Dynamics of efflux pumps in antimicrobial resistance, persistence, and community living of Vibrionaceae.

The marine bacteria of the Vibrionaceae family are significant from the point of view of their role in the marine geochemical cycle, as well as symbionts and opportunistic pathogens of aquatic animals and humans. The well-known pathogens of this group, Vibrio cholerae, V. parahaemolyticus, and V. vulnificus, are responsible for significant morbidity and mortality associated with a range of infections from gastroenteritis to bacteremia acquired through the consumption of raw or undercooked seafood and exposure to seawater containing these pathogens. Although generally regarded as susceptible to commonly employed antibiotics, the antimicrobial resistance of Vibrio spp. has been on the rise in the last two decades, which has raised concern about future infections by these bacteria becoming increasingly challenging to treat. Diverse mechanisms of antimicrobial resistance have been discovered in pathogenic vibrios, the most important being the membrane efflux pumps, which contribute to antimicrobial resistance and their virulence, environmental fitness, and persistence through biofilm formation and quorum sensing. In this review, we discuss the evolution of antimicrobial resistance in pathogenic vibrios and some of the well-characterized efflux pumps' contributions to the physiology of antimicrobial resistance, host and environment survival, and their pathogenicity.

Functional Roles of the Conserved Amino Acid Sequence Motif C, the Antiporter Motif, in Membrane Transporters of the Major Facilitator Superfamily.

The biological membrane surrounding all living cells forms a hydrophobic barrier to the passage of biologically important molecules. Integral membrane proteins called transporters circumvent the cellular barrier and transport molecules across the cell membrane. These molecular transporters enable the uptake and exit of molecules for cell growth and homeostasis. One important collection of related transporters is the major facilitator superfamily (MFS). This large group of proteins harbors passive and secondary active transporters. The transporters of the MFS consist of uniporters, symporters, and antiporters, which share similarities in structures, predicted mechanism of transport, and highly conserved amino acid sequence motifs. In particular, the antiporter motif, called motif C, is found primarily in antiporters of the MFS. The antiporter motif's molecular elements mediate conformational changes and other molecular physiological roles during substrate transport across the membrane. This review article traces the history of the antiporter motif. It summarizes the physiological evidence reported that supports these biological roles.

Comparative efficiency of native and non-native starter culture in the production of bio-silage using composite waste from fish and vegetables.

The efficiency of native and non-native starter cultures in the production of bio-silage using composite waste from fish and vegetables was studied. An ensilage experiment was conducted in a natural way (without starter culture) of composite waste (fish to vegetable at 80 to 20%) to isolate the native fermentative microflora. An Enterococcus faecalis strain isolated from the natural ensilage of composite waste showed higher efficiency over other commercial LAB strains generally used for ensilation. A total of 60 isolates were screened and characterized biochemically from ensilaged composite waste. Among them, 12 proteolytic and lipolytic positive isolates were identified as Enterococcus faecalis, based on a BLAST search of the 16S rRNA gene sequences. Subsequently, composite bio-silage was prepared by inoculating starter cultures with three (3) treatments T1 (native-Enterococcus faecalis), T2 (non-native-Lactobacillus acidophilus), T3 (a mixture of E. faecalis and L. acidophilus) and compared with control (composite bio-silage without starter culture). The highest non-protein nitrogen (0.78 ± 0.01 mg of N /100 g) and degree of hydrolysis (70.00 ± 0.06% of protein/100 g) was seen in the T3 sample, and the lowest (0.67 ± 0.02 mg of N/100 g and 50.40 ± 0.04% of protein/100 g) was seen in the control. At the end of ensilation, the pH fell (5.95-3.88) in conjunction with the formation of lactic acid (0.23-2.05 g of lactic acid/100 g), and the lactic acid bacteria count nearly doubled (log 5.60-10.60). The lipid peroxidation products PV (0.11-0.41 milli equivalent of oxygen/kg of fat) and TBARs (1.64-6.95 mg of malonaldehyde/kg of silage) were changed within a reasonable range in the following pattern Control > T2 > T3 > T1, which led to oxidatively stable products. The findings revealed that native starter culture E. faecalis, which can be employed as a single or in combination with non-native L. acidophilus, performed better in the bio-ensilation process. Additionally, the finished composite bio-silage can be used as a novel, protein-carbohydrate rich feed component to help manage wastes from both sectors.

Inhibition of Multidrug Efflux Pumps Belonging to the Major Facilitator Superfamily in Bacterial Pathogens.

Bacterial pathogens resistant to multiple structurally distinct antimicrobial agents are causative agents of infectious disease, and they thus constitute a serious concern for public health. Of the various bacterial mechanisms for antimicrobial resistance, active efflux is a well-known system that extrudes clinically relevant antimicrobial agents, rendering specific pathogens recalcitrant to the growth-inhibitory effects of multiple drugs. In particular, multidrug efflux pump members of the major facilitator superfamily constitute central resistance systems in bacterial pathogens. This review article addresses the recent efforts to modulate these antimicrobial efflux transporters from a molecular perspective. Such investigations can potentially restore the clinical efficacy of infectious disease chemotherapy.

Physiological characteristics and virulence gene composition of selected serovars of seafood-borne Salmonella enterica.

Background and Aim: All serotypes of Salmonella enterica are considered potentially pathogenic. However, the non-typhoidal Salmonella (NTS) serotypes vary considerably in terms of pathogenicity and the severity of infections. Although diverse serotypes of NTS have been reported from tropical seafood, their sources, physiological characteristics, and virulence potentials are not well understood. This study aimed to compare the physiological characteristics of selected serovars of Salmonella from seafood and investigate possible variations in the distribution of known genes within the pathogenicity islands. Materials and Methods: A series of biochemical tests, including carbohydrate fermentation and amino acid decarboxylation tests were performed to physiologically compare the isolates. The genetic characterization with respect to putative virulence genes was done by screening for genes associated with Salmonella pathogenicity island (SPI) I-V, as well as the toxin- and prophage-associated genes by polymerase chain reaction. Results: Irrespective of serotypes, all the isolates uniformly harbored the five SPIs screened in this study. However, some virulence genes, such as the avrA, sodC, and gogB were not detected in all Salmonella isolates. The biochemical profiles of Salmonella serotypes were highly conserved except for variations in inositol fermentation and citrate utilization. All the isolates of this study were weak biofilm formers on polystyrene surfaces. Conclusion: The pathogenicity profiles of environmental NTS isolates observed in this study suggest that they possess the virulence machinery necessary to cause human infections and therefore, urgent measures to contain Salmonella contamination of seafood are required to ensure the safety of consumers.

The Major Facilitator Superfamily and Antimicrobial Resistance Efflux Pumps of the ESKAPEE Pathogen Staphylococcus aureus.

The ESKAPEE bacterial pathogen Staphylococcus aureus has posed a serious public health concern for centuries. Throughout its evolutionary course, S. aureus has developed strains with resistance to antimicrobial agents. The bacterial pathogen has acquired multidrug resistance, causing, in many cases, untreatable infectious diseases and raising serious public safety and healthcare concerns. Amongst the various mechanisms for antimicrobial resistance, integral membrane proteins that serve as secondary active transporters from the major facilitator superfamily constitute a chief system of multidrug resistance. These MFS transporters actively export structurally different antimicrobial agents from the cells of S. aureus. This review article discusses the S. aureus-specific MFS multidrug efflux pump systems from a molecular mechanistic perspective, paying particular attention to structure-function relationships, modulation of antimicrobial resistance mediated by MFS drug efflux pumps, and direction for future investigation.

Influence of polyamine production and proteolytic activities of co-cultivated bacteria on histamine production by Morganiella morganii.

Consumption of temperature-abused marine fish containing elevated levels of histamine results in histamine poisoning. Histamine is a biogenic amine produced in fish by the action of certain groups of bacteria which are capable of producing an exogenous enzyme called histidine decarboxylase (HDC). Morganella morganii is one of the major causative organisms of histamine poisoning. In this study, the histamine forming potential of M. morganii (BSS142) was evaluated when it was co-incubated with proteolytic as well as polyamine forming bacteria. This experiment was designed to examine whether biotic factors such as proteolysis and the presence of other amines influenced histamine forming ability of BSS142. The study showed that the proteolytic activity of Aeromonas hydrophila as well as Pseudomonas aeruginosa greatly enhanced the histamine forming ability of M. morganii. Psychrobacter sangunis, a non proteolytic polyamine producer, negatively influenced histamine production by M. morganii.

First report of a multidrug-resistant Salmonella enterica Serovar Infantis carrying pESI megaplasmid isolated from marine shrimp in India.

BACKGROUND: Non-typhoidal Salmonella enterica (NTS) in seafood is an important human health concern. An emerging strain of NTS serovar Infantis carrying a megaplasmid pESI and resistant to multipe drugs has been responsible for frequent food-borne human infections worldwide. METHODS: S. enterica strain JS5 was isolated from a sample of shrimp from the retail market on XLD agar after enrichment in the RV medium. The genomic DNA was isolated and sequenced using the Illumina platform. The draft whole genome sequence of Salmonella Infantis JS5 revealed the presence of a plasmid. RESULTS: The genome size was 4,977,731 bp with 4663 open reading frames. The bacterium harboured a megaplasmid similar to the pESI plasmid reported in the emerging S. Infantis. The 285 kb plasmid contained the characteristic genes of the pESI plasmids, such as the mercury operon, yersiniabactin siderophore operon, fimbriae, toxin-antitoxin systems and the hypothetical protein backbone. The antibiotic resistance genes tet(A), dfrA14, aadA, qacEdelta1, and sul1 were detected. To the best of our knowledge, this is the first report on pESI plasmid carrying S. Infantis from India.

Membrane Efflux Pumps of Pathogenic Vibrio Species: Role in Antimicrobial Resistance and Virulence.

Infectious diseases caused by bacterial species of the Vibrio genus have had considerable significance upon human health for centuries. V. cholerae is the causative microbial agent of cholera, a severe ailment characterized by profuse watery diarrhea, a condition associated with epidemics, and seven great historical pandemics. V. parahaemolyticus causes wound infection and watery diarrhea, while V. vulnificus can cause wound infections and septicemia. Species of the Vibrio genus with resistance to multiple antimicrobials have been a significant health concern for several decades. Mechanisms of antimicrobial resistance machinery in Vibrio spp. include biofilm formation, drug inactivation, target protection, antimicrobial permeability reduction, and active antimicrobial efflux. Integral membrane-bound active antimicrobial efflux pump systems include primary and secondary transporters, members of which belong to closely related protein superfamilies. The RND (resistance-nodulation-division) pumps, the MFS (major facilitator superfamily) transporters, and the ABC superfamily of efflux pumps constitute significant drug transporters for investigation. In this review, we explore these antimicrobial transport systems in the context of Vibrio spp. pathogenesis and virulence.

Bacterial Resistance to Antimicrobial Agents.

Bacterial pathogens as causative agents of infection constitute an alarming concern in the public health sector. In particular, bacteria with resistance to multiple antimicrobial agents can confound chemotherapeutic efficacy towards infectious diseases. Multidrug-resistant bacteria harbor various molecular and cellular mechanisms for antimicrobial resistance. These antimicrobial resistance mechanisms include active antimicrobial efflux, reduced drug entry into cells of pathogens, enzymatic metabolism of antimicrobial agents to inactive products, biofilm formation, altered drug targets, and protection of antimicrobial targets. These microbial systems represent suitable focuses for investigation to establish the means for their circumvention and to reestablish therapeutic effectiveness. This review briefly summarizes the various antimicrobial resistance mechanisms that are harbored within infectious bacteria.

Survival of the blaNDM-harbouring Escherichia coli in tropical seawater and conjugative transfer of resistance markers.

Anthropogenic contamination of coastal-marine water is responsible for introducing multidrug-resistant bacteria such as the pNDM-harbouring Escherichia coli into the seafood chain. This study was conducted to understand the survivability of a multidrug-resistant, the New Delhi Metallo-ß-lactamase-producing E. coli (AS-EC121) in tropical seawater at room temperature (28-32 °C) compared to E. coli K12 strain. The experimental and control strains were inoculated at 6 log CFU/ml level into seawater. After an initial sharp decline in counts, AS-EC121 and K12 strains showed a gradual loss of viability after week-1 of inoculation. AS-EC121 was undetectable after day-56, while K12 colonies disappeared a week later, from day-63. The conjugation experiment revealed that pNDM was transferable to a recipient E. coli strain in seawater. This study suggests that the multidrug-resistant, pNDM-harbouring E. coli is able to survive in seawater for over 2 months stably maintaining the resistance plasmid. The resistance genotypes do not seem to compromise the survivability of MDR E. coli and the stability of plasmid provides ample opportunities for dissemination of plasmids among co-inhabiting bacteria in the coastal-marine environments.

Development of a reverse transcription (RT) polymerase chain reaction (PCR) method for the detection of human norovirus in bivalve molluscs.

Noroviruses are significant seafood-borne pathogens, commonly associated with the consumption of filter feeding bivalve molluscs. Here, we report the development of a reverse transcription polymerase chain reaction (RT-PCR) method using primers based on the RNA-dependent RNA polymerase gene of norovirus genogroup II (NoV GII). Samples of bivalves were processed for the concentration of virus and extraction of RNA, followed by reverse transcription PCR. A total of 50 molluscan shellfish samples were analyzed, of which 16 samples yielded positive amplifications of norovirus nucleic acid. The PCR method described here, involving a single set of primers, is useful for rapid screening of shellfish for NoV GII.

Isolation and Identification of Cronobacter spp. from Fish and Shellfish Sold in Retail Markets.

In this study, we investigated the incidence of Cronobacter spp. in seafood collected from retail fish markets of Mumbai, India. A total of 50 samples comprising fresh finfish (n = 32), shellfish (n = 6), dried fish (n = 9) and water (n = 3) were analyzed for Cronobacter spp. by selective enrichment, isolation and biochemical tests. Of 145 isolates presumptively identified as Cronobacter spp. by biochemical tests, 37 were confirmed as Cronobacter spp. by Polymerase Chain Reaction (PCR) specific to the internal transcribed spacer (ITS) regions. Based on the partial ITS gene sequence analysis, 35 isolates were identified as Cronobacter malonaticus and two as Cronobacter sakazakii. The highest incidence of Cronobacter spp. was in dried fish (55.6%), followed by shellfish (33.3%). The virulence gene ompA was detected in two Cronobacter sakazakii isolates. This is the first report of the incidence of Cronobacter spp. in fresh and dried seafood from India, which highlights the need to focus on this emerging pathogen in tropical seafood.

Performance evaluation of a selective-enrichment-isolation protocol for Salmonella enterica from seafood.

In this study using 57 finfish samples of marine origin, selective enrichment in Rappaport-Vassiliadis (RV) broth followed by isolation on the Hektoen enteric agar (HEA) yielded 50 (53.2%) of 94 isolates. The results suggest RV-HEA as the most suitable media combination for the recovery of Salmonella from tropical seafood.

Salmonella Contamination of Seafood in Landing Centers and Retail Markets of Mumbai, India.

BACKGROUND: Seafood contamination with Salmonella enterica is not only a public health concern, but can also lead to economic losses due to import rejections. Continuous monitoring of seafood for Salmonella is necessary for risk assessment and to establish suitable control measures. OBJECTIVE: The objective of this study was to determine the extent of Salmonella contamination of seafood in fish landing centers and retails markets. METHODS: In this study, we analyzed seafood samples from three fish landing centers and seven retail markets of Mumbai, India for S. enterica contamination. Salmonella was isolated using multiple selective enrichment broths and selective agars, and identified by conventional biochemical tests followed by polymerase chain reaction (PCR). RESULTS: Of 82 samples of seafood, comprising both finfish and shellfish, Salmonella was detected in 17 (20.7%) finfish samples. None of the shellfish samples (shrimps, clams, and cephalopods) were found to be contaminated with Salmonella. Samples from both landing centers and fish markets, as well as from pelagic and demersal sources, harbored Salmonella. Source-wise analysis showed that 11 of 57 (19.3%) samples from retail fish markets and 6 of 25 (24%) samples from fish landing centers were contaminated with Salmonella. CONCLUSIONS: The study emphasizes the need to implement measures to prevent anthropogenic contamination of coastal waters and improve the hygiene of retail fish markets in Mumbai. HIGHLIGHTS: Seafood from landing centers and retail markets, as well as from pelagic and demersalhabitats were similarly contaminated with Salmonella. Fish species widely consumed in the region of this study were found highly contaminated with Salmonella.

Functional and Structural Roles of the Major Facilitator Superfamily Bacterial Multidrug Efflux Pumps.

Pathogenic microorganisms that are multidrug-resistant can pose severe clinical and public health concerns. In particular, bacterial multidrug efflux transporters of the major facilitator superfamily constitute a notable group of drug resistance mechanisms primarily because multidrug-resistant pathogens can become refractory to antimicrobial agents, thus resulting in potentially untreatable bacterial infections. The major facilitator superfamily is composed of thousands of solute transporters that are related in terms of their phylogenetic relationships, primary amino acid sequences, two- and three-dimensional structures, modes of energization (passive and secondary active), and in their mechanisms of solute and ion translocation across the membrane. The major facilitator superfamily is also composed of numerous families and sub-families of homologous transporters that are conserved across all living taxa, from bacteria to humans. Members of this superfamily share several classes of highly conserved amino acid sequence motifs that play essential mechanistic roles during transport. The structural and functional importance of multidrug efflux pumps that belong to the major facilitator family and that are harbored by Gram-negative and -positive bacterial pathogens are considered here.

Survival of Methicillin-Resistant Staphylococcus aureus in Fish and Shrimp under Different Storage Conditions.

ABSTRACT: Foods that are extensively handled during preparation and stored without refrigeration are often associated with staphylococcal food poisoning. This problem is more confounding when contaminating strains belong to the methicillin-resistant Staphylococcus aureus (MRSA) group. In this study, we investigated the survivability of MRSA in two seafood matrices under different storage conditions. MRSA was inoculated at 6 and 3 log CFU/g into all sample groups of peeled shrimp (Parapeneopsis stylifera) stored at -20°C, Bombay duck fish (Harpadon nehereus) stored in ice, and dried Bombay duck fish stored at 30 ± 2°C. The populations of MRSA in frozen peeled shrimp inoculated with MRSA at 6 log CFU/g were reduced by 1.52 log CFU/g, whereas in samples inoculated with 3 log CFU/g levels remained stable after 60 days of storage. In fresh Bombay duck fish inoculated with 6 log CFU/g and stored in ice for 18 days, MRSA levels decreased by 2.75 log CFU/g. In contrast, in fresh fish inoculated with 3 log CFU/g the total viable count increased by 3.02 log CFU/g over 16 days of ice storage. In dried fish stored at 30 ± 2°C, MRSA levels declined by 3.27 log CFU/g in samples inoculated with 6 log CFU/g and by 0.91 log CFU/g in samples inoculated with 3 log CFU/g. These results suggest that the survival of MRSA depends on the temperature of storage and the inoculum level. In our study, MRSA survival was higher when inoculated at 3 log CFU/g regardless of the seafood matrix and storage temperature.

Incidence of norovirus in tropical seafood harbouring fecal indicator bacteria.

The occurrence of human norovirus (HuNoV) in fresh seafood was studied by reverse transcription-polymerase chain reaction (RT-PCR). Samples (n = 104) comprising of oysters, clams, shrimps and finfish were collected from 3 retail markets, a landing center and a rocky intertidal zone. The samples were subjected to virus concentration, followed by RNA extraction and RT-PCR. Of these, 43 (41.34%) samples were positive for HuNoV Group II (GII). The fecal contamination status of the samples was determined by the Most Probable Number (MPN) method. All (100%) the samples were fecally contaminated, with the fecal coliform counts ranging from 10 to >1400 MPN/100 g. The study shows that fresh seafood, especially the bivalve mollusks, can act as vehicles for transmission and spread of HuNoV in the community. The high level of HuNoV contamination of seafood not only constitutes a health risk to the domestic consumers, but also compromises the microbiological qualities of seafood meant for international trade.

Occurrence of Human Enteric Adenoviruses in Fresh Tropical Seafood from Retail Markets and Landing Centers.

Human adenoviruses (HAdVs) are the foodborne enteric pathogens transmitted by the consumption of contaminated shellfish. In this study, the occurrence of enteric adenoviruses in finfish and shellfish was investigated by virus concentration and polymerase chain reaction (PCR). Total plate count, total coliform, and fecal coliform levels were determined and correlated with the presence of adenovirus. Samples of fish, bivalve mollusks, crustaceans, and cephalopods were collected from supermarkets, landing centers, and retail fish markets of Mumbai, India for the study. Overall, the adenovirus DNA was detected in 21.27% of all the samples analyzed. The highest incidence was detected in clams (14.89%), followed by oysters, shrimps, and finfish (2.13% each). High prevalence of enteric adenovirus in filter-feeding bivalves, such as clams and oysters, as well as in fish suggests persistent fecal contamination of coastal waters in the region of study. The occurrence of adenoviruses in samples showed a positive correlation with the bacteriological indicators of fecal contamination, suggesting that fecal indicator bacteria may be used to monitor the presence of adenoviruses in seafood. PRACTICAL APPLICATION: This research demonstrates the occurrence of human adenoviruse (HAdV) in fresh seafood and the utility of fecal coliforms as indicators of HAdV presence in seafood. The study emphasizes the need to identify HAdV in seafood as a human health hazard and implement measures to prevent sewage pollution of fish and shellfish harvesting areas in India.

Effect of Sodium Hypochlorite on Biofilm-Forming Ability of Histamine-Producing Bacteria Isolated from Fish.

Histamine poisoning occurs when temperature-abused marine fish containing elevated levels of histamine are consumed. Histamine-producing bacteria found in fish can colonize processing surfaces and form biofilms. In this study, the biofilm-forming abilities of histamine-producing bacteria from Indian mackerel (Rastrelliger kanagurta) and the effect of hypochlorite treatment on biofilm formation were studied. The isolates of this study produced histamine in the range of 471 to 2,126 ppm. The histidine decarboxylase gene hdc was detected in all isolates producing histamine except in one strain each of Psychrobacter pulmonis and Proteus vulgaris. All isolates tested in this study produced moderate biofilms under control conditions, whereas exposure to 1 and 3 ppm of sodium hypochlorite significantly enhanced biofilm formation. However, exposure to 5 ppm of sodium hypochlorite showed an inhibitory effect on biofilm formation by all the isolates except Klebsiella variicola. The results of this study suggest that histamine-producing bacteria can form stable biofilms and that this activity may be enhanced by the application of low levels of sodium hypochlorite, a phenomenon that might influence the persistence of histamine-producing bacteria in fish processing areas.