Characterization of the low-salinity stress in Vibrio vulnificus

Vibrio vulnificus is a marine pathogenic bacterium commonly found in seawater or seafood. This organism encounters low-salinity stress in its natural environment and during food processing. This study was designed to investigate the response of V. vulnificus YJ03 to lethal low salinity (0.04% NaCl) and its adaptation to sublethal salinity (0.12% NaCl with 20 amino acids added). A short period in the nonculturable state was induced by lethal low-salinity stress followed by cell death after 30 min of stress. Addition of 1 mM glycine betaine or 0.5 mM sucrose reduced the damage. Low-salinity adaptation was achieved in the exponential-phase cells but not in the stationary-phase cells. Significant protection against lethal low-salinity stress was attained when the cells were adapted for as little as 1.5 min. The adapted cells were significantly protected against lethal low salinity and 2.4% sodium sorbate but sensitized to the challenge of heat (52 degrees C) and acid (pH 3.2). Nonlethal low-salinity treatment of seafood should be avoided to prevent stress adaptation of V. vulnificus.     

Variable repeat regions in the genome of Vibrio vulnificus and polymorphism in one of the loci in strains isolated from oysters

Vibrio vulnificus an estuarine bacterium is associated with severe wound infections and fatal septicemia related to consumption of raw shellfish. In this study we screened the two whole genome sequences available for V. vulnificus in GenBank for the presence of variable number of tandem repeat (VNTR) regions. Five potential VNTR loci with unit repeat size ranging from 6-7 nucleotides were identified for V. vulnificus genome. One of the loci designated Vv1 was selected to detect the repeat number present in V. vulnificus strains isolated from oyster samples in India. Twenty six of the thirty samples tested were found to be highly polymorphic for the Vv1 locus. Copy numbers for the hexanucleotide motif ranged from 4-55, giving rise to a total of 17 polymorphic groups. Our analysis, shows that different genotypic variants exist in the environment and the VNTR loci studied can be used as a marker for strain discrimination and in epidemiological study of this organism.     

Antimicrobial susceptibility of Staphylococcus aureus isolated from bovine mastitis in Europe and the United States

Minimum inhibitory concentrations were determined for 811 strains of Staphylococcus aureus isolated from cases of bovine mastitis in 11 countries. The countries and number of isolates included Denmark (105), England (92), Finland (95), Germany (103), Iceland (22), Ireland (42), Norway (101), Sweden (123), Switzerland (69), United States (53), and Zimbabwe (6). The antimicrobial agents tested were penicillin, ampicillin, oxacillin, cephalothin, ceftiofur, amoxicillin + clavulanate, penicillin + novobiocin, enrofloxacin, premafloxacin, erythromycin, clindamycin, lincomycin, pirlimycin, neomycin, lincomycin + neomycin, and sulfamethazine. The MIC90 for these antimicrobial agents for all strains were 0.5, 1.0, 1.0, 0.5, 1.0, < or =0.06, 0.125, 0.125, < or =0.0078, 0.5, 1.0, 16.0, 1.0, 2.0, 0.5, and 4.0 microg/ml, respectively. Overall, only small variations between countries were seen in the MIC90 for the majority of compounds tested. Of the strains tested, 35.6% were positive for beta-lactamase production on initial testing, with an additional 21.3% positive after induction by penicillin. In conclusion, the overall level of resistance was generally low for all antimicrobial agents tested regardless of country. Given the differences in antimicrobial use in various countries, the widespread adoption of mastitis control programs to prevent infections limits the exposure of S. aureus infected animals to antimicrobial drugs.     

Heterogeneity of environmental, retail, and clinical isolates of Vibrio vulnificus as determined by lipopolysaccharide-specific monoclonal antibodies

The opportunistic pathogen Vibrio vulnificus expresses lipopolysaccharide (LPS) antigens on its outer membrane surface. A serological typing system was developed for these antigens, utilizing the discriminatory recognition of monoclonal antibodies (MAb) by ELISA. MAb were used to recognize five unique types of LPS-associated antigens for examination of clinical. environmental, and retail isolates of V. vulnificus. The overall serotype profile of the clinical isolates was significantly different (P < 0.05) from that of the environmental and retail isolates. A higher percentage of clinical isolates were typable (61%) compared to the environmental isolates (41%) and retail isolates (44%). In particular, the percentage of serotype 1/5 among clinical isolates (33%), compared to that of environmental (9%) and retail (4%), was highly significant (P < 0.0001). Among the environmental Gulf Coast isolates, there were differences in the prevalence of serotypes 2 and 3 (P < 0.05), depending on whether isolates were obtained from Louisiana or Alabama harvest sites. There were no statistically significant differences between the serotype profiles of Gulf and Atlantic Coast retail isolates despite the absence of serotype 1/5 from the Atlantic Coast. While some serotype diversity was detected in V. vulnificus isolated during different seasons, from different geographic locations, and at retail versus at harvest, there was no apparent concordance between any of the serotype distributions obtained from oysters versus that isolated clinically. The heterogeneity of environmental isolates and relative homogeneity among clinical isolates suggest that human risk may not be predicted on quantitative exposure alone.     

食品和临床来源金黄色葡萄球菌对Caco-2细胞侵袭力的比较研究

目的 比较研究食品和临床来源金黄色葡萄球菌不同克隆系(Clonal complex,CC)菌株对Caco-2细胞的侵袭力和菌膜形成能力。方法 采用Caco-2细胞体外培养方法、庆大霉素与溶葡萄球菌酶保护试验,研究金黄色葡萄球菌12个不同来源的克隆系菌株对人体肠道表皮细胞的体外侵袭力;采用96孔板结晶紫染色法,评估不同菌株的菌膜形成能力。结果 体外侵袭能力试验结果表明CC5、CC30、CC25、CC59、CC239、 CC50和CC398等克隆系都具有细胞强侵袭株,与牲畜特异相关的CC9也展现出细胞强侵袭力,而CC1、CC72 、CC20和CC121等克隆系则明显具有较弱的细胞侵袭能力。此外,不同克隆系菌株菌膜形成的能力也具有一定差异,其中CC5、CC9、CC25和CC239等克隆系菌膜形成能力相比较强,而CC1、CC72、CC59和CC121等则菌膜形成能力较弱。进一步分析发现不同克隆系菌株的菌膜形成能力与细胞侵袭力呈现正相关(R=0.743)。结论 金黄色葡萄球菌不同克隆系菌株的细胞侵袭能力、菌膜形成能力具有一定差异,本研究结果能够为有效防控金黄色葡萄球菌临床感染提供重要支持。     

Evaluation of the polymerase chain reaction method for identification of Vibrio vulnificus isolated from marine environments.

The polymerase chain reaction (PCR) method for identification of Vibrio vulnificus in the marine environment was evaluated by comparing it to both the conventional and DNA-DNA hybridization methods. Of 13,325 isolates obtained from seawater and sediment samples, and oyster and goby specimens collected from the coastal waters of Tokyo Bay, Japan, only 61 isolates were identified as V. vulnificus on the basis of phenotypic characteristics and the amplification of the cytotoxin-hemolysin gene by the PCR method. All 61 isolates were further confirmed to be V. vulnificus by a DNA-DNA hybridization method and the API 20E system although they were divided into 13 groups on the basis of their API 20E profiles. These results strongly suggest that the PCR method is useful for identification of this organism.     

A rapid and improved method for the detection of Vibrio parahaemolyticus and Vibrio vulnificus strains grown on hydrophobic grid membrane filters

DNA probe-based detection methods were developed and characterized as an alternative to time-consuming and less specific conventional protocols. Digoxigenin-labeled probes were prepared by polymerase chain reaction amplification of the targeted sequences in the specific amplicons generated from genomic DNA. Specific probes with high yields were generated for the detection of the tlh gene of Vibrio parahaemolyticus and the cth gene of V. vulnificus. Colony (Southern) hybridization analyses were carried out using hydrophobic grid membrane filters (HGMFs) to allow biotype-specific differentiation of the two species. Eight strains of V. vulnificus and five strains of V. parahaemolyticus, including one standard (ATCC) strain of each biotype, were examined. Colony lysis, hybridization, and nonradioactive detection parameters were optimized for identification of the target biotypes arranged on the same HGMF and also on a conventional nylon membrane, thereby confirming the specificity of the probes and the comparative usefulness of the HGMFs. The experimental procedure presented here can be completed in 1 day. The protocol was designed specifically to identify the target Vibrio spp. and could potentially be used for the enumeration and differentiation of V. parahaemolyticus and V. vulnificus in foods.     

Antimicrobial resistance of Vibrio parahaemolyticus and Vibrio alginolyticus strains isolated from farmed fish in Korea from 2005 through 2007

The antimicrobial resistance patterns to 15 antimicrobial agents of Vibrio parahaemolyticus and Vibrio alginolyticus isolated from farmed fishes, including olive flounder (Paralichthys olivaceus), black rockfish (Sebastes schlegeli), red sea bream (Pagrus major), and sea bass (Lateolabrax japonicus), were investigated from 2005 through 2007. A total of 218 V. parahaemolyticus isolates and 153 V. alginolyticus isolates were obtained from the 180 fish samples collected from fish farms located along the southern coast of Korea. We found that 65.1% of V. parahaemolyticus and 85.6% of V. alginolyticus isolates showed antimicrobial resistance against more than one antimicrobial agent. The prevalence of resistance in V. parahaemolyticus isolates to ampicillin was highest (57.8%), followed by resistance to rifampin (11.9%), streptomycin (8.7%), and trimethoprim (6.4%). V. alginolyticus isolates were also most resistant to ampicillin (75.2%), followed by tetracycline (15.0%), trimethoprim (12.4%), and rifampin (9.8%). The prevalence of multiresistance to four or more antimicrobials was higher in V. alginolyticus (11.1%) than in V. parahaemolyticus (5%). Antimicrobial resistance rates per isolate of V. parahaemolyticus and V. alginolyticus possessing virulence genes were not different from those of the rest of the isolates.     

2015年我国食源性副溶血性弧菌毒力基因及 耐药特征研究

目的 探究我国2015年食品来源的副溶血性弧菌毒力基因分布情况和耐药特征。方法 通过聚合酶链式反应,研究1046株副溶血性弧菌携带毒力基因tlh、tdh和trh情况,并采用微量肉汤稀释法,测定副溶血性弧菌对氨苄西林、阿莫西林/克拉维酸、头孢唑林、头孢噻肟、头孢他啶、亚胺培南、庆大霉素、四环素、环丙沙星、左氧沙星、氯霉素、复方新诺明等抗生素的敏感性。结果 本研究所用1046株副溶血性弧菌中有965株（92.3%）对一种或多种抗生素耐药,总耐药率为92.2%。全部菌株对庆大霉素和左氧氟沙星敏感,对其余10种抗生素有不同程度耐药,耐药率最高的前三位抗生素分别为头孢唑林（85.4%）、氨苄西林（59.4%）、阿莫西林/克拉维酸（49.3%）。1046株副溶血性弧菌全部携带tlh基因,其中19株（1.8%）trh基因阳性,4株（0.4%）tdh基因阳性。毒力基因阳性的所有菌株均未发现多重耐药现象,部分毒力基因阳性菌株对青霉素类和第一代头孢类抗生素耐药。结论 我国2015年食品来源的副溶血性弧菌毒力基因携带率较低,但对第一代头孢类和青霉素类抗生素耐药率较高;水产养殖地区应在养殖环节应加强抗生素的管理并规范使用。     

Susceptibility of the heat-, acid-, and bile-adapted Vibrio vulnificus to lethal low-salinity stress

As a marine pathogenic bacterium that inhabits seawater or seafood, Vibrio vulnificus encounters low salinity and other stresses in the natural environment and during food processing. This investigation explores the cross-protective response of sublethal heat-, acid-, or bile-adapted V. vulnificus YJ03 against lethal low-salinity stress. Experimental results reveal that the acid (pH 4.4)- and heat (41 degrees C)-adapted V. vulnificus were not cross-protected against the lethal low-salinity challenge (0.04% NaCl). The bile (0.05%)-adapted exponential- and stationary-phase cells were cross-protected against low salinity, whereas low-salinity (0.12% NaCl)-adapted stationary cells were sensitized against 12% bile stress. Results of this study provide further insight into the interaction between low salinity and other common stresses in V. vulnificus.     

Antibiotic susceptibility patterns of Enterococcus strains isolated from Turkish Tulum cheese

The aim of this study was to detect the plasmid profiles, haemolytic activity and antibiotic susceptibility patterns of 47 Enterococcus strains isolated from Turkish Tulum cheese. Enterococcus strains were found to comprise 1–9 plasmids with molecular weight from 2.0 to 47.6 kb. None of the Enterococcus strains displayed haemolytic activity. The Enterococcus strains were found mostly resistant to tetracycline (30 μg) followed by high‐level streptomycin (300 μg). The Enterococcus faecalis strains were found to be highly resistant to antibiotics than Enterococcus faecium and Enterococcus durans strains. In total, 4.3% of the strains exhibited multiple antibiotic resistance patterns.     

Short communication: Fluoroquinolone susceptibility of Staphylococcus aureus strains isolated from caprine clinical mastitis in southeast Spain

The antibiotic susceptibility of 32 strains of Staphylococcus aureus isolated from the mastitic milk of dairy goats was evaluated. The antibiotics tested were 3 fluoroquinolones that have been developed especially for use in veterinary medicine: danofloxacin, marbofloxacin, and orbifloxacin. Minimum inhibitory concentration (MIC) tests were performed according to the microdilution broth method. The MIC₉₀ (concentration at which 90% inhibition is achieved) values obtained were 0.5, 1, and 1 mg/L for danofloxacin, marbofloxacin, and orbifloxacin, respectively. Danofloxacin was the most active fluoroquinolone tested against Staph. aureus strains isolated from milk; however, specific testing is required before using these drugs as therapy for the control of clinical mammary infections in goats.     

Comparison of two AFLP methods and PFGE using strains of Listeria monocytogenes isolated from environmental and food samples obtained from Piedmont, Italy

Listeria monocytogenes ranks among the most frequent causes of death due to foodborne illness (20-30% case fatality rate).Discriminative subtyping methods are important to detect the relatedness of isolates and verify epidemiologic associations. AFLP analysis is a DNA fingerprinting technique based on the selective amplification of genomic restriction fragments. In this study, two AFLP methods and PFGE were compared in regard to discriminatory power, typeability and concordance.A total of 103 unrelated L. monocytogenes strains isolated from different environmental and food sources were analyzed. Strains were isolated from samples obtained from food-production plants, supermarkets and small food markets in Piedmont, Italy.All methods clustered L. monocytogenes strains into two genetic lineages, Lineage I and II. The three methods were compared using the 82 isolates which were typeable with all techniques. The calculated pair-wise Pearson's correlation coefficients (r) showed close agreement between all three methods.Our findings suggest that the AFLP II method can be successfully used to subtype L. monocytogenes strains isolated from foods and food processing facilities.     

Virulence factors and genetic variability of Staphylococcus aureus strains isolated from raw sheep's milk cheese

Contamination of dairy products with Staphylococcus aureus can be of animal or human origin. The host pathogen relationship is an important factor determining genetic polymorphism of the strains and their potential virulence. The aim of the present study was to carry out an extensive characterization of virulence factors and to study the genetic variability of S. aureus strains isolated from raw ewe's milk cheese. A total of 100 S. aureus strains isolated from cheese samples produced in 10 artisan cheese factories were analyzed for the presence of enterotoxins (sea-see) and enterotoxins-like genes (seh, sek, sel, sem, seo, sep), leukocidins, exfoliatins, haemolysins, toxic shock syndrome toxin 1 (TSST-1) and the accessory gene regulator alleles (agr). Strains were also typed using pulsed-field gel electrophoresis (PFGE). AMOVA analysis carried out on PFGE and PCR data showed that the major component explaining genetic distance between strains was the dairy of origin. Of the total isolates 81% had a pathogenicity profile ascribable to "animal" biovar while 16% could be related to "human" biovar. The biovar allowed to estimate the most likely origin of the contamination. Minimum inhibitory concentrations (MICs) of nine antimicrobial agents and the presence of the corresponding genes coding for antibiotic resistance was also investigated. 18 strains carrying blaZ gene showed resistance to ampicillin and penicillin and 6 strains carrying tetM gene were resistant to tetracycline. The presence of mecA gene and methicillin resistance, typical of strains of human origin, was never detected. The results obtained in the present study confirm that S. aureus contamination in artisan cheese production is mainly of animal origin.     

Molecular characterization of Salmonella enterica serovar Saintpaul isolated from imported seafood, pepper, environmental and clinical samples

A total of 39 Salmonella enterica serovar Saintpaul strains from imported seafood, pepper and from environmental and clinical samples were analyzed for the presence of virulence genes, antibiotic resistance, plasmid and plasmid replicon types. Pulsed-field gel electrophoresis (PFGE) fingerprinting using the XbaI restriction enzyme and plasmid profiling were performed to assess genetic diversity. None of the isolates showed resistance to ampicillin, chloramphenicol, gentamicin, kanamycin, streptomycin, sulfisoxazole, and tetracycline. Seventeen virulence genes were screened for by PCR. All strains were positive for 14 genes (spiA, sifA, invA, spaN, sopE, sipB, iroN, msgA, pagC, orgA, prgH, lpfC, sitC, and tolC) and negative for three genes (spvB, pefA, and cdtB). Twelve strains, including six from clinical samples and six from seafood, carried one or more plasmids. Large plasmids, sized greater than 50 kb were detected in one clinical and three food isolates. One plasmid was able to be typed as IncI1 by PCR-based replicon typing. There were 25 distinct PFGE-XbaI patterns, clustered to two groups. Cluster A, with 68.5% similarity mainly consists of clinical isolates, while Cluster C, with 67.6% similarity, mainly consisted of shrimp isolates from India. Our findings indicated the genetic diversity of S. Saintpaul in clinical samples, imported seafood, and the environment and that this serotype possesses several virulent genes and plasmids which can cause salmonellosis.     

Climate anomalies and the increasing risk of Vibrio parahaemolyticus and Vibrio vulnificus illnesses

We examined the potential influence of climate anomalies in expanding the geographical and seasonal range of seafood-borne illnesses from Vibrio parahaemolyticus and Vibrio vulnificus. Archived climate data from areas of implicated seafood production were obtained from various sources, including in situ monitoring devices and satellite imagery. The geographical expansion of V. parahaemolyticus outbreaks into Peru and Alaska corresponded closely with climate anomalies such as El Niño, which brought large masses of abnormally warm water into these regions. Seasonal expansion of V. vulnificus illnesses associated with oysters harvested from the Gulf of Mexico in April and November correspond with warmer water temperatures (>20 °C) recorded during these months since 1998. This retrospective review indicates that climate anomalies have already greatly expanded the risk area and season for vibrio illnesses and suggest that these events can be forecasted. Certainly, when similar circumstances occur in the future, adjustments in industry practices and regulatory policy should be considered, especially for seafood that is consumed raw, such as bivalve mollusks.     

Genetic mechanisms contributing to reduced tetracycline susceptibility of Campylobacter isolated from organic and conventional dairy farms in the midwestern and northeastern United States

Campylobacter is one of the most common causes of gastroenteritis and can be acquired through contact with farm animals or the consumption of raw milk. Because of concerns over the role of food-producing animals in the dissemination of antimicrobial resistance to humans, we evaluated the prevalence of antimicrobial resistance in Campylobacter isolates from dairy farms and the genetic mechanism conferring the observed resistance. Evaluation of antimicrobial resistance was completed on 912 isolates from conventional and 304 isolates from organic dairy farms to eight drugs (azithromycin, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, gentamicin, nalidixic acid, and tetracycline) with microbroth dilution. Resistance to seven of eight drugs was very low and did not differ by farm type. However, tetracycline resistance was common in Campylobacter isolated from both organic and conventional dairy farms, with 48 and 58% of isolates affected, respectively. By multiplex PCR, we determined that tetracycline resistance was highly associated with the carriage of tetO in Campylobacter isolates (X2 = 124, P < 0.01, kappa = 0.86).     

Assessment of antibiotic susceptibility within lactic acid bacteria strains isolated from wine

Susceptibility to 12 antibiotics was tested in 75 unrelated lactic acid bacteria strains of wine origin of the following species: 38 Lactobacillus plantarum, 3 Lactobacillus hilgardii, 2 Lactobacillus paracasei, 1 Lactobacillus sp, 21 Oenococcus oeni, 4 Pediococcus pentosaceus, 2 Pediococcus parvulus, 1 Pediococcus acidilactici, and 3 Leuconostoc mesenteroides. The Minimal Inhibitory Concentrations of the different antibiotics that inhibited 50% of the strains of the Lactobacillus, Leuconostoc and Pediococcus genera were, respectively, the following ones: penicillin (2, < or =0.5, and < or =0.5 microg/ml), erythromycin (< or =0.5 microg/ml), chloramphenicol (4 microg/ml), ciprofloxacin (64, 8, and 128 microg/ml), vancomycin (> or =128 microg/ml), tetracycline (8, 2, and 8 microg/ml), streptomycin (256, 32, and 512 microg/ml), gentamicin (64, 4, and 128 microg/ml), kanamycin (256, 64, and 512 microg/ml), sulfamethoxazole (> or =1024 microg/ml), and trimethoprim (16 microg/ml). All 21 O. oeni showed susceptibility to erythromycin, tetracycline, rifampicin and chloramphenicol, and exhibited resistance to aminoglycosides, vancomycin, sulfamethoxazole and trimethoprim, that could represent intrinsic resistance. Differences were observed among the O. oeni strains with respect to penicillin or ciprofloxacin susceptibility. Antibiotic resistance genes were studied by PCR and sequencing, and the following genes were detected: erm(B) (one P. acidilactici), tet(M) (one L. plantarum), tet(L) (one P. parvulus), aac(6')-aph(2") (four L. plantarum, one P. parvulus, one P. pentosaceus and two O. oeni), ant(6) (one L. plantarum, and two P. parvulus), and aph(3')-IIIa (one L. plantarum and one O. oeni). This is the first time, to our knowledge, that ant(6), aph(3')-IIIa and tet(L) genes are found in Lactobacillus and Pediococcus strains and antimicrobial resistance genes are reported in O. oeni strains.     

Perfluorochemicals in pooled serum samples from United States residents in 2001 and 2002

Manufacturers have used perfluorochemicals (PFCs) since the 1950s in many industrial and consumer products, including protective coatings for fabrics and carpet, paper coatings, insecticide formulations, and surfactants. Some PFCs are persistent ubiquitous contaminants in the environment and in humans. Exposures to PFCs result in potential developmental and other adverse effects in animals. The sources of human exposure to PFCs and the potential health risks associated with exposure are still unclear, and differences in patterns of human exposure may vary. We measured the serum concentrations of perfluorooctane sulfonic acid (PFOS), perfluorooctanoic acid (PFOA; C8), perfluorohexane sulfonic acid (PFHxS), and 8 other PFCs in 54 pooled serum samples collected from 1832 participants of the 2001-2002 National Health and Nutrition Examination Survey. Participants were 12 years of age and older. The pools represented three major racial groups/ethnicities (non-Hispanic blacks, non-Hispanic whites, and Mexican Americans), four age categories (12-19 years, 20-39 years, 40-59 years, and 60 years and older), and both genders. PFCs were extracted from 100 microL of serum using on-line solid-phase extraction coupled to isotope dilution-high performance liquid chromatography-tandem mass spectrometry. The limits of detection ranged from 0.05 ng/mL to 0.2 ng/mL. The concentrations of most PFCs were similar among the four age groups. For PFOS, the estimated least-squares mean (LSM) concentrations among non-Hispanic white males (40.19 ng/mL) and females (23.97 ng/mL) were greater than among non-Hispanic black males (18.27 ng/mL) and females (17.93 ng/mL) or Mexican American males (13.71 ng/mL) and females (10.40 ng/ mL). Similarly, for PFOA, the LSM concentrations among non-Hispanic white males (6.98 ng/mL) and females (3.97 ng/ mL) were greater than among non-Hispanic black males (3.62 ng/mL) and females (2.85 ng/mL) or Mexican American males (2.89 ng/mL) and females (2.08 ng/mL). Non-Hispanic whites had also greater LSM concentrations of PFHxS than non-Hispanic blacks and Mexican Americans. These findings indicate different patterns of human exposure to PFCs among the population groups examined and stress the importance of conducting research to identify the environmental sources and pathways of human exposure to PFCs.