Diversity and Virulence Profile of Aeromonas From Potable Water

Aeromonas spp. are autochthonous in the aquatic ecosystem and some of them has been increasingly found, in patients with various diseases like enteritis, wound infection and even septicemia in amphibians, reptiles, frog, fish and in patients with impaired immunity. There are different virulence factors like aerolysin, hemolysins, cytotoxins, enterotoxins, proteolytic activity, lipolytic activity, gelatinase, slime production, DNases, and adhesions. These virulence factors are used as survival means, self defense mechanism and establishment of pathogenicity. They are also capable of forming biofilm and are found multiresistant, although free cells of Aeromonas may be relatively susceptible to disinfection; populations associated with biofilms may survive high chlorine dosing. Thus water distribution systems should be monitored to check the quality of water and for the purpose of public health. BMR Microbiology www.bmrjournals.com 2 BMR Journals| bmrjournals.com Aeromonas human pathogens: Popoff, Kluyver and Niel (2009); Carnahan and Joseph (1991) suggested that 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. There have been reports by Abbott (1998) of mistyping Aeromonas isolates as Vibrio cholera. Janda and Abbott (1998); Gavriel (1998) reported that it has been useful to divide the genus into non-motile that are commonly fish pathogens (e.g., A. salmonicida) and motile mesophiles (especially A. hydrophila, A. caviae, and A. sobria, A. veronii) that are considered opportunistic human pathogens and the causative agents in gastroenteritis and wound infections. 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 (1995) suggested that Aeromonas spp. might be indicators of nutrient loading in surface waters rather than fecal contamination. However, they are present in feces of healthy animals and humans (U.S. EPA Office of Water 2006). Percival (2004) stated that Aeromonas spp. may be normal fecal micro flora and concluded that "it is possible that Aeromonas may not even be a true enteric pathogen". Messi (2002) thought A. hydrophila should "not be considered a normal inhabitant of the human gastrointestinal tract". Edberg (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 by Moyer and Larew (1996) 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. Pablos (2010) reported that the infectious dose is high enough that waterborne transmission is unlikely except in persons with fragile gastrointestinal tracts, e.g. small children. Virulence factors: Several studies by Albert (2000) have identified toxins or other virulence factors in Aeromonas strains. The virulence factors include hemolysins, cytotoxins, enterotoxins, proteases, elastase, lipases, DNases, and adhesins (type IV pili, lateral and polar flagella) (Agarwal et al. 1998; Cascon et al. 2000; Rabaan et al. 2001; Sen et al. 2007). Chopra and Houston (1991) 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), virulenceassociated 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). Aguilera-Arreola (2005) reported a high degree of genetic diversity within the species A. hydrophila, based on the distribution of virulence factors in environmental and clinical isolates. Virulence factors such as aerolysin, haemolysin, cytosine, enterotoxin, proteolytic activity, lipolytic activity, gelatinase, slime production and antimicrobial peptides have been identified in A. hydrophila. (Asmat and Gires, 2002; Castro Escarpulli et al., 2003; Martins et al., 2002; Illanchezian et al., 2010). These virulence factors are used as survival means, self defense mechanism and establishment of pathogenicity. These are mostly found in bacteria including Aeromonas spp. (Singh et al., 2010). Aeromonads have been attributed to human infections like gastroenteritis, septicemia and wound infections (Illanchezian et al., 2010). In 2004, Subashkumar and colleagues, stated protease, aerolysin, hemolysin, enterotoxins, lipases, gelatinase and biofilm formation as virulence factors in Aeromonas spp. Biofilm is an irreversible growth of aggregated bacterial microcolonies on surfaces embedded in extracellular polysaccharide matrix. Biofilm formation results into resistance of bacteria to conventional antibiotics and persistent infections (Rodney 2008). Anne and Elizabeth (2003) attributed increase of antibiotics resistance in Aeromonas spp., hence posing threat to human health and environment. In the study made by BMR Microbiology www.bmrjournals.com 3 BMR Journals| bmrjournals.com Chacon (2003), the detection of virulence factors of Aeromonas hydrophila is a key component in determining potential pathogenicity because these factors act multifunctionally and multifactorially. For rapid detection of two virulence factors of isolated Aeromonas hydrophila, a polymerase chain reaction assay was used. The detected virulence factors include aerolysin (aer A) and haemolysin (hyl H). Gonza ́lez (2001) reported that because of Aeromonas hydrophila’s structure, it is very toxic to many organisms. When it enters the body of its victim, it travels through the bloodstream to the first available organ. It produces Aerolysin Cytotoxic Enterotoxin (ACT), a toxin that can cause tissue damage. Aeromonas hydrophila, Aeromonas caviae, and Aeromonas sobria are all considered to be opportunistic pathogens, meaning they rarely infect healthy individuals. Neyts (2000) studied that Aeromonas hydrophila is widely considered a major fish and amphibian pathogen, and its pathogenicity in humans has been recognised for decades. It was proved by Yogananth (2009) that the pathogenicity of Aeromonas spp. is mediated by a number of extracellular proteins such as aerolysin, lipase, chitinase, amylase, gelatinase, hemolysins and enterotoxins. However the pathogenic mechanisms of Aeromonas spp. are unknown. The recently proposed type III secretion system (TTSS) has been linked to Aeromonas pathogenesis. The TTSS is specialized protein secretion machinery that exports virulence factors directly to host cells. Albert (2000) found that Aeromonads are causative agents of a number of human infections. Even though aeromonads have been isolated from patients suffering from diarrhea, their etiological role in gastroenteritis is unclear. In spite of a number of virulence factors produced by Aeromonas species, their association with diarrhea has not been clearly linked. Recently, he has characterized a heat-labile cytotonic enterotoxin (Alt), a heat-stable cytotonic enterotoxin (Ast), and a cytotoxic enterotoxin (Act) from a diarrheal isolate ofAeromonas hydrophila. Alt and Ast are novel enterotoxins which are not related to cholera toxin; Act is aerolysin related and has hemolytic, cytotoxic, and enterotoxic activities. For the first time, Aeromonas eucrenophila was isolated from two children, one with diarrhea and another without diarrhea. Prevalence in Drinking Water: Prevalence and abundance in Drinking Water, the occurrence of Aeromonas spp. in a municipal drinking water system was monitored for 1 year in Leon, Spain (Pablos et al. 2009). 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. About 50 % of 171 strains were hemolytic against human erythrocytes (Ghenghesh et al. 2001). Of the 21 environmental strains isolated from these waters that were regularly used for domestic purposes, 81% showed cytotoxicity, 71 % produced 27 hemolysin, 90 % demonstrated human serum resistance and all were multiple drug resistant. Some of the isolates were able to induce fluid accumulation (enterotoxic) (Bhowmik et al. 2009). A PCR assay detected the aer gene (a marker for aerolysin) in 80 % of the isolates (n=445) (Ormen et al 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). Antibiotic susceptibility of Aeromonas: Hazen (1978); Janda and Abbott (1996) reported the in vitro susceptibility of the isolates was studied by disk diffusion method using discs (Oxoid) containing the following antibiotics were used: penicillin G (10 U), tetracycline (30 μg), gentamycin (30 μg), azitromycin (15 μg), trimethoprim-sulfamethoxazole (25 μg), naladixic acid (30 μg) and norfloxacin (10 μg). All bacterial strains studied in the research showed high degree of resistance to antibiotics. Resistance rate to tetracycline and penicillin was more than other antibiotics. Multiple drug resistance was also observed in all Aeromonas hydrophila isolates. The development of resistant or even multidrug resistant pathogens in recent years has become a major problem in Iran and many countries. Antimicrobial resistance of Aeromonas has been studied by many authors BMR Microbiology www.bmrjournals.com 4 BMR Journals| bmrjournals.com (Thayumanavan et al., 2003). Some of the authors Poobalane (2008) indicated that A. hydrophila isolated from water, food and clinical samples was not susceptible to many antimicrobial agents. However, 30 antibiotics resistance was found in 91.6% of studied isolates and in all A. hydrophila strains. The results also show that resistance to penicillin and tetracycline was observed in 79 and 83% of the isolates. In addition, all A. hydrophila strains were multiresistant, what may be the result of the spread of resistance genes among the isolated bacteria. Castro-Escarpulli (2003) reported that the best antimicrobial effect on Aeromonas is obtained by applying the first-generation quinolone and the second and third generation cephalosprins. Although, Stojanov (2010) indicated that a high percentage of the Aeromonas strains were resistant to Flumequine (over 35%) and Olaquindox (around 20%), as a representative of Quinolone. Resistance profile of antibiotics observed in decreasing order of resistance of the isolates was tetracycline, penicillin, gentamycin, norfloxacin, azitromycin, trimethoprim-sulfamethoxazole and nalidixic acid. Biofilm formation and chlorine sensitivity: Various types of drinking-water demonstrated large differences in biofilm formation rates. Aeromonas in drinking-water in distribution systems has been controlled by increased disinfection, and it appears that free cells of Aeromonas are relatively susceptible to the common chlorine-based disinfectants. Knochel (1991) found that strains of A. hydrophila, A. sobria, A. caviae, and A. veronii were generally more susceptible to chlorine and monochloramine, and Edge et al, 1987 found that laboratory-grown and environmental Aeromonas were also susceptible to chlorine dioxide. Despite this relative susceptibility to chlorine-based disinfectants, controlling the numbers of aeromonads in a distribution system may require some considerable time and chlorine concentrations in excess of 0.2 mg/l (Edge et al, 1987). This is probably due to association of the organisms with biofilms. Langsrud (2003) proved that there was evidence that the biofilm-associated A. hydrophila would also survive 0.6 mg/l monochloramine, which was sufficient to eradicate biofilm-associated E. coli. These data indicate that, although free cells of Aeromonas may be relatively susceptible to disinfection, populations associated with biofilms may survive high chlorine dosing.