Pathogenic potential of meat-borne coagulase negative staphylococci strains from slaughterhouse to fork.

This study aimed to determine the prevalence of coagulase-negative staphylococci (CoNS) in meat processing lines for their pathogenic potential associated with biofilm formation, staphylococcal toxin genes, and antibiotic resistance in obtained isolates. Out of 270 samples, 56 isolates were identified as staphylococcal with their species level, and their antimicrobial resistance profiles were also determined with the BD Phoenix™ system. Among these, CoNS were found in 32 isolates, including S. epidermidis (22%), S. warneri (22%), S. cohnii (9%), S. schleiferi (9%), S. capitis (6%), S. haemolyticus (6%), S. lugdunensis (6%), S. chromogenes (6%), S. kloosii (3%), S. sciuri (3%), S. lentus (3%), and S. caprae (3%). Biofilm formation was observed in 78.1% of CoNS isolates, with 56% being strong biofilm producers; and the frequency of the icaA, fnbA, and fnbB genes were 43.7% and 34.3%, and 9.3% in isolates, respectively. Twenty-five (78.1%) of these strains were resistant to at least one antimicrobial agent, 20 (80%) of which exhibited multidrug resistance (MDR). Regarding genotypic analyses, 15.6%, 22.2%, 87.5%, and 9% of isolates, were positive for blaZ, ermC, tetK, and aacA-aphD, respectively. In 8 (25%) of all isolates had one or more staphylococcal toxin genes: the sed gene was the most frequent (12.5%), followed by eta (9.3%), tst-1 (6.25%), and sea (3.1%). In conclusion, this study highlights meat; and meat products might be reservoirs for the biofilm-producing MDR-CoNS, which harbored several toxin genes. Hence, it should not be ignored that CoNS may be related to foodborne outbreaks.

Profile of Aliarcobacter spp. from edible giblets: Genetic diversity, antibiotic resistance, biofilm formation.

Aliarcobacter spp. are recognized as emerging foodborne pathogens and consumption of foods contaminated with them can be a hazard to human and animal health. This study was conducted to investigate the prevalence of Aliarcobacter spp. in edible internal organs of different animal species from retail markets and giblet sellers. Additionally, this study was focused on the antimicrobial resistance, virulence profiles, biofilm-forming capabilities, and phylogenetic relationships of obtained isolates. A total of 270 samples were analyzed from which, 28 (10.4 %) were isolated as Aliarcobacter spp. by conventional methods. Within the 28 Aliarcobacter spp. isolates, 17 (60.7 %) were identified as A. butzleri, 10 (35.7 %) were A. cryaerophilus and one (3.5 %) was A. skirrowii by PCR method. The disc diffusion method showed that the highest resistance rate of Aliarcobacter spp. was seen against oxacillin (78.5 %), and 20 (71.4 %) out of the 28 isolates exhibited multidrug resistance (MDR). Out of the 28 isolates, mviN, pldA, tlyA, and hecB virulence genes were detected in 85.7 %, 46.4 %, 46.4 %, and 3.5 %, respectively, but irgA, Cj1349, ciaB, cadF, and hecA genes were not detected. According to the microplate test, 27 (96.4 %) isolates had weak biofilm ability while one A. cryaerophilus isolate (3.6 %) exhibited strong biofilm formation. ERIC-PCR band patterns suggested that isolated Aliarcobacter spp. from giblets, have different contamination sources. The presence of pathogenic and multidrug-resistant Aliarcobacter spp. in food poses a potential risk to public health and control measures throughout the food chain are necessary to prevent the spread of these strains.

Green Synthesis of Gold Nanoflowers Using Rosmarinus officinalis and Helichrysum italicum Extracts: Comparative Studies of Their Antimicrobial and Antibiofilm Activities.

This study was concerned with the green synthesis of gold nanoflowers (AuNFs) using the bioactive constituents of Rosmarinus officinalis (rosemary) and Helichrysum italicum (immortelle) extracts, as reducer and stabilizer agents along with the determination of their antibacterial and antibiofilm activity against E. coli, S. aureus, and S. epidermidis. The AuNFs were characterized using STEM, UV-Vis, DLS, ZETA, FESEM-EDX, and FTIR techniques. The antibacterial and antibiofilm activity of the AuNFs were evaluated by microdilution broth and microtiter plate (MTP) tests, respectively. STEM and DLS analysis confirmed the flower-like morphology of gold nanoparticle clusters of R. officinalis-AuNFs (R-AuNFs) and H. italicum-AuNFs (H-AuNFs) with a size of 20-130 nm and 15-90 nm, respectively. The MICs of R-AuNFs were found to be 40 µg/mL for E. coli and S. epidermidis and 160 µg/mL for S. aureus. The MICs of H-AuNFs against all bacterial strains were 20 µg/mL. All tested AuNFs exhibited a strong dose-dependent antibiofilm activity against the test strains, and H-AuNFs was more effective than R-AuNFs. The green synthesis of AuNFs from the rosemary and immortelle extracts can be applied as a potential agent to overcome the growth of biofilm-producing microorganisms in food industries.

Prevalence and molecular characterization of Listeria monocytogenes isolated from wastewater of cattle slaughterhouses in Turkey.

AIM: The study aimed to investigate the role of cattle slaughterhouse wastewater as a possible source for the environmental distribution of Listeria monocytogenes. METHODS AND RESULTS: Listeria spp. isolation was performed by collecting 117 wastewater samples from four different cattle slaughterhouses in Turkey. Species-specific identification was performed biochemically, and L. monocytogenes isolates were confirmed with polymerase chain reaction (PCR). In all, 71 (62.2%) of the wastewater samples were found to be positive for Listeria spp., and 17 (14.9%) of these samples were contaminated with L. monocytogenes. Pulsed field gel electrophoresis (PFGE) analysis revealed that all L. monocytogenes isolates were of different pulsotypes and isolates belonged to seven different phylogenetic clusters. Multiplex PCR analysis for genoserotypes and lineage determination showed that the isolates were divided into genoserotypes IVb and IIc in Lineages I and II. Also, it has been investigated with SYBR-Green Real-time PCR whether the L. monocytogenes isolates harboured virulence genes (hly, sigB, plcA, plcB, inlA, inlB, inlC and inlJ), and it was found that all isolates were substantially positive. Antibiotic resistance profiles and MIC values of the isolates were determined, and all L. monocytogenes isolates were found susceptible to ampicillin. In contrast, two isolates were resistant to meropenem and erythromycin, and three isolates were resistant to trimethoprim/sulfamethoxazole. CONCLUSION: L. monocytogenes, which pose a threat to public health and resists to antibiotics effectively used in treatments, can environmentally spread via wastewater of cattle slaughterhouses. The wastewater of the food industry, which has rich microbiota, should be treated carefully, and possible environmental contamination should be prevented. SIGNIFICANCE AND IMPACT OF STUDY: This is the first study that investigates the molecular characterization of L. monocytogenes isolated from cattle slaughterhouse wastewater and the findings represent the importance of cattle wastewater in the epidemiology of L. monocytogenes in Turkey.

Mycotoxigenic and phylogenetic perspective to the yeasts and filamentous moulds in mould-matured Turkish cheese.

This study was conducted to determine the diversity of yeasts and filamentous moulds in mould-matured cheese (MMC) consumed in Turkey. Overall, 120 samples were collected from 12 different geographical locations between March 2016 and April 2017. The morphological observation was applied in combination with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and molecular analyses to determine yeasts and filamentous moulds in the cheeses. High-performance liquid chromatography (HPLC) technique was used to evaluate the ability of mycotoxins production of fungal isolates and the presence of mycotoxins in cheese samples. A total of 241 fungi (81 filamentous moulds and 160 yeast) were recovered, and Penicillium roqueforti and Debaryomyces hansenii were the most frequently isolated species in all cheese samples. The rep-PCR results indicated a high level of genetic diversity among fungal isolates, regardless of isolation source or geographical origin. Filamentous mould strains isolated from MMC were found to synthesize at least one mycotoxin (Aflatoxin B1, B2, G1 and G2, citrinine, cyclopiazonic acid, mycophenolic acid, ochratoxin A, penicillic acid and roquefortine C). Although mycotoxin producing ability was observed from all isolates, none of the cheese samples were found positive for these mycotoxins. AFM1 was detected in 8 (6.6%) MMC samples from which 2 (1.6%) were above the legal limits (0.05 µg/kg) set by the Turkish Food Codex (TFC) and European Commission (EC). In conclusion, Turkish MMCs were found to be contaminated with toxigenic fungi, so a potential public health risk, while low, exists. Therefore, the selection of nontoxigenic filamentous mould strains for cheese manufacturing and control of the ripening conditions is a critical need to ensure the quality and safety of Turkish MMC.

Peroxidase-like activity and antimicrobial properties of curcumin-inorganic hybrid nanostructure.

For the first time in this study, curcumin was utilized as an organic component reacting with Cu (II) ion (Cu2+) as an inorganic component for fabrication of curcumin based Cu hybrid nanostructure (Cu-hNs). We also systematically examined the catalytic effect towards guaiacol and antimicrobial activities of Cu-hNs towards fish pathogen bacteria. For the characterization of Cu-hNs, Scanning Electron Microscopy (SEM), Energy Dispersive X-ray spectroscopy (EDX) and Fourier transform infrared spectrometry (FT-IR) analysis were used. We claimed that hydroxyl group might react with Cu2+ in phosphate solution (PO4 -3) to form Cu-hNs. However, more uniform and spherical Cu-hNs were not seen owing to absence of more reactive functional groups like amine and carboxyl groups on structure of curcumin. In addition to our findings, synthesis of Cu-hNs were carried out in the various pH values to evaluate the effect of pHs on formation of Cu-hNs. The Cu-hNs exhibited remarkable catalytic activity throught the Fenton reaction in the presence of hydrogen peroxide (H2O2) and effective antimicrobial activities against Gram-positive/negative fish pathogen bacteria. In this study, cheap and efficient synthesis of nanoflowers (NFs) using plant extracts is proposed for biomedical applications rather than expensive molecules such as amino acids and DNA.

Green synthesis of allicin based hybrid nanoflowers with evaluation of their catalytic and antimicrobial activities.

OBJECTIVE: Although organic-inorganic hybrid nanoflowers (hNFs) with much enhanced catalytic activity and stability were fabricated using proteins and enzymes, in this study, for the first time, we report synthesis of allicin and copper ion (Cu2+) coordinated NFs and investigate their peroxidase-like and antimicrobial activities. RESULTS: The allicin (active ingredient of Allium sativum) and Cu2+ was acted as an organic and inorganic part, respectively for synthesis of the Cu-hNFs. The hNFs were characterized by various techniques. Spherical, uniform, mono-dispersed and flower-like-shaped morphology of the hNFs (synthesized at pH 5) were imaged by scanning electron microscopy. The presence of Cu metal in the hNFs was detected by energy dispersive X-ray spectroscopy. Characteristic bonds stretching and bending for structural analysis of the hNFs were carried out by Fourier transform infrared spectrometry. In terms of applications, the hNFs showed quite effective peroxidase-like activity towards to guaiacol (used as a model substrate) in the presence of hydrogen peroxide (H2O2) through Fenton reaction. We demonstrated that the NFs exhibited ~ 200% and ~ 500% higher catalytic activities in 1 h (hr) and 3 h (hrs) than their initial catalytic activity measured in 5 minute (min). Additionally, effective antibacterial properties of the Cu-hNFs were observed against fish pathogen bacteria (Aeromonas hydrophila, Vibrio parahaemolyticus, and Lactococcus garvieae). CONCLUSIONS: We finally demonsrated that allicin based hybrid nanomaterial can be prepared by a relatively cheap, one step, easy and eco-friendly method. The allicin hNFs can be considered as novel Fenton agent for peroxidase like activity and bactericidal.

[Virulence Genes, Antibiotic Susceptibility Profiles and Molecular Characterization of Campylobacter Species Isolated from Different Slaughterhouses]. / Farkli Kesimhanelerden Izole Edilen Campylobacter Türlerinin Virülans Genleri, Antibiyotik Duyarlilik Profilleri ve Moleküler Karakterizasyonu.

The aim of this study was to investigate the frequency of Campylobacter species, to detect the antibiotic resistance profiles and the virulence genes and to determine the clonal proximity of the isolates in the samples of cutting board, slaughterhouse waste water, wall, knife and carcass from three different slaughterhouses in Kayseri region. For this purpose, a total of 150 samples, 10 of each from knife, wall, cutting board, carcass smear sample and slaughterhouse wastewater were collected from each of the three types of slaughterhouses in 2018 in Kayseri. For the isolation of the Campylobacter species, following preenrichment, the suspensions were inoculated onto modified charcoal cefoperazone desoxycholate (CCD) agar and were incubated at 37°C under microaerophilic condition for 48-72 hours. Suspicious colonies with gray-white color were recovered and subjected to phenotypical (Gram staining, oxidase, catalase test, and motion test) tests. Multiplex polymerase chain reaction (mPCR) was used for the molecular identification of the Campylobacter species. Antimicrobial susceptibilities of the isolates identified at the species level were detected by using the disk diffusion test and antibiotic gradient test. Virulence genes (iam, cadF, cdtA, flaA, ceuE, cdtC, cdtB and virB11) among the isolates were evaluated by PCR. The molecular typing of the isolates determined at species level was performed by Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR). In the study, 17 (11.3%) of the 150 samples taken from the slaughterhouse were found to be suspicious in terms of Campylobacter spp. and as a result of phenotypic identification tests, all of the isolates were verified as Campylobacter spp.. As a result of mPCR; eight of the isolates were identified as Campylobacter jejuni, eight as Campylobacter fetus and one as Campylobacter coli. The isolation of the Campylobacter species from different sources was found to be higher in slaughterhouse wastewater than those of others (p<0.001) and the difference in the proportional distribution of the Campylobacter species obtained from various sources was statistically significant (p<0.05). As a result of the disk diffusion test, while, all C.jejuni isolates were resistant to ciprofloxacin, 87.5%, 25%, 25% and 12.5% of C.jejuni isolates were resistant to enrofloxacin, neomycin, amoxicillin/clavulanic acid, and erythromycin, respectively. In addition, 25%, 25% and 12.5% of C.fetus isolates were resistant to amoxicillin/clavulanic acid, neomycin and gentamicin, respectively. C.coli isolate was not resistant to any of the antibiotics tested. Antibiotic gradient test results were found to be compatible with the disc diffusion test results. One of the virulence genes examined, virB11, was not detected in any of the isolates. Moreover, iam gene was not present in C.fetus and C.coli isolates, but only in one C.jejuni isolate. The flaA gene was detected in six C.jejuni isolates. C.coli isolate and seven C.jejuni and seven C.fetus isolates were positive in terms of the cdtC gene. The cdtA, cdtB, ceuE and cadF genes were found to be positive in all C.jejuni isolates. All isolates analyzed in the study demonstrated different ERIC-PCR profiles. In conclusion, it was shown that Campylobacter strains isolated from slaughterhouses were resistant to the most of the current antibiotics. Moreover, the presence of highly virulent Campylobacters in the slaughterhouse environment threatens public health due to the risk of contamination of the humans via carcasses and foods. Therefore, it is recommended that strict hygiene rules should be followed to reduce Campylobacter species contamination in slaughterhouses.

Occurence and antimicrobial resistance of Staphylococcus aureus and Salmonella spp. in retail fish samples in Turkey.

The aims of this study were to investigate the presence of Staphylococcus aureus and staphylococcal enterotoxins, as well as Salmonella spp. and to determine the antimicrobial susceptibilities of the isolates from fish samples. A total of 100 fish samples were analysed consisting of 30 anchovy, 35 trout and 35 sea bream. The presence of SEs was detected using ELISA and its genes confirmed by mPCR. Also, S. aureus and Salmonella spp. were detected in 9 (9%) and 5 (5%) samples, respectively. None of the S. aureus isolates had SEs and SEs genes. The resistance rates of the S. aureus isolates to erythromycin, tetracycline, and penicillin G were found to be 33% while Salmonella spp. isolates were resistant to trimethoprim-sulfamethoxazole, gentamicin and neomycine in 20%, 20% and 80%, respectively of the samples. It is of utmost important for public health that retail fish markets need to use hygienic practices in handling and processing operations.