Distribution, Prevalence, and Antibiotic Susceptibility Profiles of Infectious Noncholera Vibrio Species in Malaysia

Background The noncholera Vibrio spp. which cause vibriosis are abundantly found in our water ecosystem. These bacteria could negatively affect both humans and animals. To date, there is a paucity of information available on the existence and pathogenicity of this particular noncholera Vibrio spp. in Malaysia in comparison to their counterpart, Vibrio cholera. Methods In this study, we extracted retrospective data from Malaysian surveillance database. Analysis was carried out using WHONET software focusing noncholera Vibrio spp. including Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio fluvialis, Vibrio alginolyticus, Vibrio hollisae (Grimontia hollisae), Vibrio mimicus, Vibrio metschnikovii, and Vibrio furnissii. Results Here, we report the first distribution and prevalence of these species isolated in Malaysia together with the antibiotic sensitivity profile based on the species. We found that V. parahaemolyticus is the predominant species isolated in Malaysia. Noticeably, across the study period, V. fluvialis is becoming more prevalent, as compared to V. parahaemolyticus. In addition, this study also reports the first isolation of pathogenic V. furnissii from stool in Malaysia. Conclusion These data represent an important step toward understanding the potential emergence of noncholera Vibrio spp. outbreaks.


Introduction
Vibrio spp. are ubiquitous in freshwater, estuarine, and saltwater aquatic milieus [1]. Tey are curved Gram negative bacilli, oxidase and catalase positive, and are generally sensitive to vibriostatic agents. Teir motility is conferred by polar fagella [2]. Vibrio genus consists of about 150 species which can be further categorized into halophilic and nonhalophilic Vibrio spp. based on their sodium requirement. Although nonhalophilic Vibrio spp. in humans usually cause moderate infection, they are also known to cause outbreaks in humans, fsh, and other aquatic animals with undesirable economical and public health implications [3,4].
Human vibriosis can be classifed into two major groups: cholera and noncholera. Vibrio cholerae is an important human pathogen responsible for cholera. Cholera is both waterborne and foodborne causing approximately 1.3 to 5 million cases reported annually with the death toll of 21,000-143,000 lives/year [5,6]. V. cholerae serotypes O1 and O139 are exclusively accountable for cholera epidemics. Upon entry into the human host, V. cholerae adhere to intestinal epithelium followed by secretion and accumulation of cholera toxin (CT), which is responsible for intense watery diarrhea that potentially led to fatality. Virulence genes located in the ToxR regulon within V. cholerae genome are in particular responsible for severe symptoms of cholera [7]. Meanwhile, V. cholerae serotypes non-O1 and non-O139 are responsible for sporadic choleralike infections, bacteremia, and septicemia [8][9][10]. Te V. cholerae non-O1/non-O139 are categorized as noncholera Vibrio cholerae, as well as V. cholerae serotypes O1 and O139 that do not produce cholera toxin [11]. Such serotypes have been associated with clonal outbreak among diarrheal patients in Kolkata [12].
Other Vibrio spp., such as V. mimicus, V. metschnikovii, V. vulnifcus, V. alginolyticus, V. parahaemolyticus, and V. furnissii, can also cause illnesses in humans known as vibriosis, which commonly is presented with gastrointestinal infection, wound infections, and septicemia [13]. Certain species such as V. metschnikovii have been isolated from a range of conditions such as septicemia and wound [14,15]. Diferently, cholera epidemics are highly associated with sanitary quality and socioeconomic status (typically transmitted from human to human or contaminated water). However, vibriosis caused by other Vibrio spp. is closely related to the consumption of contaminated shellfsh or contaminated water and is independent of the sanitation status [6,16,17].
Te presence and compositions of Vibrio spp. in aquatic habitats are dynamic and infuenced by the geographical factor, climate, and salinity [18]. Terefore, analysis of local clinical and environmental data is necessary to develop an understanding of occurrences of vibriosis as a result of interaction between human health and other domains over time. Tis article aims to describe the current Vibrio spp. isolated from clinical samples in Malaysia from 2014 to 2020.

Materials and Methods
Nation-wide hospital antimicrobial susceptibility data between 2014 and 2020 were retrieved from Malaysia's local surveillance program. Tese data were catalogued in a downloadable database software developed by the World Health Organization (WHO), known as WHONET. Te program converts various types of data from the diferent laboratory information systems of each hospital into a standardized common code and fle format, enabling centralized data monitoring and analysis.
From 2014 to 2020, an average of 42 Malaysian hospitals participated in the WHONET-Malaysia network, contributing laboratory data consistently throughout the year. All antimicrobial tests and respective bacterial identifcation tests were performed in each hospital's clinical laboratories. Tis study focused on the available data associated with all noncholera Vibrio spp. reported within these seven surveillance years and their antimicrobial susceptibility profles. In addition, V. cholerae nonO1/nonO139 was not included in this survey. Te WHONET software provides basic statistical analysis of the input hospital data, with a special focus on the analysis of the antimicrobial susceptibility test results.
Te antimicrobial susceptibility pattern from our data showed that Vibrio spp. isolated from clinical isolates during the period of the study remained susceptible to the common antibiotic regimes ( Table 2). For instance, 91-100% of the isolates were sensitive to ceftazidime, gentamicin, imipenem, and meropenem. Similarly, for ciprofoxacin, 94-100% of the isolates were sensitive, except the V. parahaemolyticus (82%). On the other hand, in the absence of ß-lactamase inhibitor, the sensitivity of V. parahaemolyticus and V. fuvialis towards ampicillin was only 9% and 16%, respectively. In addition, high percentage of resistance was observed in V. parahaemolyticus against cefuroxime (68%) and V. fuvialis towards cefotaxime (50%), respectively. Across the 7-year surveillance (2014-2020), the average isolation of noncholera Vibrio was 37.3 cases/year (29-43 cases) (Figure 1). Te data revealed that V. parahaemolyticus predominate the landscape of human infection during the period of the study. Surprisingly, V. fuvialis was increasingly isolated from clinical specimens from 3% in 2014 to 32% in 2020. Meanwhile, V. furnissii, V. hollisae, V. metschnikovii, and V. mimicus were not routinely isolated every consecutive year.

Discussion
V. cholerae is rarely isolated in Malaysia, yet it remains an important threat to human health in many developing countries [19]. Steadily, noncholera Vibrio spp. such as V. fuvialis, V. mimicus, V. metschnikovii, and V. furnissii become emerging causes of human infections often associated with foodborne diarrheal outbreaks. Te clinical manifestations depend on the host susceptibility, route of infections, and inoculum size [20,21]. In this study, it is shown that V. parahaemolyticus was the main causative agent of vibriosis in Malaysia, consistent with the other part of the world [22]. V. parahaemolyticus is an aquatic zoonotic pathogen that not only infects humans but is also a threat to aquaculture animal health. In aquaculture animals, V. parahaemolyticus causes early mortality syndrome (EMS) in shrimps and vibriosis in fshes with a high mortality rate and cause signifcant economic losses [23,24]. Following the infection alarm in humans, isolates of V. parahaemolyticus from seafood in Selangor Malaysia have been found and characterized to be resistant to several antibiotics and possess at least one plasmid and positive ToxR genes [25].
In this study, V. fuvialis show an increasing trend during the study period. To date, there is scarce information available on human infections caused by V. fuvialis [26,27].    Only this organism contains >50 isolates.

Journal of Tropical Medicine
V. fuvialis is similar to other noncholera Vibrio spp., they cause mild self-limiting gastrointestinal infections. Oral fuid replacement therapy is the mainstay of symptomatic treatment in mild cases. However, antimicrobial therapy using doxycycline or ciprofoxacin can be initiated in severe or prolonged cases [6]. On the other hand, V. vulnifcus infection is associated with a higher mortality rate, especially in high-risk patients with treatment delay [28]. Tis study also had shown V. vulnifcus as the second causative agent of vibriosis human infection in Malaysia. In the US, Brazil, Italy, and India, the species become a healthcare burden with a mortality rate more than 30% [29]. Tis organism is a zoonotic pathogen that can cause septicemia in fsh, where iron in the blood signals the production of toxins that elevate the severity [30]. Te acquirement of genes to evade host immune systems and antibiotic resistance are aggravated by uncontrolled usage of antibiotics in the aquaculture sectors [31]. Multidrug-resistantVibrio spp. in the aquatic environment was common in fsh farming [32,33]. In Malaysia, it was reported that up to 80% of the Vibrio isolates from the west coast of Peninsular Malaysia were found to be resistant to one or more classes of antibiotics [34]. However, in this study, the vast majority of the clinical strains from human cases were sensitive to the cephalosporin and carbapenem class of antibiotics. Despite this observation, the retrieved surveillance data from human infections are limited by the number of isolates that were tested, hence may not represent the real clinical epidemiology. In some countries such as Bangladesh, China, and Vietnam, vibriosis is a signifcant public health problem, of which, outbreaks occur regularly, associated with wet season or summer with high dynamicity [35,36]. For instance, V. parahaemolyticus serotype O10:K4, which have been frequently associated with outbreak in China, has spread to Tailand, with high sensitivities (89-100%) toward ß-lactam antibiotic such as ampicillin/ sulbactam and ceftazidime [37,38]. Overall, the incidence of noncholera vibriosis in Asia and Southeast Asia is likely to be underreported, as many cases are not diagnosed or notifable to health authorities [6].
In summary, this study highlights Vibrio parahaemolyticus as a dominant Vibrio spp. causing vibriosis in Malaysia. Further analysis of the clinical signifcance of other Vibrio species that show emerging trends will require clinical data analysis and further laboratory characterization study.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author on request.

Disclosure
All authors meet the ICMJE authorship criteria.