Bacteremia Caused by a Serotype Ob5 Vibrio cholerae Strain in a Cirrhotic Patient in China

ABSTRACT The increasing incidence of non-O1/non-O139 Vibrio cholerae (NOVC) has been observed worldwide. However, septicemia caused by NOVC remains a rare condition that has received limited attention. Currently, there are no established treatment guidelines for bloodstream infections caused by NOVC, and the understanding of this condition mainly relies on individual case reports. Although NOVC bacteremia can be fatal in a small percentage of cases, knowledge about its microbiological features remains limited. Here, we present a case of V. cholerae septicemia caused by NOVC in a 46-year-old man with chronic viral hepatitis and liver cirrhosis. The isolated strain, named V. cholerae VCH20210731 and classified as a new sequence type (ST), ST1553, was found to be susceptible to most of the antimicrobial agents tested. O-antigen serotyping of V. cholerae VCH20210731 revealed that it belonged to serotype Ob5. Interestingly, the ctxAB genes, which are typically associated with V. cholerae, were absent in VCH20210731. However, the strain possessed 25 other potential virulence genes, such as hlyA, luxS, hap, and rtxA. The resistome of V. cholerae VCH20210731 included several genes, including qnrVC4, crp, almG, and parE. Nevertheless, susceptibility testing demonstrated that the isolate was susceptible to most of the antimicrobial agents tested. Phylogenetic analysis indicated that the closest strain to VCH20210731 was strain 120 from Russia, differing by 630 single-nucleotide polymorphisms (SNPs). Our findings contribute to the understanding of the genomic epidemiological characteristics and antibiotic resistance mechanisms of this invasive bacterial pathogen. IMPORTANCE This study highlights the discovery of a novel ST1553 V. cholerae strain in China, providing valuable insights into the genomic epidemiology and global transmission dynamics of V. cholerae. It is important to note that clinical presentations of NOVC bacteremia can vary significantly, and the isolates demonstrate genetic diversity. Consequently, health care professionals and public health experts should remain vigilant about the potential for infection with this pathogen, particularly considering the elevated prevalence of liver disease in China.

extraintestinal disease and bacteremia in individuals with liver disease or immunocompromised status (3). To our knowledge, the first case of NOVC bacteremia was reported from the United States in 1974 (4). Few studies have focused on the clinical, epidemiological, and genetic characteristics of NOVC bacteremia in China. However, due to the various clinical manifestations of NOVC bacteremia and the genetic diversity among isolates, it is crucial for clinicians and public health experts to be more vigilant to prevent the possibility of infection, especially for Chinese individuals with a high prevalence of liver disease. Our study provides new insights into the genomic epidemiological characteristics and global transmission dynamics of V. cholerae.
After a blood culture tested positive, b-hemolytic, oxidase-positive colonies were observed after 24 h of culture on blood agar. Subsequently, colonies were subcultured on thiosulfatecitrate-bile salts-sucrose (TCBS) agar and appeared as large yellow colonies. Identification of V. cholerae was confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (Vitek MS; bioMérieux, France). NOVC was identified using polyvalent O1 and O139 antisera in slide agglutination tests. Vitek 2 Compact (bioMérieux) antimicrobial susceptibility testing was used to determine the susceptibility of V. cholerae VCH20210731 to most antimicrobial agents. Antimicrobial resistance testing was performed for V. cholerae VCH20210731 using the broth microdilution method with the following antimicrobial agents: amikacin, ampicillin, gentamicin, ceftazidime, ceftriaxone, imipenem, ciprofloxacin, levofloxacin, cefuroxime, cefazolin, and azithromycin. The specimen was initially inoculated overnight at 37°C on Columbia blood agar, and a single colony of the target strain was grown overnight at 37°C in Mueller-Hinton broth (Oxoid Ltd., Basingstoke, UK). The bacterial species was identified using MALDI-TOF MS (Vitek MS; bioMérieux) and verified using 16S rRNA gene sequencing. MICs of antimicrobial agents were interpreted using Clinical and Laboratory Standards Institute (CLSI) guidelines (5).
This report describes the detection of V. cholerae VCH20210731 from a blood sample obtained in July 2021 from a 46-year-old man who was hospitalized with chronic viral hepatitis and liver cirrhosis. The patient had been experiencing fatigue and stomach distension for 3 days and had a history of hepatitis B cirrhosis for more than 5 years. On admission, his temperature was 36.9°C, heart rate 86 beats/min, and blood pressure 100/64 mm Hg. Physical examination revealed icteric sclera, minor ascites, splenomegaly, tremor in both upper limbs, spider nevus, and liver palms. Laboratory examination results were as follows: alanine aminotransferase activity, 17 U/L; aspartate aminotransferase activity, 23 U/L; total bilirubin level, 33.5 mmol/L; direct bilirubin level, 12.3 mmol/L; total protein level, 54.2 g/L; albumin level, 24.5 g/L; prothrombin time, 20.90 s; D dimer level, 8.44 mg/L; fibrinogen level, 1.02 g/L; white blood cell count, 1.50 Â 10 9 cells/L; hemoglobin level, 48.0 g/L; platelet count, 34 Â 10 9 cells/L. Cirrhosis with splenomegaly, gallbladder wall edema, splenic vein wall thrombosis, and abdominal fluid buildup were found on ultrasonography. After admission, the patient received treatment to improve blood circulation with infusion of 2 U red bloods cells. On July 15, the patient reported fever with chills, and his body temperature increased to 38.1°C.
NOVC infections in humans are usually associated with exposure to contaminated water or seafood (11). A large majority of patients report no such exposure, however, implying other plausible routes of transmission (1). Liver cirrhosis was more prevalent among patients with primary bacteremia than among those with other types of infections (3), and such patients should be closely monitored. This finding may due to various factors, such as high intestinal mucosal permeability caused by inflammation and edema, portal hypertension resulting in bypassing of the hepatic reticuloendothelial system, complement deficiencies, impaired phagocytosis, alterations in iron metabolism, and/or inefficient chemotaxis (12). It has also been hypothesized that liver disease and hematological malignancies frequently result in low platelet counts or poor coagulation function, which could facilitate bacterial entry into the systemic circulation and cause bacteremia. Additionally, specific strains of NOVC that generate hemolysin may be involved in bacterial invasion by promoting cell vacuolation and hemolysis (13).
In immunocompetent patients, NOVC gastroenteritis is usually self-limited and does not require the use of antibiotics. For complicated infections and immunocompromised patients, however, antimicrobial therapy is recommended (1). Dual-agent therapy for NOVC bacteremia has been proposed by several researchers (14). Combining a third-generation cephalosporin with a tetracycline or a fluoroquinolone appears to be effective, depending on susceptibility data. Treatment duration for septicemia is also debatable, ranging from 3 to 75 days in various publications (median, 14 days) (1).
Our patient displayed an atypical clinical manifestation of NOVC bacteremia, with only abnormal liver function test results, which were determined by ultrasonography to be due to liver cirrhosis, and only mild digestive symptoms, with diagnosis using blood culture. In addition, our patient achieved remission after only 2 days of treatment with ceftriaxone. The clinical manifestations of NOVC bacteremia have further proved to be diverse.
The draft genome sequence of V. cholerae VCH20210731 consists of 95 contigs, totaling 4,032,143 bp, and could be classified as a new sequence type (ST), ST1553. The overall G1C content of the strain was 47.6%, and a total of 3,623 coding sequences (CDSs) and 113 RNA genes (93 tRNA, 16 rRNA, and 4 noncoding RNA genes) were identified. O-antigen serotyping revealed that V. cholerae VCH20210731 belonged to serotype Ob5. The ctxAB genes are absent in V. cholerae VCH20210731, but the strain has 25 other putative virulence genes, such as hlyA, luxS, hap, and rtxA. Comparative genomic analysis also revealed that the isolate was nontoxigenic and possessed the CTX-RS1 2 VPI 1 VSP 1 genotype. The resistome of V. cholerae VCH20210731 included several genes, such as qnrVC4, crp, almG, and parE. However, susceptibility testing showed that the isolate was susceptible to most of the antimicrobial agents tested, including amikacin, ampicillin, gentamicin, ceftazidime, ceftriaxone, ciprofloxacin, levofloxacin, cefuroxime, cefazolin, amoxicillin-clavulanic acid, piperacillin-tazobactam, trimethoprim-sulfamethoxazole, cefoperazone-sulbactam, and azithromycin (Table 1)  assembly strains (which were removed) and 3 Vibrio paracholerae strains. The phylogenetic connections between V. cholerae VCH20210731 and the total of 170 NOVC strains currently deposited in the NCBI GenBank database were examined to evaluate the genomic epidemiological features of V. cholerae strains in a global context (Fig. 1) (Fig. 2). Moreover, V. cholerae VCH20210731 presents a distinctive ST, which we have submitted to the PubMLST database and designated ST1553. Unfortunately, the current availability of V. cholerae genomes in the NCBI database is limited, posing a challenge to conducting comprehensive analyses on the source tracking of this isolate. Phylogenetic analysis showed that the closest strain to our isolated strain VCH20210731 was strain 120 from Russia (NCBI assembly accession number GCF_008269365.1), with 630 SNPs.
In conclusion, we report a ST1553 V. cholerae strain from China that produced NOVC bacteremia. The global increase in sea surface temperatures poses a potential threat for the proliferation of pathogenic vibrios in aquatic environments, highlighting the need for awareness and vigilance. Our findings contribute to the understanding of the genomic epidemiological traits and antibiotic resistance mechanisms of this bacterial pathogen.
Ethical approval. This study was conducted in accordance with the Declaration of Helsinki, and approval was obtained from the Medical Ethics Committee at the Sanmen People's Hospital, China. Written informed consent was provided by the patient to allow the case details to be published.
Data availability. The genome sequences of the chromosome of V. cholerae VCH20210731 have been deposited in NCBI GenBank under accession number JAJQMP000000000.

ACKNOWLEDGMENTS
No funding sources were involved. We declare no competing interests.