Introduction

The Vibrionales of the phylum proteobacteria are represented by Gram-negative, curved bacteria that inhabit coastal marine environments and are either free-living or live in association with marine crustaceans (Ghenem et al. 2017; Baker-Austin et al. 2018). The cholera epidemic caused by Vibrio cholerae is as ancient as the human race. This bacterial microorganism caused seven known epidemics between 1816 and the present, killing millions (Sherman 2007; Deen et al. 2020). As it was known in ancient times, the blue death is associated with the massive cholera-associated diarrhea that turns an infected individual’s body into a blue tinge owing to extensive dehydration and consequential capillary rupture (Morris 2007; Wade 2022). The earliest documented cholera-like epidemic could be traced back to Greece in 400 BC (Kaper et al. 1995; Ramamurthy and Ghosh 2021). The true causes of pandemics that ravaged humanity from time immemorial were unknown until Louis Pasteur disproved the theory of abiogenesis in 1862 and unequivocally established the role of microbes in food spoilage and disease, and his counterpart Robert Koch in Germany discovered the causative agents of cholera, anthrax, diphtheria, and tuberculosis (Kaper et al. 1995; Blevins and Bronze 2010).

The causative agent of cholera, V. cholerae, is a non-halophilic member of the family Vibrionaceae with over 100 species, 12 of which are associated with human infections more often (Baker-Austin et al. 2018). The Vibrio species are oxidase- and catalase-positive, ferment glucose, produce indole from tryptophan, and are motile by polar flagella. The other important species that cause food-borne infections are V. parahaemolyticus and V. vulnificus. Vibrio spp. can cause opportunistic infections such as in wounds, ears, and eyes and septicemia. V. parahaemolyticus causes gastrointestinal infections associated with consuming raw or undercooked fish and shellfish. In contrast, V. vulnificus causes wound infections and septicemia. These Vibrio spp. are widely distributed in coastal-marine water, sediment, plankton, and aquatic animals (Colwell et al. 1977). Other Vibrio spp. that are involved in sporadic food-borne intestinal or extraintestinal infections include V. furnissi, V. mimicus, V. anguillarum, V. fluvialis, V. damsele, V. furnissi, and V. metschnikovii (Colwell et al. 1977; Morris 2003; Baker-Austin et al. 2018). V. fischeri is a well-known endosymbiont of light organs of squids, and V. harveyi is a pathogen of marine crustaceans, particularly shrimp. Other Vibrio species are also opportunistic pathogens of different life stages of fish and shellfish.

The vibrios, except V. cholerae and V. mimicus, are halophilic. Their numbers increase with increasing temperature in temperate waters, with the high numbers recorded during warm summer months when the temperature is > 25 °C, which also correlates with the higher rates of infections (Motes et al. 1998; Hounmanou et al. 2023). Infections are often associated with the consumption of raw seafood containing high (> 105 CFU/g) numbers of vibrios, particularly the filter-feeding bivalve mollusks, which accumulate bacteria to concentrations several folds higher than that in the surrounding water (Baker-Austin et al. 2018). Vibrios respond uniquely to unfavorable environmental conditions such as very low temperatures and high salinity by entering into a dormant state called “viable but non-culturable” (VBNC) state (Colwell 2000; Oliver 2010). In the VBNC state, bacteria are metabolically inactive. These bacteria do not grow on standard laboratory media but retain their ability to infect and cause disease (Huq and Colwell 1996).

Epidemiology of Vibrio spp.

Vibrio cholerae

V. cholerae remains the most devastating Vibrio spp., still responsible for water-borne infections and deaths in developing countries. About 3–5 million cholera cases with 100,000 deaths are reported annually (Deen et al. 2020; Saha and Ganguly 2021; CDC 2023). Cholera is endemic in 51 countries, particularly in Sub-Saharan Africa and Southeast Asia (Harris et al. 2012; Ali et al. 2015; Guillaume et al. 2018). Although over 200 serotypes of V. cholerae have been identified based on the lipopolysaccharide antigens (Morris 2003), the severe form of cholera known as “cholera gravis” is caused by V. cholerae O1, identified by its ability to agglutinate with the O1 antiserum. All other V. cholerae that do not agglutinate with O1 antiserum are termed “non-O1.” The pandemic strains of O1 are further grouped into “Classical” and “El Tor” biotypes (Kaper et al. 1995; Reidl and Klose 2002). Epidemiological studies suggest a succession of these biotypes in cholera pandemics. The Classical biotype caused the first six pandemics, while the ongoing pandemic involves the El Tor biotype (Mukhopadhyay et al. 2014). Based on the antigenic differences, these are further subdivided into Ogawa, Inaba, and Hikojima serotypes (Kaper et al. 1995). The severity of cholera caused by V. cholerae O1 is due to cholera toxin production (Kaper et al. 1995). The non-O1 strains do not produce cholera toxin; hence, they are non-pathogenic or cause mild gastrointestinal infections. However, certain strains are occasionally involved in severe cases of intestinal and extraintestinal infections (Bag et al. 2008; Mukherjee et al. 2014).

In 1992, a non-O1 serotype involved in a cholera outbreak similar to that caused by O1 V. cholerae was attributed to the serotype O139, which also produced the cholera toxin (Ramamurthy et al. 1993). This strain is the only non-O1 V. cholerae reported thus far to be capable of causing a severe form of cholera as the O1 serotype. Although V. cholerae O139 reportedly originated from the El Tor strain, they are quite distinct antigenically (Albert et al. 1993; Faruque et al. 2003b). Large cholera outbreaks occurred in India and Bangladesh due to the O139 serotype in 1992 (Albert et al. 1993; Nair et al. 1994). However, subsequently, this serotype has not been responsible for any new cholera cases, and the El Tor biotype has caused almost all cholera infections worldwide. Recently, Yemen has been the focal point of large outbreaks involving over 364,000 cases and 639 deaths in 2019 (Deen et al. 2020; UNHCR), while countries such as Afghanistan, DR Congo (Ingelbeen et al. 2019), Haiti (Rubin et al. 2022), Ethiopia, and Cameroon continue to report the most cases (Deen et al. 2020; WHO 2022; ECDC 2023).

The virulence of V. cholerae, by far, relies on its ability to elaborate cholera toxin, CT, an A-B type toxin with one A subunit and five B subunits, which exerts its lethal effects on the physiology of intestinal epithelial cells by NAD-ribosylation of the adenylate cyclase complex leading to uncontrolled production of cyclic AMP (cAMP) molecules (Kaper et al. 1995). This results in continuous loss of water and electrolytes from the cells, which manifests as the classical rice water stool of cholera. In addition to CT, the Zonula occludens toxin (Zot), the accessory cholera enterotoxin (Ace), and a core encoded pilin (Cep) are important for the virulence of V. cholerae. Other accessory virulence factors that contribute to the ability of V. cholerae successfully to colonize the intestinal epithelium include the toxin-coregulated pilus (TCP), regulatory proteins like ToxR, membrane porins, iron-regulated outer membrane proteins, the O-antigen of the lipopolysaccharide, and accessory colonization factors (Faruque et al. 2003a).

The non-O1, non-O139 V. cholerae (NOVC) serotypes are involved in intestinal and extraintestinal infections (Dutta et al. 2013; Deshayes et al. 2015; Vezzulli et al. 2020). Although NOVC lacks CT and TCP, they possess other putative virulence factors such as a heat-stable toxin (NAG-ST), a hemolysin (Hly), RTX toxins (Repeats in ToXin), hemagglutinin protease (HapA), toxin regulatory protein (ToxR), outer membrane proteins (Omp), and the type III (T3SS) and type VI (T6SS) secretion systems. However, these factors are not consistently present in all NOVC isolates (Bag et al. 2008; Ceccarelli et al. 2015; Neogi et al. 2019). Reported cases of NOVC bacteremia involve persons with predisposing conditions such as liver cirrhosis, diabetes, and malignancy, with seafood and water as major sources of infection (Chen et al. 2015; Deshayes et al. 2015). Most reported cases of NOVC involved acute gastroenteritis, biliary tract infection, and primary bacteremia. In contrast, occasionally, peritonitis, skin and soft tissue infection, and urinary tract infection have been reported (Chen et al. 2015).

In 1998, investigation of an unusual surge in the incidence of cholera-like infections involving non-O1 V. cholerae in Calcutta, India, revealed the involvement of serotypes such as O144, O11, O6, O8, O12, O19, O39, and O58 that exhibited distinct cytotoxic effect on CHO and HeLa cells, but uniformly lacked any virulence factor associated with O1/O139 V. cholerae (Sharma et al. 1998). Case studies have reported the involvement of non-O1 V. cholerae in pulmonary infection (Shannon and Kimbrough 2006), pneumonia (Marinello et al. 2017), septicemia with meningitis (Hao et al. 2015), necrotizing fasciitis (Ottaviani et al. 2011), and traumatic wound infection (Hirota et al. 2010).

Vibrio parahaemolyticus

V. parahaemolyticus is a Gram-negative, halophilic bacterium widespread in coastal waters and seafood. Fujino first discovered the microorganism following an outbreak of food poisoning in 1950 associated with consuming partially cooked sardine (shirasu) in Japan (Chakraborty et al. 1997). These microorganisms are natural inhabitants of inshore marine waters of temperatures above 15 °C, varying salinity and estuaries, and prevalent in warm summer (DePaola et al. 2003; Ndraha and Hsiao 2019; Fernández-Vélez et al. 2023).

V. parahaemolyticus is a major cause of seafood-borne illness worldwide, characterized by gastroenteritis and wound infections. The disease is distinguished by diarrhea, headache, vomiting, nausea, abdominal cramps, and low fever. Consumption of raw or partially cooked seafood, especially bivalves like oysters and clams, is frequently associated with food-borne infections caused by V. parahaemolyticus. The pathogenic strains of V. parahaemolyticus are distinguished by the presence of hemolysins, thermostable direct hemolysin (TDH) factor, and/ a TDH-related hemolysin (TRH) encoded by tdh and trh genes with about 70% nucleotide sequence similarity (Nishibuchi et al. 1985, 1989). A pandemic clone of O3:K6 harboring the tdh gene but not trh has been responsible for many outbreaks in Asia and the US since its first report from Kolkata, India, in the mid-nineties (Okuda et al. 1997).

Subsequently, other serotypes such as O1:KUT, O4:K68, and O1:K25 were also found to belong to the pandemic group of V. parahaemolyticus, presumably evolved from O3:K6 serotype since all these serovars were genetically indistinguishable based on their arbitrarily primed PCR, ribotyping and pulsed-field gel electrophoresis (PFGE) patterns (Okuda et al. 1997; Matsumoto et al. 2000; Okura et al. 2003; Chowdhury et al. 2004). The pandemic clones are characterized by specialized genetic traits that distinguish them from pre-pandemic strains of the same serovars, such as the presence of the tdh and the absence of the trh gene and a characteristic toxRS sequence (toxRS/new) with point mutations that can be used to detect the pandemic clones using a group-specific PCR (GS-PCR) (Matsumoto et al. 2000). Recently, a new type of V. parahaemolyticus, “O4:KUT-recAin,” with a new type of K antigen and a 25 kb genomic island inserted in the housekeeping gene recA has been reportedly a predominant cause of infections in China (Chen et al. 2020).

The tdh- and trh-positive V. parahaemolyticus are frequently encountered in marine fish and shellfish. Bivalve mollusks such as clams, oysters, scallops, etc., are important sources of human infections since these filter-feeding organisms accumulate V. parahaemolyticus in their muscle to levels much higher than in the surrounding water and sufficient to cause human infections (Deepanjali et al. 2005; HongYou et al. 2014; Mok et al. 2019; Hong To et al. 2020). Wild-caught and farmed shrimp are also important vehicles of pathogenic V. parahaemolyticus (Ayyappan et al. 2018; Changsen et al. 2023). V. parahaemolyticus has been recognized as the causative agent of acute hepatopancreatic necrosis disease in shrimp, further emphasizing this bacterium’s increasing importance as a zoonotic pathogen (Hong To et al. 2020).

Vibrio vulnificus

V. vulnificus can cause a range of infections, including gastroenteritis, sepsis, necrotizing fasciitis, and cellulitis, with a case fatality rate as high as 50%, particularly when susceptible individuals with underlying debilitations such as chronic liver disease and iron overloaded-condition (Kathleen et al. 2016; Heng et al. 2017). V. vulnificus is responsible for over 95% of the fatal cases of seafood-borne infections in the US (Kathleen et al. 2016). Nearly 25% of V. vulnificus infections are associated with the exposure of open wounds to seawater containing this pathogen (Bross et al. 2007).

Like all other Vibrio spp., V. vulnificus is widely distributed in coastal-marine water associated with the chitinous exoskeleton of marine crustaceans and gets concentrated to infectious levels in filter-feeding bivalve mollusks (Heng et al. 2017; Bonnin-Jusserand et al. 2019). While infections occur with raw or undercooked seafood consumption, wound infections leading to bacteremia can occur when exposed to seawater containing these bacteria or while handling seafood. The abundance of V. vulnificus increases with seawater warmer above 20 °C and at a moderate salinity of 15–25 ppt in temperate waters (Motes et al. 1998).

In contrast, in the tropics, V. vulnificus abundance is influenced more by salinity than the water temperature (Parvathi et al. 2004). At lower temperatures (< 13 °C), the bacterium enters into a viable but non-culturable (VBNC) state (Oliver 2010). Of three known biotypes of V. vulnificus, biotype 1 is predominantly responsible for human infections, biotype 2 is an eel pathogen, and biotype 3, a hybrid between biotypes 1 and 2 first reported in Israel, is a pathogen of freshwater fish such as tilapia and carps. However, the microorganism has been reported to cause human infections, such as severe soft tissue infections, with a relatively lower mortality rate than biotype 1 (Zaidenstein et al. 2008; Jones and Oliver 2009; Oliver et al. 2012). Climate change leading to an increase in seawater surface temperature is predicted to dramatically increase the cases of V. vulnificus infections in the future (Archer et al. 2023).

Unlike V. cholerae and V. parahaemolyticus, no specific virulence factor has been associated with the pathogenesis of V. vulnificus. Some of the important virulence factors include a cytotoxic pore-forming toxin (VvhA; V. vulnificus hemolysin), capsular polysaccharide (CPS), and a multifunctional autoprocessing repeats-in-toxin (MARTX) (Jones and Oliver 2009; Yuan et al. 2020; Li and Wang 2020). Other putative virulence factors include metalloproteases, collagenases and phospholipases, siderophores, fimbriae, and others (Gulig et al. 2005; Jones and Oliver 2009; Horseman and Surani 2011).

Antimicrobial resistance in Vibrio spp.

Antimicrobial agents are indispensable in human clinical medicine and have saved humankind from life-threatening microbial infections and continue to do so. Apart from being a therapeutic drug, these agents are also administered in sub-therapeutic doses among livestock, poultry, and aquaculture to prevent disease and promote growth (Lekshmi et al. 2017). Consequentially, imprudent use of these antimicrobial agents leads to the development and spread of bacterial antimicrobial resistance (Loo et al. 2020). While the use of antibiotics in aquaculture for the treatment of bacterial infections or as a prophylactic measure can promote the development of antibiotic resistance in Vibrio spp., the antibiotic residues in farm effluents can have a similar effect on the bacteria in the environment surrounding aquaculture farms. Although vibrios are usually sensitive to antimicrobials of veterinary and human importance (Kumar et al. 2017), there are incidence reports of multidrug-resistant vibrios with different antibiotic susceptibility profiles in clinical settings (Reyhanath and Ranjeet 2014; Tan et al. 2020). The antimicrobial resistance in vibrios in wild-caught and farmed fish and shellfish is a serious threat due to their zoonotic potential and the dissemination of such bacteria in the consumer community through the food chain (Martínez 2008).

The massive diarrheal condition observed in cholera patients causes dehydration and could be fatal if not treated early. Effective management of cholera involves oral rehydration, and antibiotic treatment is generally not warranted. However, antibiotics can drastically reduce the fecal shedding of V. cholerae and help prevent environmental contamination and transmission of cholera. V. cholerae is generally sensitive to frequently prescribed antimicrobials, including β-lactams, aminoglycosides, tetracycline, and quinolone antibiotics.

However, the incidences of antimicrobial resistance in cholera and non-cholera vibrios have increased (Costa et al. 2021). This condition can be attributed to their antibiotic exposure and the acquisition of antibiotic-resistance genes from other Gram-negative bacteria in the environment. Tetracycline and azithromycin have been the drugs of choice for treating cholera for close to four decades until the first emergence of tetracycline resistance in Bangladesh in 1979, in which the O1 isolates were also resistant to ampicillin, kanamycin, streptomycin, and trimethoprim-sulfamethoxazole (Glass et al. 1980).

On the other hand, several prior studies reported the isolation of antibiotic-resistant V. cholerae, such as biotype El Tor, from cholera cases (Kuwahara et al. 1967; Prescott et al. 1968). Rahal et al. (1973) reported V. cholerae resistant to tetracycline, chloramphenicol, sulphonamides, ampicillin, streptomycin, and kanamycin in Algeria (Rahal et al. 1973). Nearly 76% of V. cholerae El Tor strains isolated during the fourth epidemic outbreak in Tanzania were resistant to tetracycline, a phenomenon attributed to the extensive therapeutic and prophylactic use of tetracycline. (Towner et al. 1980). Following this, resistance to tetracycline and other commonly employed antibiotics in cholera therapy was reported from different geographical regions worldwide (Threlfall et al. 1980; Ichinose et al. 1986; Ouellette et al. 1988; Tabtieng et al. 1989; Saraswathi and Deodhar 1990; Dalsgaard et al. 2000).

V. cholerae O1 El Tor serotype Ogawa isolated in Honduras were resistant to tetracycline, trimethoprim-sulfamethoxazole, kanamycin, gentamicin, chloramphenicol, ampicillin, cephalothin, and doxycycline (Dubon et al. 1997). Over 75% of the V. cholerae O1 strains isolated in Kolkata, India, were resistant to tetracycline (Bhattacharya et al. 2011). Multidrug-resistant V. cholerae El Tor Biotype, Ogawa serotype resistant to tetracycline, trimethoprim, co-trimoxazole, nalidixic acid, polymyxin B, spectinomycin, streptomycin, and sulfamethoxazole was reported from a cholera outbreak from Odisha, Eastern India in 2010 (Jain et al. 2016). Similarly, V. cholerae O1 El Tor Ogawa isolated from 2012 to 2015 cholera outbreaks in Mozambique were 100% resistant to sulphamethoxazole-trimethoprim, while more than 90% of the isolates were resistant to ampicillin, nalidixic acid, chloramphenicol, and nitrofurantoin, and were susceptible to azithromycin and streptomycin (Dengo-Baloi et al. 2017). In recent times, frequent isolation of V. cholerae O1 and O139 strains resistant to all clinically employed drugs has been reported from different parts of the world (Verma et al. 2019; Chatterjee et al. 2020).

Antimicrobial resistance is being reported regularly among vibrios of pathogenic importance. The β-lactamase-associated resistance mechanisms were identified in V. harveyi, V. alginolyticus, and V. parahaemolyticus associated with seafood in Italy (Ottaviani et al. 2001). In Mexico, 70% of Vibrio isolates from penaeid shrimp were resistant to carbenicillin and ampicillin and harbored resistance plasmids (Molina-Aja et al. 2002). Multiple antimicrobial resistance was also reported in Vibrio strains from wastewater final effluents, indicating the spread of resistance genes in a sewage plant, posing a threat to communities dependent on such water bodies (Okoh and Igbinosa 2010).

An investigation on farmed fishes from South Korea during 2005–2007 reported drug resistance in V. parahaemolyticus and V. alginolyticus with maximum resistance against ampicillin (Oh et al. 2011). Multi-drug resistance was also observed in both strains, with a higher incidence in V. alginolyticus (Oh et al. 2011). Studies from across the globe have reported plasmid-mediated resistance against clinically relevant antibiotics in vibrios from wild-caught and farmed fish and shellfish (Manjusha and Sarita 2011; Reyhanath and Ranjeet 2014; Letchumanan et al. 2015; Loo et al. 2020). Antibiotic resistance with an average MAR index of 0.77 among the Vibrio isolates from crustaceans of retail markets has been reported from Egypt (Ahmed et al. 2018).

Several reports suggest that V. parahaemolyticus is becoming increasingly resistant to previously effective antibiotics such as tetracycline, aminoglycosides, cephalosporins, fluoroquinolones, and others (Elmahdi et al. 2016; Dutta et al. 2021; Grudlewska-Buda et al. 2023). Irrespective of the geographic region, resistance to ampicillin, penicillins, and tetracyclines is observed in V. parahaemolyticus worldwide (Elmahdi et al. 2016). V. parahaemolyticus isolated from seafood in Malaysia exhibits resistance to ampicillin, cefazolin, and penicillin. Resistance to more than one antibiotic was found in 90% of the isolates, with the MAR index ranging from 0.04 to 0.71 (Tan et al. 2020). As high as 24% of the seafood samples in Bulgaria were found contaminated with V. parahaemolyticus resistant to ampicillin, cefepime, and ceftazidime, with the MAR index ranging from 0.10 to 0.30. (Stratev et al. 2023). A study on the market samples of seafood sold in Vietnam reported 86% of V. parahaemolyticus isolates to be resistant to at least one antibiotic tested, with the highest resistance observed against ampicillin, followed by cefotaxime, ceftazidime, trimethoprim-sulfamethoxazole, and tetracycline (Vu et al. 2022).

Numerous studies have reported the isolation of V. parahaemolyticus resistant to antibiotics such as ampicillin and cephalothin (Silva et al. 2018), ampicillin, cefazolin, and penicillin (Tan et al. 2020), ampicillin, gentamicin, tetracycline, and fluoroquinolones (Lei et al. 2020), ampicillin, ampicillin/sulbactam, cefuroxime, cefoxitin, ceftazidime, cefepime, and colistin (Coutinho et al. 2019), cephalothin, cefoxitin and ceftazidime (da Silva et al. 2021), imipenem (Lee et al. 2018), amoxicillin/clavulanate, cefoxitin, cefotaxime, ceftazidime, cefuroxime, cefepime and trimethoprim/sulfamethoxazole in a New Delhi metallo-β-lactamase (blaNDM)-positive isolate (Briet et al. 2018), ampicillin, cefpodoxime, cefotaxime, ceftizoxime, tetracycline, ceftriaxone, ciprofloxacin and nalidixic acid (Parthasarathy et al. 2021).

V. vulnificus is susceptible to most antimicrobial agents such as β-lactams, third-generation cephalosporins, carbapenems, aminoglycosides, tetracyclines, fluoroquinolones, trimethoprim-sulfamethoxazole, and chloramphenicol. Nevertheless, the antimicrobial resistance in this pathogen is gradually increasing, with reports emerging on the isolation of strains resistant to clinically employed antibiotics such as ampicillin, doxycycline, tetracycline, aminoglycosides, macrolides, ciprofloxacin, amoxicillin, imipenem, aztreonam, and cephalosporins (Han et al. 2007; Baker-Austin et al. 2009; Elmahdi et al. 2016).

Mechanisms of antimicrobial resistance in Vibrio spp.

Early reports suggested the presence of a transmissible resistance (R) factor in V. cholerae (Prescott et al. 1968; Rahal et al. 1973; Hedges and Jacob 1975; Yokota and Kuwahara 1977). Subsequent studies on the antibiotic susceptibility of V. cholerae, a self-transmissible 62-kb, chromosomally integrating genetic element called the SXT element, which carried resistance genes to sulfamethoxazole, trimethoprim, and streptomycin was found in serotype O139 (Waldor et al. 1996). The genetic mechanisms of antimicrobial resistance identified in clinical and environmental isolates of V. cholerae include mobile genetic elements such as plasmids, transposons, integrons, and integrating conjugative elements (ICEs) (Verma et al. 2019; Das et al. 2020). V. cholerae O1 El Tor strains isolated from outbreaks in Siberia and the Russian Far East carried SXT/R391 ICEs and genes such as strA, strB (streptomycin), catB9/floR (chloramphenicol/ florfenicol), sul2 (sulfonamide), dfrA1/ dhfR (trimethoprim, and cotrimoxazole), and tet(A) (tetracycline) (Gladkikh et al. 2020). The multidrug-resistant (MDR) V. cholerae El Tor Biotype Ogawa serotype isolates from the 2010 outbreak in Odisha, India, carried elements like class I integron, SXT element, aadA2 (aminoglycoside resistance), and dfrA1 (trimethoprim resistance) genes (Jain et al. 2016). The whole genome sequence (WGS) of an MDR V. cholerae strain contained genetic elements coding for resistance to multiple antibiotics such as sulfonamide (sul2), trimethoprim (dfrA1), chloramphenicol (catB9), florfenicol (floR), and tetracycline [tet(34)] (Mevada et al. 2023). Novel genomic islands identified in V. cholerae non-O1/non-O139 carried mobile genetic elements such as Class 1 integrons and quinolone resistance determinants (Morita et al. 2020).

The β-lactam genes such as blaCTX-M, blaTEM, and blaSHV have been detected in Vibrio isolates recovered from bivalves sold in retail markets (Dahanayake et al. 2020). The ESBL (extended-spectrum β-lactamase) genes blaCTX-M (87.5%), blaTEM (40.6%), and blaSHV (21.8%) were reported in Vibrio isolates from raw mussels in Korea (Hossain et al. 2020; Dahanayake et al. 2020). In another study, the β-lactamase encoding blaSHV genes were detected in Vibrio isolates from water samples coast off North-East Sardina. At the same time, none of the Vibrio spp. harbored the blaTEM gene (Zanetti et al. 2001). In India, the blaTEM gene in Vibrio isolates from estuaries, shrimp farms, and seafood samples has been reported (Silvester et al. 2019). While the blaCTX-M was not detected in any isolates, the blaNDM gene was found in 13.3% of the strains. A recent study made the first report of plasmid-encoded colistin resistance mcr-1 in a virulent strain of V. parahaemolyticus isolated from a shrimp sample in Hong Kong, China (Lei et al. 2019). The isolate carried blaCARB17 and quinolone resistance gene qnrVC5. Ampicillin resistance is widely prevalent in clinical and environmental isolates of V. parahaemolyticus, which, in addition to the production of β-lactamases, has been attributed to decreased synthesis of penicillin-binding proteins (Meng et al. 2023).

The quinolone resistance-determining regions (QRDR) of gyrA and parC and the plasmid-mediated quinolone resistance (PMQR) genes have been reported from fluoroquinolone-resistant V. parahaemolyticus with Ser-83-Ile, Ser-83-Phe substitutions in GyrA, and a Ser-85-Leu substitution in ParC (Lei et al. 2020). A novel class of chloramphenicol acetyltransferase, type C CAT or CATC, encoded by the catC gene identified in V. parahaemolyticus, confers varying levels of intrinsic resistance to chloramphenicol (Zhang et al. 2019). Recent studies have reported the occurrence of the blaNDM-harboring V. parahaemolyticus and V. vulnificus strains from seafood and the environment (Briet et al. 2018; Oyelade et al. 2018). The blaTEM and blaCMY genes are also found in V. parahaemolyticus and V. vulnificus isolates (Oyelade et al. 2018). The WGS of NDM-positive V. parahaemolyticus revealed the presence of multiple antibiotic resistance genes such as sul1, sul2, dfrA16, strA, strB and aadA2, floR and tet(A) (Briet et al. 2018). A study showed the multiple mediators of tetracycline resistance in V. parahaemolyticus involving five different types of tet genes, viz. tet(34), tet(A), tet(B), tet(M), and tet(E) (Ye et al. 2023). Three different mutations (Q513K, S522L, and H526Y) in the rpoB gene of V. vulnificus have been attributed to high resistance to rifampicin (Cutugno et al. 2020). The ESBL-producing V. vulnificus isolated from imported seafood harbored a plasmid encoding ISEc9 upstream of the blaCTX-M-55 and qnrS2 genes (Nakayama et al. 2023).

Antimicrobial efflux pumps

The known species of Vibrio are resistant to multiple structurally distinct antimicrobial agents using a variety of disparate solute transporter systems. The transport of antimicrobial solutes across the membranes of living cells, including those of Vibrio spp., is conferred by integral membrane proteins, collectively called solute transporters (Stein 2012). Passive transport systems do not use biological energy to drive the passage of solutes and water across the biological membrane of the Vibrios (Sten-Knudsen 1978). Such transport mechanisms are driven by the energy of the solute or substrate gradients across the membrane. Active solute transporters, however, are driven by the energy stored in ATP released upon hydrolysis, as seen in primary active transport (Stein 1986), and the release of energy inherent in the membrane gradients of ions, as documented in secondary active transport, or co-transport, systems (West 1980; Stein 1986).

The efforts from the laboratory of Saier and colleagues led to the establishment and maintenance of the transporter classification database (TCD) https://www.tcdb.org/ (Saier et al. 2021). The TCD platform compiles transporters based on their functional properties and phylogenetic relationships between groups of homologous and related proteins. In general, transporters in living cells have been grouped into large superfamilies harboring sub-families with members that share similarities in sequence and structure, Fig. 1. Solute transporters are often evolutionarily conserved across all living taxa (Morita and Li 2016). The membrane transport proteins of Vibrio species fall into several large superfamilies (Table 1, Fig. 1). The ATP-binding cassette (ABC) superfamily encompasses the primary active transporters that utilize the hydrolysis of ATP to mediate the “uphill” transport of solutes across the membrane (Huda et al. 2003; Stephen et al. 2023). The efflux pumps of the secondary active transport type belong to the major facilitator superfamily (MFS), the resistance-nodulation-cell division (RND) superfamily, and the multidrug/oligosaccharidyl- lipid/polysaccharide (MOP) superfamily, the proteins of which function to extrude diverse solutes/substrates out of the cell and play critical roles in the antimicrobial resistance, virulence, and environmental persistence of Vibrio spp.

Fig. 1
figure 1

Efflux proteins belonging to different superfamilies/families in Vibrio spp. The ABC superfamily of efflux pumps are primary active transporters energized by hydrolysis of ATP. In contrast, all other groups of proteins belong to the secondary active transporters, which utilize an electropotential gradient of H+/Na+ (cations) across the membrane to power the movement of solutes

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Table 1 Antimicrobial transporters, substrates, superfamilies, and transmembrane segment numbers in transporters of Vibrio spp.
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The multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) superfamily of exporters includes members of the multidrug and toxic compound extrusion (MATE) (Kuroda and Tsuchiya 2009; Upadhyay et al. 2019; Claxton et al. 2021), the prokaryotic polysaccharide transporter (PST), the oligosaccharidyl-lipid flippase (OLF) from eukaryotes, and the mouse virulence factor (MVF) families of proteins (Hvorup et al. 2003). Likewise, the drug/metabolite transporter (DMT) superfamily contains related members of other transporter families, like the small multidrug resistance (SMR) family (Jack et al. 2001). The SMR family members QacEΔ1 and QacH have been studied in various V. cholerae non-O1 and V. parahaemolyticus isolates and have been shown to confer resistance to several quaternary ammonium compounds (Kazama et al. 1999; Ceccarelli et al. 2006). The SMR transport proteins have about 110 amino acids and four transmembrane domains, and they are postulated to oligomerize to form fully intact drug transporters (Jack et al. 2001).

The SMR and rhamnose transporter (RhaT) families from bacteria and the triose phosphate transporter (TPT) and the nucleotide-sugar transporter (NST) families from organelles of eukaryotic organisms and are discussed in detail elsewhere (Jack et al. 2001). More recently, the proteobacterial antimicrobial compound efflux (PACE) superfamily of proteins was discovered, harboring many multidrug efflux pumps energized by ion/substrate antiport, most uncharacterized (Hassan et al. 2021).

The major facilitator superfamily (MFS) consists of members that are passive and secondary active transporters from bacteria to humans (Henderson 1990, 1993; Griffith et al. 1992). The proteins of the resistance-nodulation-cell division (RND) class of secondary active transporters confer efflux of antimicrobial agents in bacteria, especially from the cells of the Vibrio genus of bacteria (Bina and Bina 2023). Together, the MFS and the RND transporters represent the most well-studied transporters of V. cholerae. This review article focuses on recent findings regarding the MFS, RND, and MATE transporters.

RND efflux pumps and their role in antimicrobial resistance and virulence

The RND efflux pumps are indispensable to the physiology of Vibrio spp. and play a critical role in their environmental persistence and pathogenesis (Stephen et al. 2022). V. cholerae, V. parahaemolyticus, and V. vulnificus genomes encode six, 12, and 11 RND efflux pumps, respectively (Taylor et al. 2012; Liu et al. 2022; Stephen et al. 2022). The unique structural architecture of the RND pumps consists of a tripartite protein assembly consisting of an outer membrane protein (OMP) and an inner membrane protein (IMP) connected by a periplasmic membrane fusion protein (MFP) (Fig. 2). The entire setup is organized into a continuous transmembrane channel, allowing the movement of substrates in an inward-outward fashion (protoplasm to the outside) without entering the periplasmic space (Nikaido 1994, 2011; Destoumieux-Garzón et al. 2014; Symmons et al. 2015). The antiport activity is vested with the inner membrane protein (IMP) energized by proton (H+) gradients. Although this membrane channel is expected to transport lipophilic substrates preferentially, the RND efflux pumps are generally non-specific and allow the extrusion of diverse substrates of all charge types (Nikaido 1998, 2011). As an exception to all known RND pumps, VexEF is energized by the electrochemical gradient of Na+ ions instead of H+ (Rahman et al. 2007).

Fig. 2
figure 2

Transport proteins from Vibrio bacteria. Depicted are known protein structures from species of the Vibrio genus, representative examples of which include a AcrAB-TolC of V. vulnificus (PDB 5V5S), b MsbA from V. cholerae (PBD 5TTP), c NorM (PDB 7PHP) from V. cholerae, d YddG, (PDB 5I20) a known DMT protein that is homologous to a putative permease from V. vulnificus, and e EmrD (PDB 2GFP), which is homologous to EmrD-3 of V. cholerae

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The Vex group of the RND efflux pumps (VexAB, VexCD VexEF, VexGH, VexIJK, VexLM) from V. cholerae O1 are a group of bile-regulated proteins that confer resistance to bile and certain antimicrobial compounds. A whole-genome comparison of a non-O1 V. cholerae strain PS15 with V. cholerae O1 strain N16961 identified all of these efflux pumps except VexE (Mukherjee et al. 2014). The vexAB, vexCD, vexEF, vexGH, and vexIJK elements are on the large chromosome. At the same time, the vexLM gene is located on the small chromosome. All are organized into operons with an MFP-encoding gene upstream of the efflux pump (IMP) gene, and the operon does not contain the OMP-encoding gene (Bina and Bina 2023). Since bile acids are antimicrobial, bile resistance is necessary for successful intestinal colonization by a pathogenic bacterium such as V. cholerae. Early insights into the role of RND efflux pumps in bile resistance suggested the functional role of VexAB in intrinsic bile resistance in V. cholerae O1. At the same time, VexCD complemented this function of VexAB (Bina et al. 2006). The importance of RND efflux pumps in bile and antimicrobial resistance has been demonstrated using deletion mutants of V. cholerae. V. cholerae El Tor strain N16961 lacking all six RND efflux pumps was hypersensitive to bile, detergents, and antibiotics, produced significantly less cholera toxin and TCP protein, and was unable to colonize infant mouse intestines, suggesting the synergistic role of RND efflux pumps in the physiology and pathogenesis of V. cholerae (Bina et al. 2008).

Further, the absence of RND-mediated efflux resulted in the accumulation of metabolites in the periplasmic space, leading to the activation of periplasmic sensors involving two-component systems and the ToxR regulator as an adaptive response (Bina et al. 2018). This study conclusively showed the role of RND efflux pumps in regulating the expression of ToxR-mediated virulence factors, including TCP and the cholera toxin. However, this mechanism involves a complex network of proteins responding to stress in V. cholerae (Bina et al. 2018). Another study demonstrated the role of VexGH in Vibriobactin secretion in V. cholerae. Deleting the vexGH locus impaired homeostasis and reduced fitness under iron-limiting conditions (Kunkle et al. 2017; Du et al. 2018).

Without an identified OMP-encoding gene within the vex operon, researchers attempted to express V. cholerae Vex RND proteins in E. coli with the TolCvc outer membrane factor from V. cholerae non-O1 (Rahman et al. 2007). The VexAB and VexCD components could function using TolCvc and confer elevated MICs of cholates. VexAB-TolCvc also increased the MICs of erythromycin, novobiocin, rhodamine 6G, and TPPCl (Rahman et al. 2007). Similarly, VexEF-TolCvc was functional in an E. coli background and could efflux ethidium bromide in the presence of Na+. No definite antimicrobial substrate has been reported for the VexEF and VexLM pumps. However, VexEF showed efflux activity for bile, detergents, erythromycin, and novobiocin in an E. coli background (Rahman et al. 2007; Taylor et al. 2012; Bina and Bina 2023).

The efflux pumps homologous to VexAB, VexCD, and E. coli AcrAB (Fig. 2) encoded in the V. vulnificus genome have been shown to alter the bacterium’s antimicrobial susceptibilities. Increased susceptibility to erythromycin, ethidium bromide, acriflavine, and bile salts was observed in VexAB deletion mutants. Further, increased susceptibility was seen only to acriflavine in an AcrAB homolog mutant. No change in antimicrobial susceptibility was observed in a VexCD homolog deletion mutant, suggesting that these efflux pumps differ substantially in their preferred substrates and might contribute to other important physiological activities of the bacterium needing further investigation (Lee et al. 2015).

Twelve RND pumps of V. parahaemolyticus have been cloned in a hypersensitive E. coli background and characterized (Matsuo et al. 2007, 2013). Although VmeAB conferred significantly higher MICs of different antimicrobial agents in E. coli, the mutant strain of V. parahaemolyticus lacking VmeAB did not show the same resistance levels. However, a slight increase in the MICs was observed (Matsuo et al. 2007). In contrast, other efflux pumps, VmeCD, VmeEF, and VmeYZ, showed higher MICs in E. coli, and the effect was similar in deletion mutants of these efflux pump-encoding genes (Matsuo et al. 2013). Further, VmeAB, VmeCD, VmeEF, and VmeYZ co-expressed with VpoC, an outer membrane component in V. parahaemolyticus in E. coli, exhibited significantly higher MICs of various antimicrobials compared to the same efflux pumps functional with TolC of E. coli (Matsuo et al. 2013). VmeGHI, VmeJK, VmeLM, and VmeTUV were functional and could confer higher MICs only with VpoC. A null mutant lacking all 12 RND efflux pumps was highly sensitive to various antimicrobials. It showed a significantly lower fluid accumulation in rabbit ileal loops, suggesting that RND efflux pumps in V. parahaemolyticus contribute to its pathogenicity. These RND efflux pumps function to resist deoxycholates and other antimicrobials, are involved in the production or transport of siderophore vibrioferrin, and contribute to the pathogenicity of V. parahaemolyticus.

Proteomic profiling of the integral membrane proteins in V. parahaemolyticus revealed the involvement of the periplasmic adaptor subunit and the permease subunit of the RND efflux pump in virulence (Pérez-Acosta et al. 2018). The deletion of a membrane fusion protein, VmeL, belonging to the RND efflux pumps of V. parahaemolyticus resulted in lowered cytotoxicity towards HeLa cells, reduced virulence in a murine intraperitoneal infection assay, and an absence of lateral flagella hindering the motility (Liu et al. 2022). A ΔvmeL transcriptome analysis revealed a downregulation of genes related to the Type III Secretion 1 system (Liu et al. 2022), an important virulence factor involved in adhesion to the host cell (Wu et al. 2020; Liu et al. 2022). Similar studies in V. vulnificus involving the deletion of three RND efflux pumps VexBHZ showed increased cytotoxicity and biofilm formation (Lo et al. 2017). Analysis of whole genome sequences of Vibrio spp. can help identify new RND-type efflux pumps contributing to antimicrobial resistance (Lloyd et al. 2019).

MATE efflux pumps

The MATE group of ion/drug antiport systems is widely encountered in Vibrio spp. as well as in other Gram-negative and -positive bacteria. The first MATE-type transporter to be described was NorM from V. parahaemolyticus (Morita et al. 2000). Subsequently, proteins belonging to this group were reported from all domains of life performing essential tasks of detoxification and maintaining cellular homeostasis. These proteins actively extrude cationic substrates, including xenobiotics, drugs, toxic metabolites, hormones, and organic acids in plants (Kusakizako et al. 2020). The MATE efflux proteins fold into 12 transmembrane helices similar to the MFS transporters (Fig. 2). However, the topology of MATE proteins is distinctly different from the MFS family of efflux proteins, suggestive of different evolutionary origins of these two groups of membrane proteins (He et al. 2010; Kusakizako et al. 2020). Homologous MATE-type efflux pumps in other bacteria are NorM-VC, VcmA, and VcrM of V. cholerae (Huda et al. 2003), YdhE of Escherichia coli (Brown et al. 1999; Morita et al. 2000), BexA of Bacteroides thetaiotaomicron (Miyamae et al. 2001), PmpM from P. aeruginosa (He et al. 2004), NorM-NG of Neisseria gonorrhoeae (Rouquette-Loughlin et al. 2003), AbeM of Acinetobacter baumannii (Su et al. 2005), NorM of Erwinia amylovora (Burse et al. 2004), and KetM of K. pneumoniae (Ogawa et al. 2015). These transporters extrude cationic compounds such as DNA-intercalating dyes, detergents, aminoglycoside, and fluoroquinolone antibiotics. Although a majority of these are Na+-dependent, efflux pumps of this category either use H+ and Na+ simultaneously or H+ alone. For example, V. cholerae NorM can simultaneously couple with Na+ and H+, while AbeM of Acinetobacter baumannii is uniquely H+-dependent (Su et al. 2005; Jin et al. 2014). Similarly, VcmB, VcmD, and VcmH efflux pumps are Na+-dependent, while VcmN is Na+-independent (Begum et al. 2005). The homologous MATE efflux pumps are grouped into three clusters (Brown et al. 1999). Cluster 1 contains NorM of V. parahaemolyticus (NorM-VP), V. cholerae (NorM-VC), and YdhE of E. coli, while DinF of E. coli and its homologues such as VcmN of V. cholerae and PfMATE of Pyrococcus furiosus are in cluster 3 (Tanaka et al. 2013; Ogawa et al. 2015). Cluster 2 consists of eukaryotic MATE-type transporters. Mutational analyses have revealed that Asp-371 of NorM from V. cholerae is essential for substrate transport, and this residue is widely conserved across the Na+-dependent MATE transporters of cluster 1 (Otsuka et al. 2005; Ogawa et al. 2015). The cation-bound crystal structure of NorM-VC showed two amino acid residues, Glu-255 and Asp-371, located at the center of C-terminal lobes formed by TM7-12 constitute the substrate-binding pockets (He et al. 2010; Kusakizako et al. 2020). (Otsuka et al. 2005) showed that Asp-32, Glu-251, and Asp-367 in NorM-VP play important roles in the Na+-dependent drug transport process. Asp-367 in NorM-VP corresponds to Asp-371 of NorM-VC, and the replacement of Asp-367 with either alanine or asparagine severely impaired the drug transport activity of NorM-VP (Otsuka et al. 2005).

Lu and colleagues determined the crystal structure of Na+-coupled NorM-NG from Neisseria gonorrhoeae bound to three different substrates. They showed that Asp-41, Phe-265, Gln-284, Asp-355, and Asp-356 are essential for transport function, and these are supposedly conserved across prokaryotic and eukaryotic MATE-type transporters (Lu et al. 2013). Na+ and substrate bind distinct amino acids in NorM and bind simultaneously, in contrast to the general notion that Na+ and the substrate bind at two different stages corresponding to two different conformational statuses of the efflux protein (Lu et al. 2013). Asp-377 in NorA-NG and its corresponding residues Asp-367 and Asp-371 in NorA-VP and NorA-VC, respectively, have been shown to be critically important for substrate transport (Otsuka et al. 2005; Lu et al. 2013).

The crystal structure of another H+/drug antiport protein from V. cholerae, VcmN, and the subsequent molecular simulation studies have provided significant insights into the dynamics of ion coupling in this as well as other proteins of the DinF subfamily (Kusakizako et al. 2020; Castellano et al. 2021; Claxton et al. 2021). VcmN, originally proposed to be an H+/drug antiport with water bound to the N-lobe cavity, was later shown to exhibit Na+-dependent changes in conformation, suggesting that it could be Na+-driven like a majority of other MATE transporters (Kusakizako et al. 2020; Castellano et al. 2021; Claxton et al. 2021).

Two MATE efflux pumps of H- and D-type characterized from clinical isolates of V. fluvialis were dependent on Na+/K+ ions, and of these, the H-type pump extruded fluoroquinolones, ethidium bromide, and safranin, while the D-type extruded ethidium bromide only (Mohanty et al. 2012, 2021). Interestingly, unlike other MATE-type transporters, the H- and D-type pumps of V. fluvialis are made up of 10 and 11 TMS, respectively. However, molecular docking studies revealed a similar transport mechanism to NorM, with an aspartic acid residue in TM-1 acting as an ion binding site (Mohanty et al. 2021).

MFS efflux pumps

The multidrug efflux pumps of the MFS constitute a large group of related transporters from all known taxa (Pao et al. 1998; Saier et al. 1999). The MFS proteins share conserved sequences and structures, suggesting they share similar transport mechanisms across the membrane (Griffith et al. 1992; Varela and Griffith 1993). These transport systems confer resistance to antimicrobial and anticancer agents (Kumar et al. 2020; Hou et al. 2022). As such, these integral membrane proteins constitute suitable targets for modulation to restore the efficacy of chemotherapy for recalcitrant infections and cancer (Kumar et al. 2013b, 2016a; Stephen et al. 2022; Dhakne et al. 2023).

MFS efflux pumps of Vibrio spp.

According to the complete genome sequence of the V. cholerae O1 biovar El Tor toxigenic strain N16961, 18 genetic elements are thought to encode major facilitator superfamily transporters (Heidelberg et al. 2000). Several putative efflux pump-encoding genes from Vibrio spp. have been characterized physiologically (Stephen et al. 2022). The first MFS multidrug efflux pump from V. cholerae to be cloned, isolated, and studied for antimicrobial resistance is VceB, an integral membrane protein with 14 predicted transmembrane (TM) helices (Colmer et al. 1998). The vceB gene is encoded on the V. cholerae genome as part of the VceCRAB operon, where VceA and VceB are homologous to Escherichia coli proteins EmrA and EmrB, respectively (Lomovskaya and Lewis 1992). The VceCRAB locus encodes a multi-component membrane protein that effluxes multiple antimicrobial agents. VceC is considered an outer membrane protein by its functional similarity to OprM from Pseudomonas aeruginosa (Bai et al. 2010). VceR plays a functional role as a transcriptional regulator (Alatoom et al. 2007). VceA is a periplasmically-located membrane-fusion protein (Woolley et al. 2005). Therefore, the VceCRAB regulon encodes a tripartite antimicrobial efflux system consisting of VceC, VceA, and VceB (Woolley et al. 2005; Alatoom et al. 2007; Bai et al. 2010).

The second MFS multidrug efflux pump system discovered in V. cholerae is EmrD-3, physiologically studied in our laboratory (Smith et al. 2009). We found the emrD-3 gene on the chromosome of an O395 V. cholerae (Kumar et al. 2013a). The predicted structure of EmrD-3 encases another integral membrane protein, largely hydrophobic but with 12 TMs (Smith et al. 2009). We found that EmrD-3 confers transport of ethidium bromide across the membrane in a proton-driven manner and confers elevated resistance to multiple structurally distinct antimicrobial agents, such as linezolid, rifampin, erythromycin, and chloramphenicol, among others. Our laboratory performed a genomic comparative study of the emrD-3 gene from O395 with a toxigenic strain of V. cholerae N16961 and environmental isolate from Puget Sound, a strain denoted as PS15 (Kumar et al. 2013a; Mukherjee et al. 2014). We observed that emrD-3 was located on chromosome II of V. cholerae N16961 but absent from V. cholerae PS15 (Mukherjee et al. 2014). Due to the presence of EmrD-3 in toxigenic V. cholerae but missing in non-toxigenic counterparts, we anticipate that this multidrug efflux pump is a suitable target for antimicrobial resistance modulation to restore and enhance clinical treatment of severe cholera infections (Kumar et al. 2016a). Toward this, our laboratory discovered that both antimicrobial resistance levels and membrane drug transport activities by EmrD-3 are reduced by extracts of Allium sativum (garlic) and its pure bioactive component, allyl sulfide (Bruns et al. 2017). In the same study, we observed that A. sativum extract synergistically affects the V. cholerae growth with multiple antimicrobial agents.

From V. parahaemolyticus, the MFS transporter PvsC was discovered, showing the export of the iron siderophore vibrioferrin (Tanabe et al. 2003). This system is thought to enhance microbial virulence. The gene for PvsC is encoded on the pvsABCDE operon, and it is thought to play a role in the metabolism and efflux of Vibrio-specific siderophores (Tanabe et al. 2006).

Another set of MFS transporters from V. cholerae denoted as Mfs-1 through Mfs-5, five determinants totally, lose resistance to tetracycline and bile when the ORFs are deleted by mutation (Chen et al. 2013). The same study observed that expression programs of the mfs1-5 genes are controlled by a protein homologous to members of a family of well-characterized transcriptional regulators, namely LysR (Maddocks and Oyston 2008).

Role of efflux pumps in the physiology of Vibrio spp.

Using the deduced primary sequence (amino acid) of EmrD-3 as a query sequence in genomic databases, we found homologs in distantly related bacteria, like Proteus mirabilis and Bacillus cereus, but also in other species of the Vibrio genus, such as V. harveyi, V. vulnificus, and V. parahaemolyticus, indicating that these bacteria harbor conserved MFS multidrug resistance-conferring transporters as putative virulence factors (Smith et al. 2009; Mukherjee et al. 2014; Stephen et al. 2022).

Interestingly, the MFS VceB, EmrD-3, and Mfs1-5 transporters harbor elements of highly conserved sequence motifs, such as motif A, present on the loop between helices two and three of virtually all members of the MFS, and the antiporter motif, also called motif C, that resides in the fifth helix of MFS drug and multidrug efflux pumps (Henderson 1990; Varela et al. 1995; Colmer et al. 1998). The functional roles conferred by motifs A and C residues have been extensively studied in transporters of the MFS (Kumar et al. 2016b; Kakarla et al. 2017).

The signature sequence of motif A, “Gly-(X)3-Asp-Arg/Lys-X-Gly-Arg-Arg/Lys,” is highly conserved in virtually all members of the MFS, attesting to the functional importance of its residues in a variety of solute transporters from all living species including multidrug efflux pumps from known species of Vibrio bacteria (Griffith et al. 1992, 1994; Varela and Griffith 1993; Stephen et al. 2022). Thus, the homologous nature of the MFS transporters strongly suggests that molecular physiological studies of proteins from various species apply to the MFS transporters from the Vibrio genus. Structure–function studies of residues belonging to motif A have demonstrated critical roles in bacterial tetracycline efflux pumps, such as antimicrobial binding in TetA(C) of E. coli (McMurry et al. 1980), the substrate channel pathway (Yamaguchi et al. 1992), and elements of the transporter gate in TetA(B) (Someya et al. 2000). Residues of motif A were demonstrated to play important roles in stabilizing the transporter structure by salt-bridge formation in the YajR efflux pump of E. coli (Jiang et al. 2013). In various MFS transporters, motif A residues are proposed to mediate an essential role in conformational changes during substrate transport, such as in the LacY lactose symporter of E. coli (Varela and Wilson 1996). The LmrP drug transporter of Lactococcus lactis, with the protonation of acidic residues, influences a conformational switching system (Masureel et al. 2014). In the same study, the structure formed by residues of motif A appears to detect the energy status of the respiratory chain. This energy-sensing system should be relevant to the secondary active transporters of the MFS.

The laboratories of Skurray and Henderson identified a highly conserved consensus sequence in MFS efflux pumps that operate by an antiport process (Henderson 1990; Rouch et al. 1990). The signature sequence is “Gly-(X)8-Gly-(X)3-Gly-Pro-(X)2-Gly-Gly,” called the “antiporter motif” and later motif C (Varela and Griffith 1993; Varela et al. 1995; Ginn et al. 2000). Interestingly, when multiple amino acid sequence alignments were performed manually, the motif C appeared in symporters of the MFS, implicating a critical role for this conserved sequence in most proteins of the MFS (Yaffe et al. 2013). We showed by mutational analysis that the highly conserved glycine residue of the Gly-Pro dipeptide confers antimicrobial resistance in the TetA(C) tetracycline efflux pump encoded on the cloning plasmid pBR322 in host E. coli (Varela et al. 1995). The functional importance of the Gly-Pro dipeptide was demonstrated in TetA(L) and TetA(K) tetracycline efflux pumps from Staphylococcus aureus and Bacillus subtilis and shown to form a barrier to proton leakage, thus preventing extraneous ion-substrate energy coupling (Konishi et al. 1999; Jin and Krulwich 2002; De Jesus et al. 2005). Residues of motif C were shown to mediate conformational changes during the transport cycle of TetA(K) (Ginn et al. 2000), possibly influencing the direction of antimicrobial transport as predicted (Maiden et al. 1987; Pao et al. 1998; Saier et al. 1999). In the QacA multidrug efflux pump of S. aureus, residues of motif C bind antimicrobial substrates as they are translocated across the membrane (Hassan et al. 2006). An analysis of the structure–function relationship regarding motif C revealed its participation in forming a centrally-located substrate-binding cavity in the CaMdr1p drug transporter of antifungal agents from cells of Candida albicans, a eukaryote (Pasrija et al. 2007). Structurally speaking, in the mammalian vesicular monamine pump VMAT2, motif C forms an interface between the two large global bundles characteristic of the MFS transporters (Yaffe et al. 2013). Along these lines, residues of motif C form a molecular hinge structure that influences conformational changes during drug/ion antiport (Luo and Parsons 2010; Yaffe et al. 2013). Thus, the molecular structure formed by residues of motif C serves as a regulator of conformational switching during antimicrobial efflux in MFS transporters, a process relevant to virulence in cells of Vibrio species (Luo and Parsons 2010; Stephen et al. 2022).

Efflux pumps in biofilm formation and quorum sensing

The toxin-coregulated pilli (TCP) in V. cholerae is known to play a role in biofilm differentiation. The ΔTcpA (TCP pilin subunit) mutant in biofilms on the chitinous surface was undifferentiated, leading to high sensitivity to biocide like sodium dodecyl sulfate (SDS). Upon exposure to 0.2% SDS, the ΔTcpA mutant cells disassociated from the undifferentiated biofilm within 5 min, while the wildtype V. cholerae El Tor biofilm remained unaffected even after 30 min (Reguera and Kolter 2005). The RND efflux pump, like VexH in V. cholerae, is vital for the production of TCP (Taylor et al. 2012). Thus, the RND efflux pumps might also form biofilm on chitinous surfaces like copepods and zooplankton, which are associated with cholera outbreaks (Lipp et al. 2002). The biofilm formation capacity of V. parahaemolyticus was compromised upon deletion of VmeL, a membrane fusion protein belonging to the RND family of efflux pumps, conserved with more than 90% sequence similarity in V. diabolicus, V. harveyi, V. alginolyticus, V. chemaguriensis, and V. rotiferianus (Liu et al. 2022). The disruption of RND efflux pumps via deletion of the tolC-encoded component in V. cholerae leads to the activation of the family of LysR-type transcriptional regulator leuO (Weng et al. 2021); leuO is associated with biofilm formation in V. cholerae (Moorthy and Watnick 2005).

The relation between RND efflux pumps and quorum sensing (QS) has been investigated and proven in pathogens like Pseudomonas aeruginosa (Evans et al. 1998; Maseda et al. 2004), Escherichia coli (Rahmati et al. 2002), Salmonella enterica (Dawan et al. 2022), Acinetobacter baumannii (Abd El-Rahman et al. 2023). The QS molecules are known to act as a substrate for the efflux pumps, particularly those that cannot diffuse across the membrane passively (Rahmati et al. 2002; Piddock 2006). For V. cholerae, CqsA produces cholera autoinducer-1(CAI-1) (Wei et al. 2011), and LuxS produces autoinducer-2 (AI-2) (Bassler et al. 1993), known to play an essential role in virulence (Higgins et al. 2007) and biofilm formation (Hammer and Bassler 2003). A definitive part of RND efflux pumps in V. cholerae QS is yet to be established.

Conclusions

Cholera remains a serious healthcare concern on a global scale (Mandal et al. 2017). The causative agent of cholera, V. cholerae, and related species have acquired resistance to antimicrobial agents that are shared amongst them and unrelated microorganisms (Kitaoka et al. 2011). Of these resistance mechanisms, integral membrane transporters represent key multidrug resistance systems (Stephen et al. 2022). These bacterial multidrug efflux pumps make suitable targets for modulation to restore the clinical efficacy of antimicrobial agents in cases of severe infection (Kumar et al. 2016a; Stephen et al. 2022). Unfortunately, agents of resistance modulation are poorly understood, indicating that such studies are needed.

Future research programs can consider investigating new strategies for treating multidrug-resistant variants that cause severe disease (Varela and Kumar 2019). Thus, new modulators of antimicrobial efflux and other antimicrobial resistance mechanisms are needed. Putative modulators require translation to the clinical setting in regions with high cholera incidence and prevalence. Furthermore, studies of the physiological and molecular mechanisms of resistance modulation will provide new insights into the efficacy restoration of unrelated infections in which similar targets confound treatment.

Continued structure–function investigations are needed to fully understand the basic mechanisms of multidrug efflux in bacterial pathogens. For instance, the molecular mechanisms of energy transduction during primary and secondary active efflux pumps are not fully understood for antimicrobial efflux pumps in Vibrio species and related pathogens. Along these lines, a clear understanding is lacking of the relationships between the conformational changes that occur during transport and the mechanisms responsible for antimicrobial specificity. Interestingly, investigations of the highly conserved amino acid sequences shared by members of the various transporter superfamilies have provided insights into these mechanisms of solute translocation across the membrane. The MFS molecular hinge structure and its mode of operation during transport appear to provide a universal mechanism of solute transport. This characteristic may be beneficial not only for our basic understanding of transporter physiology but also as a means of addressing the problem of clinical resistance to antimicrobial-resistant bacterial pathogens.

Another field of study that is seemingly neglected involves our understanding of the transporter mechanisms that dictate substrate specificity. Some transporters are specific for a narrow range of substrates, like the MFS efflux pumps for the tetracycline class of antimicrobials (Nelson and Levy 2011). In contrast, many transporters are relatively more promiscuous, harboring many structurally dissimilar substrates (Alvarez-Ortega et al. 2013; Delmar et al. 2014; Ranaweera et al. 2015). It is poorly understood how these disparate transporter systems determine whether a given transporter possesses a single drug class of substrate specificity versus those of multiple drugs while simultaneously preventing leakage of ions or water that would dissipate the energetic driving forces of primary and secondary active transport.

Lastly, more work needs to be conducted regarding the nature of the pathway of substrates through the various transporters. How much multidrug transporters can accommodate the structurally distinctive antimicrobial agents is unclear. At the molecular physiological level, it remains fascinating how individual multidrug transporters appear to have a list of unique substrates for each. This issue of antimicrobial specificity remains relevant in the clinical setting and is a focus of future investigation.