Clinical Isolates Of Methicillin-resistant S. Aureus Research Articles

Unmasking a looming crisis: Escalating MIC of last resort drugs against MRSA isolates from a tertiary care hospital in Central India

PurposeThe Centers for Disease Control and Prevention has classified methicillin-resistant S aureus (MRSA) as a serious public health threat. The escalating minimum inhibitory concentration (MIC) of standard anti-methicillin-resistant S aureus (MRSA) drugs within the susceptible range, known as "MIC creep," jeopardizes their effectiveness against MRSA infections, posing additional challenges in managing MRSA infections. This cross-sectional study was conducted in a tertiary care hospital in Central India to assess the susceptibility trends of clinical MRSA isolates against commonly used anti-MRSA drugs and to observe MIC creep, if any, over three years (2020–2022). MethodsThe study included 158 non-repetitive clinical MRSA isolates. The MICs of vancomycin, teicoplanin, and linezolid were determined in MRSA strains using agar dilution, while the MIC of daptomycin was performed by broth microdilution. MIC creep was assessed by calculating MIC50, MIC90, Modal MIC, G-mean MIC, and susceptible and resistant percentages for the fiscal years 2020, 2021, and 2022. ResultsOf the 158 MRSA isolates, none were resistant to vancomycin, teicoplanin, and daptomycin, but two showed resistance to linezolid (LRSA). However, fifteen isolates showed intermediate resistance to vancomycin (VISA), and five showed intermediate resistance to teicoplanin (TISA). MIC of these anti-MRSA drugs increased in 2021 and 2022 compared to 2020. G-mean MIC for vancomycin, teicoplanin, and linezolid in MRSA strains increased significantly over the study period, while daptomycin MIC remained relatively stable, with a slight increase in 2021 and 2022. There was a high resistance rate for clindamycin, doxycycline, and chloramphenicol among VISA, TISA, and LRSA isolates compared to MRSA. ConclusionsDuring the three years of the study, “MIC creep” was observed in vancomycin, teicoplanin, and linezolid and, to some extent, for daptomycin in MRSA strains. The recovery of VISA, TISA, and linezolid-resistant MRSAs is worrisome, suggesting possible MRSA treatment failure and being a forerunner of resistant strains.

Machine learning assisted identification of antibiotic-resistant Staphylococcus aureus strains using a paper-based ratiometric sensor array

Staphylococcus aureus, a versatile human pathogen, significantly impacts global health causing a broad spectrum of medical conditions that range from minor skin infections to life-threatening diseases. The clinical importance of S. aureus is underscored by its resistance to multiple antibiotics and formation of biofilms, providing protection against antimicrobials and immune responses. To date, the identification of antimicrobial-resistant (AMR) S. aureus strains, such as methicillin-resistant S. aureus (MRSA) and vancomycin-intermediate S. aureus (VISA), requires time-consuming and expensive methodologies, including culture-based, molecular, and phenotypic techniques. Previously, we developed a paper-based ratiometric sensor array composed of fluorescent sensor dyes (3-hydroxyflavone derivatives) pre-adsorbed on paper microzone plates. Combined with machine learning algorithms such as neural networks, this sensor effectively discriminated 16 bacterial species and determined their Gram status. In this study, we evaluate its ability to distinguish antibiotic-resistant S. aureus strains and their biofilms. Our results demonstrate that the sensor array, in conjunction with LDA and neural networks, successfully differentiated three common laboratory MRSA strains from three methicillin-susceptible S. aureus (MSSA) strains with 82.5% accuracy. Furthermore, using support vector machines, this sensor was able to distinguish and categorically classify MRSA, MSSA, and VISA clinical isolates with 97.5% accuracy. Remarkably, beyond distinguishing planktonic cultures, this sensor array demonstrated a formidable capability to discriminate AMR S. aureus biofilms, achieving over 80% accuracy. Combined, the results of this study highlight the paper-based sensor array’s significant potential as a robust diagnostic tool to accurately, rapidly, and easily identify drug-resistant S. aureus strains in clinically relevant settings.

Antibacterial and Antibiofilm Potential of Bacterial Cellulose Hydrogel Containing Vancomycin against Multidrug-Resistant Staphylococcus aureus and Staphylococcus epidermidis.

The present study aimed to evaluate the in vitro antibacterial and antibiofilm activity of bacterial cellulose hydrogel produced by Zoogloea sp. (HYDROGEL) containing vancomycin (VAN) against bacterial strains that cause wound infections, such as multidrug-resistant (MDR) Staphylococcus aureus and Staphylococcus epidermidis. Initially, HYDROGEL was obtained from sugar cane molasses, and scanning electron microscopy (SEM) was performed to determine morphological characteristics. Then, VAN was incorporated into HYDROGEL (VAN-HYDROGEL). The antibacterial activity of VAN, HYDROGEL, and VAN-HYDROGEL was assessed using the broth microdilution method to determine the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) against methicillin-sensitive S. aureus (MSSA) ATCC 25923, methicillin-resistant S. aureus (MRSA) ATCC 33591, S. epidermidis INCQS 00016 (ATCC 12228), five clinical isolates of MRSA, and nine clinical isolates of methicillin-resistant S. epidermidis, following the Clinical and Laboratory Standards Institute (CLSI) guidelines. Additionally, the antibacterial activity of VAN, HYDROGEL, and VAN-HYDROGEL was studied using the time-kill assay. Subsequently, the antibiofilm activity of VAN, HYDROGEL, and VAN-HYDROGEL was evaluated using crystal violet and Congo red methods, as well as SEM analysis. VAN and VAN-HYDROGEL showed bacteriostatic and bactericidal activity against MRSA and methicillin-resistant S. epidermidis strains. HYDROGEL did not show any antibacterial activity. Analysis of the time-kill assay indicated that HYDROGEL maintained the antibacterial efficacy of VAN, highlighting its efficiency as a promising carrier. Regarding antibiofilm activity, VAN and HYDROGEL inhibited biofilm formation but did not demonstrate biofilm eradication activity against methicillin-resistant S. aureus and S. epidermidis strains. However, it was observed that the biofilm eradication potential of VAN was enhanced after incorporation into HYDROGEL, a result also proven through images obtained by SEM. From the methods carried out in this study, it was possible to observe that HYDROGEL preserved the antibacterial activity of vancomycin, aside from exhibiting antibiofilm activity and enhancing the antibiofilm effect of VAN. In conclusion, this study demonstrated the potential of HYDROGEL as a candidate and/or vehicle for antibiotics against MDR bacteria that cause wound infections.

Effect of Bioactive Peptide, KR-12-a5, on Growth of Clinical Methicillin-resistant Staphylococcus aureus Isolates

Staphylococcus aureus is one of the most frequent infectious agents causing hospitals and community associated infections. Developing the treatment for methicillin resistant S. aureus (MRSA) is a challenging arena for effective therapeutics. Application of antimicrobial peptides is an alternative option to control MRSA infection. The potential of the bioactive peptide against MRSA was previously revealed; however, the study on the effect of KR-12-a5 on the various clinical MRSA growth was not elucidated. This study aimed to investigate the effect of bioactive peptide KR-12-a5 on the growth of MRSA isolated from patients. Evaluation of KR-12-a5 peptide using antimicrobial susceptibility test was performed. Antimicrobial activity assay showed that the KR-12-a5 at 2-64 μM could inhibit the growth of S. aureus ATCC 29213 in the range of 55.0±0.8%-81.0±6.2%. In case of KR-12-a5 treatment at 2, 4, and 8 μM, growth of all twenty clinical MRSA isolates could be inhibited less than 90% in the range of 0.0-75.9±1.6%, 0.0-73.0±4.2%, and 0.0-77.1±1.7%, respectively. Whereas the treatment of those at 16, 32, and 64 μM, growth of clinical MRSA could be significantly inhibited more than 90% (p < 0.05) in the range of 94.3±6.7%-100.4±0.4% in 3 isolates (15%), 97.3±0.3%-99.5±0.2% in 4 isolates (20%), and 91.6±0.8%-99.0±0.1% in 9 isolates (45%), respectively. These findings indicated that the KR-12-a5 effectively controls growth of both S. aureus ATCC 29213 and clinical MRSA isolates

Antibacterial Efficacy of Linezolid Alone and in Combination with Zinc Oxide Nanoparticles against Methicillin-Resistant S. Aureus Clinical Isolates

Skin and soft tissue infections caused by methicillin-resistant S. aureus (MRSA) are among the most common bacterial infections. Linezolid is a cortisone drug for the treatment of infections caused by MRSA. However, developing resistance to linezolid creates a hurdle in the treatment of these infections. The present study aimed to determine the activity of linezolid alone and in combination with zinc oxide nanoparticles (ZnO-NPs) for the purpose of reducing resistance and enhancing its efficacy. For this study, MRSA isolates were taken and confirmed by using the antibiotic susceptibility testing method. The minimum inhibitory concentration (MIC) of both antibiotics and nanoparticles against MRSA clinical isolates was done by using the broth microdilution method. A checkerboard assay has used the determination of the combined activity of linezolid and ZnO-NPs. ZnO-NPs displayed a spherical shape with smooth surface morphology and had a mean size of 10 nm to 20 nm, with a zeta potential of 3.57 mV. The activity of ZnO-NPs against MRSA clinical isolates was 200 µg/ml. Almost 81% of isolates were found sensitive to linezolid with MIC lower than 4 µg/ml, and 19% were resistant, having MIC greater than 4 µg/ml. The combination of an antibiotic and nanoparticles reduced the activity of each of them twofold. The current study revealed that both linezolid and ZnO-NPs have antimicrobial activity against MRSA when used alone. The combination of both medications reduces each other's MIC twofold and has an antagonistic impact. Further research is needed to determine the mechanism through which these medications inhibit each other's activity.

Combination with a FtsZ inhibitor potentiates the in vivo efficacy of oxacillin against methicillin-resistant Staphylococcus aureus.

Oxacillin is a first-line antibiotic for the treatment of methicillin-sensitive Staphylococcus aureus (MSSA) infections but is ineffective against methicillin-resistant S. aureus (MRSA) due to resistance. Here we present results showing that co-administering oxacillin with the FtsZ-targeting prodrug TXA709 renders oxacillin efficacious against MRSA. The combination of oxacillin and the active product of TXA709 (TXA707) is associated with synergistic bactericidal activity against clinical isolates of MRSA that are resistant to current standard-of-care antibiotics. We show that MRSA cells treated with oxacillin in combination with TXA707 exhibit morphological characteristics and PBP2 mislocalization behavior similar to that exhibited by MSSA cells treated with oxacillin alone. Co-administration with TXA709 renders oxacillin efficacious in mouse models of both systemic and tissue infection with MRSA, with this efficacy being observed at human-equivalent doses of oxacillin well below that recommended for daily adult use. Pharmacokinetic evaluations in mice reveal that co-administration with TXA709 also increases total exposure to oxacillin. Viewed as a whole, our results highlight the clinical potential of repurposing oxacillin to treat MRSA infections through combination with a FtsZ inhibitor.

A fusidic acid-resistant (PVL+) clone is associated with the increase in methicillin-resistant Staphylococcus aureus in New Caledonia

ObjectivesSince 2014, Staphylococcus aureus methicillin resistance has been rapidly increasing in New Caledonia and is associated with potential serious clinical repercussions. In the present study, we investigated the epidemiology of methicillin-resistant S. aureus (MRSA) in New Caledonia and the possible emergence of a particular clonal strain. MethodsAn overview of the distribution of MRSA in New Caledonia in 2019 is presented. We collected and analysed 171 clinical MRSA isolates from New Caledonia medical laboratories during August and September 2019. Among this collection, 49 representative isolates were analyzed by the French National Reference Center for Staphylococci using the StaphyType DNA microarray, allowing genetic characterization of the isolates. ResultsAmong the 1144 S. aureus isolated over the year 2019, 442 isolates (39%) were resistant to methicillin, and 62% of these isolates were resistant to fusidic acid (FA). During the inclusion period, FA resistance rate was similar (60%). Genetic characterization evidenced CC6 as the predominant clonal complex (70%) with 26 isolates (53%) identified as CC6-MRSA-[IV+fus] (PVL+). ConclusionsThese findings demonstrated a low diversity of MRSA in New Caledonia, with the dominance of a clonal complex not reported previously. The frequent fusidic acid (FA) resistance in MRSA was associated with a high prevalence of fusC gene, suggesting that FA misuse contributed to driving the selection of this clone. Our findings suggest the recommendation to stop the topical use of FA to control the emergence of this severe MRSA clone and decrease the rate of MRSA in New Caledonia.

Comparison of peaks in the matrix-assisted laser desorption ionization time-of-flight mass spectrometry spectra of Staphylococcus aureus grown on various blood agar plates

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is routinely used for bacterial identification in clinical laboratories. Bacterial protein expression may differ according to their growth conditions, especially the culture medium composition. We aimed to study the peak variations of Staphylococcus aureus grown on various blood agar plates (BAP), especially phenol-soluble modulin-mec (PSM-mec) peak (m/z 2409) associated with mecA gene conferring methicillin resistance. Methicillin-resistant S. aureus (MRSA) ATCC 43300 and eight clinical MRSA isolates were cultured on various commercial BAPs including tryptic soy agar-based BAPs, Columbia agar-based BAP and in-house BAPs with the addition of yeast extract. Analysis of the MALDI-TOF peaks of S. aureus, cultured on various BAPs, revealed the peak intensities of low-molecular weight proteins to vary depending on the composition of BAPs, especially the presence or absence of yeast extract. Especially, the PSM-mec and delta-toxin peaks showed low intensity for S. aureus ATCC 43300 and clinical isolates. No significant differences were found in the number of peaks, but some peaks had lower intensity, corresponding to the medium containing yeast extract, in low-mass region (<m/z 4000). BAPs based on tryptic soy agar rather than Columbia agar seems to be appropriate for the detection of PSM-mec, a methicillin resistance marker of S. aureus and delta-toxin, an agr function indicator.

Detection of capsular genotypes of methicillin-resistant Staphylococcus aureus and clonal distribution of the cap5 and cap8 genes in clinical isolates.

Up until now, the capsular polysaccharides of Staphylococcus aureus have been classified into 11 types, of which only 2 types 5 and 8; (encoded by the genes cap5 and cap8, respectively) are present in 80–90% of clinically significant strains. The aim of the present study was to detect the capsular genotypes of methicillin-resistant S. aureus (MRSA) clinical isolates and determined their clonal distribution. A total of 262 MRSA clinical isolates from different hospitals in Mexico were analyzed by PCR to determine the genetic characteristics of their capsule expression. Pulsed-field gel electrophoresis and multilocus sequence typing were used to characterize the isolates. The analysis of the capsular genotypes among MRSA isolates showed that 245 isolates (93.5%) contained the cap5 gene, and that the remaining 17 (6.5%) encoded the cap8 gene. The MRSA isolates were grouped into four clonal groups. The identification of the capsular genotypes of clinical isolates of MRSA is important information because potential vaccine formulations against S. aureus involve capsular polysaccharides.

Functional Characterization of Type III-A CRISPR-Cas in a Clinical Human Methicillin-R Staphylococcus aureus Strain.

CRISPR with its cas genes is an adaptive immune system that protects prokaryotes against foreign genetic elements. The type III-A CRISPR-Cas system is rarely found in Staphylococcus aureus, and little is known about its function in S. aureus. Here, we describe the genome characteristics of the clinical methicillin-resistant S. aureus (MRSA) strain TZ0912, carrying a type III-A CRISPR-Cas system. Phylogenetic analysis of 35 reported CRISPR-Cas-positive S. aureus strains revealed that the CRISPR-Cas system is prevalent in CC8 clones (10/35) and is located in the staphylococcal cassette chromosome mec (SCCmec) V, which confers methicillin resistance. Plasmid transformation and phage infection assays reveal that the type III-A CRISPR-Cas system protects TZ0912 against foreign DNA with sequence homology to the spacers located in the CRISPR array. We observed that the CRISPR-Cas immune system could effectively protect MRSA against phage attacks in both liquid culture and solid medium. In accordance with previous reports, using RNA-seq analysis and plasmid transformation assays, we find that the crRNAs close to the leading sequence of the CRISPR array are more highly expressed and are more effective at directing plasmid elimination compared to the distant spacers. This study established a model for evaluating the efficiency of naive CRISPR-Cas system in MRSA against phage, which could contribute to future research on the function of CRISPR-Cas in clinical MRSA isolates and improve phage therapy against MRSA infections.

Corynebacterium accolens Has Antimicrobial Activity against Staphylococcus aureus and Methicillin-Resistant S. aureus Pathogens Isolated from the Sinonasal Niche of Chronic Rhinosinusitis Patients.

Corynebacterium accolens is the predominant species of the healthy human nasal microbiota, and its relative abundance is decreased in the context of chronic rhinosinusitis (CRS). This study aimed to evaluate the antimicrobial potential of C. accolens isolated from a healthy human nasal cavity against planktonic and biofilm growth of Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA) clinical isolates (CIs) from CRS patients. Nasal swabs from twenty non-CRS control subjects were screened for the presence of C. accolens using microbiological and molecular techniques. C. accolens CIs and their culture supernatants were tested for their antimicrobial activity against eight S. aureus and eight MRSA 4CIs and S. aureus ATCC25923. The anti-biofilm potential of C. accolens cell-free culture supernatants (CFCSs) on S. aureus biofilms was also assessed. Of the 20 nasal swabs, 10 C. accolens CIs were identified and confirmed with rpoB gene sequencing. All isolates showed variable antimicrobial activity against eight out of 8 S. aureus and seven out of eight MRSA CIs. Culture supernatants from all C. accolens CIs exhibited a significant dose-dependent antibacterial activity (p < 0.05) against five out of five representative S. aureus and MRSA CIs. This inhibition was abolished after proteinase K treatment. C. accolens supernatants induced a significant reduction in metabolic activity and biofilm biomass of S. aureus and MRSA CIs compared to untreated growth control (p < 0.05). C. accolens exhibited antimicrobial activity against S. aureus and MRSA CIs in both planktonic and biofilm forms and holds promise for the development of innovative probiotic therapies to promote sinus health.

Genome-wide comparison of four MRSA clinical isolates from Germany and Hungary.

Staphylococcus aureus is a drug-resistant pathogen, capable of colonizing diverse ecological niches and causing a broad spectrum of infections related to a community and healthcare. In this study, we choose four methicillin-resistant S. aureus (MRSA) clinical isolates from Germany and Hungary based on our previous polyphasic characterization finding. We assumed that the selected strains have a different genetic background in terms of the presence of resistance and virulence genes, prophages, plasmids, and secondary metabolite biosynthesis genes that may play a crucial role in niche adaptation and pathogenesis. To clarify these assumptions, we performed a comparative genome analysis of these strains and observed many differences in their genomic compositions. The Hungarian isolates (SA H27 and SA H32) with ST22-SCCmec type IVa have fewer genes for multiple-drug resistance, virulence, and prophages reported in Germany isolates. Germany isolate, SA G6 acquires aminoglycoside (ant(6)-Ia and aph(3’)-III) and nucleoside (sat-4) resistance genes via phage transduction and may determine its pathogenic potential. The comparative genome study allowed the segregation of isolates of geographical origin and differentiation of the clinical isolates from the commensal isolates. This study suggested that Germany and Hungarian isolates are genetically diverse and showing variation among them due to the gain or loss of mobile genetic elements (MGEs). An interesting finding is the addition of SA G6 genome responsible for the drastic decline of the core/pan-genome ratio curve and causing the pan-genome to open wider. Functional characterizations revealed that S. aureus isolates survival are maintained by the amino acids catabolism and favor adaptation to growing in a protein-rich medium. The dispersible and singleton genes content of S. aureus genomes allows us to understand the genetic variation among the CC5 and CC22 groups. The strains with the same genetic background were clustered together, which suggests that these strains are highly alike; however, comparative genome analysis exposed that the acquisition of phage elements, and plasmids through the events of MGEs transfer contribute to differences in their phenotypic characters. This comparative genome analysis would improve the knowledge about the pathogenic S. aureus strain’s characterization, and responsible for clinically important phenotypic differences among the S. aureus strains.

Catechin isolated from cashew nut shell exhibits antibacterial activity against clinical isolates of MRSA through ROS-mediated oxidative stress.

Staphylococcus aureus causes severe infections and among all methicillin-resistant S. aureus (MRSA) remains a great challenge in spite of decade research of antibacterial compounds. Even though some synthetic antibiotics have been developed, they are not effective against MRSA, and hence, there is a search for natural, alternative and plant-based antibacterial compound. In this connection, catechin isolated from cashew nut shell was investigated for its antibacterial potential against MRSA. Catechin exhibited zone of inhibition (ZOI) and minimum inhibitory concentration (MIC) in a range of 15.1-19.5mm and 78.1-156.2μg/ml, respectively, against ATCC and clinical isolates of MRSA. Among all clinical isolates, clinical isolate-3 exhibited highest sensitivity to catechin. Catechin has arrested the growth of MRSA strains and also caused toxicity by membrane disruption which was illustrated by AO/EB fluorescence staining. Increased nucleic acid leakage (1.58-28.6-fold) and protein leakage (1.40-23.50-fold) was noticed in MRSA due to catechin treatment when compared to methicillin. Bacteria treated with catechin at its MIC showed 1.52-, 1.87- and 1.74-fold increase of ROS production in methicillin susceptible S. aureus (MSSA), MRSA and clinical isolate-3 strains, respectively, as compared to control. Superoxide dismutase (5.31-9.63U/mg protein) and catalase (1573-3930U/mg protein) were significantly decreased as compared to control in catechin-treated S. aureus. Thus, catechin exhibited antibacterial activity through oxidative stress by increased production of ROS and decreased antioxidant enzymes. Altogether results suggest that catechin is a promising lead compound with antibacterial potential against MRSA. KEY POINTS: • Catechin was isolated and identified as active compound in cashew nut shell. • Catechin exhibited antimicrobial activity against clinical isolates of MRSA. • Bacterial cell wall damage was caused by catechin in MRSA strains. • Catechin increased the oxidative stress in MRSA by intracellular ROS production.

Evolving MRSA: High-level β-lactam resistance in Staphylococcus aureusis associated with RNA Polymerase alterations and fine tuning of gene expression.

Most clinical MRSA (methicillin-resistant S. aureus) isolates exhibit low-level β-lactam resistance (oxacillin MIC 2-4 μg/ml) due to the acquisition of a novel penicillin binding protein (PBP2A), encoded by mecA. However, strains can evolve high-level resistance (oxacillin MIC ≥256 μg/ml) by an unknown mechanism. Here we have developed a robust system to explore the basis of the evolution of high-level resistance by inserting mecA into the chromosome of the methicillin-sensitive S. aureus SH1000. Low-level mecA-dependent oxacillin resistance was associated with increased expression of anaerobic respiratory and fermentative genes. High-level resistant derivatives had acquired mutations in either rpoB (RNA polymerase subunit β) or rpoC (RNA polymerase subunit β') and these mutations were shown to be responsible for the observed resistance phenotype. Analysis of rpoB and rpoC mutants revealed decreased growth rates in the absence of antibiotic, and alterations to, transcription elongation. The rpoB and rpoC mutations resulted in decreased expression to parental levels, of anaerobic respiratory and fermentative genes and specific upregulation of 11 genes including mecA. There was however no direct correlation between resistance and the amount of PBP2A. A mutational analysis of the differentially expressed genes revealed that a member of the S. aureus Type VII secretion system is required for high level resistance. Interestingly, the genomes of two of the high level resistant evolved strains also contained missense mutations in this same locus. Finally, the set of genetically matched strains revealed that high level antibiotic resistance does not incur a significant fitness cost during pathogenesis. Our analysis demonstrates the complex interplay between antibiotic resistance mechanisms and core cell physiology, providing new insight into how such important resistance properties evolve.

First report of mecC gene in clinical methicillin resistant S. aureus (MRSA) from tertiary care hospital Islamabad, Pakistan

BackgroundStaphylococcus aureus (S. aureus) is one of the leading causes of community and hospital acquired infections globally. The objective of the study was to assess the prevalence, study the carriage of antibiotic resistance genes and evaluate the molecular typing of S. aureus isolates from a tertiary care hospital in Islamabad. MethodsA total of 1528 staphylococci isolates were included in this study. Standard microbiological procedures were applied to identify S. aureus. Antimicrobial susceptability was evaluated using the disk diffusion method and Minimum Inhibitory Concentrations (MICs) were determined using microbroth dilution method following Clinical and Laboratory Standards Institute (CLSI) guidelines. Multiplex PCR was used to detect antibiotic resistance genes, and molecular typing was performed using agr, SCCmec, spa, and Multi-Locus Sequence Typing (MLST) and clonal relatedness by Pulse Field Gel Electrophoresis (PFGE) methods. ResultsOverall 65% were MRSA and 35% were methicillin sensitive Staphylococcus aureus (MSSA). Among MRSA isolates, 83% were multi-drug resistant and mecA was found in 54% isolates, mecC was in 3% while 1 MRSA carried both mecA and mecC genes. agrI (22%) was most prevalent group in MRSA, while agrIII (16%) was observed in MSSA. SCCmec types I, II, III, IV, and VI were detected, with high prevalence of type III while type V was absent. The prevelant spa type in MRSA was t657 with SCCmecIII elelments while in MSSA it was t021. One NEW spa type identified in MSSA isolates. In a subset of isolates, ST772 with SCCmecIV, ST1 carrying PVL marker, and ST1535 was reported first time from Pakistan. ConclusionsThe study presents a comprehensive analysis of prevalent S. aureus types and their antibiotic resistance profiles. It also reports for the first time SCCmec type VI and clinical MRSA isolates with mecC alone and in combination with mecA from Islamabad, Pakistan. This calls for further detailed investigations in other hospital settings in the region.

Identification of a novel phage targeting methicillin-resistant Staphylococcus aureus In vitro and In vivo

IntroductionStaphylococcus aureus is a common human pathogen that causes various diseases including infections on the skin, in the bloodstream and the lower respiratory tracts. The emergence of methicillin-resistant S. aureus (MRSA) made the treatment of the bacterial infection more difficult, calling for development of new therapeutics. Compared with conventional antibiotic therapy, phage therapy offers a promising alternative to combat infections caused by MRSA. ResultsHere we showed that phage VB_SauS_SH-St 15644 isolated from sewage inhibited MRSA isolates in vitro and in the murine skin infection model. Phage VB_SauS_SH-St 15644 belongs to Siphoviridae. The genome of the phage is a linear, 45,111 bp double-stranded DNA with GC content of 33.35%. Among the 37 clinical MRSA isolates tested, 12 (32%) were lysed by the phage in vitro. The phage was relatively stable at temperatures up to 40 °C or between pH 6 and 9. However, the phage was sensitive to UV light. 80% of the phage was approximately adsorbed to the host MRSA isolate in 4 min. The one-step growth curve showed that the latent period was about 12 min followed by the growth period (about 9 min). The burst size was estimated at 13 PFU per infected cell. Furthermore, in a murine skin infection model, the phage significantly inhibited MRSA infection. ConclusionsOur study suggested that phage VB_SauS_SH-St 15644 has a potential to inhibit MRSA skin infection.

In vitro Evaluation of the Antimicrobial Activity of Leaf Extracts of Litsea iteodaphne Against a Selected Group of Bacteria Including Methicillin-Resistant Staphylococcus aureus.

Background:The quest for scientific endorsement of new drugs from plants continues due to the rising antibiotic resistance against pathogenic bacteria. Litsea iteodaphne is used in Sri Lanka in the treatment of infectious diseases. Therefore, in vitro antibacterial activity of L. iteodaphne plant extracts were evaluated against selected human pathogenic bacteria.Materials and Methods:Antibacterial activity of 400, 40, and 4 mg/ml concentrations of hexane, ethanol, and aqueous leaf extracts of L. iteodaphne were evaluated against Staphylococcus aureus, Psedomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, and methicillin-resistant S. aureus (MRSA) clinical isolates using disc diffusion method. Minimum inhibitory concentration (MIC) was identified, and phytochemical screening was carried out.Results:Significant zones of inhibition ranging from 5.7 mm to 8.1 mm, 7.1 mm to 8.0 mm, and 7.6 mm were obtained for ethanol, hexane, and aqueous extracts at 400 mg/ml, respectively, against above four bacteria. For MRSA clinical isolates, zones of inhibition ranging from 6.1 mm to 10.9 mm, 6.7 mm to 10.8 mm, and 6.4 mm to 8.6 mm were obtained for ethanol, hexane, and aqueous extracts at 400 mg/ml, respectively. Ethanol extract of L. iteodaphne showed the lowest MIC value (0.0256 mg/ml). Phytochemical screening revealed the presence of tannins, cardiac glycosides, reducing sugars, phenolic compounds, saponins, and flavonoids.Conclusions:L. iteodaphne crude leaf extracts showed promising antibacterial activity against Gram-positive and Gram-negative bacteria and clinical isolates of MRSA. Further investigations toward fractionation and the identification of an active compound will enhance the antimicrobial potential of L. iteodaphne.

Propionate Ameliorates Staphylococcus aureus Skin Infection by Attenuating Bacterial Growth.

Staphylococcus aureus causes various diseases including skin and soft tissue infections, pneumonia, gastroenteritis, and sepsis. Antibiotic-resistant S. aureus such as methicillin-resistant S. aureus (MRSA) and multidrug-resistant S. aureus is a serious threat in healthcare-associated settings and in the communities. In this study, we investigated the effects of short-chain fatty acids, metabolites produced by commensal bacteria, on the growth of S. aureus both in vitro and in vivo. Sodium propionate (NaP) most potently inhibited the growth of MRSA and multidrug-resistant clinical isolates. Of note, only NaP, but not sodium acetate (NaA) or sodium butyrate (NaB), ameliorated MRSA skin infection, significantly lowering bacterial load, excessive cytokine production, and the size and weight of abscesses approximately by twofold. In addition, interestingly, S. aureus deficient of lipoteichoic acids (LTA) or wall teichoic acids (WTA), which are important in bacterial physiology and antimicrobial susceptibility, was more susceptible to NaP than the wild-type. Furthermore, S. aureus deficient of D-alanine motifs common in LTA and WTA was more susceptible to NaP, its growth being almost completely inhibited. Concordantly, MRSA treated with an inhibitor of D-alanylation on LTA and WTA was more susceptible to NaP, and co-treatment of NaP and a D-alanylation inhibitor further decreased the pathology of MRSA skin infection. Collectively, these results demonstrate that NaP ameliorates MRSA skin infection by attenuating the growth of S. aureus, and suggest an alternative combination treatment strategy against S. aureus infection.

Detection of the Panton-Valentine Leukocidin Gene in Swedish Isolates of Methicillin-Resistant Staphylococcus aureus using a Multiplex PCR Assay

Background: Panton-Valentine leukocidin (PVL) is a bicomponent leukotoxin produced by <2% of Staphylococcus aureus (S. aureus) strains. PVL forms transmembrane pores that cause cell disruption and death. PVL is associated with skin necrotic lesions and severe necrotizing pneumonia. The methicillin-resistant S. aureus (MRSA) strains that harbor the lukS-PV gene (the gene encoding PVL toxin) are considered highly pathogenic since they can cause infections that are difficult to treat. Objective: This study aimed to optimize a multiplex PCR assay that can detect both methicillin-resistant S. aureus strains and their ability to produce PVL toxin isolated from Swedish patients. Methods: An optimized multiplex PCR-assay was developed to detect both nuc and lukS-PV genes and was applied to a collection of 80 MRSA clinical isolates collected between 1999 and 2004. Results: The results showed that 30/80 (40%) of all MRSA isolates were PVL-positive. The highest PVL prevalence (86%) was among abscess isolates. When PVL was correlated to staphylococcal protein A (spa) typing, the highest PVL positivity was among spa-type 44. Conclusion: The results of this study showed that the lukS-PV gene is common among Swedish community of the MRSA isolates.

Cephalosporin-Glycopeptide Combinations for Use against Clinical Methicillin-Resistant Staphylococcus aureus Isolates: Enhanced In vitro Antibacterial Activity.

The empirical combination of both a beta-lactam and glycopeptide to counter potential staphylococcal pathogens may improve the clinical outcomes for cases of Staphylococcus aureus bacteremia. We reported comparative in vitro studies of combination effects of different cephalosporins (i.e., cefazolin, cefmetazole, cefotaxime, and cefepime) combined with glycopeptides for 34 randomly selected methicillin-resistant S. aureus (MRSA) isolates by three methods, including the checkerboard, time-killing, and combination MIC measurement methods. Thirteen SCCmec type III isolates with a cefazolin MIC of ≥ 128 μg/mL were classified as the high-cefazolin MIC (HCM) group, whereas 13 SCCmec type IV and 8 SCCmec type V isolates were classified as the low-cefazolin MIC (LCM) group. With the checkerboard method, synergism was present for vancomycin-based combinations at 30.8–69.2 and 13.6–66.7%, as well as teicoplanin-based combinations of 38.5–84.6 and 0–47.6%, of the HCM and LCM isolates, respectively. No antagonism was noted. The in vitro inhibitory activity was evident even at a low concentration of 1/512x MIC of cephalosporin combined with sub-inhibitory concentrations (1/2x MIC) of a glycopeptide. With time-killing assays, synergism was noted at 1/2x or 1x susceptible breakpoint concentrations (SBCs) of a cephalosporin combined with 1/4 or 1/2 MIC of a glycopeptide. In the presence of 1/2 SBC of a cephalosporin, vancomycin or teicoplanin MICs decreased an average of 2.0- to 6.6- or 1.6- to 5.5-fold, respectively. With 8 μg/mL cephalosporin, the decline of glycopeptide MICs was most obvious in the presence of cefmetazole. In conclusion, cephalosporin-glycopeptide combinations at clinically achievable concentrations can exhibit in vitro synergistic antibacterial activity against clinical MRSA isolates. Such combinations require more clinical data to support their application for use in human MRSA infections.