Heavy metal and antibiotic co-resistance in Vibrio parahaemolyticus isolated from shellfish.

Vibrio parahaemolyticus is a major gastroenteritis-causing pathogen in Korea. Recent studies have reported that heavy metal and antimicrobial resistance in bacteria are related. In this study, we investigated heavy metal and antimicrobial resistance in wild strains of V. parahaemolyticus. First, we isolated and characterized 38 V. parahaemolyticus strains (toxR-positive) from shellfish collected from the West Sea of Korea between May and November 2018. Antibiotic and heavy metal resistance in the 38 strains were tested by disk diffusion assay and broth dilution assay, respectively. Then, we selected seven strains that showed resistance to cobalt (Co2+) and copper (Cu2+), to examine the relationship between heavy metal resistance and antimicrobial resistance. After heavy metal (Co2+ and Cu2+) pretreatment, the seven strains exhibited increased resistance to kanamycin, streptomycin, tetracycline, and gentamycin. Likewise, antimicrobial pretreatment resulted in increased heavy metal tolerance.

Heat Adaptation Improved Cell Viability of Probiotic Enterococcus faecium HL7 upon Various Environmental Stresses.

The production of viable functional probiotics presupposes stability of strain features in the final product. In previous studies, Enterococcus faecium HL7 was found to have relatively higher cell viability after freeze-drying and the long-lasting resistance to heat (60 °C) as well as higher antimicrobial activities against some of fish and human pathogens among isolated strains. For heat adaptation, E. faecium HL7 cells were exposed to 52 °C for 15 min. After adaption, slight decreases of unsaturated membrane fatty acid ratios were confirmed through fatty acid analysis. Upon subsequent exposure to various stress conditions such as H2O2 (0.01%), ethanol (20%), acid (pH 3), and alkali (pH 12), the survival rate of heat-adapted HL7 was 103-105-fold higher than that of non-adapted one. These results highlight the potential of preconditioning treatments for maximizing survival of probiotic bacteria during development of probiotic functional foods. The cross-protection afforded by acid against thermal stress may indicate that certain common protective mechanisms are induced by both heat and acid stress. These results can be applied to enhancing the cell viability during live cell formulation of E. faecium HL7 to be used as a potential probiotics in aquaculture.

Antibiotic and heavy-metal resistance of Vibrio parahaemolyticus isolated from oysters in Korea.

Vibrio parahaemolyticus, found frequently in oysters and other seafoods, is the most prevalent gastroenteritis-causing pathogen in Korea and other Asian countries. It is associated exclusively with the consumption of raw or improperly cooked contaminated seafood, especially oysters. In this study, we isolated and characterized 59 V. parahaemolyticus strains (toxR-positive) from May to October 2016 in shellfish-harvesting areas off the west coast of Korea. The results revealed that none of the isolates contained the tdh and trh toxicity genes. The multiple antibiotic resistance (MAR) value of most isolates was 0.32, but it was as high as 0.69 in one isolate strain. Moreover, when resistance to heavy metals was examined, the majority of the isolates displayed resistance to Ba2+ (98.3%), Co3+ (28.8%), Cd2+ (16.9%), and Cu2+ (13.6%). Interestingly our data revealed that tolerance to heavy metals was prevalent in the V. parahaemolyticus strains with more than two antibiotic resistance phenotypes.

In vitro probiotic properties of vaginal Lactobacillus fermentum MG901 and Lactobacillus plantarum MG989 against Candida albicans.

Candida albicans is the most important Candida species causing vulvovaginal candidiasis (VVC). We investigated the potential of the probiotic strains Lactobacillus fermentum MG901 and L. plantarum MG989 towards control of C. albiacns. Cell viability tests following co-culturing with lactobacilli revealed that C. albicans cells lost metabolic activity and were eventually killed. Further studies revealed that MG901 and MG989 had high surface hydrophobicity that enhanced its adhesion ability to epithelial cell. The MG901 and MG989 showed coaggregation with E. coli and C. albicans to affect their adhesion and colonization. The adhesion of MG901 and MG989 to HT-29 cell and its inhibition of E. coli and C. albicans adherence to these cells were demonstrated. These incidences provided evidence of the possible colonization of MG901 and MG989 that would prevent binding and growth of E. coli and C. albicans onto intestinal epithelial cells. Following daily administration of 108 CFU of viable MG901 and MG989 orally, the animals' feces were examined for bacterial excretion. The potential probiotic MG901 and MG989 were found to persist for up to 6 days in the feces of mice. In conclusion, L. fermentum MG901 and L. plantarum MG989 have the potential to inhibit the yeast growth, which could possibly have played an important role in helping to clear VVC in vivo.

In Vitro Probiotic Properties of Lactobacillus salivarius MG242 Isolated from Human Vagina.

Vulvovaginal candidiasis (VVC) is a very common infection worldwide that is mainly caused by Candida albicans. In a previous study, we showed that Lactobacillus salivarius MG242 has anti-Gardnerella vaginalis activity. In this study, we investigated the potential of using L. salivarius MG242 for biocontrol of C. albicans. In line with the results from a spot overlay assay, MG242 inhibited the growth of C. albicans by 99.99 ± 0.01% in co-culture, suggesting that L. salivarius MG242 has the potential to be developed into a probiotic formula to treat or prevent VVC. Accelerated storage tests using dehydrated live cell powder at 50, 60, and 70 °C were performed, and the results showed that immobilization with 10% skim milk effectively increased the thermal resistance of entrapped microorganisms, resulting in sevenfold longer shelf-life than the control (in PBS). Lower storage temperatures also increased the shelf-life up to 8.31 months.

Possible Probiotic Lactic Acid Bacteria Isolated from Oysters (Crassostrea gigas).

We attempted to isolate lactic acid bacteria (LAB) from the marine oyster (Crassostrea gigas) and selected several environmental stress-resistant isolates for the development of a future probiotic adjuvant for marine aquaculture. Twenty-six presumptive LAB isolates were extracted from oysters and screened (by an agar diffusion assay) for antimicrobial activity toward various pathogens: Vibrio parahaemolyticus, Streptococcus iniae, and Edwardsiella tarda. Eight isolates had an antibacterial activity toward V. parahaemolyticus; in particular, 6 isolates showed a growth-inhibitory activity, with inhibition zone diameters > 15 mm. Of these, 5 isolates (JL17, JL18, JL28, HL7, and HL32) were also active against S. iniae and E. tarda. Enterococcus faecium HL7 was selected as the isolate most resistant to environmental stressors: the minimum NaCl, ethanol, and hydrogen peroxide concentrations at which HL7 cells lost their viability were 1.9 M, 11%, and 0.013%, respectively. When an antibiotic sensitivity test was performed on E. faecium HL7, this isolate was found to be resistant to trimethoprim/sulfamethoxazole, cephalothin, ampicillin, rifampin, gentamicin, cefotaxime, cefepime, cefotetan, nalidixic acid, and kanamycin. While the oyster model studies provided indication that E. faecium HL7 could be a good candidate as biocontrol agent against V. vulnificus, further optimization is needed in the actual animal rearing situation.

Biocementation of Concrete Pavements Using Microbially Induced Calcite Precipitation.

In this study, the feasibility of introducing calcite-forming bacteria into concrete pavements to improve their mechanical performance was investigated. Lysinibacillus sphaericus WJ-8, which was isolated in a previous study and is capable of exhibiting high urease activity and calcite production, was used. When analyzed via scanning electron microscopy (SEM) and X-ray diffraction, WJ-8 showed a significant amount of calcite precipitation. The compressive strength of cement mortar mixed with WJ-8 cells and nutrient medium (urea with calcium lactate) increased by 10% compared with that of the controls. Energy dispersive x-ray spectroscopy analyses confirmed that the increase in strength was due to the calcite formed by the WJ-8 cells.

Characterization of Vibrio parahaemolyticus isolated from oysters in Korea: Resistance to various antibiotics and prevalence of virulence genes.

Vibrio parahaemolyticus, found frequently in oysters, is the most prevalent gastroenteritis-causing pathogen in Korea and in several other Asian countries. This study monitored changes in the environmental parameters and occurrence of V. parahaemolyticus in oyster aquaculture sites. Of the 44 presumed V. parahaemolyticus isolates obtained, when tested against 16 antibiotics, 90.9, 86.4, and 75.0% of the 44 isolates exhibited resistance to vancomycin, ampicillin, and streptomycin, respectively. PCR analysis for the presence of the toxR gene confirmed 31 of the 44 isolates as being positive V. parahaemolyticus strains. The toxR positive isolates were tested for the presence of thermostable direct hemolysin (tdh) and tdh-related hemolysin (trh) virulence genes. Only 9.1% toxR positive isolate exhibit the trh gene and none of the isolates were tested positive for tdh. The occurrence of multi drug resistance strains in the environment could be an indication of excessive usage of antibiotics in agriculture and aquaculture fields.

Antibiotic and heavy metal resistance in Shewanella putrefaciens strains isolated from shellfishes collected from West Sea, Korea.

The aim of the present study was to determine the antibiotic resistance patterns and distribution of heavy-metal resistance in Shewanella putrefaciens strains isolated from shellfishes collected from West Sea; and to determine the relationship, if any, between antibiotic and heavy-metal resistance in these strains. Among the 15 strains isolated, two strains, SY1 and SY2, showed heavy-metal resistance in addition to high resistance to seven antibiotics: cephalothin, gentamicin, erythromycin, vancomycin, ampicillin, rifampicin, and streptomycin. We conclude that heavy-metal contamination imposes long-term, widespread, and recalcitrant selection pressure, which potentially contributes to the maintenance and spread of antibiotic resistance factors in bacteria. Moreover, this fact holds both environmental and clinical importance.

Antimicrobial susceptibility of Vibrio alginolyticus isolated from oyster in Korea.

Pathogenic Vibrio alginolyticus, a cause of severe infection in shellfish, as well as in humans, has been found at high frequency around all coastal areas of Korea. The aim of this study was to determine the occurrence of V. alginolyticus, to identify the strains isolated from oysters in West Sea, and to investigate their antimicrobial resistance profiles. Biochemical analyses of the 90 initially recovered presumptive V. alginolyticus colonies indicated that 16 isolates were V. alginolyticus. PCR analysis to detect the presence of the gyrB gene confirmed that 15 (93.8 %) of the 16 isolates were V. alginolyticus. These 15 isolates had the following profiles of resistance against 16 antibiotics: all isolates were resistant to ampicillin and vancomycin, and 26.7 % of the isolates exhibited resistance to cephalothin. A large number of isolates showed intermediate resistance to erythromycin (100 %) and rifampin (73.3 %). Five (33.3 %) of the V. alginolyticus isolates demonstrated multiple resistance to at least three antimicrobials.

Biosequestration of copper by bacteria isolated from an abandoned mine by using microbially induced calcite precipitation.

Abandoned mine sites are frequently polluted with high concentrations of heavy metals. In this study, 25 calcite-forming bacteria were newly isolated from the soil of an abandoned metal mine in Korea. Based on their urease activity, calcite production, and resistance to copper toxicity, four isolates were selected and further identified by 16S rRNA gene sequencing. Among the isolates, Sporosarcina soli B-22 was selected for subsequent copper biosequestration studies, using the sand impermeability test by production of calcite and extracellular polymeric substance. High removal rates (61.8%) of copper were obtained when the sand samples were analyzed using an inductively coupled plasma-optical emission spectrometer following 72 h of incubation. Scanning electron microscopy showed that the copper carbonate precipitates had a diameter of approximately 5-10 µm. X-ray diffraction further confirmed the presence of copper carbonate and calcium carbonate crystals.

Formations of calcium carbonate minerals by bacteria and its multiple applications.

Biomineralization is a naturally occurring process in living organisms. In this review, we discuss microbially induced calcium carbonate precipitation (MICP) in detail. In the MICP process, urease plays a major role in urea hydrolysis by a wide variety of microorganisms capable of producing high levels of urease. We also elaborate on the different polymorphs and the role of calcium in the formation of calcite crystal structures using various calcium sources. Additionally, the environmental factors affecting the production of urease and carbonate precipitation are discussed. This MICP is a promising, eco-friendly alternative approach to conventional and current remediation technologies to solve environmental problems in multidisciplinary fields. Multiple applications of MICP such as removal of heavy metals and radionuclides, improve the quality of construction materials and sequestration of atmospheric CO2 are discussed. In addition, we discuss other applications such as removal of calcium ions, PCBs and use of filler in rubber and plastics and fluorescent particles in stationary ink and stationary markers. MICP technology has become an efficient aspect of multidisciplinary fields. This report not only highlights the major strengths of MICP, but also discusses the limitations to application of this technology on a commercial scale.

Prevalence and antimicrobial susceptibility of Vibrio parahaemolyticus isolated from oysters in Korea.

Vibrio parahaemolyticus is the most prevalent gastroenteritis-causing pathogen in Korea and in some other Asian countries. It is frequently found in oysters and other seafood. This study monitored changes in the prevalence of V. parahaemolyticus and environmental parameters in oyster aquaculture environments in Korea. From June to October 2014, we tested oysters (Crassostrea gigas) from shellfish-harvesting areas off the west coast of Korea. These 71 isolates were the sum of 16 (22.5%), 19 (26.8%), 23 (32.4%), and 13 (18.3%) isolates collected in July, August, September, and October, respectively. These 71 isolates had the following profiles of resistance against 16 antibiotics: all isolates were resistant to ampicillin and vancomycin, and 52.2, 50.7, and 50.7% of isolates exhibited resistance to cephalothin, rifampin, and streptomycin, respectively. PCR analysis for the presence of the species-specific toxR gene confirmed that 38 (53.5%) of the total 71 isolated strains were positive for V. parahaemolyticus. In PCR analysis for virulence of V. parahaemolyticus, of the 71 isolates tested in the present study, only 38 (53.5%) were positive for the trh virulence gene and 71 (100%) was negative for the tdh virulence gene.

Effects of the Bradyrhizobium japonicum waaL (rfaL) Gene on Hydrophobicity, Motility, Stress Tolerance, and Symbiotic Relationship with Soybeans.

We cloned and sequenced the waaL (rfaL) gene from Bradyrhizobium japonicum, which infects soybean and forms nitrogen-fixing nodules on soybean roots. waaL has been extensively studied in the lipopolysaccharide (LPS) biosynthesis of enteric bacteria, but little is known about its function in (brady)rhizobial LPS architecture. To characterize its role as O-antigen ligase in the LPS biosynthesis pathway, we constructed a waaL knock-out mutant and its complemented strain named JS015 and CS015, respectively. LPS analysis showed that an LPS structure of JS015 is deficient in O-antigen as compared to that of the wild type and complemented strain CS015, suggesting that WaaL ligates the O-antigen to lipid A-core oligosaccharide to form a complete LPS. JS015 also revealed increased cell surface hydrophobicity, but it showed decreased motility in soft agar plates. In addition to the alteration in cell surface properties, disruption of the waaL gene caused increased sensitivity of JS015 to hydrogen peroxide, osmotic pressure, and novobiocin. Specifically, plant tests revealed that JS015 failed to nodulate the host plant soybean, indicating that the rhizobial waaL gene is responsible for the establishment of a symbiotic relationship between soybean and B. japonicum.

Role of the extracytoplasmic function sigma factor CarQ in oxidative response of Bradyrhizobium japonicum.

As a nitrogen-fixing bacterium, Bradyrhizobium japonicum can establish a symbiotic relationship with the soybean plant (Glycine max). To be a successful symbiont, B. japonicum must deal with plant defense responses, such as an oxidative burst. Our previous functional genomics study showed that carQ (bll1028) encoding extracytoplasmic function (ECF) sigma factor was highly expressed (107.8-fold induction) under oxidative stress. Little is known about the underlying mechanisms of how CarQ responds to oxidative stress. In this study, a carQ knock-out mutant was constructed using site-specific mutagenesis to identify the role of carQ in the oxidative response of B. japonicum. The carQ mutant showed a longer generation time than the wild type and exhibited significantly decreased survival at 10 mM H(2)O(2) for 10 min of exposure. Surprisingly, there was no significant difference in expression of oxidative stress-responsive genes such as katG and sod between the wild type and carQ mutant. The mutant also showed a significant increase in susceptibility to H(2)O(2) compared to the wild type in the zone inhibition assay. Nodulation phenotypes of the carQ mutant were distinguishable compared to those of the wild type, including lower numbers of nodules, decreased nodule dry weight, decreased plant dry weight, and a lower nitrogen fixation capability. Moreover, desiccation of mutant cells also resulted in significantly lower percent of survival in both early (after 4 h) and late (after 24 h) desiccation periods. Taken together, this information will provide an insight into the role of the ECF sigma factor in B. japonicum to deal with a plant-derived oxidative burst.

Isolation and identification of a novel fibrinolytic Bacillus tequilensis CWD-67 from dumping soils enriched with poultry wastes.

A newly isolated strain, CWD-67, which exhibited high fibrinolytic activity, was screened from dumping soils enriched with poultry wastes. The strain was identified as Bacillus tequilensis (KF897935) by 16Sr RNA gene sequence analysis and biochemical characterization. A fibrinolytic enzyme was purified to homogeneity from the culture supernatant using ammonium sulfate precipitation, membrane concentration, dialysis, ion-exchange, and gel filtration chromatography. SDS-PAGE analysis showed that the purified enzyme was a monomeric protein with an apparent molecular weight of 22 kDa, which is the lowest among Bacillus fibrinolytic enzymes reported to date. The purified enzyme was confirmed to have fibrinolytic activity by a fibrin zymogram. The optimal pH and temperature values of the enzyme were 8.0 and 45 °C, respectively. The enzyme was completely inhibited by PMSF and significantly inhibited by EDTA, TPCK, Co(2+), Zn(2+), and Cu(2+), suggesting a chymotrypsin-like serine metalloprotease. In vitro assays revealed that the purified enzyme could catalyze fibrin lysis effectively, indicating that this enzyme could be a useful fibrinolytic agent.

Microbially induced calcite precipitation-based sequestration of strontium by Sporosarcina pasteurii WJ-2.

Contamination by radioactive strontium ((90)Sr) is a significant environmental problem. Ureolytically driven calcium carbonate precipitation has been proposed for use in geotechnical engineering for soil remediation applications. In this study, 68 ureolytic bacterial strains were newly isolated from various environments. Of these, 19 strains were selected based on ureolytic activity shown when cultured on urea agar plates and identified through 16S rRNA gene sequencing. From these selected strains, Sporosarcina pasteurii WJ-2 (WJ-2) was selected for subsequent study. A simple method was developed to determine the effectiveness of microbially induced calcite precipitation (MICP). Unlike any other methods, it does not require advanced skills and sophisticated tools. Using this method, we were able to determine the ability of the bioconsolidated sand to retard the flow of crystal violet through the 25-mL column. Also, MICP by WJ-2 was evaluated for its potential to counteract Sr contamination in column experiments using natural sand. WJ-2-induced precipitation led to successful sequestration of approximately 80 % of the Sr from the soluble fraction of the sand. The utility of MICP in bioremediation was further confirmed through X-ray diffraction, scanning electron microscopy, and inductively coupled plasma mass spectrometry.

Antibiotic resistance of Vibrio harveyi isolated from seawater in Korea.

Vibrio harveyi is an opportunistic human pathogen that may cause gastroenteritis, severe necrotizing soft-tissue infections, and primary septicemia, with a potentially high rate of lethality. In this study, we isolated and characterized V. harveyi from seawater collected from the West Sea in Korea, including sites located near shellfish farms. For the initial isolation of putative V. harveyi, isolates were incubated on thiosulfate citrate bile salt sucrose agar plates for 24h, followed by selection of greenish colonies. Gram-negative and oxidase-positive colonies were subsequently confirmed by biochemical assays and the API 20E kit test system. Species-specific 16S rRNA and hemolysin genes were used to design V. harveyi-specific PCR primers. From 840 seawater samples, a total of 2 strains of V. harveyi were isolated from shellfish farm seawater. The two isolates were subjected to profiling against 16 antibiotics and found to be resistant to cephalothin, vancomycin, ampicillin, cefepime, cefotetan, and streptomycin.

Bioremediation of Cd by microbially induced calcite precipitation.

Contamination by Cd is a significant environmental problem. Therefore, we examined Cd removal from an environmental perspective. Ureolysis-driven calcium carbonate precipitation has been proposed for use in geotechnical engineering for soil remediation applications. In this study, 55 calcite-forming bacterial strains were newly isolated from various environments. Biomineralization of Cd by calcite-forming bacteria was investigated in laboratory-scale experiments. A simple method was developed to determine the effectiveness of microbially induced calcite precipitation (MICP). Using this method, we determined the effectiveness of biomineralization for retarding the flow of crystal violet through a 25-mL column. When the selected bacteria were analyzed using an inductively coupled plasma optical emission spectrometer, high removal rates (99.95%) of Cd were observed following incubation for 48 h. Samples of solids that formed in the reaction vessels were examined using a scanning electron microscope. The CdCO3 compounds primarily showed a spherical shape. The results of this study demonstrate that MICP-based sequestration of soluble heavy metals via coprecipitation with calcite may be useful for toxic heavy metal bioremediation.