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Characterization of aeromonads isolated from the river Narmada, India
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Int. J. Hyg. Environ.-Health 208 (2005) 425–433 www.elsevier.de/ijheh
Characterization of aeromonads isolated from the river Narmada, India Anjana Sharma, Nidhi Dubey, Bandana Sharan Bacteriology Laboratory, Department of Post Graduate Studies and Research in Biological Sciences, R.D. University, Jabalpur (M.P.), India Received 2 September 2004; received in revised form 4 March 2005; accepted 15 March 2005
Abstract Thirty isolates of Aeromonas species were isolated from river Narmada at Jabalpur during the period of January 2002–December 2002. Diversity of these fresh water isolates were determined by randomly amplified polymorphic DNA (RAPD) analysis. These environmental isolates were found to be positive for virulence factors, i.e. protease, amylase, lipase and DNase. Isolates were also positive for b-hemolytic activity. All Aeromonas species were tested for antibiotic resistance patterns and were found to be resistant to ampicillin and sensitive to gentamycin. r 2005 Elsevier GmbH. All rights reserved. Keywords: Aeromonas; Fresh water; RAPD; Virulence factors
Introduction Aeromonas species are ubiquitous aquatic organisms, primary pathogens for cold-blooded animals and opportunistic pathogens for warm-blooded animals. The health consequences of the presence of motile species of the genus Aeromonas in water are the subject of much debate (Schubert, 1991; Janda and Abbott, 1998; Joseph and Carnahan, 2000). Recent studies have demonstrated that the presence of Aeromonas species in drinking water is a potential risk since these microorganism can produce a wide range of virulence factors (Schubert, 1991; Kersters et al., 1996; Kuhn et al., 1997; Ormen and Ostensvik, 2001). Aeromonas associated gastroenteritis has been reported from all over the world but the incidence is relatively low in developed countries as compared to that in developing countries (Altwegg Corresponding author. Tel.: +91 761 2416667; fax: +91 761 2603752. E-mail address: [email protected] (A. Sharma).
1438-4639/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijheh.2005.03.007
and Geiss, 1989). In India, Aeromonas associated diarrhoea has been reported from Bombay, Calcutta, Goa, Vellore, Pondicherry and Chennai (Chatterjee and Neogy, 1972; Bhat et al., 1974; Deodhar et al., 1991; Sujatha and Rao, 1993; Verenkar et al., 1995; Alavandi et al., 1998; Alavandi and Ananthan, 2003; Komathi et al., 1998). People from India and other developing countries use river water directly not only for drinking but also for various recreational purposes. Narmada is the fifth largest river of India and originates from Amarkantak (M.P.) and merges in the Arabian Sea at Bharuch (Gujarat). The total length of the river is 1312 km. Information about the occurrence of Aeromonas in fresh water environments in India, especially in Central India (Sharma et al., 2002), prompted this study. Biochemical and molecular characterization along with antibiograms of environmental Aeromonas strains that were isolated from river Narmada near Jabalpur (Madhya Pradesh) in Central India are described.
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Materials and methods
Screening for virulence genes of aeromonads
Bacterial strains
Detection of genes encoding for heat labile cytotonic enterotoxin (alt), cytotoxic enteroxin (act) and hemolysin (hly A) was performed following the method of Granam et al. (1998) and Heuzenroeder et al. (1999).
Fresh water samples were collected from four different sites of river Narmada: Gwarighat, Lamhetaghat, Tilwaraghat and Jelharighat at Jabalpur during January 2002–December 2002. Samples of 500 ml were brought to the laboratory under ice-cold conditions once a month. They were inoculated in glutamate starch phenol red agar (GSP) medium (Kielwein, 1969) and incubated at 37 1C for 24–48 h. Yellow coloured colonies on GSP medium were maintained on nutrient agar slants at 4 1C, after subculturing several times.
Physiological and biochemical characterization of aeromonads
Antibiotic resistance Resistance to 14 different antibiotics was tested by the agar diffusion method on nutrient agar medium with test discs from SPAN diagnostics, combi disc, code 14346 (PSAMDST, 1993).
Results Biochemical differentiation and identification
Cultures were grown at 37 1C. Cell morphology and Gram stains were determined after 24 h of incubation on nutrient agar. Motility was determined after 18 h in motility test medium (Mac Faddin, 1980). Phenotypic characterization of each strain was determined by the method described by Popoff (1984).
Molecular characterization Randomly amplified polymorphic DNA (RAPD) analysis was done by following the method described by Brocius and Raker (1981).
Screening for extracellular virulence factors of aeromonads The enzymes amylase, gelatinase, lipase and DNase were detected on the following media: starch agar medium (containing 1% starch, 5 g peptone, 3 g beef extract, 5 g NaCl, 20 g agar and 1000 ml distilled water), gelatin agar medium (containing 0.4% gelatin, 5 g peptone, 3 g beef extract, 5 g NaCl, 20 g agar and 1000 ml distilled water), tributyrin agar medium (containing 1% tributyrin, 5 g peptone, 3 g yeast extract, 20 g agar and 1000 ml distilled water) and DNase agar medium (containing 0.2% DNA, 5 g peptone, 15 g tryptone, 5 g NaCl and 1000 ml distilled water) (Mac Faddin, 1980; Vermelho et al., 1996; Thomas et al., 2003).
Bioassay for hemolytic and cytotoxic activity Aeromonads were screened for their hemolytic activity on blood agar medium using human erythrocytes (Honda and Miwatani, 1988). Cytotoxic activity was determined on the on HeLa cells by the method of Kuhn et al. (1997).
Thirty strains isolated from 168 water samples had phenotypic characteristics of the genus Aeromonas (Popoff, 1984). All isolates were Gram negative, motile, rod shaped and facultatively anaerobes. The strains were oxidase and catalase positive and did not tolerate NaCl concentrations higher than 3.0%. Out of 30 isolates, six were identified as A. hydrophila (BGCC # 174, 175, 176, 177, 178, 179), four as A. sobria (BGCC # 180, 181, 182, 183), three as A. eucrenophila (BGCC # 184, 185, 186), two as A. jandaei (BGCC # 187, 188), three as A. caviae (BGCC # 189, 190, 191), six as A. veronii bv veronii (BGCC # 192, 193, 194, 195, 196, 197) and six as A. veronii bv sobria (BGCC # 198, 199, 200, 201, 202, 203) using biochemical tests (Table 1). These isolates were maintained in the bacteriology lab, Department of Biosciences, RDVV, Jabalpur and were given BGCC (Bacterial Germplasm Collection Centre) numbers.
Molecular characterization RAPD analysis of all 30 isolates exhibited numbers of bands ranging from 4 to 11 with molecular weight between 400 bp and 4.0 kb (Fig. 1).
UPGMA cluster analysis on the basis of biochemical and molecular characterization UPGMA cluster analysis on the basis of biochemical tests depicted that the strains formed several clusters at a Euclidean distance mostly of 1–2.67, which related them at species level (Fig. 2). Genetic differentiation among the 30 isolates on the basis of RAPD analysis is presented in Fig. 3, which shows two major clusters I and II. Cluster I represented A. sobria (BGCC # 183), while cluster II was further
Table 1.
Identification of aeromonads isolated from river Narmada at Jabalpur into different genomospecies based on morphological, cultural and biochemical characteristics
Isolate no.
Morphological characteristics
Cultural Biochemical characteristics characteristics
1
3
2
4
5
6
7
8
9
Identified species
10 11 12 13 14 15 16 17 18 19 I
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + +
+ + +
+
+ + + + + +
+ + + + +
+ + + + +
+ + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + +
+ + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + +
+ + + + +
A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A.
hydrophila hydrophila hydrophila hydrophila hydrophila hydrophila sobria sobria sobria sobria eucrenophila eucrenophila eucrenophila jandaei jandaei caviae caviae caviae veronii bv veroni veronii bv veroni veronii bv veroni veroni bv veronii veroni bv veronii veroni bv veronii veronii bv sobriai veronii bv sobria veronii bv sobria veronii bv sobria veronii bv sobria veronii bv sobria
1—Gram staning, 2—shape, 3—motility, 4—salt tolerance test (6% NaCl), 5—oxidase, 6—lysine decarboxylase, 7—ornithine decarboxylase, 8—arginine dihydrolase, 9—arabinose fermentation, 10—mannitol fermentation, 11—sorbitol fermentation, 12—salicin fermentation, 13—sucrose fermentation, 14—inositol fermentation, 15—indole, 16—Voges prousker, 17—catalase, 18—citrate utilization, 19—acid production (I—glucosefermentation (TSI), II—glucose, lactose and sucrose fermentation (TSI)), 20—gas production (TSI), 21—lead sulphide production (TSI), 22—H2S production (TSI), 23—starch hydrolysis, 24—gelatin hydrolysis, 25—bile esculine hydrolysis (Aesculin).
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Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod
II
A. Sharma et al. / Int. J. Hyg. Environ.-Health 208 (2005) 425–433
BGCC # 174 BGCC # 175 BGCC # 176 BGCC # 177 BGCC # 178 BGCC # 179 BGCC # 180 BGCC # 181 BGCC # 182 BGCC # 183 BGCC # 184 BGCC # 185 BGCC # 186 BGCC # 187 BGCC # 188 BGCC # 189 BGCC # 190 BGCC # 191 BGCC # 192 BGCC # 193 BGCC # 194 BGCC # 195 BGCC # 196 BGCC # 197 RGPP # 198 RGPP # 199 BGCC # 200 BGCC # 201 BGCC # 202 BGCC # 203
20 21 22 23 24 25
427
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8 kb 7 kb 6 kb 5 kb 4 kb
8 kb 7 kb 6 kb 5 kb 4 kb
3 kb
3 kb
2 kb
2 kb 1.5 kb
1.5 kb
1 kb
1 kb
500 bp
500 bp
M 1 2 3 4 5 6 7 8 9 10 11 1213141516171819202122 23 24 25 26 27 28 29 3031 M
Fig. 1. Randomly amplified polymorphic DNA (RAPD) of aeromonads isolated from the river Narmada at Jabalpur.
x
x2 x3
c1 c
f
f1
y
f2
a1
b1
d
d1 d2 d3
b2 A
a2
h i
j I
A2
k
c a B
d b
0.00
x1
1 4 23 29 27 6 21 19 20 28 7 9 10 8 14 15 30 13 18 25 11 12 16 17 24 2 31 3 5 22 0.67
1.34 Coefficient
2.00
2.67
Fig. 2. UPGMA cluster analysis of aeromonads isolated from the river Narmada at Jabalpur on the basis of biochemical tests.
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a2 A1
c1 c b
c2 c3 d
d1 d2
A II
f1 f A2
f2 e
i h1 I
h
j h2 0.99 Coefficient
1.49
B
0.50
g
g1 g2
0.00
a
a1
1 2 5 11 24 25 27 30 9 12 28 13 14 23 6 7 8 21 22 16 18 17 3 29 4 15 19 10 26 20
429
1.98
Fig. 3. UPGMA cluster analysis of aeromonads isolated from the river Narmada at Jabalpur on the basis of RAPD analysis.
divided into clusters A and B. A was represented by 22 isolates and B was represented by eight isolates.
Screening for extracellular virulence factors Out of 30 isolates, 26 were positive for protease production, all 30 isolates were positive for lipase production. Twelve isolates were positive for amylase production and 10 for DNase production (Table 2).
Bioassay for hemolytic and cytotoxic activity Out of 30 isolates, 14 isolates produced clear zones of b-hemolysis on blood agar plates (Table 2). None of the isolates exhibited any change or lysis on HeLa cell lines.
Screening of virulence genes All isolates were found to be negative for alt and act gene. Five isolates, A. hydrophila (BGCC # 177, 178), A. sobria (BGCC # 180) and A. veronii bv veronii (BGCC # 196, 198), exhibited very faint bands for the hly A gene (Fig. 4).
Antibiotic resistance patterns All 30 isolates were resistant to the b-lactam antibiotic ampicillin but sensitive to gentamycin. Sensitivity to the rest of the 12 antibiotics varied with strains (Table 3).
Discussion The most abundant species isolated from the water of river Narmada at Jabalpur were A. hydrophila, A. veronii bv veronii, A. veronii bv sobria, which are most frequently isolated from water without faecal pollution. A. schubertii and A. trota were not detected in this study, and they are unlikely to be detected in significant numbers in environmental water samples (Joseph and Carnahan, 2000). The biochemical differentiation revealed results similar to those of Millership (1996) and Abbott et al. (2003). Fluctuations in the coliform density were observed during the period of investigation with no seasonal correlation. Also, no correlation was observed between faecal coliforms and aeromonads (data not shown). The absence of correlation could be due to multiplication of aeromonads in these habitats as a result of external sources of nutrients of non-faecal origin (Araujo et al., 1989, 1991; Huys et al., 1997). Specific biochemical reactions are usually not sufficient to assign Aeromonas strains to different genomic species (Carnahan and Altwegg, 1996). Compared with other subtyping methods that have shown a considerable heterogenecity among strains of similar origin, biotyping is certainly not sufficiently sensitive (Altwegg, 1996). In the present study, RAPD analysis was chosen because it is reliable and can be used to differentiate between strains within a species (Austin and Adams, 1996). Different RAPD profiles were recorded for different species of Aeromonas although UPGMA
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Table 2.
Screening of extracellular enzymes and b-hemolysis production by aeromonads isolated from river Narmada
Isolate no.
Isolates
Protease
Lipase
BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC
A. hydrophila A. hydrophila A. hydrophila A. hydrophila A. hydrophila A. hydrophila A. sobria A. sobria A. sobria A.sobria A. eucrenophila A. eucrenophila A. eucrenophila A. jandaei A. jandaei A. caviae A. caviae A. caviae A. veronii bv veronii A. veronii bv veronii A. veronii bv veronii A. veronii bv veronii A. veronii bv veronii A. veronii bv veronii A. veronii bv sobria A. veronii bv sobria A. veronii bv sobria A. veronii bv sobria A. veronii bv sobria A. veronii bv sobria
+ + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
# # # # # # # # # # # # # # # # # # # # # # # # # # # # # #
174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203
+ + + + + + + +
Amylase
DNase +
+ + + +
+ + +
+ + + +
+
+
+ + + +
+ + +
+ + + +
+ +
+
+
+
+ + +
+ + +
23kb 9kb 6kb 4kb
23kb 9kb 6kb 4kb
2.4kb 2.0kb
2.4kb 2.0kb
500bp
500bp
MC (+)
b-hemolysis
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 M
Fig. 4. Screening of hemolysin (hly A) gene of aeromonads isolated from the river Narmada at Jabalpur.
analysis revealed that the collection of Aeromonas under study comprises a closely related population. UPGMA cluster analysis on the basis of biochemical characteristics as well as RAPD patterns of Aeromonas depicted
all the isolates into two major clusters, suggested that RAPD pattern is species specific. The strains showed highly active hydrolytic enzymes like protease, amylase, lipase and DNase. Isolates were
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431
Table 3. Antibiotic resistance patterns of aeromonads isolated from the river Narmada at Jabalpur (zone size in mm in parentheses) Code
Name of isolates
BGCC # 174
A. hydrophila
BGCC # 175
A. hydrophila
BGCC # 176
A. hydrophila
BGCC # 177
A. hydrophila
BGCC # 178
A. hydrophila
BGCC # 179
A. hydrophila
BGCC # 180
A. sobria
BGCC # 181
A. sobria
BGCC # 182
A. sobria
BGCC # 183
A. sobria
BGCC # 184
A. eucrenophila
BGCC # 185
A. eucrenophila
BGCC # 186
A. eucrenophila
BGCC # 187
A. jandaei
BGCC # 188
A. jandaei
BGCC # 189
A. caviae
BGCC # 190
A. caviae
BGCC # 191
A. caviae
BGCC # 192
A. veronii bv veronii
BGCC # 193
A. veronii bv veronii
BGCC # 194
A. veronii bv veronii
BGCC # 195
A. veronii bv veronii
BGCC # 196
A. veronii bv veronii
BGCC # 197
A. veronii bv veronii
BGCC # 198
A. veronii bv sobria
BGCC # 199
A. veronii bv sobria
BGCC # 200
A. veronii bv sobria
Antibiotics CZ
CP
J
K
PF
AK
CX
NF
CR
Cl
C
NA
Q
I
(19) S (22) S (21) S (22) S (8) R (20) S (21) S (23) S (24) S (15) I (0) R (7) R (6) R (24) S (22) S (0) R (0) R (0) R (18) S (17) I (20) S (19) S (18) S (17) I (20) S (14) R (0) R
(15) I (5) R (17) I (11) R (7) R (16) I (20) S (19) S (21) S (19) S (0) R (3) R (5) R (18) S (12) R (0) R (0) R (0) R (22) S (15) I (22) S (24) S (17) I (21) S (2) R (12) R (0) R
(16) S (28) S (24) S (22) S (18) S (25) S (22) S (20) S (19) S (21) S (17) S (18) S (14) I (20) S (23) S (17) S (19) S (15) I (20) S (19) S (15) S (21) S (20) S (20) S (21) S (20) S (22) S
(14) I (16) I (16) I (17) I (18) S (17) I (17) I (15) I (19) S (16) I (0) R (15) R (17) I (18) S (4) R (0) R (0) R (0) R (0) R (17) I (17) I (14) I (16) I (15) I (17) S (19) S (0) R
(17) S (15) I (18) S (16) I (13) I (16) I (19) S (17) S (10) R (18) S (18) S (17) S (19) S (15) I (18) S (6) R (0) R (7) R (21) S (22) S (18) S (20) S (20) S (19) S (24) S (21) S (0) R
(19) S (18) S (21) S (20) S (20) S (20) S (21) S (21) S (20) S (22) S (22) S (20) S (14) R (10) R (12) R (18) S (14) R (12) R (22) S (20) S (17) S (17) S (23) S (18) S (26) S (20) S (5) R
(20) I (20) I (24) S (16) I (16) I (19) I (22) S (16) I (25) S (11) R (5) R (16) I (17) I (0) R (5) R (0) R (0) R (0) R (24) S (24) S (14) R (19) I (16) I (16) I (25) S (20) R (5) R
(16) I (18) R (20) S (15) I (14) I (18) S (19) S (17) S (20) S (15) I (19) S (22) S (19) S (0) R (7) R (19) S (0) R (17) S (20) S (28) S (20) S (21) S (20) S (19) S (22) S (24) S (0) R
(14) R (12) R (18) I (13) R (9) R (17) I (21) S (22) S (22) S (17) I (7) I (6) I (7) I (0) R (0) R (0) R (0) R (0) R (24) S (0) R (3) R (23) S (10) S (9) R (23) S (11) R (0) R
(17) I (21) S (15) I (19) I (17) I (14) I (23) S (24) S (22) S (22) S (24) S (25) R (18) I (0) R (4) R (10) R (18) I (16) I (26) S (14) I (18) I (25) S (11) R (20) I (25) S (20) I (18) I
(12) R (18) S (14) I (21) S (12) R (10) R (6) R (8) R (0) R (4) R (7) R (9) R (12) R (4) R (18) S (18) S (12) R (14) R (10) R (7) R (20) S (13) I (4) I (8) R (23) S (21) S (0) R
(11) R (8) R (9) R (11) R (7) R (12) R (8) R (7) R (0) R (11) R (0) R (4) R (5) R (12) R (14) I (15) S (17) I (16) R (11) R (24) S (17) I (15) I (8) R (11) R (26) S (20) S (0) R
(14) I (18) S (12) I (20) S (8) R (10) R (0) R (4) R (0) R (7) R (0) R (0) R (7) R (16) S (18) S (11) I (12) I (13) I (6) R (20) S (19) S (19) S (11) I (14) I (14) I (21) S (0) R
(0) R (0) R (4) R (2) R (0) R (0) R (11) R (10) R (5) R (0) R (0) R (2) R (0) R (0) R (6) R (0) R (0) R (0) R (4) R (0) R (0) R (5) R (0) R (2) R (2) R (0) R (0) R
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Table 3. (continued ) Code
Name of isolates
BGCC # 201
A. veronii bv sobria
BGCC # 202
A. veronii bv sobria
BGCC # 203
A. veronii bv sobria
Antibiotics CZ
CP
J
K
PF
AK
CX
NF
CR
Cl
C
NA
Q
I
(0) R (17) I (14) I
(0) R (20) S (17) I
(22) S (24) S (21) S
(7) R (21) S (20) S
(11) S (18) S (17) S
(0) R (21) S (12) R
(0) R (18) R (14) R
(12) R (22) S (17) S
(0) R (14) R (19) R
(17) I (17) I (12) I
(22) S (16) I (17) I
(23) S (27) S (19) S
(0) R (18) R (20) S
(0) R (0) R (7) R
S—sensitive, I—intermediate, R—resistant. CZ—ceftazidime, CP—cephalexin, J—gentamycin, K-kanamycin, PF—pefloxacin, AK—amikacin, CX—ceftaxime, NF—norfloxacin. CR—cefuroxime sodium, CI—ciprofloxacin, C—chloramphenicol, NA—nalidixic acid, Q—co-trimaxazole, I—ampicillin.
also found to produce b-hemolysin. From this result it can be concluded that the isolated Aeromonas species may be potentially pathogenic. Its marked resistance to frequently used antibiotics like b-lactam antibiotics may aggravate the infection with this pathogen. Aeromonads were found to be sensitive to gentamycin. Fresh water is known to be a source and reservoir of Aeromonas species. The occurrence of potentially pathogenic aeromonads in our fresh water samples is cause of concern and persistence of Aeromonas in the river Narmada should not be ignored. These observations suggest that strains of aeromonads possess potential virulence factors such as extracellular enzymes and hemolysin, which play a major role in enhancing the virulence of this organism. Since fresh water is an important source of human infections and such evidence points to the existence of pathogenic species among the genus Aeromonas, it has been argued that limits on the number of aeromonads in drinking water should be specified (Rippey and Cabelli, 1985; Kooji, 1988). If such antibiotic resistant aeromonads, which are true human and aquatic pathogens, are able to multiply within fresh water systems in developing countries, they obviously may cause problems to public health.
Acknowledgments The authors are thankful to the Head, Department of Post Graduate Studies and Research in Biological Science, R.D. University, Jabalpur (M.P.), India for providing lab facilities, Dr. T. Ramamurthy, Assistant Director, NICED, Kolkata (W.B.), India for molecular studies and M.P.C.S.T., Bhopal (M.P.), India for financial assistance.
References Abbott, S.L., Wendy, K.W., Janda, J.M., 2003. The genus Aeromonas biochemical characteristics, atypical reactions
and phenotypic identification schemes. J. Clin. Microbiol. 41, 2348–2357. Alavandi, S., Ananthan, S., Kang, G., 1998. Prevalence, invitro secretory activity and cytotoxicity of Aeromonas species associated with childhood gastroenteritis in Chennai (Madras). India Jpn. J. Med. Sci. Biol. 51, 1–12. Alavandi, S.V., Ananthan, S., 2003. Biochemical, serogroups and virulence factors of aeromonads species isolated from cases of diarrhoea and domestic water sample in Chennai. Indian J. Med. Microbiol. 21 (4), 233–238. Altwegg, M., 1996. Subtyping method for Aeromonas species. The Genus Aeromonas. Wiley, Chichester, UK. Altwegg, M., Geiss, H.K., 1989. Aeromonas as a human pathogen. CRS. Crit. Rev. Microbiol. 16, 253–286. Araujo, R.M., Arribas, R.M., Lucena, F., Pares, R., 1989. Relation between Aeromonas and faecal coliforms in fresh water. J. Appl. Bacteriol. 67, 213–217. Araujo, R.M., Arribas, R.M., Pares, R., 1991. Distribution of Aeromonas species in waters with different levels of pollution. J. Appl. Bacteriol. 71, 182–186. Austin, B., Adams, C., 1996. Fish pathogen. In: Austin, B., Altwegg, M., Gosling, P.J., Joseph, S. (Eds.), The Genus Aeromonas. Wiley, Chichester, pp. 109–122. Bhat, P., Shanthakumari, S., Rajan, D., 1974. The Characterization and significance of Plesiomonas shigelloides and Aeromonas hydrophila isolated from an epidemic of diarrhoea. Indian J. Med. Res. 62, 1051–1060. Brocius, J.A., Raker, U.M.A., 1981. Construction and fine mapping of recombinant plasmid containing rrnB ribosomal RNA operon of E. coli. Plasmid 6, 112–118. Carnahan, A.M., Altwegg, M., 1996. In: Austin, B., Altwegg, M., Gosling, P.J., Joseph, S. (Eds.), The Genus Aeromonas. Wiley, Chichester, UK. Chatterjee, B.D., Neogy, K.N., 1972. Studies on Aeromonas and Plesiomonas species isolated from cases of choleraic diarrhoea. Indian J. Med. Res. 60, 520–524. Deodhar, L.P., Sarawathi, K., Varudhkar, A., 1991. Aeromonas spp. and their association with human diarrheal disease. J. Clin. Microbiol. 29, 853–856. Granam, P.E., Sullivan, K.O’., Toman, J.M., Ormen, O., 1998. Possible virulence factors of Aeromonas sp. from food and water. FEMS Immun. Med. Microbiol. 21, 131–137.
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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 sp. correlation with virulence in a suckling mouse model. FEMS Microbiol. Lett. 174, 131–136. Honda, T.N.Y., Miwatani, T., 1988. Purification and characterization of a hemolysin production by a clinical isolates of Kanagawa phenomenon-negative Vibrio parahaemolyticus and related to the thermostable direct hemolysin. Infect. Immun. 56, 961–965. Huys, G., Kampfer, P., Altwegg, M., Coopman, R., Jansean, J., Gillis, M., Kersters, K., 1997. Inclusion of Aeromonas DNA hybridization group 11 in Aeromonas encheleia and extended description of the species Aeromonas eucrenophila and Aeromonas encheleia. Int. J. Syst. Bacteriol. 47, 1157–1164. 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. Joseph, S.W., Carnahan, A.M., 2000. Update on the genus Aeromonas. ASM News 66, 218–223. Kersters, I., Huys, G., Van Duffel, H., Vancanneyt, H., Kersters, K., Verstraete, W., 1996. Survival potential of Aeromonas hydrophila in fresh waters in comparison with other bacteria. J. Appl. Bactriol. 80, 266–270. Kielwein, G., 1969. Ein Na¨hrboden zur selektiven Zu¨chtung von Pseudomonaden und Aeromonaden. Arch. F. Lebensmittelyg. 20, 131–133. Komathi, A.G., Ananthan, S., Alavandi, S.V., 1998. Incidence and enteropathogenecity of Aeromonas spp. in children suffering from acute diarrhoea in Chennai. Indian J. Med. Res. 107, 252–256. Kooji, V.M.D.I.N., 1988. Properties of aeromonads and their occurrence and hygienic significance in drinking water. Zentrabl. Bacteriol. Microbiol. Hyg. Ser. B. 187, 1–17. Kuhn, I., Allestam, G., Huys, G., Jansen, P., Kerters, K., Krovacek, K., Stenstram, T.A., 1997. Diversity, persistence, and virulence of Aeromonas strains isolated from drinking water distribution system in Sweden. Appl. Environ. Microbiol. 63 (7), 2708–2715.
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Mac Faddin, J.F., 1980. Biochemical Tests for Identification of Medical Bacteria. Williams and Wilkins Co., Baltimore, USA. Millership, S.E., 1996. Identification. In: Austin, B., Altwegg, M., Gosling, P.J., Joseph, S. (Eds.), The Genus Aeromonas. Wiley, Chichester. Ormen, O., Ostensvik, O., 2001. The occurrence of aerolysin positive Aeromonas spp. and their cytotoxicity in Norwegian water sources. J. Appl. Microbiol. 90, 797–802. Popoff, M.Y., 1984. Genus III. Aeromonas. In: Kreig, N.R., Holt, J.G. (Eds.), Bergey’s Manual of Systemic Bacteriology. Williams and Wilkins, pp. 545–548. PSAMDST, 1993. Performance Standards for Antimicrobial Disc Susceptibility Test, fourth ed., vol. 13. National Committee for Clinical Laboratory Standards. p. 24. Rippey, S.R., Cabelli, S.C., 1985. Growth characteristics of Aeromonas hydrophila in limnetic water of varying tropic state. Arch. Hydrobiol. 104, 311–319. Schubert, R.H.W., 1991. Aeromonads and their significance as potential pathogen in water. J. Appl. Bacterial. Symp. Suppl. 70, 1315–1355. Sharma, A., Khokale, D., Budholia, M., Sharma, A., Sharma, V.K., Rajput, S., 2002. Prevalence and distribution of aeromonads in different chlorinated water samples. Indian J. Environ. Health 40, 314–319. Sujatha, R., Rao, S., 1993. Comparison of toxigenicity in Aeromonas strains isolated from different sources. Indian J. Med. Microbiol. 11, 197–202. Thomas, A., Mathew, M., Valsa, A.K., Mohan, S., Manjula, R., 2003. Optimisation of growth conditions for the production of extracellular lipase by Bacillus mycoides. Indian J. Microbiol. 43 (1), 67–69. Verenkar, M., Rodrigues, V., Naik, S., Singh, I., 1995. Aeromonas species and Plesiomonas shigelloides in diarrhoea in Goa. Indian J. Pathol. Microbiol. 38, 169–171. Vermelho, A.B., Meirelles, M.N.L., Lopes, A., Pentinate, S.D.G., Chaia, A.A., Branquinha, M.H., 1996. Detection of extracellular protease from microorganisms on agar plates Mem. Inst. Oswaldo. Gus. Rio. De Janerio. 91 (6), 755–760.
Int. J. Hyg. Environ.-Health 208 (2005) 425–433 www.elsevier.de/ijheh
Characterization of aeromonads isolated from the river Narmada, India Anjana Sharma, Nidhi Dubey, Bandana Sharan Bacteriology Laboratory, Department of Post Graduate Studies and Research in Biological Sciences, R.D. University, Jabalpur (M.P.), India Received 2 September 2004; received in revised form 4 March 2005; accepted 15 March 2005
Abstract Thirty isolates of Aeromonas species were isolated from river Narmada at Jabalpur during the period of January 2002–December 2002. Diversity of these fresh water isolates were determined by randomly amplified polymorphic DNA (RAPD) analysis. These environmental isolates were found to be positive for virulence factors, i.e. protease, amylase, lipase and DNase. Isolates were also positive for b-hemolytic activity. All Aeromonas species were tested for antibiotic resistance patterns and were found to be resistant to ampicillin and sensitive to gentamycin. r 2005 Elsevier GmbH. All rights reserved. Keywords: Aeromonas; Fresh water; RAPD; Virulence factors
Introduction Aeromonas species are ubiquitous aquatic organisms, primary pathogens for cold-blooded animals and opportunistic pathogens for warm-blooded animals. The health consequences of the presence of motile species of the genus Aeromonas in water are the subject of much debate (Schubert, 1991; Janda and Abbott, 1998; Joseph and Carnahan, 2000). Recent studies have demonstrated that the presence of Aeromonas species in drinking water is a potential risk since these microorganism can produce a wide range of virulence factors (Schubert, 1991; Kersters et al., 1996; Kuhn et al., 1997; Ormen and Ostensvik, 2001). Aeromonas associated gastroenteritis has been reported from all over the world but the incidence is relatively low in developed countries as compared to that in developing countries (Altwegg Corresponding author. Tel.: +91 761 2416667; fax: +91 761 2603752. E-mail address: [email protected] (A. Sharma).
1438-4639/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijheh.2005.03.007
and Geiss, 1989). In India, Aeromonas associated diarrhoea has been reported from Bombay, Calcutta, Goa, Vellore, Pondicherry and Chennai (Chatterjee and Neogy, 1972; Bhat et al., 1974; Deodhar et al., 1991; Sujatha and Rao, 1993; Verenkar et al., 1995; Alavandi et al., 1998; Alavandi and Ananthan, 2003; Komathi et al., 1998). People from India and other developing countries use river water directly not only for drinking but also for various recreational purposes. Narmada is the fifth largest river of India and originates from Amarkantak (M.P.) and merges in the Arabian Sea at Bharuch (Gujarat). The total length of the river is 1312 km. Information about the occurrence of Aeromonas in fresh water environments in India, especially in Central India (Sharma et al., 2002), prompted this study. Biochemical and molecular characterization along with antibiograms of environmental Aeromonas strains that were isolated from river Narmada near Jabalpur (Madhya Pradesh) in Central India are described.
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Materials and methods
Screening for virulence genes of aeromonads
Bacterial strains
Detection of genes encoding for heat labile cytotonic enterotoxin (alt), cytotoxic enteroxin (act) and hemolysin (hly A) was performed following the method of Granam et al. (1998) and Heuzenroeder et al. (1999).
Fresh water samples were collected from four different sites of river Narmada: Gwarighat, Lamhetaghat, Tilwaraghat and Jelharighat at Jabalpur during January 2002–December 2002. Samples of 500 ml were brought to the laboratory under ice-cold conditions once a month. They were inoculated in glutamate starch phenol red agar (GSP) medium (Kielwein, 1969) and incubated at 37 1C for 24–48 h. Yellow coloured colonies on GSP medium were maintained on nutrient agar slants at 4 1C, after subculturing several times.
Physiological and biochemical characterization of aeromonads
Antibiotic resistance Resistance to 14 different antibiotics was tested by the agar diffusion method on nutrient agar medium with test discs from SPAN diagnostics, combi disc, code 14346 (PSAMDST, 1993).
Results Biochemical differentiation and identification
Cultures were grown at 37 1C. Cell morphology and Gram stains were determined after 24 h of incubation on nutrient agar. Motility was determined after 18 h in motility test medium (Mac Faddin, 1980). Phenotypic characterization of each strain was determined by the method described by Popoff (1984).
Molecular characterization Randomly amplified polymorphic DNA (RAPD) analysis was done by following the method described by Brocius and Raker (1981).
Screening for extracellular virulence factors of aeromonads The enzymes amylase, gelatinase, lipase and DNase were detected on the following media: starch agar medium (containing 1% starch, 5 g peptone, 3 g beef extract, 5 g NaCl, 20 g agar and 1000 ml distilled water), gelatin agar medium (containing 0.4% gelatin, 5 g peptone, 3 g beef extract, 5 g NaCl, 20 g agar and 1000 ml distilled water), tributyrin agar medium (containing 1% tributyrin, 5 g peptone, 3 g yeast extract, 20 g agar and 1000 ml distilled water) and DNase agar medium (containing 0.2% DNA, 5 g peptone, 15 g tryptone, 5 g NaCl and 1000 ml distilled water) (Mac Faddin, 1980; Vermelho et al., 1996; Thomas et al., 2003).
Bioassay for hemolytic and cytotoxic activity Aeromonads were screened for their hemolytic activity on blood agar medium using human erythrocytes (Honda and Miwatani, 1988). Cytotoxic activity was determined on the on HeLa cells by the method of Kuhn et al. (1997).
Thirty strains isolated from 168 water samples had phenotypic characteristics of the genus Aeromonas (Popoff, 1984). All isolates were Gram negative, motile, rod shaped and facultatively anaerobes. The strains were oxidase and catalase positive and did not tolerate NaCl concentrations higher than 3.0%. Out of 30 isolates, six were identified as A. hydrophila (BGCC # 174, 175, 176, 177, 178, 179), four as A. sobria (BGCC # 180, 181, 182, 183), three as A. eucrenophila (BGCC # 184, 185, 186), two as A. jandaei (BGCC # 187, 188), three as A. caviae (BGCC # 189, 190, 191), six as A. veronii bv veronii (BGCC # 192, 193, 194, 195, 196, 197) and six as A. veronii bv sobria (BGCC # 198, 199, 200, 201, 202, 203) using biochemical tests (Table 1). These isolates were maintained in the bacteriology lab, Department of Biosciences, RDVV, Jabalpur and were given BGCC (Bacterial Germplasm Collection Centre) numbers.
Molecular characterization RAPD analysis of all 30 isolates exhibited numbers of bands ranging from 4 to 11 with molecular weight between 400 bp and 4.0 kb (Fig. 1).
UPGMA cluster analysis on the basis of biochemical and molecular characterization UPGMA cluster analysis on the basis of biochemical tests depicted that the strains formed several clusters at a Euclidean distance mostly of 1–2.67, which related them at species level (Fig. 2). Genetic differentiation among the 30 isolates on the basis of RAPD analysis is presented in Fig. 3, which shows two major clusters I and II. Cluster I represented A. sobria (BGCC # 183), while cluster II was further
Table 1.
Identification of aeromonads isolated from river Narmada at Jabalpur into different genomospecies based on morphological, cultural and biochemical characteristics
Isolate no.
Morphological characteristics
Cultural Biochemical characteristics characteristics
1
3
2
4
5
6
7
8
9
Identified species
10 11 12 13 14 15 16 17 18 19 I
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + +
+ + +
+
+ + + + + +
+ + + + +
+ + + + +
+ + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + +
+ + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + +
+ + + + +
A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A.
hydrophila hydrophila hydrophila hydrophila hydrophila hydrophila sobria sobria sobria sobria eucrenophila eucrenophila eucrenophila jandaei jandaei caviae caviae caviae veronii bv veroni veronii bv veroni veronii bv veroni veroni bv veronii veroni bv veronii veroni bv veronii veronii bv sobriai veronii bv sobria veronii bv sobria veronii bv sobria veronii bv sobria veronii bv sobria
1—Gram staning, 2—shape, 3—motility, 4—salt tolerance test (6% NaCl), 5—oxidase, 6—lysine decarboxylase, 7—ornithine decarboxylase, 8—arginine dihydrolase, 9—arabinose fermentation, 10—mannitol fermentation, 11—sorbitol fermentation, 12—salicin fermentation, 13—sucrose fermentation, 14—inositol fermentation, 15—indole, 16—Voges prousker, 17—catalase, 18—citrate utilization, 19—acid production (I—glucosefermentation (TSI), II—glucose, lactose and sucrose fermentation (TSI)), 20—gas production (TSI), 21—lead sulphide production (TSI), 22—H2S production (TSI), 23—starch hydrolysis, 24—gelatin hydrolysis, 25—bile esculine hydrolysis (Aesculin).
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Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod
II
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BGCC # 174 BGCC # 175 BGCC # 176 BGCC # 177 BGCC # 178 BGCC # 179 BGCC # 180 BGCC # 181 BGCC # 182 BGCC # 183 BGCC # 184 BGCC # 185 BGCC # 186 BGCC # 187 BGCC # 188 BGCC # 189 BGCC # 190 BGCC # 191 BGCC # 192 BGCC # 193 BGCC # 194 BGCC # 195 BGCC # 196 BGCC # 197 RGPP # 198 RGPP # 199 BGCC # 200 BGCC # 201 BGCC # 202 BGCC # 203
20 21 22 23 24 25
427
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8 kb 7 kb 6 kb 5 kb 4 kb
8 kb 7 kb 6 kb 5 kb 4 kb
3 kb
3 kb
2 kb
2 kb 1.5 kb
1.5 kb
1 kb
1 kb
500 bp
500 bp
M 1 2 3 4 5 6 7 8 9 10 11 1213141516171819202122 23 24 25 26 27 28 29 3031 M
Fig. 1. Randomly amplified polymorphic DNA (RAPD) of aeromonads isolated from the river Narmada at Jabalpur.
x
x2 x3
c1 c
f
f1
y
f2
a1
b1
d
d1 d2 d3
b2 A
a2
h i
j I
A2
k
c a B
d b
0.00
x1
1 4 23 29 27 6 21 19 20 28 7 9 10 8 14 15 30 13 18 25 11 12 16 17 24 2 31 3 5 22 0.67
1.34 Coefficient
2.00
2.67
Fig. 2. UPGMA cluster analysis of aeromonads isolated from the river Narmada at Jabalpur on the basis of biochemical tests.
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a2 A1
c1 c b
c2 c3 d
d1 d2
A II
f1 f A2
f2 e
i h1 I
h
j h2 0.99 Coefficient
1.49
B
0.50
g
g1 g2
0.00
a
a1
1 2 5 11 24 25 27 30 9 12 28 13 14 23 6 7 8 21 22 16 18 17 3 29 4 15 19 10 26 20
429
1.98
Fig. 3. UPGMA cluster analysis of aeromonads isolated from the river Narmada at Jabalpur on the basis of RAPD analysis.
divided into clusters A and B. A was represented by 22 isolates and B was represented by eight isolates.
Screening for extracellular virulence factors Out of 30 isolates, 26 were positive for protease production, all 30 isolates were positive for lipase production. Twelve isolates were positive for amylase production and 10 for DNase production (Table 2).
Bioassay for hemolytic and cytotoxic activity Out of 30 isolates, 14 isolates produced clear zones of b-hemolysis on blood agar plates (Table 2). None of the isolates exhibited any change or lysis on HeLa cell lines.
Screening of virulence genes All isolates were found to be negative for alt and act gene. Five isolates, A. hydrophila (BGCC # 177, 178), A. sobria (BGCC # 180) and A. veronii bv veronii (BGCC # 196, 198), exhibited very faint bands for the hly A gene (Fig. 4).
Antibiotic resistance patterns All 30 isolates were resistant to the b-lactam antibiotic ampicillin but sensitive to gentamycin. Sensitivity to the rest of the 12 antibiotics varied with strains (Table 3).
Discussion The most abundant species isolated from the water of river Narmada at Jabalpur were A. hydrophila, A. veronii bv veronii, A. veronii bv sobria, which are most frequently isolated from water without faecal pollution. A. schubertii and A. trota were not detected in this study, and they are unlikely to be detected in significant numbers in environmental water samples (Joseph and Carnahan, 2000). The biochemical differentiation revealed results similar to those of Millership (1996) and Abbott et al. (2003). Fluctuations in the coliform density were observed during the period of investigation with no seasonal correlation. Also, no correlation was observed between faecal coliforms and aeromonads (data not shown). The absence of correlation could be due to multiplication of aeromonads in these habitats as a result of external sources of nutrients of non-faecal origin (Araujo et al., 1989, 1991; Huys et al., 1997). Specific biochemical reactions are usually not sufficient to assign Aeromonas strains to different genomic species (Carnahan and Altwegg, 1996). Compared with other subtyping methods that have shown a considerable heterogenecity among strains of similar origin, biotyping is certainly not sufficiently sensitive (Altwegg, 1996). In the present study, RAPD analysis was chosen because it is reliable and can be used to differentiate between strains within a species (Austin and Adams, 1996). Different RAPD profiles were recorded for different species of Aeromonas although UPGMA
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Table 2.
Screening of extracellular enzymes and b-hemolysis production by aeromonads isolated from river Narmada
Isolate no.
Isolates
Protease
Lipase
BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC BGCC
A. hydrophila A. hydrophila A. hydrophila A. hydrophila A. hydrophila A. hydrophila A. sobria A. sobria A. sobria A.sobria A. eucrenophila A. eucrenophila A. eucrenophila A. jandaei A. jandaei A. caviae A. caviae A. caviae A. veronii bv veronii A. veronii bv veronii A. veronii bv veronii A. veronii bv veronii A. veronii bv veronii A. veronii bv veronii A. veronii bv sobria A. veronii bv sobria A. veronii bv sobria A. veronii bv sobria A. veronii bv sobria A. veronii bv sobria
+ + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
# # # # # # # # # # # # # # # # # # # # # # # # # # # # # #
174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203
+ + + + + + + +
Amylase
DNase +
+ + + +
+ + +
+ + + +
+
+
+ + + +
+ + +
+ + + +
+ +
+
+
+
+ + +
+ + +
23kb 9kb 6kb 4kb
23kb 9kb 6kb 4kb
2.4kb 2.0kb
2.4kb 2.0kb
500bp
500bp
MC (+)
b-hemolysis
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 M
Fig. 4. Screening of hemolysin (hly A) gene of aeromonads isolated from the river Narmada at Jabalpur.
analysis revealed that the collection of Aeromonas under study comprises a closely related population. UPGMA cluster analysis on the basis of biochemical characteristics as well as RAPD patterns of Aeromonas depicted
all the isolates into two major clusters, suggested that RAPD pattern is species specific. The strains showed highly active hydrolytic enzymes like protease, amylase, lipase and DNase. Isolates were
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431
Table 3. Antibiotic resistance patterns of aeromonads isolated from the river Narmada at Jabalpur (zone size in mm in parentheses) Code
Name of isolates
BGCC # 174
A. hydrophila
BGCC # 175
A. hydrophila
BGCC # 176
A. hydrophila
BGCC # 177
A. hydrophila
BGCC # 178
A. hydrophila
BGCC # 179
A. hydrophila
BGCC # 180
A. sobria
BGCC # 181
A. sobria
BGCC # 182
A. sobria
BGCC # 183
A. sobria
BGCC # 184
A. eucrenophila
BGCC # 185
A. eucrenophila
BGCC # 186
A. eucrenophila
BGCC # 187
A. jandaei
BGCC # 188
A. jandaei
BGCC # 189
A. caviae
BGCC # 190
A. caviae
BGCC # 191
A. caviae
BGCC # 192
A. veronii bv veronii
BGCC # 193
A. veronii bv veronii
BGCC # 194
A. veronii bv veronii
BGCC # 195
A. veronii bv veronii
BGCC # 196
A. veronii bv veronii
BGCC # 197
A. veronii bv veronii
BGCC # 198
A. veronii bv sobria
BGCC # 199
A. veronii bv sobria
BGCC # 200
A. veronii bv sobria
Antibiotics CZ
CP
J
K
PF
AK
CX
NF
CR
Cl
C
NA
Q
I
(19) S (22) S (21) S (22) S (8) R (20) S (21) S (23) S (24) S (15) I (0) R (7) R (6) R (24) S (22) S (0) R (0) R (0) R (18) S (17) I (20) S (19) S (18) S (17) I (20) S (14) R (0) R
(15) I (5) R (17) I (11) R (7) R (16) I (20) S (19) S (21) S (19) S (0) R (3) R (5) R (18) S (12) R (0) R (0) R (0) R (22) S (15) I (22) S (24) S (17) I (21) S (2) R (12) R (0) R
(16) S (28) S (24) S (22) S (18) S (25) S (22) S (20) S (19) S (21) S (17) S (18) S (14) I (20) S (23) S (17) S (19) S (15) I (20) S (19) S (15) S (21) S (20) S (20) S (21) S (20) S (22) S
(14) I (16) I (16) I (17) I (18) S (17) I (17) I (15) I (19) S (16) I (0) R (15) R (17) I (18) S (4) R (0) R (0) R (0) R (0) R (17) I (17) I (14) I (16) I (15) I (17) S (19) S (0) R
(17) S (15) I (18) S (16) I (13) I (16) I (19) S (17) S (10) R (18) S (18) S (17) S (19) S (15) I (18) S (6) R (0) R (7) R (21) S (22) S (18) S (20) S (20) S (19) S (24) S (21) S (0) R
(19) S (18) S (21) S (20) S (20) S (20) S (21) S (21) S (20) S (22) S (22) S (20) S (14) R (10) R (12) R (18) S (14) R (12) R (22) S (20) S (17) S (17) S (23) S (18) S (26) S (20) S (5) R
(20) I (20) I (24) S (16) I (16) I (19) I (22) S (16) I (25) S (11) R (5) R (16) I (17) I (0) R (5) R (0) R (0) R (0) R (24) S (24) S (14) R (19) I (16) I (16) I (25) S (20) R (5) R
(16) I (18) R (20) S (15) I (14) I (18) S (19) S (17) S (20) S (15) I (19) S (22) S (19) S (0) R (7) R (19) S (0) R (17) S (20) S (28) S (20) S (21) S (20) S (19) S (22) S (24) S (0) R
(14) R (12) R (18) I (13) R (9) R (17) I (21) S (22) S (22) S (17) I (7) I (6) I (7) I (0) R (0) R (0) R (0) R (0) R (24) S (0) R (3) R (23) S (10) S (9) R (23) S (11) R (0) R
(17) I (21) S (15) I (19) I (17) I (14) I (23) S (24) S (22) S (22) S (24) S (25) R (18) I (0) R (4) R (10) R (18) I (16) I (26) S (14) I (18) I (25) S (11) R (20) I (25) S (20) I (18) I
(12) R (18) S (14) I (21) S (12) R (10) R (6) R (8) R (0) R (4) R (7) R (9) R (12) R (4) R (18) S (18) S (12) R (14) R (10) R (7) R (20) S (13) I (4) I (8) R (23) S (21) S (0) R
(11) R (8) R (9) R (11) R (7) R (12) R (8) R (7) R (0) R (11) R (0) R (4) R (5) R (12) R (14) I (15) S (17) I (16) R (11) R (24) S (17) I (15) I (8) R (11) R (26) S (20) S (0) R
(14) I (18) S (12) I (20) S (8) R (10) R (0) R (4) R (0) R (7) R (0) R (0) R (7) R (16) S (18) S (11) I (12) I (13) I (6) R (20) S (19) S (19) S (11) I (14) I (14) I (21) S (0) R
(0) R (0) R (4) R (2) R (0) R (0) R (11) R (10) R (5) R (0) R (0) R (2) R (0) R (0) R (6) R (0) R (0) R (0) R (4) R (0) R (0) R (5) R (0) R (2) R (2) R (0) R (0) R
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Table 3. (continued ) Code
Name of isolates
BGCC # 201
A. veronii bv sobria
BGCC # 202
A. veronii bv sobria
BGCC # 203
A. veronii bv sobria
Antibiotics CZ
CP
J
K
PF
AK
CX
NF
CR
Cl
C
NA
Q
I
(0) R (17) I (14) I
(0) R (20) S (17) I
(22) S (24) S (21) S
(7) R (21) S (20) S
(11) S (18) S (17) S
(0) R (21) S (12) R
(0) R (18) R (14) R
(12) R (22) S (17) S
(0) R (14) R (19) R
(17) I (17) I (12) I
(22) S (16) I (17) I
(23) S (27) S (19) S
(0) R (18) R (20) S
(0) R (0) R (7) R
S—sensitive, I—intermediate, R—resistant. CZ—ceftazidime, CP—cephalexin, J—gentamycin, K-kanamycin, PF—pefloxacin, AK—amikacin, CX—ceftaxime, NF—norfloxacin. CR—cefuroxime sodium, CI—ciprofloxacin, C—chloramphenicol, NA—nalidixic acid, Q—co-trimaxazole, I—ampicillin.
also found to produce b-hemolysin. From this result it can be concluded that the isolated Aeromonas species may be potentially pathogenic. Its marked resistance to frequently used antibiotics like b-lactam antibiotics may aggravate the infection with this pathogen. Aeromonads were found to be sensitive to gentamycin. Fresh water is known to be a source and reservoir of Aeromonas species. The occurrence of potentially pathogenic aeromonads in our fresh water samples is cause of concern and persistence of Aeromonas in the river Narmada should not be ignored. These observations suggest that strains of aeromonads possess potential virulence factors such as extracellular enzymes and hemolysin, which play a major role in enhancing the virulence of this organism. Since fresh water is an important source of human infections and such evidence points to the existence of pathogenic species among the genus Aeromonas, it has been argued that limits on the number of aeromonads in drinking water should be specified (Rippey and Cabelli, 1985; Kooji, 1988). If such antibiotic resistant aeromonads, which are true human and aquatic pathogens, are able to multiply within fresh water systems in developing countries, they obviously may cause problems to public health.
Acknowledgments The authors are thankful to the Head, Department of Post Graduate Studies and Research in Biological Science, R.D. University, Jabalpur (M.P.), India for providing lab facilities, Dr. T. Ramamurthy, Assistant Director, NICED, Kolkata (W.B.), India for molecular studies and M.P.C.S.T., Bhopal (M.P.), India for financial assistance.
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