Biochemical characteristics, serogroups, and virulence factors of aeromonas species isolated from cases of diarrhoea and domestic water samples in Chennai.

PURPOSE: The objective of the present study was to delineate the differences between the clinical and environmental Aeromonas species with respect to their biochemical characteristics, serogrouping and virulence factors, in order to find a phenotypic marker of enteropathogenicity. METHODS: A total of 55 Aeromonas spp. inclusive of 19 isolates from cases of diarrhoea, and 36 from water samples comprising, 10 isolates of A. hydrophila, 21 isolates each of A. sobria, and A. caviae, two isolates of A. jandaei and one isolate of A. veronii were subjected to analysis of their biochemical characteristics, serogrouping, and virulence factors. RESULTS: Among the differences recorded in the biochemical characteristics in the three major species, the most striking characteristic was fermentation of lactose, which was observed in all the 11 A. caviae isolates recovered from water samples. None of the 10 clinical isolates of A. caviae tested fermented lactose. The clinical Aeromonas isolates belonged to seven typable serogroups, O:13, O:14, O:16, O:21, O:27, O:32 and O:35. The environmental isolates belonged to eight different serogroups, such as, O:3, O:11, O:14, O:16, O:18, O:28, O:64 and O:78 and were predominated by serotypes O:18 and O:64. Among the virulence factors tested, 89% of the environmental isolates produced b haemolysin, while only 62.3% of clinical isolates were able to do so. There was no significant difference between the clinical and environmental aeromonads with respect to their enterotoxigenicity in suckling mice in vivo, cytotoxicity in vitro in Vero cell monolayers, and ability to produce siderophores. CONCLUSION: Efforts to delineate the differences between the clinical and environmental Aeromonas spp. did not reveal significant difference between them. However, difference was observed with respect to their ability to produce b haemolysin, wherein, higher percentage of environmental isolates was haemolytic. The results also suggest that all the haemolytic environmental isolates need not be enteropathogenic. Further, serogroups O:18 and O:64 may not be involved in aeromonal diarrhoea in children in this geographic region.

Extended spectrum beta lactamase production & multidrug resistance in Klebsiella species isolated from children under five with intestinal & extraintestinal infections.

BACKGROUND & OBJECTIVES: Extended spectrum beta-lactamases (ES beta L) are enzymes produced in some Gram-negative bacilli that mediate resistance to third generation cephalosporins (3GC) and aztreonam. These are common in Klebsiella spp. and Escherichia coli and in other members of the family enterobacteriaceae. ES beta L production is accompanied by resistance to other antibiotics as these are encoded by multi drug resistance conjugative plasmids. The present study was undertaken to study the incidence of multi drug resistant and ES beta L producing Klebsiella spp. in children under five years of age suffering from intestinal and extraintestinal infections. METHODS: A total of 90 strains of Klebsiella spp. (76 isolates of K. pneumoniae and 14 of K. oxytoca) were tested for resistance to 3GC antibiotics (ceftazidime, cefotaxime, ceftriaxone), amikacin, ampicillin, erythromycin, gentamycin and streptomycin by disc diffusion method. Isolates found resistant to 3GC antibiotics were tested for the production of ES beta L by double disc diffusion synergy test. Transconjugation experiments were done to study the transfer of drug resistance and ES beta L production from Klebsiella isolates to an Esch. coli strain (K12 J62-2). RESULTS: All the 90 isolates showed multi drug resistance; 87 (96.6%) isolates showed resistance or decreased susceptibility to at least one of the three 3GC. ES beta L production was detected in four strains of K. pneumoniae and two K. oxytoca. ES beta L activity could be experimentally transferred to recipient Esch. coli in all the 6 isolates. Resistance to beta-lactam antibiotics was co-transferred along with resistance to gentamycin. INTERPRETATION & CONCLUSION: This study has shown the incidence of ES beta L producing Klebsiella strains in children in Chennai, and possibly poses a threat in the treatment and management of Klebsiella associated infections. The incidence of ES beta L producing strains of Klebsiella and other members of enterobacteriaceae should be carefully monitored in children to prevent unnecessary use of antibiotics especially 3GC and aminoglycoside antibiotics. Hence, tests for the detection of ES beta L producing Klebsiella strains should be carried out routinely for better therapeutic management.

Typing of Aeromonas isolates from children with diarrhoea & water samples by randomly amplified polymorphic DNA polymerase chain reaction & whole cell protein fingerprinting.

BACKGROUND & OBJECTIVES: Aeromonas spp. are water-borne organisms, often associated with childhood diarrhoea. The present study was conducted to examine the epidemiological relationship among the Aeromonas spp. isolated from water and children with acute diarrhoea in Chennai. METHODS: Thirty six Aeromonas isolates inclusive of 16 from children with diarrhoea, 15 from domestic water samples and 5 reference strains were studied by randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR). Twenty eight Aeromonas isolates, 15 from children with diarrhoea, 10 from domestic water samples and three reference strains were analysed by SDS-PAGE for their whole cell protein profiles. RESULTS: The 36 Aeromonas isolates examined by RAPD-PCR generated RAPD fingerprints with majority of the bands ranging from about 250 to 2800 bp. The RAPD fingerprints did not correspond with the phenospecies and varied greatly among the strains within the phenospecies. Cluster analysis revealed two major groups at 75 per cent hierarchical level, comprising 18 Aeromonas isolates, mainly recovered from domestic water samples, while the clinical isolates were scattered in different hierarchical levels in the dendrogram. The whole cell protein fingerprints examined by SDS-PAGE did not correspond with the phenospecies. Only four isolates of A. caviae were found to produce similar protein fingerprints allowing them to form a cluster at about 90 per cent hierarchical level, while the rest of the isolates were scattered at various hierarchical levels in the dendrogram. INTERPRETATION & CONCLUSIONS: In the present study, RAPD fingerprinting was found to be useful in distinguishing Aeromonas isolates recovered from clinical and domestic water supplies. However, RAPD-PCR could not distinguish the phenospecies of the genus Aeromonas. Whole cell protein fingerprinting and cluster analysis could neither differentiate isolates from clinical and domestic water sources nor the phenospecies of the genus Aeromonas.

Production of siderophores & effect of iron restriction on the protein profiles of Aeromonas species isolated from water & patients suffering from acute diarrhoeal disease.

Among 48 strains of Aeromonas species, 21 isolates from patients suffering from acute diarrhoea and 27 from metropolitan water samples grown under iron-restricted conditions, 45 strains produced siderophores. Forty one of the 46 strains tested produced siderophores on chrome azurol S (CAS) agar, while 43 isolates did so when the culture supernatants of the bacterial isolates grown in minimal medium were assayed with chrome azurol S assay solution. The whole cell protein profiles of A. hydrophila strains grown under iron restricted conditions expressed new proteins that were not detected in those cultured in iron rich conditions. Five high molecular weight proteins ranging from 70 to 96 kDa were distinctly absent in cultures grown in the presence of iron, indicating their role in iron acquisition by the aeromonads.

Occurrence of haemolytic & cytotoxic Aeromonas species in domestic water supplies in Chennai.

A study on the occurrence of Aeromonas species in the domestic water supplies in Chennai showed that as much as 37.9 per cent of the water samples analyzed from various sources harbored Aeromonas spp. Majority of the isolates belonged to Aeromonas sobria (13.7%), A. caviae (11.6%) and A. hydrophila (9.5%). Among the 37 metropolitan water samples analyzed, 11 samples yielded Aeromonas spp. inclusive of three isolates of A. hydrophila, four of A. sobria and two isolates each of A. caviae and A. jandaei. From a total of 28 bore well water samples analyzed, Aeromonas spp. were recovered from 15 samples, comprising five isolates of A. hydrophila, six of A. sobria and four isolates of A. caviae. Aeromonas spp. inclusive of one isolate of A. hydrophila, five of A. caviae, three of A. sobria and one isolate of A. veronii were isolated from 10 of the 30 water packets of various commercial brands sold in Chennai. Of a total of 36 isolates obtained, 32 (89%) produced beta-haemolysin with the titres ranging from 2-32 and 20 isolates (56%) were cytotoxic to vero cell monolayers. All the Aeromonas isolates were resistant to ampicillin and polymyxin B. All A. hydrophila and A. caviae isolates were also resistant to cephalothin and erythromycin and 83.3 per cent of Aeromonas isolates were resistant to erythromycin. Aeromonads resistant to tetracycline, gentamycin, co-trimoxazole and nalidixic acid appear to be emerging. The study revealed that Aeromonas spp. occur in the potable and domestic water supplies and even in the chlorinated water supplies in Chennai city, which are potentially enteropathogenic and hence may be hazardous to public health. In view of these findings drinking and domestic water quality standards need to be re-evaluated.

Biochemical characteristics & secretory activity of Aeromonas species isolated from children with gastroenteritis in Chennai.

An attempt was made to delineate the phenotypic markers for the detection of enterotoxigenic strains of Aeromonas. Eighteen Aeromonas species comprising one isolate of A. hydrophila, six isolates of A. sobria and 11 isolates of A. caviae were obtained from 379 children suffering from acute diarrhoea in Chennai. Nine of these isolates inclusive of three A. sobria and six A. caviae were found to produce secretory response in vitro in the rabbit intestinal mucosa mounted in the Ussing chambers as revealed by significant increases in the short circuit current. Eleven strains hydrolysed aesculin, 8 fermented arabinose, 6 produced acetyl methyl carbinol, 14 produced lysine decarboxylase, 3 fermented salicin, 9 produced beta-haemolysin, 9 produced CAMP-like factor and only two isolates took up congo red dye. None of these phenotypic traits were found to correlate with the in vitro secretory activity.

Isolation of nalidixic acid resistant Campylobacters from cases of paediatric diarrhoea in Chennai.

A short term investigation on the Campylobacter enteritis among children under 10 years of age was carried out in Chennai. The study revealed an isolation rate of 11 per cent in 100 patients suffering from acute diarrhoea comprising C. jejuni (8%) and C. coli. (3%). Among the two culture methods used, the candle jar method was found to be superior to plastic bag incubation system in recovering campylobacters on charcoal cefeperazone deoxycholate agar. While all the isolates were sensitive to ciprofloxacin, all of them exhibited resistance to nalidixic acid.

Incidence & enteropathogenicity of Aeromonas spp in children suffering from acute diarrhoea in Chennai.

A total of 200 stool samples from children below 10 yr suffering from diarrhoea were screened for enteric pathogens with special interest on Aeromonas. Aeromonas spp were isolated from 6.5 per cent of the patients, comprising 4 per cent A. hydrophila, 2 per cent A. sobria and 0.5 per cent A. caviae. Among the 13 isolates obtained, 10 isolates produced enterotoxin in ligated rabbit ileal loops, and 11 produced cytotoxin in HEp 2 cells. Many of the Aeromonas isolates exhibited resistance to commonly used antibiotics such as trimethoprim, sulphdiazine, chloramphenicol and tetracycline. None of the stool samples obtained from 52 age matched control children yielded Aeromonas species. Four isolates of Salmonella typhi, 7 of S. paratyphi A, 6 of Shigella flexneri, 4 of Sh. dysenteriae and 3 isolates of Vibrio cholerae (Ogawa) were also recovered during the study. Among the samples analyzed, one from a 7 yr old female patient, had A. hydrophila with S. paratyphi A. The results of this study indicate that drug resistant enteropathogenic Aeromonas is also an important etiological agent of childhood diarrhoea in Chennai.