J Med Microbiol Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sinha, S.
Right arrow Articles by Nair, G. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sinha, S.
Right arrow Articles by Nair, G. B.
Agricola
Right arrow Articles by Sinha, S.
Right arrow Articles by Nair, G. B.
J Med Microbiol 53 (2004), 527-534; DOI: 10.1099/jmm.0.05269-0
© 2004 Society for General Microbiology
ISSN 0022-2615

Prevalence, serotype distribution, antibiotic susceptibility and genetic profiles of mesophilic Aeromonas species isolated from hospitalized diarrhoeal cases in Kolkata, India

S. Sinha1, T. Shimada2, T. Ramamurthy1, S. K. Bhattacharya1, S. Yamasaki3, Y. Takeda4 and G. Balakrish Nair1,5

1National Institute of Cholera and Enteric Diseases, Beliaghata, Kolkata - 700 010, India 2National Institute of Infectious Diseases, Shinjuku-ku, Tokyo 162, Japan 3Laboratory of International Prevention of Epidemics, Department of Veterinary Sciences, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, Sakai-shi, Osaka 599-8531, Japan 4Faculty of Human Life Sciences, Jissen Women's University, Tokyo 181-8510, Japan 5International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B), Dhaka - 1212, Bangladesh

Correspondence G. Balakrish Nair gbnair{at}icddrb.org

Received March 31, 2003
Accepted January 9, 2004

A comprehensive study was performed to examine incidence, species distribution, drugs sensitivity, virulence genes and molecular fingerprints of Aeromonas species isolated from patients with acute diarrhoea over a period of 2 years in Kolkata, India. Following the Aerokey II scheme, more than 95 % of strains were identified to species level. Seven different species were encountered in this study, with Aeromonas caviae being dominant, followed by Aeromonas hydrophila and Aeromonas veronii biovar sobria. Thirty different serotypes were encountered, with O16, O83 and O85 being dominant, but no serotype was associated specifically with a single species. The majority of Aeromonas strains exhibited multidrug resistance. The alt and act genes, which encode heat-labile cytotonic and cytotoxic enterotoxins, were respectively found in 71.9 and 20.1 % of strains examined. Only 2.4 % of strains carried the heat-stable cytotonic enterotoxin (ast) gene. The hlyA gene was found in 28 % of Aeromonas strains. With few exceptions, genomic diversity of Aeromonas strains belonging to the same serotype was observed by random amplification of polymorphic DNA PCR and ribotyping. Different species of Aeromonas and different clones of Aeromonas species seem to be associated with hospitalized cases of diarrhoea in Kolkata, India.

Abbreviations: RAPD, random amplification of polymorphic DNA; UPGMA, unweighted pair group method using arithmetic averages.


   INTRODUCTION
TOP
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Aeromonas species comprise mesophilic motile and psychrophilic non-motile Gram-negative organisms. Many studies have demonstrated that Aeromonas species are distributed universally in fresh-water environments and are widely isolated from clinical, environmental and food samples, where they can survive and multiply even at low temperatures (Pin et al., 1996). Among bacterial aetiological agents of diarrhoea, Aeromonas is increasingly recognized as an enteric pathogen (Khan & Cerniglia, 1997). A strong association between gastroenteritis and Aeromonas species has been shown in children (Gracey et al., 1982; Agger et al., 1985), adults older than 60 years (Echeverria et al., 1981) and in cases of ‘traveller's diarrhoea’ (Echeverria et al., 1981). It is estimated that aeromonads may cause up to 13 % of reported gastroenteritis cases in the United States (Buchanan, 1984).

The taxonomy of aeromonads has been in a state of constant flux. Studies conducted over the past few years have provided some clarification in the systematics of Aeromonas species with respect to the number of DNA hybridization groups (genospecies) and phenotypic species (phenospecies) (Carnahan et al., 1991b). Currently, the number of species recognized within the genus has increased to 14 (Janda & Abbott, 1998). Despite this increase in the number of genospecies, only seven are currently recognized as human pathogens (Carnahan et al., 1991b). Aerokey II is a reliable and accurate system for identification of most of the currently recognized Aeromonas species isolated from clinical specimens (Carnahan et al., 1991b). A significant number of virulence genes have been described among Aeromonas species, including aerolysin, haemolysin, enterotoxins, proteases and haemagglutinins (Thornley et al., 1997). These virulence markers are useful to distinguish between potentially pathogenic and non-pathogenic strains. About 6.5 % of diarrhoeal cases in the southern part of India have been attributed to Aeromonas (Komathi et al., 1998), which indicates an urgent need for information on the causal role of this pathogen in other parts of the country. However, in India, information on the incidence and the phenotypic and genotypic characteristics of aeromonads is scanty (Misra, 1990). The present study reports on the aetiology, species distribution, antibiotic-susceptibility patterns and virulence gene markers of Aeromonas species isolated from hospitalized patients with diarrhoea in Kolkata, India.


   METHODS
TOP
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Isolation and presumptive identification.

Stool samples or rectal swabs collected from diarrhoeal patients admitted to the Infectious Diseases Hospital and the B. C. Roy Memorial Hospital for children in Kolkata, India, were screened for Aeromonas between January 2000 and December 2001. Clinical details of hospitalized patients in the Infectious Diseases Hospital were recorded on a standard form, and included type of diarrhoea, fever and dehydration status. Stool specimens were first inoculated into alkaline peptone water [1 % bacto peptone (Difco), 1 % NaCl, pH 9.0] and incubated overnight with shaking at 100 r.p.m. (Firstek Scientific). One loopful of enriched sample was plated onto xylose/deoxycholate/citrate agar (XDCA) (Millership & Chattopadhyay, 1984) and sheep-blood agar (5 % sheep blood) supplemented with 30 µg ampicillin (ASBA), followed by incubation at 37 °C overnight. Non-xylose-fermenting colourless colonies on XDCA and ampicillin-resistant haemolytic colonies on ASBA were tested for cytochrome c oxidase activity by the Kovacs method (Cowan, 1979). Oxidase-positive colonies were examined using a multitest medium (Kaper, 1979). Strains yielding alkaline slant and an acid butt reaction (mannitol- and ornithine decarboxylase-positive and inositol-negative) were further tested for resistance to 150 µg O/129 vibriostatic agent (pteridine) ml–1 (Sigma). Presumptively identified Aeromonas strains were stored in nutrient agar as stabs at room temperature. Owing to the reported increased incidence of pteridine-resistant Vibrio cholerae (Ramamurthy et al., 1992), all presumptively identified Aeromonas strains were examined by V. cholerae-specific ompW PCR (Nandi et al., 2000) to exclude the possibility of misidentification of V. cholerae as Aeromonas sp. Stool/swab samples were also examined for other common enteric pathogens including V. cholerae, Vibrio parahaemolyticus, Salmonella species, Shigella species, diarrhoeagenic Escherichia coli, parasites and rotavirus following standard procedures (WHO, 1987).

Serotyping.

Aeromonas strains were further characterized by serotyping using the antigenic typing scheme of Sakazaki & Shimada (1984), which currently recognizes 97 different somatic (O) antigens of Aeromonas species.

Speciation.

Serotyped strains were identified by biochemical tests advocated by the Aerokey II identification scheme for Aeromonas species (Carnahan et al., 1991b). All strains were examined for hydrolysis of aesculin, acid production from arabinose and sucrose, production of gas from glucose, indole formation, Voges–Proskauer reaction by conventional tube test method and resistance to cephalothin (30 µg) (MacFaddin, 2000) using commercially available antibiotic disc (HiMedia) on Muller–Hinton agar (Difco).

Antimicrobial susceptibility.

Aeromonas strains were examined for resistance to ampicillin (10 µg), chloramphenicol (30 µg), cotrimoxazole (25 µg), ciprofloxacin (5 µg), furazolidone (100 µg), gentamicin (10 µg), neomycin (30 µg), nalidixic acid (30 µg), norfloxacin (10 µg), streptomycin (10 µg) and tetracycline (30 µg) using commercial discs (HiMedia). E. coli strain ATCC 25922, sensitive to all the antibiotics used, was included for quality control. Characterization of strains as susceptible, resistant or having reduced susceptibility was done in accordance with the manufacturer's instructions on sizes of inhibition zones around each disc, which matched the interpretive criteria recommended by the NCCLS (2002).

PCR assay.

PCR assays were performed to detect various genes encoding heat-labile cytotonic enterotoxin (alt) (Granam et al., 1998), heat-stable cytotonic enterotoxin (ast) (A. K. Chopra, personal communication), cytotoxic enterotoxin (act) (A. K. Chopra, personal communication), haemolysin (hlyA) (Heuzenroeder et al., 1999) and aerolysin (aer) (Pollard et al., 1990) (primers designed specifically for Aeromonas hydrophila). Each reaction mixture (25 µl) contained 2.5 µl 10x PCR buffer (Takara), 2.5 µl dNTPs (Takara), 10 pmol of each primer, 3 µl boiled template DNA from overnight broth culture of each strain in LB and 1 U r-Taq DNA polymerase (Takara). The strain A. hydrophila SSU (Albert et al., 2000), which harbours alt, ast and act, was used as positive control. PCR mixture without template DNA was used as a negative control. PCR was performed in an automated thermal cycler (Perkin Elmer). Amplicons were visualized after electrophoresis in a 2 % agarose gel stained with ethidium bromide (0.5 µg ml–1).

DNA extraction.

A modification of the method of Murray & Thompson (1980) was used for DNA extraction from Aeromonas strains. In brief, cells from an 18 h LB culture were collected by centrifugation (12 000 r.p.m. for 10 min) and resuspended in TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 8.0), treated with 10 % (w/v) SDS and freshly prepared proteinase K (Sigma) and incubated at 37 °C for 1 h. After incubation, 10 % cetyl trimethyl ammonium bromide in 0.7 M NaCl was added and the mixture was incubated at 65 °C for 10 min. The aqueous phase was treated with phenol/chloroform and the DNA pellet was washed with 70 % ethanol. Extracted nucleic acid was suspended in TE and treated with RNase at 37 °C for 30 min.

Ribotyping.

DNA (10 µg) from each of 42 strains was digested individually with the restriction enzyme BglI (Takara) according to the conditions recommended by the manufacturer, supplementing the restriction mixture with 16 U enzyme for 18 h at 37 °C. Restriction fragments were separated by electrophoresis through a 1.0 % gel at 30 V cm–1 for 12 h at room temperature and transferred onto a Hybond-N+ membrane (Amersham) as described previously (Faruque et al., 1999). The 7.5-kb BamHI fragment of plasmid pKK3535 containing the 16S and 23S rRNA genes of E. coli was used as the rRNA probe (Brosius et al., 1981), and was labelled using the ECL nucleic acid detection system (Amersham). Southern blots were hybridized with labelled probe and autoradiographs were developed as described previously (Faruque et al., 1999).

DNA fingerprinting.

Random amplification of polymorphic DNA (RAPD)-PCR fingerprinting was performed with primer 1281 (5'-AACGCGCAAC) (Akopyanz et al., 1992) in 25 µl reaction mixture containing 2.5 µl 10x PCR buffer, 20 ng Aeromonas genomic DNA, 2.5 µl of 25 µM MgCl2, 20 pmol primer, 1.5 U r-Taq DNA polymerase and 2.5 µl of 2.5 mM dNTPs using an automated thermal cycler (Perkin Elmer). The cycling program was 94 °C for 1 min, 36 °C for 1 min and 72 °C for 2 min, continued for 45 cycles. After completion, 8 µl aliquots of products were electrophoresed in 1 % agarose gel containing 0.5 µg ethidium bromide ml–1 and photographed under UV light using Gel Doc 2000 (Bio-Rad). A 1-kb ladder (New England Biolabs) was used as a size marker in all gels.

Interpretation of ribotyping and RAPD results.

Using Gel Doc 2000 (Bio-Rad), gel images were stored in a Windows PC. All images were retrieved and aligned using Adobe software and analysed with the Diversity Database fingerprinting software (Bio-Rad). Comparison of differences in patterns of ribotype and RAPD profiles was made to ascertain the clonal relationship between the Aeromonas strains. A dendrogram was constructed with the unweighted pair group method using arithmetic averages (UPGMA) available in the software.


   RESULTS
TOP
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Of 1648 and 1853 stool samples examined from diarrhoeal patients admitted, respectively, to the Infectious Diseases and B. C. Roy Hospitals during 2000 and 2001, 107 (6.5 %) and 57 (3.1 %) were positive for Aeromonas spp. A seasonal trend in incidence of Aeromonas species was evident in 2000, with more frequent isolation during the summer months. Such seasonality was not evident in 2001; instead, few isolations of Aeromonas species were observed in that year. Of 78 diarrhoeal patients admitted to the Infectious Diseases Hospital in 2000 and 2001 from whom Aeromonas were isolated, 45 (57.7 %) were infected with Aeromonas species alone, while the remainder had mixed infections with other enteric pathogens (Table 1). Among these 78 patients, 75.6 % had watery diarrhoea, 21.8 % had fever and 44.9 % of patients suffered from severe dehydration. Thirty different serotypes were found, with O16, O83 and O85 being the dominant types (Table 2). In this study, we encountered four rough and five serologically untypable strains.


View this table:
[in this window]
[in a new window]
  		Table 1. Aetiological agents associated with diarrhoeal patients admitted to the Infectious Diseases Hospital ETEC, Enterotoxigenic E. coli; EAEC, enteroaggregative E. coli; EPEC, enteropathogenic E. coli.
 

View this table:
[in this window]
[in a new window]
  		Table 2. Serotypes of Aeromonas strains isolated from hospitalized patients in Kolkata in 2000 In addition to the serotypes listed, single strains of O2, O3, O6, O7, O9, O24, O25, O28, O32, O33, O35, O38, O44, O48, O50, O54, O81 and O97 were identified.
 

The Aerokey II identification scheme, which includes a battery of seven biochemical tests, identified the majority of Aeromonas isolates (95.3 % in 2000 and 98.2 % in 2001) to the species level. Of seven different species identified, Aeromonas caviae dominated in both years, followed by A. hydrophila and Aeromonas veronii biovar (bv.) sobria (Table 3). In 2000, there was a higher percentage of A. veronii bv. sobria (17.7 %) compared with 2001 (10.5 %). In both years, there were few isolates of A. veronii bv. veronii (Table 3). Strains identified as Aeromonas trota by Aerokey II showed unusual phenotypic characteristics. Six strains were negative for gas production from glucose and two strains were positive for sucrose and arabinose fermentation. One A. trota strain was positive for sucrose fermentation. The typical characteristics of A. schubertii are negative for sucrose and arabinose fermentation and no gas production from glucose. In this study, four A. schubertii strains exhibited unusual reactions; one strain fermented both sucrose and arabinose, one produced gas from glucose and two fermented sucrose and produced gas from glucose. Of 74 A. caviae strains, one was negative for acid production from arabinose and another was negative for indole production. Aerokey II did not identify six strains at the species level that were presumptively identified as Aeromonas.


View this table:
[in this window]
[in a new window]
  		Table 3. Distribution of various Aeromonas species identified by Aerokey II isolated from hospitalized patients in Kolkata
 

All 164 strains isolated in this study were tested for antibiotic susceptibility. The drug resistance patterns of strains isolated during 2000 showed remarkable variation compared with strains isolated during 2001 (Fig. 1). Resistance to ampicillin (94.4 and 91.2 % in 2000 and 2001), ciprofloxacin (22.4 and 12.3 %), nalidixic acid (62.8 and 54.4 %) and norfloxacin (19.0 and 14.0 %) was recorded. In 2001, strains resistant to furazolidone (7.0 %) were infrequent compared with 2000 (73.8 %). However, resistance to streptomycin was high (71.9 %) in 2001 compared with 2000 (44.9 %). In 2001, more strains exhibited reduced susceptibility to furazolidone (52.6 %). In both years, the Aeromonas strains also showed reduced susceptibility to ciprofloxacin and norfloxacin.



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 1. Antibiotic sensitivity of Aeromonas strains isolated during the study period 2000–2001. Isolates showing resistance (open bars) and reduced susceptibility (filled bars) are shown.

 

The distribution of various enterotoxin genes among Aeromonas species during the study period is shown in Table 4. Of 164 strains examined, the alt and act genes were respectively found in 71.9 and 20.1 %. However, only 18.9 % of strains had both genes, while 2.4 % carried ast. None of the strains had ast alone. One A. veronii bv. sobria (AE29) strain had all three enterotoxin genes. The presence of alt was common in A. caviae and A. hydrophila, whereas act was detected more frequently in A. veronii bv. sobria. Interestingly, a high percentage of A. veronii bv. veronii had both genes. The hlyA gene was present in 28 % of Aeromonas strains, most of which belonged to the O83 serotype. As shown in Table 4, 10 A. hydrophila, three A. caviae, and one A. veronii bv. sobria strains were positive for the aer gene.


View this table:
[in this window]
[in a new window]
  		Table 4. Presence of toxin genes in Aeromonas strains isolated from diarrhoeal patients
 

Strains representing similar and dissimilar combinations of serotypes and species were selected to determine genetic relatedness by RAPD-PCR (41 strains) and ribotyping (42 strains). With few exceptions, dendrograms generated from the gel profiles of ribotyping and RAPD-PCR showed heterogeneous distributions of strains with respect to species, serotypes, virulence gene profile and antibiogram (Figs 2 and 3). In cluster A in Fig. 2, five A. caviae strains of the O16 serotype clustered together and, except for one, all carried alt. Similarly, all A. caviae strains of cluster A in Fig. 3 also belonged to the O16 serotype and carried alt. In cluster B, 50% of Aeromonas strains belonged to the O85 serotype (Fig. 2). Strains belonging to the O34 serotype (AE57, AN1 and AN11) formed the same cluster by ribotyping (cluster C, Fig. 2) and RAPD (cluster B, Fig. 3). In both clusters, the majority of strains harboured alt and hlyA.



View larger version (71K):
[in this window]
[in a new window]
  		Fig. 2. Dendrogram generated by UPGMA summarizing the similarity of ribotyping profiles of Aeromonas strains. Entries on branches of the tree give strain number, serotype, species (AH, A. hydrophila; AC, A. caviae; AVbS, A. veronii bv. sobria; AS, A. schubertii; At, A. trota and AJ, A. jandaei), antibiogram (Ch, cephalothin; A, ampicillin; Fz, furazolidone; T, tetracycline; N, neomycin; Nx, norfloxacin; Na, nalidixic acid; Co, chloramphenicol; S, streptomycin; G, gentamicin; Cf, ciprofloxacin and C, cotrimoxazole) and gene profile.

 


View larger version (70K):
[in this window]
[in a new window]
  		Fig. 3. Dendrogram generated by UPGMA summarizing the similarity of RAPD-PCR profiles of Aeromonas strains. See legend to Fig. 2 for details.

 


   DISCUSSION
TOP
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGEMENTS
 REFERENCES
 
Aeromonads 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. In this study, we demonstrated that Aeromonas species were associated with acute diarrhoea at least in 57.7 % of patients. It is difficult to predict the importance of Aeromonas in 33 (42.3 %) cases, as these patients were co-infected with other pathogens.

One of the complex problems concerning Aeromonas species is the uncertainty surrounding their taxonomy. Since 1987, a number of novel Aeromonas species has been proposed (Janda & Abbott, 1998). However, the addition of novel species to the genus Aeromonas has contributed to and even exacerbated the existing confusion in the taxonomy. Various taxonomic studies conducted on Aeromonas, and the development of Aerokey II (Carnahan et al., 1991b), have allowed identification of most of the mesophilic clinical aeromonads to phenospecies level. In the present study, more than 95 % of the Aeromonas strains could be characterized into seven different phenospecies.

The prevalence of different species of Aeromonas is likely to vary with geographical locations. A. hydrophila and A. veronii bv. sobria are the dominant species in Australia and Thailand (Altwegg & Geiss, 1989). European and American studies have revealed that the majority of isolates were A. caviae (Altwegg & Geiss, 1989). Like European and American trends, we found that A. caviae is the dominant species in Kolkata, India. However, A. hydrophila and A. veronii bv. sobria were also isolated in significant numbers. A study in southern India has revealed that A. hydrophila is the predominant species (Komathi et al., 1998). In Bangladesh, A. trota was isolated from a large number of diarrhoeal patients (Albert et al., 2000); however, this species was not found in hospitalized diarrhoeal cases in Kolkata. It can be said that variation in geographical distribution may, to a certain extent, reflect the tentativeness of Aeromonas taxonomy and, as unified identification keys were not being followed, the possibility of misidentification of species should not be excluded.

Previous studies unquestionably established the role (>85 % of isolates) of A. hydrophila, A. caviae and A. veronii bv. sobria in diarrhoea (Janda, 1991; Janda et al., 1995). The other diarrhoea-associated isolates were A. trota, A. veronii bv. veronii and Aeromonas jandaei (Janda, 1991; Janda et al., 1995). We have also isolated A. schubertii from hospitalized diarrhoeal patients. In Kolkata, three predominant serotypes were recorded among 105 strains examined. Each species was found to be serologically heterogeneous and no serotype was uniquely associated with any of these species, indicating that Aeromonas-associated diarrhoea is sporadic, similar to infections caused by different V. cholerae non-O1, non-O139 serogroups.

Most of the Kolkata Aeromonas strains showed resistance to nalidixic acid, cephalothin, streptomycin and furazolidone. Variation in resistance to cephalothin can affect identification of aeromonads by Aerokey II (Carnahan et al., 1991b), as resistance to cephalothin is used to differentiate between A. veronii bv. veronii and A. hydrophila. As a result, increasing resistance to this drug will lead to increasing isolations of A. hydrophila and may lead to taxonomic inconsistency. Except for A. trota, ampicillin resistance is characteristic of Aeromonas species (Carnahan et al., 1991a). Use of XDCA enabled us to identify 10 A. trota strains in this study. The detection of reduced susceptibility to norfloxacin, ciprofloxacin and neomycin indicated that aeromonads are becoming resistant to these drugs. Generally, it was observed that the majority of strains exhibited a multidrug-resistance profile. This increased drug resistance presents a significant threat to management of Aeromonas-mediated diarrhoea.

A toxin expression assay was not performed in this study because of the large number of isolates. Instead, we screened all the strains by PCR for different virulence genes. In the PCR assay, Aeromonas strains were found with different virulence gene combinations. The dominant combination of enterotoxin genes in our Kolkata strains was alt (71.9 %) and act (20.1 %); this is in contrast to an earlier study in Bangladesh (Albert et al., 2000), where none of the Aeromonas isolates in Bangladesh was positive for act. The increased presence of ast in Bangladesh might be related to larger numbers of A. trota isolated compared with Kolkata, where only 2.4 % of strains were positive for ast in combination with other virulence genes. In Bangladesh, one A. hydrophila strain had all three enterotoxin genes (Albert et al., 2000), whereas, in Kolkata, all three genes were found only in one A. veronii bv. sobria strain. Generally, alt was predominant in all species isolated in Kolkata, and 84 % of A. veronii bv. sobria harboured act. The act gene is known to stimulate proinflammatory cytokine and eicosanoid cascades in macrophages in the rat intestinal epithelial cell line ICE-6, leading to tissue damage and fluid secretory response (Chopra et al., 2000). There is a good correlation between the cytotonic enterotoxins Alt and Ast and elongation of Chinese hamster ovary cells and production of C-AMP, which is typical enterotoxic activity (Chopra et al., 1994). It was evident from a previous study that Act was the major enterotoxin contributing to fluid secretory response, followed by Alt and Ast in A. hydrophila (Sha et al., 2002). In this study, we found that the majority of the A. veronii bv. sobria strains harboured act. The presence of three enterotoxins in various combinations in different Aeromonas strains could increase or decrease expression of the specific enterotoxin gene and thus dictate the severity of diarrhoea (Sha et al., 2002).

Two haemolytic toxins, haemolysin (Hirono & Aoki, 1991) and aerolysin (Howard et al., 1987), have been described in A. hydrophila. When PCR was performed to detect hlyA and aer, we found that hlyA was mainly associated with A. hydrophila, while 70 % of Aeromonas O83 serotypes harboured hlyA. It was interesting to note that primers designed from the aer gene sequence of A. hydrophila were found to give the expected size of the amplicon with three A. caviae and one A. veronii bv. sobria isolates. Pollard et al. (1990) have shown that PCR amplification with these primers is consistently negative for haemolytic A. sobria and non-haemolytic A. hydrophila and A. caviae. However, in our study, the 10 A. hydrophila and one A. veronii bv. sobria strains exhibited haemolysis on sheep blood agar but the three PCR-positive A. caviae strains were non-haemolytic.

The rationale for performing molecular typing was to understand whether any particular clone of Aeromonas species was more often associated with diarrhoea. Except for a few strains, both ribotyping and RAPD showed that the Kolkata Aeromonas strains were genetically heterogeneous and no particular clone was predominant.


   ACKNOWLEDGEMENTS
TOP
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
REFERENCES
 
This work was supported by the Japan International Cooperation Agency (JICA/NICED project 054-1061-E.O).


   REFERENCES
TOP
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 

  • Agger, W. A., McCormick, J. D. & Gurwith, M. J. (1985). Clinical and microbiological features of Aeromonas hydrophila-associated diarrhea. J Clin Microbiol 21, 909–913.[Abstract/Free Full Text]

  • Akopyanz, N., Bukanov, N. O., Westblom, T. U., Kresovich, S. & Berg, D. E. (1992). DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting. Nucleic Acids Res 20, 5137–5142.[Abstract/Free Full Text]

  • Albert, M. J., Ansaruzzaman, M., Talukder, K. A. & 7 other authors (2000). Prevalence of enterotoxin genes in Aeromonas spp.isolated from children with diarrhea, healthy controls, and the environment. J Clin Microbiol 38, 3785–3790.[Abstract/Free Full Text]

  • Altwegg, M. & Geiss, H. K. (1989). Aeromonas as a human pathogen. Crit Rev Microbiol 16, 253–286.[Medline]

  • Brosius, J., Ullrich, A., Raker, M. A., Gray, A., Dull, T. J., Gutell, R. R. & Noller, H. F. (1981). Construction and fine mapping of recombinant plasmids containing the rrnB ribosomal RNA operon of E.coli. Plasmid 6, 112–118.[CrossRef][Medline]

  • Buchanan, R. L. (1984). The new pathogens.An update of selected examples. Assoc Food Drug Off Q Bull 48, 142–155.

  • Carnahan, A. M., Chakraborty, T., Fanning, G. R., Verma, D., Ali, A., Janda, J. M. & Joseph, S. W. (1991a). Aeromonas trota sp.nov., an ampicillin-susceptible species isolated from clinical specimens. J Clin Microbiol 29, 1206–1210.[Abstract/Free Full Text]

  • Carnahan, A. M., Behram, S. & Joseph, S. W. (1991b). Aerokey II: a flexible key for identifying clinical Aeromonas species. J Clin Microbiol 29, 2843–2849.[Abstract/Free Full Text]

  • Chopra, A. K., Pham, R. & Houston, C. W. (1994). Cloning and expression of putative cytotonic enterotoxin-encoding genes from Aeromonas hydrophila. Gene 139, 87–91.[CrossRef][Medline]

  • Chopra, A. K., Xu, X.-J., Ribardo, D., Gonzalez, M., Kuhl, K., Peterson, J. W. & Houston, C. W. (2000). The cytotoxic enterotoxin of Aeromonas hydrophila induces proinflammatory cytokine production and activates arachidonic acid metabolism in macrophages. Infect Immun 68, 2808–2818.[Abstract/Free Full Text]

  • Cowan, S. T. (1979). Manual for the Identification of Medical Bacteria, 2nd edn. Edited by S. T. Cowan & K. J. Steel. Cambridge: Cambridge University Press.

  • Echeverria, P., Blacklow, N. R., Sanford, L. B. & Cukor, G. C. (1981). Travellers’ diarrhea among American Peace Corps volunteers in rural Thailand. J Infect Dis 143, 767–771.[Medline]

  • Faruque, S. M., Siddique, A. K., Saha, M. N., Asadulghani, Rahman, M. M., Zaman, K., Albert, M. J., Sack, D. A. & Sack, R. B. (1999). Molecular characterization of a new ribotype of Vibrio cholerae O139 Bengal associated with an outbreak of cholera in Bangladesh. J Clin Microbiol 37, 1313–1318.[Abstract/Free Full Text]

  • Gracey, M., Burke, V. & Robinson, J. (1982). Aeromonas-associated gastroenteritis. Lancet ii, 1304–1306.

  • Granam, P. E., O'Sullivan, K., Tomas, J. M. & Ormen, O. (1998). Possible virulence factors of Aeromonas spp.from food and water. FEMS Immunol Med Microbiol 21, 131–137.[CrossRef][Medline]

  • Heuzenroeder, M. W., Wong, C. Y. F. & Flower, R. L. P. (1999). Distribution of two hemolytic toxin genes in clinical and environmental isolates of Aeromonas spp.: correlation with virulence in a suckling mouse model. FEMS Microbiol Lett 174, 131–136.[CrossRef][Medline]

  • Hirono, I. & Aoki, T. (1991). Nucleotide sequence and expression of an extracellular hemolysin gene of Aeromonas hydrophila. Microb Pathog 11, 189–197.[CrossRef][Medline]

  • Howard, S. P., Garland, W. J., Green, M. J. & Buckley, J. T. (1987). Nucleotide sequence of the gene for the hole-forming toxin aerolysin of Aeromonas hydrophila. J Bacteriol 169, 2869–2871.[Abstract/Free Full Text]

  • Janda, J. M. (1991). Recent advances in the study of the taxonomy, pathogenicity, and infectious syndromes associated with the genus Aeromonas. Clin Microbiol Rev 4, 397–410.[Abstract/Free Full Text]

  • Janda, J. M. & Abbott, S. L. (1998). Evolving concepts regarding the genus Aeromonas: an expanding panorama of species, disease presentations, and unanswered questions. Clin Infect Dis 27, 332–344.[Medline]

  • Janda, J. M., Abbott, S. L. & Carnahan, A. M. (1995). Aeromonas and Plesiomonas. In Manual of Clinical Microbiology, 6th edn, pp. 477–482. Edited by P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover & R. H. Yolken. Washington, DC: American Society for Microbiology.

  • Kaper, J. B. (1979). Isolation, ecology and taxonomy of human pathogens in an estuary. PhD thesis, University of Maryland, College Park, MD, USA.

  • Khan, A. A. & Cerniglia, C. E. (1997). Rapid and sensitive method for detection of Aeromonas caviae and Aeromonas trota by polymerase chain reaction. Lett Appl Microbiol 24, 233–239.[CrossRef][Medline]

  • Komathi, A. G., Ananthan, S. & Alavandi, S. V. (1998). Incidence and enteropathogenicity of Aeromonas spp.in children suffering from acute diarrhoea in Chennai. Indian J Med Res 107, 252–256.[Medline]

  • MacFaddin, J. F. (2000). Biochemical Tests for Identification of Medical Bacteria, 3rd edn. Baltimore: Williams & Wilkins.

  • Millership, S. E. & Chattopadhyay, B. (1984). Methods for the isolation of Aeromonas hydrophila and Plesiomonas shigelloides from faeces. J Hyg 92, 145–152.

  • Misra, S. K. (1990). Distribution, speciation, serotype prevalence and toxigenicity of Aeromonas species isolated from clinical and environmental sources in Calcutta, India. PhD thesis, University of Calcutta, India.

  • Murray, M. G. & Thompson, W. F. (1980). Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8, 4321–4325.[Abstract/Free Full Text]

  • Nandi, B., Nandy, R. K., Mukhopadhyay, S., Nair, G. B., Shimada, T. & Ghose, A. C. (2000). Rapid method for species-specific identification of Vibrio cholerae using primers targeted to the gene of outer membrane protein OmpW. J Clin Microbiol 38, 4145–4151.[Abstract/Free Full Text]

  • NCCLS (2002). Performance standards for antimicrobial susceptibility testing, 12th informational supplement. NCCLS document M100-S12. Wayne, PA: National Committee for Clinical Laboratory Standards.

  • Pin, C., Benito, Y., Carcia, M. L., Selgas, D., Tormos, J. & Casas, C. (1996). Influence of temperature, pH, sodium chloride, and sodium nitrate on the growth of clinical and food motile Aeromonas sp.strains. Arch Lebensmittelhyg 47, 35–56.

  • Pollard, D. R., Johnson, W. M., Lior, H., Tyler, S. D. & Rozee, K. R. (1990). Detection of the aerolysin gene in Aeromonas hydrophila by the polymerase chain reaction. J Clin Microbiol 28, 2477–2481.[Abstract/Free Full Text]

  • Ramamurthy, T., Pal, A., Pal, S. C. & Nair, G. B. (1992). Taxonomical implications of the emergence of high frequency of occurrence of 2,4-diamino-6,7-diisopropylpteridine-resistant strains of Vibrio cholerae from clinical cases of cholera in Calcutta, India. J Clin Microbiol 30, 742–743.[Abstract/Free Full Text]

  • Sakazaki, R. & Shimada, T. (1984). O-serogrouping scheme for mesophilic Aeromonas strains. Jpn J Med Sci Biol 37, 247–255.[Medline]

  • Sha, J., Kozlova, E. V. & Chopra, A. K. (2002). Role of various enterotoxin in Aeromonas hydrophila-induced gastroenteritis: generation of enterotoxin gene-deficient mutants and evaluation of their enterotoxic activity. Infect Immun 70, 1924–1935.[Abstract/Free Full Text]

  • Thornley, J. P., Shaw, J. G., Gryllos, I. A. & Eley, A. (1997). Virulence properties of clinically significant Aeromonas species: evidence for pathogenicity. Rev Med Microbiol 8, 61–72.

  • WHO (1987). Manual for Laboratory Identification of Acute Enteric Infections, CDD/83-3/Rev 1987.113.




    This article has been cited by other articles:


    		Home page
    		 J Med MicrobiolHome page
    P. Bhowmik, P. K. Bag, T. K. Hajra, R. De, P. Sarkar, and T. Ramamurthy
    Pathogenic potential of Aeromonas hydrophila isolated from surface waters in Kolkata, India
    J. Med. Microbiol., December 1, 2009; 58(12): 1549 - 1558.
    [Abstract] [Full Text] [PDF]

    		Home page
    		 J Med MicrobiolHome page
    A. Andelova, I. Porazilova, and E. Krejci
    Aeromonas agar is a useful selective medium for isolating aeromonads from faecal samples.
    J. Med. Microbiol., November 1, 2006; 55(Pt 11): 1605 - 1606.
    [Full Text] [PDF]

    		Home page
    		 J. Biol. Chem.Home page
    Y. Rosenfeld, D. Barra, M. Simmaco, Y. Shai, and M. L. Mangoni
    A Synergism between Temporins toward Gram-negative Bacteria Overcomes Resistance Imposed by the Lipopolysaccharide Protective Layer
    J. Biol. Chem., September 29, 2006; 281(39): 28565 - 28574.
    [Abstract] [Full Text] [PDF]

    This Article
    Right arrow Abstract Freely available
    Right arrow Full Text (PDF)
    Right arrow Alert me when this article is cited
    Right arrow Alert me if a correction is posted
    Right arrow Citation Map
    Services
    Right arrow Email this article to a friend
    Right arrow Similar articles in this journal
    Right arrow Similar articles in PubMed
    Right arrow Alert me to new issues of the journal
    Right arrow Download to citation manager
    Right arrow reprints & permissions
    Citing Articles
    Right arrow Citing Articles via HighWire
    Right arrow Citing Articles via CrossRef
    Right arrow Citing Articles via Google Scholar
    Google Scholar
    Right arrow Articles by Sinha, S.
    Right arrow Articles by Nair, G. B.
    Right arrow Search for Related Content
    PubMed
    Right arrow PubMed Citation
    Right arrow Articles by Sinha, S.
    Right arrow Articles by Nair, G. B.
    Agricola
    Right arrow Articles by Sinha, S.
    Right arrow Articles by Nair, G. B.

    HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
    INT J SYST EVOL MICROBIOL J MED MICROBIOL MICROBIOLOGY J GEN VIROL ALL SGM JOURNALS