Evidence of fish and human pathogens associated with doctor fish (Garra rufa, Heckel, 1843) used for cosmetic treatment.

Doctor fish (Garra rufa, Heckel, 1843) are increasingly used for cosmetic treatment raising particular concerns regarding the potential transmission of infections to clients. Investigations of microbial causes undertaken in two outbreaks of mortality among G. rufa used for cosmetic treatment revealed the presence of multiple bacteria, including both fish and human pathogens such as Aeromonas veronii, A. hydrophila, Vibrio cholerae, Shewanella putrefaciens, Mycobacterium marinum and M. goodii. This range of bacteria indicates an intense microbial proliferation involving multiple pathogens, most likely induced by the poor health condition of the fish. Most of the detected pathogens are well-known agents of zoonosis. Indeed, M. goodii is an emerging nosocomial human pathogen that has never been detected in fish to date, nor in other animals. This first detection of M. goodii associated with fish infection points out a new zoonotic potential for this pathogen. These findings point out that handling, poor environmental conditions and the presence of fish pathogens, that can compromise the immune system of fish, can result in a mixed microbial proliferation and increase the spread of waterborne bacteria, including zoonosis agents. Accordingly, the microbiological surveillance of fish used for cosmetic treatment is extremely important, particularly in association with mortality outbreaks.

Preliminary investigation on the microbiological quality of edible marine gastropods of the Adriatic Sea, Italy.

According to the European Legislation, marine gastropods placed unprocessed on the market must comply with the same requirements established for live bivalve molluscs but, being considered not filterfeeding and unable to concentrate fecal contaminants, they may be harvested outside the classified areas. Despite this statement, little scientific information is available on the microbiological quality of these animals. The aim of the present study was to investigate 28 batches of edible snails of the Adriatic Sea, namely Nassarius mutabilis and Bolinus brandaris, with respect to i) smell and viability, by a method here reported; ii) the bacterial component of the whole body referred to E. coli, Vibrio spp., V. parahaemolyticus, V. vulnificus, V. cholerae and V. alginolyticus. A total of 21 batches of N. mutabilis and 7 batches of B. brandaris were analyzed. Batches of both species retrieved from the primary production were all largely composed of viable animals, had saltwater/neutral smell, and showed mean value of Vibrio spp. of 5,34 and 5,79 log10 UFC g-1 in N. mutabilis and B. brandaris respectively. 47% of the batches of N. mutabilis retrieved from the market, were largely composed of dead animals, had acrid/nasty smell, and showed mean value of Vibrio spp. of 6,53 log10 UFC g-1. E. coli, V. vulnificus and V. cholerae were never detected, but all samples were positive for V. alginolyticus. One sample of B. brandaris was positive for V. parahaemolyticus genotyped by PCR at the specie level (ToxR+) and positive for the thermostable direct hemolysin gene (tdh+).

Molecular characterization and drug susceptibility of non-O1/O139 V. cholerae strains of seafood, environmental and clinical origin, Italy.

Toxigenic and antimicrobial susceptibility patterns and genetic relatedness of 42 non-O1/O139 V. cholerae strains, the majority of them isolated from seafood and marine water of the Adriatic sea, Italy, and 9 clinical strains, two of which with seawater of the Adriatic as the source of infection, were studied. All strains had hlyA El Tor gene but lacked ctxA gene. Four and two isolates, respectively, also had stn/sto and tcpA Class genes. More than 90% of strains showed susceptibility to cefotaxime, ciprofloxacin, cloramphenicol, tetracycline, trimethoprim + sulfamethoxazole and intermediate or full resistance to tetracycline and erythromycin. Six strains of seafood and clinical source were multi-drug resistant. PFGE analysis allowed to type all the strains with 50 banding patterns. Twenty-one strains, 11 and 8 from seafood and seawater, respectively, and 2 of clinical origin, were grouped into 9 different clusters. We report the presence of toxigenic and multidrug resistant non-O1/O139 V. cholerae strains in Adriatic, some of which genetically related, and support that they represent a potential reservoir of toxin and antibiotic resistance genes.

Preliminary study on the antimicrobial susceptibility pattern related to the genotype of Vibrio vulnificus strains isolated in the north-western Adriatic Sea coastal area.

V. vulnificus is a Gram-negative bacterium, commonly found in estuarine and coastal habitats, that can infect humans through seafood consumption or wound exposure. This study represents the first attempt to correlate the genotype of Vibrio vulnificus strains isolated in the north-western Adriatic Sea coastal area, with their antimicrobial susceptibility patterns. On the whole, 40 V. vulnificus strains, isolated from shellfish (n=20), different coastal water bodies (n=19), and the blood of a Carretta carretta turtle (n=1), were utilized. All strains were positive for the species-specific genes vvhA and hsp, with high variability for other markers: 55% (22 out of 40) resulted of the environmental (E) genotype (vcgE, 16S rRNA type A, CPS2 or CPS0), 10% (4 out of 40) of the clinical (C) genotype (vcgC, 16S rRNA type B, CPS1), and 35% (14 out of 40) of the mixed (M) genotype, possessing both E and C markers. The antimicrobial susceptibility was assayed by the diffusion method on agar, according to the Clinical Laboratory Standards Institute (CLSI), utilizing the following commercial disks (Oxoid): ampicillin (AMP), ampicillin- sulbactam (SAM), piperacillin (PRL), cefazolin (KZ), cefotaxime(CTX), ceftazidime (CAZ), imipenem (IPM), meropenem (MEM), amikacin (AK), gentamicin(CN), tetracycline(TE), ciprofloxacin (CIP), levofloxacin (LEV), trimethoprim-sulfamethoxazole (SXT), and chloramphenicol (C). 75% of the strains, (n=30) including all C strains, was sensitive to all the tested antibiotics, whereas E strains showed intermediate sensitivity to AK (2 strains), CIP and CAZ (1 strain), TE (1 strain) and resistance to KZ (1 strain), and 4 M strains showed I to AK.

Occurrence of Vibrio Parahaemolyticus, Vibrio Cholerae and Vibrio Vulnificus in the Clam Ruditapes Philippinarum (Adams & Reeve, 1850) from Emilia Romagna and Sardinia, Italy.

Marine vibrios, Vibrio parahaemolyticus, V. vulnificus and V. cholerae are responsible of the majority of food-borne human infections by consumption of bivalve shellfish. The aim of the present study was to ascertain the occurrence of these bacteria, and their potential pathogenicity, in the Manila clam R. philippinarum from Emilia Romagna (ER) and Sardinia (SR) regions, Italy. Isolation was performed on CHROMagarTM vibrio with subculture on (thiosulfate-citrate-bile salts-sucrose) Agar and m-modified-cellobiose-polymyxin b-colistin (-CPC) Agar. Suspected strains were purified, biochemically characterized and genotyped by simplex polymerase chain reaction (PCR) for the specie-specific and pathogenic gene markers: V. parahaemolyticus (toxRP, tdh and trh); V. vulnificus (vvhA, hsp, vcgC, vcgE, CPS operon allele 1, CPS operon allele 2, 16s-rRNA operon allele A, 16s-rRNA operon allele B; V. cholerae (toxRC, hlya, tcpI, tcpA, ctxA, ctxB, stn/sto). Moreover a multiplex PCR was applied to the SR bivalve shellfish, for the simultaneous detection of the three targets directly on homogenate samples, targeting the species-specific gene for V. cholerae (toxRC), V. parahaemolyticus (toxRP) and V. vulnificus (vvhA). As a result of phenotyping and genotyping of isolates, bivalve shellfish from ER resulted positive for V. parahaemolyticus (27.8%) and V. vulnificus (10.1%), but negative for V. cholerae. Shellfish from SR resulted positive for V. parahaemolyticus (30.3%), V. vulnificus (6.1%) and V. cholerae (3%). No significant differences emerged between the two areas (P>0.05).

First Multi-Year Retrospective Study on Vibrio Parhaemolyticus and Vibrio Vulnificus Prevalence in Ruditapes Philippinarum Harvested in Sacca Di Goro, Italy.

The present work describes a retrospective study aiming to verify a possible correlation between the environmental conditions (temperature, salinity and dissolved oxygen), the abundance of Vibrio spp., and the prevalence of V. parahaemolyticus and V. vulnificus in the Manila clam R. philippinarum harvested in Sacca di Goro, Emilia-Romagna Region, Northern Italy. On the whole, 104 samples, collected in the period 2007-2015 and submitted to microbiological analyses (isolation and genotyping), have been reconsidered for Vibrio spp. load, V. parahaemolyticus prevalence (total, gene marker toxRP; potentially pathogenic, gene markers tdh and/or trh) and V. vulnificus prevalence (total, gene markers vvhA and hsp) together with environmental data obtained from the monitoring activity of the Emilia-Romagna Regional Agency for the Prevention, the Environment and the Energy. Environmental data have been processed to calculate the median of each, assessing the seasonal range of seawater temperature (warmer months: April-October, T°C >16.45°C; cooler months November-March, T°C <16.45°C), salinity (27 psu), and dissolved oxygen (< or >8.2 mg/L). Total V. vulnificus, total and potentially pathogenic V. parahaemolyticus were present respectively in the 11.5, 29.8 and 6.7% of the samples. The Vibrio spp. load (mean value of 4.69±0.65 log10 colony forming unit g-1) and the prevalence of potentially pathogenic V. parahaemolyticus, were not significantly correlated to the environmental conditions (P>0.05), whereas the prevalence of both total V. vulnificus and total V. parahaemolyticus was significantly higher in the warmer period (P<0.05), without correlation with salinity and dissolved oxygen values (P>0.05).