TCBS Agar Research Articles

우리나라 양식 강도다리, Platichthys stellatus에서 분리된 Photobacterium damselae subsp. piscicida의 특성

본 연구에서는 2012년 8월, 우리나라 울산 소재의 강도다리 양식장에서 뚜렷한 증상 없이 강도다리 폐사가 발생하여 그 원인을 밝히고자 하였다. 기생충, 세균, 바이러스 검사를 수행하였으며, 내부 장기에서 균이 순수 분리되어 해당 분리균주의 표현형 및 유전적 특성을 분석하였다. 순수 배양된 균체를 확인하여 계대 배양하여 생화학적 성상과 16S rRNA 유전자와 capsular polysaccharide (CPS) 유전자의 염기서열 분석 결과 Photobacterium damselae subsp. piscicida으로 동정되었다. Photobacterium damselae subsp. piscicida와 Photobacterium damselae subsp. damselae는 TCBS agar 배지의 균주 배양 유무, 16S rRNA 및 CPS 유전자 분석을 통해 구분할 수 있었다. 실험 균주는 ofloxacin과 gentamycin에 대해서 감수성을 나타냈으며, 배양 온도에 따른 발육시험 시 <TEX>$18^{\circ}C$</TEX> 및 <TEX>$25^{\circ}C$</TEX>에서 시험한 다른 균주에 비해 유의적으로 높은 생장율을 나타내었다. Starry flounder, Platichthys stellatus (body length <TEX>$4.4{\pm}0.51cm$</TEX>) that became sick during an outbreak of disease at mariculture facilities at Ulsan, Korea in August of 2012, were examined to identify the cause of the disease. Diseased fish didn't show a unique sign, but the oxidase-positive and gram negative rod was isolated from moribund fish. The bacterium was revealed as Photobacterium damselae subsp. piscicida by biochemical analysis and sequence analysis of the 16S rRNA and capsular polysaccharide (CPS) genes. The isolates (AD5) was carrying susceptible to ofloxacin and gentamycin and showed high growth value at <TEX>$18^{\circ}C$</TEX> and <TEX>$25^{\circ}C$</TEX> compared to four other P. damsela strains.

서해안에서 분리한 비브리오균의 항생제 감수성 특성

Bacteria of genus Vibrio are Gram-negative, curved, halophilic, nonspore-forming bacteria, autochthonous inhabitans of the marine and estuarine environments. Some of the Vibrio species such as V. parahaemolyticus, V. vulnificus, and V. cholerae are associated with human disease. Each year many people have been suffering from food-borne disease caused by the ingestion of seafood. In this study, we have monitored antibiotic resistance of this microorganism in 6 coastal areas of West Sea by sampling shellfish monthly. Vibrio spp. were detected from 23.3% of 120 samples analyzed using TCBS agar plates as well as API 20E kit. Among 16 antibiotics tested, resistance to vancomycin and ampicillin was observed in 82.1% of the isolates, and Vibrio spp. resistant to rifampin (71.4%) and cephalothin (53.6%) were also high. Most of the isolates were sensitive to chloramphenicol (92.9%), sulfamethoxazole/trimethoprim (92.9%), and tetracycline (96.4%). About 71.4% of the isolates showed multiple drug resistance toward 3 antibiotics including vancomycin and ampicillin.

Impact of arachidonic acid enrichment of live rotifer prey on bacterial communities in rotifer and larval fish cultures

Rotifers (Brachionus plicatilis), commonly used at first feeding in commercial fish hatcheries, carry a large bacteria load. Because they are relatively poor in essential fatty acids, it is common practice to enrich them with fatty acids, including arachidonic acid (AA). This study aims to determine whether prey enrichment with AA may act as a prebiotic and modify the microbial community composition either in AA-enriched rotifer cultures or in larval-rearing water using winter flounder (Pseudopleuronectes americanus) as a larval fish model. AA enrichment modified the bacterial community composition in both the rotifer culture tanks and the larval-rearing tanks. We observed an increase in the number of cultivable bacteria on TCBS (thiosulfate-citrate-bile salts-sucrose) agar, used as a proxy for the abundance of Vibrio sp. The results suggest that AA may also play an indirect role in larval health.

Survivability of Vibrio cholerae O1 in Cooked Rice, Coffee, and Tea.

This study aimed to investigate the survival of Vibrio cholerae O1 in 3 types of preparation for cooked rice, Oryza sativa L., (plain rice, rice with coconut milk, and rice with ginger); coffee, Coffea canephora, (plain coffee, coffee with sugar, and coffee with sweetened condensed milk); and tea, Camellia sinensis, (plain tea, tea with sugar, and tea with sweetened condensed milk) held at room temperature (27°C). The survival of V. cholerae O1 was determined by spread plate method on TCBS agar. Initial cultures of 8.00 log CFU/mL were inoculated into each food sample. After 6 h incubation, significant growth was only detected in rice with coconut milk (9.67 log CFU/mL; P < 0.05). However, all 3 types of rice preparation showed significant growth of V. cholerae after 24 h (P < 0.05). For coffee and tea preparations, V. cholerae survived up to 6 h in tea with condensed milk (4.72 log CFU/mL) but not in similar preparation of coffee. This study showed evidence for the survivability of V. cholerae in rice, coffee, and tea. Thus, holding these food and beverages for an extended period of time at room temperature should be avoided.

The role of protein extracts in the induction of disease in Acanthaster planci

Thiosulfate–citrate–bile–sucrose agar (TCBS) has been shown to induce pathogenesis in the crown-of-thorns starfish (COTS), Acanthaster planci, providing potentially novel options to control COTS outbreaks, but the mechanistic basis for this effect is unknown. This study explores reactions of COTS to individual ingredients of TCBS, testing for allergic reactions versus pathogenesis. Four out of nine TCBS chemical ingredients tested induced allergic reactions and death in injected COTS. Peptone 10gl−1 and oxgall 8gl−1 induced 100% mortality, while yeast extract and agar induced death in 40% and 20% of COTS, respectively, 48h after injection. Peptone was evaluated at three different concentrations (10g, 5g, and 1gl−1). Peptone 10gl−1 induced 100% mortality, peptone 5gl−1 killed 60% of injected COTS, and peptone 1gl−1 induced death in only 20% of challenged COTS, indicating that toxicity of peptone is concentration-dependent. Sodium citrate induced moderate mucus production in all COTS challenged, but disease did not progress and COTS completely recovered after 52h. The remaining ingredients in TCBS did not produce any kind of clinical signs of disease. Peptone, oxgall, and yeast are potentially useful in controlling outbreaks because these protein extracts can be safer to use compared to previously used noxious chemicals. In addition, lowered concentrations are required to kill COTS, therefore increasing efficiency by saving time, money, and effort in COTS control programs.

The international standard ISO/TS 21872–1 to study the occurence of total and pathogenic Vibrio parahaemolyticus and Vibrio cholerae in seafood: ITS improvement by use of a chromogenic medium and PCR

During two surveys conducted in 2008 and 2009, the culture method described in the international standard ISO/TS 21872–1 was applied to the detection of Vibrio parahaemolyticus and Vibrio cholerae in 112 living bivalve mollusc samples, with a chromogenic medium used in addition to the TCBS agar, as second selective isolation medium and for enumeration of V. parahaemolyticus and V. cholerae by surface inoculation. A PCR method for detection of these 2 Vibrio species and the hemolysin genes tdh and trh, was applied in parallel. In 2009, the survey was extended to finfish fillets and crustaceans. PCR was also used for species confirmation of characteristic colonies. The identity of the PCR products, specifically targeting V. parahaemolyticus, was checked by sequencing.Occurrence of V. parahaemolyticus and V. cholerae isolates in living bivalve molluscs ranged from 30.4% to 32.6% and from 1.4% to 4.7% respectively. In frozen crustaceans (2009 survey) V. parahaemolyticus and V. cholerae isolates were respectively found in 45% and 10% of the samples. No V. parahaemolyticus or V. cholerae was detected in frozen fish fillets, neither by the ISO method nor by PCR. In 2009, enteropathogenic V. parahaemolyticus (trh+) was isolated from 4 out of 43 oyster samples while the trh gene was present in V. alginolyticus strains and in samples where V. parahaemolyticus was not detected (9 over 112 samples).The ISO method failed to isolate V. parahaemolyticus in 44% to 53% of the living bivalve molluscs where PCR detected the toxR gene specific of V. parahaemolyticus (Vp-toxR). Our results highlighted the need for a revision of the ISO/TS 21872–1 standard, at least, for analysis of living bivalve molluscs, and confirmed the increasing concern of enteropathogenic V. parahaemolyticus in French bivalve molluscs. Enrichment at 41.5°C was questioned and some reliable solutions for the improvement of the ISO/TS 21872–1 method, such as the PCR method for screening of positive samples and confirmation of colonies, were pointed out.

경기인천 연안에서 분리된 장염비브리오균의 항생제 내성 및 플라스미드 보유 현황

장염비브리오는 우리나라에서 하절기에 발생하는 식중독의 주요 원인균으로 주로 해수 및 어패류를 통해 인체에 감염된다. 최근 지구 온난화로 해수의 온도가 상승하면서 해양환경 중 세균들의 분포도가 증가하고 또 다양한 항생제에 대한 내성을 보이는 병원균들이 확산되고 있어 국민보건 및 수산양식 등의 분야에 큰 문제로 대두되고 있다. 이에 본 연구에서는 경기, 인천지역 7곳에서 해수 및 어패류 시료를 채취하여 장염비브리오균의 분포도를 모니터링하고 이들의 항생제 내성 양상을 분석하였다. 총 966건의 시료를 <TEX>$CHROMagar^{TM}$</TEX> 및 TCBS 평판배지와 multiplex PCR기법으로 동정했을 때 전체 해수 시료의 61.9%, 어패류 시료의 41.8% 등 총 47.7%의 시료에서 장염비브리오균이 검출되었다. 이들 분리균주들의 항생제 내성을 13종의 항생제에 대해 분석한 결과 97.3%와 87.3%가 각각 vancomycin과 ampicillin에 대해 내성을 보였고 cephalothin (48.8%)과 rifampin (46.1%)에 대한 내성비율도 기타 다른 항생제들에 비해 상대적으로 높은 것으로 나타났다. 분리균주들의 항생제 감수성은 chloramphenicol (91.7%)과 trimethoprim-sulfamethoxazole (91.8%)에 대해 가장 높았고, gentamycin (82.3%), tobramycin (74.8%), nalidixic acid (71.6%), tetracyclin (69.4%), cefotaxime (63.0%) 등의 순서로 감수성 비율이 높았다. 분리균주들의 약 69%가 vancomycin과 ampicillin을 포함하여 3개 이상의 항생제에 대해 다중내성을 보였고 특히 11개 항생제에 대해 내성을 보이는 균주도 2개 검색되었다. 항생제 내성 장염비브리오 분리균주들의 플라스미드 프로파일을 분석한 결과 55.1%가 플라스미드를 보유하고 있었으며, 이들은 총 24가지 유형으로 분류되었으나 플라스미드 프로파일과 항생제 내성 간의 뚜렷한 상관관계는 관찰되지 않았다. Vibrio parahaemolyticus is one of the major agents responsible for food poisoning during summer in Korea, which is transmitted via seawater or seafoods. Recently, distribution of the bacteria in the marine environment has been increased due to global warming. Great concern also has been raised regarding public hygiene as well as marine culture by the emergence of pathogens with antibiotic resistance. Therefore, distribution of V. parahaemolyticus and antibiotic resistance of the isolates were monitored in 7 coastal areas of Kyonggi Province and Incheon by sampling seawater, fishes and clams monthly. V. parahaemolyticus was detected from 47.7% of 966 samples (seawater 61.9%, seafoods 41.8%) analyzed using <TEX>$CHROMagar^{TM}$</TEX> and TCBS agar plates as well as multiplex PCR. Among 13 antibiotics tested, resistance to vancomycin and ampicillin was observed in 97.3% and 87.3% of the isolates, respectively, and the ratios of them resistant to cephalothin (48.8%) and rifampin (46.1%) were also high. The isolates were most highly sensitive to chloramphenicol (91.7%) and trimethoprim-sulfamethoxazole (91.8%). The ratio of sensitivity for other antibiotics was also high in the descending order of gentamycin (82.3%), tobramycin (74.8%), nalidixic acid (71.6%), tetracyclin (69.4%), cefotaxime (63.0%). About 69% of the isolates showed multiple drug resistance toward 3 antibiotics including vancomycin and ampicillin. Two of them exhibited resistance for 11 antibiotics used in this study. Plasmid profile analysis of the isolates with antibiotic resistance revealed that 55.1% of them retained plasmids of 24 different types. However, no clear inter-relationship between the resistance and the plasmid profile has been observed.

Phenotypic and genetic diversity of coexisting Listonella anguillarum, Vibrio harveyi and Vibrio chagassi recovered from skin haemorrhages of diseased sand smelt, Atherina boyeri, in the Gulf of Trieste (NE Adriatic Sea)

This study identified and characterized coexisting Vibrios associated with haemorrhagic skin lesion bearing sand smelt fishes (Atherina boyeri) in north-eastern Adriatic Sea. Bacteria were isolated from external skin lesions of four samples, and representative morphotypes grown on thiosulfate-citrate-bile salt-sucrose agar were isolated. In total 25 isolates, presumptively assigned to Vibrio genus, were biochemically characterized and were grouped in 10 phenotypic profiles. Phenotypes were heterogeneously distributed among the diseased sand smelt analysed; only one phenotype was recovered from all the samples. Sequencing of 16S rRNA was performed to identify representatives of all phenotypes. Phylogenetic analysis using the neighbour-joining method revealed six isolates clustered within the Vibrio harveyi group, three clustered with known Vibrio chagasii strains and three clustered with Listonella anguillarum. Vibrios with a broad phenotypic variability were found in the external lesions of diseased A. boyeri. In total three species of Vibrio were identified: V. harveyi showed the wider phenotypical and ribotypical heterogeneity while L. anguillarum shared similar biochemical characteristics with typical strains. Previously unreported coexistence of potential pathogenic species colonizing diseased A. boyeri has ecological as well as epidemiological significance.

Diagnostic Microbiology in Post-Genocide Cambodia

The microbiology laboratory that Nhem Somary supervises in the Kampong Cham Provincial Hospital along the Mekong River, three hours northeast of Phnom Penh, serves as a model for labs in other Cambodian hospitals. She began supervising the laboratory in 1998, but did not offer diagnostic microbiology laboratory services until 2009 when the Diagnostic Microbiology Development Program established a microbiology laboratory there. Early that year, she used her only bottle of TCBS agar to isolate more than 60 strains of Vibrio cholerae during an outbreak of “acute watery diarrhea.” Somary gave physicians at the Kampong Cham Hospital an accurate diagnosis.

Occurrence of potentially pathogenic vibrios and related environmental factors in Songkhla Lake, Thailand

Vibrios are halophilic bacteria that are ubiquitous in marine environments. Their occurrence in tropical lakes has rarely been investigated. In this study, the predominance and diversity of Vibrio spp. was investigated over a 12-month period in a coastal lagoon, Songkhla Lake, in southern Thailand. Water samples were collected at 2 stations in the estuary near Yor Island in Songkhla Lake. The predominant vibrios were detected by a culture-based method, using thiosulfate-citrate-bile salt-sucrose agar and CHROMagar Vibrio. The diversity of Vibrio spp. was evaluated using denaturant density gradient electrophoresis (DGGE). The highest numbers of total vibrios and Vibrio parahaemolyticus in both areas were observed during the summer. There was no significant correlation between the numbers of vibrios, including V. parahaemolyticus, and either the water temperature or plankton density. Variations in Vibrio species were observed with changes in salinity. Vibrio parahaemolyticus and V. cholerae non-O1/non-O139 were detected during the rainy season when the salinity dropped to nearly 0 parts per thousand. In both areas, V. alginolyticus was the most prominent species detected by the culture method, whereas Vibrio parahaemolyticus was detected by DGGE, every month. Other Vibrio spp. of potential public health concern were also detected by the culture method; they included V. vulnificus , V. fluvialis , and V. mimicus .

Occurrence of Vibrio spp. in fish and shellfish collected from the Swiss market.

The genus Vibrio includes gram-negative bacteria that inhabit estuarine ecosystems. V. cholerae, V. parahaemolyticus, and V. vulnificus pose a considerable public health threat as agents of sporadic and epidemic foodborne infections associated with the consumption of raw or undercooked contaminated fish or shellfish. In this study, we analyzed 138 fish and shellfish samples collected from the Swiss market (fish fillets [n = 102], bivalves [n = 34], and squid [n = 2]). Microbiological analysis was done according to International Organization for Standardization method 21872-1/21872-2:2007, using thiosulfate citrate bile sucrose agar and chromID Vibrio agar as selective agar. Presumptive-positive colonies on thiosulfate citrate bile sucrose agar or chromID Vibrio agar were picked and were identified by the API 20E and species-specific PCR systems. V. cholerae isolates were tested further by PCR for the presence of the cholera toxin A subunit gene (ctxA). V. parahaemolyticus isolates were tested by PCR for genes encoding for thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh). V. cholerae was isolated from three samples and V. parahaemolyticus from eight samples. None of these strains harbored species-specific virulence factors. Further, V. alginolyticus was isolated from 40 samples, and V. fluvialis was isolated from 1 sample. Our study provides, for the first time, data for the assessment of exposure to Vibrio spp. in raw fish and bivalves consumed in Switzerland.

Diversity of Vibrionaceae associated with Ruditapes decussatus hatchery in Tunisia

The diversity of Vibrionaceae was investigated in a Ruditapes decussatus hatchery located in the Tunisian coastal marine waters. Quantitative study of the bacterial flora, conducted in different compartments of the hatchery, revealed that the total bacterial count (TVC) and the presumptive Vibrio (PVC) increased during the summer, due to the rise of temperature. The treatment of breeders with chloramphenicol (8 mg/l/day) for 4 days and the usage of open circuit sea water as a larva-rearing technique significantly reduced the density of the presumptive Vibrio in larva. Vibrionaceae strains were isolated on TCBS agar plates and identified using some bacteriological tests (“API 20 E strips” standardized micro-method for biochemical identification, vibriostatic agent O/129, growth at different temperatures and salinity, string test and antibiotics susceptibility). Vibrio alginolyticus was the predominant species, followed by V. fluvialis, V. vulnificus, V. cholerae and V. parahaemolyticus. Furthermore, the antibiotic susceptibilities patterns of Vibrio isolates showed the presence of a number of resistant strains.

Antimicrobial Effects of Essential Oils ofCinnamosma fragranson the Bacterial Communities in the Rearing Water ofPenaeus monodonLarvae

Farmed shrimps are vectors of various Vibrio species that are considered a potential health hazard. Previous study has shown that Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio alginolyticus dominated in the water and larval samples of shrimp hatchery (Randrianarivelo et al. 2010 ). The effects of two essential oils (EOs) of Cinnamosma fragrans, an endemic plant to Madagascar (B8: linalool-type and B143: 1,8-cineole-type), were determined on the total heterotrophic aerobic bacteria and the Vibrio concentrations in the rearing water of Penaeus monodon hatchery. The assays took place in OSO Farming's shrimp hatchery in Madagascar. EOs were directly added to the water tank. The bacterial concentrations of water tank were assessed on marine agar and thiosulfate citrate bile sucrose agar. The larvae culture corresponded to four replicates each of B8, B143, erythromycin (E), and control (oil and antibiotic free). The bacterial concentration of the rearing water in B8, B143, and antibiotic (E) tanks were significantly lower (p < 0.05) than in the control. Further, there was no significant difference (p > 0.05) between the three treatments B8, B143, and E. This study demonstrated that both EOs of C. fragrans, like antibiotic, inhibited bacterial growth in the rearing water of P. monodon larvae. The potential of C. fragrans EO to control the bacterial load in in vivo conditions of P. monodon hatchery makes it a relevant option for producers to minimize risk of Vibrio growth in the rearing water of larvae, which is the primary source of colonization of shrimp larvae.

Comparative evaluation of a chromogenic agar medium – PCR protocol with a conventional method for isolation of Vibrio parahaemolyticus strains from environmental and clinical samples

Screening for pathogenic Vibrio parahaemolyticus has become routine in certain areas associated with food-borne outbreaks. This study is an evaluation of the CHROMagar Vibrio (CV) medium-PCR protocol and the conventional method (TCBS (thiosulfate-citrate-bile salts-sucrose) agar plus biochemical and Wagatsuma agar tests) for detection of V. parahaemolyticus in shrimp, water, sediment, and stool samples collected for biosurveillance in an endemic area of northwestern Mexico. A total of 131 environmental and clinical samples were evaluated. The CV medium-PCR protocol showed a significantly improved ability (P < 0.05) to isolate and detect V. parahaemolyticus, identifying isolates of this bacteria missed by the conventional method. Although some other bacteria, distinct from pathogenic V. parahaemolyticus, produced violet colonies similar to that of V. parahaemolyticus on CV medium, we were able to detect a superior number of samples of V. parahaemolyticus with the CV medium-PCR protocol than with the conventional method. The Kanagawa phenomenon is routinely determined on Wagatsuma agar for the diagnosis of V. parahaemolyticus (pathogenic) positive for thermostable direct hemolysin (TDH) in developing countries. In our results, Wagatsuma agar showed low sensitivity (65.4% at 24 h and 75.6% at 48 h) and specificity (52.4% at 48 h) for identifying V. parahaemolyticus positive for TDH. Overall, our data support the use of the CV medium-PCR protocol in place of the conventional method (TCBS-biochemical tests-Wagatsuma agar) for detection of pathogenic V. parahaemolyticus, both in terms of effectiveness and cost efficiency.

Kualitas Perairan di Selat Makassar Ditinjau dari Aspek Bakteriologi

Makassar strait is one of the important and strategic straits in Indonesia as it constitutes interislands and international sealanes. Its water condition is under the influence of both Kalimantan and Sulawesi islands, so that the dynamics in the region of marine and coastal environment becomes more complex. A study on water quality of bacteriological points of view in Makassar strait was conducted in October 2003. Seawater and sediment samples were collected by using the Research Vessel Baruna Jaya VIII at 16 stations. The aims of this study is to find out the water quality based on the contents of coliform bcteria and pathogenic bacteria Salmonella and Vibrio . The analysis of coliform bacteria was conducted by using membrane filter technique, while the pathogenic bacteria Salmonella and Vibrio were cultivated in the specific media Selenith broth and TCBS agar. The results showed that coliform bacteria at the surface layers depth varied from 80– 824 CFU/100mL with an average of 245 CFU/100mL, while those at the middle layer varied from 0–60 CFU/100mL with an average of 23 CFU/100mL. Seven pathogenic bacteria were isolated from seawater sample, i.e. Citrobacter spp ., Edwardsiella spp ., Proteus spp ., Pseudomonas spp ., Providencia spp ., Shigella spp . and Vibrio spp, while eight pathogenic bacteria were obtained at sediment sample, i.e. Aeromonas spp ., Citrobacter spp. , Proteus spp ., Pseudomonas spp ., Providencia spp ., Shigella spp ., Yersenia spp. and Vibrio spp. These results indicated that coliform bacteria content in Makassar strait is under the threshold of Indonesian standard, i.e. Ministry Decree of Environment No: 51, 2004. Vibrio spp. was the pathogenic bacteria found both from seawater and sediment samples. In general, however, pathogenic bacteria was dominated by low pathogenic bacteria. Therefore, it can be concluded that from bacteriological point of view, water quality of Makassar strait is still relatively good.

Occurrence and seasonality of Vibrio aestuarianus in sediment and Crassostrea gigas haemolymph at two oyster farms in France

Vibrio aestuarianus is frequently found in coastal areas and can infect and induce mortalities in the pacific oyster Crassostrea gigas. However, nothing is known about its distribution and seasonality in the estuarine environment, especially where oyster farming is practiced. Its occurrence was investigated in sediment and oyster haemolymph at 2 oyster farms in Brittany (France) over 2 yr during 2 periods, from June to September 2007 and from February to June 2008. Total heterotrophic bacteria (HB) were cultured on marine agar while total Vibrio spp. and V aestuarianus were selectively numerated using thiosulfate citrate bile salts sucrose agar (TCBS agar) and the species-specific hybridisation method, respectively. PCR was performed to detect V aestuarianus in sediment when it became unculturable. Both total Vibrio spp. and V aestuarianus had a seasonal trend. The highest concentrations were recovered in the warmest months. Its abundance ranged from 10(2) to 4 x 10(5) CFU ml(-1) in haemolymph and from 10(3) to 1 x 10(4) CFU g(-1) in the sediment. Temperature was the main factor influencing the concentration of Vibrio spp. and V. aestuarianus in the sediment. Thus V aestuarianus might subsist during the cold seasons in the sediment, from which it can emerge when environmental conditions became favourable.

Occurrence of Vibrio vulnificus in mussel farms from the Varano lagoon environment

Monitoring the occurrence of the human pathogen Vibrio vulnificus in a mussel farm located in the lagoon of Varano (Italy). A total of 72 samples of mussel, water and sediment, collected from two locations of Varano lagoon in the Gargano peninsula, during a 7-month survey, were analysed. Isolation and PCR characterization of six V. vulnificus environmental genotype strains revealed that this pathogen was isolated when with T was above 22 °C and salinity ranged between 22.7 and 26.4‰. No significant correlation of the occurrence of V. vulnificus with water pH or salinity was observed. Moreover, 8% of mussel samples were found to be contaminated by V. vulnificus. All of that positive mussel samples originated from the same sampling station. It is suggested that warmer season are risky to eat raw or undercooked bivalve molluscs in the local area. To increase knowledge about environmental conditions that may affect the occurrence of waterborne pathogen Vibrio vulnificus in seafood.

Distribution of type III secretion systems in Vibrio parahaemolyticus from the northern Gulf of Mexico

Two well-characterized Vibrio parahaemolyticus pathogenicity factors - thermostable direct haemolysin (TDH) and TDH-related haemolysin - are produced by strains containing the tdh and trh genes, respectively. Most strains of V. parahaemolyticus contain two nonredundant type III secretion systems (T3SS), T3SS1 and T3SS2, both of which contribute to pathogenicity. Furthermore, a recent study has revealed two distinct lineages of the V. parahaemolyticus T3SS2: T3SS2α and T3SS2β. The aim of this study was to determine the incidence of these pathogenicity factors in environmental isolates of V. parahaemolyticus. We collected 130 V. parahaemolyticus isolates (TCBS agar) containing tdh and/or trh (determined by colony hybridization) from sediment, oyster and water in the northern Gulf of Mexico and screened them and 12 clinical isolates (PCR and agarose gel electrophoresis) for pathogenicity factors tdh, trh, T3SS1, T3SS2α and T3SS2β. The majority of potential pathogens were detected in the sediment, including all tdh(-) /trh(+) isolates. T3SS2α components were detected in all tdh(+) /trh(-) isolates and zero of 109 trh(+) isolates. One T3SS2α gene, vopB2, was found in all tdh(+) /trh(-) clinical strains but not in any of the 130 environmental strains. Fluorescence in situ hybridization adapted for individual gene recognition (RING-FISH) was used to confirm the presence/absence of vopB2. T3SS2β was found in all tdh(-) /trh(+) isolates and in no tdh(+) /trh(-) isolates. The combination of haemolysins found in each isolate consistently corresponded to the presence and type of T3SS detected. The vopB2 gene may represent a novel marker for identifying increased virulence among strains. This is the first study to confirm the presence of T3SS2β genes in V. parahaemolyticus strains isolated from the Gulf of Mexico and one of the few that examines the distribution and co-existence of tdh, trh, T3SS1, T3SS2α and T3SS2β in a large collection of environmental strains.

Bacterial accumulation by the Demospongiae Hymeniacidon perlevis: A tool for the bioremediation of polluted seawater

Sponges can filter large amounts of water, which exerts an important grazing impact on free bacteria, an important component of the diet of sponges. We examined the accumulation of bacteria in the Demospongiae (Hymeniacidon perlevis). Analyses were performed on homogenates from unstarved and starved sponges in seawater from their sampling site (the Ionian Sea). Culturable heterotrophic bacteria (22°C), total culturable bacteria (37°C) and vibrios densities were measured on marine agar 2216, plate count agar and TCBS agar, respectively. Total and fecal coliforms, as well as fecal streptococci, were determined by the most probable number method (MPN). H. perlevis was able to accumulate all of the six microbiological groups. Bacterial groups differed in their resistance to digestion by H. perlevis. Our data suggest that H. perlevis may accumulate, remediate and metabolize bacteria and that they may be employed as a useful bioindicator and bioremediator.

Novel alternative to antibiotics in shrimp hatchery: effects of the essential oil of Cinnamosma fragrans on survival and bacterial concentration of Penaeus monodon larvae

The activity of two essential oils (EOs) of Cinnamosma fragrans, an endemic plant to Madagascar (B8: linalool-type and B143: 1,8-cineole-type), against bacterial isolates from a shrimp hatchery of Penaeus monodon and their effects on the survival and bacterial concentration of larvae were determined. Minimum inhibitory concentrations were determined using a broth dilution technique. The bacterial concentrations of both larvae and water tank were assessed on Marine agar and Thiosulfate Citrate Bile Sucrose agar. The assays took place in OSO Farming's shrimp hatchery in Madagascar. EOs were directly added to the water tank. Regarding the survival, the assays in larval culture (four replicates each of B8, B143, E and control) showed that B8 oil had a similar effect (P > 0.05) as the antibiotic (Erythromycin) and was more active than B143 (P < 0.05). A negative correlation was observed between the bacterial concentration and the survival of larvae for all assays. Both C. fragrans essential oils, as antibiotic, exhibited significantly higher survival rates and lower bacterial concentrations of the larvae than the control (oil and antibiotic free). The potential of C. fragrans essential oil to control the bacterial load in in vivo conditions, thereby enhancing survival rate of P. monodon larvae, makes it a relevant option for developing a novel alternative to antibiotics in shrimp hatchery culture.