Antibiotic Susceptibility Patterns of Vibrio cholerae isolates

Introduction: Cholera is one of the most common diarrhoeal diseases in Nepal. Etiological agent of cholera is Vibrio cholerae which removes essential body fl uids, salts and vital nutrients, which are necessary for life causing dehydration and malnutrition. Emerging antimicrobial resistant is common. The aim of the present study was to determine the antibiotic susceptibility pattern of cholera patients in Nepal.


INTRODUCTION
Cholera is a disease caused by Vibrio cholerae.Since 1817, six pandemics have swept over the world, and the seventh one is in progress. 1Seventh pandemic was started from an Indonesian island which was caused by the E1 Tor biotype and it spread across Asia and Africa. 2 In Nepal, owing to the low socioeconomic status and poor hygienic condition of the people, Cholera constitute an important cause of morbidity and mortality. 3The case fatality rate in Nepal from diarrhoea is highest (2%) in the age group of 1-4 and is lowest (1%) among the infants. 4here are several serogroups of V. cholera, the O1 serogroup being the cause of endemic and epidemic cholera.Using specifi c antisera to detect different fractions of the O antigen, the O1 serogroup is divided into Inaba (AC), Ogawa (AB) and Hikojima (ABC) serotypes. 5Drug resistant V. cholera have been reported since 1977 and some of these strains were multidrug resistant. 6,7Due to the development of these resistant strains, it is now important to know the local antimicrobial susceptibility patterns of V. cholerae.

METHODS
This observational study was conducted in the bacteriology section of National Public Health Laboratory (NPHL), Teku, Kathmandu from March to September 2005, following standard microbiological techniques.Each patient suffering from diarrhoea was directly interviewed for his or her demographic profi le (name, age, sex, address and hospital treated) and signifi cant clinical history (sign, symptoms, stool passage frequency, stool consistency, presence of blood and mucus in the stool) during sample collection.Data was entered on a standard excel worksheet.Data analysis was performed by using Microsoft Excel 2007 and statistical package of social sciences (SPSS).
Stool sample was primarily inoculated on the plate of MacConkey Agar (MA) medium and Thiosulphate Citrate Bile Salt Sucrose (TCBS) medium, at the same time a loopful sample was inoculated in Alkaline Peptone Water (APW) for enrichment and incubated for 4-6 hours.From APW, sample was subcultured on second plate of MA and TCBS.All inoculated plates were incubated aerobically at 37ºC for 24 hours.Vibrio suspected non-lactose fermenting colonies on MA and sucrose fermenting yellow colonies on TCBS were subcultured on Nutrient Agar (NA) medium and incubated for 24 hours at 37ºC.Further processing was performed from the colonies on NA plate.Antibiotic susceptibility testing was done on Mueller-Hinton Agar plate by Kirby-Bauer disc diffusion method.Sensitivity of Ampicillin, Nalidixic acid, Ciprofl oxacin, Cotrimoxazole, Erythromycin, Furazolidone, and Tetracycline were tested for Vibrio cholerae.The results were categorized as sensitive, resistant and partially sensitive based on standardized zones of inhibition.

RESULTS
Out of 340 patients, 82.94% were from Shukraraj Tropical Hospital and 17.05% were from different locality of Kathmandu valley attending NPHL by requesting stool culture.There were total of 53 (15.58%) strains of V. cholerae and all of them were EI Tor, Inaba.Association of isolation between male and female was not statistically signifi cant.Majority of the patients were belonged to age group 21-30 years (Table 1).Isolation rate was found highest in August (45.28%)(Figure 1).Vomiting, dehydration, nausea and passage of rice watery stool with more than 10 times per day were reported as the main clinical features of the cholera (Table 2 & 3).All isolates were sensitive to Ciprofl oxacin, Ampicillin, Erythromycin and Tetracycline.Only 15.1% isolates were sensitive towards Furazolidon but all isolates were resistant to Nalidixic acid and Cotrimoxazole (Table 4).Hundred percent V. cholerae isolates were Multi-Drug resistant.

DISCUSSION
Cholera is an important cause of acute diarrhoeal diseases in Nepal.Poor environmental hygiene, poor water sanitation and improper management of sewage wastes are the predominant factors that V. cholerae are established in Kathmandu valley.V. cholerae is one of the most important enteropathogens in all countries. 8he sixth pandemic of cholera and, presumably, the earlier pandemics were caused by the classical biotype of Vibrio cholerae O1, which was progressively replaced by the El Tor biotype representing the seventh cholera pandemic. 9Eighth pandemic in cholera history, could be due to Vibrio cholerae O1 El Tor, Inaba serotype, infection. 10Our result also support this view.
It was reported that for the last six years V. cholerae El Tor O1 Ogawa was responsible for the cholera endemic in Kathmandu, but in 1990 it was V. cholerae El Tor O1 Inaba, so it can be said that after 15 years later V. cholerae O1 Inaba emerged hugely in Kathmandu. 11ome previous outbreaks of cholera in Kathmandu valley like, in 1994 and in 2004 were due to V. cholerae, El Tor, O1 Ogawa. 12,13Although Ogawa was predominant, frequently Hikojima and to less often Inaba serotypes was also isolated in Nepal. 14brio cholerae O1 strains with a periodic shift of different serotypes (Inaba and Ogawa) may be a consequence of genetic reversion which is known to take place in vitro and vivo. 15,16The only changes observed between the Ogawa and Inaba serotypes are related to a mutation in the wbeT region on a 21.6kb SacI fragment of DNA. 16,17The epidemic strains had undergone a serotype conversion, possibly because of immune pressure in the population; or due to seletive pressure of anti-Ogawa antibodies and the emergence of anti-Inaba antibodies. 15,18 the present study, the highest cholera cases were seen among the age group between 21-30 years and were predominant among male patients.Although children are the primary victims of cholera, the present result may be due to higher frequency of adult patients visiting NPHL.The incidence of cholera are frequently reported in August and September. 14,19Vomiting, dehydration, nausea with rice watery stool and stool passage frequency ≥10 were found the important clinical manifestations exhibited by cholera patients which are the universal clinical features of cholera.
Antibiotics shorten the course and diminish the severity of the illness, but they are not as important as rehydration, which is the cornerstone of therapy for cholera.The susceptibility pattern of the isolates, against seven different antibiotics, belonging to different groups were studied and found all with multiple antibiotic resistant capacity.It appears that antibiotic-resistant strains are increasingly being found worldwide. 20,21nce, Tetracycline is the drug of choice for cholera because it is more effective, non-toxic, easily available and comparatively cheap.In our study and in some other studies, all isolates were sensitive to Tetracycline and Cotrimoxazole. 22But Tetracycline, Cotrimoxazole and Furazolidon resistant V. cholerae strains were also reported. 11,23Resistance to Cotrimoxazole, Furazolidone and Streptomycin suggests the possibility of the presence of the SXT element in Inaba strains. 24Genes and their mechanisms of transfer may be important in the maintenance and transfer of resistance among V. cholerae and other enteric pathogens.In 1989, Erythromycin was reported to be effective for the treatment of Tetracycline resistant strains. 25All isolates in this study were found sensitive towards Erythromycin.
This study can help health authorities to review the cholera treatment guideline.Health education campaigns about prevention of diarrhoeal diseases and proper disposal of sewage, good sanitation and hygiene may be effective practice to control the cholera in Kathmadu Valley.

CONCLUSIONS
In Kathmandu Valley there is an incidence of cholera in the rainy season almost every year.The marked ability of V. cholera isolates to resist the inhibitory action of antibiotics has become a global problem.Increasing antibiotic resistance against commonly prescribed drugs signify that treatment options have become diffi cult in cases of cholera.The results indicate a continuing need for resistance surveillance and rational use of antimicrobial agents to reduce the multi-drug resistant strains of Vibrio.

Table 1 . Age and gender wise distribution of samples and V. cholera cases
Shrestha et al.Antibiotic Susceptibility Patterns of Vibrio Cholerae isolates in Kathmandu Valley

Table 3 . Physical characteristics of stool exhibited by cholera patients
Shrestha et al.Antibiotic Susceptibility Patterns of Vibrio Cholerae isolates in Kathmandu Valley