Molecular evidence of ß-lactam resistant Staphylococcus aureus in equids with respiratory tract infections: Frequency and resistance modulation strategy.

The emergence of antimicrobial-resistant strains in Staphylococcus aureus (ß-lactam and methicillin-resistant) is an overwhelming issue worldwide. Using the purposive sampling technique, 217 equids samples were collected from district Layyah which were subjected to culturing followed by genotypic identification of mecA and blaZ genes by PCR. This study revealed that by phenotypic methods, a prevalence of 44.24%, 56.25%, and 47.92% was found for S. aureus, MRSA, and ß-lactam resistant S. aureus in equids. While genotypically, MRSA was found in 29.63% and ß-lactam resistant S. aureus in 28.26% of equids. In-vitro antibiotic susceptibility testing against S. aureus isolates harboring both mecA and blaZ genes showed a high resistance against Gentamicin (75%), followed by Amoxicillin (66.67%) and Trimethoprim+sulfamethoxazole (58.34%). In an attempt to re-sensitize the resistant bacteria to antibiotics, a combination of antibiotics and non-steroidal anti-inflammatory drugs (NSAIDs) was used which revealed synergistic effect of Gentamicin and Trimethoprim+sulfamethoxazole with Phenylbutazone; and Amoxicillin with Flunixin meglumine. Analysis of risk factors revealed significant association with the S. aureus-associated respiratory infection in equids. Phylogenetic analysis of mecA and blaZ genes showed a high resemblance of study isolate's sequences with each other and variable resemblance with already reported isolates obtained from different samples of neighboring countries. This study reports the first molecular characterization and phylogenetic analysis of ß-lactam and methicillin resistant S. aureus in equids in Pakistan. Moreover, this study will help in the resistance modulation of resistant antibiotics (Gentamicin, Amoxicillin, Trimethoprim+sulfamethoxazole) and provide a good insight into planning an effective therapeutic regime.

Molecular characterization of vancomycin-resistant Staphylococcus aureus isolated from bovine milk.

Vancomycin-resistant Staphylococcus aureus (VRSA) is a zoonotic life-threatening pathogen. Vancomycin exhibits anti-bacterial activity by inhibiting peptidoglycan synthesis by binding to the D-ala-D-ala terminus of the peptidoglycan. But in VRSA, D-ala-D-ala is replaced by D-ala-D-lactate due to the presence of vanA, vanB or vanD genes. This study was intended to identify the molecular prevalence of VRSA in 768 bovine milk samples, risk factor association, antibiogram profile and bioinformatics analysis of VRSA by targeting vanB gene. Out of a total of 248 S. aureus isolates from mastitic milk samples, the phenotypic and genotypic prevalence of VRSA was estimated to be 17.74% and 10.89%, respectively. Farm-level risk factors including use of improper milking technique, lack of milker's care during milking, unhygienic conditions during milking and no dry cow therapy were found to be significantly associated (p < 0.05). Anti-microbial susceptibility testing of VRSA isolates exhibited the highest resistance to oxytetracycline, followed by oxacillin and Trimethoprim+sulfamethoxazole. The current study sequences showed more resemblance with reported sequences from Iraq (MN747834) and Egypt (MK095504, MK087830), which belong to vanB gene from S. aureus as compared to sequences from other countries, which belong to vanB gene from the genus Enterococcus. The Genetic Algorithm for Recombination Detection (GARD) found 234 potential breakpoints, translating into a search room of 123,883,305 models with up to 4 breakpoints. The phylogenetic motif profiling method discovered evolutionarily conserved residues across target genes' homologous protein sequences. These residues were discovered to be conserved in drug-resistant target proteins over the evolutionary process and may play a key role in their function. The current study revealed a molecular prevalence of VRSA in dairy animals, along with molecular analysis of vancomycin resistance in S. aureus by targeting the vanB gene using standard bioinformatics tools. The occurrence of VRSA in animals requires serious attention because this pathogen has zoonotic potential, so it can become a greater risk to consumer health.

Drug repurposing strategy: An emerging approach to identify potential therapeutics for treatment of bovine mastitis.

The current study was designed to characterize methicillin-resistant Staphylococcus aureus (MRSA) isolated from bovine milk, along with its response to antibiotics, and ultimately reverse its mechanism of resistance by modulation with non-antibiotics. The synergistic combination of antibiotics with NSAIDs were tested in-vivo by giving MRSA challenge to rabbits. The current study reported an overall 23.79% prevalence of MRSA. The BLAST alignment of current study sequences revealed 99% similarity with mecA gene of MRSA from NCBI database. The current study isolates were more similar to each other and also with reference sequences as compared to other mecA gene sequences from Turkey, India, and Russia. Antibiogram of MRSA isolates showed a highly resistant response to cefoxitin, amoxicillin, and gentamicin. Amoxicillin, gentamicin, tylosin, vancomycin, and ciprofloxacin elicited a significant response (p < 0.05) in combination with non-antibiotics against tested MRSA isolates. The highest zone of inhibition (ZOI) increase was noted for vancomycin in combination with flunixin meglumine (145.45%) and meloxicam (139.36%); gentamicin with flunixin meglumine (85.71%) and ciprofloxacin with ivermectin (71.13%). Synergistic behavior was observed in the combination of gentamicin with ketoprofen; sulfamethoxazole and oxytetracycline with meloxicam. Hematological analysis showed significant differences (p < 0.05) among lymphocyte count and bilirubin. On histopathological examination of skin tissue, hyperplasia of epithelium, sloughed off epidermis, hyperkeratosis, infiltration of inflammatory cells, and hemorrhages were observed. The highest cure rate was observed in case of gentamicin in combination with ketoprofen as compared to other treatment groups. The current study concluded antibiotics in combination with non-antibiotics as potential therapeutic agents for resistance modulation against MRSA. This study will help to devise treatment and control strategies against bovine mastitis. Although the prospect of using NSAIDs to manage infections caused by MRSA appears to be a promising direction, further studies should be conducted to test these medications using suitable in-vivo models in controlled clinical trials to justify their repurposing as a treatment for MRSA infections.

Pathological insights into camel mastitis.

Camel is a multipurpose animal bred to produce milk, meat, and transport and serves as a financial reserve for pastoralists by playing an important role in social prestige and prosperity. Camel milk is a good substitute for human milk because of its exceptional nutritional properties. Udder infections are considered one of the main limitations to camel farming. In recent decades, the disease has been reported by numerous camel-producing countries in Africa and Asia, such as Egypt, Somalia, Sudan, Kenya, Saudi Arabia, and Iraq. The current review provides an overview of the forms of camel mastitis, which can be clinical mastitis characterized by hardening and swelling of the breast, pain on palpation, and visible changes in the colour and texture of the milk or subclinical mastitis refers to the presence of inflammation with no obvious signs and it can be detected by indirect tests such as the California mastitis test (CMT), somatic cell count (SCC), and microbiological examination. Major pathogens of camel mastitis are Staphylococcus aureus, Streptococcus agalactiae, Escherichia coli, and Corynebacterium bovis. Regarding the risk factors for camel mastitis, this study provides an overview of the most important risk factors such as severe tick infestation, teat injuries, hygienic milking protocols, and physiological disorders causing mastitis. The use of indirect tests and bacteriological studies as diagnostic tools and their values for detecting camel mastitis will also be reviewed. Based on the above, further epidemiological studies on camel mastitis are needed to have solid scientific data on disease transmission, pathogen characterization, other possible risk factors or diagnostic methods, and the impact of the disease on public health. Proper control strategies should be adopted through early diagnosis, treatment and by avoiding potential risk factors to get good quality milk from camels.

MRSA compendium of epidemiology, transmission, pathophysiology, treatment, and prevention within one health framework.

Staphylococcus aureus is recognized as commensal as well as opportunistic pathogen of humans and animals. Methicillin resistant strain of S. aureus (MRSA) has emerged as a major pathogen in hospitals, community and veterinary settings that compromises the public health and livestock production. MRSA basically emerged from MSSA after acquiring SCCmec element through gene transfer containing mecA gene responsible for encoding PBP-2α. This protein renders the MRSA resistant to most of the ß-lactam antibiotics. Due to the continuous increasing prevalence and transmission of MRSA in hospitals, community and veterinary settings posing a major threat to public health. Furthermore, high pathogenicity of MRSA due to a number of virulence factors produced by S. aureus along with antibiotic resistance help to breach the immunity of host and responsible for causing severe infections in humans and animals. The clinical manifestations of MRSA consist of skin and soft tissues infection to bacteremia, septicemia, toxic shock, and scalded skin syndrome. Moreover, due to the increasing resistance of MRSA to number of antibiotics, there is need to approach alternatives ways to overcome economic as well as human losses. This review is going to discuss various aspects of MRSA starting from emergence, transmission, epidemiology, pathophysiology, disease patterns in hosts, novel treatment, and control strategies.

Bringing resistance modulation to epidemic methicillin resistant S. aureus of dairy through antibiotics coupled metallic oxide nanoparticles.

The current study probed methicillin resistant S. aureus from milk of different dairy farms along with its response to multiple antibiotics, assessment of risk factors, and response to antibiotic coupled nanoparticle. XRD of Np was confirmed as miller indices (hkl) values i.e. (101), (100), (002), (110), (012) and (013) while STEM finally revealed 40-60 nm nanorods in aggregated form. Total of 6 preparations viz a viz gentamicin (G), chloramphenicol (C), zinc oxide nanoparticle (Np), gentamicin coupled Np (GNp), chloramphenicol coupled Np (CNp), and simultaneously coupling of gentamicin and chloramphenicol on Np (GCNp) were formulated for their potential to bring resistance modulation. Data analysis of this study revealed 24.59% MRSA from dairy milk appearing potentially associated (OR> 1, p < 0.05) with most of assumed risk factors. MRSA in response to various antibiotics showed highest resistance against amoxicillin (100%), penicillin (100%), vancomycin (100%), and linezolid (90%). Zone of inhibitions were increased by 249.76% (GNp), 184.86% (CNp), and 279.76% (GCNp) in case of coupled preparations. Significant reduced minimum inhibitory concentration was observed in case of GCNp (7.8125 ± 0.00 µg/mL) followed by GNp (15.00 ± 0.00 µg/mL) and CNp (41.67 ± 18.042 µg/mL) as compared to Np alone (125.00 ± 0.00 µg/mL). Minimum bactericidal concentrations for GCNp, GNp, and CNp, and Np were 31.125, 62.5, 125, and 500 µg/mL, respectively. The study thus concluded increased prevalence of MRSA while coupling of ZnO nanoparticles with antibiotics significantly brought resistance modulation to MRSA.

Phylogeny of hospital acquired MRSA, and its comparative phenotypic clinico-epidemiology with vancomycin resistant S. aureus (VRSA).

Staphylococcus aureus is emerging as complicated pathogen because of its wide-ranging origin, multiple variants, and compromised antibiotic susceptibilities. Current study was planned to find lineage of hospital acquired methicillin resistant Staphylococcus aureus (HA-MRSA), and its comparative phenotypic clinico-epidemiology with vancomycin resistant S. aureus (VRSA). A total of (n = 200) samples were aseptically collected from wound, nose, and cerebrospinal fluid of patients from metropolitan and rural background hospitals along with on spot filling in of questionnaire. Phylogenetic analysis of HA-MRSA was identified by targeting mecA gene in S. aureus. At optimal tree branch length of 1.91 and evolutionary distance 0.1, high level sequence similarity (97%-99%) was observed with different strains of S. aureus isolated from both human and animal. Non-descriptive statistics at 5% probability found 61% S. aureus, while 43.44% of them were HA-MRSA, 92.62% VRSA, and 42.62% were both MRSA and VRSA. Among assumed risk factors, use of antibiotics, venous catheterization, chronic disease, pre-hospital visits, and ICU admitted patients showed significant association (p<0.05) with pathogen. HA-MRSA was 37.50%, 80%, and 37.50% sensitive to chloramphenicol, gentamicin, and oxacillin, respectively. While <50% of VRSA were sensitive against oxacillin, enoxacin, and chloramphenicol. A significant difference (p<0.05) of percentage responses of MRSA and VRSA at resistant, intermediate, and sensitive cadre against all antibiotics except chloramphenicol was obvious in this study. The Current study concluded higher prevalence of MRSA & VRSA, significant association of risk factors, limiting antibiotic susceptibility profile, and genetic transfer at animal-human interface which suggests further studies cum preventive strategies to be planned.

Antimicrobial resistance modulation of MDR E. coli by antibiotic coated ZnO nanoparticles.

We evaluated three types of total six preparations against multidrug resistant E. coli i) three antibiotic coated ZnO nanoparticles (gentamicin coated nanoparticle-GNp; chloramphenicol coated nanoparticles-CNp; and both gentamicin & chloramphenicol coated nanoparticle-GCNp), ii) ZnO nanoparticle alone-Np, and iii) two antibiotics used in single (Gentamicin-G; and Chloramphenicol-C). A total of n = 200 sub-clinically positive mastitic milk samples of bovine origin were processed for isolation of MDR E. coli using microbiological and clinical laboratory & standard institute's protocols. ZnO Nps were prepared from zinc acetate dihydrate (Zn (CH3COO)2. 2H2O), polyethylene glycol (C2nH4n+2On+1), and urea (CH4N2O) by standard chemical protocol. Nps were characterized by XRD and STEM analyses while coating of antibiotics on Nps was confirmed by UV-Visible spectrophotometric analysis. Analysis of variance and student t-test were applied at 5% probability using SPSS version 22 statistical software for inferences on obtained data. There was significantly (p < 0.05) lowest minimum inhibitory concentrations (MICs) and highest zone of inhibitions (ZOIs) in case of GCNp (10.42 ± 4.51 µg/mL & 22.00 ± 1.00 mm) followed by GNp (20.79 ± 8.95 µg/mL & 20.00 ± 1.00 mm) and then CNp (25.96 ± 8.95 µg/mL & 12.33 ± 0.57 mm). Percentage increase in ZOI were expressed as 135.8, 78.43, and 312.76% by GCNp when compared with that of G, C, and Np, respectively. GNp and CNp coated preparations exhibited 114.36 and 275.73% increase in ZOI than to that of G and C, respectively. Similar trend was found in percentage reduction of MICs of preparations. Highest filamentation, indicator of bacterial damage, of E. coli was noted at MIC of GCNp followed by GNp and CNp. The study concluded antibiotic coated ZnO nanoparticles significant candidates modulating antibiotic resistance in MDR E. coli.