Adriana Morar

Zoonotic Cryptosporidium parvum in Romanian newborn lambs (Ovis aries).

This study was undertaken to investigate the occurrence and public health significance of Cryptosporidium species/genotypes and subtypes in a newborn lambs. A total of 175 diarrheic fecal samples from lambs (younger than 21 days) were collected in seven sheep flocks located in western Romania, and were microscopically examined for the presence of Cryptosporidium oocysts after staining with modified Ziehl-Neelsen technique. Twenty-four (13.7%) fecal samples were tested Cryptosporidium positive by microscopy and were subjected for molecular characterization. All positive samples were successfully amplified through a nested polymerase chain reaction (PCR) of the small subunit (SSU) rRNA gene (18S). Cryptosporidium species were determined by restriction fragment length polymorphism (RFLP) analysis of the secondary PCR products using the conventional SspI and VspI restriction enzymes. The identified species were: Cryptosporidium parvum (20/24), C. ubiquitum (2/24) and C. xiaoi (2/24), respectively. PCR-RFLP results for C. ubiquitum and C. xiaoi isolates were confirmed by DNA sequencing. Subsequently, subtyping of seven randomly selected C. parvum isolates, based on sequence analysis of the GP60 gene, revealed the presence of five different subtypes (IIaA17G1R1, IIaA16G1R1, IIdA20G1, IIdA24G1 and IIdA22G2R1) belonging in two zoonotic subtype families (IIa and IId). These findings may suggest the potential role of the newborn lambs as a source for human cryptosporidiosis. This is the first published report about the presence of C. ubiquitum and C. xiaoi in lambs from Romania.

Environmental Occurrence and Antibiotic Susceptibility Profile of Listeria monocytogenes at a Slaughterhouse Raw Processing Plant in Romania

This survey was conducted to investigate the occurrence and antimicrobial susceptibility of Listeria monocytogenes isolates in the environment of a pig slaughterhouse raw processing plant complex in western Romania. A total of 97 environmental samples from food contact (n = 60) and nonfood contact (n = 37) surfaces were examined with standard methods. The susceptibility of the isolates to 17 antimicrobial agents was determined with the VITEK 2 automated system. Overall, 25 (25.8%) samples were positive for L. monocytogenes , with recovery rates of 23.3% (n = 14) and 29.7% (n = 11) for food contact and nonfood contact surfaces, respectively. All 25 tested isolates were resistant to benzylpenicillin, imipenem, and fusidic acid. Resistance was observed to oxacillin (23 isolates; 92%), fosfomycin (23 isolates; 92%), clindamycin (22 isolates; 88%), rifampin (14 isolates; 56%), trimethoprim-sulfamethoxazole (12 isolates; 48%), tetracycline (11 isolates; 44%), and ciprofloxacin (1 isolate; 4%). No resistance was found to seven tested agents: gentamicin, moxifloxacin, erythromycin, linezolid, teicoplanin, vancomycin, and tigecycline. All tested L. monocytogenes isolates were resistant to four to nine antimicrobial agents. These results indicate a high prevalence of multidrug-resistant L. monocytogenes isolates in this pork-processing environment, providing baseline information for hygienists and public health specialists.

Phytochemical Screening and Biological Activity of Mentha × piperita L. and Lavandula angustifolia Mill. Extracts

The present study aimed to investigate the phytochemical composition of Mentha × piperita L. (MP) and Lavandula angustifolia Mill. (LA) extracts in terms of hydroxycinnamic acid (HCAs) content, in particular, caffeic (CA), p-cumaric (CU), ferulic (FE), and rosmarinic (RS) acids using LC-MS. Also, the in vitro antimicrobial effect against Staphylococcus aureus and the antiproliferative activity against two cancerous cell lines (A375 and MDA-MB-231) using the MTT assay were tested. The extracts were prepared using aromatic water which resulted from the extraction of oils from plants as extraction medium, with/without acid. The results showed that RS and FE represent the majority of HCAs compounds; the highest content of FE is found in LA (7.47 mg·g–1d.m.), and the maximum content of RS in MP (6.36 mg·g–1d.m.). Regarding the antimicrobial effect against Staphylococcus aureus, the two extracts showed a simulative role on the growth rate of Staphyloccocus aureus, but a slightly inhibitory effect (69.12%) can be attributed to the acidic environment. In terms of biological activity against MDA-MB-231 breast carcinoma cell line, and A375 human melanoma cell line, at the highest employed concentration, 150 μg·mL–1, the tested extracts present a weak antiproliferative effect.

The red fox (Vulpes vulpes) plays a minor role in the epidemiology of the domestic cycle of Trichinella in Romania.

Nematode worms of the genus Trichinella are zoonotic parasites with a worldwide distribution. The majority of the biomass of these nematodes circulates among wildlife, but when humans fail in the proper management of domestic animals and wildlife, Trichinella infections are transmitted from the sylvatic to the domestic environment. Such failures occur in Romania, where a high prevalence of Trichinella spiralis has been detected in domestic pigs. The aim of the present study was to provide data about the prevalence of Trichinella spp. infections in red foxes (Vulpes vulpes) that are hunted in Romanian counties, in which the prevalences of Trichinella spp. infection in backyard and free-ranging pigs range from 0.17 to 2.5%, to determine the role played by this carnivore species in the transmission of the parasite to domestic cycle. A total of 121 animals from 45 hunting grounds of three counties were screened to detect Trichinella spp. larvae by the digestion method. Infections were detected in 26 (21.5%) foxes from 18 (40%) hunting grounds of the three counties (13/67 in Arad, 1/3 in Hunedoara, and 12/51 in Timiş). The mean larval density was 10.5 larvae per gram. Of the 25 successfully tested samples, the Trichinella larvae from 24 isolates were identified as T. britovi (96%), and the larvae from one isolate were identified as T. spiralis (4%). No mixed infections were recorded. The present results revealed that the red fox should be considered an important T. britovi reservoir in the sylvatic cycle; in contrast, the detection of only a single T. spiralis-positive isolate suggests that red foxes play a minor role in the epidemiology of the domestic cycle in the investigated area of western Romania.

Salmonella in raw chicken meat from the Romanian seaside: frequency of isolation and antibiotic resistance.

The purpose of this study was to investigate the occurrence of Salmonella in raw chicken meat samples collected at the Romanian seaside and to evaluate the antimicrobial susceptibility of the isolates. In 2012, 317 chicken meat samples from slaughterhouses (n = 289) and retail markets (n = 28) were evaluated. Overall, 13.2% (42) of the samples contained Salmonella; 12.8% (37) and 17.8% (5) from the chicken carcasses at slaughterhouses and the fresh meat from retail markets, respectively. Eight serotypes of Salmonella enterica subsp. enterica were identified: Infantis (18 isolates), Bredeney (7), Virchow (6), Djugu (4), Grampian (4), Brandenburg (1), Derby (1), and Ruzizi (1). The isolates were resistant to tetracycline (66.6% of isolates), nalidixic acid (64.3%), sulfamethoxazole (64.3%), ciprofloxacin (61.9%), streptomycin (59.5%), trimethoprim (33.3%), ampicillin (9.5%), chloramphenicol (7.1%), and gentamicin (2.4%). No resistance was found to cefotaxime and ceftazidime. Thirty (71.4%) of the 42 tested isolates had multidrug resistance patterns to at least two antimicrobials. This survey highlighted a multidrug-resistant Salmonella contamination rate in raw chicken meat in this area of Romania, which can seriously threaten human health.

Occurrence and antimicrobial susceptibility of Salmonella isolates recovered from the pig slaughter process in Romania.

INTRODUCTION Reported human salmonellosis cases have increased in Romania. Antibiotic susceptibility testing of Salmonella strains isolated from pork and chicken meat indicate a worrying multidrug resistance pattern. This study aimed to investigate the occurrence of Salmonella and to evaluate the antibiotic resistance of Salmonella strains in a pig slaughterhouse-processing complex, which receives animals from 30% of the large industrialized swine farms in Romania. METHODOLOGY A total of 108 samples, including pork (n = 47), packaged pork products (n = 44), scald water sludge (n = 8), and detritus from the hair removal machine of the slaughterhouse (n = 9) were examined for the presence of Salmonella through standard methods. The antibiotic susceptibility of the isolated strains to 17 antibiotics was tested using the Vitek 2 system. RESULTS Twenty-six (24.1%) samples were found to be Salmonella positive; this included 25.5% of meat samples and 15.9% of packaged products, as well as samples from two different points of the slaughter (41.2%). Resistance was observed against tetracycline (61.5%), ampicillin (50%), piperacillin (50%), trimethoprim-sulfamethoxazole (34.6%), amoxicillin/clavulanic acid (26.9%), nitrofurantion (23.1%), cefazolin (15.4%), piperacillin/tazobactam (7.7%), imipenem (3.8%), ciprofloxacin (3.8%), and norfloxacin (3.8%). No resistance towards cefoxitin, cefotaxime, ceftazidime, cefepime, amikacin, and gentamicin was found. CONCLUSIONS Our study demonstrated the occurrence of multidrug-resistant Salmonella strains in the investigated pork production complex and highlighted it as a potential source of human infections. The results demonstrate the seriousness of antibiotic resistance of Salmonella in Romania, while providing a useful insight for the treatment of human salmonellosis by specialists.

Chemical Composition and Antimicrobial Potential of Satureja hortensis L. in Fresh Cow Cheese

This study presents data about the chemical composition and antimicrobial effect of Satureja hortensis L. used as both dry plant and essential oil, on fresh cow’s cheese, in order to extend its shelf-life. The proximate and elemental composition of dry plant of Satureja hortensis L. highlights important level of microelements. The content of microelements increases even when small amounts of Satureja hortensis in fresh cheese were added. The addition of Satureja hortensis dry plant leads to an increase in Fe (13.46–65.54%) and Mn (8.33–88.33%) content of fresh cheese, depending on the amount of plant added. The composition of essential oil isolated from Satureja hortensis L. was analyzed by GC-MS and the main compounds found were carvacrol (19.68%), o-cymene (30.86%), and p-cymene (28.07%). In order to use Satureja hortensis L. as natural preservative in food industry, in vitro effect of plant extract and essential oil against Staphylococcus aureus Gram-positive bacteria was tested. The oil of Satureja hortensis L. showed antimicrobial activity at 0.50–1.5%, while the alcoholic extract does not inhibit Staphylococcus aureus mycelial growth. The antimicrobial effect of Satureja hortensis L. dry plant in various proportions (0.5–1.5%) and essential oil (0.1%; 0.25%; 0.5%), on fresh cow’s cheese, was assessed after 3 and 7 days by counting colonies obtained at 30°C. Results have shown that the addition of Satureja hortensis L. dry plant and essential oil led to a reduction in the total number of germs, this reduction being more significant when the essential oil was used. Regarding the effect of Satureja hortensis L. essential oil against Staphylococcus aureus inoculated in fresh cow’s cheese, the results highlight that the essential oil of Satureja hortensis L. may be a natural solution to prevent the development of this bacteria, while the ethanol extract does not prove to be effective.

Occurrence and first molecular characterization of Sarcocystis spp. in wild boars (Sus scrofa) and domestic pigs (Sus scrofa domesticus) in Romania: Public health significance of the isolates

Domestic and wild pigs, as intermediate hosts, can harbor tissue cysts of three Sarcocystis species namely S. miescheriana, S. suihominis and S. porcifelis. Out of them, S. suihominis is zoonotic. Romania is a country with high consumption of raw and/or undercooked traditional pork products. This fact may greatly favor the acquiring of the zoonotic Sarcocystis infections by humans, as definitive host. Based on this consideration and in order to investigate the occurrence and public health significance of Sarcocystis spp. in two western counties (Caraş-Severin and Timiş) of Romania, a total of 165 heart samples from hunted wild boars (Sus scrofa, n=101) and home slaughtered domestic pigs (Sus scrofa domesticus, n=64) were screened using microscopic fresh examination and molecular methods. Microscopic examination revealed the presence of sarcocysts in 60.4% of wild boars, and 23.4% of domestic pigs. Genetic characterization of isolates through the PCR-RFLP procedure, targeting the 18S rRNA gene, was successfully achieved for all microscopically positive samples, indicating the presence of a single species, S. miescheriana, in both hosts. The identity of 13 selected S. miescheriana isolates was also confirmed through sequencing. The tested hosts older than 27 months were found to be significantly higher infected (p<0.05) with Sarcocystis than the 6 to ≤27months age group. Although the human infective S. suihominis has not been registered, for a more reliable epidemiological picture, further molecular studies enrolling a larger number of animals and diagnosis on human intestinal Sarcocystis infections are still necessary.