Bovine mastitis caused by multidrug resistant Staphylococcus aureus is a huge problem reported worldwide, resulting in prolonged antibiotic treatment and death of livestock. The current study is focused on surveillance of antibiotic susceptibility along with genotypic and phenotypic characterization of the pathogenic S. aureus strains causing mastitis in India. One hundred and sixty seven milk samples were collected from mastitis-affected cows from different farms in India resulting in thirty nine isolated S. aureus strains. Antibiotic sensitivity profiling revealed the majority of the strains (n = 24) to be multidrug resistant and eleven strains showed reduced susceptibility to vancomycin (MICs = 2μg/ml). All strains were oxacillin sensitive, but 19 strains were positive for the mecA gene, which revealed the occurrence of oxacillin susceptible mecA positive strains (OS-MRSA) for the first time from India. Additionally, 32 strains were positive for the pvl gene, a virulence determinant; of these 17 were also OS-MRSA strains. Molecular characterization based on multilocus sequence typing (MLST), spa typing, agr typing and SCCmec classification revealed strains belonging to different groups. Moreover, strains showed spa types (t2526, t9602) and MLST sequence types, ST-72, ST-88 and ST-239 which have been earlier reported in human infections. The prevalence of OS-MRSA strains indicates the importance of including both the genetic and phenotypic tests in characterizing S. aureus strains. Increased genotypic variability with strain related to human infections and pvl positive isolates indicates a worrisome situation with the possibility of bilateral transfer.

Methicillin resistant Staphylococcus aureus causing bovine mastitis has been very well investigated worldwide. However, there are only limited reports on the characterization of methicillin resistant and sensitive coagulase negative staphylococci (CoNS) across the globe. Hence, in the present study, we aim to determine the phenotypic traits based on antimicrobial susceptibility profile and genotypic characterization by verifying the presence of resistance determinants, virulence and toxin genes present in the CoNS causing clinical mastitis. We obtained 62 CoNS isolates from 167 mastitic milk samples collected from three different states of India. The 62 isolates comprises of 10 different CoNS species S. sciuri, S. haemolyticus, S. chromogenes, S. saprophyticus, S. xylosus, S. simulans, S. agnetis, S. epidermidis, S. gallinarum, and S. cohinii. Susceptibility screening against 11 antibiotics determined 45.16% isolates as multidrug resistant (resistant to more than two class of antibiotic), 46.74% resistant (one or two antibiotic class) and 8.06% isolates were pan-sensitive (sensitive to all drugs). High resistance was observed against oxacillin and cefoxitin, whereas all isolates were susceptible toward vancomycin and linezolid. Fifty three isolates were methicillin resistant and 9 isolates were sensitive as determined by oxacillin susceptibility assay. The methicillin resistance gene, mecA was found in 95.16% isolates and staphylococcal cassette chromosome mec (SCCmec) typing predominantly revealed Type III (n = 34) and Type V (n = 18). Interestingly, 11.9% of mecA positive isolates were oxacillin susceptible and referred as oxacillin susceptible mecA positive staphylococci (OS-MRS). Additionally, genes encoding for enterotoxin, (sea, seb, seh, see) toxic shock syndrome (tsst), exfoliatin (eta, etb, etd) and virulence (pvl, Y-hlg) were also screened. Of all the genes examined, 67.74% of isolate were positive for the Y-hlg gene, followed by the sea gene in 25.8% whereas in none of the isolates the eta and the etb gene was amplified. The study also highlights the incidence of clinical isolates of CoNS, which are harboring the toxin and the virulence genes rendering them as a more potential threat. This is the first report of animal origin OS-MRS from India, which emphasizes on the inclusion of both the genetic and phenotypic test for proper characterization of CoNS and preventing resistant strain misidentification.

The clinical success of CRISPR therapies is dependent on the safety and efficacy ofCas proteins. The Cas9 from Francisella novicida (FnCas9) has negligible affinity formismatched substrates enabling it to discriminate off-targets in DNA with very highprecision even at the level of binding. However, its cellular targeting efficiency is low,limiting its use in therapeutic applications. Here, we rationally engineer the protein todevelop engineered(enFnCas9) variants with enhanced activity and expand its cellularediting activity to genomic loci previously inaccessible. Notably, some of the variantsrelease the protospacer adjacent motif (PAM) constraint from NGG to NGR/NRGmaking them rank just below SpCas9-RY and SpCas9-NG in their accessibility acrosshuman genomic sites. The enFnCas9 proteins, similar to Cas12a and Cas12f, harborhigh intrinsic specificity and can diagnose single nucleotide variants accurately.Importantly, they provide superior outcomes in terms of editing efficiency, knock-inrates, and off-target specificity over other engineered high-fidelity versions of SpCas9(SpCas9-HF1 and eSpCas9). Broad targeting range coupled with remarkablespecificity of DNA interrogation underscores the utility of these variants for safe andefficient therapeutic gene correction across multiple cell lines and target loci.

The clinical success of CRISPR therapies is dependent on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) has negligible affinity for mismatched substrates enabling it to discriminate off-targets in DNA with very high precision even at the level of binding. However, its cellular targeting efficiency is low, limiting its use in therapeutic applications. Here, we rationally engineer the protein to develop enhanced (enFnCas9) variants and expand its cellular editing activity to genomic loci previously inaccessible. Notably, some of the variants release the protospacer adjacent motif (PAM) constraint from NGG to NGR/NRG making them rank just below SpCas9-RY and SpCas9-NG in their accessibility across human genomic sites. The enFnCas9 proteins, similar to Cas12a and Cas12f, harbor high intrinsic specificity and can diagnose single nucleotide variants accurately. Importantly, they provide superior outcomes in terms of editing efficiency, knock-in rates, and off-target specificity over other engineered high-fidelity versions of SpCas9 (SpCas9-HF1 and eSpCas9). Broad targeting range coupled with remarkable specificity of DNA interrogation underscores the utility of these variants for safe and efficient therapeutic gene correction across multiple cell lines and target loci.

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The authors incorrectly denoted ATCC 25923 as a methicillin resistant control strain in the penultimate sentence of the Antibiotic Susceptibility (Disc Diffusion, Micro-Broth Dilution) subsection of the Methods. The correct sentence is: ATCC 29213 (methicillin sensitive control strain) and ATCC 25923 (a strain used as a control in some antibiotic susceptibility testing, not methicillin resistant) were used as a control for micro broth dilution and disk diffusion assay, respectively. The authors would like to acknowledge the importance of using a genuinely positive MRSA control strain in phenotypic testing and interpreting results. There are errors in Table 1. The authors have amended the data to correct their dilution series and reflect the actual concentrations used. The authors apologize for the errors in the table. Please see the correct Table 1 here.