Diarrhoea due to Aeromonas in children 75J DIARRHOEAL DIS RES 1999 Jun;17(2):75-80 © 1999 ICDDR, B: Centre for Health and Population Research0253-8768/99 $ 1.00+0.10

Characterization of Virulence Factors ofAeromonas isolated from Children with and

without Diarrhoea in Tripoli, Libya

Khalifa Sifaw Ghenghesh1, Fauzi Bara2, Belqis Bukris2,Awatif El-Surmani 2, and Salaheddin S. Abeid1

1Department of Medical Microbiology, Faculty of Medicine, Al-Fateh University of MedicalSciences, PO Box 80013, Tripoli; and 2Aljalla Children Hospital, Tripoli, Libya

ABSTRACT

During September 1992-August 1993, stool samples from 157 children with diarrhoea and 157matched healthy controls were examined for the presence of Aeromonas and otherenteropathogens. Aeromonas strains were tested for haemolytic activity, haemagglutinationpatterns, and antibiotic susceptibility. In total, 62 Aeromonas were isolated, of which 27 (17.2%)were from children with diarrhoea and 35 (22.3%) from healthy controls. Only 23 (14.6%) ofthe diarrhoeal children and 28 (17.8%) of the healthy controls were positive for Aeromonas; ofwhich, 4 (2.5%) of the diarrhoeal children and 6 (3.8%) of the healthy controls showed multiplespecies. Aeromonas hydrophila was isolated from 5 (3.2%) children with diarrhoea and from 9(6.4%) controls, A. veronii bv sobria from 8 (5.1%) and 7 (4.5%), A. caviae from 13 (8.3%) and17 (10.8%), and A. schubertii from 1 (0.6%) and 2 (1.3%) respectively. No significant differencein the haemolytic activity of Aeromonas was found between diarrhoeal and healthy children.However, a significant difference (p<0.002) was observed in mannose-resistanthaemagglutination (MRHA) by diarrhoeal isolates of Aeromonas (7/27, 26%) compared to thehealthy controls (1/35, 3%). Aeromonas strains were uniformly sensitive to ciprofloxacin,gentamicin, and nalidixic acid. The results of this study suggest that A. caviae strains may beassociated with diarrhoea in children and MRHA may be used as one of the virulence markersfor distinguishing between Aeromonas isolated from diarrhoeal children and healthy controlsor environmental isolates.

Key words: Aeromonas; Diarrhoea, Infantile; Haemolysin; Haemagglutination; Microbialsensitivity tests

Correspondence and reprint request should be addressedto: Dr. Khalifa Sifaw GhengheshAssociate Professor, Department of Medical MicrobiologyFaculty of Medicine, PO Box 80013, Tripoli, LibyaFax: +218 21 333 4474 and +218 21 490 6397Tel: +218 21 444 7343

INTRODUCTION

Aeromonas species are oxidase-positive, Gram-negative,facultative anaerobic, rod-shaped bacteria, and areassociated with acute diarrhoea in children (1-3).

Aeromonas hydrophila and A. sobria (4, 5) and, recently,A. caviae (6,7) have been described as causative agentsof gastroenteritis. It is reported that there is no significantdifference in isolation rates of Aeromonas species fromchildren with diarrhoea compared to controls (8,9).

Aeromonas species have been reported to elaboratea number of virulence factors, such as exotoxins(haemolysins, cytotoxins, enterotoxins), haemag-glutinins, and invade tissue in culture (10-15). Burke etal. (16) found that when haemolysin production alone

76 J DIARRHOEAL DIS RES Jun 1999 Ghenghesh KS et al.

was used for characterizing Aeromonas species, 97% ofthe strains concurrently produced enterotoxins. Otherworkers also reported a correlation between haemolysinand cytotoxin production (9). However, enterotoxigenicisolates of A. hydrophila showed haemagglutination(HA) which was not sensitive to mannose and fucose,but Aeromonas strains that were HA-sensitive tomannose or no haemagglutination (NHA) were non-toxicstrains of A. caviae commonly isolated from non-diarrhoeal infection or the environment (10).

To understand the prevalence of Aeromonas speciesand their potential to cause gastroenteritis, someinvestigators (17) suggested to isolate these organismsfrom well-defined populations. This study was, therefore,carried out to determine the prevalence of Aeromonasstrains in children with diarrhoea and in age-and sex-matched controls in Tripoli area (population>1.250,000), to identify the species and to investigatetheir haemolytic activity, haemagglutination patterns, andantibiotic susceptibility.

MATERIALS AND METHODS

During September 1992-August 1993, stool samplesfrom 157 children (85 males) with acute diarrhoea (threeor more liquid stools per day), admitted to the AljallaChildren Hospital in Tripoli, Libya, were examined.During the same period, stool samples from 157 age-and sex-matched children without diarrhoea (controls)were obtained. Control samples were from patients whohad not had diarrhoea or received antibiotics within 2weeks of specimen collection. The age of the studychildren ranged from few days to 3 years, but most(84.7%) were aged less than one year.

Clinical data obtained included: age, sex, date ofonset of diarrhoea, presence of frank mucus or blood instools, presence of fever or vomiting, and antibioticstaken before stool collection. Whenever possible,information on the source of water (treated or untreated)used for drinking and recent travel abroad was alsoobtained.

Stool samples were also examined for Salmonella,Shigella, and Yersinia enterocolitica.

To isolate Aeromonas, faecal specimens were sub-cultured on blood agar, containing 15 mg/L ampicillinafter enrichment in alkaline peptone water (pH 8.6)

overnight at 37oC. Plates were incubated at 37oC for 18-24 hours. Suspected colonies were tested for the presenceof oxidase growth in nutrient broth with 0% and 6%NaCl, resistance to the compound O/129 (2, 4-diamino-6, 7-diisopropylpteridine), and were further characterizedby the API 20E System (bioMerieux, France). Isolateswere further characterized to the species level, using aset of biochemical tests in the Aerokey II as describedby Carnahan et al. (18). Based on the seven biochemicaltests, such aesculin hydrolysis, gas from glucose, acidfrom arabinose, indole production, acid from sucrose,Voges-Proskauer reaction, and resistance to cephalothin(30 µg), Aeromonas were identified as A. hydrophila, A.veronii bv sobria, A. caviae, and A. schubertii.

Haemolysin production test was performed asdescribed by Burke et al. (11), using human erythrocytes.Haemagglutination tests were carried out, using humanerythrocytes in absence of mannose [mannose-sensitivehaemagglutination (MSHA)] and in presence of mannose[mannose-resistant haemagglutintion (MRHA)] asdescribed by Evans et al. (25).

Antibiotic susceptibility tests were performed by thedisc-diffusion method (20). Antibiotics (Oxoid, UK)tested included: amoxycillin-clavulanic acidcombination (30 µg), carbenicillin (100 µg), cephalothin(30 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg),gentamicin (10 µg), kanamycin (30 µg), nalidixic acid(30 µg), tetracycline (30 µg), and trimethoprim-sulphamethoxazole (25 µg).

RESULTS

During the 12-month , Aeromonas species were isolatedfrom 23 (14.6%) children with diarrhoea and from 28(17.8%) healthy controls. A. hydrophila was isolatedfrom 5 (3.2%) children with diarrhoea and 9 (6.4%)controls, A. veronii bv. sobria from 8 (5.1%) and 7(4.5%), A. caviae from 13 (8.3%) and 17 (10.8), and A.schubertii from 1 (0.6%) and 2 (1.3%) respectively.Multiple Aeromonas were isolated from 4 (2.5%)diarrhoeal children and 6 (3.8%) controls (Table 1). Ofthe 23 diarrhoeic children with Aeromonas in their stools,4 had Salmonella, and 3 had Shigella. No significantdifference was found in the frequency of isolation ofAeromonas between diarrhoeal children and matchedhealthy controls.

Table 1. Distribution of Aeromonas species in diarrhoeal children and healthy controlsNo. of strains (%) belonging to the species

Subjects No. tested A. hydrophila A. veronii bv sobria A. caviae A. schubertii TotalDiarrhoeal children 157 5 (3.2) 8 (5.1) 13 (8.3) 1 (0.6) 27 (17.2)Healthy controls 157 9 (6.4) 7 (4.5) 17 (10.8) 2 (1.3) 35 (22.3)

Diarrhoea due to Aeromonas in children 77

Aeromonas were isolated from 9.6% of the83 children aged less than 6 months, 20% ofthe 50 diarrhoeal children aged between 6months and one year, and 20.8% of thechildren with diarrhoea aged over one year.Aeromonas were detected in 20.8%, 11.9%,11.4%, and 12.5% of the specimens collectedduring autumn, winter, spring, and summerseasons respectively. Information on thesource of water used for drinking wasavailable for 12 diarrhoeal children and 10controls, of whom 83% and 90% respectivelyused untreated drinking water supplies. Theorganisms were found in 18.1% and 11.8%of the female and male children withdiarrhoea and in 23.6% and 12.9% of thefemale and male controls respectively. Noneof the children with diarrhoea or controlstravelled abroad in the last 30 days prior tostool collection.

Of the 23 diarrhoeal children with faecalAeromonas, 43.5% had mucus, and 26.1%had blood in their stools; 34.8% and 39.1%presenting with fever and vomitingrespectively. Clinical data on diarrhoealchildren with positive Aeromonas are shownin Table 2.

Results of the haemolysin andhaemagglutination assays are shown in Table3. Regardless of whether isolated fromchildren with diarrhoea or controls, nosignificant differences in the haemolyticactivity of A. hydrophila, A. veronii bv sobria,or A. schubertii were found. Of the 14 A.hydrophila, 15 A. veronii bv sobria, and 3 A.schubertii strains, 71%, 67% and 66% wererespectively positive in the haemolysin assay.None of the 30 A. caviae strains were positivein this test. Of the 27 Aeromonas strains fromthe diarrhoeal children and 35 strains fromthe controls, 56% and 43% showed MSHA(p>0.05, Fisher’s exact test) respectively.Whereas 26% of the strain s from the childrenwith diarrhoea and 3% from the controlsshowed MRHA (p<0.002, Fisher’s exacttest).

Ninety-seven percent of the 62Aeromonas strains, in the present study wereresistant to amoxycillin-clavulanic acidcombination, 95% to carbenicillin, 64% tocephalothin, 6% to chloramphenicol, 3% tokanamycin, 11% to tetracycline, and 23% toTa

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78 J DIARRHOEAL DIS RES Jun 1999 Ghenghesh KS et al.

trimethoprim-sulphamethoxazole. All strains were,however, susceptible to ciprofloxacin, gentamicin, andnalidixic acid.

DISCUSSION

In this study, Aeromonas were isolated with nearly equalfrequencies from children with diarrhoea (15%) andchildren without diarrhoea (18%). This observation isin agreement with earlier workers who reported that thereis no difference in isolation rates of Aeromonas speciesbetween children with diarrhoea and healthy controls(8,9).

Many workers reported Aeromonas-associatedgastroenteritis to be distinctly seasonal, with a sharpsummer peak, and it is suspected to be water-borne (21).Most of our children with diarrhoea (83%) and controls(90%), from whom information on the source of drinkingwater was available, used untreated drinking water.Aeromonas were isolated from 55% of the 600 watersamples obtained from untreated drinking water suppliesin Tripoli area, and their prevalence, in such supplies,was highest during autumn and winter months (22). Thismay explain the high isolation rates of Aeromonasobtained in our study, especially during autumn.

Some studies (3,9) reported A. caviae to be the mostprevalent species in faeces from children with andwithout diarrhoea, while others (23) found A. hydrophilato be the most common. A. caviae was the most oftenisolated species in our study from children with andwithout diarrhoea. This organism is also the second mostcommon species, after A. hydrophila from water samplesobtained from wells in Tripoli and submitted to ourlaboratory for bacteriological analysis (K.S. Ghenghesh,unpublished observation). This observation supports thefindings of earlier workers who demonstrated that A.caviae are diarrhoeagenic suggested by the fact that these

strains were potentially enterotoxigenic (24), and wereoften found in the environment (6).

Reports on the prevalence of A. schubertii in casesof childhood gastroenteritis and in controls are lacking.Studies are, thus, required to determine their role indiarrhoeal diseases. In the population we studied, thisorganism was not an important enteric pathogen.

It has been reported that children with A. sobria (A.veronii bv sobria)-associated infection were more likelyto have associated systemic manifestations (3). In ourstudy, 56% and 67% of the children with diarrhoea dueto A. veronii bv sobria had fever and vomitingrespectively. Also mucus and blood were found more instools (50% and 33% respectively) of cases with A.sobria bv sobria.

Contrary to reports by other workers (25), we foundno significant differences in the haemolytic activity ofAeromonas species regardless of whether they wereisolated from diarrhoeal children or controls. The resultsof our study showed that haemolysin production wassignificantly associated with A. veronii bv sobria, A.hydrophila, and A. schubertii, but not with A. caviaeisolates, regardless of their isolation from diarrhoealchildren or controls. These findings are consistent withthose of other investigators (8).

Human diarrhoeal isolates and enterotoxigenic strainsof Aeromonas species are strong haemagglutinators ofhuman blood cells (26). In the present study, we found asignificant association between Aeromonas isolated fromchildren with diarrhoea and MRHA. Others (10) reporteda strong association between diarrhoea and agglutinationnot inhibited by fucose, galactose, or mannose. Weobserved that MRHA was markedly species-dependent,66% (2 of 3) of A. schubertii, 50% (4 of 8) of A. veroniibv sobria, 23% (3 of 13) of A. caviae, and none of A.

Table 3. Haemolytic and haemagglutination activities of Aeromonas species isolated from diarrhoeal children andhealthy controls

Species Subjects No. studied No. of strains (%) positiveHaemolysin Haemagglutination

MS MRA. hydrophila Diarrhoeal children 5 4(80) 3(60) 0.0

Healthy controls 9 6(67) 5(56) 0.0A. veronii bv sobria Diarrhoeal children 8 6(75) 6(75) 4(50)

Healthy controls 7 4(57) 3(42) 0.0A. caviae Diarrhoeal children 13 0.0 5(38) 3(21)

Healthy controls 17 0.0 6(35) 0.0A. schubertii Diarrhoeal children 1 1(100) 1(100) 0.0

Healthy controls 2 1(50) 1(50) 1(50)Aeromonas sp. (Total) Diarrhoeal children 27 11(41) 15(56) 7(26)†

Healthy controls 35 11(31) 15(43) 1(3)MS=mannose-sensitive; MR=mannose-resistant; †(p<0.002, Fisher’s exact test)

Diarrhoea due to Aeromonas in children 79

hydrophila strains. Furthermore, our findings are inagreement with the recently published reports (12 , 27,28) which showed a strong association ofhaemagglutination activity and enteropathogenicity ofAeromonas isolated from clinical and environmentalsources.

With very few exceptions, Aeromonas are known tobe uniformly resistant to ampicillin (29). Nearly 50% ofour patients took ampicillin prior to stool collection forreasons other than diarrhoea. This may have predisposedthem to infection with these organisms. Moyer (30)reached to the same conclusion and reported thatantibiotic therapy and drinking of untreated water aretwo significant risk factors for the susceptible host.Although gastrointestinal infections with Aeromonas aregenerally self-limiting, marked alleviation or resolutionof gastrointestinal symptoms of patients takingantimicrobials to which Aeromonas strains aresusceptible has been reported (31). The results of theantibiotic sensitivity test indicate that ciprofloxacin,gentamicin, and nalidixic acid might be effective agentsfor the treatment of diarrhoea due to Aeromonas.

In conclusion, A. caviae strains may have thepotential to cause diarrhoea in children, and MRHA maybe used as one of the virulence markers to distinguishbetween diarrhoeal and healthy control or environmentalisolates.

The failure of exotoxin-producing strains ofAeromonas to cause diarrhoea in human volunteers (32)suggests that exotoxins are not the only determinants ofpathogenicity (33). Future studies with human volunteersshould include Aeromonas that possess multiplevirulence factors, particularly mannose-resistantadhesion.

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