Resistance Gene mecA Research Articles

ANTIBIOTIC RESISTANCE PROFILE AND MOLECULAR CONFORMATION AND MRSA ISOLATE BY MULTIPLEX PCR FROM CLINICAL SAMPLE AT A TERTIARY CARE HOSPITAL IN INDIA

Methicillin Resistant Staphylococcus aureus (MRSA) is an important human pathogen. They are emerging as a serious public-health issue. It causes nosocomial and community acquired infections. MRSA is present throughout hospitals in many countries. Nowadays it is the most commonly isolated antimicrobial resistant pathogen worldwide. The study include Material Method – d 450 isolates of Staphylococcus species from various clinical samples. The samples received from outpatient departments (OPDs), inpatient departments (IPDs) & various intensive care units (ICUs) included urine, blood, pus, sputum, vaginal swab, tissue, pleural uid. All clinical samples were processed as per standard bacteriological techniques. Molecular identication of Antibiotic Resistance Gene-mecA gene was also performed. Out of 450 Result– Staphylococcus species, 150 isolates were Staphylococcus aureus & 300 isolates were Coagulase Negative Staphylococci (CONS). Conclusion – Methicillin resistance is caused by the presence of mec- A gene, which encodes a low afnity penicillin binding protein (PBP)-2a or PBP2' which has a low afnity for β-lactam antibiotics. Therefore, presence of mec-A gene indicates methicillin resistance in Staphylococci. Detecting mec-A gene by polymerase chain reaction is now considered the gold standard for identifying methicillin resistance in S. aureus.

Антибиотикорезистентность штаммов грамположительных кокков, выделенных из секрета предстательной железы мужчин с хроническим бактериальным простатитом

to characterize the antibiotic resistance of gram-positive cocci strains isolated from the prostate secretion in men with chronic bacterial prostatitis at the level of phenotype and genotype.Bacteria were isolated from the prostate secretion of men of reproductive age (20-45 years) with chronic bacterial prostatitis by conventional bacteriological method. The type of microorganisms was determined by MALDI-TOF mass spectrometry. Resistance to 16 antibiotics of 31 strains of E. faecalis and 91 cultures of coagulase-negative staphylococci (CoNS) was determined by the disk diffusion method. Antibiotic resistance genes (mecA; blaZ; aac(6)- aph (2); ant (4)-Ia; aph (3)-IIIa; gyrA, grlA) were detected using polymerase chain reaction (PCR ) using selected primers.A high resistance of enterococci to antibacterial drugs was revealed: fluoroquinolones, carbapenens, cephalosporins (with the exception of cefoperazone), gentamicin and oxacillin. It has been established that CoNS are characterized by variable antibiotic resistance, while: isolates of S. epidermidis and S. haemolyticus are resistant to all studied fluoroquinolones and carbapenems; S. warneri to carbapenems and the vast majority of studied cephalosporins; S. saprophyticus - to aminoglycosides. Amoxiclav and cefoperazone are characterized by the highest activity against clinical isolates. Using PCR, the presence of genetic determinants of resistance to aminoglycosides and -lactams was established in the isolates, with a predominance of the studied genes in CoNS.For effective antibiotic therapy in chronic bacterial prostatitis, it is necessary to conduct regional monitoring of the resistance of microorganisms to antibacterial drugs.

Colonization and Antibiotic Resistance of Nasal Staphylococcus Aureus among Healthcare Workers in Southwestern Iran: Occurrence of OS-MRSA

Background & Objectives: Staphylococcus spp. is a resident flora of the skin and mucosa of humans that can colonize the anterior nares of individuals. This cross-sectional study was conducted to determine the rate and antibiotic resistance pattern of nasal Staphylococcus aureus (S. aureus) carriers among the staff of Fasa hospital, southern Iran.
 Materials & Methods: Nasal swab samples were collected from 117 hospital staff working in 12 wards. Microbiological culture method was applied for S. aureus identification. The isolates were confirmed by tuf gene identification using PCR assay. Five isolates were randomly sequenced and phylogenetically analysed using MEGA software. The antimicrobial resistance pattern of the isolates was evaluated using the disc diffusion assay and the amplification of the methicillin resistance (mecA) gene.
 Results: The prevalence of S. aureus nasal carriers included 10.26% (n=12). The nasal carriers were identified in the wards of surgery ICU, gynecologic surgery, NICU, pediatric, internal surgery, and emergency. Among them, gynecologic surgery staff had the highest rate of nasal colonization (33.33%). Phylogenetic analysis showed that of five isolates, four had high similarities with each other. Also, the highest rate of resistance was related to penicillin (83.3%), followed by cefazolin (75%), and cephalexin (75%). However, the highest level of susceptibility (100%) was found for vancomycin, cefoxitin, and oxacillin. Furthermore, the methicillin resistance gene (mecA) was highly detected (75%) from the isolates, elucidating oxacillin-susceptible or cefoxitin-susceptible mecA-positive S. aureus (OS-MRSA).
 Conclusions: The high rates of OS-MRSA can lead to antibiotic resistance among health care workers tremendously. Moreover, the high similarity probability in phylogenetic analysis shows the possibility of cross-infection between these health care workers, warning to exert effective strategies to control infection spread, especially in the surgery ward.

Resistance profile and biofilm production capacity of Staphylococcus spp. beef slaughterhouse isolates and their sensitivity to Rosmarinus officinalis essential oil.

Microorganisms can interfere with meat quality, being a public health problem. The aim of this study was to characterize the antimicrobial susceptibility profile of Staphylococcus spp. isolated from utensils of a bovine slaughterhouse and to evaluate the antibacterial and antibiofilm activity of the essential oil of Rosmarinus officinalis L. (rosemary). Samples of surfaces and utensils used during slaughter in the northwest of the state of Paraná, Brazil were collected. After isolation and differentiation of the isolates by the coagulase test, the antimicrobial susceptibility test, Staphylococcus aureus identification and mecA gene research were performed. The study for biofilm production was carried out by the method of adhesion in borosilicate tube and by adhesion in polystyrene plate. Subsequently, the inhibitory activity of theR. officinalis essential oiland its ability to inhibit biofilm were investigated. Twenty-two of the samples collected were identified as coagulase-negative Staphylococcus and five as coagulase-positive Staphylococcus. There was resistance to all antibiotics tested, with clindamycin (33.33%) and rifampicin (29.6%) showing the highest rate. None of the samples was confirmed as Staphylococcus aureus or for the presence of the mecA resistance gene. The essential oil inhibited the growth of 48% of the isolates at a concentration of 16,000µg/mL. Of these isolates, 33% were positive for biofilm production and this biofilm was also inhibited by the essential oil. This work revealed that multidrug-resistant Staphylococcus and biofilm producers are present in the slaughter environment and are susceptible to the essential oil of R. officinalis.

Detection of Some Virulence Factors in Staphylococci Isolated from Mastitic Cows and Ewes

Abstract About 150 million families around the world are engaged in milk production. However, inflammation of the mammary gland (mastitis) remains a major problem in dairy ruminants that affects the quality of milk worldwide. The aim of this study was the examination of udder health with detection of contagious and environmental pathogens causing mastitis in 960 and 940 dairy cows and ewes, respectively. The presence of selected virulence factors such as: the formation of haemolysis, gelatinase, biofilm, hydrolyse DNA, and resistance to antibiotics with detection of methicillin resistance gene (mecA), were determined in selected virulence factors associated with isolated staphylococci. These isolated staphylococci with selected virulence factors can have untoward effects on the severity of mastitis. The results of our study indicated that, in addition to the major udder pathogens (S. aureus, S. uberis, and S. agalactiae) causing mastitis, non-aureus staphylococci (NAS), is a major risk to dairy cows and ewes. NAS, such as S. chromogenes, S. warneri, and S. xylosus isolated from infected animals with clinical and chronic mastitis, had the highest representation of virulence factors in comparison to less virulent strains. In addition, the isolates of S. aureus and NAS demonstrated 77.0 % and 44.2 % resistance to one or more antimicrobial classes from mastitic milk samples obtained from dairy cows and ewes, respectively. Due to the high resistance to β-lactamantibiotics in two isolates of S. aureus and two species of NAS isolated from cows’ mastitic milk samples, the presence of a methicillin-resistant gene mecA poses serious complications for the treatment and a serious health risk to milk consumers.

Prevalence and Molecular Characterization of Methicillin-Resistant Staphylococcus aureus from Nasal Specimens: Overcoming MRSA with Silver Nanoparticles and Their Applications.

Staphylococcus aureus is a cause of high mortality in humans and therefore it is necessary to prevent its transmission and reduce infections. Our goals in this research were to investigate the frequency of methicillin-resistant S. aureus (MRSA) in Taif, Saudi Arabia, and assess the relationship between the phenotypic antimicrobial sensitivity patterns and the genes responsible for resistance. In addition, we examined the antimicrobial efficiency and application of silver nanoparticles (AgNPs) against MRSA isolates. Seventy-two nasal swabs were taken from patients; MRSA was cultivated on Mannitol Salt Agar supplemented with methicillin, and 16S rRNA sequencing was conducted in addition to morphological and biochemical identification. Specific resistance genes such as ermAC, aacA-aphD, tetKM, vatABC and mecA were PCR-amplified and resistance plasmids were also investigated. The MRSA incidence was ~49 % among the 72 S. aureus isolates and all MRSA strains were resistant to oxacillin, penicillin, and cefoxitin. However, vancomycin, linezolid, teicoplanin, mupirocin, and rifampicin were effective against 100% of MRSA strains. About 61% of MRSA strains exhibited multidrug resistance and were resistant to 3-12 antimicrobial medications (MDR). Methicillin resistance gene mecA was presented in all MDR-MRSA strains. Most MDR-MRSA contained a plasmid of > 10 kb. To overcome bacterial resistance, AgNPs were applied and displayed high antimicrobial activity and synergistic effect with penicillin. Our findings may help establish programs to control bacterial spread in communities as AgNPs appeared to exert a synergistic effect with penicillin to control bacterial resistance.

First Finding of a Methicillin-Resistant Staphylococcus aureus (MRSA) t304/ST6 from Bovine Clinical Mastitis.

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) comprises a global threat to humans and animals. Here, we report and characterize the MRSA t304/ST6 variant which, to our knowledge, represents the first case found in bovine clinical mastitis. In general, the MRSA t304/ST6 variant is rarely described in livestock, contrary to humans where it is widely recognized. Phenotypic and genotypic resistance profiling showed that the bovine-MRSA t304/ST6 isolate expressed low susceptibility toward cefoxitin (MICcefoxitin = 16 µg/mL) and carried the mecA resistance gene in the SCCmec IVa. The bovine-MRSA t304/ST6 isolate carried a plasmid similar to that which has been frequently observed among human-MRSA t304/ST6 isolates in Denmark (GenBank accession no. NZ_CP047022). In addition, a Staphylococcus prophage 3 (ϕSA3) was detected, encoding an immune evasion cluster (IEC) of putative virulence genes associated with human host-specificity (sea, sak, and scn). Taken together, these findings suggest that the MRSA t304/ST6 found in this study represents a recent host-jump event, with human to cow transmission. This study emphasizes the importance of and the need for performance of antimicrobial resistance surveillance among bovine mastitis pathogens, including S. aureus and MRSA.

Prevalence of Methicillin and β-Lactamase Resistant Pathogens Associated with Oral and Periodontal Disease of Children in Mymensingh, Bangladesh.

Oral and periodontal diseases (OPD) is considered one of the main problems of dentistry worldwide. This study aimed to estimate the prevalence of oral and periodontal pathogenic bacteria along with their antimicrobial resistance pattern in 131 children patients aged between 4–10 years who attended in Mymensingh Medical College Hospital during October 2019 to March 2020. OPD pathogens were identified through isolation, cultural and biochemical properties, and nucleic acid detection. The isolates were subjected to antimicrobial susceptibility to 12 antibiotics commonly used in dentistry. In addition, the isolates were analyzed molecularly for the presence of six virulence and three antibacterial resistance genes. Five pathogens were identified, of which Staphylococcus aureus (S. aureus) (49%) and S. salivarius (46%) were noticed frequently; other bacteria included S. mutans (16.8%), S. sobrinus (0.8%) and L. fermentum (13.7%). The virulence genes—clumping factor A (clfA) was detected in 62.5% isolates of S. aureus, and gelatinase enzyme E (gelE) gene was detected in 5% isolates of S. salivarius, while other virulence genes were not detected. All the tested isolates were multidrug-resistant. The overall prevalence of MDR S. aureus, Streptococcus spp. and L. fermentum was 92.2%, 95.1% and 100%, respectively. It was observed that a high proportion of isolates were found resistant to 5–8 antibiotics. A majority of S. aureus, Streptococcus spp., and L. fermentum isolates tested positive for the β−lactamase resistance genes blaTEM and cfxA, as well as the methicillin resistance gene mecA. Phylogenetically, the resistance genes showed variable genetic character among Bangladeshi bacterial pathogens. In conclusion, S. aureus and S. salivarius were major OPD pathogens in patients attended in Mymensingh Medical College Hospital of Bangladesh, and most were Beta-lactam and methicillin resistant.

Microbiological evaluation of stethoscopes used in the clinical routine of Veterinarians in the Metropolitan Region of Recife, Pernambuco, Brazil

We evaluated the level of contamination of stethoscopes used in the clinical routine of veterinarians. Specimens from 15 stethoscopes were cultured and analyzed before (M0) and after (M1) disinfecting the stethoscope diaphragm with alcohol at 70ºGL. At moment M0, bacterial growth was observed in 73.33% (11/15) of the stethoscopes. Seventeen strains were isolated, and the majority (41.2%, 7/17) were Bacillus spp. followed by Staphylococcus spp. (35.3%, 6/17) and Micrococcus spp. (23.5%, 4/17). In samples collected after disinfection with alcohol (M1), only Bacillus spp. were isolated in 13.3% (2/15) of the stethoscopes. Coagulase phenotypic testing of the Staphylococcus spp. revealed that all the strains were negative. Polymerase chain reaction was performed for the mecA resistance gene in coagulase-negative Staphylococcus strains. Amplification of mecA gene was observed in only 16.66% (1/6) of the samples. Stethoscopes are instruments that transfer pathogens, especially bacteria that may carry resistance genes. However, simple cleaning and disinfection measures in the veterinary medical clinic routine reduce contamination and minimize the risk of spread of resistant microorganisms to animals and the environment.

Sink survey to investigate multidrug resistance pattern of common foodborne bacteria from wholesale chicken markets in Dhaka city of Bangladesh

Antimicrobial resistance (AMR) among foodborne bacteria is a well-known public health problem. A sink survey was conducted to determine the AMR pattern of common foodborne bacteria in cloacal swab of broiler chickens and sewage samples from five wholesale chicken markets of Dhaka city in Bangladesh. Bacteria were identified by culture-based and molecular methods, and subjected to antimicrobial susceptibility testing. Resistance genes were identified by multiplex PCR and sequencing. Multidrug resistance (MDR) was observed in 93.2% of E. coli, 100% of Salmonella spp., and 97.2% of S. aureus from cloacal swab samples. For sewage samples, 80% of E. coli, and 100% of Salmonella and S. aureus showed MDR. Noteworthy, 8.3% of S. aureus from cloacal swab samples showed possible extensively drug resistance. Antimicrobial resistance genes (beta-lactamase—blaTEM, blaSHV; quinolone resistance gene—qnrS) were detected in a number of E. coli and Salmonella isolates from cloacal swab and sewage samples. The methicillin resistance gene (mecA) was detected in 47.2% and 25% S. aureus from cloacal swab and sewage samples, respectively. The findings envisage the potential public health risk and environmental health hazard through spillover of common foodborne MDR bacteria.

Whole-Genome Sequence of Multidrug-Resistant Methicillin-Resistant Staphylococcusepidermidis Carrying Biofilm-Associated Genes and a Unique Composite of SCCmec.

Staphylococcus epidermidis is part of the normal human flora that has recently become an important opportunistic pathogen causing nosocomial infections and tends to be multidrug-resistant. In this investigation, we aimed to study the genomic characteristics of methicillin-resistant S. epidermidis isolated from clinical specimens. Three isolates were identified using biochemical tests and evaluated for drug susceptibility. Genomic DNA sequences were obtained using Illumina, and were processed for analysis using various bioinformatics tools. The isolates showed multidrug resistance to most of the antibiotics tested in this study, and were identified with three types (III(3A), IV(2B&5), and VI(4B)) of the mobile genetic element SCCmec that carries the methicillin resistance gene (mecA) and its regulators (mecI and mecR1). A total of 11 antimicrobial resistance genes (ARGs) was identified as chromosomally mediated or in plasmids; these genes encode for proteins causing decreased susceptibility to methicillin (mecA), penicillin (blaZ), fusidic acid (fusB), fosfomycin (fosB), tetracycline (tet(K)), aminoglycosides (aadD, aac(6′)-aph(2′’)), fluoroquinolone (MFS antibiotic efflux pump), trimethoprim (dfrG), macrolide (msr(A)), and chlorhexidine (qacA)). Additionally, the 9SE strain belongs to the globally disseminated ST2, and harbors biofilm-formation genes (icaA, icaB, icaC, icaD, and IS256) with phenotypic biofilm production capability. It also harbors the fusidic acid resistance gene (fusB), which could increase the risk of device-associated healthcare infections, and 9SE has been identified as having a unique extra SCC gene (ccrB4); this new composite element of the ccr type needs more focus to better understand its role in the drug resistance mechanism.

Occurrence and Distribution of Antibiotic-Resistant Staphylococcus aureus in a Brazilian Pork Production Chain

Here we characterized the distribution and the antibiotic resistance of staphylococci from a Brazilian pork production chain. Samples (n = 1,114) from pig farms, pig lots, and slaughterhouses, located in two Brazilian states (Minas Gerais and Paraná), were subjected to coagulase-positive Staphylococcus enumeration. S. aureus isolates (n = 251) from this collection were further characterized for their resistance to oxacillin, cefoxitin, vancomycin, and tetracycline through phenotypic and molecular assays. Coagulase-positive Staphylococcus counts from pig farms were higher compared with other samples (P < 0.05). Other counts were relatively low but were present in all production stages. S. aureus isolates were commonly resistant to oxacillin and cefoxitin (54 of 73, 74.0%), qualifying them as methicillin-resistant S. aureus, but PCR assays indicated that few harbored the expected antimicrobial resistance genes (femB, mecA, and mecC). Lower frequencies of vancomycin and tetracycline resistance were found (6.8 to 37.0%). PCR sensitivity (34.5 to 86.7%) and specificity (26.6 to 85.0%) for detection of antibiotic resistance genes varied based on the assessed antibiotic. Antibiotic-resistant staphylococci are widely distributed in the Brazilian pork production chain, and methicillin-resistant S. aureus can become a potential health and economic impediment for the Brazilian pork industry.

Whole-genome sequencing to assess clonality in a series of prosthetic joint Staphylococcus epidermidis isolates – WITHDRAWN

Background: Prosthetic joint infections (PJIs) are costly and cause increased morbidity and mortality for patients. Staphylococcus epidermidis is a common cause of both early postoperative and late-presenting PJIs. Although S. epidermidis is a normal part of the human skin microflora, its ability to form biofilm on implanted medical devices make it an important causative pathogen of PJIs. We investigated genetic, epidemiologic, and environmental factors contributing to S. epidermidis PJIs by performing whole-genome sequencing and clinical epidemiologic investigation of isolates collected from infected patients between 2017 and 2020. Methods: Patients with S. epidermidis isolated from a prosthetic joint that was placed at our orthopedic specialty hospital were identified using the microbiology laboratory records and electronic medical records. Whole-genome sequencing and single-nucleotide polymorphism (SNP)–based clonality analyses were performed using the epiXact service at Day Zero Diagnostics. These analyses included species identification, in silico MLST typing, phylogenomic analysis, as well as genotypic assessment of the prevalence of specific antibiotic resistance genes, virulence genes, and other relevant genes. For clonal isolates, additional reviews of surgical history and clinical data were performed. Results: In total, 62 S. epidermidis joint isolates were identified from 46 patients. Among these isolates, 52 were of sufficient purity to be used for genomic analysis (Fig. 1). A number of genes appeared in every isolate including sepA, smr, cap, sesB, sesG, and embp. Also, 6 S. epidermidis samples had a discrepancy between phenotypic resistance to oxacillin and the presence of the mecA resistance gene. We also identified 6 distinct clusters of isolates, all of which had SNP distances &lt;10 base pairs (Fig. 2). Each cluster consisted of 2–4 patients. Cluster isolates accounted for 29.8% of all S. epidermidis prosthetic joint isolates. Most clonal isolates occurred in patients who were heavily exposed to different healthcare settings. Further epidemiologic investigation showed that some of these clonal isolates had ties to aspirations or procedures, whereas no clear connection could be determined for others. Conclusions:S. epidermidis isolated from clinical prosthetic joint samples contains a high degree of genetic resistance, including a mismatch between presence of mecA and phenotypic oxacillin resistance and genetic propensity for chlorhexidine resistance. Mupirocin resistance was not observed. Of all isolates, 29.8% belonged to multiple clusters, confirming hospital spread of this commensal organism in some cases.Funding: NoneDisclosures: None

Mec-Positive Staphylococcus Healthcare-Associated Infections Presenting High Transmission Risks for Antimicrobial-Resistant Strains in an Equine Hospital.

Healthcare-associated infections caused by Staphylococcus, particularly Staphylococcus aureus, represent a high risk for human and animal health. Staphylococcus can be easily transmitted through direct contact with individual carriers or fomites, such as medical and non-medical equipment. The risk increases if S. aureus strains carry antibiotic resistance genes and show a phenotypic multidrug resistance behavior. The aim of the study was to identify and characterize methicillin resistant coagulase-positive staphylococci (MRSA) and coagulase-negative staphylococci (MRCoNS) in equine patients and environmental sources in an equine hospital to evaluate the genetic presence of multidrug resistance and to understand the dissemination risks within the hospital setting. We explored 978 samples for MRSA and MRCoNS using Oxacillin Screen Agar in an equine hospital for racehorses in Chile, which included monthly samples (n = 61–70) from equine patients (246) and hospital environments (732) in a one-year period. All isolates were PCR-assessed for the presence of methicillin resistance gene mecA and/or mecC. Additionally, we explored the epidemiological relatedness by Pulsed Field Gel Electrophoresis (PFGE) in MRSA isolates. Phenotypic antibiotic resistance was evaluated using the Kirby-Bauer disk diffusion method. We estimated the unadjusted and adjusted risk of acquiring drug-resistant Staphylococcus strains by employing logistic regression analyses. We identified 16 MRSA isolates and 36 MRCoNS isolates. For MRSA, we detected mecA and mecC in 100% and 87.5 % of the isolates, respectively. For MRCoNS, mecA was detected among 94% of the isolates and mecC among 86%. MRSA and MRCoNS were isolated from eight and 13 equine patients, respectively, either from colonized areas or compromised wounds. MRSA strains showed six different pulse types (i.e., A1–A3, B1–B2, C) isolated from different highly transited areas of the hospital, suggesting potential transmission risks for other patients and hospital staff. The risk of acquiring drug-resistant Staphylococcus species is considerably greater for patients from the surgery, equipment, and exterior areas posing higher transmission risks. Tackling antimicrobial resistance (AMR) using a One Health perspective should be advocated, including a wider control over antimicrobial consumption and reducing the exposure to AMR reservoirs in animals, to avoid cross-transmission of AMR Staphylococcus within equine hospitals.

Molecular Diversification and Frequency of mecA Gene in the MRSA Infections of Hospitalized Patients at Islamabad, Pakistan

Objective: To determine the molecular diversification and frequency of mecA Gene in MRSA infections of hospitalised patients at Islamabad, Pakistan.&#x0D; Study design: Cross-sectional study.&#x0D; Place and Duration: Pakistan Institute of Medical Science, Islamabad, Pakistan, from Sep 2017 to Sep 2018.&#x0D; Methodology: To explore the occurrence of pathogenic S. aureus in the hospitalised patients of Islamabad, 500 samples were collected. All the isolates were further characterised by gram staining, catalase test and DNAse media analysis validating the presence of S.aureus. The overall frequency of S.aureus was 19.5% in all the isolates. Six highly distinguished colonies were further evaluated through colony PCR by 16S r RNA analysis, and results revealed 99.9% homology of isolates with S.aureus. The degree of resistance was further evaluated for 12 isolates based on the previous MRSA resistance pattern. The extraction and concentration of DNA were done by the CTAB and Nanodrop methods. Then primers were designed for the presence of the mecA gene of S. aureus.&#x0D; Results: Results showed 51 (52.7%) MSSA and 45 (47.4%) MRSA among all the isolates. Colony PCR by 16S r RNA analysis results revealed 97 (99.9%) homology of isolates with S.aureus. PCR of all the twelve extracted DNAs of isolates showed the frequency of the mecA gene in all the isolates.&#x0D; Conclusion: The study is the first report the presence of resistance mecA gene in the hospitalised patients of Islamabad. It has depicted a resistant gene's presence and showed the rising trend of resistance in pathogenic S. aureus.

Invastigation of 16S rRNA, mecA and nuc genes in coagulase-positive and negative Staphylococci by Real-Time PCR

Staphylococcus aureus is a Gram-positive, round-shaped bacterium that is a member of the Firmicutes. It is often positive for catalase and nitrate reduction. Pathogenic strains often promote infections by producing virulence factors such as potent protein toxins, and the expression of a cell-surface protein that binds and inactivates antibodies. Sepsis-related to methicillin-resistant S. aureus (MRSA) has significant morbidity and high mortality rates (15-30%). The methicillin resistance for S. aureus is coded with the mecA gene, while the methicillin sensitivity is coded with the nuc gene, and they are chromosomal. Similarly, it is coded with the coagulase gene for S. aureus (Coa). In this study, the 16S rRNA gene identification by Real-Time PCR was investigated in forty S. aureus isolates, which were cultured at different times in terms of MIC and SIR tests. The isolates used in the study were determined at the gene level in terms of their differences in methicillin resistance gene (MecA), methicillin susceptibility gene (Nuc), coagulase gene (Coa) and in terms of intra-species differences were examined. As a result of the study, Staphylococcus spp. yielded positive results with 16S rRNA gene-specific primers in all isolates. Real-Time PCR analysis of the isolates with SYBRGreen-based PCR analysis was performed with 16S rRNA gene-specific primers, and the samples were confirmed to be Staphylococcus. Analysis at the family level was followed by Coa, Nuc, and MecA gene Real-Time PCR results, and it was found that, in terms of Coa and Nuc genes, 19 isolates were positive and 21 isolates were negative. In terms of MecA gene, 16 isolates were positive according to the positive sigmoidal curves and to the single peak melting values, whereas 24 isolates were found to be negative. It was thought that this study will benefit the community by contributing to the rapid and effective diagnosis and treatment of infections caused by coagulase positive and negative Staphylococci.

Survey of Staphylococcus aureus carriage by free-living red deer (Cervus elaphus): Evidence of human and domestic animal lineages.

Staphylococcus aureus is a pathogen that can affect multiple host species. Evidence of transmission between humans and animals and among different animal species has been reported in recent years. In this study, we investigated 284 free-living red deer (Cervus elaphus) in the Central Italian Alps to assess the prevalence and molecular characteristics of S. aureus in nasal and intestinal samples in relation to host features and environmental factors. A prevalence of 90%, 26.2% and 10.7% of S. aureus was detected in nasal rectal swabs and faeces, respectively. Calves had a higher probability of being S. aureus intestinal carriers than adults, especially in females when considering faecal samples. Clonal complex (CC) 425 was the most prevalent lineage (61.5%). This is a lineage known to be widespread in both domestic and free-living animals. It was followed by CC2671 (15.4%) and CC350 (6.4%). A high rate of the phage-borne virulence factor lukM/lukF-P83 was detected in CC425 and CC350. Further lineages, which are known to occur in both humans and animals, were detected sporadically in red deer faeces only, that is, CC7, CC9, CC121 and CC707, harbouring the genes of the penicillinase operon and a gene for macrolide resistance (CC9 and CC121). Methicillin resistance genes mecA and mecC were not found. Our results suggest that free-living red deer may be reservoir for S. aureus in Alpine habitats.

Co-occurrence of antibiotic resistance and virulence Genes in Methicillin Resistant Staphylococcus aureus (MRSA) Isolates from Pakistan.

Methicillin resistant Staphylococcus aureus (MRSA) is one of the major human pathogen that is associated with hospital as well as community acquired infections and is responsible for huge amount of life-threatening diseases. Objective of the study was to determine MRSA prevalence, their antibiotic sensitivity patterns, frequency of virulence genes (sea, seb, sed, tst, hla, hld) and their co-occurrence with resistance marker mecA among Rawalpindi and its nearby regions of Pakistani clinical isolates. The present study was carried out to identify the virulence and antibiotic resistance genes that co-occur in MRSA through polymerase chain reaction. Antibiotic sensitivity, presence of virulence genes and their co-occurrence with resistance marker mecA were analyzed. These isolates were found resistant to number of antibiotics i.e. Amoxicillin (16.1%), Cefixime (48.38%), Doxycycline (27.415), Trimethoprim/sulfamethoxazole (37.09%), Clindamycin (30.64%), Erythromycin (83.87%), Penicillin (100%), Vancomycin (4.83%), Ciprofloxacin (70.96%), Tetracycline (20%), Linezolid (3.22%) and Fusidic acid (11.295). The frequency of antibiotic resistant gene (mecA) was 69.35% and that of virulence genes hla, hld, sea, seb, sed and tst was 100, 100, 53.2, 30.6, 3.2 and 24.2% respectively. Amongst all examined genes, hla and hld genes had the highest and sed gene had the lowest frequency. The maximum coexistence of genes was observed for hla+hld+mecA gene combination (42 out of 62 isolates). This study reports the presence of multidrug resistant, vancomycin-resistant and mecA negative MRSA isolates in infected patients of Rawalpindi and nearby regions of Pakistan that may have attributed to treatment failures, adaptability of new virulence characteristics and spread of antibiotic resistance.

Frequently used therapeutic antimicrobials and their resistance patterns on Staphylococcus aureus and Escherichia coli in mastitis affected lactating cows

ABSTRACT Mastitis is one of the most frequent and costly production diseases of dairy cattle. It is frequently treated with broad-spectrum antimicrobials. The objectives of this work were to investigate the prevalence of Staphylococcus aureus and Escherichia coli, find out the antimicrobials used in mastitis treatment, and explore the antimicrobial resistance profile including detection of resistance genes. Bacterial species and antimicrobial resistance genes were confirmed by the polymerase-chain reaction. A total of 450 cows were screened, where 23 (5.11%) and 173 (38.44%) were affected with clinical and sub-clinical mastitis, respectively. The prevalence of S. aureus was 39.13% (n = 9) and 47.97%(n = 83) while, E. coli was 30.43% (n = 7) and 15.60% (n = 27) in clinical and sub-clinical mastitis affected cows, respectively. The highest antimicrobials used for mastitis treatment were ciprofloxacin (83.34%), amoxycillin (80%) and ceftriaxone (76.67%). More than, 70% of S. aureus showed resistance against ampicillin, oxacillin, and tetracycline and more than 60% of E. coli exhibited resistance against oxacillin and sulfamethoxazole-trimethoprim. Selected antimicrobial resistance genes (mecA, tetK, tetL, tetM, tetA, tetB, tetC, sul1, sul2 and sul3) were identified from S. aureus and E. coli. Surprisingly, 7 (7.61%) S. aureus carried the mecA gene and were confirmed as methicillin-resistant S. aureus (MRSA). The most prevalent resistance genes were tetK 18 (19.57%) and tetL 13 (14.13%) for S. aureus, whereas sul1 16 (47.06%), tetA 12 (35.29%), sul2 11 (32.35%) and tetB 7 (20.59%) were the most common resistance genes in E. coli. Indiscriminate use of antimicrobials and the presence of multidrug-resistant bacteria suggest a potential threat to public health.

In Vitro Antibacterial Experiments of Qixingjian Decoction and Its Synergistic Interaction with Oxacillin against Clinical Isolates of Methicillin-Resistant Staphylococcus aureus.

Background With the widespread use and abuse of antimicrobial drugs, the problem of bacterial resistance is becoming increasingly prominent. The clinical detection rate of drug-resistant bacteria is increasing year by year, so there is an urgent need to develop new antimicrobial drugs. Qixingjian Decoction (QXJT) is a formula commonly used in Chinese medicine for the treatment of sepsis caused by acute purulent infections of the face, hands, and feet. There are many compounds with antimicrobial effects that are available, but little is known about their mode of action. In this study, we mainly evaluated the antimicrobial activity of QXJT and explored its synergistic interaction with oxacillin (OX) and the mechanism of its antimicrobial activity. Methods The antimicrobial activity of QXJT against methicillin-resistant Staphylococcus aureus (MRSA) was determined by the microdilution method, the broth macrodilution method, and the time-kill curve method. The main compounds in QXJT were analyzed by ultra-performance liquid chromatography. The synergistic interaction of QXJT and oxacillin (OX) was determined by checkerboard assay, and the antimicrobial mechanism of QXJT, OX, and QXJT + OX was evaluated by transmission electron microscopy (TEM) technique. The expression of MRSA superantigen virulence factors (sea, seb, and tst), and drug resistance gene (mecA) was detected to provide a new strategy for new antibiotic drugs. Results QXJT exhibited antimicrobial activity against both clinical isolates of MRSA, MICs ranging from 18.75 to 37.5 mg/mL. Active substances such as Scutellarein, Scutellarin, Apigenin, and Wogonin 7-O-glucuronide were detected in the phytochemical analysis that may be associated with the antimicrobial activity of QXJT. The synergistic effect of QXJT and OX was determined by checkerboard assay (FICI = 0.5), and TEM images showed that QXJT could cause the disruption of MRSA cell wall, and QXJT + OX could produce greater disruption of MRSA cell wall, elucidating the synergistic effect of the two together on cell wall disruption by microscopic mechanisms. Our study shows that the combination of QXJT and OX can inhibit the expression of MRSA virulence factor, reduce the virulence of MRSA, and have no significant effect on the expression of MRSA resistance gene mecA. Conclusion The results of this study provide scientific experimental data for the traditional application of QXJT and initially explore the mechanism of action of QXJT combined with OX.