Nuc Gene Research Articles

Subclinical mastitis in crossbred dairy cows: Staphylococcus spp. and risk factors in small holder dairy farms

Subclinical mastitis (SCM) is one of the major economic diseases of dairy cows. In the present cross-sectionalstudy, dairy cows (n=205) of small holding dairy farms were screened using California mastitis test (CMT) andprevalence of 12.19% was recorded. Holstein Friesian and Jersey crossbred cows showed highest CMT score of 2followed by 3, 1 and T; linear scores of somatic cell counts (SCC) of 3.14±0.08, 4.72±0.25, 6.17±0.12, 7.99±0.11and 9.84±0.45 were observed for CMT scores N, T, 1, 2 and 3, respectively. Risk factors associated with SCM werestudied in 25 randomly selected cows. Staphylococcus species were detected in 54% of quarters based on PCRamplification of 16s rRNA gene; thermonuclease (nuc gene) PCR categorized Staphylococcus species as S. aureus(SA, 37%) and non-aureus Staphylococci (NAS, 26%). Significant association between CMT score 2 andStaphylococcus species was established. Staphylococcus species, SA and NAS, were detected in 30.7, 20.5 and17.9% of SCM negative as well as 68.85, 47.54 and 31.14% SCM positive quarters, respectively. Significantassociation between SCM and Staphylococcus species and Staphylococcus aureus was observed. Study indicatedstrong association between SCM, Staphylococcus aureus and certain risk factors among cows reared under smallholding dairy farms of the tropical region.

Simultaneous detection of Bacillus cereus and Staphylococcus aureus by teicoplanin functionalized magnetic beads combined with triplex PCR

Bacillus cereus (B. cereus) and Staphylococcus aureus (S. aureus) are two typical foodborne pathogens. In this study, we proposed a new strategy that combined antibiotic with magnetic beads (MBs) and multiplex PCR (mPCR) to detect B. cereus and S. aureus in juice sample with high sensitivity and specificity. Teicoplanin (TEI) is a glycopeptide antibiotic which can identify Gram-positive bacteria. The polyethylene glycol (PEG) and bovine serum albumin (BSA) double-mediated teicoplanin functionalized magnetic beads (TEI-BPBs) were prepared for separation of target bacteria, and the triplex PCR was used for specific detection by nuc, cesB and flic gene. Under optimal conditions, the TEI-BPBs showed a good ability to simultaneously capture S. aureus (>83%) and B. cereus (>81%). The TEI-BPBs-mPCR assay efficiently detected target bacteria even in the presence of 105 CFU/mL non-target bacteria. The results showed that the limit of detection (LOD) of B. cereus and S. aureus in PBS and juice sample could reach 100 CFU/mL within 4 h. Compared with conventional methods, this method has lower LOD and shorter detection time. The results demonstrate that the constructed TEI-BPBs capture probe and triplex PCR could be successfully applied to the rapid separation and detection of B. cereus and S. aureus or other Gram-positive pathogens in food samples.

Characterization of Staphylococcus aureus isolates from pastry samples by rep-PCR and phage typing.

Background and Objectives:Staphylococcus aureus is one of the most common causes of food poisoning. This study aimed to identify S. aureus isolated from pastries, the virulence factors, antimicrobial resistance patterns, biofilm formation, and then classification based on SCCmec types, phage types, and also Rep types.Materials and Methods:In this study, 370 creamy and dried pastry samples have been randomly collected from different confectioneries in Hamadan city. The S. aureus isolates were identified by conventional microbiological methods and nuc gene amplification. The virulence factors and prophage genes were detected. After that, the biofilm production and antibiotic susceptibility assay of S. aureus isolates were examined. Finally, the isolates were classified by rep-PCR typing.Results:Among 370 samples, 97 creamy (34.64%) and 3 dried (3.33%) pastry samples were contaminated with S. aureus. Antibiotic sensitivity results showed the highest resistance to penicillin (90%) but none of them were MRSA. According to biofilm formation assay, 14 strains (45%) were strongly adhesive. The dominant phage among isolates was SGF, especially SGFa subgroup. About half of the isolates carried SCCmec Types I and III. Analysis of the genetic linkage between isolates by rep-PCR showed ≥80% genetic similarity and also different rep-types of S. aureus isolates.Conclusion:The presence of different prophage encoded virulence factors and antibiotic resistance enable S. aureus strains to produce a broad range of diseases. Thus, consumption of creamy pastries increases the risk of infection with S. aureus and it is a serious warning to the health system.

A portable CRISPR Cas12a based lateral flow platform for sensitive detection of Staphylococcus aureus with double insurance

Foodborne pathogens cause many food safety problems and Staphylococcus aureus ( S. aureus ) is one of them that seriously threaten human health through the ages. The conventional methods for S. aureus detection basically include culture-based methods and polymerase chain reaction, which are somewhat time-consuming and laborious, and require infrastructure and professional operations. In this article, a portable and visible detection platform for S. aureus was established based on CRISPR/Cas12a, which integrated with nucleic acid isothermal amplification, CRISPR/Cas12a detection, and lateral flow strips for final signal readout. The nuc gene specific in S. aureus and conservative 16S rDNA fragment in Staphylococcus genus were selected as the detection targets. The CRISPR detection platform achieved the sensitivity of 1 × 10 0 CFU/reaction of S. aureus in pure culturing samples and performed well in the specificity assay, while the sensitivity of 2 × 10 1 CFU/mL of S. aureus was obtained in artificially contaminated food samples. Consequently, this platform combined ultra-sensitivity, specificity, accuracy, portability, user-friendliness, and time-saving, which fulfills the actual requirements in the point-of-care testing of S. aureus and was also promising for further applications in the detection of other foodborne pathogens. • CRISPR/Cas12a based lateral flow method for Staphylococcus aureus was established. • Nuc and 16S rDNA were selected as the targets for double insurance. • Results can be sensitively observed by naked eyes within an hour.

Occurrence of Methicillin Resistant Staphylococcus Aureus in Cats and Man

This study was carried out to isolate Methicillin-resistant Staphylococcus aureus (MRSA) strains from cats as well as human. Nasal, ear and skin swabs were collected from 50 cats either diseased or apparently healthy animals as well as 50 swabs were obtained from diseased and apparently healthy individuals with or without cats’ contact. Swabs were streaked on Oxacillin Resistance Screening Agar Base (a selective medium for detection of MRSA). It was found that the overall rate of isolation of MRSA from cats was 20% with significant association between the rates of isolation from diseased cats (21.4%) and apparently healthy cats (18.4%). In addition, the highest rate of isolation was recorded in the age group from 1 to 2 years (29.4%) followed by the age group > 2 years (26.7%) and finally, the age group ≤ 1 year (5.6%) with non-significant association between the rates of isolation. On the other side, the overall rate of isolation of MRSA from human was 32% with significant association between the rates of isolation from diseased individuals (28.6%) and apparently healthy individuals (40%). Moreover, individuals exceed 60 years old showed the highest rate (40.9%) followed by those of the age group extend from 30 to 60 years (30.8%) and lastly the age group less than 30 years old (20%) with significant association between the rates of isolation. Molecular characterization of MRSA strains recovered (n=26) from cats and human for detection of nuc and mecA genes by PCR was performed. It was recorded that nuc gene was detected in 65.4% of isolates while mecA gene was detected in 80.8% of the recovered isolates. Finally, it was found that the presence of human with animals in the same community and in close contact increases the opportunity of zoonotic transmission of MRSA between cats, humans, and the environment.

Prevalence, Molecular Characterization, Antibiotics Resistance of Staphylococcus aureus Isolated from Egyptian Soft Cheese, and Effect of Rosemary Essential Oil on its Viability

Staphylococcus aureus has the ability to yield heat stable enterotoxins resulting in food intoxication in human with variable intensity. So, the objectives of this research are to investigate the incidence of Staphylococcus aureus in Egyptian Soft Cheese (Talaga cheese), molecular identification of nuc and mecA genes in isolated strains by Multiplex PCR, assess the antimicrobial resistance profile using 16 different antibiotics as well as evaluate the Antimicrobial activity of rosemary oil on the viability of S. aureus. Sixty random samples of Talaga soft cheese were collected from different market places and dairy shops in various districts of Mansoura town, Dakahlia governorate. Egypt. S. aureus was discovered in 30% (18/60) from examined samples where 4 isolates (22.2%) were proved to be enterotoxigenic strains. All S. aureus isolates contained on nuc gene, however the mecA gene was found only in 5 S. aureus isolates (27.7%). S. aureus isolates showed a higher multiple antibiotic resistance (MAR) where the average MAR index was 0.479 and this leads to an increase in the public health hazard. The antimicrobial properties of rosemary essential oil as a natural antimicrobial agent were evaluated against S. aureus strain (ATCC 25923). It could be concluded that using rosemary essential oil with 2% concentration was highly effective in reducing and inhibiting S. aureus.

Prevalence, Virulence Genes and Antibiogram Susceptibility Pattern of Staphylococcus aureus and Streptococcus agalactiae Isolated from Mastitic Ewes

Clinical mastitis is a common disease found in dairy ewes worldwide that results in great economic losses. This study was designed to investigate the prevalence rate of Staphylococcus aureus and Streptococcus agalactiae isolated from clinically mastitic ewes, determine the virulence determinant genes, and identify antimicrobial sensitivity assay of the isolated bacteria. A total of 37 milk samples were collected from 27 ewes diagnosed with clinical mastitis. These revealed 12 isolates of S. aureus (32.4%), followed by 7 isolates of S. agalactiae (18.9%). Real-time polymerase chain reaction was used to identify eight strains of S. aureus as possessing the thermonuclease (nuc) gene, and five isolates of S. agalactiae were positive for the presence of the cell surface protein (cfb) gene. Eight isolates of S. aureus and five isolates of S. agalactiae isolated from dairy ewes with clinical mastitis in El-Sharkia, Egypt, possessed several virulence determinants. The antibiotic sensitivity pattern was performed and a large proportion of isolates of S. aureus were found to be sensitive to Vancomycin, Kanamycin, Ciprofloxacin, and Gentamycin, whereas large proportions were highly resistant to Penicillin G and Tetracycline. S. agalactiae isolates revealed full sensitivity to Vancomycin, Ciprofloxacin, and Oxacillin, but they were highly resistant to Kanamycin and Penicillin G. The virulence gene should be considered when developing prevention and treatment program for clinical mastitis in ewes in regional areas of isolation. It is necessary that the antibiogram findings of drugs with the highest sensitivity be used to replace the currently used antibiotics with those that are more effective.

Molecular characterisation of virulence genes in Staphylococcus aureus associated with clinical bovine mastitis

Staphylococcus aureus is the most frequently isolated pathogen from bovine mastitis including subclinical, clinical, and chronic infections. Virulence factors possessed by S. aureus aid in causing infection and inflammation by producing toxins and proteins, which are responsible for the pathogenesis of the disease. In the current study out of 51 animals presented with clinical mastitis, S. aureus was isolated from the milk of 18 animals. S. aureus was confirmed by genus and species level identification using polymerase chain reaction. Molecular characterization of selected virulence genes including thermonuclease (nuc) and Panton Valentine Leucocidin (PVL) was performed in all the S. aureus isolates. Presence of nuc gene was observed in all the isolates (100 %) of S. aureus. No isolates were found to be positive for the presence of PVL gene. Profiling the virulence genes is an important tool for epidemiological studies of mastitis, which can be employed for the prevention and control of the disease.

Prevalence of Staphylococcus aureus and Staphylococcus sciuri Isolated from Apparently Healthy and Hospitalized Patients in Ekiti State, Nigeria

The emergence of microbial resistance to antibiotics on daily basis has become a major global challenge. The increasing prevalence of methicillin resistant Staphylococcus aureus (MRSA) infections among hospitalized and non-hospitalized patients due to emergence of unique community-associated strains has become a great problem in Nigeria in particular and the entire world in general. In this study, a total of four hundred (400) clinical specimens were collected from hospitalized patients at Ekiti State University Teaching Hospital (EKSUTH), Ado-Ekiti and Federal Teaching Hospital (FETHI), Ido-Ekiti and from apparently healthy individuals from both communities. Standard bacteriological procedures were employed to isolate and identify these organisms. Antibiotic susceptibility test was carried out using the modified Kirby Bauer disc diffusion method. Polymerase chain reaction (PCR) assay was used to identify S. aureus nuc gene, as well as mecA and aac resistance genes. Seventy seven (19.3%) S. aureus isolates were phenotypically identified; 43 (21.5%) hospital-associated (23 from EKSUTH and 20 from FETHI) and 34 (17.0%) community-associated (27 from Ado-Ekiti and 7 from Ido-Ekiti communities). No S. sciuri was phenotypically isolated in both locations. Fifty five (71.4 %) of the 77 S. aureus isolates were nuc gene positive, while 11 (20%) of the 55 isolates were mecA gene positive, implying that they were MRSA. Of the 11 isolates, 6 (19.4%) were hospital-associated MRSA while 5 (20.8 %) were community-associated MRSA. Phenotypic resistance of S. aureus to cefoxitin in the two locations ranged between 77.8 – 100%, but 80% of cefoxitin-resistant nuc gene positive S. aureus lacked mecA gene. S. aureus isolates exhibited high phenotypic resistance to tetracycline, erythromycin and fusidic acid but susceptible to clindamycin and gentamycin. The prevalence of clinical MRSA infection of 20% in this study is relatively high. However, there was 0% prevalence of S. sciuri colonization. Cefoxitin disc test demonstrated low specificity as a phenotypic marker of methicillin resistance.There is need to institute control measures for MRSA infections and colonization in this environment.

Prevalence of coagulase‐positive methicillin‐resistant Staphylococcus aureus and Staphylococcus pseudintermedius in dogs in Bangladesh

BackgroundThe emergence of methicillin‐resistant Staphylococcus aureus (MRSA) and methicillin‐resistant Staphylococcus pseudintermedius (MRSP) have a significant health impact on people with direct or supportive occupations in veterinary medicine including veterinarians, animal handlers, laboratory personnel and pet owners.ObjectivesThis cross‐sectional survey was conducted to determine the prevalence of and risk factors for S. aureus, S. pseudintermedius, MRSA and MRSP in dogs in Bangladesh.MethodsA total of 358 swab samples were collected from different body sites of 150 dogs attending a university teaching veterinary hospital between January and June 2018. Standard bacteriological procedures were followed to isolate Staphylococcus, and identification was confirmed to the species level by PCR to detect the nuc gene. MRSA and MRSP were confirmed by the presence of the mecA gene.ResultsThe prevalence of coagulase‐positive S. aureus and S. pseudintermedius in dogs were 16% and 45.3%, respectively. S. aureus and S. pseudintermedius isolates displayed the highest resistance against nalidixic acid (95.2% and 91%, respectively) and erythromycin (89.3% and 84.7%, respectively). Notably, all the staphylococcal isolates showed resistance to ≥3 antimicrobial classes. The prevalence of MRSA and MRSP in dogs was 8.7% and 6%, respectively. Multivariable logistic regression analysis identified the following variables as risk factors for MRSA colonisation in dogs: dogs with dermatitis (odds ratio [OR], 12.24, 95% CI: 3.12–57.33; p < 0.001) and history of antibiotic use (OR 8.73, 95% CI: 2.23–43.10; p < 0.001). Presence of otitis (OR 14.22; 95% CI: 1.64–103.58; p = 0.008) and oral lesions (OR 9.48, 95% CI: 1.14–64.82; p = 0.002) were identified as the significant risk factors for the carriage of MRSP.ConclusionsThe circulation of multidrug‐resistant S. aureus and S. pseudintermedius is a serious concern to dogs and humans. To our knowledge, this is the first report of S. pseudintermedius and MRSP affecting dogs in Bangladesh.

Prevalence and Characterization of Methicillin-Resistant Staphylococcus aureus from Meat Retail Shops and Meat Handlers in the Buea Municipality, Cameroon

Aim: Methicillin-resistant Staphylococcus aureus (MRSA) is a notorious multidrug-resistant biovar of S. aureus associated with infections that result in high morbidity and mortality in humans and animals. Its persistence in both host and non-host environments adds a major ecological dimension to the problem, thereby making it a one health challenge. This study determined the prevalence and antibiotic resistance patterns of MRSA from meat retail shops and meat handlers in the Buea municipality in addition to basic hygiene practices implemented in the meat retail shops.&#x0D; Study Design: In this cross-sectional study, a total of 260 samples (comprising 52 swabs each from weighing balances, butchering slabs, cutting knives, hands of meat handlers and meat samples) were randomly obtained and subjected to microbiological analysis. Data on sociodemographic characteristics of meat handlers and basic hygiene practices were captured in a questionnaire.&#x0D; Methodology: After enrichment, each sample was inoculated on mannitol salt agar and DNA extracted from presumptive S. aureus isolates for detection of the nuc gene. MRSA was identified by oxacillin disk diffusion and confirmed by amplification of the mecA gene. All MRSA isolates were challenged with 10 commonly used antibiotics using the Kirby-Bauer disk diffusion method.&#x0D; Results: A total of 27 MRSA isolates were recovered from hand swabs (5.8%), meat (15.4%), knives swabs (21.2%) and butchering slabs (9.6%). All (100%) MRSA were resistant to ampicillin and cefepime followed by ofloxacin (92.6%), ciprofloxacin (81.5%) and vancomycin (37%). High susceptibility was observed for gentamicin (100%) and amikacin (100%). All isolates were multidrug-resistant and comprised 11 antibiotypes. Most meat handlers (71.2%) did not have basic meat safety training, while 90.4% of the meat retail shops did not have a nearby water source.&#x0D; Conclusion: Our results indicate that meat retail shops could be a potential reservoir of MRSA in the environment and meat handlers need more basic meat safety training.

Characterization of Staphylococcus pseudintermedius Isolates from Skin Infections of Dogs

Background: Staphylococcus pseudintermedius is a part of the canine skin microflora and an opportunistic pathogen. It plays a central role in canine pyoderma, otitis and surgical wound infections. These conditions correlate with virulence genes distributed in the bacterial genome. These genes determine strain variability on typing, in turn aiding epidemiological surveillance. The aim of this study was to isolate, identify and characterize Staphylococcus pseudintermedius (SP) and Methicillin resistant Staphylococcus pseudintermedius (MRSP) from dogs with skin infections in Chennai, India. Methods: SP and MRSP positive isolates were identified by multiplex PCR for nuc and mecA genes respectively. Characterization of the isolates for virulence genes responsible for biofilm formation (icaA, icaD), cell wall adherence (SpsO, SpsK, SpsP, SpsQ, SpsF), toxins (ExpA, ExpB, SIET, Sel, Se-int, LukS, LukF) and gene regulation (Agr, SarA) was performed. Result: Out of 275 samples, 120 SP and 8 MRSP positive isolates were identified. Only one isolate could be typed as SCCmec Type V whereas other MRSP isolates were non typeable. Agr typing of MRSP isolates revealed type II in 7 isolates and type III in one isolate. Our study revealed that there was no significant difference in the detection of virulence genes between MSSP and MRSP.

Virulence, agr groups, antimicrobial resistance and epidemiology of Staphylococcus aureus isolated from bovine subclinical mastitis

Fifty-two Staphylococcus aureus recovered from papillary ostium and milk samples collected from cows with subclinical mastitis and milking environments in three small dairy herds located in southeastern Brazil were subjected to PCR identification based on the thermonuclease (nuc) gene. All the strains were submitted to in vitro antimicrobial susceptibility testing, and we investigated the sequence types (STs), agr groups (I-IV), virulence genes encoding for Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs), biofilm-associated proteins, bi-component toxins, pyrogenic toxin superantigens, and enterotoxins. Screening for oxacillin resistance (2-6 μg/ml oxacillin), beta-lactamase activity assays, and PCR for the mecA/mecC genes detected 26 methicillin-susceptible S. aureus (MSSA) and 26 mec-independent oxacillin-nonsusceptible S. aureus (MIONSA). While MSSA isolates were found to be susceptible to all antimicrobial agents tested, or only resistant to penicillin and ampicillin, MIONSA isolates were multidrug-resistant. ST126-agr group II MSSA isolates were prevalent in milk (n=14) and carried a broad set of virulence genes (clfA, clfB, eno, fnbA, fiB, icaA, icaD, lukED, hla, and hlb), as well as the ST126-agr group II MIONSA isolated from milking liners (n=1), which also carried the eta gene. ST1-agr group III MIONSA isolates (n=4) were found in papillary ostium and milk, but most MIONSA isolates (n=21), which were identified in both papillary ostium and milking liners, were agr-negative and assigned to ST126. The agr-negative and agr group III lineages showed a low potential for virulence. Studies on the characterization of bovine-associated MSSA/MIONSA are essential to reduce S. aureus mastitis to prevent economic losses in dairy production and also to monitor the zoonotic potential of these pathogens associated with invasive infections and treatment failures in healthcare.

Isolation and molecular detection of enterotoxigenic Staphylococcus aureus from raw milk of cows

Staphylococcus aureus is a microbe associated with human’s food poisoning. It is caused by isolates producing different heat stable enterotoxins which act as one of the most spread worldwide gastroenteritis causes. The goal of the current study was to assess the incidence of S. aureus (enterotoxigenic) in the raw milk samples of cows which were collected from various places of Baghdad city. Isolation and identification of S. aureus were done by conventional laboratory method and the diagnoses were confirmed by using molecular method RT-PCR to detect the specific gene of S. aureus nuc gene. In addition, we investigate the occurrence of some of Staphylococcal enterotoxins genes such as SEA, SEB, SEC, SED, and SEE. We found that 12/50 (24%) of the isolates were S. aureus and these isolates carried one or more of the enterotoxin genes such as enterotoxin type SEC 12/12 (100) followed by enterotoxin type SEA 7/12 (58.33%) and only 1/12 (8.33%) was type SED, while type SEB and SEE were negative 0%. Some of the isolates have genes that coded for two enterotoxins patterns such as SEA and SEC 7/12 (58.33%) and SEC and SED 1/12 (8.33%). Fewer of the isolates expressed only one genotype of enterotoxin gene like SEC 4/12 (33.3%). This study has proven that there was a high chance of occurrence for the enterotoxigenic S. aureus at the raw milk of cows in Baghdad city.

A point-of-care test device for MRSA rapid detection

Staphylococcus aureus (SA) is one of the most common pathogenic bacteria, and methicillin-resistant SA (MRSA) is an equally common drug-resistant bacteria. MRSA detection is of great significance for clinical diagnosis, medication guidance, and prevention of antibiotic abuse. Traditional MRSA detection using the culture method is time-consuming, laborious, and difficult to conduct rapid on-site detection. In this research, we developed a device for rapid MRSA detection, which can detect the nuc gene in SA and mecA gene in MRSA simultaneously for 30–40 min. After simple sample processing, the mixture can be directly loaded onto the chip device. The detection results can be directly determined by a color change, with a limitation of approximately 102 copies. This isothermal amplification chip device can be widely applied in many fields, with simple operation and low contamination.

Detection, antibiogram and molecular characterization of MRSA and MSSA isolated from swine

The emergence of bacteria with antimicrobial resistance traits such as methicillin-resistant Staphylococcus aureus (MRSA) in food-producing animals is a significant public health concern. The aim of this study is to determine the prevalence rate, antibiotic susceptibility and the molecular characteristics of Staphylococcus aureus (S. aureus) in swine from selected swine farms in Peninsular Malaysia. The oral and nasal swabs of 200 live swine were collected. Screening and isolation of S. aureus isolates were carried out using phenotypic identification techniques. The identity of the bacteria isolates was confirmed by detection of nuc gene (S. aureus) and mecA gene (MRSA). Antibiogram of S. aureus against 19 antibiotics was developed using Kirby-Bauer test. Molecular detections of antimicrobial resistance and virulence genes as well spa typing of S. aureus was carried out. 25% of swine were carrying S. aureus with 9% being MRSA carriers. S. aureus and MRSA have shown high resistance against clindamycin, tetracycline, chloramphenicol, oxacillin, penicillin, erythromycin, cefoxitin and amoxicillin/clavulanate. Antimicrobial resistance and virulence genes, including tetK, tetL, tetM, ermA, ermB, ermC, msrA and scn genes were detected. 93% of S. aureus were multiple-drug resistant (MDR), suggesting the emergence of MDR S. aureus in swine in Malaysia.

Prevalence, antibiotic resistance, and enterotoxin production of Staphylococcus aureus isolated from retail raw beef, sheep, and lamb meat in Turkey

The main objective of this study was to isolate and identify Staphylococcus aureus from retail raw red meat samples and evaluate their enterotoxin gene and antibiotic resistance profiles. A total of 452 retail raw meat samples, including beef (n = 200), sheep (n = 125), and lamb (n = 127) randomly purchased from various supermarkets and butchers in Ankara between July 2019 and November 2020, were tested for the prevalence of S. aureus. The S. aureus strain was identified using morphological and molecular (16S rRNA and nuc gene) methods. Moreover, nine Staphylococcal enterotoxin (SE) genes were screened using polymerase chain reaction. Antibiotic resistance of S. aureus was determined using the phenotypic disc diffusion method. The overall prevalence of S. aureus among screened samples was 21.23%. Additionally, 65.62% of S. aureus strains contained SE gene regions. The predominant SEs in the S. aureus strains were sea (50.79%), followed by sed (25.39%) and seb (23.80%). However, sec, see, seg, seh, sei, and sej genes were never detected. A substantial proportion (40–100%) of the isolates were found resistant to kanamycin, telithromycin, penicillin G, streptomycin, erythromycin, cloxacillin, ampicillin, pristinamycin, nalidixic acid, azithromycin, and ciprofloxacin. Multi-drug resistance (MDR) was observed in 96.87% of the S. aureus strains. These results show a low prevalence of S. aureus in raw red meat samples in Turkey. However, a high rate of SEA raises serious health concerns. Due to the high levels of MDR observed in this study, there is a need to strictly control antibiotic use in animals in Turkey.

Development of multiplex TaqMan qPCR for simultaneous detection and differentiation of eight common swine viral and bacterial pathogens.

It is laborious to diagnose the infections of classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), and Suid herpesvirus 1 (SuHV-1) because of the similar clinical symptoms in piglets. Staphylococcus aureus (S. aureus), Streptococcus suis (S. suis), Salmonella choleraesuis (S. choleraesuis, serotype: 6,7:c:1,5), and Escherichia coli (E. coli) are common secondary bacterial pathogens in viral infections. Furthermore, the mixed infection of these viral and bacterial pathogens is more and more common in practical swine breeding. Therefore, a TaqMan multiplex qPCR method for simultaneous detection and differentiation of their pathogen was established in this study by designing specific primers and probes for the E2 gene of CSFV, the ORF7 gene of PRRSV, the ORF1 gene of PCV2 and the gE gene of SuHV-1, the nuc gene of S. aureus, the ef-tu gene of S. suis, the ivnA gene of S. choleraesuis, and the 23S rRNA gene of E. coli, and its specificity, sensitivity, and reproducibility were subsequently tested. The results showed that TaqMan multiplex qPCR method showed a high specificity with no cross reaction between different viruses, and a good repeatability with its coefficient of variation lower than 5%. Besides, the sensitivity of this method was also at least 10 times higher compared with conventional PCR. Overall, this study provided a reliable multiplex TaqMan qPCR method for the diagnosis and differentiation of the mentioned pathogens in pigs, laying a certain technical basis for disease prevention and control.

Molecular Identification and antibiotic susceptibility pattern of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA)

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is an emerging bacterium associated with a much higher incidence of clinical infection worldwide than any other strains of S. aureus. The misappropriation of non-prescribing drugs amongst university students in Pakistan has become a severe problem. The present study aims to evaluate the nasal carriage rates of Staphylococcus aureus and methicillin resistant Staphylococcus aureus (MRSA) in university students and described the history of associated risk factors. Among the 350 samples, 300 (85.71%) were S. aureus positive and 50 (14.28%) were other isolates. The positive individual includes 295 (98.33%) males and 5 (1.66%) females. A total of 16 (5.3%) S. aureus out of the total 300 were resistant to cefoxitin (MRSA) and 284 (94.66%) isolates were MSSA (Methicillin Susceptible Staphylococcus aureus). The strains were confirmed by the amplification of nuc gene analysis as S. aureus and the MRSA was confirmed with mecA gene analysis. All the samples were catalase positive, whereas 72.33% was coagulase positive and 27.66% were found as coagulase negative. Among the coagulase positive 7.37% were resistant to methicillin. The antibiotic resistance patterns showed no MRSA isolates to be resistant to Vancomycin and Linzolids. In most cases the MRSA was less susceptible to other antibiotics. It was concluded that Vancomycin and Linzolid can be used as drug of choice as no MRSA was found resistant to them. Furthermore, molecular PCR diagnosis is suggested as rapid and sensitive technique for the identification of MRSA isolates based on their mecA gene analysis.

Mastitisli Sığırlardan İzole Edilen Stafilokoklarda Antibiyotik Direnci ve Virülens Gen Profilleri

Staphylococcus spp. are the important bacterial agents of subclinical and clinical mastitis cases. This study was aimed to determine the vancomycin resistances, antibacterial resistance profiles, some virulence genes, and pheno- and genotyping of staphylococci from mastitis. For this aim, 121 staphylococcal isolates were analyzed. The identifications of isolates were confirmed with PCR for being Staphylococcus spp. and Staphylococcus aureus. The antibiotic resistance patterns were determined by Kirby bauer Disc Diffusion Tests and according to the resistance profiles, the isolates were antibiotyped. The vancomycin resistance genes were determined by PCR for investigating the vanA, vanH, vanR, vanS, vanZ, vanY and vanX genes. The vancomycin resistant isolates were genotyped with RAPD-PCR. The nuc gene was detected in 86 of 121 staphylococcal isolates examined and named as Staphylococcus aureus. The remaining 35 isolates were defined as Staphylococcus spp. S. aureus isolates were found to be resistant to penicillin G, amoxicillin/clavulanic acid, oxacillin, tetracycline, cefaperazone, teicoplanin, vancomycin and trimethoprim-sulfomethaxazole at 50% (43/86), 40.7% (35/86), 34.9% (30/86), 23.3% (20/86), 22.1% (19/86), 18.6% (16/86) 10.5% (9/86) and 8.1% (7/86) respectively. On the other hand, 4 (11.4%) of 35 Staphylococcus spp. isolates were resistant to penicillin G, 3 (8.6%) to amoxicillin/clavulanic acid, 2 (5.7%) to trimethoprim-sulfometaxazole, 1 (2.9%) to oxacillin, cefaperazone, teicoplanin, while all isolates were sensitive to vancomycin and tetracycline. Of the 9 S. aureus isolates that were phenotypically resistant to vancomycin, vanA gene was detected in 2 and vanR gene in 3 isolates. Multiple resistances to three or more antibiotics were determined in 42 of 86 S. aureus isolates. In addition, coa gene was detected in 61 (70.9%) of 86 S. aureus isolates. 10 different gene polymorphisms were detected in coa gene positive isolates. While the spa gene was determined in all S. aureus isolates, it was revealed that they had 4 spa gene polymorphisms. Nine different genotypes with a similarity between 51-75% were detected in the genotyping of vancomycin resistant 9 isolates. In conclusion, multiple antibiotic resistance rates in S. aureus isolates investigated were found to be important for mastitis treatment. The results obtained from this study show that milk and dairy products containing these factors pose a public health risk due to the determination of vancomycin resistance in mastitis-derived Staphylococcus strains.