Advertisement
Free access
29 May 2015

The Biology of Vibrio vulnificus

ABSTRACT

Vibrio vulnificus, carrying a 50% fatality rate, is the most deadly of the foodborne pathogens. It occurs in estuarine and coastal waters and it is found in especially high numbers in oysters and other molluscan shellfish. The biology of V. vulnificus, including its ecology, pathogenesis, and molecular genetics, has been described in numerous reviews. This article provides a brief summary of some of the key aspects of this important human pathogen, including information on biotypes and genotypes, virulence factors, risk factor requirements and the role of iron in disease, association with oysters, geographic distribution, importance of salinity and water temperature, increasing incidence associated with global warming. This article includes some of our findings as presented at the “Vibrios in the Environment 2010” conference held in Biloxi, MS.

DISEASE CHARACTERISTICS

Vibrio vulnificus is the single most fatal foodborne pathogen in the United States, and possibly in the world, accounting for 95% of all seafood-related deaths in the US with a fatality rate of ca. 50% (1). This greatly exceeds that of other foodborne pathogens (2), such as Salmonella (0.6%), Escherichia coli (3–5%), and Clostridium botulinum (<8%). The bacterium occurs naturally in estuarine waters worldwide and the food vehicle is primarily raw/undercooked oysters, which account for 93% of ingestion cases. While the concentration of V. vulnificus in estuarine waters is typically quite low (<10 CFU/ml), it becomes concentrated in such molluscan shellfish as oysters and clams due to their efficient use of filter-feeding to obtain food. The resultant levels in shellfish can reach 105 CFU/g of tissue or more (3, 4, 5). While the infectious dose is not known, it has been estimated to be as few as 100 cells or less (6). Most cases (86%) occur in males over the age of 40, with 95% of victims having one or more preexisting risk factors (see below). These statistics, based on US cases, appear not to differ from those in other countries, even where raw seafood is widely consumed. For example, Ito et al. (7) recently reported that the average age of patients of 166 V. vulnificus cases is 60 years, with approximately 90% being male; 42% had liver cirrhosis and 11% diabetes mellitus. The majority of cases (85%) reported in the US occur in the summer months of May to October, which correlates to the higher water temperatures common during that period. The incubation period for ingestion cases is quite rapid, averaging only 26 h. Symptoms of infection following ingestion of infected food include fever (94%), chills (86%), nausea (60%), abdominal pain (44%), hypotension (43%), and the development of secondary lesions (69%), which typically develop on the extremities (1). The reason for the latter may be the temperature optimum for growth of the pathogen (ca. 30°C) and the lower temperature of such extremities as the legs (8).
It has been estimated that 80,000 people contract Vibrio infections each year in the USA, contributing to 500 hospitalizations and 100 deaths (9). Despite at least 12 Vibrio spp. being human pathogens (10), the great majority of hospitalizations and deaths are due to V. vulnificus.
The reason for the highly significant gender difference (ca. 86% of infections are in males) is due, at least in part, to the role that estrogen plays in protecting against the bacterium’s endotoxin (11). However, why those over the age of 40 are predominantly affected is not known (Fig. 1). It might be thought that older people are the prime consumers of raw oysters, but in fact this honor goes to those under 29 years (12), and this aspect of the infection remains a mystery.
FIGURE 1
FIGURE 1 Age distribution of people developing Vibrio vulnificus septicemia (J.D. Oliver, unpublished data).
In addition to primary septicemia resulting from V. vulnificus infection, this bacterium is also capable of causing potentially fatal wound infections (13, 14, 15, 16). Most of these involve exposure of a preexisting wound to seawater or shellfish, or acquisition and infection of a wound during saltwater-associated recreational activity. Like ingestion cases, some 89% of wound cases are in males. This may reflect a greater occurrence of commercial and recreational fishermen of this gender, but given the large number of females involved in coastal swimming activities, such a gender difference is surprising. Major symptoms include fever (85% of patients), chills (68%), with edema (91%) and cellulitis (94%) at the wound site. The incubation period for this form of infections is even more rapid, averaging only 16 h. Chronic disease does not appear to be a prerequisite to these infections (over 80% have no underlying syndrome), but the 22% fatality rate that occurs in wound infections is likely a consequence of the fact that these victims generally have some form of liver or other serum iron-elevating condition. Nonfatal cases are often serious enough to warrant surgical intervention, including limb amputation. Interestingly, the incidence of wound infections is increasing in the USA; between 1988 and 1999 there was an average of 24 cases/year reported to the Cholera and Other Vibrio Illness Surveillance (COVIS) system, whereas between 2000 and 2010, the rate had increased to 52/year. Indeed, wound infections caused by V. vulnificus are now the predominant form of infection caused by this pathogen in the USA, exceeding nonwound isolations (17, 18).

BIOTYPES AND GENOTYPES

Despite the need for underlying chronic disease, a national health survey estimates that as many as 36 million Americans have one or more of these predisposing syndromes (19), so the question arises, why are there not more cases? There are undoubtedly several reasons for this, including the need to consume raw shellfish harboring this pathogen, human physiological conditions both natural (male, age over 40 years) and pathological (liver or immunocompromising disease), and the production of essential virulence factors by the infecting V. vulnificus cells. There are likely more human host factors we have yet to discern, but it is certainly the case that we currently understand little of the essential virulence traits needed. Despite extensive study by many laboratories around the world, we know very little of this critical aspect. We do now know, however, that there are at least three biotypes, and of the major human biotype, the existence of two genotypes. Biotype 1 was the first described and is the type found in virtually all human infections (15). Biotype 2 causes a rapidly fatal septicemia in eels, especially those raised in aquaculture farms (20, 21, 22, 23, 24), and it has, on rare occasions, been isolated from human cases. Biotype 3 was the most recently described and it has been proposed to be a genetic mosaic of biotype 1 and 2 strains (25). To date, this biotype has only been isolated from human wound infections associated with tilapia aquaculture, and only in Israel (26). The latter observation is rather incredible, given the ease of disease spread around the world.
In 1999, we reported results of a RAPD-PCR analysis of numerous clinical and environmental (oyster, seawater) strains of V. vulnificus. This revealed the presence of a PCR amplicon, which appeared to be unique to the human clinical isolates (27). On sequencing this DNA fragment, we discovered that the base sequence of the human isolate form was dramatically different (up to 30% of the bases) from that found in a homologous gene in the environmental isolates (28). A similar separation of V. vulnificus isolates using 16S rRNA sequencing was reported by Aznar et al. (29), and subsequently, by Nilsson et al. (30) and Gutacher et al. (31), and a 100% correlation between the B/C and C/E genotyping schemes has been reported (28). Realizing that two different genotypes of this pathogen existed, we examined over 50 strains of V. vulnificus and determined that 90% of isolates with the “C” (clinical) genomic pattern had come from human clinical cases, while 93% of those possessing the “E” (environmental) pattern had come from oysters or water (28). We subsequently designed a multiplex PCR protocol that simultaneously identifies an isolate as V. vulnificus (through analysis of the vvhA gene, which is unique to this bacterium) and reveals whether it possesses the C or E genomic pattern (33). Compared to conventional phenotypic analyses, this PCR method is highly accurate and requires only 3 h from colony selection to species confirmation and genotype determination, which is much faster than other typing methods (34). Subsequent studies using extensive phenotypic analysis (35), multilocus sequence typing (34, 36), pulsed field genomic analysis (34, 37), and ultimately, whole genome sequencing (38), confirmed the existence of the two biotype 1 genotypes, and the highly significant correlation of the C-genotype with human disease-causing ability. That two distinct genotypes are present in this species was confirmed by the genomic studies of Gulig et al. (39) and when we recently sequenced three E-genotype strains and compared them to three previously published C-genotype strains (38). We found numerous genes to be unique to the E-genotype, likely allowing their enhanced survival in the environment, as well as in the C-genotype strains, undoubtedly in some cases responsible for the increased human virulence of this genotype. While strains of the C-genotype are better able to resist serum killing (40, 41), why this genotype is better able to cause disease is only beginning to be understood.
This striking and consistently observed difference in genome structure in multiple V. vulnificus strains led us to propose that V. vulnificus exists as two distinct ecotypes (42). How such diversity in a single species evolved is a fascinating question but may well have involved horizontal gene transfer that is ubiquitous among vibrios, including V. vulnificus (43, 44).

VIRULENCE FACTORS

The one absolute requirement for virulence that is known is possession of an antiphagocytic capsule (45, 46, 47). Several capsule types exist (48, 49), and these are capable of undergoing phase variation/conversion (50, 51, 52). Interestingly, phase variation occurs at a very high rate (50), and on occasion, cells spontaneously mutate. In both instances capsule loss, either temporary or permanent, results. Whether the cells produce a capsule or not is easily determined, as encapsulated cells produce an opaque colony, while those lacking capsular polysaccharide appear translucent. Such a colony morphology change is easily seen on routine media (Fig. 2). Only the opaque (encapsulated) strains are virulent (45, 46) but capsule serotype does not appear to correlate with virulence (53).
FIGURE 2
FIGURE 2 Opaque (encapsulated) and translucent (nonencapsulated) colonies of Vibrio vulnificus on heart infusion agar. Reprinted from reference 10.
Our studies have indicated that the factor that is the likely cause of human fatality is the lipopolysaccharide (LPS) endotoxin (54). This is not unusual for a Gram-negative pathogen (50), and when the nitric oxide-inducing role of LPS was inhibited, we no longer observed death in laboratory animals (56).
In contrast to the significant roles played by capsule and LPS to pathogenesis, most of the numerous factors that have been proposed to be important to virulence have typically been found not to be essential for disease production, at least in animal models (1). An exception may be the toxin, which several studies have recently shown to be essential to virulence in mouse models (57, 58, 59, 60, 61).
Several excellent reviews exist on the known or suspected virulence factors possessed by V. vulnificus and the reader is referred to these (1, 62, 63, 64).

RISK FACTOR REQUIREMENTS AND ROLE OF IRON IN DISEASE

While possessing an overwhelming case fatality rate, infections caused by V. vulnificus are quite rare, averaging ca. 50 ingestion and 50 wound infections per year in the US (1, 19). This is likely due to the fact that V. vulnificus is an opportunistic pathogen, requiring one or more of several predisposing factors in order to initiate disease. Bross et al. (65) stated that 97% of patients have some chronic disease, including liver disease (80%), alcoholism (65%), diabetes (35%), malignancy (17%), or renal disease (7%). A major reason liver disease is such a prominent factor is the elevated serum iron that results from the chronic damage to this major reservoir of iron in the human body. Iron has been shown in numerous studies to be critical to the ability of V. vulnificus to survive and grow in the body, and the serum iron overload that results from chronic liver disease is central to its ability to cause fatal infections (40, 41, 66, 67, 68, 69). It must be noted, however, that the likely importance of such underlying syndromes has been described almost exclusively in primary septicemia (ingestion) cases. The situation might be quite different in wound infections (63).

ASSOCIATION OF V. VULNIFICUS WITH OYSTERS

V. vulnificus occurrence is associated with all estuarine organisms (15, 70, 71, 72), as well as particulates and plankton (73). It occurs in the highest levels in molluscan bivalves (including mussels and clams) (74) due to their filter-feeding of particles from seawater. However, oysters are of greatest importance in the transmission of this most fatal seafood-born pathogen in the US and considerable effort has been made to understand the interactions of V. vulnificus with the Eastern oyster, Crassostrea virginica; for a recent review, see Froelich and Oliver (75). We examined the uptake and depuration of both genotypes of V. vulnificus by adult oysters, but found no significance differences in these interactions (76). In a novel variation, we obtained aseptically removed eggs and sperm from female and male oysters and, following in vitro fertilization, examined the uptake of both V. vulnificus genotypes at the various larval stages as development progressed. Again, no significant differences in uptake were observed, at least through the veliger larval stage (K. Doyle, A.H. Ringwood, J.D. Oliver, unpublished data).
In such studies, researchers have invariably added V. vulnificus cells to tanks containing oysters, and examined uptake/depuration of these cells. In fact, oysters undertake a sophisticated size discrimination of particles they ingest, and particles of bacterial size are filtered out with extremely low efficiency (ca. 10%). We recently reported that if V. vulnificus cells are allowed to incorporate into marine aggregates (“snow”) and then added to oyster tank water, the efficiency of uptake is greatly increased, and C-/E-genotype differences are seen (77).
It is well known that great oyster-to-oyster variations exist in the levels of V. vulnificus (3), but the reasons for this are not as obvious. It may be that, as in all organisms, significant variation exists in the genetic makeup, and thus the physiological and innate immune response, of individual oysters. Indeed, we (78) have identified several genes that appear to correlate to V. vulnificus levels. Similarly, we have found V. vulnificus loads to correlate with oyster size, but interestingly, not with level of Perkinsus infection (79).
When we employed our multiplex PCR method to determine which genotype was present in oysters, we were surprised to find that ∼85% of the ∼900 V. vulnificus cells we isolated from 85 different oysters were of the (relatively avirulent) E-genotype (80). This finding is likely one more factor accounting for the relative rarity of these infections; in order to develop a V. vulnificus infection, a person presumably must consume a sufficient number of C-genotype cells, and these are a minority of those found in oysters.
The question of how many oysters must be consumed to put a person at risk of V. vulnificus infection can only be indirectly addressed. Using Food and Drug Administration data on the number of oysters eaten by people who developed V. vulnificus infection led us to conclude that a single oyster may contain enough V. vulnificus cells to cause disease, and to cause death. While we cannot know the number of V. vulnificus cells present in an oyster that is consumed, it is likely that there must be a sufficient number of C-genotype cells. It may be significant that, in our study examining the occurrence of C- and E-genotypes in oysters (80), we found that only two of the 85 oysters examined had more C than E-type cells, suggesting it may be sufficient to consume a “more dangerous” oyster to allow initiation of infection (1). Interestingly, Jackson et al. (81) provided evidence that, despite there being 103 V. vulnificus cells/g of oyster associated with infection, presumably of significant genetic variety, only a single V. vulnificus strain was subsequently isolated from human tissue. A recently developed real-time PCR method (77) capable of detecting and enumerating C-genotype cells in oysters may allow a more rapid and definitive method to help define the numbers of such cells needed for human infection.

GEOGRAPHIC DISTRIBUTION

V. vulnificus and V. vulnificus infections have been reported throughout Europe, Scandinavia, South America, the Far East, South Pacific, as well as on all coasts of the United States (15). Along with oysters and other molluscan bivalves and estuarine/coastal waters, V. vulnificus has been reported in fish (82, 83, 84), sediments (85, 86), and plankton (73). The presence of this pathogen appears to be spreading to areas where it was not previously reported, and this is likely due to global warming (see below).

IMPORTANCE OF SALINITY

A number of environmental parameters have been examined for their role in determining the ecology of V. vulnificus in estuarine waters, including dissolved oxygen, coliform levels, pH, turbidity, and dissolved organic carbon (see references 15, 70, 71, 87, 88). The two factors that are routinely reported to have the greatest significance, however, are salinity and temperature (88, 89, 90, 91, 92). These two parameters are discussed here.
Although its salt requirements are not high, V. vulnificus is an obligate halophilic bacterium, restricted to estuarine/brackish waters of moderate salinity. An ongoing study conducted in the Neuse River estuary of North Carolina indicates that the salinity limits for isolation of this pathogen are ca. 2‰ to 25‰, with an optimum of ca. 10‰ to 18‰ (C. Taylor and J.D. Oliver, unpublished data). Such findings are consistent with the numerous and multiyear studies we have conducted in estuarine waters (80, 87, 88). The organism is not isolated from open ocean waters, suggesting that the high salinities found in such waters (typically ca. 35‰) are not permissive to the growth of this organism. Indeed, during the period 2007 to 2009, a severe drought occurred in NC, resulting in an increase in the salinity of the estuarine waters we had sampled for many years to increase from a normal of ca. 15‰ to ≥22‰. The result was a nearly total loss of our ability to isolate V. vulnificus from these waters and from oysters taken from those waters (93) as might be predicted from Fig. 3. When the drought ended in 2010, the salinity returned to ca. 15‰, and we were again able to routinely isolate V. vulnificus from these waters and oysters (93). Such findings are consistent with studies suggesting “relaying” oysters from low to high salinity waters significantly reduces the V. vulnificus load (94, 95).
FIGURE 3
FIGURE 3 Distribution of Vibrio vulnificus cells in the Neuse River Estuary of North Carolina, as determined by salinity. Note different axes for E- and C-genotypes (C. Taylor and J.D. Oliver, unpublished data).
We have conducted several studies (96, 97) wherein cells of V. vulnificus were incubated in environmental chambers placed in natural estuarine waters of various salinities (11‰ to 31‰) in order to examine in situ gene expression of a variety of genes involved in stress responses and virulence, as they might be regulated by this critical parameter. Expression of rpoS, the “stress” sigma factor, was observed for up to 108 h at 11‰, suggesting that even lower salinities may present a stress to the cells. Another gene of interest was viuB, which is required for siderophore-mediated iron acquisition. As noted earlier, the ability of V. vulnificus to acquire iron is essential for growth of this pathogen in the human host, and the fact that expression of viuB was no longer observed by 12 h in both salinity environments may again help explain the relative rarity of V. vulnificus infections. If cells in the environment are unable to rapidly scavenge iron on entrance into the human host, they might be unable to cause human infection. However, whether cells within oysters respond in a similar manner to salinities ≥21‰ is not known. Such in situ studies have also identified other genes that are differentially expressed, and which respond to the natural estuarine environment in a quite different manner (Fig. 4).
FIGURE 4
FIGURE 4 Gene expression by Vibrio vulnificus cells in membrane diffusion chambers incubated in situ (estuarine waters of North Carolina coast). Shown are duration of expression (h) for vvhA (hemolysin), trkA (encodes a surface component of the constitutive K+-uptake system), viuB (siderophore), relA (ppGpp synthetase), and groEL (a chaperonin) (T.C. Williams and J.D. Oliver, unpublished data).

IMPORTANCE OF WATER TEMPERATURE

Water temperature is a parameter critical to the ecology of V. vulnificus and in the incidence of human infection (15, 90, 91, 92). As shown in Fig. 5, cases of V. vulnificus over a 12-year period demonstrate a distinct seasonality, with almost all cases (97%) occurring in the months of April through November when water temperatures in the Gulf of Mexico are at or above 20°C. Indeed, our field studies have regularly suggested that water temperatures of 20°C indicate the point where a human health concern resulting from elevated Vibrio levels may exist (88). In contrast, temperatures of 13°C or lower induce the viable but nonculturable state in this pathogen (see below).
FIGURE 5
FIGURE 5 Seasonal distribution of Vibrio vulnificus cases (n=380) reported by the Food and Drug Administration in the United States over the 12-year period, 2000 to 2011. Shown are numbers of cases reported by month. Also plotted is mean monthly surface water temperatures recorded at Dauphin Island, AL. Horizontal lines show 13°C, at which temperature V. vulnificus cells enter the viable but nonculturable state and few cases occur (eight each in February and March and two in December, out of the 380 total cases), and 20°C, which represents the critical temperature at which most V. vulnificus cases occur (J.D. Oliver, unpublished data).
The two V. vulnificus genotypes appear to respond in a similar manner to seasonal temperature variations (Fig. 6), although again the level of the E-genotype is consistently 5- to 6-fold greater than the C-genotype, both in estuarine water and oysters. It is evident that V. vulnificus prefers warmer temperatures (>20°C) but appears to be adversely affected by temperatures above 30°C.
FIGURE 6
FIGURE 6 Distribution of Vibrio vulnificus cells in the Neuse River Estuary of North Carolina, as determined by temperature. Note different axes for E- and C-genotypes (C. Taylor and J.D. Oliver, unpublished data).

TEMPERATURE AND THE VIABLE BUT NONCULTURABLE STATE

The lower line in Figure 5 temp indicates 13°C, which is the temperature that our in situ studies indicate is the lower limit of growth for V. vulnificus. In fact, below this point, cells of V. vulnificus become dormant, undertaking a physiological response known as the viable but nonculturable (VBNC) state. VBNC bacteria are organisms that fail to grow and develop colonies on the media they are normally cultured on, but their metabolic activity capabilities indicate that they are still alive (98). Several reviews of this dormancy state have been published (99, 100, 101, 102, 103, 104, 105, 106, 107, 108), and the list of bacteria known to enter this state is constantly growing. We have also employed diffusion chambers to demonstrate that this phenomenon occurs not only in the lab, but also in situ in estuarine waters (105). We have also found cells of V. vulnificus, when present in the VBNC state, to be more resistant to a variety of potentially lethal environmental factors than are the culturable cells of this pathogen (109).

INCREASING INCIDENCE DUE TO GLOBAL WARMING

Recent dramatic rises in seawater temperature may well lead to significant increases in the geographic distribution of estuarine Vibrio spp., and thus, a likely increase in the incidence of infection (110, 111, 112, 113). Increases in Vibrio infections have already been noted in northwest Spain and the Baltic Sea (114), as well as in Israel (115) and New Caledonia (116), with correlations made to global warming. In the United States, while food-borne Vibrio infections are increasing, this does not appear to be due to global climate change. However, wound infections caused by V. vulnificus are increasing worldwide, including in the US (19), and in these cases, global warming and the spread of vibrios may be a factor. It also cannot be excluded that strains of V. vulnificus, as well as other pathogens in this genus (e.g., V. parahaemolyticus), may be exhibiting increased virulence (117, 118). Significantly more study is needed to elucidate the factors causing this rise.

CONCLUSIONS

V. vulnificus is an exceptional bacterial pathogen, carrying the highest case-fatality rate of any food-borne pathogen, and causing extremely rapid infections in those consuming raw oysters. Of interest is its gender specificity, its predilection for those over the age of 40 years, and the need for underlying disease in its victims; liver cirrhosis is an especially dangerous predisposing factor. Added to this is its ability to cause potentially fatal wound infections. While the virulence factors essential to the disease are little understood, we now realize there are more than one biotype and genotype, and this should prove of value in deciphering its ecology. Temperature plays a critical role in its occurrence and distribution, including in its entrance into the viable but nonculturable state. Its incidence, both in the US and worldwide, is increasing and global warming is undoubtedly a factor in its worldwide spread. Along with temperature, salinity is a determining factor in its ecology, and extreme weather events, such as droughts, have proven to at least temporarily eliminate this pathogen from regions stricken with high salinities. Recent genome sequencing is likely to help us understand both its pathogenesis and ecology and the near future promises to be a most exciting time in this study of this fascinating bacterium.

ACKNOWLEDGMENT

I declare no conflicts of interest with regard to the manuscript.

REFERENCES

1.
Jones MK, Oliver JD. 2009. Vibrio vulnificus: disease and pathogenesis. Infect Immun 77:1723–1733.
2.
Mead PS, Slutsker L, Dietz V, McCaig LF, Bress JS, Shapiro C, Griffin PM, Tauxe RV. 1999. Food-related illness and death in the United States. Emerg Infect Dis 5:607–625.
3.
Motes ML, DePaola A, Cook DW, Veazey JE, Hunsucker JC, Garthright WE, Blodgett RJ, Chirtel SJ. 1998. Influence of water temperature and salinity on Vibrio vulnificus in Northern Gulf and Atlantic Coast oysters (Crassostrea virginica). Appl Environ Microbiol 64:1459–1465.
4.
DePaola A, Nordstrom JL, Dalsgaard A, Forslund A, Oliver J, Bates T, Bordage KL, Gulig PA. 2003. Analysis of Vibrio vulnificus from market oysters and septicemia cases for virulence markers. Appl Environ Microbiol 69:4006–4011.
5.
Birkenhauer JB, Oliver JD. 2003. Use of diacetyl to reduce the load of Vibrio vulnificus in the Eastern oyster, Crassostrea virginica. J Food Protect 66:38–43.
6.
Food and Drug Administration. 2012. Vibrio vulnificus. In Bad Bug Book. http://www.fda.gov/Food/FoodborneIllnessContaminants/CausesOfIllnessBadBugBook/ucm070473.htm.
7.
Ito H, Khibayama A, Abe M, Antoku S, Nawata H, Isonishi M, Fujita M, and Kato S. 2012. Vibrio vulnificus septicemia and necrotizing fasciitis in the patients with liver cirrhosis and diabetes mellitus. J Diab Mell 2:122–125.
8.
University of Notre Dame. 2003. Physics in Medicine. http://isnap.nd.edu/Lectures/mphysics/index.htm.
9.
Scallan E, Hoekstra RM, Angulo FJ, Tauxe RV, Widdowson M-A, Roy SL, Jones JL, Griffin P. 2011. Foodborne illness acquired in the United States – major pathogens. Emerg Infect Dis 17:7–15.
10.
Oliver JD, Pruzzo C, Vezzuli L, Kaper JB. 2013. Vibrio species, p 401–439. In Doyle MP, Buchanan RL (eds), Food Microbiology: Fundamentals and Frontiers, 4th Ed. The American Society for Microbiology Press, Washington, DC.
11.
Merkel SM, Alexander S, Oliver JD, Huet-Hudson YM. 2001. Essential role for estrogen in protection against Vibrio vulnificus induced endotoxic shock. Infect Immun 69:6119–6122.
12.
Posadas BC, Posadas RA. 2011. Consumer preferences for postharvest-processed raw oyster products in coastal Mississippi. Mississippi Agricultural and Forestry Experiment Station Bulletin 1192. http://msucares.com/pubs/bulletins/b1192.pdf.
13.
Oliver JD. 2005. Wound infections caused by Vibrio vulnificus and other marine bacteria. Epidemiol Infect 133:383–391.
14.
Oliver JD. 2006. Vibrio vulnificus, p 349–366. In Thompson FL, Austin B, Swing J (eds), Biology of Vibrios. The American Society for Microbiology Press, Washington, DC.
15.
Oliver JD. 2006. Vibrio vulnificus, p 253–276. In Belkin S, Colwell RR (ed), Oceans and Health: Pathogens in the Marine Environment. Springer Science, New York, NY.
16.
Inoue Y, Ono T, Matsui T, Miyasaka J, Kinoshita Y, Ihn H. 2008. Epidemiological survey of Vibrio vulnificus in Japan between 1999 and 2003. J Dermatol 35:129–130.
17.
Dechet AM, Yu PA, Koram N, Painter J. 2008. Nonfoodborne Vibrio infections: an important cause of morbidity and mortality in the United States, 1997–2006. Clin Infect Dis 46:970–976.
18.
Weis KE, Hammond RM, Hutchinson R, Blackmore CGM. 2011. Vibrio illness in Florida, 1998–2007. Epidemiol Infect 139:591–598.
19.
Pleis J, Lethbridge-Cejku M. 2007. Summary health statistics for U.S. adults: National Health Interview Survey, 2006. Vital Health Stat. ser. 10 (235). http://www.cdc.gov/nchs/data/series/sr_10/sr10_235.pdf.
20.
Amaro C, Biosca EG, Fouz B, Alcaide E, Esteve C. 1995. Evidence that water transmits Vibrio vulnificus biotype 2 infections to eels. Appl Environ Microbiol 61:1133–1137.
21.
Amaro C, Biosca EG. 1996. Vibrio vulnificus biotype 2, pathogenic for eels, is also an opportunistic pathogen for humans. Appl Environ Microbiol 62:1454–1457.
22.
Biosca EG, Oliver JD, Amaro C. 1996. Phenotypic characterization of Vibrio vulnificus biotype 2, a lipopolysaccharide-based homogeneous O serogroup within Vibrio vulnificus. Appl Environ Microbiol 62:918–927.
23.
Amaro C, Hor LI, Marco-Noales E, Bosque T, Fouz B, Alcaide E. 1999. Isolation of Vibrio vulnificus serovar E from aquatic habitats in Taiwan. Appl Environ Microbiol 65:1352–1355.
24.
Fouz B, Larsen JL, Amaro C. 2006. Vibrio vulnificus serovar A: an emerging pathogen in European anguilliculture. J Fish Dis 29:285–291.
25.
Bisharat N, Cohen DI, Harding RM, Falush D, Crook DW, Peto T, Maiden MC. 2005. Hybrid Vibrio vulnificus. Emerg Infect Dis 11:30–35.
26.
Bisharat N, Agmon V, Finkelstein R, Raz R, Ben-Dror G, Lerner L, Soboh S, Colodner R, Cameron DN, Wykstra DL, Swerdlow DL, Farmer JJ 3rd. 1999. Clinical, epidemiological, and microbiological features of Vibrio vulnificus biogroup 3 causing outbreaks of wound infection and bacteraemia in Israel. Israel Vibrio Study Group. Lancet 354:1421–1424.
27.
Warner JM, Oliver JD. 1999. Randomly amplified polymorphic DNA (RAPD) analysis of clinical and environmental strains of Vibrio vulnificus and other Vibrio species. Appl Environ Microbiol 65:1141–1144.
28.
Rosche TM, Yano Y, Oliver JD. 2005. A rapid and simple PCR analysis indicates there are two subgroups of Vibrio vulnificus which correlate with clinical or environmental isolation. Microbiol Immunol 49:381–389.
29.
Aznar R, Ludwig W, Amann RI, Schleifer KH. 1994. Sequence determination of rRNA genes of pathogenic Vibrio species and whole-cell identification of Vibrio vulnificus with rRNA-targeted oligonucleotide probes. Int J Syst Bacteriol 44:330–337.
30.
Nilsson WB, Paranjpye RN, DePaola A, Strom MS. 2003. Sequence polymorphism of the 16S rRNA gene of Vibrio vulnificus is a possible indicator of strain virulence. J Clin Microbiol 41:442–446.
31.
Gutacker M, Conza N, Benagli C, Pedrollil A, Bernasconi MV, Permin L, Aznar R, Piffaretti JC. 2003. Population genetics of Vibrio vulnificus: identification of two divisions and a distinct eel-pathogenic clone. Appl Environ Microbiol 69:3203–3212.
32.
Chatzidaki-Livanis M, Hubbard MA, Gordon K, Harwood VJ, Wright AC. 2006. Genetic distinctions among clinical and environmental strains of Vibrio vulnificus. Appl Environ Microbiol 72:6136–6141.
33.
Warner EB, Oliver JD. 2008. Multiplex PCR assay for detection and simultaneous differentiation of genotypes of Vibrio vulnificus biotype 1. Foodborne Path Dis 5:691–693.
34.
Gonzalez-Escalona N, Whitney B, Jaykus LA, DePaola A. 2007. Comparison of direct genome restriction enzyme analysis and pulsed-field gel electrophoresis for typing Vibrio vulnificus and their correspondence with multilocus sequence typing data. Appl Environ Microbiol 73:7494–7500.
35.
Sanjuan E, Fouz B, Oliver JD, Amaro C. 2009. Evaluation of genotypic and phenotypic methods to distinguish clinical from environmental Vibrio vulnificus strains. Appl Environ Microbiol 75:1604–1613.
36.
Cohen ALV, Oliver JD, DePaola A, Feil EJ, Boyd EF. 2007. Emergence of a virulent clade of Vibrio vulnificus and correlation with the presence of a 33-kilobase genomic island. Appl Environ Microbiol 73:5553–5565.
37.
Wong HC, Chen SY, Chen MY, Oliver JD, Hor LI, Tsai WC. Pulsed-field gel electrophoresis analysis of Vibrio vulnificus strains isolated from Taiwan and the United States. 2004. Appl Environ Microbiol 70:5153–5158.
38.
Morrison SS, Cain A, Froelich B, Williams T, Taylor C, Baker-Austin C, Verner-Jeffreys D, Hartnell R, Oliver JD, Gibas CJ. 2012. Pyrosequencing-based comparative genome analysis of Vibrio vulnificus environmental isolates. PLoS ONE 7:e37553.
39.
Gulig PA, de Crécy-Lagard V, Wright AC, Walts B, Telonis-Scott M, McIntyre LM. 2010. SOLiD sequencing of four Vibrio vulnificus genomes enables comparative genomic analysis and identification of candidate clade-specific virulence genes. BMC Genomics 11:512.
40.
Bogard R, Oliver JD. 2007. Role of iron in human serum resistance of the clinical and environmental Vibrio vulnificus genotypes. Appl Environ Microbiol 73:7501–7505.
41.
Kim HY, Ayrapetyan M, Oliver JD. 2014. Survival of Vibrio vulnificus genotypes in male and female serum, and production of siderophores in human serum and seawater. 2014. Foodborne Path Dis 11:119–125.
42.
Rosche TM, Binder EA, Oliver JD. 2010. Vibrio vulnificus genome suggests two distinct ecotypes. Environ Microbiol Rep 2:128–132.
43.
Urbanczyk H, Ast JC, Kaeding AJ, Oliver JD, Dunlap PV. 2008. Horizontal transfer of lux genes in Vibrionaceae. J Bacteriol 190:3494–3504.
44.
Gulig PA, Tucker MS, Thiaville PC, Joseph JL, Brown RN. 2009. USER friendly cloning coupled with chitin-based natural transformation enables rapid mutagenesis of Vibrio vulnificus. Appl Environ Microbiol 75:4936–4949.
45.
Yoshida S, Ogawa M, Mizuguchi Y. 1985. Relation of capsular materials and colony opacity to virulence of Vibrio vulnificus. Infect Immun 47:446–451.
46.
Simpson LM, White VK, Zane SF, Oliver JD. 1987. Correlation between virulence and colony morphology in Vibrio vulnificus. Infect Immun 55:269–272.
47.
Wright AC, Simpson LM, Oliver JD, Morris JG, Jr. 1990. Phenotypic evaluation of acapsular transposon mutants of Vibrio vulnificus. Infect Immun 58:1769–1773.
48.
Simonson JG, Siebeling RJ. 1993. Immunogenicity of Vibrio vulnificus capsular polysaccharides and polysaccharide-protein conjugates. Infect Immun 61:2053–2058.
49.
Grau BL, Henk MC, Pettis GS. 2005. High-frequency phase variation of Vibrio vulnificus 1003: isolation and characterization of a rugose phenotypic variant. J Bacteriol 187:2519–25.
50.
Hilton T, Rosche T, Froelich B, Smith B, Oliver JD. 2006. Capsular polysaccharide phase variation in Vibrio vulnificus. Appl Environ Microbiol 72:6986–6993.
51.
Rosche TM, Smith B, Oliver JD. 2006. Evidence for an intermediate colony morphology of Vibrio vulnificus. Appl Environ Microbiol 72:4356–4359.
52.
Neiman J, Guo Y, Rowe-Magnus DA. 2011. Chitin-induced carbotype conversion in Vibrio vulnificus. Infect Immun 79:3195–203.
53.
Linkous DA, Simpson LM, Oliver JD. 1997. Comparison of pathogenicity among Vibrio vulnificus strains based on capsular and LPS serotypes. Abstracts of the General Meeting of the American Society For Microbiology 97:65.
54.
McPherson VM, Watts JA, Simpson LM, Oliver JD. 1991. Physiological effects of the lipopolysaccharide of Vibrio vulnificus on mice and rats. Microbios 67:141–149.
55.
Toda K. 2012. Bacterial endotoxin. In Todar’s Online Textbook of Bacteriology. http://textbookofbacteriology.net/endotoxin.html.
56.
Elmore SP, Watts JA, Simpson LM, Oliver JD. 1992. Reversal of hypotension induced by Vibrio vulnificus lipopolysaccharide in the rat by inhibition of nitric oxide synthase. Micro Pathogen 13:391–397.
57.
Lee JH, Kim MW, Kim BS, Kim SM, Lee BC, Kim TS, Choi SH. 2007. Identification and characterization of the Vibrio vulnificus rtxA essential for cytotoxicity in vitro and virulence in mice. J Microbiol 45:146–152.
58.
Kim YR, Lee SE, Kook H, Yeom JA, Na HS, Kim SY, Chung SS, Choy HE, Rhee JH. 2008. Vibrio vulnificus RTX toxin kills host cells only after contact of the bacteria with host cells. Cell Microbiol 10:848–862.
59.
Kwak JS, Jeong HG, Satchell KJ. 2011. Vibrio vulnificus rtxA gene recombination generates toxin variants with altered potency during intestinal infection. PNAS 108:1645–1650.
60.
Jeong HG, Satchell KJ. 2012. Additive function of Vibrio vulnificus MARTX Vv and VvhA cytolysins promotes rapid growth and epithelial tissue necrosis during intestinal infection. PLoS Pathog 8:e1002581.
61.
Lee CT, Pajuelo D, Llorens A, Chen YH, Keiro JM, Padrós F, Hor LI, Amaro C. 2013. MARTX of Vibrio vulnificus biotype 2 is a virulence and survival factor. Environ Microbiol 15:419–432.
62.
Strom MS, Paranjpye RN. 2000. Epidemiology and pathogenesis of Vibrio vulnificus. Microbes Infect 2:177–188.
63.
Gulig PA, Bourdage KL, Starks AM. 2005. Molecular pathogenesis of Vibrio vulnificus. J Microbiol 43:118–131.
64.
Oliver JD. 2013. Vibrio vulnificus: death on the half shell. A personal journey with the pathogen and its ecology. Microb Ecol 65:793–799.
65.
Bross MH, Soch K, Morales R, Mitchell RB. 2007. Vibrio vulnificus infections: diagnosis and treatment. Amer Fam Phys 76:539–544.
66.
Wright AC, Simpson LM, Oliver JD. 1981. Role of iron in the pathogenesis of Vibrio vulnificus infections. Infect Immun 34:503–507.
67.
Morris JG Jr, Wright AC, Simpson LM, Wood PK, Johnson DE, Oliver JD. 1987. Virulence of Vibrio vulnificus: association with utilization of transferrin-bound iron, and lack of correlation with levels of cytotoxin or protease production. FEMS Microbiol Lett 40:55–59.
68.
Zakaria-Meehan Z, Massad G, Simpson LM, Travis JC, Oliver JD. 1988. Ability of Vibrio vulnificus to obtain iron from hemoglobin-haptoglobin complexes. Infect Immun 56:275–277.
69.
Brennt CE, Wright AC, Dutta SK, Morris JG Jr. 1991. Growth of Vibrio vulnificus in serum from alcoholics: association with high transferrin iron saturation. J Infect Dis 164:1030–1032.
70.
Oliver JD, Warner RA, Cleland DR. 1982. Distribution and ecology of Vibrio vulnificus and other lactose-fermenting marine vibrios in coastal waters of the southeastern United States. Appl Environ Microbiol 44:1404–1414.
71.
Oliver JD, Warner RA, Cleland DR. 1983. Distribution of Vibrio vulnificus and other lactose-fermenting vibrios in the marine environment.Appl Environ Microbiol 45:985–998.
72.
Tao Z, Bulard S, Arias C. 2011. High numbers of Vibrio vulnificus in tar balls collected from oiled areas of the North-Central Gulf of Mexico following the 2010 BP Deepwater Horizon oil spill. EcoHealth 8:507–511.
73.
Heidelberg JF, Heidelberg KB, Colwell RR. 2002. Bacteria of the ϒ-subclass Proteobacteria associated with zooplankton in Chesapeake Bay. Appl Environ Microbiol 68:5498–5507.
74.
Beneduce L, Vernile A, Spano G, Massa S, Lamacchia F, Oliver JD. 2010. Occurrence of Vibrio vulnificus in mussel farms from the Varano Lagoon environment. Lett Appl Microbiol 51:443–449.
75.
Froelich B, Oliver JD. 2013. The interactions of Vibrio vulnificus and the oyster Crassostrea virginica. Microb Ecol 65:807–816.
76.
Froelich B, Ringwood A, Sokolova I, Oliver JD. 2010. Uptake and depuration of the C- and E-genotype of Vibrio vulnificus by the Eastern oyster (Crassostrea virginica). Environ Microbiol Rep 2:112–115.
77.
Froelich B, Ayrapetyan M, Oliver JD. 2013. Integration of Vibrio vulnificus into marine aggregates and subsequent uptake by Crassostrea virginica oyster. Appl Environ Microbiol 79:1454–1458.
78.
Sokolova IM, Oliver JD, Leamy LJ. 2006. An AFLP. approach to identify genetic markers associated with resistance to Vibrio vulnificus and Perkinsus marinus in eastern oysters. J Shellfish Res 25:95–100.
79.
Sokolova IM, Leamy L, Harrison M, Oliver JD. 2005. Intrapopulational variation in Vibrio vulnificus levels in Crassostrea virginica is associated with the host size but not with disease status or developmental stability. J Shellfish Res 24:503–508.
80.
Warner EB, Oliver JD. 2008. Population structure of two genotypes of Vibrio vulnificus in oysters (Crassostrea virginica) and sea water. Appl Environ Microbiol 74:80–85.
81.
Jackson JK, Murphree RL, Tamplin ML. 1997. Evidence that mortality from Vibrio vulnificus infection results from single strains among heterogeneous populations in shellfish. J Clin Microbiol 35:2098–2101.
82.
Baker-Austin C, Gore A, Oliver JD, Rangdale R, McArthur JV, Lees DN. 2010. Rapid in situ detection of virulent Vibrio vulnificus strains in raw oyster matrices using real-time PCR. Env Microbiol Rep 2:76–80.
83.
DePaola A, Capers GM, Alexander D. 1994. Densities of Vibrio vulnificus in the intestines of fish from the U.S. Gulf Coast. Appl Environ Microbiol 60:984–988.
84.
Tao Z, Larsen AM, Bullard SA, Wright AC, Arias CR. 2012. Prevalence and population structure of Vibrio vulnificus on fishes from the Northern Gulf of Mexico. Appl Environ Microbiol 78:7611–7618.
85.
Vanoy RW, Tamplin JL, Schwarz JR. 1992. Ecology of Vibrio vulnificus in Galveston Bay oysters, suspended particulate matter, sediment and seawater: detection by monoclonal antibody-immunoassay-most probable number procedures. J Ind Microbiol 9:219–223.
86.
Aono E, Sugita HI, Kawasaki J, Sakakibara H, Takahashi T, Endo K, Deguchi Y. 1997. Evaluation of polymerase chain reaction method for identification of Vibrio vulnificus isolated from marine environments. J Food Prot 60:81–83.
87.
Pfeffer CS, Hite MF, Oliver JD. 2003. Ecology of Vibrio vulnificus in estuarine waters of eastern North Carolina. Appl Environ Microbiol 69:3526–3531.
88.
Blackwell KD, Oliver JD. 2008. The ecology of Vibrio vulnificus, Vibrio cholerae, and Vibrio parahaemolyticus in North Carolina estuaries. J Microbiol 46:146–153.
89.
Banakar V, Constantin de Magny G, Jacobs J, Murtugudde R, Huq A, Wood RJ, Colwell RR. 2011. Temporal and spatial variability in the distribution of Vibrio vulnificus in the Chesapeake Bay: a hindcast study. Ecohealth 8:456–467.
90.
Johnson CN, Bowers JC, Griffitt KJ, Molina V, Clostio RW, Pei S, Laws E, Paranjpye RN, Strom MS, Chen A, Hasan NA, Huq A, Noriea NF 3rd, Grimes DJ, Colwell RR. 2012. Ecology of Vibrio parahaemolyticus and Vibrio vulnificus in the coastal and estuarine waters of Louisiana, Maryland, Mississippi, and Washington (United States). Appl Environ Microbiol 78:7249–7257.
91.
Johnson CN. 2013. Fitness factors in vibrios: a mini-review. Microb Ecol 65:826–851.
92.
Randa MA, Polz MF, Lim E. 2004. Effects of temperature and salinity on Vibrio vulnificus population dynamics as assessed by quantitative PCR. Appl Environ Microbiol 70:5469–5476.
93.
Froelich B, Williams T, Nobel R, Oliver JD. 2012. Apparent loss of Vibrio vulnificus from North Carolina oysters coincides with a drought-induced increase in salinity. Appl Environ Microbiol 78:3885–3889.
94.
Motes ML, DePaol A. 1996. Offshore suspension relaying to reduce levels of Vibrio vulnificus in oysters (Crassostrea virginica). Appl Environ Microbiol 62:3875–3877.
95.
Audemard C, Kator HI, Rhodes MW, Gallivan T, Erskine AJ, Leggett AT, Reece KS. 2011. High salinity relay as a postharvest processing strategy to reduce Vibrio vulnificus levels in Chesapeake Bay oysters (Crassostrea virginica). J Food Prot 74:1902–1907.
96.
Smith BE, Oliver JD. 2006. In situ gene expression by Vibrio vulnificus. Appl Environ Microbiol 72:2244–2246.
97.
Jones MK, Warner E, Oliver JD. 2008. Survival and in situ gene expression of Vibrio vulnificus at varying salinities in estuarine environments. Appl Environ Microbiol 74:182–187.
98.
Oliver JD. 2005. The viable but nonculturable state in bacteria. J Microbiol 43:93–100.
99.
Colwell RR, Brayton PR, Grimes DJ, Roszak DB, Huq SA, Palmer LM. 1985. Viable but not culturable Vibrio cholerae and related pathogens in the environment. Biol Technol 3:817–820.
100.
Oliver JD, Hite F, McDougald D, Andon NL, Simpson LM. 1995. Entry into, and resuscitation from, the viable but nonculturable state by Vibrio vulnificus in an estuarine environment. Appl Environ Microbiol 61:2624–2630.
101.
Oliver JD. 2000. Public health significance of viable but nonculturable bacteria, p 277–300. In Colwell RR, Grimes DJ (ed), Non-Culturable Microorganisms in the Environment. The American Society for Microbiology Press, Washington, DC.
102.
Colwell RR. 2000. Viable but nonculturable bacteria: a survival strategy. J Infect Chemother 6:121–125.
103.
Oliver JD. 2005. Viable but nonculturable bacteria in food environments, p 99–112. In Fratamico PM, Bhunia AK, Smith JL (ed), Food-borne Pathogens: Microbiology and Molecular Biology. Caister Academic Press, Norfolk, UK.
104.
Karunasagar I, Karunasagar I. 2005. Retention of pathogenicity in viable nonculturable pathogens p 361–371. In Belkin S, Colwell RR (ed), Oceans and Health: Pathogens in the Marine Environment. Springer, New York, NY.
105.
Smith BE, Oliver JD. 2006. In situ and in vitro gene expression by Vibrio vulnificus during entry into, persistence within, and resuscitation from the viable but nonculturable state. Appl Environ Microbiol 72:1445–1451.
107.
Oliver JD. 2009. Recent findings on the viable but nonculturable state in pathogenic bacteria. FEMS Microbiology Rev 34:415–425.
108.
Trevors JT. 2011. Viable but non-culturable (VBNC) bacteria: gene expression in planktonic and biofilm cells. J Microbiol Meth 86:266–73.
109.
Nowakowska J, Oliver JD. 2013. Resistance to environmental stresses by Vibrio vulnificus in the viable but nonculturable state. FEMS Microbiol Ecol 84:213–222.
110.
Halpern BS, Walbridge S, Selkoe KA, Kappel CV, Micheli F, D’Agrosa C, Bruno JF, Casey KS, Ebert C, Fox HE, Fujita R, Heinemann D, Lenihan HS, Madin EM, Perry MT, Selig ER, Spalding M, Steneck R, Watson R. 2008. A global map of human impact on marine ecosystems. Science 319:948–952.
111.
Martinez-Urtaza J, Bowers JC, Trinanes J, DePaola A. 2010. Climate anomalies and the increasing risk of Vibrio parahaemolyticus and Vibrio illnesses. Food Res Intern 43:1780–1790.
112.
Newton A, Kendall M, Vugia DJ, Henao OL, Mahon BE. 2012. Increasing rates of vibriosis in the United States, 1996–2010: review of surveillance data from 2 systems. Clin Infect Dis 54:S391–S395.
113.
Lindgren E, Andersson Y, Suk JE, Sudre B, Semenza JC. 2012. Monitoring EU emerging infectious disease risk due to climate change. Science 336:418–419.
114.
Baker-Austin C, Trinanes JA, Taylor NGH, Hartnell R, Siitonen A, Martinez-Urtaza J. 2012. Emerging Vibrio risk at high latitudes in response to ocean warming. Nat Clim Change 3:73–77.
115.
Paz S, Bisharat N, Paz E, Kidar O, Cohen D. 2007. Climate change and the emergence of Vibrio vulnificus disease in Israel. Environ Res 103:390–396.
116.
Cazorla C, Guigon A, Noel M, Quilici ML, Lacassin F. 2011. Fatal Vibrio vulnificus infection associated with eating raw oysters, New Caledonia. Emerg Infect Dis 17:136–137.
117.
Daniels NA, Ray B, Easton A, Marano N, Kahn E, McShan AL, Del Rosario L, Baldwin T, Kingsley MA, Puhr ND, Wells JG, Angulo FJ. 2000. Emergence of a new Vibrio parahaemolyticus serotype in raw oysters. A prevention quandary. J Amer Med Assoc 284:1541–1545.
118.
Boyd EF, Cohen ALV, Naughton LM, Ussery DW, Binnewies TT, Stine OC, Parent MA. 2008. Molecular analysis of the emergence of pandemic Vibrio parahaemolyticus. BMC Microbiol 8:110–123.

Information & Contributors

Information

Published In

cover image Microbiology Spectrum
Microbiology Spectrum
Volume 3Number 318 June 2015
eLocator: 3.3.01
Editor: Michael Sadowsky, University of Minnesota, St. Paul, MN

History

Received: 24 September 2014
Returned for modification: 24 February 2015
Published online: 29 May 2015

Permissions

Request permissions for this article.

Contributors

Author

James D. Oliver
Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC 28223

Editor

Michael Sadowsky
University of Minnesota, St. Paul, MN

Notes

Correspondence: James D. Oliver, [email protected]

Metrics & Citations

Metrics

Note: There is a 3- to 4-day delay in article usage, so article usage will not appear immediately after publication.

Citation counts come from the Crossref Cited by service.

Citations

If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download.

View Options

View options

PDF/ePub

PDF/ePub

Get Access

Buy Article
Microbiology Spectrum Vol.3 • Issue 3 • ASM Journals Pay Per View, PPV 25
Journal Subscription
Microbiology Spectrum
ASM members can purchase subscriptions to journals.
Join or renew

Figures and Media

Figures

Media

Tables

Share

Share

Share the article link

Share with email

Email a colleague

Share on social media

American Society for Microbiology ("ASM") is committed to maintaining your confidence and trust with respect to the information we collect from you on websites owned and operated by ASM ("ASM Web Sites") and other sources. This Privacy Policy sets forth the information we collect about you, how we use this information and the choices you have about how we use such information.
FIND OUT MORE about the privacy policy