pseudomonas aeruginosa outbreak linked to mineral water ... · three mineral water bottles...

JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 2010, p. 3146–3152 Vol. 48, No. 90095-1137/10/$12.00 doi:10.1128/JCM.00402-10Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Pseudomonas aeruginosa Outbreak Linked to Mineral Water Bottlesin a Neonatal Intensive Care Unit: Fast Typing by Use ofHigh-Resolution Melting Analysis of a Variable-Number

Tandem-Repeat Locus�†F. Naze,1 E. Jouen,2 R. T. Randriamahazo,1 C. Simac,1 P. Laurent,1 A. Bleriot,1 F. Chiroleu,2

L. Gagnevin,2 O. Pruvost,2‡ and A. Michault1‡*Service de Bacteriologie-Parasitologie-Virologie et Hygiene, Groupe Hospitalier Sud Reunion, BP350, Saint Pierre 97448,

La Reunion, France,1 and UMR Peuplements Vegetaux et Bioagresseurs en Milieu Tropical, CIRAD-Universite dela Reunion, Pole de Protection des Plantes, 7 Chemin de l’Irat, Saint Pierre 97410, La Reunion, France2

Received 26 February 2010/Returned for modification 13 April 2010/Accepted 17 June 2010

Pseudomonas aeruginosa is an opportunistic pathogen that causes nosocomial infections in intensive careunits. Determining a system of typing that is discriminatory is essential for epidemiological surveillance of P.aeruginosa. We developed a method for the typing of Pseudomonas aeruginosa, namely, multiple-locus variable-number tandem-repeat (VNTR) typing with high-resolution melting analysis (HRMA). The technology wasused to genotype a collection of 43 environmental and clinical strains isolated during an outbreak in a neonatalintensive care unit (NICU) that we report. Nineteen strains isolated in other departments or outside thehospital were also tested. The genetic diversity of this collection was determined using VNTR-HRMA, withamplified fragment length polymorphism (AFLP) analysis as a reference. Twenty-five and 28 genotypes wereidentified, respectively, and both techniques produced congruent data. VNTR-HRMA established clonal relation-ships between the strains of P. aeruginosa isolated during the outbreak in the NICU and proved, for the first time,the role of mineral water as the inoculum source. VNTR typing with one primer pair in association with HRMA ishighly reproducible and discriminative, easily portable among laboratories, fast, and inexpensive, and it demon-strated excellent typeability in this study. VNTR-HRMA represents a promising tool for the molecular surveillanceof P. aeruginosa and perhaps for molecular epidemiologic analysis of other hospital infections.

Pseudomonas aeruginosa is one of the most important op-portunistic pathogens involved in nosocomial infections world-wide. P. aeruginosa is a ubiquitous bacterium which is able tosurvive under many conditions, and many hospitals create newniches for its development. Several reservoirs are frequentlyinvolved in infections, such as medical equipment, contami-nated environments (sinks, taps, etc.), contaminated bottledwater (10), contaminated products (eye drops, soaps, etc.),health care workers, other contaminated patients, and some-times the patient him- or herself (4, 21, 39). In neonatal inten-sive care units, in addition to the previously described sources,P. aeruginosa strains were recovered in contaminated feedingbottles and were associated with breast milk feeding (5, 25, 34).

Typing of nosocomial pathogens is necessary to determinethe source of an outbreak and to take effective control mea-sures to prevent the spread of the pathogens. A good typingtool should be convenient, highly discriminatory, reproducible,fast, and inexpensive. For inoculum source investigations, itshould target loci with a rather fast molecular clock so theepidemiological relationships are not overestimated. Pheno-

typic and biochemical methods based on serotyping, bacte-riophage typing, or antimicrobial susceptibility have weakdiscriminatory power and were replaced with DNA-basedtechniques. Among these molecular methods, pulsed-field gelelectrophoresis (PFGE) of restricted genomic DNA fragmentsis commonly considered the most appropriate technique avail-able to type closely related isolates of P. aeruginosa. However,this method is time-consuming (2 or 3 days) and expensive andrequires specialized equipment and qualified operators (16,27). Consequently, other molecular methods have been used todiscriminate strains with the same accuracy while avoiding thedrawbacks inherent to PFGE.

Random amplified polymorphic DNA PCR typing is fasterand less expensive than PFGE, but it is less discriminatory(35). Multilocus sequence typing, a method based on allelicdifferences among housekeeping genes, is less discriminatorythan PFGE for detecting genetic differences among P. aerugi-nosa isolates (20). Genotyping using amplified fragment lengthpolymorphism (AFLP) analysis was applied successfully to P.aeruginosa epidemiological investigations (22, 30). Moreover,AFLP typing is comparable to PFGE (6) and is more accurateunder certain conditions (35). Other methods target short re-peated DNA sequences that are interspersed in bacterial ge-nomes. Repetitive DNA sequence-based PCR (rep-PCR) isfast and convenient but lacks interlaboratory reproducibility(36). Semiautomated rep-PCR seems to be highly reliable fortyping of P. aeruginosa but requires specialized equipment (8).Minisatellites or variable-number tandem repeats (VNTR)

* Corresponding author. Mailing address: Service de Bacteriologie-Parasitologie-Virologie et Hygiene, Groupe Hospitalier Sud Reunion,BP350, Saint Pierre Cedex 97448, La Reunion, France. Phone: 262 359100. Fax: 262 359 110. E-mail: [email protected].

‡ O.P. and A.M. contributed equally to this work.� Published ahead of print on 23 June 2010.† The authors have paid a fee to allow immediate free access to

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are appropriate tools for developing a highly discriminatoryand reproducible typing assay. Multiple-locus VNTR anal-ysis (MLVA) displays a similar clustering ability to that ofPFGE (28), and increasing the marker number was shown toenhance the typing efficiency (38).

High-resolution melting (HRM) technology is now availableon many real-time PCR devices. HRM curve analysis (HRMA)is a fast, convenient, and cost-effective screening methodfor PCR products. Such a technology could be used advanta-geously to separate polymorphic PCR products generated byVNTR amplifications, using the same equipment. The aim ofthis work was to elucidate the source of a P. aeruginosa out-break in a neonatal intensive care unit by using such an alter-native technique. Data derived from HRMA of a VNTR locuswere compared with data obtained by AFLP analysis. AFLPanalysis was used as a reference technique that requires noprior knowledge of genomic DNA sequences and targets mul-tiple interspersed sites over the whole genome. We believe thatthis is the first study to use VNTR typing with HRMA and isthe first time that mineral water used to prepare baby bottles isdescribed as the source of contamination in a neonatal inten-sive care unit.

MATERIALS AND METHODS

Strains. P. aeruginosa strains were obtained from neonates admitted to theneonatal department (28 beds, including 10 for the neonatal intensive care unitand 2 for pediatric resuscitation; 350 admissions per year) of the Groupe Hos-pitalier Sud Reunion (the largest hospital on the island, with 1,300 beds). Thisdepartment has an incidence of infections or colonizations with P. aeruginosa of�2 per 1,000 hospitalization days (14). Criteria for colonization in this outbreakreport were the presence of P. aeruginosa in peripheral samples, no clinical signsof infection, and a normal value for C-reactive protein. During the first 3 monthsof 2006, the incidence was 1.4 per 1,000 hospitalization days. From April to June2006, the incidence rose to 6.8 infections per 1,000 hospitalization days. Duringthis prospective study, to investigate the outbreak, serial stool and conjunctivalsamples were taken once a week from 100 neonates admitted to the neonataldepartment during this period. Ocular swabs were chosen because these sampleswere positive in a previous epidemic due to P. aeruginosa (14). These samples areeasy to obtain and are not invasive. Additional specimens were collected forclinical reasons. The presence of P. aeruginosa was investigated in 100 neonatesadmitted to the hospital. Forty-two had at least one sample positive for P.aeruginosa. Among these 42 patients, 40 were colonized and 2 were infected. Inthis study, 20 patients were included, among whom 19 patients were colonizedand 1 was infected. Twenty-one strains isolated from stool, 3 strains from eyes,1 strain from blood culture, and 1 strain from a tracheal aspirate were studied.Nine environmental samples positive for P. aeruginosa were also obtained duringthe study period to research the source of contamination, particularly in theaqueous environment of the department, where P. aeruginosa is usually present(3). Positive environmental samples were obtained from 2 sink drains, 4 tapwater samples, 2 faucets, and 1 sink, with potential contamination throughout theneonatal department, including the room where milk and baby bottles wereprepared. Three mineral water bottles (different brands and batches) purchasedby the hospital, 4 mineral water samples with milk (in a baby bottle or not), and1 whisk used to mix mineral water and milk were collected. Some strains wereisolated from clinical samples (13 strains) and from the environment (1 strain) inother departments of the hospital and outside the hospital (5 strains isolatedfrom sink drains in the houses of the authors) during the same period. Waterfrom taps was collected in 250-ml sterile bottles with sodium thiosulfate. Forsampling of surfaces, an agar contact technique (Count-Tact agar; bioMerieux,Marcy l’Etoile, France) was used. Other environmental samples were obtainedwith cotton swabs and were plated onto Mueller-Hinton plates (bioMerieux,Marcy l’Etoile, France). Water was concentrated by filtration through a 0.45-�mmembrane and then seeded onto Cetrimid agar (bioMerieux, Marcy l’Etoile,France). Clinical stool samples were plated onto MacConkey agar (bioMerieux,Marcy l’Etoile, France), and conjunctival samples were plated onto Polyvitexagar (bioMerieux, Marcy l’Etoile, France). The strains were identified by theanalytical profile index procedure (API 20NE test; bioMerieux, Marcy l’Etoile,

France). Sixty-two strains were identified, and a colony of each strain was frozenat �80°C on cryo-beads (AES, Combourg, France). Figure 1 summarizes theepidemiological data for the P. aeruginosa strains studied. Strain ATCC 27853was chosen as the root for tree construction.

Genotypic methods. (i) DNA preparation. Target DNA was prepared fromcells grown overnight at 37°C on Polyvitex agar (bioMerieux, Marcy l’Etoile,France). Cells were resuspended in 200 �l of RNase- and DNase-free water(Sigma).

A DNeasy blood and tissue kit (Qiagen, Courtaboeuf, France) was used forDNA extraction, with an extended lysis step (overnight at 56°C). The concen-tration of DNA was measured with Quant-iT PicoGreen dsDNA reagents andkits (Invitrogen, Cergy Pontoise, France).

(ii) AFLP analysis. The AFLP experiments were performed in 96-well platesin a GeneAmp model 9700 thermocycler (Applied Biosystems, Foster City, CA),as previously described (1). Oligonucleotides were synthesized by Applied Bio-systems. Digestions were carried out with SacI and MspI restriction enzymes.The first combination for the selective amplification step used selective primersMspI�A and 5�-6-carboxyfluorescein (FAM)-labeled SacI�C, the second com-bination used MspI�C and 5�-VIC-labeled SacI�C, the third combinationused MspI�T and 5�-NED-labeled SacI�C, and the fourth combination usedMspI�G and 5�-PET-labeled SacI�C. Samples were then prepared for capillaryelectrophoresis by adding 1 �l of each of the four final PCR products to 10.7 �lof formamide and 0.3 �l of LIZ500 DNA ladder (Applied Biosystems, FosterCity, CA), used as an internal standard. The mixture was then denatured for 5min at 95°C and placed on ice for at least 5 min. Electrophoresis was performedin an ABI Prism 3130 genetic analyzer (Applied Biosystems, Foster City, CA),using performance-optimized POP-7 polymer (Applied Biosystems, Foster City,CA) at 15,000 V for about 20 min at 60°C, with an initial injection time of 69 s.The AFLP fingerprints were analyzed visually using GeneMapper software 4.0(Applied Biosystems, Foster City, CA). The amplified fragments were scored aspresent (1) or absent (0) to create binary matrices before analysis by R software,version 2.9.0 (32). Bands with intensities above a preset level (500 relativefluorescence units) were the only ones to be scored. To test the reproducibilityof the AFLP technique, two independent DNA extractions were used for allstrains, and nonreproducible markers were discarded from the analysis. StrainPyo-7 was used as a control in each experiment. The genetic similarities werecalculated with the ade4 package (9) in R software for each AFLP condition andfor the concatenated data set, using the Dice similarity index (7).

Kendall’s coefficient of concordance (W) (23) among the four distance matri-ces was computed and tested through a permutation test (9,999 permutations)with the ape package in R software (29). A posteriori permutation tests of thecontribution of each of the four AFLP condition distance matrices to the overallconcordance of the group were carried out using Mantel tests. Permutationalprobabilities were corrected using Holm’s correction method. A weighted neigh-bor-joining tree was constructed with the ape package in R software for theconcatenated data set, using Dice similarity coefficients as distances. Bootstrapvalues (12) were computed to assess the robustness of the tree (1,000 resam-plings).

(iii) PCR amplification and HRMA. Six polymorphic tandem repeats (ms77,ms127, ms142, ms172, ms207, and ms209) with different motif lengths (39, 15,115, 54, 6, and 6 bp, respectively) and PCR product lengths (442, 210, 890, 789,146, and 148 bp, respectively, for reference strain PAO1), as described by Vu-Thien et al. (38), were tested with 15 clinical strains. Previously, 4 of these 6markers were described by Onteniente et al. (28). First, we looked for thetypeability of these VNTR. We chose tandem repeats of different motif lengths(micro- and minisatellites) which had PCR products of different sizes to comparethe discriminatory powers of VNTR-HRMA and AFLP typing and to obtain thebest discriminatory power with VNTR-HRMA. The influence of motif lengths ofVNTR on HRMA is unknown. When the VNTR was chosen, the reproducibilityof our technique was tested.

The conditions were optimized using a Light Cycler 480 high-resolution melt-ing master kit (Roche Diagnostics, Meylan, France). The 20-�l reaction mixcontained 1� master mix, 3 mM MgCl2, a 0.3 �M concentration of each VNTRprimer, and LCGreen I dye.

PCRs were performed with a Light Cycler 480 instrument (Roche Diagnostics,Meylan, France) under the following conditions: an activation cycle at 95°C for10 min; 10 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 15 s, andelongation at 72°C for 25 s; and 40 cycles of 95°C for 10 s, 65°C for 15 s, and 72°Cfor 25 s.

HRMA was performed immediately after PCR cycling. The samples wereheated to 98°C, rapidly cooled to 40°C, and heated again to 70°C, and thenfluorescence was measured with continuous acquisitions (25 per °C; 1°C/s until98°C).

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LC480 genotyping software (Roche Diagnostics, Meylan, France) was used tocalculate the melting curve data, and 3 groups were obtained (Fig. 2). The limitsof each group (premelt/postmelt slider settings) were searched and fixed (Fig. 3)to obtain the most genotypes. The difference plot was analyzed by the software,and samples with similar melting profiles were grouped.

To assess the reproducibility of the analysis, five repeats were carried out withthe chosen limits.

Index of discriminatory power. The discriminatory power of the differenttyping methods was evaluated using an index described by Hunter (19). Desirabletechniques for inoculum-tracking purposes should have a high discriminatorypower (index of �0.95) (4).

Consensus between AFLP typing and HRMA. The Rand index (15, 31) esti-mates the proportions of agreements and disagreements found for two classifi-cations over the total number of pairs. Hubert and Arabie (18) proposed acorrection for chance for this index to ensure that its maximum value is 1 andexpected value is 0 when the partitions are selected randomly (15). The correctedRand index was computed to assess the agreement between classifications ob-tained with AFLP typing and HRMA (13), using fpc package 1.2-4 (17) in Rsoftware.

RESULTS

VNTR typing. Six loci were tested on 15 clinical strains.Three loci were eliminated (ms77, ms127, and ms172) becausemany strains could not be interpreted by HRMA, probably due

to low concentrations of PCR products (data not shown).Markers ms207 and ms209 were tested on the 62 strains andwere eliminated because their discriminatory power in HRMAwas low. In addition, the discriminatory power obtained whenthese markers were associated with ms142 was not superior tothat for ms142 alone. In this study, better HRMA results wereobtained with the VNTR with the greatest motif length and thegreatest PCR product length. The influences of the PCR prod-uct length and the motif length have to be confirmed by typingother bacteria by VNTR-HRMA. The typeability with ms142primers was 98.4%. Strain P2 was the only one that was notamplified with these primers. Therefore, ms142 primers wereselected because their typeability and discriminatory powerwere high.

AFLP typing. A total of 63 P. aeruginosa strains (62 isolatedstrains and strain ATCC 27853) were typed by AFLP typing,regarded as the “gold standard” in this study, and yielded 419reproducible band positions (97.8% segregated) under the 4AFLP conditions (99, 122, 97, and 101 positions for theSacI�C/MspI�A, SacI�C/MspI�C, SacI�C/MspI�T, andSacI�C/MspI�G conditions, respectively). The number of

FIG. 1. Neighbor-joining tree derived from Dice similarities (7) computed from the AFLP binary matrix, showing the relationships between thePseudomonas aeruginosa strains related or not related to the outbreak and isolated inside or outside the hospital. Branches with bootstrap valuesof �80% are represented with bold lines. The tree was rooted with strain ATCC 27853. Bar, 0.05 substitution per site. E, environment; P, patient;End, endocrinology; Gastr, gastrology; ICU, intensive care unit; NN, neonatal unit; NS, neurosurgery; ORL, otorhinolaryngology; OS, orthopedicsurgery; Ped, pediatric; PDS, protected distal sampling; NAM, no amplification; SNN, suspected outbreak-related strains isolated in the neonatalunit; NSIH, nonsuspected strains isolated inside the hospital; NSOH, nonsuspected strains isolated outside the hospital.

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markers per strain ranged from 42 to 110. Twenty-nine geno-types (Fig. 1) were identified from the concatenated data set(genotypes A1 to A29). Dice’s coefficient of similarity betweengenotypes ranged from 0.08 to 0.85 (median � 0.65). Kendall’scoefficient of concordance between the four distance matricesrevealed a high degree of correlation among them (W � 0.85;P � 0.0001). The mean of the Mantel correlations for eachdistance matrix with the others ranged from 0.74 (for theSacI�C/MspI�C condition) to 0.83 (for the SacI�C/MspI�Acondition). The probability of these a posteriori permutationtests, corrected with Holm’s method, was 0.0004, which indi-cated that the four distance matrices were also highly congru-ent with one another. Therefore, subsequent analyses wereperformed on a pooled data set.

The weighted neighbor-joining tree (Fig. 1) showed that 32strains, which were all outbreak related, of the 63 strains clus-tered within a genetic cluster containing 31 strains identified asgenotype A6 and one strain (P8) identified as genotype A7.This genotype differed from genotype A6 only by the absenceof a marker (71 markers for A7 versus 72 markers for A6). TenAFLP markers were found to be specific for these genotypes.This genetic cluster was supported by a bootstrap value of100%.

VNTR-HRMA versus AFLP typing. The interreproducibilityof VNTR-HRMA on the 61 strains tested five times was 100%.Based on a reference index of discriminatory power of 1.00 forAFLP typing, the index of discriminatory power calculated for61 strains was 0.99 for VNTR-HRMA. The corrected Randindex between AFLP and HRMA classifications (0.95) showeda high level of agreement between these two classifications.

All of the suspected outbreak-related strains were identifiedas genotype H5 by VNTR-HRMA. The strain (P8) with geno-type A7 (by AFLP typing) was classified into group H5. Amongthe 26 strains isolated from 21 neonatal patients during thestudy, three genotypes were identified by AFLP typing (A6,A7, and A27) and two others (H5 and H23) were identified byVNTR-HRMA, with H5 corresponding to A6 and A7 and H23corresponding to A27. Genotypes A6 and H5 were isolated themost often, in 92.3% (24/26 strains) and 96% (25/26 strains) ofstrains, respectively.

AFLP typing showed that most patients (20/21) were colo-nized by strains with the A6 genotype; the same results wereobtained by VNTR-HRMA for the H5 genotype. For 2 pa-tients (patients 17 and 19), P. aeruginosa was isolated fromseveral samples: it was isolated from 2 samples for patient 17(conjunctival and stool samples) and from 4 samples forpatient 19 (conjunctival, stool, blood culture, and trachealaspiration samples), and these different isolates all exhibitedgenotype A6 by AFLP typing and genotype H5 by VNTR-HRMA. The death of patient 19 was attributed directly to a P.aeruginosa infection with a genotype A6 and H5 strain. P.aeruginosa was isolated from two stool samples from patient20, and the 2 isolates exhibited two different genotypes (geno-type A6 or the closely related genotype A7, which is probablya variant of the same strain, and genotype H5). Seventeenstrains were isolated from the environment of the neonataldepartment during the study. AFLP typing of these strainsrevealed 8 genotypes, including the A6 genotype (47% of thesestrains), and 8 genotypes, including the H5 genotype (47% ofthese strains), were revealed by VNTR-HRMA. The classifi-

FIG. 2. Melting temperature analysis with LC480 software. Three groups were defined: groups 1 and 2 have one melting temperature, andgroup 3 has two melting temperatures.



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cation of genotypes determined by AFLP typing and VNTR-HRMA had a concordance of 100%. Strains exhibiting geno-types A6 and H5 were isolated either from commercial bottledmineral water (bottled at 15:40 and 15:41 ratios), from milk

prepared with this water, or from whisks used to mix water andmilk. None of the strains isolated in other departments of thehospital (with 14 AFLP genotypes and 13 VNTR-HRMA ge-notypes; one strain [P2] was not amplified) or outside the

FIG. 3. Gene scanning analysis. Signal variations for the three groups are shown for analyses in which the limits were fixed before or after theexponential phase.



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hospital exhibited the AFLP genotype A6 or H5. Outside thehospital, five new genotypes were identified by AFLP typingand VNTR-HRMA.

DISCUSSION

Outbreak. The role of P. aeruginosa in outbreaks caused bycontaminated water has been well described (2, 33, 37). Re-cently, a P. aeruginosa outbreak provoked by the contamina-tion of feeding bottles was reported (34). To our knowledge,there has not been any reported outbreak of P. aeruginosacaused by mineral water used to prepare milk in a neonatalintensive care unit.

The integration of molecular genotyping in the epidemicinvestigations of P. aeruginosa in the neonatal intensive careunit established the role of two batches of a brand of mineralwater. When another brand of mineral water was used, theoutbreak stopped. The contamination of the mineral water wasreported to health authorities and to the manufacturer. Anal-ysis of the process of fabrication by the manufacturer haspermitted an understanding of the reasons for the presence ofP. aeruginosa in these two batches: the decontamination pro-cess after a stop of production due to a factory mechanicalfailure was not realized, and the length of the control culturewas only 2 days (it was later increased to 5 days to avoidfalse-negative results).

The pathogen associated with the outbreak was rapidly iden-tified, and the role of water was suspected. The source of theoutbreak was more difficult to identify. During the outbreak,five patients died: four deaths were not attributed to a P.aeruginosa infection, but one death was attributed to a P.aeruginosa infection. The other patient infected by P. aerugi-nosa had a septicemia. Forty patients were colonized.

Discriminatory power. At first, two phenotypic methodswere used, but the indices of discriminatory power were tooweak (�95%) to determine the source of the outbreak (forantibiotyping, 60%; for serotyping, 82%; and for antibiotypingplus serotyping, 92% [data not shown]).

AFLP typing. The high discriminatory power of AFLP typ-ing permitted us to define genotype A6, which was found onlyin the environmental samples of mineral water, in preparationsmade with that water, and in neonates admitted to the neona-tal department during the outbreak.

Although AFLP typing can be suitable for outbreak inves-tigations of P. aeruginosa (11), easier and cheaper techniquescould improve the real-time monitoring of an outbreak.

MLVA. MLVA is now used widely for the genotyping ofmany different bacteria. Consequently, we tested the effective-ness of this technique for the genotyping of P. aeruginosa (28,38). Onteniente et al. (28) showed with a set of 7 loci thatMLVA has a high discriminatory power. Vu-Thien et al. (38)concluded by use of 15 VNTR that MLVA is an efficient, easy,and rapid typing method.

In this study, we targeted a single locus (ms142). The dis-criminatory power of the locus, analyzed by polyacrylamide gelelectrophoresis, was 0.95 (17 genotypes [data not shown]). Inthe study of Vu-Thien et al. (38), agarose gel electrophoresisanalysis showed a Hunter discriminatory index of 0.81 withms142.

HRMA. HRMA has been applied for mutation scanning andgenotyping (24, 26). However, in this study, we used HRMA asa post-PCR analysis.

HRMA combined with VNTR typing permitted an increaseof the discriminatory power of the typing system to 25 geno-types for the 62 strains tested, which is very close to the numberof genotypes obtained by AFLP typing (28 genotypes). Theindex of discriminatory power obtained was 0.99, which is veryclose to that of AFLP typing. The same number of genotypeswas identified by VNTR-HRMA and AFLP typing for dis-tances superior to 0.5 (data not shown). In this study, VNTR-HRMA had a high discriminatory power and an excellenttypeability and reproducibility.

In our study, VNTR-HRMA confirmed the role of twobatches of a brand of mineral water. The technique has theadvantages of requiring only PCR reagents, LCGreen I dye, 2unlabeled primers hybridizing with the flanking region of aVNTR locus, and 2 min of post-PCR analysis. No other anal-ysis, such as electrophoresis, is necessary, and the risk of con-tamination is reduced.

This technique is simple, rapid (amplification, 42 min; andHRMA, 2 min), and very cost-efficient and requires only melt-ing instrumentation. One strain was not amplified, perhaps dueto mutations in the VNTR locus or the deletion, in whole or inpart, of the VNTR locus.

In this study, outbreak strains and other strains isolated inLa Reunion, France, were tested. The ms142 locus was testedwith 90 European strains, suggesting a good typeability for thistechnique (38). However, the high discriminatory power ofHRMA with other strains must be confirmed.

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