Abstract: Of the samples examined, 25.7% of children diarrhoeal samples harbored Aeromonas spp. Among them 16.7% were A. hydrophila. Though all the isolates were resistant to all β-lactam antibiotics (except carbapenem) and non β-lactam antibiotics tested and further studied for the presence of plasmid in all the strains. The majority of isolates were resistant to clarithromycin and vancomycin. Thirty one (77.5%) of the 40 isolates harbored plasmids, with sizes ranging from 2.6 to 15.2 kb. These results indicate that multiple antibiotic resistant and genetically diverse aeromonads are easily available among the children diarrhoeal samples collected from the study area. It indicates that the practice of frequent application of therapeutics for the treatment of diseases might have enhanced occurrence of drug resistance and carrying the resistant plasmids.
INTRODUCTION
Aeromonas spp. comprises mesophilic, motile organisms, which are distributed universally in fresh water, estuarine, coastal water bodies and food samples. They are associated with wide spectrum of infection in human diseases especially diarrhoea in children, aged individuals and immunocompromised patients (Gracey et al., 1982; Agger et al., 1985). Among Aeromonas species, A. hydrophila is widely studied and most commonly involved in causing human infections (Agger et al., 1985; Ananthan and Alavandi, 1999). Clinical and environmental strains secrete many extracellular products, such as haemolysins, enterotoxins, aerolysin, haemagglutinins and protease (Kuhn et al., 1997). Occurrence of A. hydrophila from water and food sources, are widely increasing the resistance to various antimicrobial agents and chlorine in potable water, which presents a significant threat to public health (Thayumanavan et al., 2003). About 6.5 to 13% of the diarrhoeal cases of south India have been attributed to Aeromonas (Alavandi and Ananthan, 2003) and the frequency of diarrhoea is more in children than adults. It compels the necessity to investigate the incidence of pathogenic Aeromonas spp.
The strains of A. hydrophila were highly susceptible to tetracycline, chloramphenicol, polymyxin-B, gentamicin and trimethoprim-sulfamethoxazole (Ramteke et al., 1993). Increased incidence of multiple antibiotic resistant Aeromonas isolated from clinical and environmental sources have been reported worldwide and they may pose a serious problem in chemotherapy (Pettibone et al., 1996).
The present work was aimed to evaluate in vitro susceptibilities for wide range of antimicrobial agents, against the strains of A. hydrophila isolated from diarrhoeal samples of children. During the treatment of infections with multi drug resistant bacterial pathogens is a serious problem faced by the clinicians. This may also help to know the emergence of drug resistant A. hydrophila and regular up-taking of antibiotic resistant pattern will be useful for formulate the hospital policy.
MATERIALS AND METHODS
Bacterial Strain
A total of 239 stool samples were collected from children, who had affected
with acute diarrhoea in the hospitals. The samples were procured just before
starting treatment with chemotherapy from hospitals located in Coimbatore, Tirupur,
Kovilpalayam and Pattukottai of South India, during the period of January 2003
to December 2003. The stool samples were processed in Alkaline Peptone Water
(APW) (Cat. No. M6181, HiMedia, India) for pre-enrichment and then cultured
on starch ampicillin agar medium and Rimler-Shotts medium (Cat. No. M576, HiMedia,
India) and Rippey Cabelli Agar medium (Cat. No. M859, HiMedia, India). The presumptive
identification was performed by oxidase reaction, fermentation of glucose, presence
of lysine, arginine decarboxylation and absence of ornithine decarboxylase,
proposed by Cowan and Steel (1970). Kaper’s multi test medium was used
for the confirmation of A. hydrophila (Kaper et al., 1979).
Further, the strains were identified on the basis of the restriction fragment
length polymorphism patterns (RFLP) obtained by the 16s rDNA following the method
described by Borrel et al. (1997) and Figueras et al. (2000).
Statistical Analysis
The χ2 and Fisher’s exact tests were used to compare
the diarrhoeagenic A. hydrophila between the four sampling stations in
all the months. A p-value <0.05 was considered as significant. The statistical
analysis was performed using SPSS 10.0 for Windows XP.
Antimicrobial Susceptibility
A. hydrophila (n = 40) strains were examined for their resistance
to 42 antibiotics (β-lactam and non β-lactam) (HiMedia, India). The
resistance pattern was determined by the disc diffusion method of Bauer et
al. (1966), using Mueller Hinton agar medium (Cat. No. M1084, HiMedia, India).
The plates were incubated for 24 h at 37°C, the size of inhibition zones
were measured and classified as resistant, intermediate and sensitive according
to manufacturer’s instruction. The intermediate strains were also scored
as resistant.
MAR Indexing
The MAR index, when applied to a single isolate is defined as a/b, where
a represents the number of antibiotics to which the isolate was resistant and
b represents the number of antibiotics to which the isolate was exposed (Krumperman,
1983). MAR index value higher than 0.2 is considered to have originated from
high-risk sources of contamination like human, commercial poultry farms, swine
and dairy cattle where antibiotics are very often used. MAR index value of less
than or equal to 0.2 considered the origination of strain from animals in which
antibiotics are seldom or never used.
Detection of Plasmids
Plasmid DNA was extracted, followed by electrophoresis, essentially as described
by Sambrook et al. (1989). The approximate molecular mass of each plasmid
was determined by comparison with plasmids of known standard molecular mass
(Cat # MBD 22, Genei, India).
RESULTS AND DISCUSSION
In recent years, Aeromonas spp. have been recognized with increasing frequency as a cause of gastroenteritis in children and adults. Several investigators have determined the asymptomatic carrier rate to assess the role of Aeromonas species in diarrhoeal diseases (Janda, 1991; Janda and Abbott, 1998; Joseph and Carnahan, 2000; Alavandi and Ananthan, 2003; Sinha et al., 2004). Significant number of diarrhoeal samples (25.7%) (p<0.05) were found to harbor Aeromonas species with a highest (47.2%) at Coimbatore and followed by Pattukottai (26.4%), Kovilpalayam (20.8%) and Tirupur (21.4%). Among them 16.7% of A. hydrophila were noticed in the above-mentioned four places (Table 1). Higher percentage of incidence (17.6%) was observed from the samples collected from Coimbatore, followed by 16.6, 15.1 and 14.3% in Kovilpalayam, Pattukottai and Tirupur, respectively, where the diarrhoeal stool samples were found to harbor in a significantly higher number of A. hydrophila in all the sampling areas (p<0.05).
The samples were also found with other enteropathogens (Vibrio, Salmonella, Shigella, Proteus, Escherichia and Staphylococcus) and Pseudomonas and Proteus (data not shown). But the present investigation was mainly focused on the incidence of A. hydrophila. In Coimbatore, 17.6% of incidence was higher than the findings observed in Chennai, Mumbai and Goa, India, with 4.7, 1.4 and 7.7%, respectively (Alavandi and Ananthan, 2003). Numerous cases of incidence of Aeromonas from patients with diarrhoea and the bacterium isolated from stool of healthy persons have been reported (Janda and Abbott, 1998; Albert et al., 2000; Joseph and Carnahan, 2000). To our knowledge this is the first study describing the Aeromonas in clinical aspects in this study area.
All the (n = 40) A. hydrophila strains were tested against 42 (17 β-lactam and 25 non β-lactam) antimicrobial agents. Resistance of A. hydrophila isolates towards β-lactam antibiotics are given in Table 2. The study showed the existence of multiple antibiotic resistant strains of A. hydrophila. Most of the isolates were resistance towards β-lactam antibiotics, such as penicillins and cephalosporins. It was observed that 22.5% of the A. hydrophila isolates showed resistance towards carbapenem (imipenem). Bravo et al. (2003) stated that the genus Aeromonas has been considered to be dependant on chromosome-mediated β-lactamases. First, second and third generation cephalosporins were found to be more active against A. hydrophila. The similar findings have been reported by several authors (Ko et al., 1996; Nwosu and Ladapo, 1999; Joseph and Carnahan, 2000). Most of the strains were resistant towards cephalexin, cephaloridine, oxacillin, amoxycillin, amoxycillin plus clavulanic acid (amoxyclav) and cefoperazone. As a result, there was increasing isolation of drug resistant A. hydrophila and taxonomic inconsistency present in diarrhoeal sources (Carnahan et al., 1991). Generally, β-lactam group of antibiotics are not prescribed for the treatment of diarrhoea. Our study also indicate that most of the β-lactam antimicrobial agents were ineffective to A. hydrophila, it is not recommended for treatment of Aeromonas infection in human. In contrast Ko et al. (1998) reported the β-lactam agents could be used for the treatment of invasive A. hydrophila infections.
Among non β-lactam antibiotics tested, netilmicin and bacitracin were
found more susceptible to all the isolates followed by norfloxacin (95%) (Table
3). It was also reported that 12.5% of strains were resistant to chloramphenicol.
Chloramphenicol resistant A. hydrophila is an emerging and high risk
levels among the species (Pettibone et al., 1996; Vivekanandhan et
al., 2002; Radu et al., 2003; Subashkumar et al., 2006b).
| Table 1: | Percentage of incidence of Aeromonas spp. and A. hydrophila |
| Table 2: | Antibiotic susceptibility of isolates of A. hydrophila to β-lactam antibiotics |
| Table 3: | Antibiotic susceptibility of isolates of A. hydrophila to non β-lactam antibiotics |
Lincosamides resistant (87.5%) A. hydrophila were noticed in all the strains, such type of lincosamides resistant A. hydrophila were reported in the diarrhoeal strains from Kollkata, India (Sinha et al., 2004). In this study, 80 and 60% of the strains were resistant to rifampicin and tetracycline. Chang and Bolton (1987) found that greater percentage of Asian isolates of A. hydrophila were resistant to tetracycline and rifampicin than Australian isolates.
Increasing ciprofloxacin resistance (60%) observed in all the strains with those of Sinha et al. (2004) who found ciprofloxacin resistant A. hydrophila have been 22.4%. Most of the A. hydrophila strains were exhibited lesser sensitivity to non β-lactam antibiotics. Generally, it was observed that the majority of the strains exhibited a multidrug resistant profile. The increased drug resistance is a significant threat to management of Aeromonas mediated diarrhoea (Sinha et al., 2004).
The Multiple Antibiotic Resistance (MAR) was calculated and the percentage occurrence of A. hydrophila MAR index values are presented in Fig. 1. It was observed that all the strains showed MAR index value of more than 0.2. This clearly indicates that the choice of a MAR index of 0.2 to differentiate between low and high risk source contamination is arbitrary. Indices of between 0.2 and 0.25 are in a range of ambiguity and the samples in this range require careful and specific scrutiny.
Bacteria resistant to β-lactam and non β-lactam antibiotics, may occur by selective pressure or because of antibiotic abuse by humans or over use in animals (White et al., 2000). In the present study, it was observed that all the strains isolated from children diarrhoeal samples showed multiple antibiotic resistances. A MAR index value of 0.2 or above is said to be originated from high-risk source of contamination (Krumperman, 1985). In the present study, all the strains showed a MAR index value more than 0.2, which implies that the organism might have been originated from high risk-sources like sewage, animal husbandry waste, biomedical waste, faecal contaminated drinking water. Survival of such MAR A. hydrophila strains in the food of animal origin may become contaminated with bacteria of intestinal origin and consequently animals may act as a potential source of resistant bacteria for humans. Multiple antibiotic resistance of A. hydrophila has been reported by several researchers throughout the world (De Vicente et al., 1990; Davies, 1992; Vivekanandhan et al., 2002; Radu et al., 2003; Thayumanavan et al., 2003; Lakshmanaperumalsamy et al., 2005; Subashkumar et al., 2006a). The release of such type of organisms through faeces may ultimately pave the way for the contamination of aquatic environments (Lakshmanaperumalsamy et al., 2005; Subashkumar et al., 2006b). In addition, antibiogram is considered as one of the useful techniques in the characterization of pathogens (Obi et al., 2004).
Plasmids were detected in all the isolates and it was ranged from 2.6 to 15.2
kb. The overall prevalence of plasmids was 77.5%. The frequency of occurrence
and the detection of small size plasmids among the Aeromonas were in
broad agreement with those of other workers who found plasmid prevalence in
Aeromonas have been 15 to 94%, with most of the isolates harbouring
small sized plasmids (Chang and Bolton, 1987; Alabi and Odugbemi, 1990; Vadivellu
et al., 1995; Chaudhury et al., 1996; Brown et al., 1997).
| Fig. 1: | Percentage occurrence of MAR index value of A. hydrophila |
Chang and Bolton (1987) have suggested that the plasmid-mediated antibiotic resistant is not frequent. Thus the observation that the plasmid containing isolates were devoid of large plasmid and together with the finding of isolates not containing plasmids but being multi-resistant to the antibiotics is of chromosomal origin.
Consequently, the contamination of the environment with bacterial pathogens resistant to various antimicrobial agents is a real threat not only as source of infection but also a source from which R plasmids can easily spread to other pathogens of diverse origin (Linton et al., 1996). In the study area, the clinical practitioners are administering tetracycline, gentamycin, amoxycillin, nalidixic acid and cotrimoxazole and streptomycin for the treatment of diarrhoea in children. Increased level of resistances in clinical isolates of A. hydrophila to commonly used antibiotics has been observed. Like Gram-negative bacilli, the emergence of resistance among aeromonads will be accelerated by the extensive clinical use of antibiotics (Chaudhury et al., 1996). Such high level of multiple drug resistance may arise from selective pressure due to the indiscriminate use of antibiotics. As previously reported, plasmids conferring resistance may also play an important role in linking resistance to more than one drug (Lacey et al., 1974). The emergence of resistance among aeromonads may also be increased by the frequent usage of antibiotics. These multiple antibiotic resistance strains from the hospital environments may cause serious health hazards
ACKNOWLEDGEMENT
The authors thank Bharathiar University, Coimbatore-641 046, India for providing necessary facilities.