Histopathology of Marine Vibrio Wound Infections

Histopathology of Marine Vibrio Wound Infections EDWIN N. BECKMAN, M.D., GEORGE L. LEONARD, M.D., LUIS E. CASTILLO, M.D., CHARLES F. GENRE, M.D., AND GEORGE A. PANKEY, M.D.

HALOPHILIC VIBRIOS, found in seawater and other marine sources, fall into five principal groups: V. parahaemolyticus, V. alginolyticus, V. vulnificus, V. metschnikovii (CDC enteric group 16), and an unnamed group designated group F (CDC EF-6). 12 V. parahaemolyticus, recognized as a human pathogen less than three decades ago, is a well known cause of gastroenteritis. However, it is an unusual cause of soft tissue infection and bacteremia. V. alginolyticus is a rare cause of marine wound infections, otitis, and sepsis. It has not been implicated in outbreaks of gastroenteritis. 212 V. metschnikovii, an oxidase-negative organism which has only recently been classified, is not associated with human disease. The EF-6 group has been isolated from numerous cases of diarrhea, but its etiologic role in these illnesses has not been confirmed.712 V. vulnificus, the previously unnamed "lactose-positive marine vibrio," has recently been recognized as a human pathogen.1,6 The species resembles V. parahaemolyticus and V. alginolyticus but differs in its ability to ferment lactose and production of beta D-galactosidase. It is further differentiated from V. parahaemolyticus by a lower NaCl tolerance and from V. alginolyticus by failure to ferment sucrose and lack of acetyl methyl carbinol production (Voges-Proskauer reaction). 612 The lactose fermentation may be weak or delayed for three Received February 20, 1981; received revised manuscript and accepted for publication March 26, 1981. Address reprint requests to Dr. Beckman: Ochsner Medical Institutions, 1516 Jefferson Highway, New Orleans, Louisiana 70121.

Departments of Pathology and Internal Medicine, Section on Infectious Diseases, Ochsner Medical Institutions, New Orleans, Louisiana

to seven days in the lactose-positive vibrios, and some of the infections previously reported as lactose negative are now included in the lactose positive group. 6 ' 0 1 3 The clinical features of lactose-positive marine vibrio infections have been detailed by Blake.1 The two distinct clinical syndromes are infection of skin and soft tissue and bacteremia presumably originating in the gastrointestinal tract. Though marine vibrio infections have only been recognized since 1970,10 reports of them are now appearing with increasing frequency. Description of the histologic changes of these marine vibrio wound infections have been rare and limited, however. This presentation is of the histopathology in three patients whose clinical aspects have been previously reported2,5 (Table 1). Case Reports Case 1 A 60-year-old man was referred to the Ochsner Medical Institutions for chills, fever, vomiting, and myalgia. Four days previously, after wading off the Mississippi Gulf Coast, he had received multiple fire ant stings on the legs. He was treated with antibiotics at an outside hospital for an infected right leg. When seen at our institution, he was hypotensive and dehydrated. The right leg was edematous with hemorrhagic bullae. Peripheral pulses were decreased. Despite administration of antibiotics, fasciotomy, and ultimately amputation of the leg, he died 45 hours after admission. A gram-negative rod was isolated from the leg and venous blood. It was sent to the Center for Disease Control (CDC) in Atlanta, Georgia and confirmed as a lactose-positive vibrio. (This strain was one of those discussed by Hollis6 and is now considered to have been V. vulnificus.) At autopsy all acute changes were nonspecific. There were no localized foci of metastatic infection. The autopsy revealed advanced Laennec's cirrhosis which was the presumed basis for the patient's fulminant course. The right leg amputation specimen had numerous

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Beckman, Edwin N., Leonard, George L., Castillo, Luis E., Genre, Charles F., and Pankey, George A.: Histopathology of marine vibrio wound infections. Am J Clin Pathol 76: 765-772, 1981. Although marine vibrio wound infections and septicemia are being reported with increasing frequency, description of the histopathologic changes has been scanty. The histologic alterations in three patients with primary marine vibrio wound infections are presented. The lesions are characterized by intense acute cellulitis of the subcutis with much tissue destruction and extension into the adjacent dermis. The superficial dermis is devitalized and lacks an inflammatory cellular infiltrate. Subepidermal noninflammatory bullae are formed. Many organisms are seen both within the areas of intense acute inflammation and in devitalized areas. Organisms and inflammation are especially oriented around vessels, with associated acute vasculitis. It is concluded that the morphologic picture in marine vibrio wound infections is nonspecific yet characteristic. (Key words: Marine vibrios; Vibrio vulnificus.)

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Table 1. Clinical Features and Therapy Patient

l5

22

35

Age (years), sex

60 M

68 M

41 M

Prior health

Laennec's cirrhosis

On prednisone for chronic active hepatitis and postnecrotic cirrhosis

Good, but (?) alcohol abuse and prior "mild hepatitis"

Exposure

Fire ant stings after wading in sea

Shrimp wound

Stingray wound

Month of onset

September

July

June

Incubation (hrs)

48

%L'- •>- -* v.*

bullae was non-necrotic. There were many organisms in the dermis, both in areas of inflammation and in edematous superficial dermis without inflammation. Case 3 A 41-year-old man was stung on the dorsum of the left foot by a stingray while wading in the Gulf of Mexico. He was otherwise healthy, although there was a questionable history of excessive alcohol consumption, and he had been told in the past that he had "mild hepatitis." Systemic symptoms appeared 12 hours after the sting, and he came to the Ochsner Medical Insti-

tutions. The puncture site was edematous and erythematous, and tenderness extended up to the mid-thigh. He was successfully treated with antibiotics, debridement, and a skin graft. The CDC identified the organism as a halophilic "vibrio species (unable to speciate with certainty)". This lactose-negative halophilic vibrio had biochemical characteristics which distinguished it from V. parahaemolyticus, V. alginolyticus, V. vulnifius, Vibrio group F, and V. metschnikovii.5 Antibiotic sensitivities were identical to those of the lactose-positive vibrio. The exact classification of this halophilic marine vibrio remains unresolved.


FIG. 2 Case I. Bands of neutrophils and bacteria transverse the subcutaneous adipose tissue, principally in the pattern of a septal panniculitis. Focally the inflammatory infiltrate extends into the adjacent deep dermis. Hematoxylin and eosin. X32.

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FIG. 3 Case I. The cellulitis consisting of neutrophils and bacteria extends from the subcutaneum into adjacent muscle. Major areas (above) are necrotic. Hematoxylin and eosin. X40.

Morphologic changes in the debridement specimen were principally those of an intense necrotizing cellulitis with a large zone of acute suppurative inflammation and edema filling the subcutaneum. Much of the subcutaneum was necrotic and devitalized, some portions intensely inflamed and others resembling coagulation necrosis. Focally within the latter regions were gram-negative coccobacilli. Acute inflammation and organism surrounded a few vessels. Focally neutrophils extended into the media. An occasional vessel contained thrombus, but there was no fibrinoid necrosis. There was a large region of ulceration

of the skin. The tissue immediately adjacent to the area of ulceration consisted of necrotic debris with intense acute inflammation. Otherwise the cellulitis of the subcutaneum dominated the picture with only minimal extension of the inflammation into the adjacent dermis. Discussion The morphologic changes in these cases of marine vibrio wound infections are nonspecific, yet there are common elements that suggest a characteristic pattern (Table 2). The principal change is one of an intense

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acute cellulitis transversing the subcutaneum with much tissue destruction. The cellulitis may extend into adjacent skeletal muscle and into the adjacent dermis. Some dermal areas may be intensely inflamed, but there is a distinct tendency for relative sparing of the dermis when compared to the degree of inflammatory infiltrate of the subcutaneum. Regions of the subcutaneous tissue and of the dermis may be devitalized and contain numerous organisms, yet lack inflammatory cellular infiltration. Bullae are formed by a split at the dermalepidermal interface. Numerous organisms are seen in the base of the bullae and in the bullae fluid. The bullae

are of the noninflammatory type, lacking inflammation within the fluid or at the base. There is a tendency for organisms and inflammation to be centered around vessels of the subcutaneum. Neutrophils transverse the media, but fibrinoid necrosis is not a feature. Endothelial cells are hyperplastic and swollen. These may be fibrin thrombus formation in the lumen. The appearance is one suggesting vasculitis as a result of perivascular orientation of organisms as opposed to the picture of primary fibrinoid necrosis seen in a collagen vascular disease. Few reports in the literature present the histopa-


FIG. 4 Ca^e / . A massive number of bacteria surround this medium size artery of the subcutaneous tissue. Endothelial cells are hyperplastic and swollen. A few neutrophils transverse the media, but this artery lacks the picture of fibrinoid necrosis seen in collagen vascular diseases. Hematoxylin and eosin. X320.

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The patient of Zide was a 46-year-old male with hemochromatosis who died of a fulminating vibrio septicemia.13 The organism was originally considered to be a form of V. parahaemolyticus though it did not ferment lactose or sucrose and had a positive Voges-Proskauer reaction. As lactose fermentation may be weak or delayed in the lactose-positive halophilic vibrios, Hollis now considers the organism from Zide's patient to fall in the V. vulnificus group. 6 After first having systemic features of septicemia, the patient developed a maculopapular urticarial eruption over the entire body which was considered possible erythema multiforme. The histopathologic features of these eruptions are consistent with our findings. There were broad zones of fat necrosis in the subcutaneous tissue. The dermis was infiltrated by many gram-negative organisms, but there was no inflammatory cellular response. The pancreas and liver had features of hemochromatosis. Thorsteinsson's Patient 3 was a 66-year-old man who presented with sepsis and developed bilateral lower limb cellulitis." Two weeks earlier he had waded in the Gulf of Mexico, without known skin trauma. The portal of entry was presumed to be the gastrointestinal tract because at autopsy he was found to have chronic duodenitis. Skin lesions had large bullae containing organisms in the fluid. There was intense cellulitis, and inflammation resulted in arterial occlusion. They state that halophilic vibrios can cause vasculitis and thrombophlebitis leading to tissue swelling and necrosis. Such a pathologic process may have been occurring in our Patient 1. A similar pathogenesis seems to have resulted in gangrene of the leg in Roland's patient who initially


thology of marine vibrio infections, either as local wound infections or in metastatic foci. Matsuo's patient was a 62-year-old male with alcoholic cirrhosis who developed bullous skin lesions apparently as metastatic foci from a primary lactose-positive vibrio septicemia.8 The morphologic changes in the skin were similar to our findings. There was intense cellulitis of the subcutaneous tissue and adjacent dermis. Vessels of the subcutaneum had transmural acute inflammation, but without thrombosis, and were surrounded by numerous organisms and acute inflammation. The mid and superficial dermis appeared necrotic and contained many gram-negative rods, but lacked an inflammatory infiltrate. Subepidermal noninflammatory bullae were formed without necrosis of the overlying epidermis. In the testis was neutrophilic arteritis with secondary parenchymal necrosis. Multiple splenic foci of necrosis and many organisms with associated plasma cells and phagocytes were interpreted as acute splenitis. The patient had alcoholic cirrhosis and esophageal varices.

FIG. 5 Case 2. Intense cellulitis of neutrophils and bacteria has extended from the subcutaneum into the dermis. The superficial dermis is edematous but relatively spared of inflammatory cells. Hematoxylin and eosin. X40.

presented with sepsis two days after wading in Narragansett Bay.10 (Hollis now considers this infection to have been of the lactose-positive marine vibrio type. 6 ) An arteriogram indicated occlusion of vessels of the leg, and pathologic examination of these vessels revealed "transmural acute necrotizing inflammation of large veins and arteries. . . ." The subcutaneum and muscular tissue had suppurative inflammation with massive necrosis.10

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Table 2. Summary of Histopathology Features Seen in at Least Two of the Three Patients Epidermis

Subepidermal, noninflammatory bullae

Dermis

Devitalized, edematous dermis Many bacteria in dermis, without inflammation

Subcutaneum

Intense acute cellulitis Extension into dermis and/or muscle

Blood vessels

Perivascular orientation of inflammation and organisms with associated vasculitis.

limited sampling of pathologic material, or limited surgical material available for pathologic evaluation. There may be variation in the intensity of some of the morphologic features present. Yet there appears to be a constellation of similar histopathologic findings in skin infections from halophilic vibrios, whether the skin infection is the result of a primary wound or is a secondary focus as a result of a primary septicemia (Table 2). We believe that the infection results in a nonspecific yet characteristic histopathologic picture. Acknowledgments. Catherine S. Dickinson, M(ASCP), and Dean L. Winslow, M.D. reviewed the manuscript.

References 1. Blake PA, Merson MH, Weaver RE, et al: Disease caused by a marine vibrio: clinical characteristics and epidemiology. N Engl J Med 300:1-5, 1979 2. Castillo LE, Winslow DL, Pankey GA: Wound infection and septic shock due to Vibrio vulnificus. Am J Trop Med Hyg 309:844-848, 1981 3. Craig DB, Stevens DL: Halophilic vibrio sepsis. South Med J 73:1285-1287, 1980 4. English VL, Lindberg RB: Isolation of Vibrio alginolyticus from wounds and blood of a burned patient. Am J Med Technol 43:989-993, 1977 5. Fernandez CR, Pankey GA: Tissue invasion by unnamed marine vibrios. JAMA 233:1173-1176, 1975 6. Hollis DG, Weaver RE, Baker CN, et al: Halophilic vibrio species isolated from blood cultures. J Clin Microbiol 3:425-431, 1976 7. Huq MI, Alam AKMJ, Brenner DJ, et al: Isolation of vibrio-like group, EF-6, from patients with diarrhea. J Clin Microbiol 11:621-624, 1980 8. Matsuo T, Kohno S, Ikeda T, et al: Fulminating lactose-positive vibrio septicemia. Acta Pathol Jap 28:937-948, 1978 9. Mertens A, Nagle J, Hansen W, et al: Halophilic, lactose-positive vibrio in a case of fatal septicemia. J Clin Microbiol 9:233235, 1979 10. Roland FP: Leg gangrene and endotoxin shock due to Vibrio parahaemolyticus—an infection acquired in New England coastal waters. N Engl J Med 282:1306, 1970 11. Thorsteinsson SB, Minuth JN, Musher DM: Clinical manifestations of halophilic non-cholera vibrio infections. Lancet 2:1283-1284, 1974 12. Wachsmuth IK, Morris GK, Feeley JC: Vibrio, Manual of Clinical Microbiology, Edited by EH Lennette, A Balows, WJ Hausler, et al. Third edition, Washington, DC, American Society of Microbiology, 1980, pp 226-234 13. Zide N, Davis J, Ehrenbranz J; Fulminating Vibrio parahemolyticus septicemia. Arch Intern Med 133:479-481, 1974


The pathogenesis of the bullous skin lesions may include toxin formation by the vibrio. Craig has pointed out the similarity of the histologic changes to those in streptococcal gangrene and in the staphylococcal scalded skin syndrome, two processes in which bacterial toxins have been implicated in the bullae formation.3 Craig's patient, a 67-year-old male with postnecrotic cirrhosis, who had no history of trauma, contact with the sea, or ingestion of seafood, died from lactose-positive halophilic vibrio sepsis. Bullous lesions on the legs resulted from separation at the dermal-epidermal interface; inflammation and organisms were centered around dermal vessels. Other descriptions in the literature of the histologic changes in halophilic vibrio infections have been brief. Mertens' patient with lactose-positive halophilic vibrio septicemia developed skin lesions which "consisted of significant fibrinous exudate and hemorrhage and contained massive numbers of bacteria and leukocytes."9 The patient of English suffered extensive burns and had been immersed in sea water to extinguish the fire. Specimens showed bacteria had invaded unburned subcutaneous tissue with associated hemorrhage.4 There is accord between the histopathologic changes described in the literature and those present in our three patients. Reported minor variations in features may have resulted from the stage of the lesions sampled,