First Record of the Rare Species Aeromonas culicicola from a Drinking Water Supply

We describe the recovery of the rare species Aeromonas culicicola,so far known only in mosquitoes in India, from a drinking watersupply in Spain. Typing, using enterobacterial repetitive intergenicconsensus-PCR, revealed that the 27 new isolates belonged to3 very closely related strains. These strains were geneticallyidentified by 16S rRNA gene sequencing. Spanish strains differedfrom the mosquito strains in three nucleotide positions. TheAHCYTOEN gene was present in these water strains, which mayhave a public health significance.

INTRODUCTION

Top

Introduction

Aeromonas species are common inhabitants of aquatic ecosystems,although they have also been described in connection with fishand human diseases (1, 2). The most important human pathogensare Aeromonas veronii bv. sobria, Aeromonas caviae, and Aeromonashydrophila, while Aeromonas salmonicida is the most importantfish pathogen. The two major diseases associated with humansare gastroenteritis and wound infections. Gastroenteritis typicallyoccurs after the ingestion of contaminated water or food, whereaswound infection results from exposure to contaminated water.The recently discovered species A. culicicola is a very rareand exotic taxon found only in the midgut of mosquitoes in India,from which so far only three isolates are available (14). Surprisingly,we have recently recovered a significant number of isolatesof this species in a very different substrate and at a distantlocation from where this species was originally isolated. Theywere found in a drinking water supply in Spain. Here, we describethe characterization of strains of this species using molecularidentification methods (16S DNA coding for RNA [rDNA]-restrictionfragment length polymorphism [RFLP] and 16S rDNA sequencing)and genotyping (enterobacterial repetitive intergenic consensus[ERIC]-PCR) (3, 8, 13, 17). We have also evaluated the potentialrisk to human health that this finding can represent by determiningthe presence of the cytolytic enterotoxin gene (AHCYTOEN) insuch isolates. This is a multivirulent gene that causes deathin mice, hemolysis, cytotoxicity, and enterotoxicity (5, 10).

Sample collection and biochemical identification of the strains.

Top

Sample collection and...

A total of 59 isolates of Aeromonas spp. were recovered from494 water samples from 50 different sites in the domestic waterpipelines during a routine monitoring sanitary survey. Theywere collected at 1- or 2-week intervals by the Instituto Municipalde Salud Publica in Zaragoza, Spain, from April to June 2002.Free chlorine residuals evaluation and recovery with ADA mediumwas performed as in a previous study (4). Tergitol agar, incubatedfor 24 h at 37°C (Difco Laboratories, Detroit, Mich.) wasused in parallel in all samples. From each sample one typicalcolony of those grown on each medium was selected for biochemicalidentification, i.e., yellow ones in ADA and red ones in tergitolagar. Oxidase, glucose fermentation (Kligler agar), gas fromsucrose, and esculin hydrolysis tests were used for this purpose.Strains were further confirmed by using API 20NE (BioMerieux,Marcy-l'Etoile, France). Once biochemically identified, theisolates were sent to our laboratory for molecular identification.

Aeromonas was found in 6.9% of the drinking water samples, correspondingto 20 different sampling sites within the drinking water supplysystem. Samples normally had concentrations of Aeromonas below10 CFU/100 ml and an absence of fecal indicators (data not shown).Free chlorine residuals in these samples were between <0.05and 1.5 ppm. The 59 isolates recovered were identified by usingAPI 20NE as follows: 47 were identified as A. veronii bv. sobria,8 were identified as A. hydrophila, and 4 were identified asA. caviae.

Molecular identification and typing.

Top

Molecular identification and...

Water isolates were reidentified using the 16S rDNA-RFLP method,which produces a unique pattern for each of the species of thegenus (3, 8). In the study, we included the three isolates ofA. culicicola known so far, i.e., the type strain (CECT 5761^T),isolated from the midgut of the mosquito Culex quinquefasciatus,and the strains SH and SLH from Aedes aegyptii, all of themfrom India (14). Our results were completely different fromthose obtained biochemically. For instance, the 27 water isolatesthat had been identified biochemically as A. veronii bv. sobriashowed an identical RFLP pattern that did not correspond toany previously identified Aeromonas species (3, 8). The patternof the Spanish isolates was also different from that of theoriginal strains of A. culicicola. The rest of the isolateswere identified as follows: 21 were identified as A. veronii,5 were identified as A. salmonicida, 4 were identified as A.hydrophila, 1 was identified as Aeromonas media, and 1 was identifiedas Aeromonas jandaei.

The 27 isolates and the 3 strains of A. culicicola were typedusing the ERIC-PCR technique as previously described (17). Thetype strains of A. salmonicida (CECT 894^T), A. jandaei (CECT4228^T), and A. hydrophila (CECT 839^T) were used as an outgroup.PCRs and image analyses have been done as previously described(17).

In the dendrogram obtained from the ERIC-PCR analysis (Fig.1), the 27 isolates were split into 3 groups with a similarityof 95 to 96%. Each group contained strains with an identicalERIC pattern. At the same time, they grouped together with thethree strains of A. culicicola at a similarity level of 51.78%,suggesting that they could be related to that species. Two ofthe isolates of A. culicicola (SH and SLH) grouped with a similaritylevel of 100% and grouped with a similarity of 72.22% with thethird isolate (the type strain). These data revealed that the27 isolates belong to 3 genetically very similar strains andthat isolates SH and SLH are identical. All the Spanish isolatesrecovered from the same sample grouped together (Fig. 1). However,within each of the three subclusters, there were isolates recoveredfrom distant sites (up to 4 km) in the water supply system.

View larger version (18K):
[in this window]
[in a new window]
FIG. 1. Dendrogram based on UPGMA clustering of Dice correlation values (S_D) of ERIC patterns of 27 isolates from drinking water and reference strains of A. culicicola (CECT 5761^T, SH, and SLH), A. jandaei (CECT 4228^T), A. hydrophila (CECT 839^T), and A. salmonicida (CECT 894^T). The scale represents S_D values converted to percentages. A final letter A in the strain designation indicates that it was recovered from ADA medium, and T indicates tergitol. Superscript "a" indicates a strain selected for 16S rDNA sequencing. Superscript "b" indicates an isolate from water leaving the drinking water treatment plant.

16S rRNA gene sequencing.

Top

16S rRNA gene sequencing.

In order to confirm the identity of the Spanish isolates, wedecided to sequence the 16S rRNA gene. A 1,303-bp fragment ofthis gene of one of the isolates (3037T) was initially sequencedin order to perform a BLAST and CLUSTAL analysis. Genomic DNAextraction, 16S rDNA amplification, and sequencing was performedas previously described (13). Sequencing was performed by usingan ABI PRISM 310 genetic analyzer (Perkin-Elmer, Foster City,Calif). The higher similarity (99%) was with A. culicicola (strainsSH and SLH). The sequence was completed, and two other isolates(2238A and 2424A) representative of the other two ERIC-subclusters(Fig. 1) and the three existing isolates of A. culicicola werealso sequenced. The obtained sequences (1,503 bp) were comparedby using CLUSTAL W with those of all the type strains of Aeromonasfrom GenBank (Fig. 2). A phylogenetic tree (Fig. 2) was drawnby using the neighbor-joining method with MEGA software, version2.1 (11).

View larger version (20K):
[in this window]
[in a new window]
FIG. 2. The phylogenetic tree of the 16S rRNA gene sequences of the Spanish strains (2238A, 2424A, and 3037T) and described Aeromonas species. The unrooted phylogenetic tree was drawn from 1,503 bp of the gene, using the neighbor-joining method in MEGA software. Numbers in parentheses are the GenBank sequence accession numbers.

The analysis of the sequences showed several differences (Table1). Although Pidiyar et al. (14) indicated that the sequencesof their three Indian strains were identical, we observed thatthe sequences of the isolates SH and SLH that they depositedin GenBank were identical to each other and differed in fivenucleotides from the type strain (Table 1). The nucleotide differencesfound between the sequences of the three strains of A. culicicolawe obtained and those of the same strain withdrawn from theGenBank (AF170914, AY130991, and AY130992) were due to double-sequencingpicks at seven positions in our sequences (Table 1). The predominatingnucleotides at these sites coincided with those that appearedin the sequences obtained from GenBank. Five of these variations(positions 457 to 476) were located at the hypervariable stem-loop,a region considered not phylogenetically informative (12), andthe other two were located at positions 1011 and 1018. The threeSpanish isolates showed less variability and presented two double-sequencingpicks at positions 649 and 1308 not observed in the other strains(Table 1). Despite these differences, the three Spanish isolateswere on the same branch as the three Indian isolates of A. culicicolain the phylogenetic tree, indicating that they belonged to thisspecies (Fig. 2). A. jandaei was the species closest to A. culicicola,in agreement with the work of Pidiyar et al. (14). However,apart from the specific single-nucleotide difference reportedby those authors for these two species at position 264, we havefound another variation at position 1503 not described before.At this position, A. culicicola shows a G instead of the A thatA. jandaei possesses.

View this table:
[in this window]
[in a new window]
TABLE 1. Base differences in the 16S rDNA sequences among original and selected strains of A. culicicola

Intraspecific variations of the 16S rRNA gene have previouslybeen described for Aeromonas popoffii (7, 12) and in other Aeromonasspp. (18). and are not uncommon in prokaryotes (6, 9). Thisvariability is due to the fact that nucleotide composition variesbetween operon copies of the ribosomal 16S multigene familyand the PCR produces a consensus of the sequences (16). Thetwo above-mentioned intraspecific variations for our strainstogether with the one encountered at position 475 for the Indianones explain the mentioned differences in RFLP pattern. Contraryto what happens with the 16S rDNA intraspecific variations describedfor A. popoffii (7), the variations encountered in A. culicicolaaffected the restriction sites of the AluI and MboI enzymes.Although two RFLP patterns were observed in A. culicicola, bothare specific to this species and different from those for otherspecies of the genus.

Detection of the AHCYTOEN gene.

Top

Detection of the ahcytoen...

The presence of the AHCYTOEN gene was investigated in the SpanishA. culicicola isolates using primers (AHCF1 and AHCR1) and conditionspreviously described by Kingombe et al. (10). An amplicon of232 bp, typical of the AHCYTOEN gene, was obtained for all the27 isolates tested. The PCR product of three strains was sequencedto demonstrate that the amplified region corresponded to thegene. This cytolytic enterotoxin gene is considered a characteristicvirulence trait in Aeromonas (10, 15), and its general presencein A. culicicola, indicates that this species may have publichealth significance.

ACKNOWLEDGMENTS

Strains SH and SLH were provided by Yogesh S. Shouche of theNational Center for Cell Science, Pune University Campus, Ganeshkhind,India.

This work was supported by grants from Fundació Ciènciai Salut and from the Spanish Ministry of Health (FIS 03/1183).

FOOTNOTES

* Corresponding author. Mailing address: Departament de Ciències Mèdiques Bàsiques, Facultat de Medicina i Ciències de la Salut, Universitat Rovira i Virgili, Sant Llorenç 21, 43201 Reus, Spain. Phone: 34-977759321. Fax: 34-977759322. E-mail: mjfs{at}fmcs.urv.es.

REFERENCES

Top