Document Detail

A molecular study on the prevalence and virulence potential of Aeromonas spp. recovered from patients suffering from diarrhea in Israel.
Jump to Full Text
MedLine Citation:
PMID:  22355306     Owner:  NLM     Status:  MEDLINE    
BACKGROUND: Species of the genus Aeromonas are native inhabitants of aquatic environments and have recently been considered emerging human pathogens. Although the gastrointestinal tract is by far the most common anatomic site from which aeromonads are recovered, their role as etiologic agents of bacterial diarrhea is still disputed. Aeromonas-associated diarrhea is a phenomenon occurring worldwide; however, the exact prevalence of Aeromonas infections on a global scale is unknown.
METHODOLOGY/PRINCIPAL FINDINGS: The prevalence and virulence potential of Aeromonas in patients suffering from diarrhea in Israel was studied using molecular methods. 1,033 diarrheal stools were sampled between April and September 2010 and Aeromonas species were identified in 17 (∼2%) patients by sequencing the rpoD gene. Aeromonas species identity and abundance was: A. caviae (65%), A. veronii (29%) and Aeromonas taiwanensis (6%). This is the first clinical record of A. taiwanensis as a diarrheal causative since its recent discovery from a wound infection in a patient in Taiwan. Most of the patients (77%) from which Aeromonas species were isolated were negative for any other pathogens. The patients ranged from 1 to 92 years in age. Aeromonas isolates were found to possess different virulence-associated genes: ahpB (88%), pla/lip/lipH3/apl-1 (71%), act/hlyA/aerA (35%), alt (18%), ast (6%), fla (65%), lafA (41%), TTSS ascV (12%), TTSS ascF-ascG (12%), TTSS-dependent ADP-ribosylating toxins aexU (41%) and aexT (6%) in various combinations. Most of the identified strains were resistant to beta-lactam antibiotics but susceptible to third-generation cephalosporin antibiotics.
CONCLUSIONS: Aeromonas may be a causative agent of diarrhea in patients in Israel and therefore should be included in routine bacteriological screenings.
Yigal Senderovich; Shifra Ken-Dror; Irina Vainblat; Dvora Blau; Ido Izhaki; Malka Halpern
Related Documents :
22569516 - Barriers to horizontal gene transfer in campylobacter jejuni.
22870986 - A putative merr family regulator involved in biofilm formation in listeria monocytogene...
15995186 - The genome of salmonella enterica serovar gallinarum: distinct insertions/deletions and...
9023176 - A role for cpeyz in cyanobacterial phycoerythrin biosynthesis.
9302906 - Bacterial flora of stethoscopes' earpieces and otitis externa.
16329986 - Bionomics of anopheles stephensi liston in the malarious area of hormozgan province, so...
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2012-02-15
Journal Detail:
Title:  PloS one     Volume:  7     ISSN:  1932-6203     ISO Abbreviation:  PLoS ONE     Publication Date:  2012  
Date Detail:
Created Date:  2012-02-22     Completed Date:  2012-06-18     Revised Date:  2013-06-26    
Medline Journal Info:
Nlm Unique ID:  101285081     Medline TA:  PLoS One     Country:  United States    
Other Details:
Languages:  eng     Pagination:  e30070     Citation Subset:  IM    
Department of Evolutionary and Environmental Biology, Faculty of Natural Sciences, University of Haifa, Mount Carmel, Haifa, Israel.
Data Bank Information
Bank Name/Acc. No.:
GENBANK/JF738005;  JF738006;  JF738007;  JF738008;  JF738009;  JF738010;  JF738011;  JF738012;  JF738013;  JF738014;  JF738015;  JF738016;  JF738017;  JF738018;  JF738019;  JF738020;  JF738021
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Aeromonas / classification,  isolation & purification,  pathogenicity*
Aged, 80 and over
Anti-Infective Agents / therapeutic use
Bacterial Proteins / genetics
Child, Preschool
Diarrhea / drug therapy,  epidemiology*,  microbiology*
Feces / microbiology
Gram-Negative Bacterial Infections / drug therapy,  epidemiology*,  microbiology*
Israel / epidemiology
Middle Aged
Molecular Sequence Data
Virulence / genetics*
Virulence Factors
Young Adult
Reg. No./Substance:
0/Anti-Infective Agents; 0/Bacterial Proteins; 0/Virulence Factors

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

Full Text
Journal Information
Journal ID (nlm-ta): PLoS One
Journal ID (publisher-id): plos
Journal ID (pmc): plosone
ISSN: 1932-6203
Publisher: Public Library of Science, San Francisco, USA
Article Information
Download PDF
Senderovich et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Received Day: 7 Month: 8 Year: 2011
Accepted Day: 12 Month: 12 Year: 2011
collection publication date: Year: 2012
Electronic publication date: Day: 15 Month: 2 Year: 2012
Volume: 7 Issue: 2
E-location ID: e30070
ID: 3280246
PubMed Id: 22355306
Publisher Id: PONE-D-11-15711
DOI: 10.1371/journal.pone.0030070

A Molecular Study on the Prevalence and Virulence Potential of Aeromonas spp. Recovered from Patients Suffering from Diarrhea in Israel Alternate Title:Prevalence of Aeromonas spp. in Diarrhea Patients
Yigal Senderovich1
Shifra Ken-Dror2
Irina Vainblat2
Dvora Blau2
Ido Izhaki1
Malka Halpern13*
Patrick C. Y. Wooedit1 Role: Editor
1Department of Evolutionary and Environmental Biology, Faculty of Natural Sciences, University of Haifa, Mount Carmel, Haifa, Israel
2Microbiology Laboratory of Clalit Health Services in Haifa, Haifa, Israel
3Department of Biology and Environment, Faculty of Natural Sciences, University of Haifa, Oranim, Tivon, Israel
The University of Hong Kong, China
Correspondence: * E-mail:
Contributed by footnote: Conceived and designed the experiments: YS SKD MH. Performed the experiments: YS SKD IV DB. Analyzed the data: YS SKD II MH. Contributed reagents/materials/analysis tools: SKD II MH. Wrote the paper: YS SKD MH.


Aeromonas species are waterborne, Gram-negative, oxidase-positive, rod-shaped bacteria that are ubiquitous in water. This includes chlorinated drinking water, as the bacteria can grow and survive in biofilms in the water distribution systems [1]. The prevalence of Aeromonas species in the aquatic environment has been recognized as a potential health risk, and some countries have adopted aeromonad counts as an additional indicator of water quality [2].

The most common clinical manifestations of Aeromonas infections are diarrhea, bacteremia and localized soft-tissue infections [3]. Patients may acquire Aeromonas infections both in community and hospital settings [3][5]. Both immunocompetent and immunocompromised patients are susceptible to Aeromonas infections [3], [6]. The gastrointestinal tract is by far the most common anatomic site from which aeromonads are recovered [3], [6]. Aeromonas have been isolated from children with acute diarrhea and from adults with traveler's diarrhea [3], [6][8]. The following species are frequently associated with diarrhea in humans: A. hydrophila, A. veronii bv. sobria and A. cavia[3], [6], [9], [10], [11].

The mechanism of Aeromonas pathogenesis is complex and not well understood. Aeromonas virulence is considered to be multifactorial. The virulence factors that were associated with Aeromonas pathogenicity are: cytotoxic enterotoxin, haemolysins, proteases [serine protease (aspA), elastase (ahpB)], lipases (pla and plc, sat), DNAses and adhesins [type IV pili and polar flagella (flaA and flaB)] [1], [6], [9], [10], [12], [13]. Several of these virulence factors have been identified in strains isolated from water [1]. In addition, genes for a type III secretion system (TTSS) were identified in this genus [14], [15]. TTSS has a role in delivering toxins directly into the host cell and in inducing apoptosis [14], [15].

In Israel, diarrhea patients are tested routinely by clinical laboratories for the presence of several bacterial pathogens, such as: Campylobacter spp., Shigella spp. and Salmonella spp., but not for Aeromonas. The aim of this research was to study the prevalence and virulence potential of Aeromonas spp. in diarrheal stools in Israel.

Materials and Methods
Ethics Statement

N/A. The data was analyzed anonymously.

We have applied to the ethics committee at Carmel Hospital, Clalit Health Services, Haifa, Israel, and the committee stated that such a research does not fall under the scope of the Helsinki Committee.

Aeromonas Prevalence in Diarrheal Stools

The presence of Aeromonas was monitored in fecal specimens from diarrheal patients submitted to the Microbiology Laboratory of Clalit Health Services in Haifa. This Laboratory provides services to a wide range of population, from the district of Haifa and West Galilee in Israel (this is a community health service, not a hospital). The surveillance was conducted between April 13 and September 15, 2010 (five months). All specimens were checked routinely for the following enteropathogens: Shigella, Salmonella and Campylobacter spp. were isolated and identified by conventional methods [16]; Rotavirus was detected by an antigen detection method (Novamed, Israel); parasites were studied according to methods described in Garcia and Isenberg [17]. For the isolation of Aeromonas spp. the fecal specimens were either enriched in alkaline peptone water (APW) containing peptone (1%, wt/vol) and NaCl (1%, wt/vol) pH 8.5, or directly streaked on a selective m-Aeromonas agar base (Havelaar Biolife, Milano, Italy). In the case of enrichment, the tubes were incubated at 37°C without shaking for 6–18 h, and then streaked on m-Aeromonas selective agar. The agar plates were incubated overnight at 37°C. Colonies that were morphologically suspected as Aeromonas (yellow, smooth and rounded) were subcultured onto LB agar (Himedia, India), and then tested for oxidase (1% tetramethyl-phenylenediamine, Sigma). The identity of the isolates with positive results was further verified by Aeromonas genus specific PCR assay in accordance with Kong et al. (1999) [18]. Reddy Mix PCR master mixture (ABgene, Epsom, UK) was used for the DNA amplification. All the isolates that were found to belong to the Aeromonas genus were maintained in LB with 30% glycerol (−80°C).

Aeromonas isolates were further identified by amplifying and sequencing the housekeeping gene rpoD, encoding σ70 factor, which is one of the sigma factors that confer promoter-specific transcription initiation on RNA polymerase [19]. The PCR products were sequenced by MCLAB (San Francisco, CA). Newly determined sequences were compared to those available in the GenBank database, using the standard nucleotide–nucleotide BLAST program (BLASTN;, to ascertain their closest relatives. The sequences were submitted to the GenBank database under accession numbers JF738005–JF738021. A phylogenetic tree was generated using the neighbor-joining method with NJPlot (MEGA 4.1) based on alignments from CLUSTAL W.

Virulence Factors and Antimicrobial Susceptibility

The presence of the following genes encoding virulence factors was determined in all Aeromonas isolates: cytotoxic enterotoxin (act)/aerolysin (aerA)/haemolysin (hlyA) by using one set of primers AHCF1/AHCR1 [12]; alt and ast genes for cytotonic enterotoxins; ahyB gene for elastase; pla/lipH3/apl-1/lip genes for phospholipase; and fla gene for flagellin [1]. The presence of the genes act/aerA/hlyA and ast; fla and alt; ahyB and pla/lipH3/apl-1/lip was tested simultaneously in the same reaction mixture, in accordance with Sen and Rodgers (2004) [1]. The presence of genes encoding the components of the type III secretion system, ascV, ascF-ascG[15], type III secretion dependent ADP-ribosylating toxins, aexT and aexU[20], and of lafA gene encoding a lateral flagella [13] was determined as well.

The disk diffusion antimicrobial susceptibility tests were performed by a standardized method [21], [22]. All disks were purchased from OXOID (UK).

Aeromonas Prevalence in Diarrheal Stools

A total of 1,033 stool specimens from patients suffering from diarrhea were monitored for the presence of Aeromonas during a five month period between April 13 and September 15, 2010. Seventeen patients (∼2%) tested positive for Aeromonas species which included 11 (65%) A. caviae, five (29%) A. veronii, and one strain, (H53AQ1). This strain showed the highest rpoD gene similarity (96%) to the deposited sequence of the type strain of A. taiwanensis, and clustered with this species in the phylogenetic tree (Figure 1).

The specimens of the diarrhea patients were also checked for other enteropathogenes. The results revealed that pathogenic bacteria, Rotavirus and parasites were recovered from about 15% (155 of 1033) of the diarrhea patients. The prevalence of the detected enterophathogenes was; Campylobacter sp. 5.2%, Shigella 3.3%, Salmonella enterica 2%, Aeromonas spp. 2%, Rotavirus 0.4%, Giardia lamblia 2.3%, and Cryptosporidium parvum 0.15%. Mixed infections were found in four patients that were positive for Aeromonas as well as for other known enteropathogens (Table S1).

Virulence Factors and Antimicrobial Susceptibility

All Aeromonas isolates were screened for the presence of virulence genes (Table 1). The most prevalent genes were ahyB (88%) and pla/lipH3/apl-1/lip (71%). The two types of flagella that were screened (polar and lateral) were quite prevalent as well (65% and 41%, respectively). In every strain that was positive for the genes encoding the TTSS, a gene for the effector aexU was present as well. The ast gene was found only in one isolate (H65AT3), which was identified as A. veronii. The virulence genotypes were found in different combinations: three isolates (18%) possessed five different genes, four (24%) possessed three or four different genes and two (12%) possessed two different genes (Table 1).

The susceptibility of Aeromonas isolates was evaluated against 15 antimicrobial agents. All isolates were susceptible to amikacin, cefotaxime, ceftazidime, ceftriaxone, ciprofloxacin and chloramphenicol; however, they varied in their susceptibility to other antimicrobial agents (Table 2). The three Aeromonas species displayed the same antibiotic sensitivity patterns.


Despite the existence of detailed case reports and epidemiological case control investigations, the role of Aeromonas as the etiological agent of bacterial diarrhea has been questioned and debated several times [3], [6], [11], . Figueras et al. [26] rebutted arguments provided by several authors against considering Aeromonas a true enteropathogenic bacterium one by one. Today it is well accepted that if Aeromonas can cause different infections like cellulitis, meningitis, pneumonia, wound infections and more in healthy humans, it can also have the capacity to produce diarrhea [3], [6], [26].

In several reported studies throughout the world, Aeromonas species have been isolated at a rate of 0.6 to 7.2% in patients with diarrhea, predominantly in infants and children [2], [27]. In the current study, Aeromonas positive patients ranged in age and only two out of 17 isolates were taken from children (Table S1). The current study relied on a limited amount of strains as it was performed only during a period of five months; however, the Aeromonas isolation rate amounted to about 2%, which is similar to the rate obtained in other studies performed in other countries [3,6 and references therein], as well as in Israel in 1990 [28]. In a recent study performed by Pablos et al. [29] in León (Spain) they found a frequency of Aeromonas of 4% (32 positive patients of the 800 investigated), mainly associated with infant or pediatric patients (68.8%). Furthermore they found mixed infections with other pathogens in 12 patients [29]. In our study, only two of the patients were infants (12%), and all four patients that had mixed infections were adults (Table S1).

In mixed infections, Aeromonas may be transient colonizers lacking a causal relationship with a disease, but in some cases, multiple pathogens may act synergistically to produce diarrhea [30]. Aeromonas species are carried asymptomatically by some individuals [3], [6] as occurs with other recognized enteropathogens like Salmonella. However, a study that was performed in 1990 in Israel compared the prevalence of Aeromonas in the stools obtained from 932 adult patients with acute diarrhea (recovered between 1986 and 1987) to 500 stools from asymptomatic controls. They found an Aeromonas prevalence of about 2% in the diarrhea cases, which conforms to our study. But no Aeromonas were detected in the controls [28]. This seems to indicate a clear association of Aeromonas with diarrhea cases in Israel, as we found in our study.

Among the recognized Aeromonas species, A. veronii bv. sobria, A. caviae and A. hydrophila are more frequently associated with diarrhea in humans, representing 85% of clinical isolates [11]. Interestingly, none of the identified strains in the current study belonged to A. hydrophila. This is in agreement with the false importance attributed to this species on the basis of phenotypic identifications [3], [26]. In the current study, A. caviae was the predominating species (65%, 11/17), followed by A. veronii that was isolated from five patients (29%). One patient carried a strain that was identified as A. taiwanensis (Figure 1). All the strains were identified using the rpoD gene sequencing method. Aeromonas identification on the basis of rpoD gene sequencing is considered to be much more accurate than 16S rRNA gene sequencing or biochemical identification methods. The fact that many studies found A. hydrophila a major species to cause diarrhea (among Aeromonas species) may be due to limitations in the identification methods that were used in those studies [3], [26].

The current study provides the first clinical record of A. taiwanensis as a diarrheal causative since this species was identified [31]. So far, the only available strain (the type strain) was recovered from an infected burn wound of a 40 years old male [31] and in the current study the strain was isolated from feces of a 35 years old diarrheal female patient.

The clinical manifestations of Aeromonas associated gastroenteritis can range from mild self-limiting watery diarrhea to a more severe and invasive dysenteric form. Chronic diarrhea episodes and isolated cases of a cholera-like illness have also been described [11]. The bacterial flagella are thought to play an important role in pathogenicity. Aeromonas produces two types of flagella: a constitutively expressed polar flagellum (fla) and multiple inducible lateral flagella (laf). Both types play a role in the attachment of the bacteria to the gastrointestinal epithelium, biofilm formation and long-term colonization [6]. Both types of flagella (fla and lafA) were common among the Aeromonas isolates from the patients in the current study (Table 1). The occurrence of genes encoding hemolytic, cytotonic, cytotoxic, and enterotoxic activities (aerA, hlyA, alt, ast, act) may contribute to diarrheal-related virulence [6], [31], [32]. In the present study, 35% of the Aeromonas isolates possessed the act/aerA/hlyA gene. The most prevalent virulence-associated genes in the isolates from our study were ahpB for elastase (88%) and pla/lip/lipH3/apl-1 for lipase (71%) (Table 1). These genes may be essential for the ability of the bacterium to adhere and invade the intestinal mucosa [1].

Type III secretion system (TTSS) plays crucial roles in host-pathogen interactions [14], [15]. One of the best-described toxins that are translocated via a TTSS is the ADP-ribosylating toxin, AexT. This toxin was found to be more common among the environmental, rather than the clinical Aeromonas strains [13]. In our study, the gene for this toxin was detected only in one strain. Recently, a novel type-three-secretion-dependent effector, AexU, was discovered in Aeromonas. AexU is an ADP-ribosylating toxin and is required for virulence of Aeromonas hydrophila in mice [20]. The gene for this toxin was quite prevalent among the strains in our study (41%). The prevalence of the genes encoding TTSS apparatus (12%) was lower than the aexU gene prevalence (41%). The TTSS is probably underrepresented, as may happen in PCR based studies. Nevertheless, the presence of aexU gene strengthens the case of Aeromonas being recognized as a stronger pathogen.

In another study that surveyed the distribution of virulence associated genes among Aeromonas species from human stool specimens in Spain, it was found that alt, ast, laf, aerA, and hlyA genes were present in 72, 19, 3, 25, and 28% of the strains, respectively. None of the strains harbored ascF – G[29]. In clinical diarrheic isolates of A. hydrophila in Spain the distribution of associated virulence genes was different: alt – 82%, ast – 96%, laf – 77%, aexT – 5%, ascV – 5% [13].

Aeromonas species are known to be intrinsically susceptible to all antibiotics active against non-fastidious Gram-negative bacilli, except for many beta–lactams, due to the production of multiple inducible, chromosomally encoded β–lactamases [33]. In our study, most strains (80%) were resistant to cephalotin and partially resistant to amoxicillin combined with clavulanic acid (46%) (Table 2). All strains were susceptible to third-generation cephalosporin antibiotics (cefotaxime, ceftazidime, ceftriaxone), second-generation fluoroquinolone antibiotics – ciprofloxacin, aminoglycoside antibiotic – amikacin, and to chloramphenicol.

Recently, it was found that the egg masses of chironomids, non-biting midges, (Diptera; Chironomidae) serve as a natural reservoir for Aeromonas pathogenic species [34], [35] as well as for Vibrio cholerae[36]. Chironomid infestations in drinking water supply systems are an existing problem in Israel [37] and worldwide [38]. Chironomids may disseminate pathogenic species of Aeromonas between drinking water reservoirs, as was suggested for V. cholerae[39].

The source of Aeromonas in diarrheal patients was not investigated in the current study. In order to investigate the route of transmission of Aeromonas pathogenic strains an extensive study on strains from various origins should be performed. Chironomid egg masses in drinking water ponds and tap waters should be screened for Aeromonas isolates and compared with isolates from diarrheal patients.

Aeromonas infections are self–limiting, but their diagnosis may be crucial in young children, old and immunocompromised patients. We conclude that Aeromonas may be a causative agent of diarrhea in patients in Israel and therefore should be included in routine bacteriological screenings.

Supporting Information Table S1

Characterization of Aeromonas isolates from diarrheal patients.Aeromonas rpoD sequences were deposited in the GenBank database under the accession numbers JF738005–JF738021 (see also Figure 1).


Click here for additional data file (pone.0030070.s001.doc)


Competing Interests: The authors have declared that no competing interests exist.

Funding: This study was supported by the Research Authority, University of Haifa, Israel. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

1. Sen K,Rodgers M. Year: 2004Distribution of six virulence factors in Aeromonas species isolated from US drinking water utilities: a PCR identification.J Appl Microbiol971077108615479425
2. Borchardt MA,Stemper ME,Standridge JH. Year: 2003Aeromonas isolates from human diarrheic stool and groundwater compared by pulsed-field gel electrophoresis.Emerg Infect Dis922422812603994
3. Figueras MJ. Year: 2005Clinical relevance of Aeromonas sM503.Rev Med Microbiol16145153
4. Essers B,Burnens AP,Lanfranchini FM,Somaruga SGE,von Vigier RO,et al. Year: 2000Acute community-acquired diarrhea requiring hospital admission in Swiss children.Clin Infect Dis3019219610913424
5. Anaissie EJ,Penzak SR,Dignani MC. Year: 2002The hospital water supply as a source of nosocomial infections: a plea for action.Arch Intern Med1621483149212090885
6. Janda JM,Abbott SL. Year: 2010The Genus Aeromonas: taxonomy, pathogenicity, and infection.Clin Microbiol Rev23357320065325
7. Echeverria P,Blocklow NR,Sanford IB,Cakor GG. Year: 1981Traveler's diarrhea among American Peace Corps volunteers in rural Thailand.J Infect Dis1437677717019355
8. Burke V,Gracey M,Robinson J,Peck D,Beaman J,et al. Year: 1983The microbiology of childhood gastroenteritis: Aeromonas species and other infective agents.J Infect Dis14868746886488
9. Ormen O,Regue MQ,Tomas JM,Granum PE. Year: 2003Studies of aerolysin promoters from different Aeromonas spp.Microb Pathog3518919614521877
10. Agarwal RK,Kapoor KN,Kumar A. Year: 1998Virulence factors of aeromonads – an emerging food borne pathogen problem.J Commun Dis3071789914671
11. Janda JM,Abbott SL. Year: 1998Evolving concepts regarding the genus Aeromonas: an expanding panorama of species, disease presentations, and unanswered questions.Clin Infect Dis273323449709884
12. Kingombe CI,Huys G,Tonolla M,Albert MJ,Swings J,et al. Year: 1999PCR detection, characterization, and distribution of virulence genes in Aeromonas spp.Appl Environ Microbiol655293530210583979
13. Aguilera-Arreola G,Hernandez-Rodrıguez C,Zuniga G,Figueras MJ,Castro-Escarpulli G. Year: 2005Aeromonas hydrophila clinical and environmental ecotypes as revealed by genetic diversity and virulence genes.FEMS Microbiol Lett24223124015621443
14. Yu HB,Srinivasa Rao PS,Lee HC,Vilches S,Merino S,et al. Year: 2004A type III secretion system is required for Aeromonas hydrophila AH-1 pathogenesis.Infect and Immun721248125614977925
15. Chacón M,Soler L,Groisman E,Guarro J,Figueras MJ. Year: 2004Type III secretion system genes in clinical Aeromonas isolates.J Clin Microbiol421285128715004096
16. Murray PR,Baron EJ,Jorgensen JH,Pfaller MA,Yolken RH. Year: 2003Manual of Clinical Microbiology, 8th edWashington, D. C.ASM press654671 and 902–914
17. Garcia LS,Isenberg HD. Year: 2010Clinical Microbiology Procedures Handbook, 3rd ed. Washington, D. C.: ASM press.Section9
18. Kong R,Pelling A,So C,Wu R. Year: 1999Identification of oligonucleotide primers targeted at the 16S–23S rDNA intergenic spacers for genus-and species-specific detection of aeromonads.Mar Pollut Bull38802808
19. Soler L,Yanez MA,Chacon MR,Aguilera-Arreola MG,Catalan VM,et al. Year: 2004Phylogenetic analysis of the genus Aeromonas based on two housekeeping genes.Int J Syst Evol Microbiol541511151915388703
20. Silver AC,Graf G. Year: 2009Prevalence of genes encoding the Type Three Secretion System and the effectors AexT and AexU in the Aeromonas veronii group.DNA Cell Bio2838338819534604
21. Miller R,Walker R,Baya A,Clemens K,Coles M,et al. Year: 2003Antimicrobial susceptibility testing of aquatic Bacteria: quality control disk diffusion ranges for Escherichia coli ATCC 25922 and Aeromonas salmonicida subsp. salmonicida ATCC 33658 at 22 and 28°C.J Clin Microbiol41431812958263
22. Jorgensen JH. Year: 2010Methods for antimicrobial dilution and disk susceptibility testing of infrequently isolated or fastidious Bacteria. Approved Guideline. Clinical and Laboratory Standards Institute. CLSI/NCCLS M45-A2.
23. Ogunsanya T,Rotimi V,Adenuga A. Year: 1994A study of the aetiological agents of childhood diarrhoea in Lagos, Nigeria.J Med Microbiol4010148289209
24. Janda JM,Abbott SL. Year: 2006New gram-negative enteropathogens: fact or fancy?Rev Med Microbiol172737
25. Morgan D,Johnson P,DuPont H,Satterwhite T,Wood L. Year: 1985Lack of correlation between known virulence properties of Aeromonas hydrophila and enteropathogenicity for humans.Infect Immun5062654044042
26. Figueras MJ,Horneman AJ,Martinez-Murcia A,Guarro J. Year: 2007Controversial data on the association of Aeromonas with diarrhea in a recent Hong Kong study.J Med Microbiol5699699817577068
27. Moyer NP. Year: 1987Clinical significance of Aeromonas species isolated from patients with diarrhea.J Clin Microbiol25204420483693537
28. Golik A,Modai D,Gluskin I,Schechter I,Cohen N,et al. Year: 1990Aeromonas in adult diarrhea: an enteropathogen or an innocent bystander?J Clin Gastroenterol121481522324479
29. Pablos M,Remacha MA,Rodríguez-Calleja JM,Santos JA,Otero A,et al. Year: 2010Identity, virulence genes, and clonal relatedness of Aeromonas isolates from patients with diarrhea and drinking water.Eur J Clin Microbiol Infect Dis291163117220549532
30. Albert MJ,Ansaruzzaman M,Talukder KA,Chopra AK,Kuhn I,et al. Year: 2000Prevalence of enterotoxin genes in Aeromonas spp. isolated from children with diarrhea, healthy controls, and the environment.J Clin Microbiol383785379011015403
31. Alperi A,Martínez-Murcia AJ,Ko WC,Monera A,Saavedra M,et al. Year: 2010Aeromonas taiwanensis sp. nov. and Aeromonas sanarellii sp. nov., clinical species from Taiwan.Int J Syst Evol Microbiol602048205519819994
32. Heuzenroeder MW,Wong CYF,Flower RLP. Year: 1999Distribution of two hemolytic toxin genes in clinical and environmental isolates of Aeromonas spp.: correlation with virulence in a suckling mouse model.FEMS Microbiol Lett17413113610234831
33. Jones BL,Wilcox MH. Year: 1995Aeromonas infections and treatment.J antimicrob chemother354534617628980
34. Senderovich Y,Gershtein Y,Halewa E,Halpern M. Year: 2008Vibrio cholerae and Aeromonas: do they share a mutual host?ISME J22768318317460
35. Figueras MJ,Beaz-Hidalgo R,Senderovich Y,Laviad S,Halpern M. Year: 2011Re-identification of Aeromonas isolates from chironomid egg masses as the potential pathogenic bacteria Aeromonas aquariorum.Environ Microbiol Rep3239244
36. Broza M,Halpern M. Year: 2001Chironomid egg masses and Vibrio cholerae.Nature4124011452294
37. Halpern M,Gasith A,Broza M. Year: 2002Does the tube of a benthic chironomid larva play a role in protecting its dweller against chemical toxicants?Hydrobiologia4704955
38. Sun XB,Cui FY,Zhang JS,Xu F,Liu LJ. Year: 2007Inactivation of chironomid larvae with chlorine dioxide.J Hazard Mater14234835316978773
39. Broza M,Gancz H,Halpern M,Kashi Y. Year: 2005Adult non-biting midges: possible windborne carriers of Vibrio cholerae non-O1 non-O139.Environ Microbiol757658515816934


[Figure ID: pone-0030070-g001]
doi: 10.1371/journal.pone.0030070.g001.
Figure 1  Phylogenetic tree of Aeromonas isolates recovered from diarrhea patients.

The tree shows the relationships based on partial sequences of rpoD gene of type strains of Aeromonas species and the isolates from the current study. The sequence alignments were performed using the CLUSTAL W program, and the tree was generated using the neighbor–joining method with Kimura 2 parameter distances in MEGA 4.1 software. Bootstrap values (from 1,000 replicates) greater than 50% are shown at the branch points. The bar indicates 2% sequence divergence.

[TableWrap ID: pone-0030070-t001] doi: 10.1371/journal.pone.0030070.t001.
Table 1  Prevalence of virulence genes in Aeromonas isolates from diarrheal patients.
Isolatename Virulence genes
ahpB pla/lip/lipH3/apl-1 act/aerA/hlyA ast alt fla lafA TTSSascV TTSSascF-ascG aexT aexU
H3TK1 + + +
H15AI+1 + + + +
H17AD1 + + + +
H22AJ4 + +
H22AG8 + + + + +
H23AM+2 + + + + +
H30AD+5 + + +
H33AJ+7 + + +
H34AA+4 + + + + +
H35AG+1 + + + +
H39AA+3 + + + +
H45AF+12 + + + + +
H45AK+3 + + +
H50AI2 + + + + + +
H53AQ1 + + + +
H65AT3 + + +
H67AJ5 + + + +

For more details on the isolates and on the patients see Table S1.

[TableWrap ID: pone-0030070-t002] doi: 10.1371/journal.pone.0030070.t002.
Table 2  Susceptibility of Aeromonas isolates to antimicrobial agents.
Antimicrobial agent Number (%) of strains
(number of strains tested) susceptible intermediate resistance resistant
amikacin (11) 11 (100) - -
cefotaxime (11) 11 (100) - -
ceftazidime (11) 11 (100) - -
ceftriaxone (E test) (11) 11 (100) - -
ciprofloxacin (11) 11 (100) - -
chloramphenicol (11) 11 (100) - -
gentamicin (11) 10 (91) - 1 (9)
piperacillin–tazobactam (11) 10 (91) - 1 (9)
trimethoprim–sulfamethoxazole (11) 10 (91) - 1 (9)
imipenem (also meropenem) (11) 8 (73) - 3 (27)
cefoxitin (10) 7 (70) 2 (20) 1 (10)
nalidixic acid (11) 7 (64) - 4 (36)
tetracycline (11) 6 (55) - 5 (45)
amoxicillin+clavulanic acid (11) 3 (27) 3 (27) 5 (46)
cephalotin (10) 2 (20) - 8 (80)

Most of the identified strains were resistant to beta-lactam antibiotics but susceptible to third-generation cephalosporin antibiotics.

Article Categories:
  • Research Article
Article Categories:
  • Biology
    • Microbiology
    • Population Biology
      • Epidemiology
Article Categories:
  • Medicine
    • Clinical Research Design
    • Epidemiology
    • Gastroenterology and Hepatology
    • Infectious Diseases

Previous Document:  The effects of terlipressin on regional hemodynamics and kidney function in experimental hyperdynami...
Next Document:  Virologic failures on initial boosted-PI regimen infrequently possess low-level variants with major ...