in total 99 isolates identified biochemically as members of the genus aeromonas randomly selected from a large collection of nearly 400 aeromonads isolated from different sources during the past two decades in tripoli , libya , were included in the present investigation .
the strains were obtained from diarrheal children ( n=23 ) , non - diarrheal children ( n=16 ) , untreated drinking water from wells ( n=32 ) , and chicken carcasses ( n=28 ) .
the organisms were isolated from stool , chicken , and water samples using standard bacteriological procedures as reported previously ( 11 ) .
all organisms were identified by biochemical tests and api 20e ( biomerieux , marcy l'etoile , france ) .
strains were then inoculated into semi - solid nutrient agar in duplicates and maintained at ambient temperature until further characterized .
genospecies was determined using a combination of 16s rdna ( 12 ) , and gyrb ( 13 ) sequencing analysis described previously ( 14 ) .
a loopful of a fresh overnight growth from each aeromonas isolate cultured on macconkey - lactose agar ( oxoid , hampshire , united kingdom ) was suspended in 400 l sterile deionized water , boiled for 10 min and transferred to ice for 5 min .
cell debris was pelleted by centrifugation at 12,000g for 3 min ( 15 ) , the supernatant was transferred to a new tube and refrigerated until use .
pcr amplicons were purified using the pcr purification kit ( qiagen , valencia , ca ) according to the manufacturer 's specifications .
nucleotide sequence was determined using dye terminator chemistry and cycle sequencing products were purified prior to loading on an abi prism 3,100 genetic analyzer ( applied biosystems , foster city , ca ) using a dyeex purification kit ( qiagen ) .
sequence files were assembled using bioedit version 7.0.1 ( 16 ) and aligned with clustal x ( 17 ) .
phylogenetic trees were constructed using the neighbor - joining method with genetic distance calculated using the kimura two - step algorithm .
bootstrap analysis ( 19 ) was performed with 2,000 samplings and values below 70% were excluded as non - significant . in total 52 aeromonads ( 12 from diarrheal children , 12 from non - diarrheal children , 17 from chicken carcasses , and 11 from untreated drinking water from wells ) were examined for the genes aer , act , ast , and alt using pcr techniques and sequencing as reported previously ( 8 , 2022 ) .
in addition , isolates were tested for their cytotoxic activity in vero cell tissue culture using a previously described procedure ( 23 ) .
in total 99 isolates identified biochemically as members of the genus aeromonas randomly selected from a large collection of nearly 400 aeromonads isolated from different sources during the past two decades in tripoli , libya , were included in the present investigation .
the strains were obtained from diarrheal children ( n=23 ) , non - diarrheal children ( n=16 ) , untreated drinking water from wells ( n=32 ) , and chicken carcasses ( n=28 ) .
the organisms were isolated from stool , chicken , and water samples using standard bacteriological procedures as reported previously ( 11 ) .
all organisms were identified by biochemical tests and api 20e ( biomerieux , marcy l'etoile , france ) .
strains were then inoculated into semi - solid nutrient agar in duplicates and maintained at ambient temperature until further characterized .
genospecies was determined using a combination of 16s rdna ( 12 ) , and gyrb ( 13 ) sequencing analysis described previously ( 14 ) .
a loopful of a fresh overnight growth from each aeromonas isolate cultured on macconkey - lactose agar ( oxoid , hampshire , united kingdom ) was suspended in 400 l sterile deionized water , boiled for 10 min and transferred to ice for 5 min .
cell debris was pelleted by centrifugation at 12,000g for 3 min ( 15 ) , the supernatant was transferred to a new tube and refrigerated until use .
pcr amplicons were purified using the pcr purification kit ( qiagen , valencia , ca ) according to the manufacturer 's specifications .
nucleotide sequence was determined using dye terminator chemistry and cycle sequencing products were purified prior to loading on an abi prism 3,100 genetic analyzer ( applied biosystems , foster city , ca ) using a dyeex purification kit ( qiagen ) .
sequence files were assembled using bioedit version 7.0.1 ( 16 ) and aligned with clustal x ( 17 ) .
phylogenetic trees were constructed using the neighbor - joining method with genetic distance calculated using the kimura two - step algorithm .
bootstrap analysis ( 19 ) was performed with 2,000 samplings and values below 70% were excluded as non - significant .
a loopful of a fresh overnight growth from each aeromonas isolate cultured on macconkey - lactose agar ( oxoid , hampshire , united kingdom ) was suspended in 400 l sterile deionized water , boiled for 10 min and transferred to ice for 5 min .
cell debris was pelleted by centrifugation at 12,000g for 3 min ( 15 ) , the supernatant was transferred to a new tube and refrigerated until use .
pcr amplicons were purified using the pcr purification kit ( qiagen , valencia , ca ) according to the manufacturer 's specifications .
nucleotide sequence was determined using dye terminator chemistry and cycle sequencing products were purified prior to loading on an abi prism 3,100 genetic analyzer ( applied biosystems , foster city , ca ) using a dyeex purification kit ( qiagen ) .
sequence files were assembled using bioedit version 7.0.1 ( 16 ) and aligned with clustal x ( 17 ) .
phylogenetic trees were constructed using the neighbor - joining method with genetic distance calculated using the kimura two - step algorithm .
bootstrap analysis ( 19 ) was performed with 2,000 samplings and values below 70% were excluded as non - significant .
in total 52 aeromonads ( 12 from diarrheal children , 12 from non - diarrheal children , 17 from chicken carcasses , and 11 from untreated drinking water from wells ) were examined for the genes aer , act , ast , and alt using pcr techniques and sequencing as reported previously ( 8 , 2022 ) .
in addition , isolates were tested for their cytotoxic activity in vero cell tissue culture using a previously described procedure ( 23 ) .
of the 99 isolates , we identified 44 isolates ( 44% ) as a. hydrophila ( 3 [ 3.0% ] subspecies anaerogenes , 23 [ 23% ] subspecies dhakensis , and 18 [ 18% ] subspecies ranae ) ; 27 isolates ( 27% ) as a. veronii ; 23 isolates ( 23% ) as a. caviae ; and 5 isolates ( 5.1% ) as other genospecies ( table 1 ) .
a. hydrophila was common in water samples ( 84.4% ) compared with diarrheal and non - diarrheal stool ( 33.3% ) and chicken ( 14.3% ) samples ; a. veronii in chicken samples ( 60.7% ) compared with diarrheal and non - diarrheal stool ( 23.1% ) and water ( 3.1% ) samples ; and a. caviae in stool samples from diarrheal and non - diarrheal children ( 41.0% ) compared with water ( 6.3% ) and chicken ( 17.9% ) samples .
the genes aer , act , and ast were detected in 45 ( 87% ) , 4 ( 7.7% ) , and 9 ( 17% ) , respectively ( table 2 ) .
cytotoxicity to vero cells was observed in 7 of 12 ( 58% ) aeromonads from diarrheal , 4 of 12 ( 33% ) from non - diarrheal children , 8 of 11 ( 73% ) from water , and 10 of 17 ( 59% ) from chicken carcasses .
genospecies of aeromonads isolated from different sources in tripoli , libya significantly higher than prevalence among diarrheal stool isolates , non - diarrheal stool isolates and chicken isolates ( p<0.0002 , or=10.13 ; p<0.0003 , or=11.88 and p<0.0000002 , or=32.40 , respectively ) . significantly higher than prevalence among water isolates ( p<0.005 , or=13.56 ) .
significantly higher than prevalence among diarrheal children stool isolates and water isolates ( p<0.04 , or=3.53 and p<000002 , or=47.91 , respectively ) .
significantly higher among diarrheal stool isolates ( p<0.007 , or=8.00 and non - diarrheal stool isolates ( p<0.0005 , or=15.00 ) than among water isolates .
virulence genes in aeromonas from libya aer = aerolysin , act = aeromonas hydrophila cytolytic enterotoxin , ast = aeromonas hydrophila isolate ssu enterotoxin , alt = aeromonas extracellular lipase .
significantly higher than in diarrheal children , non - diarrheal children and chicken samples ( p<0.03 , odds ratio [ or]=unidentified ; p<0.03 , or = unidentified and p<0.08 , or - undefined , respectively ) .
significantly higher than diarrheal children , non - diarrheal children and chicken samples ( p<0.003 , or = unidentified ; p<0.003 , or = unidentified and p<0.05 , or=5.60 , respectively ) .
previous studies conducted in libya found aeromonas species in 4.2 to 14.6% of diarrheal children ( 2426 ) . in one of these studies ( 24 )
phenotypic speciation using aerokey ii ( 27 ) showed predominance of a. caviae , followed by a. veronii , and a. hydrophila .
abdullah et al . ( 28 ) genotyped eight aeromonads from diarrheal libyan children by pcr - restriction fragment length polymorphism ( pcr - rflp ) analysis of 16s rrna genes : four ( 50% ) were a. caviae , three ( 37.5% ) were a. veronii , and one ( 12.5% ) was a. hydrophila . in the present investigation , the genospecies a. caviae predominated , followed by a. hydrophila ( mainly subspecies dhakensis and subspecies ranae ) , and a. veronii , among aeromonads from diarrheal and non - diarrheal children .
predominance of these three genospecies was also observed among aeromonads from chicken and water samples , accounting for 95% ( 94/99 ) of total isolates tested .
these genospecies account for the majority of aeromonads reported from different sources in developed and developing countries ( 6 , 7 ) .
the remaining 5% ( 5/99 ) of aeromonads investigated belonged to genospecies a. enteropelogenes clx204 , a. culicicola , and a. allosaccharophila .
these aeromonads are recognized aeromonas species with uncertain taxonomic status , with the latter two not being isolated from clinical material ( 29 ) , indicating they may have no role in human disease . in agreement with our findings ,
a previous study reported the predominance of a. caviae genospecies among aeromonads from diarrheal and non - diarrheal children attending clinical research and service centre of the international centre for diarrhoeal disease research in dhaka , bangladesh ( icddr - b ) ( 30 ) .
recently , carvalho et al . ( 31 ) identified 80 distinct aeromonas strains by gyrb - based phylogenetic analysis among a collection of 206 isolates from untreated waters used for human consumption in portugal that were discriminated by random amplified polymorphic dna - pcr ( rapd - pcr ) .
the most common genospecies detected was a. hydrophila ( 26% ) followed by a. media ( 23% ) . in agreement with their finding
, we identified the majority of aeromonads in untreated well water as a. hydrophila , but at a much higher rate ( 84% ) .
chicken carcasses may also become contaminated with aeromonads during the washing and cleaning of such carcasses with aeromonas - contaminated water .
( 32 ) found aeromonas species in more than 75% ( 218/290 ) of frozen chicken carcasses sold at retail outlets in tripoli , libya .
there is lack of data on genospeciation of aeromonads from chicken by gyrb - based phylogenetic analysis .
rflp analysis of 16s rrna genes , identified 30 of 32 ( 94% ) isolates from chicken carcasses in libya as a. veronii . in the present study , more than 60% of isolates from chicken samples were a. veronii .
therefore , it is important to detect virulence factors in aeromonads isolated from children in a matched case - control study of diarrhea in such countries .
hemagglutination ( ha ) of erythrocytes is associated with the ability of enteric bacteria to adhere to human epithelial cells .
burke et al . ( 33 ) reported that enterotoxigenic a. hydrophila isolates showed ha resistant to mannose and to fucose , whereas non - enterotoxigenic a. caviae commonly isolated from the environment or non - diarrheal individuals showed mannose - sensitive ha .
examination of aeromonas isolated in libya for genes coding for virulence factors indicated that the aer gene was commonly present across all sample sources ( 86% ) .
however , we did not find statistical significance between the presence of aer and sample source or genospecies ( data not shown ) .
aerolysin is a pore forming toxin and is regarded as the most important virulence factor in aeromonas food poisoning and one of the major virulence factors in gastroenteritis ( 8 , 34 , 35 ) .
a previous study from libya ( 28 ) reported aerolysin - like hemolysin gene sequences in 100% of 52 aeromonas isolates from children with diarrhea , chicken carcasses , and a hospital environment .
ottaviani et al . ( 36 ) observed aer , act , alt , and ast in 50 , 31 , 31 , and 34% , respectively , in 32 aeromonads from diarrheal patients . among the act , alt , ast , and aer genes , only aer ( 83% ) was detected in stools of diarrheal and non - diarrheal children in the present study .
albert et al . ( 30 ) examined alt , ast , and act genes in aeromonas isolates from children with diarrhea , children without diarrhea , and environmental sources ( including surface water ) in bangladesh .
they found that aeromonads positive just for the alt gene had similar distributions in the three sources ; aeromonads positive just for the ast gene were significantly more prevalent among environmental specimens than among diarrheal children specimens ; and aeromonads positive just for the act gene were not found in any of the three sources . in agreement with their results we detected the ast gene significantly more frequently among aeromonads from water samples than among aeromonads from three other sources ( i.e. diarrheal children , non - diarrheal children , and chicken ) .
however , contrary to their findings we did not detect the alt gene in aeromonads from the four sources examined and detected the act gene in more than 36% of aeromonas isolates from water .
differences in the reported rates of virulence genes among aeromonas species from different regions may be related to differences in geographical location . in conclusion ,
the majority of aeromonads from libya fall within three genospecies ( i.e. a. hydrophila , a. veronii , and a. caviae ) .
furthermore , genes coding for toxin production and cytotoxicity to vero cells are common features among aeromonas species isolated from food and water sources in libya , which may pose a health risk to users of such sources , particularly to immunocompromised individuals .
the authors have not received any funding or benefits from industry or elsewhere to conduct this study .
 introductionaeromonads of medical importance have been reported from numerous clinical , food , and water sources , but identification of genospecies and virulence factors of aeromonas species from countries in north africa and the middle east are few.methodsin total 99 aeromonas species isolates from different sources ( diarrheal children [ n=23 ] , non - diarrheal children [ n=16 ] , untreated drinking water from wells [ n=32 ] , and chicken carcasses [ n=28 ] ) in tripoli , libya , were included in the present investigation . 
 genus identification was confirmed by biochemical analysis , and genospecies were determined using a combination of 16s rdna variable region and gyrb sequence analysis 
 . polymerase chain reaction ( pcr ) was used to detect genes encoding toxins from 52 of the isolates.resultswe identified 44 isolates ( 44% ) as a. hydrophila ( 3 [ 3.0% ] subspecies anaerogenes , 23 [ 23% ] subspecies dhakensis , and 18 [ 18% ] subspecies ranae ) ; 27 isolates ( 27% ) as a. veronii ; 23 isolates ( 23% ) as a. caviae ; and 5 isolates ( 5.0% ) as other genospecies . 
 the genes encoding aerolysin ( aer ) , cytolytic enterotoxin ( act ) , and a. hydrophila isolate ssu enterotoxin ( ast ) were detected in 45 ( 87% ) , 4 ( 7.7% ) , and 9 ( 17% ) of the 52 isolates tested , respectively . 
 the gene encoding an extracellular lipase ( alt ) was not detected.conclusionthe majority of aeromonads from libya fall within three genospecies ( i.e. a. hydrophila , a. veronii , and a. caviae ) , and genes coding for toxin production are common among them . 
Methods
Strains
Determination of 
Whole cell lysate preparation
DNA analysis
Determination of virulence factors
Results
Discussion
Conflict of interest and funding