Detection of Psychrophilic Clostridium spp. in Fecal Samples from Cattle of Different Ages Sampled at the Slaughterhouse Level

ABSTRACT Clostridium estertheticum and C. estertheticum–like spp. are obligate anaerobic psychrophiles causing “blown pack” spoilage of chilled vacuum-packed meat. The present study aimed at detecting and isolating these spoilage bacteria in fecal samples of cattle of different ages at the slaughterhouse level. One hundred two swab fecal samples were obtained and enriched anaerobically in prereduced peptone-yeast-glucose-starch (PYGS) medium for 3 weeks at 4°C and then screened for C. estertheticum and C. estertheticum–like spp. by using a 16S rRNA gene–based real-time PCR (RT-PCR) assay. The RT-PCR–positive samples were further enriched for 3 weeks in prereduced PYGS medium and then subjected to an ethanol (50%, v/v) and lysozyme (4 mg/mL) treatment. Isolation was carried out anaerobically on Columbia agar with 5% defibrinated sheep blood at 4°C for 3 weeks. Isolated strains were identified morphologically and by the 16S rRNA gene. Forty (39%) of 102 samples were RT-PCR positive. The frequency of positive samples was the following: 9 (45%) of 20 in calves (aged ≤160 days), 23 (43%) of 54 in young cattle (aged 161 to 1,000 days), and 8 (29%) of 28 in cows or bulls (aged >1,000 days). Six strains were isolated from 6 of 40 RT-PCR–positive samples. Of these, five were from the calves (n = 1) and young cattle (n = 4). The six isolates were identified as C. estertheticum (n = 1), Clostridium frigoriphilum (n = 1), and C. estertheticum–like spp. (n = 4). The present findings confirm that feces of cattle are an important source of psychrophilic Clostridium spp. The fecal carriage among livestock animals at slaughter is strongly correlated with the risk of carcass contamination. Therefore, the maintenance of slaughter hygiene is of central importance. HIGHLIGHTS

Streptococcus agalactiae in pregnant women: serotype and antimicrobial susceptibility patterns over five years in Eastern Sicily (Italy)

Streptococcus agalactiae (also known Group B Streptococcus or GBS) represents the main pathogen responsible for early- and late-onset infections in newborns. The present study aimed to determine the antimicrobial susceptibility pattern and the capsular serotypes of GBS isolated in Eastern Sicily over 5 years, from January 2015 to December 2019. A total of 3494 GBS were isolated from vaginal swabs of pregnant women (37–39 weeks), as recommended by the Centers for Disease Control and Prevention. Capsular polysaccharide’s typing of GBS was determined by a commercial latex agglutination test containing reagents to serotypes I–IX. The antimicrobial resistance pattern of GBS was determined through the disk diffusion method (Kirby-Bauer) and the double-disk diffusion test on Mueller-Hinton agar plates supplemented with 5% defibrinated sheep blood, according to the guidelines of the Clinical and Laboratory Standards Institute. Serotypes III (1218, 34.9%) and V (1069, 30.6%) were the prevalent colonizers, followed by not typable (570, 16.3%) and serotypes Ia (548, 15.7%), Ib (47, 1.3%), II (40, 1.1%), and IV (2, 0.1%). All 3494 clinical isolates were susceptible to cefditoren and vancomycin. Resistance to penicillin, ampicillin, levofloxacin, clindamycin, and erythromycin was observed in 6 (0.2%), 5 (0.1%), 161 (4.6%), 1090 (31.2%), and 1402 (40.1%) of the strains, respectively. Most of erythromycin-resistant GBS (1090/1402) showed the cMLSB phenotype, 276 the M phenotype, and 36 the iMLSB phenotype. Our findings revealed a higher prevalence of serotype III and a relevant resistance rate, among GBS strains, to the most frequently used antibiotics in antenatal screening.

Characteristics of Nasopharyngeal Carriage of Bacterial Pathogens in Children and Adults Suffering from Recurrent Respiratory Infections in Khabarovsk City in 2015–2018

Objective. To designate the nasopharyngeal carriage of bacterial pathogens among children and adults diagnosed with recurrent respiratory diseases residing in the Khabarovsk city during a four-year period.Materials and methods. Nasopharyngeal and oral swabs obtained from 7,043 children and adults were tested using classical bacteriological methods. In order to grow “difficult-to-culture” microorganisms a columbian agar with addition of 5% defibrinated sheep blood, incubation in the atmosphere rich with CO2 (5%), bacteriological analyzer Vitek 2 Compact were used. Real-time PCR was used to confirm the identification of S. pyogenes.Results. A high level of nasopharyngeal pathogens carriage (47%) was detected. The most prevalent microorganisms were as follows: S. pneumoniae (47%), M. catarrhalis (30.4%), H. influenzaе (17.5%), S. pyogenes (5.2%). The age groups at risk were children aged 0–6 years for S. pneumoniae and children aged 7–12 years for S. pyogenes. An emerging trend it the level of nasopharyngeal carriage of S. pneumoniae observed in 2018 was followed by the increase of registered incidence of pneumococcal pneumonia.Conclusion. Nasopharyngeal carriage of S. pneumoniae imposes a high risk of community-acquired pneumonia and other pneumococci-associated diseases, predominantly in children.

THE ANTIMICROBIAL ACTIVITY OF ETHANOLIC EXTRACTS DERIVED FROM LEAVES AND PSEUDOBULBS OF COELOGYNE FLACCIDA LINDL. (ORCHIDACEAE) AGAINST DIFFERENT STAPHYLOCOCCUS AUREUSSTRAINS

The present study was conducted to investigate in vitro antimicrobial activity of ethanolic extracts obtained from leaves and pseudobulbsof Coelogyneflaccida against Staphylococcus aureus subsp. aureus Rosenbach (ATCC®25923™) (mecA negative), Staphylococcus aureus subsp. aureus Rosenbach (ATCC®29213™)(mecA negative, Oxacillin sensitive, weak β-lactamase-producing strain), Staphylococcus aureus NCTC 12493 (mecA positive, Methicillin-resistant, EUCAST QC strain for cefoxitin).The antimicrobial susceptibility testing was done on Muller-Hinton agar by the disc diffusion method (Kirby-Bauer disk diffusion susceptibility test protocol). The leaves and pseudobulbs of C. flaccida plants, cultivated under glasshouse conditions, were sampled at M.M. Gryshko National Botanic Garden (NBG), National Academy of Science of Ukraine. Freshly leaves and pseudobulbs were washed, weighed, crushed, and homogenized in 96% ethanol (in proportion 1:19) at room temperature. The extracts were then filtered and investigated for their antimicrobial activity. The cultivation medium was trypticase soy agar (Oxoid™, UK), supplemented with 10% defibrinated sheep blood. Cultures were grown aerobically for 24 h at 37°C. The cultures were later diluted with a sterile solution of 0.9% normal saline to approximate the density of 0.5 McFarland standard. The McFarland standard was prepared by inoculating colonies of the bacterial test strain in sterile saline and adjusting the cell density to the specified concentration. The examined ethanolic extracts obtained from leaves and pseudobulbs of C. flaccida showed remarkable antibacterial activities against different S. aureusstrains. The observed activity may contribute to the reasons why orchids are used for infectious and inflammatory conditions in ethnomedicine. The research showed that ethanolic extracts of C. flaccida possess the highest antibacterial potency against S. aureus NCTC 12493 strain. These findings led the authors to suggest that these extracts may be used as natural antiseptics and antimicrobial agents in medicine and veterinary practice. Nevertheless, despite the promising results, more research should be carried out to further evaluate the roles of particular compounds, isolated from all parts of orchid plants, attributable to antimicrobial activity.

Goiter parenchyma, blood vessels and lymphatics contain Staphylococcus epidermidis - saprophyte or pathogen?

BackgroundGoiter in its various clinical and histopathological forms is accompanied by an inflammatory process requiring intensive therapy. The thyroid gland is an organ specifically exposed to the microbial environment due to its close location to the mouth microbiome. A number of bacterial phenotypes has been detected in the inflamed thyroid gland. A question raises as to whether bacteria have not already been present in the thyroid gland before the clinical symptoms of goiter became evident.AimTo answer the questions: a) do the goiter tissue structures contain bacteria, b) if so, which bacterial phenotypes can be identified, c) what are the genetic similarities of the thyroid and periodontal bacterial strains.Material and methodsStudies were carried out in 60 patients with the non-toxic multinodular goiter in 40 cases, toxic multinodular goiter in 10, single adenoma in 3, Hashimoto’s disease with nodular changes in 4 and recurrent thyroid disease in 3. Tissue fragments harvested during surgery were placed on Columbia blood agar base enriched with 5% defibrinated sheep blood. In this method bacteria present in the tissue slowly proliferate in their in vivo transferred to ex-vivo environment, crawl out and form the on-plate colonies. It enables detection of single bacteria usually difficult in a standard planktonic culture.Resultsa) Coagulase-negative Staphylococci were shown growing on culture plates in above 50% of thyroid parenchyma, veins, arteries and adjacent lymphoid tissue specimens, b) tissue-originating colony-forming bacteria appeared on plates on day 3, but in some as late as after 12-21 days, c) all isolates were sensitive to the basic antibiotics, d) bacterial thyroid and oral DNA tests showed similarities indicating possibility of the oral origin, e) the on-plate time-prolonged cultures showed shrinking of the colonies and upon adding liquid medium formed the small variant colonies.ConclusionsThyroid gland tissues contained in above 50% of specimens the coagulase-negative Staphylococci. Over 88% similarity of the genetic pattern of Staphylococcus epidermidis strain from tooth, oropharyngeal and thyroid tissues, estimated with PCR MP technique, suggested their periodontium origin.

Investigation of some gastric Helicobacter species in saliva and dental plaque of stray cats by cultural and PCR methods

The purpose of this study was to explore the presence of gastric Helicobacter species in the oral cavity of stray cats in the Kars region. Saliva and dental plaque samples collected from 100 stray cats were evaluated by culture and PCR methods in terms of gastric Helicobacter species. For culture, samples were plated on 5% defibrinated-horse blood and 5% defibrinated-sheep blood enriched selective agar plates supplemented with Vancomycin (6 μg/ml), Polymyxine B (2.500 IU/l), Trimethoprim (20 μg/ml) and Amphotericin B (2.5 μg/ml). Molecular methods were also included to study by using the PCR targeting amplification of the 16S rRNA gene sequence for Helicobactergenus and urease B gene sequence for each Helicobacter species. As the results of cultural examination, Helicobacter spp. were isolated from 10 (10%) cats (10 saliva and 5 dental plaque samples) and these were further identified as H. heilmannii by PCR. Direct analysis of samples by genus-specific PCR revealed that a total of 70 (50 saliva and 20 dental plaques) samples from 65 cats were positive in terms of Helicobacter DNA. As the results of species – specific PCR analysis of these samples 34 (48.57%) (24 saliva and 10 dental plaque samples) were identified as H. heilmannii, while the remaining 36 (51.42%) were found to be negative in terms of related species (H. heilmannii, H. pylori and H. felis). It has been concluded that these bacteria, identified in the oral cavity of the cats, may play a role in transmission of infection to humans.

Przeciwbakteryjne działanie olejku rozmarynowego (Oleum Rosmarini) na bakterie beztlenowe

Introduction. Rosmarinus officinalis L. a member of family Lamiaceae is widely found in many countries of North Africa, America and Europa. It grown to 2-3 m high. The plant produced of essential oils. The composition of rosemary oil based on genotype, climate, geography, and method of preparation. The major constituents of the oil are 1-8 cineole, α-pinene, camphene, α-terpineol, borneol, camphor, β-myrcene, geraniol, eugenol, p-cymen, linalool, romarinic acid and caffeic acid. Rosmarinic acid is well adsorbed from gastrointestinal tract and from the skin. The oil is used in medicine as an anti-inflammation, anticancer, analgesic, antidiabetic, antiulcerogenic, hepatoprotective, antirheumatic, antiepileptic, diuretic and anti Alzheimer disease. The extracts and essential oil have antimicrobial activity towards bacteria, fungi, viruses and insects. Aim. The goal of this work was to test the antimicrobial activity of rosmarinic oil on anaerobic bacteria. Material and methods. The bacterial strains were isolated from oral cavity. A total 33 strains of anaerobic bacteria isolated from patients and 6 reference strains were investigated. The susceptibility (MIC) was determined by the two-fold of plate dilution method in Brucella agar supplemented with 5% defibrinated sheep blood, menadione and hemin. The rosmarinic oil (Semifarm) was dissolved at first in DMSO and afterwards in distilled water. Concentrations of oil used were 0.06, 0.12, 0.25, 0.5, 1.0 and 2.0 mg/ml. The inoculum containing 106 CFU/per spot was seeded with Steers replicator upon the surface of agar with oil and without the oil (the strains growth control). Incubation the plates was performed in anaerobic conditions in anaerobic jar, at 37°C for 48 hrs. The MIC was defined as the lowest concentrations of rosmarinic oil that completely inhibited the growth of tested anaerobic bacteria. Results. The results indicated that the tested bacteria were high sensitive to the essential oils. The most susceptible from Gram-negative bacteria were the rods from genus of Porphyromonas asaccharolytica, Prevotella levii and Bacteroides uniformis (MIC ≤ 0.06 mg/ml). The strains from genus Prevotella buccalis and Bacteroides vulgatus were less sensitive (MIC = 0.5 mg/ml). Remainded Gram-negative rods were susceptible to the oil in concentrations in range from 0.5 to 1.0 mg/ml. The rosmarinus oil was more effective against the Gram-positive bacteria. The most susceptible from the cocci were strains from the genus of Peptostreptococcus anaerobius and Parvimonas micros (MIC 0.25-≤ 0.06 mg/ml) and from rods Gram-positive rods genus of Actinomyces viscosus and Bifidobacterium breve (MIC 0.12-≤ 0.06 mg/ml). Conclusions. The results indicated that the rosmarinic oil showed high antibacterial activity against all tested anaerobic bacteria. The more susceptible to oil were the Gram-positive bacterial strains than Gram-negative anaerobic rods.

Wrażliwość bakterii beztlenowych na olejek anyżowy (Oleum anisi)

Introduction. Anise (Pimpinella anisum L.) belong to Apiaceae family. The plant is distributed in China, Iran, Indie and America. The fruits green' lanceoled leaves, white flowers and green-yellow seeds. The fruits are used to produce an essential oils. The anise oil is widely used in folk medicine, food, cosmetic and pharmaceutical industries. The fruits consists of 6-30% volatile oil. Its the major components are trans-anethole, estragol, eugenol, linalool, α-terpineol, cis-anethole, anisaldehyde, coumarins, methylchavikol, scopoletin, umbelliferone, estrols and polyacetylenes. Aim. The aim of this study was to determine the antimicrobial activity of anise oil against anaerobic bacteria isolated from oral cavity and upper respiratory tract. Material and methods. A total 54 strains of anaerobic bacteria and 8 standards strains from genera Porphyromonas, Prevotella, Bacteroides, Parabacteroides, Fusobacterium, Tannerella, Finegoldia, Peptostreptococcus, Actinomyces, Propionibacterium, Bifidobacterium and 8 standards strains Bacteroides fragilis ATCC 25285, Porphyromonas asaccharolytica ATCC 29743, Peptostreptococcus anaerobius ATCC 27337, Fusobacterium nucleatum ATCC 25586, Finegoldia magna ATCC 29328, Parabacteroides distasonis ATCC 8503, Propionibacterium acnes ATCC 11827 and Actinomyces odontolyticus ATCC 17929 were investigated. The susceptibility (MIC) was determined by the two-fold of plate dilution method in Brucella agar supplemented with 5% defibrinated sheep blood, menadione and hemin. The inoculum containing 106 CFU/spot was seeded with Steers replicator upon the surface of agar containing testing oil and free oil (the strains growth control). Incubation the plates was performed in anaerobic conditions in anaerobic jars with 10% CO2 , 10% H2 and 80% N2 . The MIC was considered to be the last dilution that did not bacterial colonial growth. Results. The results showed, that the most susceptible to oils from Gram-negative anaerobes were the strains of Tannerella forsythia (MIC 0.5-< 0.12 mg/ml), Fusobacterium necrophorum, Bacteroides vulgatus and Bacteroides uniformis (MIC = 25 mg/ml). The strains from genus of Prevotella bivia and Parabacteroides distasonis were the lowest sensitive. The growth of the strains was inhibited by concentration > 2.0 mg/ml. The anise oil was more active against Gram-positive rods and cocci then Gram-negative anaerobic bacteria. Conclusions. The anise oil was very active against all bacterial strains tested. The most susceptible to oil was the strains from genus Tannerella forsythia, Fusobacteria necrophorum, Bacteroides vulgatus and Bacteroides uniformis. The Gram-positive-rods and cocci was more susceptible to anise oil then anaerobic Gram-negative rods.

Prevalence of Aerobic Bacteria in the Hands of School-Going Children of Rural Areas of Eastern Part of Nepal

Introduction: The aim of this study was to find out the prevalence of bacteria in hands of school going children in a rural area of eastern part of Nepal. Contaminated hands play a major role in faeco-oral transmission of diseases. The students can expose themselves to infection if they do not properly wash their hands before taking food. Methods: The study was done in a remote school of eastern part of Nepal in 2013. A prospective study was done on 200 school children of age group 10 years to 15 years. Swab samples collected from all the children were transported to the laboratory within one to two hours of its collection. The samples were kept in nutrient broth for overnight incubation. They were then sub-cultured aerobically at 37°C on 5% defibrinated sheep blood agar and MacConkey agar plates for 24-48 hrs. Then the plates were examined to quantify the organisms present according to Clinical Laboratory Standard Institute (CLSI) guidelines.Results: Out of 200 dominant hand swabs of school children of age group 10 years to 15 years, 52 children (26.0%) were harboring pathogenic bacteria in their hands. The major aerobic pathogenic bacteria in hands were i.e. Staphylococcus aureus, Streptococcus spp, Enterococci spp, Escherichia coli, Klebsiellaspp, Acinetobactersppand Pseudomonas aeruginosa. In most of the hands normal flora like Diphtheroids spp, Coagulase negative Staphylococci (CONS), Micrococci spp and some yeast were found.Conclusion: The finding of the study concludes that there is high incidence of aerobic bacterial flora in the hands of school going children of eastern part of Nepal. The incidence of infection was due to lack of proper hand washing before meal. The school should be told to keep soaps in the toilets for hand washing and conduct continue health education about proper hand washing and maintain cleanliness both in the schools and at the community level.

Streptobacillus hongkongensis sp. nov., isolated from patients with quinsy and septic arthritis, and emended descriptions of the genus Streptobacillus and Streptobacillus moniliformis

Two bacterial strains, HKU33T and HKU34, were isolated in Hong Kong from the pus aspirated from the right peritonsillar abscess of a patient with quinsy and the left elbow joint fluid of another patient with tophaceous gout and left elbow septic arthritis, respectively. The bacteria were Gram-stain-negative, non-motile, non-spore-forming, non-haemolytic pleomorphic bacilli. They grew best on Columbia agar with 5 % defibrinated sheep blood in an anaerobic environment or aerobic environment with 5 % CO2. They also grew on chocolate agar but not on MacConkey agar. They were catalase- and cytochrome oxidase-negative. They showed a unique profile of enzyme activities distinguishable from their closely related species. Phylogenetic analysis of the complete 16S rRNA gene, and partial groEL, gyrB and recA gene sequences showed the two isolates formed a distinct branch within the family Leptotrichiaceae , being related most closely to Streptobacillus moniliformis . Hierarchical cluster analysis of mass spectra of whole-cell protein contents showed that strains HKU33T and HKU34 were closely related to each other, but were distinct from Streptobacillus moniliformis , Sneathia sanguinegens and ‘Leptotrichia amnionii’. The DNA G+C content of strain HKU33T was 26.0±2.1 mol% (mean±sd; n = 3). DNA–DNA hybridization demonstrated ≤45.02 % DNA relatedness between the two isolates and Streptobacillus moniliformis CCUG 13453T. A novel species, Streptobacillus hongkongensis sp. nov., is proposed to accommodate strains HKU33T and HKU34, with HKU33T ( = JCM 18691T = NCTC 13659T = DSM 26322T) designated the type strain. Emended descriptions of the genus Streptobacillus and Streptobacillus moniliformis are also given.