene (ahaI) was used as a target in a quantitative TaqMan(r) PCR (qPCR) method to detect A. hydrophila in wastewater. The assay was quantitative for initial cell concentrations over a range of 100 fg-10 ng, with 100 fg being the limit of detection. This corresponded to ~ 22 copies of ahaI (assumes one copy per genome, 4.6 fg DNA per cell) ( Shannon et al. 2007).
Random amplification of polymorphic DNA (RAPD-PCR):
- Oakey et al. 1999 performed RAPD-PCR (uses a single random primer) to amplify an array of fragments from 31 A. hydrophila strains. Two fragments were found specific for all A. hydrophila strains tested and were used to develop probes and PCR primers.
- RAPD PCR in combination with Enterobacterial repetitive intergenic consensus (ERIC) PCR (described in the next bullet) was used to determine the genetic diversity of A. hydrophila strains obtained from clinical as well as environmental sources. Both methods performed well in discriminating between strains; a consistent dendogram was obtained when results from RAPD and ERIC were combined. As expected, the strains from any given ecological origin tended to cluster together (Aguilera-Arreola et al. 2005).
Amplified fragment length polymorphism (AFLP):
AFLP analyis has been shown to support in general the classification obtained by DNA HG groups ( Huys et al. 1996). Some of the differences were that A. allosaccharophila and A. enchelia did not represent separate AFLP cluster, but were genotypically related to HG8/10 and HG6 respectively. HG6 or A. eucrenophila strains demonstrated significant heterogeneity and thus may comprise more than one species.
BOX, ERIC, Repetitive extragenic palindromic PCR (REP PCR):
These finger printing methods take advantage of the presence of repetitive sequences, that are spread throughout the genome of bacterial species. They include the REP sequence, ERIC sequence and the 154 bp BOX element. They have been shown capable of discriminating Aeromonas spp. (Szczuka and Kaznowski 2004; Tacao et al. 2005). ERIC PCR had the same discriminatory power when compared with RAPD and there was good correlation between the methods when tested with 121 strains of Aeromonas, while REP PCR was less effective in distinguishing the different species.
Probe based methods:
- An outer membrane protein-based Dioxigenin (DIG) labeled DNA probe was developed for the detection of Aeromonas in environmental and clinical samples. The probe was specific to 40 different isolates from 6 different Aeromonas species that were obtained from fish, and diarrhea patients (Khusiramani et al. 2009), when used in colony blot hybridization assays.
- Khajanchi et al. 2010 developed DNA probes directed to 11 virulence factor genes act, alt, ast, ascV, aexU, gidA, enolase gene, dam, tagA, hlyA, and ahyRI to determine the virulence profile of 227 clinical and environmental Aeromonas isolates. They showed that the heat-labile cytotonic enterotoxin and the dam gene were more prevelant in clinical isolates than in water isolates.
- Aguilera-Arreola et al. 2005 developed and used DIG labeled probes to aerolysin-hemolysin, ast and lafA genes to determine the presence of these genes from 109 Aeromonas clinical isolates in dot blot assays.
- A 237 bp DIG labeled probe was developed to target the glycerophospholipid cholesterol acetyltranserase gene (gcat) that correctly distinguished Aeromonas spp. (n=16) from other enteropathogenic bacteria (n=20) in primary isolation media ( Chacon et al. 2002).
- Dorsch et al. 1994 developed species specific DNA probes directed to the 16S rRNA gene from A. hydrophila and A. veronii for examining environmental isolates.
Fluorescent in situ hybridization (FISH)
Borno et al. 2004 used FISH to examine Aeromonas spp. in biofilms and demonstrated that none of the isolates belonged to A. caviae, A. hydrophila, or A. veronii. An ATCC strain of A. hydrophila was, however, able to establish and persist in a pre-formed biofilm.
ELISA based methods that include the use of either polyclonal or monoclonal bodies have been described mainly for identification of A. hydrophila species.
- Merino et al. 1993 demonstrated an ELISA (enzyme-linked immunoabsorbent) method that identified virulent strains of A. hydrophila and A. sobria.
- Delamare et al. 2002 produced monoclonal antibody against A. hydrophila that were specific to this species (n=12) isolated from different matrices by recognizing a 110 kDa polypeptide fragment . The antibody showed only baseline activity against 10 other species of the genus tested in ELISA reactions.
- An immuno PCR has been described for detection of A. hydrophila. Aeromonas was captured by anti-A. hydrophila antibody coupled to a microplate, which was then amplified by bacterial universal primers targeted to the 16S rRNA gene ( Peng et al. 2002). The limit of quantification was 5 CFU by this universal primer PCR (UPPCR).
- An electrochemical DNA biosensor, that had a recognition layer consisting of thiolated single stranded DNA of the aerolysin gene, could identify A. hydrophila DNA present at a concentration of 2.5 μg/cm3 among other microbial DNA ( Tichoniuk et al. 2010).
- A DNA piezoelectric biosensor coupled with PCR was used to distinguish pathogenic Aeromonas from other bacteria present in water, vegetable or clinical samples ( Tombelli et al. 2000). The aer gene of Aeromonas was amplified by PCR and the PCR products were then detected by the biosensor which contained a biotinylated capture probe immobilized on the streptavidin coated surface of a quartz crystal.
The following pictures are part of the Public Health Image Library (PHIL) from the Centers for Disease Control and Prevention (CDC). The photos are in the public domain and thus free of copyright restrictions. We would like to express our appreciation for providing these images.
Aeromonas hydrophila. Gram stain
Photo ID: 1255
Content provider(s): Centers for Disease Control and Prevention/ Dr. W.A. Clark
Aeromonas hydrophila. Leifson flagella stain (digitally colorized).
Photo ID: 1041
Content provider(s): Centers for Disease Control and Prevention/ Dr. William A. Clark
Aeromonas shigelloides. Leifson flagella stain (digitally colorized).
Photo ID: 1040
Content provider(s): Centers for Disease Control and Prevention/ Dr. William A. Clark
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