Prevalence of Vibrio Species Isolated from Fecal Specimens of Patients with Diarrhea in Siriraj Hospital during

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1 Original Article Vol. 21 No. 3 Vibrio isolated from fecal specimens in Siriraj Hospital:- Srifuengfung S, et al. 83 Prevalence of Vibrio Species Isolated from Fecal Specimens of Patients with Diarrhea in Siriraj Hospital during Somporn Srifuengfung, Ph.D. Podjanee Komolpis, M.D. Thitiya Yungyuen, M.Sc. Wanida Techachaiwiwat, M.Sc. Chanwit Tribuddharat, M.D., Ph.D. ABSTRACT This is a retrospective study on Vibrio spp. isolated from patients with diarrhea (both inpatients and outpatients) at Siriraj Hospital, Bangkok, Thailand from January 1994 to December Of the 33,334 fecal specimens sent for culture, 2,229 (6.68%) were positive for pathogenic bacteria. The most commonly isolated bacteria was Vibrio (1,183 of 2,229 isolates, 53%). Of these 1,183 isolates, there were V. parahaemolyticus (420, 35.5%), V. cholerae (Ogawa) (279, 23.6%), V. cholerae 0139 (240, 20.3%), V. cholerae non-01/non-0139 (167, 14.1%), V. fluvialis (44, 3.7%), V. alginolyticus (22, 1.9%) and V. cholerae (Inaba) (11, 0.9%). The prevalence of Vibrio spp. was highest (76.01%) in 1994 and then rapidly decreased (30.51%) in V. cholerae were susceptible to ampicillin ( %), tetracycline ( %) except V. cholerae 0139 (29.3%), trimethoprim/sulfamethoxazole ( %), norfloxacin (100%), ofloxacin ( %) and ciprofloxacin ( %). Vibrio spp. other than V. cholerae were susceptible to ampicillin ( %), tetracycline ( %), trimethoprim/sulfamethoxazole ( %), quinolones ( %) and third-generation cephalosporins (100%). (J Infect Dis Antimicrob Agents 2004;21:83-8.) INTRODUCTION Vibrio spp. are gram-negative, highly motile curved rods which are natural inhabitants of the marine environment. Pathogenic Vibrio spp. cause 3 major syndromes of clinical illnesses: gastroenteritis, wound infection and septicemia. Many species of Vibrio are considered pathogenic and capable of causing public health problems. Vibrio spp. of clinical relevance include V. cholerae, V. vulnificus, V. parahaemolyticus,v. mimicus and to a lesser extent, Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. Received for publication: October 19, Reprint request: Somporn Srifuengfung, Ph.D., Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. Keyword: Vibrio, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio fluvialis, Vibrio alginolyticus 83

2 84 J INFECT DIS ANTIMICROB AGENTS Sep.-Dec V. furnissii, V. hollisae, V. damsela and V. alginolyticus, respectively. 1-4 Early detection and initiation of treatment of Vibrio infections are very important, particularly of V. cholerae because these may rapidly progress to death 5 due to severe diarrhea induced by an enterotoxin. Our study reports on the prevalence of Vibrio spp. isolated from patients at Siriraj Hospital, a medical center, Bangkok, Thailand from 1994 to 2001 and their antimicrobial susceptibility patterns. MATERIALS AND METHODS A total of 33,334 fecal samples and rectal swabs were obtained from patients of all age groups with acute diarrhea (both inpatients and outpatients) at Siriraj Hospital during January 1994 and December 2001 according to the laboratory methods. 6 All specimens were initially cultured on blood agar, MacConkey agar, SS agar and thioglycolate citrate bile salt sucrose (TCBS) agar. They were also enriched in GN medium (semisolid agar), alkaline peptone water (APW), and incubated for hours at 35 ο C. After 6 hours of enrichment in APW, direct plating of the fecal samples was done on TCBS agar, and incubated at 35 ο C for 24 hours. For the identification of Vibrio spp., slide agglutination was performed with V. cholerae polyvalent O1, monospecific Ogawa, Inaba, Hikoshima and O139 antisera. Antimicrobial susceptibility test This was performed on Mueller Hinton agar plates by the disk diffusion method of Kirby and Bauer. 7 Ten antimicrobial agents used in this study included ampicillin, chloramphenicol, tetracycline, trimethroprim/ sulfamethoxazole, norfloxacin, ofloxacin, ciprofloxacin, cefotaxime, ceftriaxone, and ceftazidime. Escherichia coli ATCC was used as a quality control strain every time that clinical isolates were tested. RESULTS During the study period, 33,334 fecal cultures were carried out at Siriraj Hospital. Of these, 2,229 cultures (6.68%) were positive for pathogenic bacteria. The most commonly isolated bacteria was Vibrio spp. (1,183 of 2,229, 53%). The numbers of Vibrio spp. isolated from patients in each year are shown in Table 1. The prevalence of Vibrio spp. was highest (76.01%) in 1994 and then rapidly decreased to percent in 2001 (Figure 1). Of Vibrio spp., there were V. parahaemolyticus (420, 35.5%), V. cholerae (Ogawa) (279, 23.6%), V. cholerae O139 (240, 20.3%), V. cholerae non-o1/non-o139 (167, 14.1%), V. fluvialis (44, 3.7%), V. alginolyticus (22, 1.9%) and V. cholerae (Inaba) (11, 0.9%) (Table 2). Table 1. The numbers of Vibrio spp. isolates at Siriraj Hospital during 1994 and Years Total Number of total patients 5,830 3,789 4,065 4,086 4,502 3,842 3,676 3,544 33,334 Number of patients with positive ,229 for pathogenic bacteria Number of patients with positive ,183 for Vibrio spp. Percentage of Vibrio spp. (*) (*) Percentage of Vibrio spp. calculated from total positive pathogenic bacteria

3 Vol. 21 No. 3 Vibrio isolated from fecal specimens in Siriraj Hospital:- Srifuengfung S, et al Number of isolates Years Figure 1. The prevalence of Vibrio spp. isolates at Siriraj Hospital during 1994 and Table 2. The species of Vibrio spp. isolates during 1994 and Years Total % V. cholerae (Ogawa) V. cholerae (Inaba) V. cholerae O V. cholerae non-01/non-o139 V. parahaemolyticus V. alginolyticus V. fluvialis In each year during , most Vibrio spp. showed similar antimicrobial susceptibility patterns (Table 3 and 4). V. cholerae were susceptible to ampicillin ( %), tetracycline ( %) except V. cholerae 0139 (29.3%), trimethroprim/ sulfamethoxazole ( %), norfloxacin (100%), ofloxacin ( %), ciprofloxacin ( %) and ceftriaxone (100%) (Table 3). Vibrio spp. other than V. cholerae (V. parahaemolyticus, V. fluvialis and V. 85 alginolyticus) were susceptible to ampicillin ( %), chloramphenicol ( %), trimethoprim/ sulfamethoxazole ( %), quinolones ( %) and third-generation cephalosporins (100%) (Table 4). DISCUSSION In our study, we found that the most common Vibrio spp. identified were V. parahaemolyticus, V.

4 86 J INFECT DIS ANTIMICROB AGENTS Sep.-Dec Table 3. Antimicrobial susceptibility patterns of V. cholerae during 1994 and Antimicrobial agents V. cholerae V. cholerae V. cholerae O139 V. cholerae (Ogawa) (inaba) non-o1/non-0139 Ampicillin (266) 90 (10) 99.5 (218) (143) Tetracycline (80) 100 (7) 29.3 (218) (76) Trimethoprim/sulfamethoxazole (210) 100 (10) 100 (217) (110) Norfloxacin 100 (208) 100 (9) 100 (217) 100 (92) Ofloxacin 100 (205) 100 (10) 100 (80) 98.1 (109) Ciprofloxacin 100 (171) 100 (8) 100 (10) (84) Ceftriaxone 100 (235) 100 (10) 100 (218) 100 (126) ( ) denotes numbers of isolates tested. Table 4. Antimicrobial susceptibility patterns of Vibrio spp. other than V. cholerae during 1994 and Antimicrobial agents V. parahaemolyticus V. fluvialis V. alginolyticus Ampicillin 6.74 (393) 30 (43) 13.6 (22) Chloramphenicol 100 (168) 92.6 (41) 100 (22) Tetracycline (220) 61.5 (26) 100 (17) Trimethoprim/sulfamethoxazole 99.4 (167) 81.3 (33) 95 (20) Norfloxacin* 100 (278) 100 (29) 100 (20) Ofloxacin* 100 (289) 100 (29) 100 (17) Ciprofloxacin* 99.4 (278) 100 (3) 100 (16) Cefotaxime* 100 (381) 100 (42) 100 (22) Ceftriaxone* 100 (316) 100 (37) 100 (20) Ceftazidime* 100 (376) 100 (38) 100 (18) * The zone diameter interpretative standards were used according to Enterobacteriaceae (7). ( ) denotes numbers of isolates tested. cholerae (Ogawa and Inaba), V. cholerae O139, V. cholerae non-o1/non-o139, and V. fluvialis. Interestingly, the prevalence of Vibrio spp. isolates decreased from 1994 to This decreasing trend was similar to the study in Louisiana USA (1995), which showed that the most common isolated Vibrio spp. was V. parahaemolyticus, followed in order by V. vulnificus and other Vibrio spp. (V. fluvialis, V. hollisae and V. mimicus). 8 V. parahaemolyticus was first discovered in 1950, during a food poisoning outbreak in Osaka, Japan. 9 This bacterium is one of the most prevalent food-borne pathogens in Taiwan, Japan and other coastal countries. 10 It is often present in seawater and either grows on living marine organisms or exists freely. In Lousiana between 1988 and 1997, 88 percent of patients with V. parahaemolyticus gastroenteritis and 91 percent of patients with V. parahaemolyticus primary septicemia had a known

5 Vol. 21 No. 3 Vibrio isolated from fecal specimens in Siriraj Hospital:- Srifuengfung S, et al. 87 history of consumption of raw oyster. Consumption of crustacean and molluscan shellfish has commonly been implicated in the transmission of V. parahaemolyticus. A study in six countries of East Africa reported the rate of isolation of V. cholerae from rectal swabs increased from less than 20 percent in 1994 to more than 45 percent in The world now is experiencing a seventh cholera pandemic, with the El Tor biotype of V. cholerae O1 being the infectious agent. The pandemic started in Indonesia in 1961, spread to the Middle East in the late 1960s and into Africa in the 1970s, where it remains endemic. In 1991 the pandemic reached South America through Peru and spread rapidly. From 62 cases reported worldwide in 1961, the number reached a peak of 595,000 in By 1993 it decreased to 300, Cholera is now endemic in Southeast Asia, Africa and South America, with sporadic outbreaks worldwide. 13 Cholera is transmitted by the fecal-oral route. It is mainly caused by two serotypes: Inaba and Ogawa. Currently, the Center for Disease Control and Prevention of the US reported that between January 1995 and July 1999, there were 51 cases of laboratory confirmed V. cholerae O1 infections and no cases of V. cholerae O139 infection. However, in 1992 cholera caused by serogroup O139 emerged in epidemic proportions in India and Bangladesh. 14 It was suggested that this serogroup arose from an O1 strain of El Tor biotype by acquisition of foreign DNA involved in the synthesis of serogroup-determining O antigen. 15,16 Extensive outbreaks have also occurred in Pakistan, Nepal, Republic of China, Thailand, Kazakstan, Afghanistan and Malaysia So this was explained the high incidence of V. cholerae in 1994 in our study. Other Vibrio spp. may be clinically significant. These include V. vulnificus and V. alginolyticus. They cause infections in human and animals including fish. In Louisiana, 8 a total of twenty-four V. vulnificus cases were reported during 2000 and Sixty-three 87 percent of these had consumed oysters. Fifteen cases (61%) had wound infection from contact with salty blackish water or drippings from raw seafood. Consequently, Vibrio spp. isolates should be monitored carefully to detect their antimicrobial resistance especially those responsible for more pathogenic Vibrio such as V. cholerae. According to the National Committee for Clinical Laboratory Standards (NCCLS), the results of disk diffusion tests for ampicillin, tetracycline and trimethoprim/ sulfamethoxazole correlate well with those determined for minimal inhibitory concentration by the broth microdilution tests. 7 In our study, most Vibrio spp. was susceptible to ampicillin, tetracycline and trimethoprim/ sulfamethoxazole. This differs from the study in the East Africa region during 1994 and 1996 which showed that all isolates from Tanzania and Rwanda were 100 percent resistant to tetracycline, chloramphenicol and trimethopirm/sulfamethoxazole, while in South Sudan more than 70 percent of isolates were susceptible to these drugs. 12 A study in Baltimore demonstrated that the V. cholerae was highly susceptible to beta-lactam antibiotics. 13 According to the NCCLS, there are no zone diameter interpretive standards for quinolones and third-generation cephalosporins in V. cholerae. 7 We used the zone diameter interpretive standards for Enterobateriaceae for these antibiotics in V. cholerae, and for all tested antibiotics in Vibrio spp. other than V. cholerae. The clinician should aware that the susceptibility results may not be effective clinically or may not correlate well with the clinical outcome. The results of our study suggest that the prevalence of Vibrio spp. was decreasing. Cholera is endemic or epidemic in areas with poor sanitation. In Thailand, there was only one outbreak of V. cholerae 0139 in Therefore, the decreasing prevalence of isolation for Vibrio spp. may be due to good hygiene and sanitation. The results of local susceptibility patterns will provide clinicians for appropriate empirical

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