Virulence Gene- and Pandemic Group-Specific Marker Profiling of Clinical Vibrio parahaemolyticus Isolates

Abstract

Vibrio parahaemolyticus is a halophilic bacterium capable of causing food- and waterborne gastroenteritis, wound infections, and septicemia in humans. The organism has recently received increasing attention, as the emergence of a new clone, V. parahaemolyticus O3:K6, has resulted in the first documented pandemic spread of V. parahaemolyticus. We used microarray analyses to explore the presence of known virulence factors and genetic markers thought to be specific for V. parahaemolyticus O3:K6 and its clonal derivatives. Analyses of 48 human clinical isolates collected between 1997 and 2005 revealed that the V. parahaemolyticus chromosome 2 type III secretion system is not specifically associated with pandemic strains and can be found in tdh-negative (i.e., Kanagawa-negative) clinical isolates. These results highlight the genetic dynamism of V. parahaemolyticus and aid in refining the genetic definition of the pandemic group members.

Vibrio parahaemolyticus is a gram-negative halophilic bacterium that is indigenous to marine and estuarine environments around the world. While most strains and serotypes of V. parahaemolyticus are nonpathogenic, those that are pathogenic have rapidly become major etiologic agents of human gastroenteritis, wound infections, and septicemia. Since 1996, one serotype in particular, V. parahaemolyticus O3:K6 (and its clonal derivatives O4:K68, O1:K25, and O1:KUT) (7), has received increasing notoriety, as it is the first documented V. parahaemolyticus serotype to cause pandemic disease (19, 24) and has recently been linked to gastroenteritis outbreaks on the Asian (6, 11, 24, 40), North American (8, 21), South American (10), European (18, 31), and African (1) continents. To compound matters, serotype-based detection and surveillance efforts have been complicated by serotype transition and variation within the pandemic lineage, as additional serotypes (O1:K26 [26], O6:K18 [39], O1:K41, O4:K12 [16], O1:K56, O3:K75, O4:K8, O4:KUT, and O5:KUT [5]) from specific locales have now been identified as having been derived from the original pathogenic O3:K6 clone.

V. parahaemolyticus-mediated disease has traditionally been thought to be associated with two virulence factors, the thermostable direct hemolysin (TDH) and the TDH-related hemolysin (TRH). Strains producing TDH are responsible for the β-type hemolysis on Wagatsuma agar that is known as the Kanagawa phenomenon (KP) (33); most clinical strains are KP⁺, while environmental strains tend to be KP⁻. However, strains carrying either the tdh or the trh gene (or both genes) are considered virulent strains (34). In addition to these two virulence factors, recent analysis of the genome sequence of V. parahaemolyticus strain RIMD2210633 (KP⁺, serotype O3:K6, pandemic group member) suggests that another virulence factor, the type III secretion system (T3SS), may also play a role in the disease manifestation of V. parahaemolyticus infections (17). Unlike the genomes of other Vibrio species, such as V. cholerae O1 (12), V. vulnificus (4), and V. fischeri (32), which do not contain T3SS genes, or V. cholerae non-O1/non-O139 (9) and V. harveyi (13), which appear to contain one set of T3SS genes, that of the sequenced V. parahaemolyticus O3:K6 pandemic group strain contains two sets of T3SS genes (17). The first set of T3SS genes is found on chromosome 1 (T3SS1), is similar in genetic structure and organization to the Yersinia sp. T3SS, and appears to be present in all V. parahaemolyticus isolates tested, regardless of origin (17). The second set of T3SS genes is found on chromosome 2 (T3SS2) embedded within an ∼80-kb pathogenicity island that harbors two copies of the tdh gene. T3SS2 does not appear to be similar to any bacterial T3SS other than the V. cholerae non-O1/non-O139 T3SS (9) and is reported to be found only in KP⁺ isolates (17, 30). Importantly, both T3SS1 and T3SS2 in V. parahaemolyticus appear to be functional (30).

Recent research concerning the V. parahaemolyticus pandemic has focused on serotype-based epidemiological investigations, molecular assays for the identification of pandemic group members, and identification of the molecular determinants that may help to explain this group's emergence and rapid spread. In this study, we tested 48 clinical V. parahaemolyticus isolates using microarray technology to determine the specificity of known virulence factors (TDH, TRH, T3SS1, T3SS2) and other genetic markers for V. parahaemolyticus pandemic group members.

MATERIALS AND METHODS

Bacterial isolates.

The 48 V. parahaemolyticus isolates chosen for this study were all clinical in origin and collected between 1997 and 2005 at the Centers for Disease Control and Prevention (CDC; Atlanta, GA) as part of the Cholera and Other Vibrio Illness surveillance system (http://www.cdc.gov/foodborneoutbreaks/vibrio_sum/CSTE_2005.pdf). The isolates were originally received at the CDC and were confirmed by using standard biochemical testing, growth on thiosulfate citrate bile sucrose and tryptic soy agar with 5% sheep blood, and PCR for the tdh and trh genes (2). Serotypes were determined using V. parahaemolyticus Seiken typing antiserum (Denka Seiken, Tokyo, Japan). Genomic DNA from all 48 V. parahaemolyticus isolates used in this study were sent from the CDC to the Naval Research Laboratory for testing. Genotyping assays for 40 of the 48 samples were performed by the investigators in a blinded manner.

DNA amplification.

Each multiplex PCR contained 24 primer pairs (data not shown). The 24-plex PCRs were performed with 50-μl volumes containing 1× PCR buffer (QIAGEN, Valencia, CA); 1.5 mM MgCl₂; 400 μM concentrations of dATP, dGTP, and dTTP; 40 μM dCTP; 40 μM Cy3-dCTP (GE Healthcare Bio-Sciences Corp., Piscataway, NJ); 200 nM concentrations of each primer (except trhF3 and trhR3 [400 nM]); 15 U Taq DNA polymerase (QIAGEN); and 50 ng of genomic DNA. The amplification reactions were performed with an initial denaturation at 94°C for 5 min; 35 cycles of 94°C for 30 s, 59°C for 60 s, and 72°C for 90 s; and a final extension at 72°C for 7 min. Upon completion of these steps, the amplified products were spin purified using an UltraClean PCR cleanup kit (MoBio Laboratories, Carlsbad, CA) and eluted in 50 μl distilled H₂O. Pandemic member group-specific toxRS PCR (GS-PCR) was performed for each isolate as previously described (19), and the resulting amplicons were analyzed with 2% agarose gels.

Microarray hybridization and processing.

V. parahaemolyticus-specific oligonucleotide probes targeting toxin, T3SS1, T3SS2, and species-specific and pandemic group-specific genes were designed, synthesized, and covalently immobilized as previously described (36) (data not shown). Once constructed, the spotted microarrays were blocked with a 3% bovine serum albumin-casein solution for 15 min at room temperature, and the slides were outfitted with MAUI M4 hybridization chambers (BioMicro Systems, Salt Lake City, UT). Hybridization reactions (6.0 μl multiplex PCR amplicons, 2.4 μl 20× SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 2.4 μl formamide, 0.8 μl 3% bovine serum albumin-casein solution, 0.2 μl hybridization-positive control, and 0.24 μl 10% sodium dodecyl sulfate) were denatured for 5 min at 95°C, applied to the microarray, and allowed to incubate for 2 h at 63°C with a MAUI hybridization system (BioMicro Systems). After hybridization, the slides were washed twice with 4× SSC-0.2% sodium dodecyl sulfate buffer for 3 min at 63°C, washed twice with 2× SSC buffer for 1 min at room temperature, rinsed once with distilled H₂O, and dried with compressed air. Images were captured with a ScanArray Lite confocal laser scanning system (PerkinElmer, Boston, MA) at a laser power of 80 and with a Photo Multiplier Tube gain of 80. Quantitative comparisons based on fluorescence intensities were made using the accompanying QuantArray quantitative analysis software package. The signal from each microarray element was considered positive only when its fluorescence intensity was at least three times those of the neighboring background and internal negative control probes. Borderline-positive and negative microarray results were confirmed by individual PCRs that were amplified using the same conditions, analyzed on 2% agarose gels, and sequenced when appropriate. To avoid incorrect hybridization profiles due to multiplex amplification bias or mutated primer binding sites, 25% of the isolates were tested using a previously described (Klenow-plus-Klenow) random amplification method (37) to confirm the multiplex amplification/hybridization profiles.

RESULTS

Targeted genes.

We designed 32 oligonucleotide probes to enable the detection of hemolysin, T3SS1, T3SS2, and pandemic group-specific genes from clinical V. parahaemolyticus isolates (data not shown). Known hemolysins associated with V. parahaemolyticus were detected using probes targeting the thermolabile hemolysin (tl), tdh, and trh genes. To attempt to differentiate between pandemic and nonpandemic group members, we designed probes that targeted sequences, such as toxRS/new (19), the open reading frame 8 (orf8) gene from phage f237 (14, 22), VPF2/VPR2 primer product (15), and the histone-like DNA-binding protein (HU-α) gene (38), that have previously been utilized for the identification of the O3:K6 clone and pandemic group members. As the T3SS1 has been implicated in host cytotoxic activity (30), six probes were designed to target the following spatially distributed genes from within the chromosome 1 T3SS gene cluster: vopB (VP1657), yopN (VP1667), vscP (VP1670), the ORF encoding a secreted effector protein (VP1680) (28), vscJ (VP1690), and vscC1 (VP1696). Similarly, as the T3SS2 has been implicated in host enterotoxicity (30) and appears to be unique to vibrios, a special emphasis was placed on the design of probes that would permit the accurate identification of 11 T3SS genes from within the chromosome 2 pathogenicity island T3SS gene cluster (Fig. 1A) that had recognized T3SS orthologs in other gram-negative bacterial pathogens (Fig. 1B).

FIG. 1.

Targeted V. parahaemolyticus T3SS2 gene cluster. (A) Genetic organization of the 11 targeted T3SS2 genes (gray arrows) that reside within the 80-kb pathogenicity island on chromosome 2 of V. parahaemolyticus strain RIMD2210633 (17). The locus spanning VPA1334 to VPA1368 is shown with the T3SS2 gene designations listed above each gray arrow. A scale line showing the position (in base pairs) from the chromosome 2 origin of replication can be found below the gene cluster map. (B) BLAST comparison of V. parahaemolyticus T3SS2 genes with known T3SS genes in other bacteria. *, BLAST comparison with V. cholerae non-O1/non-O139 AM-19226 (9); †, BLAST comparison with bacterial systems with known and established T3SSs.

V. parahaemolyticus genotyping.

We compared the hybridization profile results generated by the microarray with the results of standard PCR and biochemical and serotyping-based identification for a set of 48 human clinical V. parahaemolyticus isolates. The partial hybridization profiles from four randomly amplified isolates are presented as representative examples in Fig. 2, and the genotypes for all 48 isolates are presented in Table 1. This test set included serotypes accepted as belonging to the pandemic group (O1:K25, O1:KUT, O3:K6, and O4:K68) as well as serotypes that are not associated with the pandemic lineage (e.g., O3:K48, O4:K4, O5:K56, and O11:K40) and contained four hemolysin genotypes (tl-positive, tdh-negative, trh-negative; tl-positive, tdh-positive, trh-negative; tl-positive, tdh-positive, trh-positive; and tl-positive, tdh-negative, trh-positive genotypes). We defined the pandemic group based on a previously described genotypic definition (GS-PCR⁺, tdh positive, trh negative) (16). Based on this definition, the microarray identified 14 of the 48 isolates as pandemic group members, and the serotypes of these isolates matched those of known pandemic group members. The same 14 isolates also harbored the orf8, VPF2/VPR2, and HU-α sequences and a complete set of targeted T3SS1 and T3SS2 genes. T3SS2 genes were also found in 8 of the 34 nonpandemic isolates that had the same hemolysin genotype as the pandemic clone (tl positive, tdh positive, trh negative). Two additional nonpandemic isolates, F8950 (serotype O11:K43) and F9837 (serotype O4:K8), presented unique profiles in that they were tl positive, tdh negative, and trh negative (and thus KP⁻), yet they still harbored a complete set (F9837) or incomplete set (F8950) of T3SS2 genes.

FIG. 2.

Representative confirmatory microarray hybridization profiles of randomly amplified clinical V. parahaemolyticus isolates. Probes targeting (row 1, left to right) VP1657, VP1667, VP1670, and VP1680; (row 2) VP1690, VP1696, negative control, and negative control; (row 3) TL, TDH, TRH, and TRH; (row 4) ToxRS/new, ORF8, VPF/R2, and HU-α; (row 5) VPA1335, VPA1338, VPA1339, and VPA1341; (row 6) VPA1342, VPA1346, VPA1349, and VPA1354; and (row 7) VPA1355, VPA1362, VPA1367, and the unrelated hybridization-positive control are shown. The strain identification number and corresponding serotype are found under each panel.

TABLE 1.

Microarray-based genotypes of nonpandemic and pandemic V. parahaemolyticus serotypes

Strain	Year	Origin	Serotype	GS-PCRa	Presence of gene encoding:
					Hemolysin			Pandemic group-specific protein				T3SS1						T3SS2
					TL	TDH	TRH	ToxRS/new	ORF8	VPF2/VPR2	HU-α	VP1657	VP1667	VP1670	VP1680	VP1690	VP1696	VPA1335	VPA1338	VPA1339	VPA1341	VPA1342	VPA1346	VPA1349	VPA1354	VPA1355	VPA1362	VPA1367
K0850	2004	LA	O1:K25		+			+				+	+	+	+	+	+
K0851	2004	LA	O1:KUT		+			+		+		+	+	+	+	+	+
F8979	2002	ME	O1:KUT		+			+				+	+	+	+	+	+
K1621	2005	CO	O3:K6		+			+				+	+	+	+	+	+
F9674	2003	LA	O3:K48		+			+		+		+	+	+	+	+	+
F9005	2002	LA	O4:K4		+			+		+		+	+	+	+	+	+
F9837	2003	CT	O4:K8		+			+		+		+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
F7979	2001	MN	O5:K17		+			+				+	+	+	+	+	+
F8131	2001	VA	O5:K30		+			+		+		+	+	+	+	+	+
K0853	2004	LA	O5:K56		+			+				+	+	+	+	+	+
F9835	2003	LA	O5:KUT		+			+		+		+	+	+	+	+	+
F6179	1998	CT	O6:K18		+			+		+		+	+	+	+	+	+
F9843	2003	CT	O6:K18		+			+		+		+	+	+	+	+	+
K1000	2004	NC	O8:K20		+			+				+	+	+	+	+	+
K0103	2003	NC	O10:KUT		+			+				+	+	+	+	+	+
F8950	2002	MD	O11:K43		+			+		+		+	+	+	+	+	+	+	+	+	+	+	+		+	+	+	+
F8837	2002	NH	O1:K38		+	+		+			+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
F7074	2000	CA	O4:K8		+	+		+				+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
F9082	2002	AZ	O4:K8		+	+		+		+	+	+	+	+	+	+	+	+	+	+		+	+	+	+	+	+	+
K0300	2004	OR	O4:K8		+	+		+		+		+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
K1610	2005	HI	O4:K8		+	+		+				+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
F7065	2000	CA	O4:K55		+	+		+				+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
K0845	2004	HI	O8:K21		+	+		+				+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
F9869	2003	NV	O9:K44		+	+		+		+		+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
F7635	2001	CA	O1:K25	+	+	+		+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
F8701	2002	MN	O1:K25	+	+	+		+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
K0071	2003	TN	O1:KUT	+	+	+		+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
K0456	2004	MA	O1:KUT	+	+	+		+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
F5835	1998	TX	O3:K6	+	+	+		+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
F5847	1998	TX	O3:K6	+	+	+		+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
F7630	2001	CA	O3:K6	+	+	+		+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
F8949	2002	MA	O3:K6	+	+	+		+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
F9083	2002	AZ	O3:K6	+	+	+		+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
F5828	1998	TX	O3:K6	+	+	+		+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
F6820	1999	GA	O3:K6	+	+	+		+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
K1533	2004	CT	O3:K6	+	+	+		+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
F7636	2001	CA	O4:K68	+	+	+		+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
F7680	2001	CA	O4:K68	+	+	+		+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
K1824	2005	WA	O1:K56		+	+	+	+				+	+	+	+	+	+
K0301	2003	TN	O1:KUT		+	+	+	+		+	+	+	+	+	+	+	+
F7279	2000	GA	O4:K12		+	+	+	+				+	+	+	+	+	+
K0082	2003	LA	O4:K12		+	+	+	+				+	+	+	+	+	+
F8314	2001	MD	O4:K53		+	+	+	+				+	+	+	+	+	+
F5165	1997	WA	O4:K63		+	+	+	+				+	+	+	+	+	+
F8199	2001	OR	O4:K63		+	+	+	+		+		+	+	+	+	+	+
F8119	2001	MD	O4:KUT		+	+	+	+				+	+	+	+	+	+
K0976	2004	AK	O6:K18		+	+	+	+				+	+	+	+	+	+
F5170	1997	WA	O11:K40		+		+	+				+	+	+	+	+	+

^aGS-PCR for the identification of pandemic group members as described in reference 19.

In accordance with previous findings, all of the isolates tested were positive for T3SS1 (17, 30) and the tl gene (2, 35). However, this data set also revealed exceptions pertaining to genetic targets that were previously thought to be specific for the identification of pandemic group members, such as the VPF2/VPR2 primer product (15) (e.g., F8199, serotype O4:K63; F9674, serotype O3:K48) and the HU-α insertion (25, 38) (e.g., F8837, serotype O1:K38; K0301, serotype O1:KUT). While both markers were found in all the pandemic strains tested, they were less specific (59% and 91%, respectively) than the orf8 gene, which was 100% specific in this data set. Our modified primer/probe combination for the detection of the toxRS/new marker lacked the previously described specificity (19).

DISCUSSION

Most currently used molecular identification methods for pandemic V. parahaemolyticus (such as pulsed-field gel electrophoresis [40], ribotyping, arbitrarily primed PCR [19, 24], toxRS PCR [or GS-PCR] [19], orf8 PCR [14, 21, 22], enterobacterial repetitive intergenic consensus PCR [15], toxRS/tdh multiplex PCR [26], PGS-PCR [27], and mass spectrometry [38]) rely on the detection of a single genetic marker and/or are unable to make any direct determination about the virulence factors that contribute to human infection and disease. In this study, we have demonstrated the use of a microarray genotyping method that enables the simultaneous interrogation of a panel of species-specific, virulence gene-specific, and pandemic group-specific markers to accurately determine the molecular identities and virulence potentials of pandemic and nonpandemic V. parahaemolyticus isolates.

A significant portion of V. parahaemolyticus-related research has revolved around the development of molecular assays for the identification of pandemic group members. In an attempt to identify pandemic group members, we chose four molecular targets (toxRS/new [19], orf8 [14, 22], VPF2/VPR2 primer product [15], and HU-α [38]) that had previously been utilized for the identification of the O3:K6 clone and pandemic group members. Our results clearly demonstrated that neither the VPF2/VPR2 primer product nor the HU-α insertion sequence was entirely specific for pandemic group members. Furthermore, although the orf8 gene was specific to the 14 pandemic isolates tested in this study, it by itself has been previously shown to be an unreliable marker for the identification of the pandemic group (3, 5, 26). The same case has been made for the toxRS/new sequence (26). Considering the dynamic genetics of V. parahaemolyticus and of Vibrio spp. in general, exceptions such as these are not surprising and underscore the importance of simultaneously monitoring more than one genetic marker for the confident identification of pandemic group members. Whether by microarray, multiplex PCR, or other means, the continued molecular surveillance of pandemic V. parahaemolyticus would likely benefit from the codetection of molecular markers such as those used in this study.

A second example of the benefit of this sort of analysis is the ability to elucidate unexpected genetic assemblages and genotypes that underlie disease causation. Previous studies have demonstrated that the V. parahaemolyticus T3SS2 is present only in KP⁺ (i.e., tdh-positive) strains (17, 30). We have found that there are exceptions to this association, as we identified two KP⁻ (tdh-negative and trh-negative) isolates that were T3SS2⁺ (F8950 and F9837) and, conversely, that many KP⁺ strains are T3SS2⁻. The absence of the tdh genes in isolates F8950 and F9837 suggests that (i) this pathogenicity island differs from that of strain RIMD2210633 (17), (ii) the T3SS2 may be acquired without the surrounding pathogenicity island (which in addition to two copies of the tdh gene may harbor other currently unrecognized accessory or virulence factors), or (iii) the tdh genes were mobile and subsequently lost from the pathogenicity island (23). A suggestive, as the V. parahaemolyticus T3SS2 is involved in enterotoxicity in the rabbit ileal loop model (30), and tdh mutants (29) and trh mutants (41) still maintain partial enterotoxic activity, the identification of two human clinical isolates that are T3SS2⁺ and tdh- and trh-negative further bolsters the contention that the Vibrio T3SS2 is a virulence factor that contributes to human infection and illness (9, 29, 30). Thus, although the T3SS2 gene cluster is not restricted to pandemic group members, the acquisition of the T3SS2 (and potentially the ∼80-kb pathogenicity island) as it pertains to potentially pathogenic V. parahaemolyticus is of concern and may warrant routine monitoring. While the exact roles of this and other V. parahaemolyticus virulence factors in overall disease manifestation and severity of illness remain to be determined, such determinations will be greatly aided by studies that correlate the presence of these virulence factors with the type of infection, severity of illness, and need for hospitalization (42).

We have designed and tested a method that simultaneously characterizes a panel of known virulence genes and pandemic group-specific markers that can be used for the accurate detection and screening of potentially epidemic environmental and clinical V. parahaemolyticus isolates. The results of this study suggest that the data generated in a single assay would be valuable not only for the rapid detection of newly emerging pandemic clones that have undergone serotype conversion but also for providing information regarding the virulence potential of nonpandemic strains that are capable of causing large outbreaks (20).