Aeromonas

Aeromonas Introduction Species of Aeromonas are Gram-negative, non-spore-forming, rod-shaped, facultatively anaerobic bacteria that occur ubiquitously and autochthonously in aquatic environments. The genus Aeromonas was part of the family Vibrionaceae until the mid-1980s when phylogenetic evidence from molecular studies supported separating out the genus as the family Aeromonadaceae. The species are principally associated with gastrointestinal infection in human. The present study was attempted to isolate and identify toxigenic Aeromonas spp. from environmental samples by conventional biochemical and molecular method in the Enteric and Food Microbiology Laboratory of International Centre for Diarrohoeal Disease Research, Bangladesh (ICDDR,B).A total six numbers (n=6) of isolates were randomly selected in this study based on their initial biochemical identification. These isolates were then subjected to extensive phenotypic, antibiogram and molecular characterization by polymerase chain reaction (PCR) for the detection of aerolysin gene (aerA). The antibotic susceptibility patterns of the isolates were determined against five commonly used antibiotics such as Ciprofloxacin, Erythromycin, Ampicillin, Gentamicin, Trimethoprim sulphamethoxazole, Furazolidone. All of the isolates were found to be resistant to Ampicillin (Amp) and showed sensitivity to other antibiotics.

The aerolysin genes of Aeromonas spp. have frequently been targeted in molecular PCR methods. The result of which indicate the presence of virulence gene (aerA) in only two isolates. Aerolysin is a class of pore-forming cytotoxins that disrupt cell membranes. It is secreted as a 52-kDa protein called proaerolysin because it is inactive until it is proteolytically activated. Finally, the toxigenic property of the isolated strains was determined by molecular method. Thus, the detection of aerA gene by PCR suggest that the isolated Aeromonas strains was toxigenic

Discussion Aeromonas species are important pathogens of human. They are ubiquitous in the environment and naturally occurring inhabitants of aquatic environments, namely fresh waters, marine waters, and estuarine waters. However, Aeromonas also have been isolated from fish, shellfish, meats, dairy products, and fresh vegetables. Aeromonas spp. are associated with life threatening diseases of humans, such as bacteremia (Ko et al.,2000), meningitis (Lin and Cheng 1998), septicemia (Tabata et al., 1999), myonecrosis (Balasco et al., 1995), lung abscess (Hur et al., 1995), pulmonary infection (Leclerc et al., 1990) etc. It is one of the important agents associated with diarrhea.

Aeromonas cause acute diarrheal disease of short duration or chronic loose stools in children, the elderly, or the immunocompromised. Aeromonas spp. associated diarrhea is usually mild and self-limiting (Holmberg et al.,1984). However, cholera like watery diarrhea (Champsaur et al., 1982) as well as dysentery-like syndrome (Rahman et al., 1980) is associated with aeromonad infection. Some Aeromonas spp. are opportunistic pathogens of humans, causing a wide variety of extra-intestinal infections and occasionally associated with gastrointestinal disease. An increasing number of epidemiological studies indicate that Aeromonas spp.may

be etiological agents in sporadic diarrhoeal illness in both developed and developing countries.

In Bangladesh, different species of Aeromonas are present in river, lake and pond ecosystems (Islam et al., 1992). However, increasing levels of pollution may result in substantially greater populations, and may also affect distribution of the organisms (Holmes, Niccolls & Sartory, 1996). Aeromonas spp. produces various virulence-associated extracellular metabolites, such as enterotoxin (Chopra and Houston 1999), cytotoxin (Alavandi et al., 1999), hemolysin (Fujii et al., 1999) etc. Aerolysin (aerA) is one of the major enterotoxin, which is considered as reasonable predictors of human diarrhoeal disease.

The present study was undertaken to identify Aeromonas spp. by cultural and molecular method, in addition to determine the presence of aerolysin gene (aerA). All the environmental isolates were collected from the rivers Sitalkhya, Buriganga, Turag, and Brahmhaputra. A total of six (6) six isolates were randomly selected based on their primary identification by cultural and biochemical method which were then further analyzed by molecular biological characterization.

Isolation and identification of Aeromonas spp. from environmental samples provide a challenge because of the presence of competing bacteria. However it is also very vital and important for the characterization purpose of Aeromonas. The colonies having typical cultural characteristics were selected as presumptive Aeromonas. Typical colonies having the circular, yellow, shiny characteristics on TCBS (1-4 mm in diameter); circular, grey, flattened opaque zone around in TTGA (1-2 mm in diameter) and circular, flat with entire margin, non mucoid, lactose non fermenter on MacConkey medium (1-4 mm in diameter) were presumptively selected as Aeromonas spp. the strains were further subcultured onto gelatinase agar medium to observe their ability to utilize gelatinase and resistance to VSC (10 an 150 Âľg) discs were observed for a more confirmative analysis. They were then subjected to biochemical tests for confirmation.

Extensive biochemical tests were performed in order to measure the variability of biochemical behavior among the strains. However, all the strains showed the typical biochemical behavior characteristics of Aeromonas spp. as compared to the control strain. All the strains were positive for indole, oxidase, as well as citrate and also unable to grow in 6.5 and 8% of NaCl. The genus Aeromonas is differentiated from Plesiomonas and Vibrio by its resistance to VSC. Other key differential characteristics include its inability to grow in the presence of 6.5% sodium chloride, gelatin liquefaction and the absence of the SXT element.

Antimicrobial susceptibility testing is a necessary prerequisite to successful therapy. The role of antibiotics in treatment of gastrointestinal Aeromonas infections is controversial, since most patients regain health without treatment. Antimicrobials are indicated for only severe and unresponsive cases of Aeromonas gastroenteritis (Phavichitr and Catto-Smith 2003). So, antimicrobials should be considered for chronic gastrointestinal disease or extra-intestinal infection for Aeromonas (Ghenghesh et al., 1999b). In addition to selection of antibiotic therapy in the clinical setting, antibiotic sensitivity patterns are sometimes useful as phenotypic characteristics for Aeromonas identification.

However, the pattern of susceptibility is not always uniform as it depends on the source of isolation. In this study, the susceptibility to antibiotics of Aeromonas isolates was also examined. All the isolates were subjected to six commonly used antibiotics such as

ciprofloxacin, erthromycin, ampicillin, gentamicin, trimethoprim sulphamethoxazole, furazolidone. All of the isolates were found to be resistant to ampicillin (Amp) and showed sensitivity to other antibiotics. Resistance to ampicllin indicates the presence of the ampicillin resistance gene ampR (also known as blaTEM1). It is responsible for the synthesis of the enzyme beta-lactamase, which neutralizes antibiotics in the penicillin group, including ampicillin. Again, in this experiment none of the isolates were showed resistance against the five most commonly used antibiotics that are commonly used as a therapeutic agent to treat and control diarrhea. This indicate that the isolates might lack the other common drug resistance markers like the plasmids, the class I integron and the SXT element (Dalsgard et al., 2001).

Aeromonas spp. have been recognized for some time (Janda & Abbott, 1998), but only during the past three decades has their role in a variety of human illness been documented. The role of Aeromonas species in bacterial gastroenteritis is not yet clearly understood owing to a paucity of long-term studies (Janda & Abbott, 1998) and the inability to differentiate pathogenic from non-pathogenic strains.

So the role of Aeromonas in gastrointestinal disease is very controversial. Increasing epidemiological data suggest that these organisms play a major role in enteric infections, but so far enteropathogenicity has not been demonstrable in experiments where volunteers were given high numbers of Aeromonas possessing different virulence factors.

Identification is the practical use of classification criteria to distinguish certain organisms from others, to verify the authenticity or utility of a strain or a particular reaction, or to isolate and identify the organism that causes a disease. Bacteria are identified routinely by morphological and biochemical tests, supplemented as needed by specialized tests such as antibiotic inhibition patterns. Newer molecular techniques permit species to be identified by their genetic sequences, sometimes directly from the environmental and clinical isolates. So for a more specific, detailed result, higher resolution identification can be done at the molecular level using genotyping method. Genotyping is analysis of the genetic material (nucleic acid) of an isolate by various forms of PCR. The PCR approach developed in this study has value in characterizing Aeromonas isolates from water. To determine whether the isolates were toxigenic or not, the isolates in this study were examined for the presence of aerA gene by PCR based method. aerA was presence in only two (2) isolates and the rest of the isolates were deprived from the gene.

The virulence of Aeromonas species is likely multifactorial. Possible virulence factors include toxins (cytotoxic and cytotonic), proteases, hemolysins, lipases, adhesins, agglutinins, pili, invasions, enterotoxins, various enzymes, and outer membrane arrays, such as an Slayer, flagella and capsule (cell associated virulence factors). It is difficult to determine which and how many aeromonas contain these putative virulence factors. In addition to the presence of virulence factors in the organism, the host immune response to infection influences the severity of infection. Although Aeromonas strains isolated from water have multiple virulence factors this studies have been limited because only one of the virulence gene (aerA) was targeted. This is because Because aerolysin has been suggested as possible contributory in the pathogenesis of diarrheal disease (Fujii et al., 2008).

The primary toxins produced by Aeromonas spp. are haemolysins, of which the most significant is aerolysin. Aerolysin is one of the major enterotoxins, which is considered as reasonable predictors of human diarrheal disease. This is a heat-labile b-haemolysin, which

exhibits phospholipase A and C activity. It is a pore-forming cytolysin able to insert into the cell membrane bilayer causing leakage of cytoplasmic contents (Chopra et al., 1991).

In this experimient, only two of the isolates were aerolysin-positive, it was found frequency of aerolysin-positive strains varied with the species. Usually aerolysin/hemolysin genes were not detected in A. media, A. allosaccharophila, and A. schubertii (Chacon et al., 2003). But it is commonly expressed by many strains of A. hydrophila, A. caviae, A. trota. and A. sobria (Janda et al., 1991).

In conclusion, it can be interpreted that, the goal of the study was to isolate toxigenic Aeromonas spp. from the surface water of the river. Among the six (6) isolated strain of Aeromonas species only two(2) were aerolysin positive. So the target of our study to isolate toxigenic Aeromonas spp. was successfully achieved. This toxigenic species of Aeromonas remain to be a threat to public health. As these species are opportunistic and transmitting infection to human, monitoring and periodic surveys are required to observe the abundance of toxigenic Aeromonas species.

Concluding Remarks Aeromonas species are found in the aqueous environment, foods, intestinal tract of animals and humans, with or without any evidence of disease. Aeromonas infection has drawn attention in recent years as causative agent of acute diarrhea (Taher et al., 2000). So, the present was employed for the isolation of toxigenic Aeromonas species from the surface water of the river. The prominent features of the present study can be delineated as follows: All the six isolates were reasonably identified as Aeromonas species by cultural and biochemical methods.

All the isolated strains showed resistance to Ampicillin and sensitivity to other antibiotics.

(Gentamicin, Furazolidone, Erythromycin, Trimethoprim Sulphamethoxazole, and Ciprofloxacin.)

Among the six (6) isolates, only two (2) (Env-2, Env-4) were found to be aerolysin positive strain.

Future plan Aeromonas spp. cause disease in poikilothermic animals, and in mammals. Aeromonas may produce a variety of extracellular products that confer virulence in some strains. So, in future, certain additional evaluation must be done. These are: Molecular detection of the most prevalence genes in Aeromonas spp. such as fla, act, alt, lip and pro gene.

Identification of Aeromonas spp. up to species level by Aerokey II procedure.

Detection and sequencing of antibiotic resistance marker.

Rapid typing method of Aeromonas strain by ERIC-PCR method.

Pathogenic Aeromonas spp. will have been characterized in animal model.

Comparative analysis between the environmental and clinical isolates.

Detection of Aeromonas spp. in RIL model based on their availability of two hemolysin genes such as alt and hly.

Molecular typing of the isolates by pulse Field Gel Electrophoresis (PFGE) and clonal relationship by Denature Gradient Gel Electrophoresis (DGGE).

Monitoring the physicochemical parameters of water and correlate it with the density of Aeromonas spp.

Measurement of the density of Aeromonas phages in water and sediment samples of sewage contaminated and uncontaminated ponds.

Isolation and Identification of toxigenic Aeromonas species from the surface water of aquatic environment 1. Introduction and Review of Literature 1.1.Background Aeromonas spp. comprises a complex group of ubiquitous bacteria. They are widely distributed and often isolated from clinical (Krovacek et al., 1994; Carnahan and Altwegg 1996; Kuhn et al., 1997), environmental (Araujo et al., 1990; Burke et al.,, 1984; Krovacec et al., 1994), and food samples (Buchanan and Palumbo 1985; Abeyta et al., 1986; Abeyta and Wekell 1988). Members of the genus are important pathogens (Austin and Austin1997; Gray et al., 1990; O'Brien et al., 1994; Kawula et al., 1996; Santos et al., 1999). In humans Aeromonas cause opportunistic infections and gastroenteritis (Krovacec et al. ,1994; Kuhn et al., 1997) .So, they have been implicated as pathogens of humans and lower vertebrates, including fish (Janda and Abbott 1998). Although a demonstrated, Aeromonas are often isolated from patients with diarrhoea. Some studies have indicated a significant association with diarrhoeal diseases diarrhea, especially amongst children (Albert et al., 1999, 2000).

Members of the genus Aeromonas are gram-negative, motile, facultative anaerobic, rod shaped, oxidase positive bacteria of the recently assigned family Aeromonadaceae (Janda JM 1991). Today, six species of Aeromonas are recognized to cause a variety of intestinal and extra-intestinal infections in humans (Janda and Abbott 1998) such as, Aeromonas viz., A. hydrophila, A. caviae, A. veronii (biovar sobria and veronii), A. schubertii, A. jandaei and A. trota (Carnahan et al., 1993). The significance of Aeromonas species as causative agent of human diarrhoea has recently been established (Farmer III JJ, Arduino MJ, Hickman-Brenner 1992). Aeromonas have been found to cause a variety of primary infections in the normal host as well as severe infections in immuno-compromised patients. (Kirov SM.1993) Aeromonas spp. are abundant in natural water bodies (Gibotti et al.,, 2000). They can be isolated from fresh water (Pasetto et al., 1998), drinking water supplies (Legnani et al., 1998) and bottled water (Warbutton et al., 1994) as well. Density of Aeromonas spp. fluctuates with the variation in physiochemical parameters of water in an aquatic ecosystem (Fliermans et

al., 1977). Aeromonas spp. show population dynamics in natural water (Pettibone 19998) and in sewage treatment pond (Monfort and Baleux 1990). Strains of Aeromonas spp. have also been isolated from polluted waters (Araujo et al., 1991).

From the conataminated water, soft tissue infection in human beings caused by this organism has been reported (Joseph et al.,,1979,Sacho et al., 1990). Besides, Aeromonas spp. are important fish pathogen (Rahim et al., 1984,Chattopadhay et al., 1991,McGray et al.,19991) and can adversely affect fish industry severely . Aeromonas infested water induces mortality of fingerlings in the hatchery (Ortega et al., 1996).

The Aeromonas from the heterogenous environment possessed several virulence factors.

Virulence of Aeromonas spp. is multifactorial and incompletely understood. Factors contributing to virulence include toxins, proteases, hemolysins, lipases, adhesins, agglutinins, and various hydrolytic enzymes (Janda and Abbott 1996). Virulence factors are present in two forms, cell-associated structures, and extracellular products. Among the cell-associated structures are pili, flagella, outer membrane proteins, lipopolysaccharide, and capsules. The major extracellular products include cytotoxic, cytolytic, hemolytic, and enterotoxic proteins.

Aeromonas have been isolated from diseased animals and fish for over 100 years, but they have been recognized as human pathogens only in past 50 years, and there is still controversy concerning their relationship to enterotoxin production and resulting gastrointestinal disease (Albert et al 2000). An environmental source of Aeromonas implicated in gastrointestinal infection was first proposed by Holmberg et al., (1986). Associations of Aeromonas with human disease were reported by von Graevenitz and Mensch (1968) in a review of 30 cases of Aeromonas infection or colonization, providing evidence for their recognition as human pathogens and suggesting that some Aeromonas may be associated with gastrointestinal disease.

1.2. Review of Literature 1.2.1. Taxonomy: The bacterium Aeromonas was first isolated by Zimmerman in 1890 in early nineteen century from tap water and he named it as Bacillus punctatus. One year later, Sanarelli isolated a similar type of bacterium from frog infected with a disease called “red leg disease� (Sanarelli 1891). Sanarelli named this bacterium as Bacillus hydrophilus fuscus .Finally, Kluyver and Van Niel (1936) proposed the genus Aeromonas to describe the bacteria the bacteria isolated by Zimmerman (1890) and Sanarelli(1891)(Md.Zeaur Rahim1997).Aeromonas have been placed under the family Vibrionaceae based on phenotypic characteristics. But molecular biological evidence,including nucleotide sequence of 16S and 5S rRNA and rRNA-DNA hybridization data , suggest that Aeromonas are different from the members of Enterbacteriaceae and Vibrionaceae. All these evidence suggest that the species of the genus Aeromonas represent a distinct family Aeromonadaceae fam.nov (Colwell et.al.1986).

Table 1.1: Current taxonomy of the species belonging to Aeromonas genus , grouped according to pathogenicity for man ( modified after Janda and Abbott , 1998). ∗ Based on frequency rather than on the severity of the disease

Species associated to disease in man Major pathogens A. hydrophila

Minor pathogens A. veronii biotype veronii

A. caviae A. jandaei A. veronii biotype sorbia

Environmental Species

A.schubertii

A. salmonicida A. sorbia A. media A. eucrenophila A.trota A.popoffii A.bestiarum

1.2.2. Serotyping of Aeromonas spp.

Serotyping is based upon somatic (O) antigen determinants (Sakazaki and Shimada 1984).

Several typing schema have been proposed (Fricker 1987; Cheasty et al.,,, 1988; Thomas et al.,,, 1990), but only one comparison study of two of these schema has been published (Shimada and Kosako 1991). The schema of Sakazaki and Shimada recognizes 44 serogroups, with an additional 52 provisional serogroups (Albert et al.,, (1995). Aeromonas spp. are found to be serologically heterogeneous, with individual serogroups found in more than one species (Janda et al.,,, 1996). Most type and reference strains were not serologically representative of a genomospecies. Three serotypes predominate in clinical specimens, O:11 (24%), O:16 (14%), and O:34 (10%). Korbsrisate et al.,, (2002) characterized the distribution of A. hydrophila serogroups in clinical specimens and developed polyclonal antibodies for rapid identification of clinical isolates by direct agglutination. Only 50% of strains fell into the common serogroups O:11, O:16, O:18, O:34, or O:83. Rough strains (15.2%) and untypable strains (2.3%) reduced the effectiveness of serotyping for identification of clinical strains. A polyvalent antiserum was produced that resulted in positive agglutination of 102 or 105 strains, for a calculated sensitivity of 97.1% and specificity of 90.7%. This test could be useful in rapid identification of Aeromonas to genus where they are isolated from samples that may also contain vibrios.

1.2.3. Classification: The genus includes at least 13 genospecies, among which are the mesophilic A. hydrophila, A. caviae, A. sobria, A. veronii, and A. schubertii, and the non-motile, psychrophilic A.

salmonicida infection. By contrast, the mesophilic species have been associated with a wide range of infections in humans (Janda & Abbott, 1996). Although members of the genus have classically been divided into three biochemically differentiated groups (typified by A. hydrophila, A. caviae, and A. sobria), these contain a number of genospecies, to which new species have been added (Carnahan & Altwegg, 1996). Currently the genus is made up of 17 DNA hybridization groups representing a range of genospecies and phenospecies The mesophilic Aeromonas have been commonly isolated from patients with gastroenteritis although their role in disease causation remains unclear. They are also associated with sepsis and wounds, and with eye, respiratory tract, and other systemic infections (Janda & Duffey, 1988; Janda & Abbott, 1996; Nichols et al.,,, 1996); Many of the systemic infections arise following contamination of lacerations and fractures with Aeromonas-rich waters The species principally associated with gastroenteritis are A. caviae, A. hydrophila, and A. veronii biovar sobria (Joseph, 1996); A. caviae is particularly associated with young children (under 3 years of age).

1.2.4. Sources of Aeromonas spp: Aeromonas are ubiquitous in aquatic environments and readily isolated from both nutrientrich and nutrient-poor environments (Holmes, Niccolls & Sartory, 1996). As they are autochthonous to fresh and marine waters their recovery is to be expected. However, increasing levels of pollution may result in substantially greater populations, and may also affect distribution of the organisms (Holmes, Niccolls & Sartory, 1996). Different physicochemical parameters of water may affect the growth and distribution of Aeromonas in aquatic environment .Two decades from nineteen seventies, effect of physicochemical parameters of water on Aeromonas in the aquatic ecosystem was extensively studied by different investigators. These studies mostly focused on the effect of dissolved oxygen (Seidler et al.,, 1980), temperature (Hazen et al.,,1978), pH (Hazen et al.,,1978) and conductivity (Burke et al.,,1984a) etc. Aeromonas spp. are also found in soil and the feces of aquatic and terrestrial animals and humans. They occur in fish and other sea foods, meat sausage, vegetables, raw milk,cheese, and processed food products (Palumbo, 1996).

1.2.5. Transmission Routes of Aeromonas spp.

Aeromonas spp. are ubiquitous in the environment and there are multiple opportunities for transmission to humans through food, water, animal contact, and direct human contact. Extraintestinal infections are typically acquired following trauma in an aquatic environment, and intestinal infections are acquired by ingestion of contaminated food or water. Intestinal infections in immunocompromised patients may disseminate resulting in septicemia with multiple organ involvement.

Transmission routes

Environmental Transmission Waterborne transmission

Foodborne transmission

Person to Person transmission

Animal to Person transmission

Fig 1.1: Transmission Routes of Aeromonas spp.

1.2.6. Worldwide Occurrence Aeromonas spp. are found worldwide in surface water, ground water, non-chlorinated drinking water, chlorinated drinking water, and bottled mineral water (Holmes et al.,,, 1996).

Aeromonas are found in a wide variety of foods (Palumbo, 1996). They are found in the intestinal tract of humans and animals, raw sewage, sewage effluents, activated sludge, and sewage-contaminated waters (Holmes et al.,,, 1996).

Aeromonas reach population densities of 106-108 CFU/mL in raw sewage and 103-105 CFU/mL remain in sewage effluents after treatment (Holmes et al.,,, 1996). Their occurrence in the environment is not dependent upon fecal pollution; however, they reach higher numbers in nutrient-rich waters contaminated by sewage. They may reach 3-5 log10 CFU/mL in surface waters during summer months. They are not common in groundwater, though they may colonize poorly constructed wells.

1.2.6.1. Occurrence in Bangladesh Mesophilic, motile Aeromonas are ubiquitous (everywhere) and autochthonous (naturally occurring) in the aquatic environment of Bangladesh. They inhabit freshwater, esturine waters, and wastewater, and have been found in chlorinated and unchlorinated drinking water (Havelaar et al.,,, 1992). They are also found in soil and the feces of aquatic and terrestrial animals and humans. They occur in fish and other seafoods, vegetables, raw milk, and processed food products. Aeromonas spp. occur in the human gastrointestinal tract both in the presence and absence of disease, but the presence of Aeromonas spp. in other body sites is usually associated with infection and disease. Aeromonas cause disease in poikilothermophiles (cold blooded animals) such as frogs, eels, and fish, where they are an economic liability to the aquaculture industry. Aeromonas have been found in association

with marine copepods and plankton, where they are present at cell densities from 4 CFU/mL to 1.3x103 CFU/100mL in seawater and from 1.5x101 CFU/100mL to 6x102 CFU/100mL on plankton.

1.2.7. Environmental Factors Affecting Survival: Environmental survival of Aeromonas is dependent upon many physical and biological factors. Temperature, pH, ionic strength, sunlight (UV irradiation), moisture, available nutrient, presence of suspended solids, cell-specific protection mechanisms, and the presence of toxic substances and predators all interact to determine survival times.

1.2.7.1. Survival in Water: Aeromonas spp. has their natural habitat in water and grow over a wide temperature range.

Because Aeromonas spp. grows between 0º C and 45º C, with a temperature optimum of 22º C to 32º C, there are few environmental habitats where they are not found. Both high (Tsai and Yu 1997; Warburton 2000; Croci et al.,,, 2001) and low (Kersters et al.,,, 1996b) survival rates have been reported. Nutrient availability, temperature, and water activity most affected growth rates.

Growth was optimal at 30º C at pH 7 and a water activity of 0.99 (Sautour et al.,,, 2003).

Imbert and Gancel (2004) studied the effect of temperature downshift on protein synthesis of A. hydrophila. While a few proteins were under-expressed, two-dimensional electrophoresis revealed that numerous new proteins appeared with a decrease in temperature and some others were over-expressed. Cold shock proteins distinct from those produced by E. coli were recognized. Additional studies are required to elucidate the nature of heat and cold shock proteins produced by Aeromonas.

Aeromonas grow best between pH 7-9 (Vivekanandhan et al.,,, 2003). Sautour et al.,, (2003) reported that variation in pH had little effect upon survival over a range of pH 5-9, and this is consistent with the growth range reported by Popoff and Lallier (1984). Aeromonas spp. are sensitive to acid conditions below pH 3.5; however, they exhibit an acid stress response in that when they are acclimated at pH 5, the kill time at pH 3.5 is extended. Treatment with protein-inhibiting antibiotics prior to exposure to low pH eliminated the acid stress response, suggesting that protein synthesis is an important part of the acid stress response.

1.2.8. Viable but Non-culturable (VNC) State of Aeromonas spp.

Nutrient deprivation of bacteria has been reported to induce physiological changes that reduce the ability to detect them using culture methods. Nutritional depletion is correlated with detachment of Aeromonas cells adsorbed to surfaces (Sawyer and Hermanowicz2000).

Maalej et al.,, (2004) studied survival of A. hydrophila in natural filtered seawater.

Populations declined below the detection limit at both 5º C and 23º C in 3-5 weeks. Cells grown at 5º C were more resistant to stress than cells grown at higher temperature. A temperature shift from 5º C to 23º C did not result in cell resuscitation. Cells lost respiratory activity before they lost membrane integrity. The shift to VNC state is associated with formation of hydrogen peroxide sensitive cells populations Sun et al.,, (2000) claims to have induced A. hydrophila into a VNC state by incubation at4º C for 45 days. Cells were resuscitated using liquid media and solid media containing catalase

or sodium pyruvate. Wai et al.,, (2000) also reported induction of VNC Aeromonas with recovery on media through addition of catalase or sodium pyruvate. Contrary to these reports, Rahman etal. (2001) reported that induction of the VNC state was not reversible in Aeromonas. Mary et al.,, (2002) reported that A. hydrophila declined to non-detectable levels in nutrient-poor filter sterilized distilled water at 4º C within 7 weeks, while the number of cells with intact membranes by the Live/Dead method decreased by 1 log10 CFU. Cells could not be resuscitated by an increase in temperature to 25º C, and neither catalase or sodium pyruvate improved recovery Whether or not Aeromonas exist in a reversible VNC state remains to be determined.

1.2.9. Human infection associated with Aeromonas spp: The mesophilic Aeromonas have been commonly isolated from patients with gastroenteritis although their role in disease causation remains unclear. They are also associated with sepsis and wounds, and with eye, respiratory tract, and other systemic infections (Janda & Duffey, 1988; Janda & Abbott, 1996; Nichols et al.,,, 1996). Many of the systemic infections arise following contamination of lacerations and fractures with Aeromonas-rich waters.

Table 1.2: Relative frequency occurrence of human infections associated with Aeromonas.

Type of infection Diarrhoea Dysenteric Chronic Diarrhoea

Characteristics Secretory Acute watery vomiting

diarrhoea,

Acute diarrhoea with blood and mucus

Common

Lasting more than 10 day

Common

Choleraic

“Rice water” stools

Systemic Cellulitis

Inflammation of connective tissue

Myonecrosis Haemorrhage Erythema Skin gangrenosum Septicaemia

Relative frequency Very common

Rare Common

Necrosis with/without gas gangrene lesions with necrotic centre, sepsis

Rare Uncommon

Fever, chills, mortality

Fairly common

hypotension,

high

Peritonitis

Inflammation of peritoneum

Uncommon

Pneumonia

Pneumonia with sometimes necrosis

septicaemia,

Rare

Osteomyelitis

Bone infection following soft-tissue infection

Rare

Cholecystitis

Acute infection of gallbladder

Rare

Eye infections

Conjunctivitis, endophthalmitis

corneal

ulcer,

Rare

(Modified from Janda & Duffey, 1988, and Nichols et al.,,, 1996).

1.2.10. Virulence Factor: Virulence of Aeromonas is multifactorial and incompletely understood despite decades of intense investigation (Trower et al.,,, 2000). Many putative virulence factors have been described, including toxins, enterotoxins, proteases, hemolysins, lipases, adhesins, agglutinins, hydrolytic enzymes, outer membrane proteins, S-layer, flagella, and pili. Janda (2002) reviewed the many virulence factors produced by Aeromonas spp.

Other virulence-associated factors of Aeromonas spp. are collagen-binding protein (Gullberg D, Terracia L, Borg TK, et al.,,1989) and haemagglutinin (Majeed KN, Macrae IC1994). In addition, Aeromonas strains having an S-layer (Kokka RP, Janda JM, Oshiro LS, et al.,,1991) could resist a bactericidal activity of 65% pooled human serum (Rahim Z, Aziz et al.,,1994).

These strains are more pathogenic to mice compared to S-layer-negative strains (Janda JM, Kokka RP, Guthertz LS, et al .1994). Moreover, Aeromonas strains of serotype 0:34 can resist complement-mediated lysis (Merino S, Alberti S, Tomas 1994). These properties help the bacteria to cause bacteraemia and septicaemia (Ko Wc et al.,,1996). Invading strains of Aeromonas spp. can be disseminated via the bloodstream to other parts of the body to infect vital organs, such as th lung (Hur T,Cheng KC, Hsieh JM, et al.,,1995 )and/or the heart (Blasco MA, Moreno R, Pardo FJ, et al ,1995) Thornley et al.,, (1997) reviewed the virulence genes of Aeromonas spp. A summary of virulence factors is shown in table: Table 1.3: Cell-Associated and Extracellular Virulence Factors of Aeromonas species Cell-Associated Virulence Factors Pili (fimbriae) Flagella Outer membrane proteins A or S layer Lipopolysaccharide Capsule

Extracellular Virulence Factors Hemolysin Enterotoxin Cytotoxin Protease Glycerophospholipid cholesterol acetyltransferase (GCAT) Other hydrolytic enzymes

From Thornley et al., 1997 1.2.10.1. Pathogenic Mechanism: Adhesion is the first step in pathogenesis of Aeromonas like other bacterial pathpgens, such as Vibrio cholerae (Finkelstein et al.,, 1983) and V. parahaemolyticus (Oishi et al.,, 1979).

Adhesion of Aeromonas spp. is mediated by hemagglutinin,pilus,adhesion for binding extracellular matrix etc.

1.2.10.2. Hemagglutinin: Hemagglutination is widely used as an indicator of adhesion mediated by hemagglutinin(s).

Aeromonas spp. posses soluble (Stewart et al.,,1986) and cell associated hemagglutinin (HA) like that of Vibrio cholerae (Finkelstein et al.,, 1983) and V. parahaemolyticus (Oishi et al.,, 1979). Hemagglutinins are classified in two types: soluble hemagglutinin and cell associated hemagglutinin.

1.2.10.3. Adhesion: Other than hemagglutination, attempt was made to correlate the pathogenic potential of Aeromonas isolates with adhesion. Cell properties promoting adhesion of Aeromonas to host cells were recognized early in the studies of the pathogenesis (Gosling 1996a).

Ascencio et al.,, (1998) reported cell surface extracts containing active mucin-binding components from 22-95 kDa from Aeromonas spp. Adhesions to HEp-2, Caco-2 and INT407 cells has been reported by several investigators (Nishikawa et al.,,, 1994; Bartkova and Ciznar 1994; Kirov et al.,,, 1995b). A correlation between high level HEp-2 cell adherence and enteropathogenicity has been reported (Kirov and Sanderson 1995).

Fig 1.3: Aeromonas hydrophila adheres to human epithelial cells 1.2.10.4. Pili of Aeromonas spp: Filamentous structure, thinner and shorter than flagella, which are projected from the periphery of the bacterial cellwall, is called pili. It is also called fimbriae. This organ helps bacteria to colonize on a particular surface. Two morphotypes of pili have been observed in Aeromonas spp., short rigid pili and long wavy flexible pili. These filamentous structures were described as potential colonization factors in A. hydrophila and A. veronii biovar sobria (Hokama and Iwanaga 1991). Kirov (1993b) reported that pili were important adhesive factors for mucosal surface attachment and described filamentous and nonfilamentous adhesins. Aeromonas spp. from cases of gastroenteritis may exhibit Type IV pili (Tap) (Barnett and Kirov 1999) or bundle-forming pili (Bfp) (Kirov and Sanderson 1996; Kirov et al.,,, 1999). Barnett et al.,, (1997) also reported the presence of two distinct families of Type IV bundle-forming pili and Tap pili in Aeromonas strains from patients with gastroenteritis.

1.2.10.5. Plasmid: It is established that virulence associated properties of Aeromonas, such as cytolytic enterotoxin (hemolysin), invasive properties, type IV pili etc. are mediated by genes located on chromosome(Lawsom et al.,,1985,Husslein 1988,Pepe et al.,,1996).But other pathogenic factors ,such as mini pilin (Ho et al.,,1992 ),Shiga-like toxin (Haque et al .1993)are encoded by genes located on the plasmids .

1.2.10.6. Lipopolysaccharide: Lipopolysaccharide comprises the major structural element of the gram-negative cell wall that is responsible for somatic antigenic specificity (O-antigen). It also plays a role in adhesion to epithelial cells (Merino et al.,,, 1996), resistance to nonimmune serum (Merino et al.,,, 1991), and virulence (Aguilar et al.,,, 1997).

Fig 1.4: Lipopolysaccharide (LP) 1.2.10.7. Outer Membrane Proteins: The literature contains several conflicting reports concerning attachment mechanisms, but it is generally accepted that outer membrane proteins (OMP) mediate bacterial adherence to host cells Nishikawa et al.,, (1994) suggested the role of an outer membrane protein in binding of Aeromonas to Caco-2 cells. The role of OMPs has been proposed for attachment to HEp-2, HeLa, Chineses hamster ovary (CHO) and Vero cells (Bartkova and Ciznar 1994).

Some OMP have hemagglutination activity, while other OMPs are thought to have poreforming capability.

1.2.10.8. Capsule: The role of capsule polysaccharide as a virulence factor since most motile strains are not encapsulated. Capsules have been shown in A. hydrophila serotypes O: 11 and O: 34 when they are grown in glucose-rich media (Martinez et al.,,, 1995). Preliminary work suggests that capsule may play a role in septicemia, as non-encapsulated strains are less virulent.

Aeromonas capsule material has the capability of protecting cells from complement-mediated serum killing activity (Zhang et al.,, 2002). Aguilar et al.,, (1999) also reports serum resistance properties of capsule polysaccharide.

1.2.10.9. S-Layer Proteins: Paracrystalline layer, which lies to the periphery of outer membrane of the bacterial cell wall,is known as surface layer or S-layer . S-layer provides protection against Aeromonas strains from bactericidal activity of 65% pooled human serum (PHS). S-layer producing Aeromonas spp. is associated with extraintestinal infection of humans (Kokka et al.,,, 1992;

Janda et al.,,, 1994). S-layer plays a role in uptake of porphyrins and shows unique immunoglobulin and extracellular matrix protein-binding capacity.

Capsules Fig 1.5: Capsules and S-layers 1.2.10.10. Flagella: Flagella allow Aeromonas to reach target cells where they colonize (Barnett et al.,,, 1997).

Flagellation in Aeromonas is usually monotrichous and polar. However, lateral flagella occur S - layers in some strains and some strains are nonmotile. Peritrichous flagella are unsheathed and they are associated with swarming movement across solid media surfaces (Kirov et al.,,, 2002).

1.2.11. Extracellular virulence factor involved in pathogenic mechanism: 1.2.11.1. Enzymes: TEM

image of

a bacterial cell with an

S - layer with

square virulence lattice symmetry(Holder attice . Bar=100nmand Haidaris 1979, Aeromonas produce different enzymes associated with Janda and Bottone 1981).Waltman et al., (1982) were able to detect different enzymes,such as caprylate esterase-lipase, leucine amino peptidase ,acid phosphatase, phosphoamidase, and N-acetyl- β-glucosaminidase. Isolates showed variability with respect to production of other enzymes, such as alkaline phosphatase, butyrate esterase, myristate lipase,trypsin, βgalactosidase,α-glucosidase and β-glucosidase(Waltman et al., 1982).

1.2.11.2. Siderophores: During infection, microbes must acquire iron from the host, and this is accomplished by production of siderophores; thus, siderophores are considered to be virulence factors.

Siderophores are iron-specific ligands of low molecular mass. The ferric siderophore gene fstA of A. salmonicida has significant sequence similarity with the fstA gene of several known pathogens (Pemberton et al., 1997). Motile Aeromonas produce either of the phenolate siderophores, enterobactin or amonabactin. Amonabactin is unique to Aeromonas spp.; whereas, many enteric bacteria produce enterobactin.

1.2.11.3 Enterotoxin: Initial idea of Aeromonas enterotoxin was evolved in the late eighties when live culture of Aeromonas isolates from diarrheal patients could induce fluid accumulation as a result of injecting it into the rabbit ileal loop (RIL)(Sanyal et al., 1975). This finding provided a preliminary idea of enterotoxic potential of this bacterium. Enterotoxin of Aeromonas spp.inhibits steroid synthesis in Y1 adrenal cell line (Ljungh et al., 1982b). This toxin increases the intracellular content of cyclic adenosine monophosphate (cAMP) without affecting the cyclic guanosine monophosphate (cGMP) ( Ljungh and Wedstrom) .Similarly in RIL, Aeromonas enterotoxin increased the contents of cAMP (Duby et al., 1980).

1.2.11.4. Classification of Aeromonas enterotoxins:

Aeromonas enterotoxins have been classified into cytotonic and cytotoxic based on their effect on Y1 and HeLa cell-line. Keusch and Donta (1975) initially proposed this classification.

1.2.11.5. Cytotonic entertoxin(CE): The nature of cytotonic entertoxin was characterized by Ljungh et al., (1981,1982b).This toxin showed rounding of cells (without killing),stimulation of cAMP synthesis and steroid secretion by Y1 cell line and stimulation of fluid accumulation in RIL.Biological activity of this toxin was not affected when heated at 56 ยบ C. This toxin was also distinct from cholera toxin and E.coli enterotoxin (Chakraborty et al., 1984).

1.2.11.6. Cytotoxic enterotoxin: Cytotoxic nature of Aeromonas enterotoxin was initially reported by Cumberbatch et al., (1979).This properties are : cell rounding before death and fluid accumulation in RIL. Cytotoxic enterotoxin is a single-chain polypeptide 52 kDa in length and is related to aerolysin, with hemolytic, cytotoxic and enterotoxic activity.It was inactivated by heating at 56 ยบ C for 5 minutes (Asao et al., 1984).

Aerolysin, a pore-forming enterotoxin of Aeromonas spp., is a known virulence factor (Krause et al., 1998). The toxin is secreted in an inactive precursor form (Parker et al., 1996), which becomes active with cleavage of a C-terminal peptide. The toxin is thought to bind to specific receptors located on host target cells (Nelson et al., 1997; Abrami et al., 1998).

Binding is thought to concentrate the toxin and facilitate polymerization into heptameric complexes that penetrate the cell membrane and form water-filled channels leading to cell lysis (Abrami et al., 1998b; Krause et al., 1998; Rossjohn et al., 1998). aerolysin is know by several other names (cytotoxic enterotoxin, Asao toxin, and cholera toxin cross-reactive cytolytic enterotoxin).

Aerolysin is a 50-52 kDa heat labile protein that causes fluid accumulation in rabbit ileal loops and lyses a wide range of cells including CHO and rabbit erythrocytes (Chopra et al., 1993). It belongs to a class of pore-forming cytotoxins that disrupt cell membranes and is highly lethal for rats and mice. Aerolysin from A. hydrophila and A. veronii biovar sobria share properties with the cardiotoxic thermostable hemolysin of V. parahaemolyticus. The structure has been determined by X-ray crystalography and the monomer protein is divided into four domains (Parker et al., 1994).

Antibodies specific for cholera toxin react with aerolysin, but there is no sequence homology and cholera antisera do not neutralize aerolysin activity. Aerolysin crosses the bacterial cell membrane as an inactive proaerolysin, which binds to the receptor glycophorin on the membrane of erythrocytes. The toxin is then activated by proteolysis, either by bacterial or host proteases, and forms a transmembrane channel. The mechanism of action has been elucidated using erythrocytes and little is know about the toxins effect on intestinal cells, though antisera to aerolysin neutralized the toxin effects in rabbit ileal loops (Ferguson et al., 1997).

1.2.11.7. Other toxins:

In addition to cytotonic and cytotoxic enterotoxin, Aeromonas produce cholera toxin crossreacting factors and Shiga–like toxins. These toxins are also considered as pathogenic factors.

1.3. Antimicrobial susceptibility Aeromonas spp. can cause both gastrointestinal and extraintestinal infectious disease. The role of antibiotics in treatment of gastrointestinal Aeromonas infections is controversial, since most patients regain health without treatment. Antimicrobials are indicated for only severe and unresponsive cases of Aeromonas gastroenteritis (Phavichitr and Catto-Smith 2003).

Antimicrobials should be considered for chronic gastrointestinal disease or extra-intestinal infection (Ghenghesh et al., 1999b).

In addition to selection of antibiotic therapy in the clinical setting, antibiotic sensitivity patterns are sometimes useful as phenotypic characteristics for species identification, especially for clinical isolates (Overman and Janda 1999). Aeromonas spp. are characteristically resistant to ampicillin (94.9%), with variable resistance to cephalexin (76.3%), trimethoprim (37.3%), tetracycline (11.9%), cefuroxime (5.1%), and ceftazidime (1.7%). All strains tested were susceptible to gentamicin, chloramphenicol, and ciprofloxacin (Murphy et al., 1995). Aeromonas spp. are typically sensitive to tetracycline, aminoglycosides, trimethoprim-sulfamethoxazole, third-generation cephalosporins, and quinolones (Koehler and Ashdown 1993; Janda and Abbott 1998).

1.4. Major Objectives of the study: It is evident from the forgoing review of literature that Aeromonas is an important pathogen, which is associated with intestinal and extra-intestinal infections of human beings.

Aeromonas associated gastroenteritis or other infections are becoming more common in Bangladesh and thus having great ecological and epidemiological significance. Therefore, the present study has been undertaken with the following objectives: Isolation of toxogenic Aeromonas spp. from surface water samples and identified by using conventional culture, biochemical, serological, and molecular methods.

Determination of antibiotic sensitivity pattern of the Aeromonas isolates.

Detection of the presence of virulence gene (aerA) by polymerase chain reaction.

Molecular characterization of Aeromonas isolates using simplex PCR technique.

Analysis and interpretation of data.

2. Material and Methods 2.1. Environmental samples collection and processing Water samples were collected monthly from June 2008 to August 2008 from four different rivers such as Sitalkhya, Buriganga, Turag, and Brahmhaputra. The samples were collected with aseptic technique using sterile glass conical flask. Isolation of Aeromonas spp. from environmental samples provides a challenge because of the presence of competing bacteria and the possibility of sample matrix interference with sample preparation and culture methods.

2.2.Isolation and Identification of Aeromonas spp.*

2.2.1. Enrichment and Plating Samples were enriched in APW (Alkaline Peptone Water) at 37º C for 6 to 8 hrs. before plating. Following enrichment, 0.1µl of the enriched APW broth were inoculated onto Thiosulphate Citrate Bile Salt (TCBS), Taurocholate Tellurite Gelatin Agar (TTGA), and MacConkey Agar media by a sterile glass rod through spread plate and about two loopful of the enriched APW broth was streaked, using an inoculating loop. The culture plates were then incubated at 37º C for 18 to 24 hrs. Colonies with the characteristic appearance of Aeromonas spp. were confirmed by biochemical, serological and by molecular method.

2.2.2. Microscopic observation of Aeromonas spp A pure colony was picked and gram staining was performed. Then the shape, arrangement and gram reactions of the isolates were observed in a microscopic field (Pelczar et al., 1993).

2.3. Identification of Aeromonas spp. by Biochemical test Various strains of Aeromonas spp. were identified by conventional biochemical tests as outlined by Popoff et al., 1998. After incubation, characteristic colonies on TCBS were selected and screened for the presence of cytochrom oxidase, and gelatinase activity by sub culturing to Gelatin Agar Plate. Colonies that are giving a positive cytochrome oxidase and gelatinase activity were then employed to vibrio-static compound (2,4-diamino-6, 7diisopropylpteridine phosphate) that showing resistance to this compounds which confirms the presence of Aeromonas spp. * Composition of the media and solution through out the research work are listed in the appendix 2.3.1. Oxidase test The presence of cytochrom oxidase is detected by Kovac’s oxidase test.The test was performed with 1% solution of N’N’N’N-tetramethyl–p-phynylenadiamine dihydrochloride which was soaked in apiece of Whatman filter paper. A portion of colony of the test organism was picked tip with a sterile toothpick and touched onto the paper with impregnated reagent .A dark purple color developed within 5-10 seconds was considered positive and no change in color was interpreted as negative for the test.

2.3.2. Salt tolerance test All isolates of Aeromonas are tested for their salt tolerance in alkaline peptone water (APW) containing 0, 6.5, and 8% (w/v) sodium chloride. Tubes containing 3.0 ml broth are inoculated with test organism grown in T1N1 broth for 3-4 hours at 37ºC. Growth was observed visually after 24 and 48 hours of incubation at 37º C. Growth was observed visually after 24 and 48 hours of incubation at 37ºC 0% (w/v) sodium chloride but no growth observed in 6.5 and 8% (w/v) sodium chloride.

2.3.3. Gelatinase test

Gelatinase activity of the test organism was observed on gelatin agar plate. The test organism was streaked onto gelatin agar plate and incubated at 37ºC for 18 to 24 hours. Cloudy hazy zone of gelatinase activity was observed around each isolated colony. No change in transparency of this medium around the colonies was interpreted as negative.

2.3.4. Kligler Iron Agar test (KIA) The test was performed to assess the mode of dextrose utilization in oxidative/fermentative test. Stabbing the butt and streaking the slant with fresh culture inoculated tubes of KIA media. After incubation at 37º C for 18 to 24 hours, results were recorded for changes in color of the butt and slant, H2S production. Formation of acid from dextrose in fermentative mode indicated by yellowing of the butt, whereas the yellowing of the slant indicated the oxidative mode. Production of hydrogen sulphide (H2S) makes blackening of the medium.

2.3.5. Motility, Indole , Urea (MIU) test Tubes containing MIU medium were inoculated with straight wire by stabbing the medium to a depth not touching the bottom. The tubes were incubated as with KIA tubes. Motile organisms dispersed through the medium leaving the stab line and made the tube turbid. Pink coloration of the MIU medium indicated the positive increase and no change in color were recorded as negative. Indole test was done separately in inoculated T1N1 broth incubated as KIA tubes. After incubation, 3-4 drops of Kovacs reagent is added to observe the production of pink zone at the top of T1N1 broth within one minute. Development of deep pink color indicated the indole production from tryptophan.

2.3.6. Citrate Utilization test Organisms were tested for utilization of citrate by stabbing the butt with fresh culture inoculated tubes of citrate agar media. After incubation at 37º C for 18 to 24 hours, results were recorded for changes in color of the butt. Prussian blue color of the butt from the green indicated the mode of citrate utilization.

2.3.7. Susceptibility to Vibrio-static compound (VSC) The test was employed to differentiate Aeromonas spp. from the genera Vibrio and Plesimonas .The vibrio-static compound (2,4-diamino-6, 7-diisopropylpteridine phosphate) impregnated discs of two different concentration (10 and 150 µg per disc) were used for the test. A uniform lawn of test organism was prepared on gelatin agar plate and the discs were placed at two different corners by means of a sterile needle. After incubation at 37º C for 18 to 24 hours, for Aeromonas, found to be resistant to this static discs of two concentrations.

2.4. Preservation of isolates All the Aeromonas strains isolated were inoculated into T1N1 soft agar medium and incubated 37º C for 18 to 24 hours. After incubation, the growth was observed into medium and paraffin oil was added on the surface of the medium. Isolated strains with in the medium were then stored at room temperature. Before use, the identities of the cultures were confirmed by biochemical reaction (Carnahan et al., 1991b).

2.5. Determination of antimicrobial susceptibility by modified Kirby-Bauer method

A commonly used agar diffusion procedure that measures antimicrobial activity is called the Kirby-Bauer method (Brown and Manning, 1985). This method is particularly valuable for tracking the emergence of antibiotic resistant strains of pathogens to detect their disease potential.

Thus, antibiotic sensitivity testing for organisms is absolutely essential for the rapid determination of the efficacy of a drug by measuring the diameter of the zone of inhibition that results from diffusion of the agent into the medium surrounding the disc. Commercially available antimicrobial discs were used for the test. The following are the antibiotics that were tested environmental isolates.

Table 2.1 : Antimicrobial agents and their disc concentrations Antimicrobial agent

Disc concentrations (Âľ g)

Ciprofloxacin (CIP) Erthromycin (E) Ampicillin(Amp) Gentamicin (CN) Furazolidone (FR) Trimethoprim sulphamethoxazole (SxT)

5 15 10 10 100 25

To determine the antimicrobial susceptibility several steps were

maintained:

A suspension of test organism was prepared in normal saline (0.85%NaCl) to match the equivalent turbidity standard to that of McFarland 0.5 Standard.

A sterile cotton swab was dipped into the suspension and excess fluid was removed by pushing and rotating the swab firmly against the internal wall of the tube, just above the fluid level .

The swab (inoculums) was then heavily inoculated over the entire surface of the MullerHinton agar medium (with a pH of 7.3) to obtain a confluent growth of the organism.

Antibiotic disc were applied aseptically to the surface of the inoculated plates at an appropriate special arrangement with the help of a sterile needle.

The plates were then inverted and incubated at 37Âş C for 24 hours.

After incubation, the plates were examined and the diameters of the zone of complete inhibition were measured in mm. Susceptibility to the specific antibiotic is interpreted by the presence of clear zone around the disc.

2.6. Molecular identification of Aeromonas spp.*

Boiled DNA template preparation This method was performed for the rapid detection of any gene, by inoculation of a single colony from GA plate to 3 ml of LB broth and incubated at 37º C with agitation (120 rpm).

Then 1.5 ml of sample was taken in an eppendorf tube and centrifuged at 13000 rpm for 10 min. After then supernatant was discarded and pellet was dissolved in the sterile normal saline and again sample was centrifuged at 13000 rpm for 10 min. The supernatant was discarded and the pellet was dissolved in 200µl of normal saline. The sample was boiled for 10 min and cooled in ice for 30 min. Then the sample was centrifuged at 13000 rpm for 10 min and supernatant was transferred to afresh eppendorf tube and store at - 20º C for use template DNA in PCR. Boiled DNA template preparation is not only simpler and easier but also requires less time than chromosomal DNA extraction.

2.6.2. Molecular detection of Aeromonas spp. by Polymerase Chain Reaction The polymerase chain reaction (PCR) is a technique widely used in molecular biology.

The purpose of a PCR is to make a huge number of copies of a gene. It derives its name from one of its key components, a DNA polymerase used to amplify a piece of DNA by in vitro enzymatic replication. This sets in motion a chain reaction in which the DNA template is exponentially amplified. With PCR it is possible to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the DNA piece.

Developed in 1983 by Kary Mullis, PCR is now a common and often-indispensable technique used in medical and biological research labs for a variety of applications. PCR is used to amplify specific regions of a DNA strand. This can be a single gene, a part of a gene, or a non-coding sequence. * Composition of the master mix for PCR is given in the appendix Simplex Polymerase Chain Reaction (PCR) Simplex PCR assay was employed to detect the virulence gene for the confirmation of the Aeromonas spp.. Sample (3 µl) were added to the PCR mixture to achieve a 25µl final volume and amplification conditions used were 5 min at 94º C for initial denaturation of DNA and 35 cycles, each consisting of denaturation at 94º C, annealing at 64º C, and extension at 72º C for 30 seconds each, with a final round of extension for 7 min at 72º C in a DNA RoboCycler gradient temperature cycler (Stratagene, La Jolla, Calif).

Agarose gel eletrophoresis Agarose gel eletrophoresis was performed for the detection of PCR product. After PCR reaction, 6µl of each reaction mixture was subjected to electrophoresis on a 1% agarose gel (11 by 14 cm) using a horizontal electrophoresis apparatus (Horizon 11.14, Life Technologies/Gibco-BRL). The gel containing the amplified DNA was stained with ethidium bromide and visualized with a UV transilluminator, and images of the transilluminator were digitized with a one –dimensional gel documentation system (Bio-Rad) and the DNA profile of the organism was determined.

Results The present study was performed for identification of Aeromonas spp. by genotyping and phenotyping characteristics. Isolates of Aeromonas were isolated from Dhaka, Narayanganj, and Tongi area.

Isolation of Aeromonas spp. by cultural method For isolation of Aeromonas spp., after 24 hours of incubation typical colonies having the following characteristics on TCBS, TTGA, and MacConkey media (Table:3.1) were presumptively selected as Aeromonas spp.

Table 3.1: Colony characteristics on TCBS , TTGA and MacConkey media Medium TCBS TTGA MacConkey

Colony morphology

Colony size (mm)

Circular, Yellow, shiny

1-4

Circular, Grey, flattened opaque zone around

1-2

Circular, flat with entire margin, non mucoid, lactose non fermenter

1-4

The colonies that appeared with typical characteristics of Aeromonas spp. in both TCBS and TTGA media (Fig: 3.1) were selected and then subcultured onto gelatinase agar medium to check their ability for gelatinase production (Table: 3.2, Fig: 3.1) and their response to Vibrio Static Compound (VSC) 10 and 150 µg.

Table 3.2: Colony characteristics of Aeromonas spp. on GA medium VSC property Medium

Gelatin Agar

Colony characteristics

Smooth, opaque, white and presence of gelatinase

10µg

150µg

R

R

Colony size (mm)

2-4

TCBS

TTGA

GA Fig 3.1: Colony characteristics of Aeromonas on TCBS, TTGA, and GA plate Microscopic observation of Aeromonas spp.

All the isolates exhibited similar staining patterns. The isolates were found to be Gramnegative, rod shaped that is characteristic of Aeromonas spp.

Fig 3.2: Gram-staining of the isolate of Aeromonas spp (Thornley et al.)

Biochemical studies Aeromonas spp. were identified by conventional biochemical tests. Initially, strains were screened by a cytochrome oxidase test. Cytochrome oxidase-positive isolates were identified to the species level by their sensitivity to vibrio-static compound (2,4-diamino-6, 7diisopropylpteridine phosphate), Kligler Iron Agar (KIA), Motility Indole Urea, and Citrate utilization test. Using these conventional biochemical reactions, environmental isolates were identified as Aeromonas spp. Table 3.3 shows the biochemical responses obtained with the isolated strains of Aeromonas.

Table 3.3: Biochemical properties of the isolated Aeromonas spp.

Strain ID

Oxidase

KIA MIU Slant Butt H2S Mot In d

Citrate VSC Urea Utilization (µg/ml 10 15 0

Salt tolerance %

Control Strain Env-01

+

K

A

-

+

+

-

+

R

R

+

6.

5 -

+

K

A

-

+

+

-

+

R

R

+

-

-

Env-02

+

K

A

-

+

+

-

+

R

R

+

-

-

Env-03

+

K

A

-

+

+

-

+

R

R

+

-

-

Env-04

+

K

A

-

+

+

-

+

R

R

+

-

-

Env-05

+

K

A

-

+

+

-

+

R

R

+

-

-

Env-06

+

K

A

-

+

+

-

+

R

R

+

-

-

KIA: Kligler Iron Agar test MIU: Motility Indole Urea test VSC: Vibrio Static compound

• K : Alkaline reaction • A: Acidic reaction • R: Resistant • +: Positive • -: Negative

Env: Environmental isolate Biochemical characteristics of Aeromonas spp.

(A)

(B)

0

8 -

(C)

(D)

1

(E)

2

Fig 3.3: Typical Biochemical Characteristics of Aeromonas spp. (A) Motility test, (B) Indole test, (C) Oxidase test, (D) Citrate test, (E) KIA test (1. Alkaline slant, acidic butt, no gas, 2. uninoculated).

3.4. Antibiotic susceptibility test All strains (n = 6) of Aeromonas spp. were tested for their antibiotic susceptibility against the five commonly prescribed antibiotics belonging to different groups was used. All of the isolates were found to be resistant to Ampicillin (Amp) and showed sensitivity to other antibiotics. These results are listed in table: Table 3.4: Antibiotic susceptibility pattern of the isolated Aeromonas strains Antibiotic susceptibility pattern Strain ID CIP

E

Env - 01

S

S

Env - 02

S

Env - 03 Env -04

AMP

CN

SXT

FR

R

S

S

S

S

R

S

S

S

S

S

R

S

S

S

S

S

R

S

S

S

Env - 05 Env - 06

S

S

R

S

S

S

S S R S S CIP = Ciprofloxacin; E = Erythromycin; AMP = Ampicillin; CN = Gentamycin; SXT = Trimethoprim Sulphamethoxazole; FR= Furazolidone

S

From the Table 3.4, it was found that antibiotic response of the strains revealed that all of the strains were uniformly sensitive to CIP, E ,CN , SXT, FR and resistant to AMP.

CIP (5) E (15) CN (10) SXT (25) FR (100) AMP(10) Fig 3.4: Antibiotic susceptibility pattern by disc diffusion assay 3.5. Detection of Aeromonas spp. for virulence genes by simplex PCR 3.5.1. Detection of gene aerA (aerolysin) by PCR The hemolysin produced by some Aeromonas species is termed ‘aerolysin’, and it posesses both hemolytic and enterotoxic activity. This hemolytic enterotoxin (aerolysin) has been shown to share significant homology with the cytotoxic enterotoxin (Act), and two cytotonic toxins (Alt and Ast). Aerolysin gene (aerA) contributes to the virulence of Aeromonas. So, the presence of aerA gene indicates that the species is pathogenic. Simplex PCR assay method showed that only 2 out of 6 Aeromonas strain were positive for aerolysin gene.

A B C D E F G M

416 bp

Fig 3.5: Agarose gel electrophoresis showing PCR amplification products of aerA genes.

Key lane: A=Env-01, B=Env-02, C=Env-03, D=Env-04, E=Env-05, F=Env-06 G= Control strain ATCC 7966 (A. hydrophila) M=1 kb plus DNA Ladder (λ DNA digested with Hind III) Table 3.5: PCR result of aerA gene of different isolates Strain ID Env-01 Env-02 Env-03 Env-04 Env-05 Env-06

Lane no A B C D E F

Symbol: +: Positive, -: Negative APPENDIX - Ι APPARATUS ∗ Autoclave ∗ Centrifuge ∗ Disposable micropipette tips ∗ Disposable syringe ∗ Distilled water plant ∗ Drier ∗ Electronic balance

aerA + + -

∗ Electrophoresis power supply ∗ Eppendorf tube ∗ Gel kit and power supply, Life Technologies ∗ Glass wares, Pyrex band, USA ∗ Gel electrophoresis, BioRad ∗ Incubator ∗ Laminar airflow ∗ Micropipette ∗ Oven ∗ Petri dishes, disposable ∗ pH meter ∗ Sterilizer ∗ Slide ∗ Shaker water bath, Germany ∗ Test tube ∗UV-transilluminator ∗ Vortex ∗ Water bath APPENDIX - ΙΙ Microbiological media Media used were prepared by standard methods using appropriate compositions.Components used were high grade and were produced either by Sigma or Difco, USA. All media were sterilized by autoclaving for 20 minutes. The composition used for different media have been shown below:

1. Luria-Bertani (LB) Broth Ingredients Baco-tryptone Baco-yeast extract NaCl Distilled water pH

Amount (g/l) 10.0 5.0 10.0 1.0 liter

adjusted to 7.4

2. Taurocholate Tellurite Gelatin Agar (TTGA)

Ingredients

Amount (g/l)

Tripticase

10.0

NaCl

10.0

Sodium-uiurocholate

5.0

Gelatin

30.0

Agar

16.0

Distilled water

pH adjusted to 8.5-9.0

3. Thiosulphate Citrate Bile Sucrose (TCBS) agar

1.0 liter

Ingredients

Amount (g/l)

Yeast extract

5.0

Peptone

10.0

Sodium thiosulohate

10.0

Sodium citrate

10.0

Bile

8.0

Sucrose

20.0

NaCl

10.0

Ferric citrate

1.0

Brornothymol blue

0.04

Thymol blue

0.04

Agar

14.0

Distilled water

1.0 liter

pH adjusted to 8.6 4. Gelatin Agar Ingredients Amount (g/l) NaCl 10.0 Trypticase (BBL)

10.0

Gelatin

30.0

Agar

10.0

Distilled water

1 liter

pH adjusted to 7.4

5. MacConkey Agar Ingredients Pancreatic digest of gelatin

Amount (g/l) 17.0

Pancreatic digest of casein

1.5

Peptic digest of animal tissue

1.5

Lactose

10.0

Bile Salt

1.5

NaCl

5.0

Agar

13.5

Neutral red

0.03

Crystal violet

0.001

pH adjusted to 7.1 6. Muller-Hinton Agar

Ingredients

Amount (g/l)

Beef infusion

2.0

Bacto casammo acid

17.5

Starch

1.5

Bacto agar

17.5

Distilled water

1 liter

pH adjusted to 8.4 7. T1N1 broth Ingredients

Amount (g/l)

Trypricase NaCl Distilled water

10.0 10.0 1 liter

pH adjusted to 7.5 8. Motility Indole Urea (MIU) Agar

Ingredients Peptone

Amount (g/l) 30.0

Potassium dihydrogen phosphate NaCl

2.0 5.0

Phenol red

2.0 ml

pH pH adjusted to 7.5adjusted to 7.5 pH adjusted to 7.5 9. Kligler Iron Agar (KIA) Ingredients Magnesium sulphate

Amount (g/l) 0.20

Ammonium dihydrogen phosphate

1.0

Dipotassium phosphate

1.0

Sodium citrate

2.0

Sodium chloride

5.0

Bromothymol blue Agar

0.08 15.0

pH adjusted to 7.4 9. Simmon’s Citrate Agar Ingredients

Amount (g/l)

Beef extract

3.0

Yeast extract

3.0

Peptone

15.0

Protease peptone

5.0

Lactose

10.0

Saccharose

10.0

Dextrose

1.0

Ferrous sulphate

0.2

NaCl

5.0

Sodium thiosulphate

0.5

Phenol red

0.024

Agar Distilled water

12.0 1 liter

pH adjusted to 7.4 APPENDIX - ΙΙΙ B Laboratory Reagents Reagents, which were used in carrying out different methods together with their sources, are mentioned below.

Normal saline Ingredients NaCl Distilled water

Amount (g/l) 8.5 1.0 liter

pH adjusted to 7.8

2. Phosphate buffer saline (PBS)

Ingredients

Amount (g/l)

NaCl (sigma)

8.56

Na2HP04

1.18

K2HPO4

0.23

KCL

0.20

Deionised water

800 ml

Gel loading buffer Ingredients Sucrose

Amount (g/l) 6.7

Bromophenol blue

0.04

Stored at 4º C 3. Kovac’s reagent Ingredients

Amount (g/l)

p-dimethyaminobenzaldehyde Amyl alcohol Hydrochloric acid (concentrated)

5.0 75 ml 25 ml

Dissolve the p-dimethyaminobenzaldehyde in the amyl alcohol and add the hydrochloric acid Oxidase reagent Ingredients N’N’N’N’-tetramethyl-pphenylenediamine dihydrochloride

Amount 100 mg/100ml of distilled water

4. Preparation of agarose gel (1.0%) Ingredients

Amount

5% TBE Buffer

75 ml

Agarose

0.75 gm

4. Ethidium bromide solution 1.0 gm of ethidiumbromide was dissolved in distilled water to a final volume of 100 ml. The container was wrapped in aluminium foil and stored at 4 C. 5. Vibro static compound 0/129 75 mg 2,4 diamino 6,7 –diisopropyl peteridine phosphate dissolved in 10 ml of distilled water, which is equivalent to 150 µg.

0.75 ml of the above standard solution was diluted with 10.5 ml of distilled water to make a equivalent of 10 µg concentration. 150 µg and 10 µg discs were prepared by impregnating 20µl standard solutions.

6. Polymerase chain reaction (PCR) reagents Life technologies reaction (PCR) reagents 10 X PCR buffer (200 mM TrisHCL, pH 8.4, 500 mM KCL)

Control primer mix

50 mM MgCl2

Sterile water

10 mM dNTP mix

Silicone oil

Taq DNA polymerase

Molecular weight marker

Reaction mixture for aerA Reagents

Quantity

10 X PCR buffer

2.5 µl

50 mM MgCl2

1.5 µl

2.5 mM dNTP

1.5 µl

Primer 1 (aerA)(10 pm/µl)

1.0 µl

Primer 2 (aerA)(10 pm/µl)

1.0 µl

Taq polymerase

0.2 µl

Template DNA

2.0 µl

Distilled water

15.5 µl

ABBREVIATIONS A APW

Aeromonas Alkaline peptone water

ATCC bp ºC c.f.u cm DNA dNTP EDTA et al e.g F GA g hr Kb KCL KIA LT LB mg ml mm MIU NA n NaCl N/A ND PBS PCR pH R rpm S spp.

ST TTGA TCBS TE CIP AMP CN FR SXT TE µg µm µl % UV

American type culture collection Base pair Degree Celsius Colony forming unit Centimeter Deoxyribonucleic acid Deoxy nucleotide phosphate Ethyltne diamine tetraacetic acid And others Example Forward Gelatin Agar Gram Hour Kilo basepair Potassium chloride Kligler Iron Agar Heat labile toxin Luria Bertani Miligram Mililitre Milimeter Motility Indole Urea Nucleic acid Total number Sodium chloride Not applicable Not done Phosphate buffered saline Polymerase chain reaction Negative logarithom of hydrogen ion Resistant Rotation per minute Sensitive Species Heat stable toxin Taurocholate Tellurite Gelatin Agar Thiosulphate Citrate Bile Salt Agar Tetracycline Ciprofloxacin Ampicillin Gentamicin Furazolidone Trimethoprim -Sulphamethoxazole Tris-EDTA Microgram Micrometer Microlitre Percentage Ultra violet

VSC Vibrio static compound +ve Positive -ve Negative w/v Weight per volume WHO World Health Organization REFERENCES Abeyta, C., Kaysner, C.A., Wekell, M.M., Sullivan, J.J. and Stelma, G.N. (1986) Recovery of Aeromonas hydrophila from oysters implicated in an outbreak of food-borne illness. Journal of Food Protection 49, 643±646.

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