Journal of Medical Microbiology (2004), 53, 527–534
DOI 10.1099/jmm.0.05269-0
Prevalence, serotype distribution, antibiotic susceptibility and genetic profiles of mesophilic Aeromonas species isolated from hospitalized diarrhoeal cases in Kolkata, India S. Sinha,1 T. Shimada,2 T. Ramamurthy,1 S. K. Bhattacharya,1 S. Yamasaki,3 Y. Takeda4 and G. Balakrish Nair1,5 Correspondence
1
National Institute of Cholera and Enteric Diseases, Beliaghata, Kolkata - 700 010, India
G. Balakrish Nair
2
National Institute of Infectious Diseases, Shinjuku-ku, Tokyo 162, Japan
3
Laboratory 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
4
Faculty of Human Life Sciences, Jissen Women’s University, Tokyo 181-8510, Japan
5
International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B), Dhaka - 1212, Bangladesh
[email protected]
Received 31 March 2003 Accepted 9 January 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.
INTRODUCTION 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),
Abbreviations: RAPD, random amplification of polymorphic DNA; UPGMA, unweighted pair group method using arithmetic averages.
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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 527
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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 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 ìg ampicillin (ASBA), followed by incubation at 37 8C 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 inositolnegative) were further tested for resistance to 150 ìg O/129 vibriostatic agent (pteridine) ml1 (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 serotyp-
ing 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 528
(30 ìg) (MacFaddin, 2000) using commercially available antibiotic disc (HiMedia) on Muller–Hinton agar (Difco). Antimicrobial susceptibility. Aeromonas strains were examined for resistance to ampicillin (10 ìg), chloramphenicol (30 ìg), cotrimoxazole (25 ìg), ciprofloxacin (5 ìg), furazolidone (100 ìg), gentamicin (10 ìg), neomycin (30 ìg), nalidixic acid (30 ìg), norfloxacin (10 ìg), streptomycin (10 ìg) and tetracycline (30 ìg) 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 ìl) contained 2.5 ìl 103 PCR buffer (Takara), 2.5 ìl dNTPs (Takara), 10 pmol of each primer, 3 ìl 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 ìg ml1 ). 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 8C for 1 h. After incubation, 10 % cetyl trimethyl ammonium bromide in 0.7 M NaCl was added and the mixture was incubated at 65 8C 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 8C for 30 min. Ribotyping. DNA (10 ìg) 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 8C. Restriction fragments were separated by electrophoresis through a 1.0 % gel at 30 V cm1 for 12 h at room temperature and transferred onto a Hybond-N þ 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 (59AACGCGCAAC) (Akopyanz et al., 1992) in 25 ìl reaction mixture containing 2.5 ìl 103 PCR buffer, 20 ng Aeromonas genomic DNA, 2.5 ìl of 25 ìM MgCl2 , 20 pmol primer, 1.5 U r-Taq DNA polymerase and 2.5 ìl of 2.5 mM dNTPs using an automated thermal cycler (Perkin Elmer). The cycling program was 94 8C for 1 min, 36 8C for 1 min and 72 8C for 2 min, continued for 45 cycles. After completion, 8 ìl aliquots of products were electrophoresed in 1 % agarose gel containing 0.5 ìg
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Characteristics of Aeromonas spp. isolated in India ethidium bromide ml1 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 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. 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. Pathogen(s) present Aeromonas spp. alone Aeromonas spp., V. cholerae O1 Aeromonas spp., V. cholerae O139 Aeromonas spp., V. cholerae non-O1, non-O139 Aeromonas spp., V. parahaemolyticus Aeromonas spp., rotavirus Aeromonas spp., Shigella spp. Aeromonas spp., ETEC Aeromonas spp., EPEC Aeromonas spp., V. cholerae O1, EAEC Aeromonas spp., V. cholerae O1, EPEC Aeromonas spp., V. cholerae O1, ETEC Aeromonas spp., V. cholerae O139, EPEC Aeromonas spp., V. parahaemolyticus, EPEC Aeromonas spp., Shigella spp., V. cholerae non-O1, non-O139, EPEC
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Cases (n) 45 13 3 3 1 1 1 3 1 2 1 1 1 1 1
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. Serotype
Strains (n)
O11 O13 O14 O16 O23 O26 O29 O34 O59 O83 O85 O86 Rough O-antigen unknown Not done
3 4 4 17 3 2 3 6 2 10 22 2 4 5 1
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
Table 3. Distribution of various Aeromonas species identified by Aerokey II isolated from hospitalized patients in Kolkata Species
A. caviae A. hydrophila A. veronii bv. sobria A. veronii bv. veronii A. trota A. schubertii A. jandaei Unidentified
Strains (%) 2000
2001
48 (44.8) 19 (17.7) 19 (17.7) 6 (5.6) 7 (6.5) 2 (1.8) 1 (0.93) 4 (3.7)
26 (45.6) 16 (28.1) 6 (10.5) 3 (5.3) 3 (5.3) 2 (3.5) 0 2 (3.5)
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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.
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. 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.
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.
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
Prevalence (%)
100 90 80 70 60 50 40 30 20 10 0
Fig. 1. Antibiotic sensitivity of Aeromonas strains isolated during the study period 20002001. Isolates showing resistance (open bars) and reduced susceptibility (filled bars) are shown.
A Ch Fz T N Nx Na Co S G Cf C A Ch Fz T N Nx Na Co S G Cf C 2000 2001 Antibiotic
Table 4. Presence of toxin genes in Aeromonas strains isolated from diarrhoeal patients Species
A. caviae A. veronii bv. sobria A. hydrophila A. veronii bv. veronii A. trota A. schubertii A. jandaei Unidentified
530
Strains (n)
74 25 35 9 10 4 1 6
Toxin gene (%) alt
act
ast
hlyA
aer
72 (97.3) 12 (48.0) 31 (88.6) 7 (77.7) 5 (50.0) 3 (75.0) 1 (100.0) 5 (83.3)
1 (1.4) 21 (84.0) 14 (40.0) 7 (77.7) 0 2 (50.0) 0 3 (50.0)
1 (1.4) 2 (8.0) 0 0 1 (10.0) 0 0 0
3 (4.1) 6 (24.0) 31 (88.6) 0 1 (10.0) 1 (25.0) 0 4 (66.7)
3 (4.1) 1 (4.0) 10 (28.6) 0 0 0 0 0
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Characteristics of Aeromonas spp. isolated in India 0·14
0·40
0·60
A
B
C
0·80
1·00 AE38,O11,AChFzCfGCoNaNz alt AE31,O16,At,ChFzNaCoC AN37,O83,AH,AS alt,act AN36,O16,AC,AChNaSF alt AE23,O16, AC AChNaCoFz alt AE3,O16,AC,AChNaS alt AE42,O16,AC,AChCfSCoNaNxTFz alt AE47,O16,AC,AChCfSCoNaNxTFz, alt,ast AE41,O85,AVbS,AFzNa act AN35,O85,AT,ChFzNaCoSC AN50,OUK,AVbS,AChNNaNxCo alt,act AN29,R,AC,AChFzNaCoS alt AE16,O83,AVbS,AFz act,hylA AN3,O16,AC,AChNaCCfGSCoNxNTFz alt AE34,O85,AC,AChFzNaCoCfNxTS alt AE48,O85,AS,ChFzNa alt AE53,O28,AH,AChNaS act,hlyA AE55,O34,AH,AChFzSCoNa alt,hlyA AE44,O85,AH,AChFzSNa alt AE39,O85,AC,AChFzCfGCoNaNxTSC alt AE37,O83,AH,AChFz alt,hlyA AE21,O85,AVbS,AFzNa alt,hylA AN25,O83,AH,AChFz act,hylA AN52,OUK,AC,AChFzSNa alt,aer AE19,O85,AC,AChFz alt AN51,OUK,AJ,AChFz alt,act,hlyA AN28,O85,AC,ACh act AN44,O16,AVbS,AFzNaCoS act AE24,O85,AC,AChFzNaCoCfNxT alt AE11,O16,AC,ACh alt AN21,O85,AC,AChFzNaC alt AN5,O16,AC,AChNaCoCfSNxFz alt AE29,R,AVbS,AFzNaS alt,act,ast AE25,O85,AVbS,AFz act,hlyA AN32,O83,AH,AChNa alt,hlyA AN24,O83,AC,AChFzNaCoT alt,hlyA AN31,O83,AH,AChFzNaCoT alt,hlyA AE57,O34,AH,AChCoS alt,hlyA AN22,O85,AC,AChFz alt AN11,O34,AH,AChNa alt,hlyA AN1,O34,AH,AChNaFz alt,hlyA AN2,O34,AH,AChNaNx alt,hlyA
DISCUSSION 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 http://jmm.sgmjournals.org
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.
(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. 531
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0·40
A
B
0·60
0·80
1·00 AN51,OUK,AJ,AChFz alt,act,hlyA AN52,OUK,AC,AChFzSNa alt,aer AE24,O85,AC,AChFzNaCoCfNxT alt AN5,O16,AC,AChNaCoCfSNxFz alt AE3,O16,AC,AChNaS alt AE23,O16,AC,AChNaCoFz alt AE11,O16,AC,ACh alt AE29,R,AVbS,AFzNaS alt,act,ast AE21,O85,AVbS,AFzNaS act,hlyA AE25,O85,AVbS,AFz act,hlyA AE34,O85,AC,AChFzNaCoCfNxTS alt AE53,O28,AH,AChNaS act,hlyA AN36,O16,AC,AChNaSFz alt AE16,O83,AVbS,AFz act,hlyA AN21,O85,AC,AChFzNaC alt AE55,O34,AH,AChFzSCoNa alt,hlyA AN44,O16,AVbS,AFzNaCoS act AN31,O83,AH,AChFzNaCoT alt,hlyA AN24,O83,AC,AChFzNaCoT alt,hlyA AE47,O16,AC,AChCfSCoNaNxTFz alt,ast AE42,O16,AC,AChCfSCoNaNxTFz alt AE19,O85,AC,AChFz alt AN50,OUK,AVbS,AChNNaNxCo alt,act AE57,O34,AH,AChCoS alt,hlyA AN25,O83,AH,AChFz alt,hlyA AN1,O34,AH,AChNaFz alt,hlyA AN11,O34,AH,AChNa alt,hlyA AN3,O16,AC,AChNaCCfGSCoNxNTFz alt AE41,O85,AC,AFzNa act AN28,O85,AC,ACh act AE37,O83,AH,AChFz alt,hlyA AN32,O83,AH,AChNa alt,hlyA AE39,O85,AC,AChFzCfGCoNaNzTSC alt AE44,O85,AH,AChFzSNa alt AE48,O85,AS,ChFzNa alt AN37,O83,AH,AS alt,act AN2,O34,AH,AChNaNx alt,hlyA AN29,R,AC,AChFzNaCoS alt AN22,O85,AC,AChFz alt AE31,O16,At,ChFzNaCoC AN35,O82,At,ChFzNaCoS
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 combi532
Fig. 3. Dendrogram generated by UPGMA summarizing the similarity of RAPD-PCR profiles of Aeromonas strains. See legend to Fig. 2 for details.
nation 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 Journal of Medical Microbiology 53
Characteristics of Aeromonas spp. isolated in India
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 nonhaemolytic 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.
Echeverria, P., Blacklow, N. R., Sanford, L. B. & Cukor, G. C. (1981).
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.
gastroenteritis. Lancet ii, 1304–1306.
ACKNOWLEDGEMENTS This work was supported by the Japan International Cooperation Agency (JICA/NICED project 054-1061-E.O).
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