Identification of the Emerging Pathogen Vibrio vulnificus Biotype 3 by Commercially Available Phenotypic Methods

Identification of the emerging pathogen Vibrio vulnificus biotype3 has become a challenge for clinical laboratories in the lastfew years. In this study, the abilities of five commercial systemsto identify this new species have been evaluated for the firsttime, using a unique collection of strains. Fifty-one well-documentedwild strains of V. vulnificus biotype 3 were processed usingAPI 20 NE, GNI+ Vitek 1 cards, ID-GNB Vitek 2 cards, Neg Combo20 Microscan panels, and NMIC/ID-5 BD Phoenix panels. The numbersof strains identified as V. vulnificus by ID-GNB, NMIC/ID-5,and GNI+ were 50 (98.0%), 46 (90.2%), and 7 (13.7%), respectively.Neg Combo 20 Microscan panels and API 20 NE were unable to identifyany of the strains of this emerging pathogen to the specieslevel and mostly misidentifies them as other species of theVibrionaceae family. Data on the phenotypic pattern of V. vulnificusbiotype 3 when processed in all five systems as presented herecould help clinical laboratories in identifying this new pathogen.

INTRODUCTION

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Introduction

Since it was first described by Reichelt et al. in 1976 (9),for many years only two biotypes or serovars of the human pathogenVibrio vulnificus have been recognized (1).

In the midsummer of 1996, we reported the first isolation ofa new pathogen, V. vulnificus biotype 3, which causes septicemiaand severe soft-tissue infections following contact with fishfrom artificial freshwater ponds (2). Since then, the identificationof this emerging pathogen has become a challenge for clinicallaboratories. With the phenotypic behavior of this new biotypebeing different from that of the V. vulnificus biotypes knownso far, most of the commercial systems in use do not includethe relevant data in their software, making correct identificationof the new bacteria problematic.

In this study, the phenotypic pattern of this new biotype whentested with five commercial systems was defined, and the abilitiesof the current software of those systems to correctly identifyV. vulnificus biotype 3 to the species level have been evaluatedfor the first time with a unique collection of strains.

MATERIALS AND METHODS

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Materials and Methods

Identification systems. Fifty-one well-documented V. vulnificus biotype 3 strains isolatedduring the years 1996 to 1997 were processed by five commercialidentification systems: API 20 NE, GNI+ Vitek 1 cards, ID-GNBVitek 2 cards (BioMerieux, Marcy l-Etoile, France), Neg Combo20 Microscan panels (Dade Behring Inc.), and NMIC/ID-5 BD Phoenixpanels (BD Biosciences).

Given the fact that previous reports have already concludedthat the use of API 20 E is inadequate for the identificationof V. vulnificus (6), only the API 20 NE system was used inthis study. API 20 NE strips were inoculated and processed followingthe manufacturer's recommendations, and the results were analyzedby using Apilab software, version 3.2.2.

Inoculated Vitek GNI+ cards were processed with a Vitek 1 system,version VTK-R 07.02.

Vitek ID-GNB cards were used with a Vitek 2 system, versionVT2-R 02.03.

Microscan Neg Combo 20 panels were inoculated according to themanufacturer's recommendations for halophilic vibrios (0.125ml of water plus Pluronic plus 0.1 ml of a 0.5 McFarland bacterialsuspension in each well) and processed in a Microscan Walkaway96 apparatus. Results were analyzed by using the Microscan DMSsystem, version 24.1.

Finally, inoculated NMIC/ID-5 panels were processed with theBD Phoenix system according to the manufacturer's instructions.

Bacterial strains. Fifty-one bacterial strains tested in the study were isolatedfrom patients during the years 1996 and 1997 in laboratoriesserving the northern region of Israel, where all cases appearfollowing contact with Tilapia fishes. The strains, which werepreviously identified at local laboratories, were eventuallyconfirmed by the Central Laboratories of the Israel Ministryof Health in Jerusalem as belonging to V. vulnificus biotype3 by using an extensive phenotypic workflow proposed by J. J.Farmer from the Centers for Disease Control and Prevention (Atlanta,Ga.).

Eventually, all the strains were confirmed as V. vulnificusat the Nuffield Department of Clinical Laboratory Sciences ofJohn Radcliffe Hospital, University of Oxford (Oxford, UnitedKingdom), using PCR for the cytotoxin-hemolysin (CTH) gene,as described by Brauns et al. (4). The DNeasy kit (QIAGEN GmbH,Hilden, Germany) was used to extract DNA, and the protocol forgram-negative bacteria was followed. Briefly, several coloniesfrom a single clone of each bacterial culture were picked offinto phosphate-buffered solution and centrifuged at 7,500 rpm(5,000 x g) for 10 min. The cell pellet was resuspended in 180µl of tissue lysis buffer, and then 20 µl of proteinaseK (10 mg/ml) was added and the sample was incubated at 55°Cuntil the tissue was completely lysed. Next, 200 µl oflysis buffer was added, and incubation was at 70°C for 10min. The DNA in the clear viscous lysates was precipitated withethanol (95% [vol/vol]) and added to DNeasy mini-columns. Ethanol(70% [vol/vol])-based buffers (AW1 and AW2) were added sequentiallyto the columns and centrifuged at 8,000 rpm (6,000 x g). Thesupernatants were discarded, and the DNA was resuspended insterile water and used for amplification.

PCR amplification was carried out with previously describedoligonucleotide primers (4). Each 50-µl amplificationreaction mixture comprised 10 ng of chromosomal DNA, 100 pmolof each PCR primer (MWG Biotech, Ebersberg, Germany), 10x PCRbuffer with 1.5 mM MgCl₂ (QIAGEN GmbH), 0.5 U of Taq DNA polymerase(QIAGEN GmbH), and 1.6 mM deoxynucleoside triphosphates (ABgene,Epsom, United Kingdom). The reaction conditions were denaturationat 94°C for 1 min, primer annealing at 68°C for 1 min,and extension at 72°C for 1 min, for 30 cycles.

A volume (5 µl) of PCR-amplified DNA was separated ona 0.8% agarose gel containing 0.5 µg of ethidium bromideml^–1. The gel was run in Tris-borate-EDTA buffer at 120V for 30 min with a 100-bp DNA ladder (ABgene, Epsom, UnitedKingdom) as a size standard. DNA fragments in the gel were visualizedunder a UV transilluminator (Gel Doc 1000; Bio-Rad LaboratoriesLtd., Bio-Rad House, Hemel Hempstead, United Kingdom).

In addition, two control strains were also tested with all systemsbut NMIC/ID-5 BD-Phoenix panels: V. vulnificus biotype 1 (CDCstrain 9028A95) and V. vulnificus biotype 2 (L. Hoi, Denmark;strain 96-7-138), both from an Israel Ministry of Health straincollection.

RESULTS

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Results

The results by PCR for 48 of the strains are shown in Fig. 1.Samples 2, 3, and 4 were tested separately and were also positivefor the CTH gene. Samples 26 and 40, shown as negative for theCTH gene, gave a positive reaction when retested using an annealingtemperature of 60°C.

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FIG. 1. PCR results for the CTH gene. Px, positive control strain; Nx, negative control strain.

The numbers of strains correctly identified by the differentsystems are shown in Table 1. The numbers of isolates misidentifiedas other Vibrionaceae or non-Vibrio species or not identifiedat all are also shown in the same table.

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TABLE 1. Percentages of strains correctly identified as V. vulnificus by all systems

ID-GNB, NMIC/ID5, and GNI+ correctly identified 50 (98.0%),46 (90.2%), and 9 (17.6%) strains, respectively. Microscan NegCombo 20 and API 20 NE were unable to correctly identify anyof the strains of this emerging pathogen.

For the single strain that ID-GNB was unable to identify, theVitek 2 system proposed Plesiomonas shigelloides and V. vulnificuswith the same probability rates and asked for further tests.

From the five strains not identified by NMIC/ID5 as V. vulnificus,only two were misidentified by the system as other Vibrio spp.

GNI+ misidentified 10 strains as other Vibrio spp. Thirty-onestrains, all of them presenting the same phenotypic pattern,were misidentified as P. shigelloides (75% probability), withV. vulnificus as the second choice (19% probability).

Microscan Neg Combo 20 misidentified all the strains as otherVibrionaceae, most of them as Vibrio mimicus and Vibrio parahaemolyticus.

Finally, API 20 NE also misidentified all the strains as otherVibrionaceae, most of them as Vibrio alginolyticus.

Tables 2 to 6 show the phenotypic profiles presented by all51 strains of biotype 3 tested with the API 20 NE, GNI+, ID-GNB,NMIC/ID5, and Microscan Neg Combo 20 systems, respectively.In addition, Tables 2, 3, 4, and 6 show the results for thecontrol strains of biotypes 1 and 2 with the respective systems.Table 2 also shows the expected result for V. vulnificus withAPI 20 NE as it appears in the kit insert.

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TABLE 2. API 20 NE profile of the 51 strains of biotype 3^a

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TABLE 6. Profile of the 51 strains of biotype 3 in Microscan system, using Neg Combo 20 panels^a

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TABLE 3. Profiles of the 51 strains of biotype 3 in Vitek 1 using GNI+ cards^a

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TABLE 4. Profile of the 51 strains of biotype 3 in Vitek 2, using ID-GNB cards^a

DISCUSSION

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Discussion

In the last few years, the routine use of molecular biologytechniques such as nested PCR (7) has been playing a more importantrole in clinical microbiology laboratories in developed countries.However, this is not the situation in the majority of the clinicallaboratories worldwide, especially in nondeveloped countries.Thus, the use of commercial phenotype-based identification systemswill continue to be the mainstream in the next years.

In this study, the abilities of five widely used phenotype-basedcommercial systems to identify the emerging pathogen V. vulnificusbiotype 3 were evaluated.

ID-GNB with Vitek 2 and NMIC/ID5 with BD Phoenix proved to bethe best systems for correctly identifying this new biotypeas V. vulnificus (98.0 and 90.2% of strains correctly identified,respectively). These two systems offer a wide range of phenotypicreactions, many of them enzymatic, and a considerable numberof them were positive with the strains tested. Microscan NegCombo 20, API 20 NE, and GNI+ with Vitek 1, however, offer asignificantly smaller number of reactions, and the number ofpositive tests was minimal with the tested strains. However,the majority of the strains presented a consistent phenotypicpattern with each of the systems. Thus, clinical laboratoriesusing these systems could tentatively identify strains showingthe phenotype presented here as that of V. vulnificus or atleast suspect the presence of this pathogen.

So far, this emerging pathogen has been reported only in Israel(2, 3, 5, 8).

However, since commercial systems may misidentify it as anothermember of the Vibrionaceae family, it is not unlikely that thepresence of this pathogen in other countries could be underestimated.Thus, clinical microbiology laboratories should be aware ofthis possibility and must meticulously check the correct identificationof any vibrio isolated from clinical sources.

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TABLE 5. Profile of the 51 strains of biotype 3 in BD-Phoenix, using NMIC/ID-5 cards^a

ACKNOWLEDGMENTS

We thank Bactlab Ltd., Caesarea Industrial Park, Israel, forsupplying the NMIC/ID5 BD Phoenix panels and Ilex Ltd., Roch-Ha’ayin,Israel, for supplying the GNI+ and ID-GNB Vitek cards.

We also thank the technical staff of the Microbiology Laboratoryof Ha'Emek Medical Center, the Microbiology Laboratory of MaccabiHealth Services in Haifa, Israel, the Microbiology Laboratoryof Assaf Harofeh Medical Center, Zerifin, Israel, and the MolecularMicrobiology Laboratory of John Radcliffe Hospital, Universityof Oxford, Oxford, United Kingdom.

FOOTNOTES

* Corresponding author. Mailing address: Clinical Microbiology Laboratory, Ha'Emek Medical Center, Afula 18101, Israel. Phone: 972 4649 4480. Fax: 972 4649 5428. E-mail: colodner_ra{at}clalit.org.il.

REFERENCES

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