Vibrio metschnikovii: Current state of knowledge and discussion of recently identified clinical case

Abstract Vibrio metschnikovii is a widespread opportunistic pathogen that rarely causes disease in human. It caused graft infection in our case. It is important to differentiate it from another water‐transmitted pathogens.


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KONECHNYI Et al. bleeding caused by the ulcer. Intestinal bleeding was managed conservatively. On the 3-rd day after hospitalization, patient with posthemorrhagic anemia was transferred to the surgical department.
According to patient's medical history: 1991-bifurcated aorto-bifemoral alloprosthesis placing due to Leriche syndrome; 1999-replacement of bifurcated aorto-bifemoral prosthesis due to thrombosis; 2001-thrombosis of the right branch of bifurcated aorto-bifemoral prosthesis, extra-anatomical axillofemoral bypass performed with early thrombosis of reconstruction which led to limb amputation. After few years, femoropopliteal autogenous venous bypass was made due to chronic limb ischemia on the other side. At the time of hospital admission, the femoropopliteal graft was occluded without chronic limb-threatening ischemia.
Clinical symptoms of gastrointestinal bleeding relapse were observed on the fifth day of treatment in the hospital. Though no source of bleeding was found during upper gastrointestinal tract endoscopy, multislice spiral computed tomography (MSCT) was performed. MSCT angiography has shown pseudoaneurysm formation of central anastomosis of aorto-bifemoral graft and aorto-small intestine fistula formation (reason of gastrointestinal bleeding). Ultrasonography has shown an exudate around the alloprosthesis. Surgery is considered to be the best option. The extra-anatomical axilofemoral bypass, exclusion of the central anastomosis aneurysm, removal of infected graft, and aorto-digestive fistula repair have been performed under general anesthesia. The blood loss was about 2250 mL, and autologous blood reinfusion was used. The hemostasis system control was made using TEG and aggregometry.
Postoperative period was complicated with: renal dysfunction; hospital-acquired bilateral pneumonia; partial, closed eventration without need of surgery; postoperative wound healing by secondary intention (treated with NPWT (negative pressure wound therapy)); and antibiotic-associated diarrhea.
After the surgical removal, Vibrio metschnikovii was found in large quantities (about 108 colony-forming units/ mL) on the surface of the removed Dacron prosthesis. The antibiotic sensitivity test is displayed in Table 1. From the same fragment, Trichosporum spp was isolated in insignificant quantities (resistant to clotrimazole and susceptible to fluconazole).
The patient's condition was significantly improved after treatment with meropenem (10 days), metronidazole (3 days), and fluconazole (7 days). After that, he was transferred to a simple ward. After 10 days of treatment, the blood culture examination has shown S. piscifermentans in small quantities (about 106 colony-forming units/mL). According to the literature review, S. piscifermentans is not pathogenic. Also, the patient was stable (no signs of infection). In that case, we suppose that the mistake was made during a blood sample collection.
The patient was discharged from the hospital in good condition after 67 days of hospital treatment. In the next 6 months, patient underwent ventral hernia repair surgery and reported significant improvement in his condition.

| Bacterial isolation and growth conditions
The isolation and identification of pathogens were carried out in a certified microbiological laboratory of the Department of Microbiology at the Danylo Halytsky Lviv National Medical University, Ukraine. A bacteriological method was used to isolate a pure pathogen culture with blood agar, meat peptone agar, and Sabouraud agar.

| Biochemical identification
The pathogen identification was performed using the MIKRO-LA-TEST NEFERM24 kit (Erba Lachema, Czech Republic) in five technical repetitions and using the corresponding codebook. CANDIDATEST 21 kit was used for Trichosporon spp identification.

| Antibiotic sensitivity test
Antibiotic sensitivity test was measured using disk diffusion method with interpretation according to genus Vibrio 1,2 ( Table 1).

| DISCUSSION
Vibrio metschnikovii is a rare human pathogen, which could be classified as a partially zoonotic microorganism with the fecal-oral route of transmission. patients were elderly people with long-term concomitant diseases, which lead to secondary immunodeficiency, for example, diabetes, chronic heart failure, and chronic kidney failure.
Patients denied having contact with the sea or consuming marine products, so it was impossible to set the infection source in all of the cases.
The vascular prosthesis infection may be a result of the not perfect aseptic regime and bacterial contamination during surgery. Also, bacterial translocation to the vascular graft may occur from wound infection or intracavity abscess or due to the formation of erosion or fistula between the digestive tract and the graft. The less common cause is transient or chronic bacteremia, which can bring about the transmission of infection to synthetic materials in the body, especially in the early postoperative period when the endothelialization of the graft is not complete. Other surgical procedures near the vascular graft or paravasal thrombus inflammation with bacterial translocation also may be the reason for vascular graft infection.
We believe that vascular alloprosthesis, in our case, was infected through the aorta-digestive fistula. But, it is important to mention a couple of genus Aeromonas pathogens selected from the intensive care unit of the same hospital within a month. The Aeromonas spp. were extracted from blood and urine and had infectious characteristics connected with providing medical care (the beginning of infection after hospitalization, multidrug resistance to antibiotics, others). It is known that Aeromonas spp. generally have water routes of transmission and are similar to genus Vibrio, according to some abilities. That is why these two types of bacteria are sometimes differentiated wrongly. 3 In 3 months, there was selected one more species from another patient of this department. It was similar to Vibrio spp. conforming to its colony growth and microscopic figure. The surgery was done due to Takayasu disease, and the biopsy from the artery was taken, which revealed the pathogen in a low amount. The antibiotic therapy was not prescribed because the amount of the pathogen was not high enough, and there were no signs of infectious process.
Trichosporon spp. might be the reason for arterial vascular prosthesis infection, 4 but in over case, Trichosporon spp. was selected in a low amount. So, it cannot be the leading cause of infection.
In our case, Vibrio metschnikovii's cultivation is simple: on blood agar, with the temperature up to 37°C, causes betahemolysis and does not have a specific smell or pigment. Colonies are nonspecific, and size is approximately 0.3 mm. (Figure 1). Initially, the pathogen looked like micrococci and only after passaging changed to rod-shaped bacteria, with slightly bent ends (Figure 2). Biochemical characteristics were the same as described in literature. 5 The result of biochemical identification is shown in Table 2.  In all described cases were no multidrug-resistant strains. Moreover, there were no similar cases with similar antibiotic resistance. For example, some of them were resistant to ampicillin; otherwise, some were susceptible to it. It could be classified as an exogenous (more iatrogenic) opportunistic infectious factor.

| Literature review
Genus Vibrio belongs to the family Vibrionaceae, class Gammaproteobacteria and includes 140 ubiquitous species. These bacteria are more common in the salty and freshwaters all over the planet. 6 It lives as plankton, associated with free chitinous material, algae. 13 species of genus Vibrio are associated with human diseases, it causes diarrhea or extraintestinal symptoms (wound and skin infections, bacteremia), particularly V. cholerae, V. parahaemolyticus, and V. vulnificus consider to be the most significant. Water routes and uncooked marine products are the main ways of transmission. It is common for genus Vibrio to cause illnesses of fish (especially trouts), shellfish, eels, and oysters. For example, eels and fish (rainbow trouts and salmonidae) could have terminal hemorrhagic septicemia caused by V. anguillarum; sea perches-gastroenteritis caused by V. harveyi; larvae of flounders-intestinal necrosis caused by V. ichthyoenteri and Japanese horse mackerel-intestinal hemorrhage caused by V. trachuri. 5 Vibrio metschnikovii is ubiquitous in water (both salty and fresh), particularly in 2020, 38 species of Vibrio metschnikovii were selected from the Norwegian Sea nearby the Norwegian coast and from the herring fish of the same Sea. 7 Vibrio metschnikovii is associated with the ability to cause human diseases, mostly diarrhea, skin infections, and bacteremia; otherwise, some authors say that Vibrio metschnikovii induce only extraintestinal symptoms. Mostly, the pathogen can be isolated from blood, urine, wound surfaces, bile, and feces. In general, it infects fish, particularly sardines that have their gills, digestive system, and ovaries in females affected (despite other Vibrio spp), with testicles in males unaffected. 8 Vibrio metschnikovii, the same as other species of genus Vibrio, belong to halophiles (organisms that thrive in high salt concentrations). It is common for vibrio to decrease in size and lose its volume (15%-67%), changing from rod-shaped to cocci (spherical ultramicrococci) when they are in a lack of nutrients. They are mobile, mostly oxidase-negative (due to cytochrome C insufficiency, but some are oxidase-positive), Gram-negative, facultative anaerobic rods. 9 The main pathogenic factors are hemolysin (tlh gene), some genes of toxic formation are present (rtxA gene, responsible for producing holotoxins). The ability to secrete cytotoxin was confirmed when Vibrio metschnikovii was indicated in aborted fetuses of pigs, cattle, and horses, moreover in different parts of the duck's and geese's brain in Germany. Also, this pathogen was found in the chicken's intestines and together with E. coli caused hepatitis. 10 38 strains indicated nearby the Norwegian seashore had the genes of resistance adeF (responsible for polypharmacological efflux mechanism), blaCARB (responsible for carbenicillin resistance), catB-related (responsible for chloramphenicol resistance), and genes that are responsible for pathogenicity factors Hem III (hemolysin type III), hylA, tlh. 7 Usually, the pathogen has huge plasmids (190-340 кB), but their function is not exactly known.
Ten strains were isolated from the lakes, specifically those situated on the graphite quarries, in Slovakia had 100% resistance to penicillin and 90% streptomycin resistance. 6 13% pathogens found in the Norwegian Sea turned out to be tobramycin-resistant and 21%-ampicillin-resistant. 7 Some authors 5 report about 91% resistance to penicillin G, 95% sulfadiazine, 86% kanamycin, and 69% ampicillin resistance.
Due to the ecological plasticity of the Vibrio metschnikovii, the cultivation is relatively simple. The optimal conditions are as follows: pH-6.0 (but can vary up to 10.0); temperature-30-40°C; NaCl concentration-1.0%-8.0%. The best culture media are as follows: Sheep blood agar, Alkaline peptone water, Marine broth, Marine agar, Photobacterium agar, Photobacterium broth, Selective media for Vibrio such as thiosulfate-citrate-bile salts-sucrose agar (TCBS) and Vibrio agar; MacConkey agar not a priority for Vibrio cultivation. 5 Vibrio metschnikovii causes RBC's hemolysis in vivo and in vitro (alpha-and beta-hemolysis on blood agar). Isolation of the pathogen is more frequent in warm seasons of the year (typically May-August). Vibrio metschnikovii is less susceptible to the cold environment in comparison to other Vibrio spp.
Vibrio metschnikovii, together with Vibrio gazogenes, are unique species of genus Vibrio. They are nitrate negative (unable to convert nitrates to nitrites) and oxidase-positive (some strains). Moreover, these two species differ from other Vibrio spp. by their immunological relation to superoxide dismutase. Finally, it is essential to mention that results of DNA-DNA hybridization may be the reason for further reclassification into separate genus. 5

| Review of case reports
There are 13 clinical cases of Vibrio metschnikovii infections, according to the PubMed database.
The first clinical case was described in 1981. 11 82-year-old woman was diagnosed with acute cholecystitis that led to peritonitis. After this case, river and wastewaters, mussels, shrimps, lobsters, crabs, and poultry were considered to be infectious factors. Besides, 5 clinical cases of acute watery or hemorrhagic diarrhea in infants were recorded in Peru. 12 The first description of postoperative wound infection with | 2243 KONECHNYI Et al.
Vibrio metschnikovii was made in 2004. It was a 64-year-old male patient who underwent bilateral saphenectomy combined with cardiac surgery. 10 All these cases did not include multidrug-resistant strains of Vibrio metschnikovii. Another interesting case was septic shock and cardiac arrest in a 78-year-old Dutch male caused by Vibrio metschnikovii in 2014. 13 A short review of all clinical cases is shown in Table 3.

| CONCLUSION
Nowadays, it is important to be aware of new pathogens, which commonly were not considered to cause an infection in humans. As new technologies of endoprosthesis develop, new measures to prevent the infection should be taken.
The use of antiseptics and hypertonic solutions during wound care is effective mostly for common pathogens that are in priority. On the other hand, they could be useless and purposeless for some species that were not tested and turned out to be resistant. This might happen due to the lowering of the human's body immune system resistibility and deterioration of the protective barriers, making it effortless for the pathogen to infect the body.