alvina 14 ABSTRACT Aeromonas species are gram-negative, motile, facultative anaerobic, rod shaped, oxidase positive bacteria of the recently assigned family Aeromonadaceae. The significance of Aeromonas species as causative agent of human diarrhoea has recently been established. The aim of the present study was to investigate the distribution, and antibiotic sensitivity of Aeromonas in nonhospitalized children with diarrhea.One hundred and seventeen rectal swabs from children with diarhhea were cultured for isolation of Aeromonas organisms as the etiological agents. In addition to Aeromonas, other enteric pathogens were also isolated. Overall, the isolates of enteric pathogens amounted to 36.8%, consisting of Salmonella, Shigella, Aeromonas, and Vibrio. Aeromonas was only found in 5.1% of cultures, with a ratio of A. caviae and A. hydrophila of 2:1, while Salmonella made up the majority of causative organisms with an isolation frequency of 18.8%, followed by Shigella with 11.1%. In this study no isolates of Vibrio cholerae O1 were found as etiological agents of diarrhea; however, V. cholerae non-O1 and V. parahaemolyticus were found in small numbers (<1%). All isolates of Aeromonas were resistant to ampicillin and tetracycline, but sensitive to ciprofloxacin and ceftriaxone, as were the other enteric pathogens. Although the frequency of isolation of these enteric pathogens was higher than for Vibrio spp., their role in infective diarrhea was less clearcut in comparison with Salmonella and Shigella. Keywords: Diarrhea, Aeromonas, resistance, children *Department of Pharmacy, **Department of Public Health, ***Department of Microbiology, Medical Faculty, Trisakti University Correspondence a dr. Meiyanti, SpFK Department of Pharmacy Medical Faculty, Trisakti University Jl. Kyai Tapa No.260 Grogol - Jakarta 11440 Phone: 021-5672731 ext.2805 Univ Med 2010;29:14-20 Isolation and antibiotic sensitivity of Aeromonas from children with diarrhea Meiyanti*a, Oktavianus Ch. Salim**, Julius E. Surjawidjaja***, and Murad Lesmana*** January-April, 2010January-April, 2010January-April, 2010January-April, 2010January-April, 2010 Vol.29 - No.1 Vol.29 - No.1 Vol.29 - No.1 Vol.29 - No.1 Vol.29 - No.1 UNIVERSA MEDICINA INTRODUCTION There are approximately 40 species of diarrhea-causing pathogens, and around half of them were only described within the last t h r e e d e c a d e s . A l l t h e s e p a t h o g e n s a r e relatively important in some age groups and/or geographical localities, both because of the chronicity of the involved pathogens as well as the duration of the disease, or because of progress of the disease from the acute to the 15 Univ Med Vol.29 No.1 chronic form.(1) To date many cases of diarrhea a r e c o n s i d e r e d t o b e c a u s e d b y v i r u s e s , because of the difficulties and failures in isolation and identification of these pathogenic bacteria.(1) Among these bacteria is the species Aeromonas, which is one of the important groups of enteropathogenic microorganisms whose etiological role in diarrhea is subject to controversy, such that it has received scant attention and is frequently ignored.(2) The s p e c i e s A e ro m o n a s c o n s i s t s o f m o t i l e mesophilic Gram-negative rods. As a result of various studies(3-6) it is recognized that the species Aeromonas is universally distributed in freshwater and other environments and may be isolated from clinical material, potable w a t e r, f o o d s a n d b e v e r a g e s . I n t h e s e environments the bacteria are capable of reproduction even at low temperatures.(7) Among the bacteria causing diarrhea the role of A e ro m o n a s a s a n e n t e r i c p a t h o g e n i s i n c r e a s i n g l y a c k n o w l e d g e d a n d t h e r e i s d e f i n i t e e v i d e n c e t h a t t h e o r g a n i s m i s associated with gastroenteritis in children, adults, and the elderly(6,7) and with cases of ’traveler ’s diarrhea’. (7) Although healthy individuals may become carriers of this bacterium, it is reported that Aeromonas is a significant cause of diarrhea.(8,9) The three phenotypes of Aeromonas that are considered to be the main pathogens and to be associated with gastrointestinal infectious disease are A.hydrophila, A.caviae, and A. veronii biovar sobria, but the most frequently associated with diarrheal infection is A. hydrophila,(5) as reported by Juan et al.(10) In addition these organisms produce virulence f a c t o r s t h a t a r e u s e f u l a s m a r k e r s f o r d i ff e r e n t i a t i n g b e t w e e n p a t h o g e n i c a n d nonpathogenic strains.(11) In Indonesia, data on the presence of Aeromona s in connection with diarrheal d i s e a s e a r e d i ff i c u l t t o o b t a i n , b e c a u s e Aeromonas is not among the bacteria that are routinely reported as etiological agents of d i a r r h e a , a s i s t h e c a s e w i t h S h i g e l l a , Salmonella or Vibrio cholerae. Actually this bacterium may cause various disorders, either intestinal (diarrhea) or extraintestinal, (11) making it necessary to pay attention to its presence. The aim of the present study was to investigate the distribution, and antibiotic sensitivity of Aeromonas in nonhospitalized children with diarrhea. METHODS Research subjects and location The study was performed in infants and children aged <14 years with diarrhea, who were visiting the Tebet and Mampang Health Centers in South Jakarta. The study was c o n d u c t e d f r o m D e c e m b e r 2 0 0 8 u n t i l November 2009. Media X y l o s e - l y s i n e - d e o x y c h o l a t e ( X L D ) , MacConkey (MAC), Salmonella-Shigella (SS), and thiosulfate citrate bile salts sucrose agar (TCBS) (DIFCO, Becton Dickinson, S p a r k s , M D ) , w e r e u s e d a n d p r e p a r e d according to standard methods. Sampling and culture Rectal swabs were collected from the study participants after informed consent had been obtained from their parents or caregivers. Personal data and data related to the diarrhea were obtained from the parents or caregivers and recorded on standard forms by special personnel. Collection of rectal swabs was done irrespective of degree of diarrhea (mild, moderate or severe). Rectal swabs were taken at the time of admission to the Health Center and before administration of antibiotics. The swabs were placed in Cary Blair transport medium and stored in a refrigerator, pending transportation to the laboratory. The samples were transported in thermos flasks to the Microbiological Laboratory, Medical Faculty, Trisakti University. The rectal swabs were plated on MAC, SS, XLD and TCBS agar and 16 the agar plates were incubated under aerobic conditions at 370C for 18-20 hours. Non-lactose fermenting colonies on MAC, SS and XLD agar, and suspected colonies of Vibrio on TCBS agar, were selected and characterized in biochemical test media, namely Kligler’s iron agar (KIA), mannitol motility ornithine (MIO), and sucrose semi-solid (SSS).(12) Preliminary screening for colonies of Aeromonas was performed by means of the oxidase test. Oxidase negative, ornithine positive (MIO positive) colonies were considered to be Aeromonas and were subjected to further testing for species determination. Confirmatory serological testing was performed using specific antisera (Difco laboratories, Detroit, MI). The bacterial isolates were subsequently tested for susceptibility to a variety of antibiotics by the disk diffusion method as described in the manual of the National Committee for Clinical Laboratory Standards (NCCLS).(13,14) The antibiotics used in these sensitivity tests were ampicillin, chloramphenicol, tetracycline, cotrimoxazole, ciprofloxacin, and ceftriaxone (Becton Dickinson and Company, Cockeysville, MA, USA). Escherichia coli ATCC 25922 was included in the tests as control strain. Data analysis Percentage analysis was done to describe the distribution and antibiotic sensitivity of Aeromonas. RESULTS Bacterial culture of 117 rectal swab samples collected from infants and children aged <14 years with diarrhea resulted in the isolation from 43 (36.8%) subjects of the f o l l o w i n g e n t e r o p a t h o g e n i c b a c t e r i a i n descending order of frequency: Salmonella 22 (18.8%), Shigella 13 (11.1%), Aeromonas 6 (5.1%) and Vibrio 2 (1.7%). The number of Aeromonas caviae isolates was higher than that of A.hydrophila (3.4% vs 1.7% or 2:1). Isolates o f S a l m o n e l l a b e l o n g e d t o s e r o g r o u p B ( 3 6 . 5 % ) , C ( 2 2 . 7 % ) , D ( 1 8 . 1 % ) , a n d E (22.7%), while isolates of Shigella comprised S.flexneri (53.8%) and S.sonnei (46.2%). Isolates of Aeromonas ranked third among the causative organisms in this study, consisting o f A . h y d ro p h i l a ( 3 3 . 3 % ) a n d A . c a v i a e (66.7%). Vibrio cholerae was not found among the children with diarrhea in the locality of the present study, but V.cholerae non-O1 and V. Age of patient/number of isolates (%) Organism Number of positives 0-1 yr >1-4 yr >4-14 yr Salmonella spp. 22 Salmonella B 8 3 (37.5) 4 (50) 1 (12.5) Salmonella C 5 2 (40) 3 (60) 0 Salmonella D 4 1 (25) 3 (75) 0 Salmonella E 5 2 (40) 3 (75) 0 Shigella spp. 13 S. flexneri 7 1 (14.3) 6 (85.7) 0 S. sonnei 6 4 (66.6) 1 (16.7) 1 (16.7) Aeromonas spp. 6 A. hydrophila 2 0 2 (100) 0 A. caviae 4 1 (25) 3 (75) 0 Vibrio spp 2 V. cholerae non-O1. 1 0 0 1 (100) V.parahaemolyticus 1 0 0 1 (100) Table 1. Distribution of enteric pathogens isolated from children with diarrhea Meiyanti, Salim, Surjawidjaja, et al Isolation of Aeromonas 17 Univ Med Vol.29 No.1 parahaemolyticus were obtained in small numbers (<1%) (Table 1). The results of the antibiotic sensitivity testing indicated that both A.hydrophila and A. c a v i a e w e r e r e s i s t a n t t o a m p i c i l l i n a n d tetracycline, but that A.hydrophila was also resistant to co-trimoxazole (Table 2). Shigella flexneri was resistant to various antibiotics, such as ampicillin, chloramphenicol, tetracycline and c o t r i m o x a z o l e , f o l l o w e d b y S a l m o n e l l a serogroup B, which was resistant to ampicillin, c h l o r a m p h e n i c o l a n d t e t r a c y c l i n e . A l l enteropathogenic organisms were still sensitive to ceftriaxone and ciprofloxacin. DISCUSSION There are still doubts as to the role of A e ro m o n a s i n t h e e t i o l o g y o f d i a r r h e a l infections in humans, which is presumably because Aeromonas is a heterogenous group of microorganisms, with only some subgroups being pathogenic, while there are still problems in subgrouping of these microorganisms.(11,15) However, within the last two or three decades the role of Aeromonas in various human diseases has been proven, particularly as a cause of gastroenteritis.(8,10,11,15) Although clinically it may be obvious that Aeromonas causes diarrhea, this organism is not looked for routinely by means of fecal culture or rectal swab, such that very little is known about the clinical evaluation of this b a c t e r i u m . T h e i s o l a t i o n o f A e ro m o n a s hydrophila as a cause of diarrhea has been reported from Taiwan,(10) India,(11)and other localities. (15-17) Juan et al. (10) reported that Aeromonas hydrophila is more prevalent in the summer, around August and September, with an isolation frequency of 24-30%. The number of affected males and females is almost equal, and is highest between the age of 1-3 years. From various localities it has been reported that the main causative organisms of diarrhea are Salmonella and Shigella,(18,19) followed by others such as Campylobacter and Aeromonas Table 2. Distribution of antibiotic sensitivity of pathogenic enteric bacteria isolated from children with diarrhea (n=43) Antibiotic / proportion of resistant bacteria Organism Total number of isolates AM C TE SXT CRO CIP NA Salmonella spp. Salmonella B 8 5 (62.5) 1 (12.5) 5 (62.5) 0 0 0 0 Salmonella C 5 1 (20) 0 1 (20) 1 (20) 0 0 1 (20) Salmonella D 4 1 (25) 0 1 (25) 0 0 0 0 Salmonella E 5 1 (20) 0 1 (20) 0 0 0 0 Shigella spp. S. flexneri 7 4 (57.1) 4 (57.1) 4 (57.1) 6 (85.7) 0 0 0 S. sonnei 6 0 0 6 (100) 6 (100) 0 0 0 Aeromonas spp. A. hydrophila 2 2 (100) 0 2 (100) 2 (100) 0 0 0 A. caviae 4 4 (100) 0 4 (100) 0 0 0 0 Vibrio spp V. cholerae non-O1. 1 1 (100) 0 0 0 0 0 0 V.parahaemolyticus 1 1 (100) 0 0 0 0 0 0 AM = ampicillin; C = chloramphenicol; TE = tetracycline; SXT = cotrimoxazole; CRO = ceftriaxone; CIP = ciprofloxacin; NA = nalidixic acid 18 hydrophila. (10, 20) In the present study the isolation of A. hydrophila ranks after that of Salmonella and Shigella in order of frequency. Their numbers are much smaller than those reported by Juan et al.,(10) but their order of frequency is similar to that reported by Yates(18) in his discussion on the etiologic agents of traveler’s diarrhea. The order of presentation of the most frequently isolated organisms may differ from one locality to another, due to s e v e r a l f a c t o r s , s u c h a s d i f f e r e n c e s i n a p p l i c a t i o n o f m i c r o b i a l i d e n t i f i c a t i o n techniques, seasonal variation and geographic location.(21) The frequency of isolation of Aeromonas obtained in the present study is less than the 9.7% reported by Subashkumar et al.(21) and the 17.7% reported by Sinha et al.,(11) but almost identical to the frequency reported by other investigators.(7, 23,24) The isolation frequency of Aeromonas might have been higher, had the study been conducted in a hospital, because patients with severe diarrhea are generally hospitalized. It has been reported that gastroenteritis due to Aeromonas, particularly A.hydrophila, is more commonly found in children and the e l d e r l y ( 1 0 , 1 7 ) a n d i n t o u r i s t s ( t r a v e l e r ’s diarrhea).(6,7,18,25) According to Vila(7) A.caviae is isolated with the highest frequency in traveler’s diarrhea, whereas A.hydrophila is predominant in the local population.(9-11,26) Although diarrhea due to Aeromonas is a self-limiting disease,(5) occasionally it may become severe and around 50% of patients with diarrhea due to this organism may develop persistent diarrhea(7) such that antibiotic therapy is indicated. The drugs of choice for diarrhea d u e t o A e ro m o n a s a r e t h e q u i n o l o n e s (ciprofloxacin). In our study, all Aeromonas species, both A.hidrophila and A.caviae, were still sensitive to ciprofloxacin. However, resistance to quinolones should receive more attention, because within the last years the emergence of quinolone-resistant Aeromonas strains have been reported in industrialized countries.(25) As to the â -lactam antibiotics, the majority of Aeromonas strains have been reported to be resistant to ampicillin, but to be still sensitive to third-generation cephalosporins such as ceftriaxone,(7,10) as is evident from the results of the present study. Resistance to chloramphenicol, tetracycline and cotrimoxazole is due to extensive usage of these antibiotics, particularly in developing countries.(7) The limitation of this study lies in the relatively small sample size that is inadequate for ascertaining that Aeromonas spp. plays an important role as an etiological agent of diarhhea in children. However, the presence of Aeromonas itself in feces is indeed less recognized and has received less attention, such that the organism is seldom looked for in patients with diarrhea.(10) Although the isolation frequency of Aeromonas is higher than that of Vibrio spp., its role in diarrheal infection is less obvious in comparison to Salmonella and Shigella. From the aspect of antibiotic therapy, the emergence of multiresistant Aeromonas strains should receive attention and antibiotic administration should be performed with care. CONCLUSIONS The main etiological agents of diarrhea are Salmonella, Shigella, Aeromonas and Vibrio, although the role of Aeromonas spp has to be put under scrutiny. All Aeromonas isolates are resistant to ampicillin and tetracycline, but are still sensitive to ciprofloxacin and ceftriaxone. ACKNOWLEDGEMENTS The investigators wish to express their gratitude to the Dean and Vice-Deans of the Medical Faculty, Trisakti University, for the funding of the present study. We also wish to extend our thanks to the staff of the Mampang and Tebet Health Centers in South Jakarta, for their support and cooperation in this study. We also thank Ms Rosma Oppusunggu for her tireless efforts in the laboratory. Meiyanti, Salim, Surjawidjaja, et al Isolation of Aeromonas 19 Univ Med Vol.29 No.1 REFERENCES 1. Thielman MT, Guerrant RL. Acute infectious diarrhea. N Engl J Med 2004;350:38-47. 2. Borchardt MA, Stemper ME, Standridge JH. Aeromonas isolates from human diarrheic stool and ground water compared by pulsed-field gel electrophoresis. Emerg Infect Dis 2003;9:224-8. 3. Wang G, Clark CG, Liu C, Puncknell C, Munro CK, Kruk TM, et al. Detection and characterization of the hemolysin genes in Aeromonas hydrophila and Aeromonas sobria by multiple PCR. J Clin Microbiol 2003;41:1048-54. 4. 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