Staphylococcal Strains Research Articles

Methicillin-Resistant Staphylococcus aureus (MRSA) and Other Methicillin-Resistant Staphylococci and Mammaliicoccus (MRNaS) Associated with Animals and Food Products in Arab Countries: A Review.

Simple SummaryStaphylococci are present in the microbiota of both humans and animal species, being recognized as the most important opportunistic pathogens. Antimicrobial resistance (AMR) has become a global public health issue presenting a significant risk because it severely limits treatment options. Methicillin resistance in staphylococci (MRS) poses a specific problem as it may cause serious human and animal infections, eventually resulting in death. The increasing observation of MRS in different animal species has raised the concern of their impact on animal health and the potential of zoonotic transmission. The availability of comprehensive data on the ecology and distribution of MRS in animals and food products worldwide is necessary to understand their relevance in the “One Health” domain. However, there is a gap in information in terms of MRS and the Arab countries. Therefore, our study aimed to provide an overview of the situation of MRS in these countries by reviewing the available data on livestock and animal products and making recommendations for the future.The prevalence of methicillin resistance in staphylococci has been increasing globally and is currently one of the major public health concerns. In particular, treating infections caused by staphylococci with acquired antimicrobial resistance is problematic, as their treatment is more difficult. The resistance is found both in human and animal staphylococcal strains. Methicillin-resistant staphylococci (MRS) have also been increasingly reported in wildlife. In Arab countries, MRS has been detected in food producing animals and food products; however, the risk this poses is somewhat unclear, and still a significant lack of information on the trend and distribution of these pathogens in these countries, which have a specific ecosystem (desert) and traditions (Muslim culture). In this manuscript, we aim to provide an overview of the prevalence and the major MRS clonal lineages circulating in these specific countries and compare to them other situations with different ecosystems and cultures.

Membrane acting Povarov-Doebner derived compounds potently disperse preformed multidrug resistant Gram-positive bacterial biofilms

The National Institute of Health (NIH) estimates that the majority of human microbial infections are either linked to or directly caused by bacterial biofilms and these infections are immune to most currently approved FDA drugs. Hence, there is a need for the development of potent antibiotics against biofilms. We have previously shown that pentafluorosulfanyl (SF5)-containing quinoline compounds, which were synthesized via the Povarov reaction, kill persister bacteria (Onyedibe et al. RSC Med Chem, 2021, 12, 1879–1893). Inspired by this earlier discovery, we expanded upon the compounds in the library to identify additional members that could have similar or better potencies, with a goal of increasing the diversity of compounds that could be further developed into therapeutics. Compounds from the Povarov derived SF5-containing compounds inhibited both clinical and laboratory strains of Gram-positive bacteria at minimum inhibitory concentration (MIC) of 0.5 μg/mL to 2 μg/mL. Interestingly, the lead compound, HSD 1919 exhibited rapid bactericidal mode of action against multidrug resistant (MDR) staphylococcal and enterococcal strains such as MRSA and VRE via bacterial membrane disruption. HSD 1919 eradicated persister MRSA in 2 h–8 h. Most remarkably, we found that HSD 1919 (newly identified compound) and HSD 1835 (previously disclosed, Onyedibe et al. RSC Med Chem, 2021, 12, 1879–1893), dispersed preformed MRSA and VRE biofilms at relatively low concentrations (8 μg/mL). Bithionol (at 1 μg/mL) or nitroxoline (at 4 μg/mL) did not appreciably disperse pre-existing biofilms but when combined with HSD 1919 or HSD 1835 (at 0.5–4 μg/mL), preformed MRSA biofilms could be dispersed, highlighting exciting synergy at reasonably low concentrations of the drugs. Biofilm dispersal was verified by scanning electron microscopy (SEM) whilst membrane disruption properties of HSD 1919 were confirmed by both transmission electron microscopy (TEM) and SEM. Further mechanistic studies showed inhibition of DNA, RNA, cell wall and protein synthesis in a macromolecular biosynthesis assay indicating that these compounds inhibit bacteria via multiple mechanisms, which is now being appreciated as an effective way to tackle resistant bacteria. Toxicity studies showed that HSD 1919 was nontoxic in-vitro to mammalian red blood cells at 10X MIC. Herein, we report HSD 1919 and analogs thereof as critical chemical scaffolds, which can be harnessed to develop highly potent antibiofilm therapeutics.

Ceftiofur efficacy in the intramammary extended treatment of staphylococcal subclinical mastitis in primiparous cows

Extended therapy is an alternative approach to treat nonresponsive staphylococcal mastitis, although it has been used mainly in adult cows. This study investigated the efficacy of intramammary ceftiofur in the treatment of staphylococcal subclinical mastitis in primiparous cows. Sixty primiparous cows Holstein were divided into four groups (G1 = Group negative to staphylococci infection, G2 = Group positive to staphylococci infection untreated with intramammary ceftiofur, Group, G3 = Group treated with intramammary ceftiofur early lactation, G4 = Group treated with intramammary ceftiofur late lactation group) with milk samples collected at five moments (M0 = diagnosis of subclinical mastitis, M1 = 7 days after diagnosis of subclinical mastitis and early extended therapy, M2 = 14 after the diagnosis of subclinical mastitis, M3 = 21 days after diagnosis of mastitis) and M4 = 28 days after diagnosis of mastitis). In groups G3 and G4, which were treated with intramammary ceftiofur, the microbiological cure was evidenced by a reduction in SCC of 73.3% and 46.7%, respectively. Thus, in the treated groups (G3 + G4) and untreated (G2), cure rates were 60% and 26.7%, respectively (P <0.05). Forty-five strains of staphylococci were isolated, with a predominance of S. aureus (51,1%) and S. intermedius (48,8%). The in vitro antimicrobial susceptibility pattern showed higher sensitivity indices of isolates for oxacillin (80%), ceftiofur (77.8%), cephalexin (77.8%), ciprofloxacin (66.7%), and gentamicin (60%). In contrast, cloxacillin (24.4%), penicillin (35.6%), and tetracycline (44.5%) were ineffective antimicrobials for staphylococci isolates. Among the milk samples taken when M0 and M1 (7 days after isolation), 13.3% were positive for the detection of the inhibiting substances (antimicrobials) in Delvotest™ and all negative in the Snap test™, whereas in the M2 (14 days after isolation), 55% were positive in Delvotest™ and 46.7% in the Snap test™. Twenty-one days after diagnosis (M3), 16.7% revealed positive results in Delvotest™ and 11.7% in the Snap test™. For M4 (28 days after isolation), 11.7% showed positive results in Delvotest™, and 8.3% revealed positive results in the Snap test™. There was a reduction in SCC among all cured animals. Significant results were identified for milk production in the M0, so G1 produced more milk than the other three groups, G2, G3, and G4. A statistical association was observed between the reduction in the volume of daily milk and production in animals with subclinical mastitis, denoting losses in milk production in primiparous bovines infected during lactation by staphylococci. Extended intramammary therapy with ceftiofur in bovine primiparous cows subclinically affected by staphylococcal mastitis is effective mainly in cases treated at the beginning of lactation, showing microbiological cure and reduction in SCC.

The occurrence of antibiotic-resistant bacteria on the clothes of nursery teachers in daycare centres.

AimsChildcare facilities act as microenvironments that facilitate and promote the selection, spread and transmission of antibiotic‐resistant micro‐organisms in the community. We focused on the study of antimicrobial resistance and genetic predispositions for β‐lactamase production in bacterial isolates from nursery teachers' clothing.Methods and ResultsAntimicrobial resistance of bacterial strains belonging to Enterobacteriaceae, Enterococcus, Staphylococcus spp., Pseudomonas spp. and Bacillus spp. isolated from 80 samples of nursery teachers' clothing was determined. The selected ESβL genes were found in 30 (44.1%) of 68 strains examined. The CTX‐M type ESβL determinants were detected in 15.4%, 71.5% and 42.5% of the Enterobacteriaceae, Pseudomonas and Bacillus isolates, respectively. The OXA‐type coding genes were detected only in strains of the genera Pseudomonas (57.1%) and Bacillus (48.6%). Thus, most B. cereus strains were sensitive to the recommended antibiotics used to treat infections caused by these bacteria. Methicillin resistance was phenotypically confirmed in 27 (14.6%) of 185 staphylococcal isolates. Four isolates (2.2%) were identified as MRSA. Vancomycin resistance was not observed in any of the staphylococcal and enterococci strains.ConclusionsThis study has shown that potential pathogens have been isolated from the clothing of nursery teachers, posing a risk of transmission to children. These clothes should be maintained and properly laundered to avoid cross‐contamination and the spread of multidrug‐resistant (MDR) bacteria in childcare centres.Significance and impact of the studyThis study provides insight into the route of transmission of MDR micro‐organisms through the clothing of nursery teachers, to which greater importance should be given in the future. Proper procedures for the cleaning and use of clothing in daycare centres should be clarified and standardized.

Phytochemical profiling, antibacterial and antioxidant properties of Crocus sativus flower: A comparison between tepals and stigmas

Abstract Several studies have demonstrated that stigmas of Crocus sativus contain several bioactive compounds with potential health-promoting properties. However, during the processing of stigmas, large amounts of floral bio-residues are normally discarded as by-products. In this study, using untargeted metabolomics, the comprehensive phytochemical composition of C. sativus stigma and tepals was investigated. Moreover, the antibacterial and anti-biofilm properties of the extracts of C. sativus stigmas and tepals were compared. The study was carried out using two methicillin-resistant staphylococcal reference strains (i.e., Staphylococcus aureus ATCC 43300 and Staphylococcus epidermidis ATCC 35984), representing important Gram-positive biofilm-forming human pathogens. The antibacterial properties were correlated with total polyphenol content, total terpenoid content, and in vitro antioxidant properties of tepals and stigmas. The results demonstrated that stigma and tepal extracts, at the sub-toxic concentrations, were able to interfere with biofilm formation by ATCC 43300 and ATCC 35984. Besides, the higher antibacterial activity of tepals than stigmas was associated with higher levels of phycompounds. Therefore, our results demonstrated that C. sativus stigmas and bio-residues, such as tepals, are potential antioxidant sources and good candidates as antibacterial agents to prevent biofilm formation. Taken together, these findings showed that C. sativus could be used as functional ingredient by the food and pharmaceutical industries.

Evaluation of Drug Activity Against Staphylococcal Biofilms – Settings of Conditions Leading to Reproducible and Robust Biofilm Formation in vitro

Staphylococcus aureus(SA)andepidermidis(SE) are the most common pathogens of the genus Staphylococcus, causing biofilm-associated infections. Bacteria in biofilms are difficult to eradicate due to their resistance and serve as a reservoir for recurring persistent infections. A variety of protocols for in vitro drug activity testing against biofilms has been introduced. However, there are often fundamental differences. In our pilot study, we developed optimal conditions for staphylococcal biofilm formation on plastic pegs in order to set a methodology for an evaluation of the antibiofilm activity of candidate molecules. The convenience of the plastic pegs lies in their removability from the lid for easy access to multiple equivalent biofilms, and in possibility of in situ detection and quantification by confocal laser microscopy. For the purpose of enhancement in staphylococcal biofilm formation, the impact of peg surface modification with 3 different coating materials was studied as well. An increase of biofilm biomass was evaluated by crystal violet staining method. The basic precondition for obtaining relevant and reproducible data regarding antibiofilm activity is the formation of robust biofilms with typical attributes such as the presence of a biofilm matrix. In our study, in vitro conditions revealed that we fully met the preconditions for the SA and methicillin-resistant SA strains. In conclusion, we demonstrated statistically significant enhancement of biofilm formation in all studied staphylococcal strains, including either strong biofilm producer phenotype (SA, methicillin-resistant SA) and weak biofilm producer phenotype (SE). Supported by the SVV Project No. 260549 and by the Czech Science Foundation project No. 20-19638Y.

Neoaortoiliac system and cryopreserved human allograft for the treatment of aortic graft infections.

To report and compare neoaortoiliac system reconstruction and cryopreserved human allograft in treating aortic graft infections. We retrospectively analysed the data of the patients treated for aorto graft infections between January 2015 and May 2021 in our hospital. The clinical data, diagnostic procedures, and surgical options were evaluated. The primary endpoint of this study was the 30-day and 1-year mortality; secondary endpoints were major postoperative complications. We retrospectively reviewed a series of 31 consecutive patients (28 males; median age 72years, range, 50-87years) with aortic graft infection treated with NAIS (n = 20, 65%) or cryopreserved allograft (n = 11, 36%). The clinical presentation included fever attacks in 18 (58%) patients, abdominal pain in 15 (48%) patients, haemodynamic instability in 6 (19%) patients, and haematemesis in 2 (7%) patients. The median operative time of the NAIS was longer than CHA without a statistically significant difference (458 min vs. 359 min, p = .505). The postoperative morbidity for all patients was 81%, with no significant difference between NAIS and CHA groups (85% vs. 73%, p = .638). There was no limb thrombosis of the new reconstructions. Limb loss occurred in 4 (13%) patients, including 2 (10%) NAIS patients and 2 (18%) CHA patients. One NAIS patient developed complications in the form of a distal (femoral) disruption of the vein 15days after surgery. There were no significant differences between NAIS and CHA groups in ICU stay (12 vs 8days, .984) but in hospitalization (22 vs 33, p = .033). The most common bacteria isolated were staphylococci strains in 15 (48%). In 13 (36%) patients, candida was positive. The in-hospital 30-day and 1-year mortality for all patients was 16% (5/31) and 29% (9/31), with no significant differences between NAIS and CHA at 30days (25% vs. 0, p = .133) or 1year (35% vs. 18%, .429). Five NAIS patients died during the hospital stay; three of them had end-of-life decisions. After a median follow-up of 16months (1-66months), 12 (39%) patients died, including 9 patients with NAIS and 3 with CHA reconstructions. The causes of death included overwhelming sepsis in 5 (42%) patients, graft disruption in one (8%) NAIS patient, non-small cell lung cancer in one (8%) patient, COVID-19 in one (8%) patient and unknown causes (8%) in one. Non-staged neoaortoiliac system reconstruction and cryopreserved human allografts show comparable short- and midterm results for treating aortic graft infections. However, both procedures remain challenging with high morbidity and mortality rates.

Description of Staphylococcal Strains from Straw-Coloured Fruit Bat (Eidolon helvum) and Diamond Firetail (Stagonopleura guttata) and a Review of their Phylogenetic Relationships to Other Staphylococci.

The phylogenetic tree of the Staphylococcus aureus complex consists of several distinct clades and the majority of human and veterinary S. aureus isolates form one large clade. In addition, two divergent clades have recently been described as separate species. One was named Staphylococcus argenteus, due to the lack of the “golden” pigment staphyloxanthin. The second one is S. schweitzeri, found in humans and animals from Central and West Africa. In late 2021, two additional species, S. roterodami and S. singaporensis, have been described from clinical samples from Southeast Asia. In the present study, isolates and their genome sequences from wild Straw-coloured fruit bats (Eidolon helvum) and a Diamond firetail (Stagonopleura guttata, an estrildid finch) kept in a German aviary are described. The isolates possessed staphyloxanthin genes and were closer related to S. argenteus and S. schweitzeri than to S. aureus. Phylogenetic analysis revealed that they were nearly identical to both, S. roterodami and S. singaporensis. We propose considering the study isolates, the recently described S. roterodami and S. singaporensis as well as some Chinese strains with MLST profiles stored in the PubMLST database as different clonal complexes within one new species. According to the principle of priority we propose it should be named S. roterodami. This species is more widespread than previously believed, being observed in West Africa, Southeast Asia and Southern China. It has a zoonotic connection to bats and has been shown to be capable of causing skin and soft tissue infections in humans. It is positive for staphyloxanthin, and it could be mis-identified as S. aureus (or S. argenteus) using routine procedures. However, it can be identified based on distinct MLST alleles, and “S. aureus” sequence types ST2470, ST3135, ST3952, ST3960, ST3961, ST3963, ST3965, ST3980, ST4014, ST4075, ST4076, ST4185, ST4326, ST4569, ST6105, ST6106, ST6107, ST6108, ST6109, ST6999 and ST7342 belong to this species.

Phenol-soluble modulins PSMα3 and PSMβ2 form nanotubes that are cross-α amyloids

Phenol-soluble modulins (PSMs) are peptide-based virulence factors that play significant roles in the pathogenesis of staphylococcal strains in community-associated and hospital-associated infections. In addition to cytotoxicity, PSMs display the propensity to self-assemble into fibrillar species, which may be mediated through the formation of amphipathic conformations. Here, we analyze the self-assembly behavior of two PSMs, PSMα3 and PSMβ2, which are derived from peptides expressed by methicillin-resistant Staphylococcus aureus (MRSA), a significant human pathogen. In both cases, we observed the formation of a mixture of self-assembled species including twisted filaments, helical ribbons, and nanotubes, which can reversibly interconvert in vitro. Cryo–electron microscopy structural analysis of three PSM nanotubes, two derived from PSMα3 and one from PSMβ2, revealed that the assemblies displayed remarkably similar structures based on lateral association of cross-α amyloid protofilaments. The amphipathic helical conformations of PSMα3 and PSMβ2 enforced a bilayer arrangement within the protofilaments that defined the structures of the respective PSMα3 and PSMβ2 nanotubes. We demonstrate that, similar to amyloids based on cross-β protofilaments, cross-α amyloids derived from these PSMs display polymorphism, not only in terms of the global morphology (e.g., twisted filament, helical ribbon, and nanotube) but also with respect to the number of protofilaments within a given peptide assembly. These results suggest that the folding landscape of PSM derivatives may be more complex than originally anticipated and that the assemblies are able to sample a wide range of supramolecular structural space.

Antibiotic Sensitivity of Staphilococcus Aureus Strains in the Clinical Hospital for Infectious Diseaseas Constanta

Abstract Introduction: Staphylococcus are gram-positive bacteria that inhabit both the skin and mucous membranes of both humans and animals. Antimicrobial susceptibility testing is one of the most important laboratory procedures for infectious disease management. Material and method: Between January 2017 and June 2019, in the Clinical Infectious Diseases Hospital (CIDH) of Constanta, 331 strains of staphylococcus isolated from various pathological products were identified and tested for antibiotic sensitivity: urine culture, stool culture, pharyngeal / nasal discharge, blood culture, sputum, vomiting fluid and various secretions. Results: During 2017, 95 strains were isolated from patients hospitalized in CIDH, in 2018: 118, and in the first half of 2019: 124 staphylococcal strains. An analysis by age groups shows that staphylococcus infection predominates in adults with a total of 196 (59.21%), and in children, less than 18 years: 135 (40.78%). Of the 196 adults, there are immunocompetent patients: 115 (58.67%), immunocompromised by HIV 40 (20.50%), and 19 were hospitalized in the intensive care unit (9.69%). Regarding the predominant sex in staph infections, the most strains were registered in males: 179 (54.07%) and 152 in females (45.92%). Out of the total strains of isolated Staphylococcus, 160 (48.33%) of Staphylococcus aureus were identified, of which methycillin resistant staphylococcus aureus (MRSA) 43 (26.87%). Testing for Staphylococcus aureus strains with Oxacillin showed that 45 (28.12%) were resistant, Rifampicin 26 (16.25%) was resistant, Fluoroquinolones 29 (18.12%), and Cotrimoxazole / Sulfamethoxazole 27 (16.87%) of the strains were resistant. Conclusions: There is a relatively high resistance to Oxacillin of Staphylococcus aureus strains and a relatively low resistance to fluoroquinolones, Rifampicin and Cotrimoxazole / Sulfamethoxazole, over the years studied.

Linearized teixobactin is inactive and after sequence enhancement, kills methicillin-resistant Staphylococcus aureus via a different mechanism.

Staphylococcus aureus is a highly adaptable pathogen that can rapidly develop resistance to conventional antibiotics such as penicillin. Recently, teixobactin was discovered from uncultivated soil bacteria by using the i-chip technology. This depsipeptide forms an ester bond between the backbone C-terminal isoleucine carboxylic acid and the hydroxyl group of threonine at position 8. Also, it contains multiple nonstandard amino acids, making it costly to synthesize. This study reports new peptides designed by linearizing teixobactin. After linearization and conversion to normal amino acids, teixobactin lost its antibacterial activity. Using this inactive template, a series of peptides were designed via hydrophobic patching and residue replacements. Three out of the five peptides were active. YZ105, only active against Gram-positive bacteria, however, showed the highest cell selectivity index. Different from teixobactin, which inhibits cell wall synthesis, YZ105 targeted the membranes of methicillin-resistant S. aureus (MRSA) based on kinetic killing, membrane permeation, depolarization, and scanning electron microscopy studies. Moreover, YZ105 could kill nafcillin-resistant MRSA, Staphylococcal clinical strains, and disrupted preformed biofilms. Taken together, YZ105, with a simpler sequence, is a promising lead for developing novel anti-MRSA agents.

Dynamics of resistance to antibiotics in nosocomial staphylococci from multidisciplinary hospital

Background. Staphylococci are still the leading causative agents of infections associated with healthcare, and the study of their antibiotic resistance is still relevant.
 Aim. The research is aimed at study of antibiotic resistance of hospital strains of staphylococci in dynamics.
 Materials and methods. Susceptibility to 16 antimicrobial agents was studied in 554 Staphylococcus strains, isolated from patients in a multidisciplinary medical centre. The method of serial microdilutions was used.
 Results. Antibiotic-resistant strains prevailed (85.4%). Methicillin-resistance and multy-resistance were found to be more typical for coagulase-negative strains 75.2% and 74.1% respectively, than for Staphylococcus aureus 14.2% and 15.4% respectively. Methicillin-resistance and poly-resistance in S. aureus was found to decrease it was 11.1% and 12.8% in 20152016 (17.1% and 17.9% respectively in 20112012). On the contrary, methicillin-resistance in coagulase-negative staphylococci strains during the same period increased 1.5 times. Totally, methicillin-resistant strains composed a half of the isolates 48.7%. The studied Staphylococcus strains were susceptible to vancomycin, daptomycin, tigecycline. Resistance to linezolid and amikacin was 2.2% and 2.7% respectively. S. aureus strains were all susceptible to linezolid, fusidic acid, rifampicin, trimethoprim-sulfamethoxazole. Minimum inhibitory concentrations (MIC) of antibiotics for staphylococci varied in wide ranges from 0.06 to 128 mg/l. For S. aureus and S. epidermidis, the MIC50 and MIC90 of only five drugs (benzylpenicillin, tigecycline, vancomycin, linezolid, and daptomycin) were the same, while the MIC50 and MIC90 of most of the other studied drugs against S. epidermidis were significantly higher compared to S. aureus.
 Conclusion. The variability of resistance of staphylococci to antimicrobial drugs in a multidisciplinary hospital confirms the need for continuous monitoring of their antibiotic resistance.

Epigenetic states of genes controlling immune responsiveness in bovine chronic mastitis

Abstract Mastitis is a common disease in dairy cows, causing substantial economic losses. The leading cause of elevated milk somatic cell count (SC), which is the best indicator for detecting mastitis, is the invasion of pathogens. A major pathogen responsible for bovine mastitis is Staphylococcus aureus, a member of the coagulase-positive staphylococci. Some strains of coagulase-negative staphylococci can also be a cause of clinical or subclinical mastitis. Our study used bisulfite sequencing PCR (BSP) to detect the methylation status of nine candidate genes (CCL2, HCK, F11R, CD8A, PDIA3, LGMN, HSPA1A, IL18 and NFKBIA). We investigated the mechanisms associated with overexpression of these genes, in the mammary gland secretory tissue of cows diagnosed with mastitis and infected with coagulase-positive or coagulase-negative staphylococci. The results showed no changes at the DNA methylation level between the mastitis (CoPS and CoNS) and control groups (H), except for in the HCK region, where the observed differences between the CoPS and H groups were statistically significant. The low methylation level of the CpG sequence seems not to correspond to the previously observed increased activity of these genes, suggesting that mechanisms other than DNA methylation may control mRNA expression at the analyzed loci.

Multiplex Detection of 24 Staphylococcal Enterotoxins in Culture Supernatant Using Liquid Chromatography Coupled to High-Resolution Mass Spectrometry

Staphylococcal food poisoning outbreaks are caused by the ingestion of food contaminated with staphylococcal enterotoxins (SEs). Among the 27 SEs described in the literature to date, only a few can be detected using immuno-enzymatic-based methods that are strongly dependent on the availability of antibodies. Liquid chromatography, coupled to high-resolution mass spectrometry (LC-HRMS), has, therefore, been put forward as a relevant complementary method, but only for the detection of a limited number of enterotoxins. In this work, LC-HRMS was developed for the detection and quantification of 24 SEs. A database of 93 specific signature peptides and LC-HRMS parameters was optimized using sequences from 24 SEs, including their 162 variants. A label-free quantification protocol was established to overcome the absence of calibration standards. The LC-HRMS method showed high performance in terms of specificity, sensitivity, and accuracy when applied to 49 enterotoxin-producing strains. SE concentrations measured depended on both SE type and the coagulase-positive staphylococci (CPS) strain. This study indicates that LC-MS is a relevant alternative and complementary tool to ELISA methods. The advantages of LC-MS clearly lie in both the multiplex analysis of a large number of SEs, and the automated analysis of a high number of samples.

PHENOTYPIC CHARACTERIZATION OF METHICILLIN-RESISTANT STAPHYLOCOCCAL STRAINS ISOLATED FROM DAIRY CATTLE IN A RURAL COMMUNITY

The emergence of methicillin-resistant staphylococci among livestock production have revealed different epidemiological dimension to the understanding of the staphylococcal infection. The aim of this study was to investigate methicillin-resistant staphylococcal strains isolated from raw milk and nasal cavity from free range dairy cattle in rural community using phenotypic profiling. A total of 283 samples were examined from free range dairy cattle (137 milk samples and 146 nasal swabs) were assessed between February and April 2015. Staphylococcal were isolated and positively identified using morphological and biochemical reactions. Of the 283 samples, 100 isolates (50 nasal and 50 milk samples) were methicillin-resistant Staphylococcus species and were further characterized by phenotypic methods. The antimicrobial resistance profiles of the isolates were determined using the phenotypic disc diffusion method. Phenotypic characterization using Modified Hodge Test showed that 45/50 (90%) and 48/50 (96%) of the isolates from nasal and raw milk revealed carbapernemase activity respectively. Also, 2/50 (4%) of the isolates from the nasal cavity and 4/50 (8%) of the isolates from raw milk were positive to Double Disc Synergistic Test and thus revealing metallo-?-lactamases (MBLs) production. Extended spectrum beta-lactamase activity, carbapenemase activity and metallo-beta-lactamase activity producing isolates are becoming prevalent in food animal settings. The detection of staphylococcal strains that are phenotypically resistant to oxacillin and methicillin are known to pose serious health concerns and are of great clinical relevance.

Laccase-Catalyzed Derivatization of Aminoglycoside Antibiotics and Glucosamine.

The increasing demand for new and effective antibiotics requires intelligent strategies to obtain a wide range of potential candidates. Laccase-catalyzed reactions have been successfully applied to synthesize new β-lactam antibiotics and other antibiotics. In this work, laccases from three different origins were used to produce new aminoglycoside antibiotics. Kanamycin, tobramycin and gentamicin were coupled with the laccase substrate 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide. The products were isolated, structurally characterized and tested in vitro for antibacterial activity against various strains of Staphylococci, including multidrug-resistant strains. The cytotoxicity of these products was tested using FL cells. The coupling products showed comparable and, in some cases, better antibacterial activity than the parent antibiotics in the agar diffusion assay, and they were not cytotoxic. The products protected mice against infection with Staphylococcus aureus, which was lethal to the control animals. The results underline the great potential of laccases in obtaining new biologically active compounds, in this case new antibiotic candidates from the class of aminoglycosides.

The study of the effectiveness of the antibacterial action of the combination of a bacteriophage with a probiotic

Intestinal infections occupy one of the leading places in diseases of the gastrointestinal tract in humans and animals. Strains of Staphylococcus spp are one of the causative agents of an intestinal infection. When treating an intestinal infection caused by staphylococcus, antibiotics are most often used, but in recent years there has been the formation of resistance in staphylococcus strains to antibiotics that have been used for many years. One of the promising ways to solve the problem of antimicrobial resistance is the use of drugs based on bacteriophages, which have a specific effect on the disease pathogens, however, they are free of toxic and allergenic side effects on the human body and do not cause resistance. Aim. To study the antibacterial effect of the combination of a staphylococcal bacteriophage and a probiotic based on L. acidophilus lactobacilli to different staphylococcal strains. Materials and methods. The following bacterial strains were used in the study: St. aureus ATCC 25923 and St. epidermidis ATCC 12228. The “Polyphag Staf” staphylococcal bacteriophage (manufactured by NVK MVK, Ukraine) and the “Lactofor” probiotic (manufactured by Ananta Medicare Limited, India) based on L. acidophilus lactobacilli were selected as objects. In the study, the antibiotic “Erythromycin” in tablets of 100 mg (the manufacturer – Borschagovsky HFZ, Ukraine) was used as a reference drug. To detect the antibacterial activity to the experimental strains of St. aureus and St. epidermidis, the methods of Appelman and agar diffusion were used. Results and discussion. It was found that when using the combination of a staphylococcal bacteriophage and a probiotic with L. acidophilus lactobacilli the level of the antibacterial activity to the experimental strains of St. aureus and St. epidermidis was higher than when using only the bacteriophage. This is probably due to the synergism of the interaction of the components of the combination of active substances proposed. It was also found that the antibacterial activity of the combination of drugs and the antibiotic erythromycin proposed was at the same level. Thus, the combination of a bacteriophage that destroys staphylococcal bacteria, has no side effects, and does not cause resistance and a probiotics that improves the microflora of the gastrointestinal tract has a number of advantages over antibioticsin the treatment of intestinal infections caused by staphylococcal strains. Conclusions. The studies conducted indicate the prospects of using the combination of a staphylococcal bacteriophage and a probiotic based on L. acidophilus lactobacilli for the treatment of intestinal infections caused by different staphylococcal strains.

Submicron topography design for controlling staphylococcal bacterial adhesion and biofilm formation.

Surface topography modification with nano- or micro-textured structures has been an efficient approach to inhibit microbial adhesion and biofilm formation and thereby to prevent biomaterial-associated infection without modification of surface chemistry/bulk properties of materials and without causing antibiotic resistance. This manuscript focuses on submicron-textured patterns with ordered arrays of pillars on polyurethane (PU) biomaterial surfaces in an effort to understand the effects of surface pillar features and surface properties on adhesion and colonization responses of two staphylococcal strains. Five submicron patterns with a variety of pillar dimensions were designed and fabricated on PU film surfaces and bacterial adhesion and biofilm formation of Staphylococcal strains (Staphylococcus epidermidis RP62A and Staphylococcus aureus Newman D2C) were characterized. Results show that all submicron textured surface significantly reduced bacterial adhesion and inhibited biofilm formation, and bacterial adhesion linearly decreased with the reduction in top surface area fraction. Surface wettability did not show a linear correlation with bacterial adhesion, suggesting that surface contact area dominates bacterial adhesion. From this, it appears that the design of textured patterns should minimize surface area fraction to reduce the bacterial interaction with surfaces but in a way that ensures the mechanical strength of pillars in order to avoid collapse. These findings may provide a rationale for design of polymer surfaces for antifouling medical devices.

Surface microbiota and associated staphylococci of houseflies (Musca domestica) collected from different environmental sources

Houseflies (Musca domestica) are important mechanical vectors for the transmission of pathogenic microorganisms. In this study, 129 houseflies (69 males and 60 females) were collected from 10 different environmental sources and a laboratory population was used. The surface microbiota of houseflies was identified by Next-Generation Sequencing. Staphylococci from the surfaces of houseflies were selectively isolated and their virulence genes, antibiotic susceptibilities, biofilm formation, and clonal relatedness were determined. Metagenomic analysis results demonstrated that Staphylococcus, Bacillus, and Enterococcus were mostly present on the surface of houseflies at the genus level. Additionally, the isolated 32 staphylococcal strains were identified as Staphylococcus sciuri (n = 11), S. saprophyticus (n = 9), S. arlettae (n = 6), S. xylosus (n = 4), S. epidermidis (n = 1) and S. gallinarum (n = 1). tetK, tetM, tetL, ermC, msrAB, and aad6 genes were found to carry by some of the staphylococcal strains. The strains were mostly resistant to oxacillin, penicillin, and erythromycin and three strains were multi-drug resistant. There was a statistical difference between housefly collection places and antibiotic resistance of isolated staphylococci to penicillin G, gentamicin, and erythromycin (p < 0.05). Biofilm test showed that 17 strains were strong biofilm formers, and it plays important role in the transmission of these bacteria on the surface of houseflies. Staphylococcal strains showed extracellular proteolytic and lipolytic activity in 31 and 12 strains, respectively. Closely related species were found in PFGE analysis from different environmental sources. By this study, surface microbiota and carriage of pathogenic staphylococci on the surfaces of houseflies and their virulence properties were elucidated.

A novel dressing with silver to treat meticillin-resistant Staphylococcus aureus biofilm infection in a pig model.

The purpose of this study was to use an in vivo biofilm porcine model to examine a new polyvinyl alcohol-based gelling fibre dressing with silver and compare it to other commercial dressings containing: polyvinyl alcohol-based gelling fibre without silver; carboxymethyl cellulose-based fibre with silver, benzethonium chloride and ethylenediaminetetraacetic acid; and untreated control. A total of 52 deep partial-thickness wounds (10x7x0.5mm) were created on each of three animals and inoculated with 25µl of meticillin-resistant Staphylococcus aureus (MRSA) (106 colony forming units (CFU)/ml). Wounds were covered for 24 hours to allow biofilm formation and were randomly designated to one of the four treatments. Samples were recovered for microbiological and histological analysis on days 3, 5 and 7 post-treatment. Polyvinyl alcohol-based gelling fibre dressing with silver was able to significantly reduce biofilm more effectively than the other treatment groups. By day 7, wounds treated with the dressing had a 2.72±0.01 log CFU/g reduction in MRSA count versus untreated control wounds and a 2.59±0.01 log CFU/g reduction versus baseline counts. For histology analysis, all wounds reached 100% re-epithelialisation by day 5. The results of this study indicated that polyvinyl alcohol-based gelling fibre dressing with silver was effective against biofilm of antibiotic-resistant staphylococcal strains without inhibiting the wound healing process, and may have important clinical implications when treating acute and/or hard-to-heal wounds.