Antimicrobial susceptibilities of enteric bacterial pathogens isolated in Kathmandu , Nepal , during 2002-2004

Introduction: The prevalence and antimicrobial susceptibility patterns of the bacterial enteropathogens Vibrio cholerae, Salmonella species and Shigella species were investigated. Methodology: A total of 877 stool samples were received for culture at the National Public Health Laboratory (NPHL), Kathmandu, Nepal, during January 2002 to December 2004, from diarrhoea patients attending Shukraraj Tropical Infectious Hospital and referral outpatients. All samples collected were processed for isolation and antibiotic susceptibility testing of Vibrio cholerae, Salmonella spp. and Shigella spp. Results: Of the 877 stool samples, 148 (16.8%) were culture positive for one of the three bacterial enteropathogens investigated. Among them, Vibrio cholerae, Shigella spp. and Salmonella spp. accounted for 98/877 (11.1%), 41/877 (4.6%), 9/877 (1.02%) of the isolates respectively. A year-to-year variation was seen in the type of predominant organism, with Shigella spp. being the most prevalent in 2002 and 2003 and Vibrio spp. in 2004. In all three years, Vibrio cholerae were encountered only during the months of April to June while Salmonella spp. and Shigella spp. were isolated throughout the whole year. All Vibrio cholerae and Salmonella isolates were susceptible to ciprofloxacin. All Shigella isolates were susceptible to ceftriaxone. Ciprofloxacin resistance was observed among isolates of Shigella dysenteriae type-1 isolated after 2003. Conclusion: Vibrio cholerae, Salmonella and Shigella infections are prevalent in Kathmandu, Nepal. A gradual increase in resistance to commonly used antimicrobials was seen among bacterial enteropathogens. Antimicrobial resistance surveillance is necessary to guide empirical treatment.


Introduction
Infectious diarrhoeal diseases are responsible for considerable morbidity and mortality, especially in developing countries [1].According to a 2009 World Health Organization (WHO) bulletin, diarrhoeal diseases account for an estimated annual 1.5 million deaths among children younger than five years old in the world, while in Nepal 1.05 % (37,000/3,535,000) mortality was reported among children younger than 5 years old [2].Owing to the low socioeconomic status and poor hygienic conditions of the people in Nepal, intestinal parasitic and bacterial infections constitute a major cause of morbidity and mortality, contributing to several epidemics each year [3].Gastroenteritis prevails throughout the year with epidemics mainly in the rainy season [4].Among the bacterial enteric-pathogens, Vibrio cholerae, Salmonella spp.and Shigella spp.are of special concern because of the severity of the illness they cause and their association with various outbreaks [5].
Though the treatment of choice for acute diarrhoea is fluid and electrolyte replacement, antibacterial agents are often recommended for treatment of suspected shigellosis, invasive salmonellosis and cholera.Since most diarrhoeal diseases are treated empirically, it is important to know the susceptibility pattern of the prevalent pathogens [6].The problem of antimicrobial resistance in bacterial pathogens causing diarrhoeal diseases continues to be alarming.Emergence and spread of antimicrobial resistance to newer and more potent agents used in treatment have been described for Salmonella, Shigella and Vibrio cholerae [7,8,9].
Information concerning enteric pathogens in each country is essential in terms of epidemiology, surveillance, and management of patients.Despite few studies on diarrhoeal diseases in Nepal, there is lack of adequate information on bacterial enteric pathogens and their antimicrobial resistance trend over a longer time period in Kathmandu valley.Hence this study aimed for the first time to investigate the prevalence of common enteric bacterial pathogens, Vibrio cholerae, Salmonella spp.and Shigella spp., and their antimicrobial susceptibility profiles in Kathmandu over a period of three years.

Methodology
The study was conducted at the National Public Health Laboratory (NPHL) in Kathmandu, the national reference laboratory of Nepal that receives referral samples for various laboratory investigations from different health-care institutions as well as from selfreferred patients.However, the majority of samples referred to NPHL come from the Shukraraj Tropical Infectious Hospital, an infectious disease hospital for adults located in close proximity to NPHL.A total of 877 stool culture samples were obtained from NPHL during a period of three years from January 2002 to December 2004.Patient's demographic data were recorded which included name, age/sex and date of specimen collection.As expected, the majority of samples were from adult diarrhoea patients from Shukraraj Tropical Infectious Hospital.The samples were processed for isolation of Vibrio cholerae, Salmonella spp.and Shigella spp.Alkaline peptone water was used for the enrichment of Vibrio cholerae, whereas Gram-negative (GN) broth (BD Diagnostic system, Sparks, MD, USA) was used for the enrichment of Samlonella spp.and Shigella spp.The antimicrobial susceptibility testing of Vibrio cholerae, Salmonella spp.and Shigella spp.were performed by Kirby Bauer's disc diffusion technique [10] using commercially available discs (Oxoid Limited, Basingstoke, England).The antibiotics tested for Vibrio cholerae were tetracycline (T, 30 µg), nalidixic acid (NA, 30 µg), ciprofloxacin (Cip, 5 µg), erythromycin (E, 15 µg), cotrimoxazole (Sxt, 25 µg) and furazolidone (F, 100 µg).The antibiotics tested for Shigella spp.were ampicillin (Amp, 10 µg), ciprofloxacin (Cip, 5 µg), nalidixic acid (NA, 30 µg), cotrimoxazole (Sxt, 25 µg), mecillinam (Mel, 25 µg) and ceftriaxone (CRO, 5 µg), while ampicillin (Amp, 10 µg), ciprofloxacin (Cip, 5 µg), chloramphenicol (Chl, 30 µg), cotrimoxazole (Sxt, 25 µg), ceftriaxone (CRO, 5 µg) and nalidixic acid (NA, 30 µg) were tested for Salmonella spp..In this study, the defining criterion for multidrug resistance (MDR) was resistance to > 2 of the antimicrobial agents belonging to different structural classes [11,12].

Results
Prevalence of Vibrio cholerae, Shigella spp.and Salmonella spp.
Out of 877 stool specimens, 148 (16.8%) were culture positive for the bacterial entero-pathogens investigated in the study.Of the 148 culture positive stool specimens, 87 were from males and 61 were from females (data not shown).There was no significant association between gender and enteropathogenic bacterial infection (p > 0.05).Of the 148 culture positive specimens, 43/148 (29.05%) were from the age group of 20 to 29 years (Table 1).However, the yearly breakdown of enteric pathogens showed that the highest isolation rates of 6/28 (21.42%) and 5/29 (17.24%), in the years 2002 and 2003 respectively, were from children aged 0-9 years.
Vibrio cholerae, Shigella spp.and Salmonella spp.were isolated from 98, 41 and 9 out of 877 stool specimens respectively (Table 2).Mixed infections of these enteric pathogens were not detected.Salmonella spp.and Shigella spp.were encountered throughout the whole year whereas Vibrio cholerae were isolated only during the months of April to June across the 3 year period.The monthly distribution of these enteropathogens is shown in Table 3.A year-to-year variation was observed in the type of prevalent organism with Shigella being the most prevalent in 2002 (12/20; 60%) and in 2003 (8/15; 53.33%).In 2004, a large increase in the number of Vibrio cholerae isolates was observed compared to those in 2002 and 2004, indicating an outbreak of cholera (Table 2).All Vibrio cholerae isolates were identified as belonging to serogroup O1, El Tor biotype and Ogawa serotype.Out of 41 Shigella spp., Shigella dysenteriae was the most common (26/41; 63.41 %) followed by Shigella flexneri (9/41; 21.95 %), while Shigella boydii and Shigella sonnei accounted for 3/41 (7%) each.Of the 26 Shigella dysenteriae isolated, 6 (24.39%) were identified to be Shigella dysenteriae type-1.Only nine isolates of Salmonella were found during the study period of which five were Salmonella Typhi, three were S. Paratyphi A and one was S. Enteritidis.

Antimicrobial susceptibility profile
All Vibrio cholerae isolated were resistant to nalidixic acid, but remained susceptible to tetracycline and ciprofloxacin (Table 4).Resistance rates for furazolidone and erythromycin varied during the study period.Cotrimoxazole resistance gradually increased from 35% in 2002 to 100% in 2004 (Table 4).All the Shigella isolates in the study were susceptible to ceftriaxone (Table 4).Nalidixic acid resistance in Shigella isolates increased from 43% in 2002 to 55% in 2004.No ciprofloxacin resistance was observed among the Shigella spp.isolated in 2002, but in the years 2003 and 2004 ciprofloxacin resistance was seen among 20% and 24% of the isolates.Serological typing identified all the ciprofloxacin resistant Shigella isolates to be Shigella dysenteriae type-1.All nine Salmonella isolates were susceptible to ciprofloxacin and ceftriaxone (Table 4).Out of five Salmonella Typhi identified, three were MDR showing simultaneous resistance to ampicillin, chloramphenicol, cotrimoxazole and nalidixic acid.

Discussion
Intestinal enteropathogens which cause gastroenteritis are major public health problems in developing countries, especially among children and the elderly.The present study showed that the enteropathogenic bacteria were almost equally distributed in both the genders.The majority of the bacterial enteropathogens encountered were from patients aged 20 to 29 years.This result is in agreement with recent outbreaks of diarrhoeal diseases in the western part of Nepal, which has reported that adults aged 15 to 44 years were most affected with equal impact on males and females [13].Another report from Kavrepalanchok district, a region near Kathmandu valley, also found the most common age group to be 11 to 20 years, followed by ages 21 to 30 years [14].However, our results contrast those of other studies [6,15] which report diarrhoeal diseases to be prevalent in children.One of the major reasons for this difference is associated with the predominance of samples received from patients above nine years old.However, the yearly breakdown showed that the majority of isolates were from the children aged 0 to 9 years in the years 2002 and 2003.This overall higher distribution of entropathogens in the adult age group is due to the cholera outbreak in Kathmandu [16] in 2004, that led to a large number of adult patients seeking treatment at Sukraraj Tropical Hospital which referred stool samples for laboratory investigation to NPHL and resulted in the high number of Vibrio cholerae isolated in the current study.Cholera outbreaks in Asian countries have been caused by V. cholerae O1 biotype El Tor, specific strains of V. cholerae O1 biotype Classical and V. cholerae O139 (17).Without adequate appropriate therapy, severe cholera kills approximately half of the affected individuals [18].In this study, Vibrio cholerae which contributed to the cholera epidemic in Kathmandu, Nepal, in 2004 [16] was isolated in the highest frequency (89/113; 78.76%) in 2004, followed by Shigella spp.(21/113; 18.5%) (Table 2).All the Vibrio cholerae isolates in our study were Vibrio cholerae O1 Ogawa biotype El Tor and were 100% susceptible to tetracycline and ciprofloxacin.Ciprofloxacin is widely used in the empirical treatment of cholera, but the emergence of ciprofloxacin resistance in Vibrio cholerae from different parts of the world has raised concern [19,20].Although no ciprofloxacin-resistant strains were encountered in this study, all the Vibrio cholerae isolates were resistant to another quinolone, nalidixic acid.Nalidixic acid resistance can be suggestive of impending ciprofloxacin resistance among Vibrio cholerae isolates in Nepal.Hence continuous monitoring is necessary to trace changes in susceptibility patterns and the emergence of resistance to new agents.

Number of isolates
Shigellosis in developing countries are commonly caused by Shigella dysenteriae and Shigella flexneri species and their presence is associated with inadequate sanitation, while Shigella sonnei is more prevalent in developed countries [21].In agreement with this notion, our results also showed that Shigella dysenteriae and Shigella flexneri were the most prevalent of the four Shigella species.Among Shigella infections, those caused by Shigella dysenteriae type-1 are of major concern because of their potential to cause outbreaks and their high mortality rates.In this regard, our finding that around one fourth of the Shigella dysenteriae isolates were Shigella dysenteriae type-1 is of special concern.Increasing antimicrobial resistance is also becoming a problem in the treatment of Shigellosis [8].The majority of Shigella species in this study, especially Shigella dysenteriae, were also MDR.Widespread use of nalidixic acid as the first-line drug for treatment of shigellosis resulted in the emergence of nalidixic acid resistant strains.After the spread of nalidixic acid resistance, ciprofloxacin was recommended as the first-line treatment for treatment of shigellosis.Multiple antibiotic resistance has been reported among Shigella spp.and lately ciprofloxacin resistance has also been reported among Shigella dysenteriae type-1 isolates from various countries [21,22].In this study, ciprofloxacin resistance was also encountered among Shigella dysenteriae type-1 strains isolated in the years 2003 and 2004.These ciprofloxacin-resistant strains were MDR, also showing co-resistance to ampicillin, chloramphenicol, cotrimoxazole and nalidixic acid, but were susceptible to ceftriaxone and mecillinam.Of the nine Salmonella spp.isolated in this study, three were MDR (showing co-resistance to ampicillin, chloramphenicol and cotrimoxazole) and were identified to be Salmonella Typhi.[23].All the Salmonella isolates in our study remained susceptible to ciprofloxacin and ceftriaxone.Multidrug resistance has been reported in Salmonella since 1989 [24] and the spread of multidrug resistance in Salmonella species is one of the major therapeutic challenges in the treatment of such infections.The incidence rates of MDR Salmonella species were 26% in the United Kingdom and 17% in the United States, but infections have been detected in patients with a recent history of travel to Asian countries [25,26].In India an even higher percentage of Salmonella Typhi reported in 1993 were multiple antibiotic resistant (64.5%) [27].

Month
The identification and management of outbreaks of cholera, Salmonella infections and shigellosis in Nepal is still challenging due to limited laboratory facilities in both the government and private sectors, as well as a lack of awareness about diarrhoeal infections and the limited practice of pathogen-directed antimicrobial therapy [28].In this context, the present study addresses some important issues about diarrhoeal infections and their most common aetiogical bacterial agents at the national reference laboratory of Nepal.In conclusion, our results showed that enteric bacterial infections caused by Vibrio, Salmonella and Shigella are prevalent in Kathmandu Valley.Considering the threat of emerging antimicrobial resistance among these enteric bacterial pathogens, it is important to continue surveillance on these organisms in terms of prevalence, clinical epidemiology, and antimicrobial susceptibility patterns obtained from different hospital and community settings throughout the country.

Table 1 .
Age-wise distribution of total and enteropathogen positive patients

Table 3 .
Monthly distribution of enteropathogenic bacteria in 3 years