Staphylococcus Aureus Strains Research Articles

Acute Prosthetic Joint Infections with Poor Outcome Caused by Staphylococcus Aureus Strains Producing the Panton-Valentine Leukocidin.

The aim of this study was to investigate whether the presence of Staphylococcus aureus (SA) producing the Panton-Valentine leukocidin (PVL) affects the outcome of Prosthetic Joint Infection (PJI). Patients with acute and chronic PJI sustained by SA were prospectively enrolled at the orthopedic unit of "Casa di Cura Santa Maria Maddalena", from January 2019 to October 2021. PJI diagnosis was reached according to the diagnostic criteria of the International Consensus Meeting on PJI of Philadelphia. Synovial fluid obtained via joint aspirations was collected in order to isolate SA. The detection of PVL was performed via real-time quantitative PCR (RT-qPCR). The outcome assessment was performed using the criteria of the Delphi-based International Multidisciplinary Consensus. Twelve cases of PJI caused by SA were included. Nine (75%) cases were acute PJI treated using debridement, antibiotic and implant retention (DAIR); the remaining three (25%) were chronic PJI treated using two-stage (n = 2) and one-stage revision (n = 1), respectively. The SA strains that tested positive for PVL genes were 5/12 (41.6%,). Treatment failure was documented in three cases of acute PJI treated using DAIR, all supported by SA-PVL strains (p < 0.045). The remaining two cases were chronic PJI treated with a revision arthroplasty (one and two stage, respectively), with a 100% eradication rate in a medium follow-up of 24 months. Although a small case series, our study showed a 100% failure rate in acute PJI, probably caused by SA PVL-producing strains treated conservatively (p < 0.04). In this setting, toxin research should guide radical surgical treatment and targeted antibiotic therapy.

Repositioning of FDA-approved drugs for the treatment of methicillin-resistant Staphylococcus aureus infection: Structure-based virtual screening and in vitro assay

Background: Computer-aided repositioning of approved drugs is an increasingly popular strategy for the discovery of effective therapies. The potency of the newly repositioned drugs can be optimized using them as a component of an effective drug combination, thereby achieving the desired therapeutic effect at a lower and more tolerable drug concentration. Aim and Objectives: The aim of the study was to perform structure-based virtual screening and repurposing of FDA-approved drugs for the treatment of methicillin resistance by Staphylococcus aureus (SA) and perform an in vitro validation of the prediction. Materials and Methods: Following ethical clearance at the Department of Pharmacology and Therapeutics, College of Health Sciences, Usmanu Danfodiyo University Sokoto, molecular docking was performed against 5 validated protein targets involved in the development of methicillin resistance by SA and an in vitro validation of the prediction was done using 3 of the top-ranking drug candidates against methicillin-resistant vancomycin-susceptible strain of the pathogen (ATCC 43300). Results: Desmopressin and docetaxel, two of the 20 top-ranking repurposed drugs discovered through virtual screening, enhanced the inhibitory effect of oxacillin against the ATCC 43300 SA strain in a ratio-dependent manner, although each of the two drugs singly was only weakly effective against the bacterial strain. The standard drug, vancomycin (also among the top-scoring candidates), alone, was effective against ATCC 43300 strain and in combination with oxacillin, the two drugs produced a ratio-dependent synergistic effect against the bacterial strain. Conclusion: These findings suggest that oxacillin-based combinations with desmopressin, docetaxel, and the standard drug vancomycin, three of the 20 top-ranking drugs, at optimum ratios, may be beneficial in reversing the resistance of the ATCC 43300 SA strain to oxacillin, thus supporting the prediction of the molecular docking results.

Transcriptomics and systems biology identify non-antibiotic drugs for the treatment of ocular bacterial infection

SummaryIncreasing antibiotic resistance among ocular pathogens often results in treatment failure for blinding infections such as endophthalmitis. Hence, newer therapeutics is needed to combat multidrug-resistant infections. Here, we show a drug repurposing approach using a connectivity map based on temporal transcriptomics of Staphylococcus aureus (SA) infected mouse retina. The analysis predicted three non-antibiotic drugs, Dequalinium chloride (DC), Clofilium tosylate (CT), and Glybenclamide (Glb) which reversed the SA infection signatures. Predicted drugs exhibited anti-inflammatory properties in human retinal cells against sensitive and resistant strains of SA. Intravitreal administration of all drugs reduced intraocular inflammation in SA-infected mouse eyes while DC and CT also reduced bacterial burden. Drug treatment improved visual function coinciding with reduced Caspase-3 mediated retinal cell death. Importantly, all drugs exhibited synergy with vancomycin in improving disease outcomes. Overall, our study identified three non-antibiotic drugs and demonstrated their therapeutic and prophylactic efficacies in ameliorating intraocular bacterial infection.

#77 Prevalence of Staphylococcus aureus bacteremia at Saint Damien Hospital, Nos Petits Frères et Sœurs January (HSD/NPFS) 2020-June 2021, Haiti

Abstract Background Staphylococcus Aureus (SA) is one of the main agents implicated in healthcare-associated infections (HAI) [1]. These infections are associated with significant morbidity and mortality, particularly methicillin-resistant staphylococcal (MRSA) infections, and their costs are also a concern. Bacteremia is regularly monitored at Saint Damien Hospital (HSD), for the year 2020 an increase in Staphylococcus aureus (SA) bacteremia was observed in certain units of the hospital. In this context, all SA infections were reviewed during eighteen months to determine the prevalence of methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) staphylococcus aureus infections, their origins: in healthcare-associated infections (HAI), community-acquired infections (CI) and the distribution of MRSA per unit Method A Descriptive retrospective study was carried out at Saint Damien Hospital from January 2020 to June 2021. It includes all hospitalized children under fourteen years old (14) who had a positive blood culture for SA per site, only the 1st blood culture was taken into account. According to NHSN/CDC, are defined as infections associated with healthcare-associated infections occurring at ≥ 72 hours of hospitalization (HAI) and as community-acquired infectious infections occurring at = &amp;lt; 48 hours of hospitalization.. Data were collected from microbiology laboratory registers and patient records Results From January 2020 to June 2021, two thousand two hundred thirty five 2235 blood cultures were performed. The positivity rate was 21.43% (n=479/2235) distributed in different units. 16.49% SA strains were isolated (n=79/479), including fifty (50) strains of MRSA (63.29%) and twenty nine (29) strains of MSSA (36.70%) (n=29/79).From the strains of MRSA, 54% (n=27/50) were HAI and 46% community infections (CI) (n=23/50). The Origin of the infections (HAI/CI) was not significantly associated with MRSA and MSSA strains (p=0.6235), MRSA mainly came from emergency 48% (n=24/50) followed by neonatology 20% (n=10/50), intensive care 10% (5/50) and oncology at 10% (5/50). (n=19/79), the mortality rate attributable to MRSA and MSSA was 28% (n=14/50) and 17.24% (n=5/29), respectively. 46% of MRSA were resistant to more than 3 families of antibiotics (n = 23/50). SA strains were resistant to gentamycin at 30.37% (n=24/79) to 54.34% to sulfametoxazole (n=43/79) and 18.98% and to vancomycin (n=15/79 Conclusion The frequency of SA infections, specifically MRSA, is of great concern. Its existence in the community and its multiresistance, in particular to vancomycin, is an alert making this bacteremia a priority at Saint Damien Hospital, the standard precautions measures, the proper use of antibiotics by clinicians and epidemiological surveillance local would be needed to help control its spread.

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.

Characterization of Staphylococcus aureus biofilms via crystal violet binding and biochemical composition assays of isolates from hospitals, raw meat, and biofilm-associated gene mutants

Staphylococcus aureus (SA) is a gram-positive coccus and an opportunistic pathogen of humans. The ability of SA to form biofilms is an important virulence mechanism because biofilms are protected from host immune responses and antibiotic treatment. This study examines the relative biofilm strength of a variety of hospital and meat-associated strains of SA, using a crystal violet (CV) staining assay. Biofilms were treated with either DNase or proteinase K prior to CV staining, and compared to mock-treated results, to better understand the biochemical composition. Biofilm polysaccharide concentration was also measured using the phenol sulfuric-acid assay which was normalized to base biofilm strength. We found that hospital-associated isolates have biofilms that bind significantly more CV than for meat isolates and are significantly more protein and polysaccharide-based while meat isolates have significantly more DNA-based biofilms. This study also investigates the effects that biofilm-related genes have on biofilm formation and composition by analyzing specific transposon mutants of genes previously shown to play a role in biofilm development. agrA, atl, clfA, fnbA, purH, and sarA mutants produce significantly weaker biofilms (bind less CV) as compared to a wild-type control, whereas the acnA mutant produces a significantly stronger biofilm. Biofilms formed from these mutant strains were treated (or mock-treated) with DNase or proteinase K and tested with phenol and sulfuric acid to determine what role these genes play in biofilm composition. The acnA, clfA, fnbA, and purH mutants showed significant reduction in biofilm staining after either proteinase K or DNase treatment, agrA and sarA mutants showed significant biofilm reduction after only proteinase K treatment, and an atl mutant did not show significant biofilm reduction after either proteinase K or DNase treatment. These data suggest that biofilms that form without acnA, clfA, fnbA, and purH are DNA- and protein-based, that biofilms lacking agrA and sarA are mainly protein-based, and biofilms lacking atl are mainly polysaccharide-based. These results help to elucidate how these genes affect biofilm formation and demonstrate how mutating biofilm-related genes in SA can cause a change in biofilm composition.

Antibacterial activity of Myrtus communis L. and Melaleuca leucadendron var. cajaputi essential oils against antibiotic-resistant bacterial strains

Background: Antimicrobial resistance (AMR) is a global threat to public health. There is a dire need for new antibiotics as AMR threatens our last-resort antibiotics efficacy.1,2 Myrtle plants have been used in folk medicine for centuries. Essential oils from these plants demonstrated therapeutics effects. This study aims to examine the antibacterial activity of essential oils extracted from Myrtus communis L and Melaleuca leucadendron against clinically relevant bacterial pathogens.3Methods: Bacterial growth curves and bactericidal assays were performed on antibiotic-sensitive Staphylococcus aureus (SA) and antibiotic low and high resistant Methicillin-resistant Staphylococcus aureus (MRSA) strains; sensitive and resistant Klebsiella pneumoniae strains (KPS and KPR). Minimum bactericidal concentration (MBC) was performed on 4 strains of colistin sensitive E. coli and 4 colistin resistant E. coli strains. Results: Myrtle essential oils demonstrated a dose-dependent antibacterial activity against all tested strains and inhibited growth even after 24 hours. The tested oils dilutions ranged from 6.15 μl/ml up to 50 μl/ml and inhibited bacterial growth of both antibiotic sensitive and resistant strains of SA, MRSA (Figure 1), and Klebsiella. Further, the incubation of colistin sensitive and colistin-resistant E. coli strains with 50 μl/ml of Myrtle oil for one hour inhibited the growth of all tested strains. The viability of bacteria was tested by spotting on agar plates and further incubation overnight. The data suggest that Myrtle essential oils can effectively kill bacterial pathogens. Conclusion: Essential oils from Myrtus communis L. and Melaleuca leucadendron var. cajaputi possess potent antibacterial activity against antibiotic sensitive and resistant bacterial pathogens. There is a potential for utilizing these antibacterial oils as topical treatment of wound infections.

The effects of Staphylococcus aureus biofilm conditioned media on 3T3 fibroblasts.

Staphylococcus aureus (SA) is the most common bacterial species in chronic wounds. However, there is a lack of understanding of how SA secretions affect the cell biology during the healing process. We studied the effects of biofilm-secretions from SA strain SA29213 on 3T3 fibroblasts. SA29213 is a chronic wound isolate and widely used as a reference strain. We used a series of concentrations of biofilm-conditioned media (BCM) and found 100% BCM is lethal within 10 h. Cells survived in ≤75% BCM but the rate of closure in scratch wound assays was reduced. Treatment with 75% and 50% BCM caused fibroblasts to change shape and develop dendrite like processes. Prolonged treatment with 75% and 50% BCM reduced cell proliferation and increased the 4n deoxyribonucleic acid cell population with cell cycle arrest. There was also an elevation in the senescence marker beta galactosidase and the number of multinucleated cells. Shorter treatments with 75% and 50% SA BCM caused an increase in cell-cell adhesion and a redistribution of β-catenin from the cell membrane to the cytoplasm along with a change in the appearance and decrease in size of ZO-1, vinculin and paxillin structures. Fibroblasts in the edge of chronic wounds exposed to the secretions of SA may suffer similar effects such as induction of senescence, reduced proliferation and migration, which may contribute to the delayed healing of these chronic infected wounds.

Updated understanding of Staphylococcus aureus in atopic dermatitis: From virulence factors to commensals and clonal complexes.

Atopic dermatitis (AD) is a common inflammatory dermatosis that has multiple contributing factors including genetic, immunologic and environmental. Staphylococcus aureus (SA) has long been associated with exacerbation of AD. SA produces many virulence factors that interact with the human skin and immune system. These superantigens and toxins have been shown to contribute to adhesion, inflammation and skin barrier destruction. Recent advances in genome sequencing techniques have led to a broadened understanding of the multiple ways SA interacts with the cutaneous environment in AD hosts. For example, temporal shifts in the microbiome, specifically in clonal complexes of SA, have been identified during AD flares and remission. Herein, we review mechanisms of interaction between the cutaneous microbiome and SA and highlight known differences in SA clonal complexes that contribute to AD pathogenesis. Detailed knowledge of the genetic strains of SA and cutaneous dysbiosis is becoming increasingly relevant in paving the way for microbiome-modulating and precision therapies for AD.

Frequency and Sensitivity Patterns of Staphylococcus Aureus in a Tertiary Care Setting

Background: Staphylococcus aureus (SA) is a major etiological pathogen causing multiple infections and broadly known as a serious public health challenge faced due to antibiotic resistance. It is the need of time that infection prevention and control strategies; and antibiotic stewardship policies have to be employed conjointly to minimize the extended rise of antibiotic resistance.&#x0D; Objectives: To determine the frequency and sensitivity patterns of Staphylococcus aureus in tertiary care setting.&#x0D; Study Design: Descriptive study&#x0D; Place &amp; Duration of Study: Pathology Laboratory of tertiary care center from 1st March’ 2020 till 28th February’ 2021.&#x0D; Materials &amp; Methods: A total 643 Staph. aureus isolated from various clinical specimens received in a tertiary care hospital; were processed and identified by culture, staining and bench tests. Sensitivity testing was done by Disc Diffusion method. Resistance to cefoxitin(30µg) was labelled as Methicillin Resistant Staphylococcus aureus (MRSA). Constitutive and Inducible Clindamycin resistance was also evaluated. (CLSI, 2020-21).&#x0D; Results: During the study period 125 (19.44%) MRSA were recovered. Statistically, gender distribution regarding MRSA was significant, most of SA was recovered from blood (53.68%), while 46.31% from pus and wound swabs. The frequency of MRSA from Surgical and allied wards was higher (52.63%) than Medicine and allied wards (47.36%). Sensitivity to vancomycin, linezolid and tigecycline was noted 100% by all the isolates. Sensitivity to clindamycin and Doxycycline was 68.42% and 64.81% respectively; while resistance to erythromycin, ciprofloxacin and trimethoprim/sulfamethoxazole was 73.68%, 60% and 57.89%, respectively.&#x0D; Conclusion: The hazardous infections due to Staphylococcus aureus are worrisome in the present therapeutic scenario. A levelheaded prescription of sensitive antibiotics has to be ensured to minimize the rising frequency of resistant strains of SA.

Study of the impact of cultivation conditions and peg surface modification on the in vitro biofilm formation of Staphylococcus aureus and Staphylococcus epidermidis in a system analogous to the Calgary biofilm device.

Introduction. Staphylococcus aureus (SA) and Staphylococcus epidermidis (SE) are the most common pathogens from the genus Staphylococcus causing biofilm-associated infections. Generally, biofilm-associated infections represent a clinical challenge. Bacteria in biofilms are difficult to eradicate due to their resistance and serve as a reservoir for recurring persistent infections.Gap Statement. A variety of protocols for in vitro drug activity testing against staphylococcal biofilms have been introduced. However, there are often fundamental differences. All these differences in methodical approaches can then be reflected in the form of discrepancies between results.Aim. In this study, we aimed to develop optimal conditions for staphylococcal biofilm formation on pegs. The impact of peg surface modification was also studied.Methodology. The impact of tryptic soy broth alone or supplemented with foetal bovine serum (FBS) or human plasma (HP), together with the impact of the inoculum density of bacterial suspensions and the shaking versus the static mode of cultivation, on total biofilm biomass production in SA and SE reference strains was studied. The surface of pegs was modified with FBS, HP, or poly-l-lysine (PLL). The impact on total biofilm biomass was evaluated using the crystal violet staining method and statistical data analysis.Results. Tryptic soy broth supplemented with HP together with the shaking mode led to crucial potentiation of biofilm formation on pegs in SA strains. The SE strain did not produce biofilm biomass under the same conditions on pegs. Preconditioning of peg surfaces with FBS and HP led to a statistically significant increase in biofilm biomass formation in the SE strain.Conclusion. Optimal cultivation conditions for robust staphylococcal biofilm formation in vitro might differ among different bacterial strains and methodical approaches. The shaking mode and supplementation of cultivation medium with HP was beneficial for biofilm formation on pegs for SA (ATCC 29213) and methicillin-resistant SA (ATCC 43300). Peg conditioning with HP and PLL had no impact on biofilm formation in either of these strains. Peg coating with FBS showed an adverse effect on the biofilm formation of these strains. By contrast, there was a statistically significant increase in biofilm biomass production on pegs coated with FBS and HP for SE (ATCC 35983).

Virulence and Drug-Resistance of Staphylococcus aureus Strains Isolated from Venous Ulcers in Polish Patients

Infected chronic venous ulcers (VUs) represent a major health problem. We analysed the aerobic microbiome in the VUs, the virulence, and drug-resistance of Staphylococcus aureus (SA) strains. Swabs from 143 outpatients and inpatients Polish subjects were collected. SA strains were tested for drug sensitivity using a phenotyping method and for methicillin-resistant SA (MRSA) and macrolide-lincosamide-streptogramin B (MLSB) resistance using PCR. We analysed virulence genes, the genetic similarity of strains, and performed Staphylococcal cassette chromosome mec typing and Staphylococcal protein A typing. SA was isolated as a single one in 34.9% of cases, 31.5% paired with another pathogen, and 33.6% S. aureus combined with at least two other strains. The majority of SA isolates (68.5%) possessed the virulence lukE gene. Drug resistance was significantly common in hospitalised than in ambulatory patients (OR 3.8; 95%CI 1.8–7.91). MLSB (altogether in 19.6% isolates) were observed mostly in non-hospitalised patients (OR 9.1; 95%CI 1.17–71.02), while MRSA was detected in 11.9% of strains equally. Hospitalisation and patient’s age group (aged > 78.0 or < 54.5 years) were significant predictors of the multi-drug resistant SA (MDR-SA). Over 30% of the infected VUs were associated with multi-species biofilms and presence of potentially highly pathogenic microorganisms. Elderly hospitalised patients with chronic venous ulcers are prone to be infected with a MDR-SA.

Endotracheal tubes coated with a broad-spectrum antibacterial ceragenin reduce bacterial biofilm in an in vitro bench top model.

Ventilator-associated pneumonia is one of the most common nosocomial infections, caused mainly by bacterial/fungal biofilm. Therefore, it is necessary to develop preventive strategies to avoid biofilm formation based on new compounds. We performed an in vitro study to compare the efficacy of endotracheal tubes (ETTs) coated with the ceragenin CSA-131 and that of uncoated ETTs against the biofilm of clinical strains of Pseudomonas aeruginosa (PA), Escherichia coli (EC) and Staphylococcus aureus (SA). We applied an in vitro bench top model using coated and uncoated ETTs that were treated with three different clinical strains of PA, EC and SA for 5 days. After exposure to biofilm, ETTs were analysed for cfu count by culture of sonicate and total number of cells by confocal laser scanning microscopy. The median (IQR) cfu/mL counts of PA, EC and SA in coated and uncoated ETTs were, respectively, as follows: 1.00 × 101 (0.0-3.3 × 102) versus 3.32 × 109 (6.6 × 108-3.8 × 109), P < 0.001; 0.0 (0.0-5.4 × 103) versus 1.32 × 106 (2.3 × 103-5.0 × 107), P < 0.001; and 8.1 × 105 (8.5 × 101-1.4 × 109) versus 2.7 × 108 (8.6 × 106-1.6 × 1011), P = 0.058. The median (IQR) total number of cells of PA, EC and SA in coated and non-coated ETTs were, respectively, as follows: 11.0 [5.5-not applicable (NA)] versus 87.9 (60.5-NA), P = 0.05; 9.1 (6.7-NA) versus 62.6 (42.0-NA), P = 0.05; and 97.7 (94.6-NA) versus 187.3 (43.9-NA), P = 0.827. We demonstrated significantly reduced biofilm formation in coated ETTs. However, the difference for SA was not statistically significant. Future clinical studies are needed to support our findings.

S. pseudintermedius and S. aureus lineages with transmission ability circulate as causative agents of infections in pets for years

BackgroundStaphylococcus pseudintermedius (SP) and Staphylococcus aureus (SA) are common colonizers of companion animals, but they are also considered opportunistic pathogens, causing diseases of diverse severity. This study focused on the identification and characterization of 33 coagulase-positive staphylococci isolated from diseased pets (28 dogs and five cats) during 2009–2011 in a veterinary hospital in Spain in order to stablish the circulating lineages and their antimicrobial resistance profile.ResultsTwenty-eight isolates were identified as SP and five as SA. Nine methicillin-resistant (MR) isolates (27%) carrying the mecA gene were detected (eight MRSP and one MRSA). The 55% of SP and SA isolates were multidrug-resistant (MDR). MRSP strains were typed as ST71-agrIII-SCCmecII/III-(PFGE) A (n=5), ST68-agrIV-SCCmecV-B1/B2 (n=2), and ST258-agrII-SCCmecIV-C (n=1). SP isolates showed resistance to the following antimicrobials [percentage of resistant isolates/resistance genes]: penicillin [82/blaZ], oxacillin [29/mecA] erythromycin/clindamycin [43/erm(B)], aminoglycosides [18–46/aacA-aphD, aphA3, aadE], tetracycline [71/tet(M), tet(K)], ciprofloxacin [29], chloramphenicol [29/catpC221], and trimethoprim-sulfamethoxazole [50/dfrG, dfrK]. The dfrK gene was revealed as part of the radC-integrated Tn559 in two SP isolates. Virulence genes detected among SP isolates were as follow [percentage of isolates]: siet [100], se-int [100], lukS/F-I [100], seccanine [7], and expB [7]. The single MRSA-mecA detected was typed as t011-ST398/CC398-agrI-SCCmecV and was MDR. The methicillin-susceptible SA isolates were typed as t045-ST5/CC5 (n=2), t10576-ST1660 (n=1), and t005-ST22/CC22 (n=1); the t005-ST22 feline isolate was PVL-positive and the two t045-ST45 isolates were ascribed to Immune Evasion Cluster (IEC) type F. Moreover, the t10576-ST1660 isolate, of potential equine origin, harbored the lukPQ and scneq genes. According to animal clinical history and data records, several strains seem to have been acquired from different sources of the hospital environment, while some SA strains appeared to have a human origin.ConclusionsThe frequent detection of MR and MDR isolates among clinical SP and SA strains with noticeable virulence traits is of veterinary concern, implying limited treatment options available. This is the first description of MRSA-ST398 and MRSP-ST68 in pets in Spain, as well the first report of the dfrK-carrying Tn559 in SP. This evidences that current transmissible lineages with mobilizable resistomes have been circulating as causative agents of infections among pets for years.

Activity of Temporin A and Short Lipopeptides Combined with Gentamicin against Biofilm Formed by Staphylococcus aureus and Pseudomonas aeruginosa.

The formation of biofilms on biomaterials causes biofilm-associated infections. Available treatments often fail to fight the microorganisms in the biofilm, creating serious risks for patient well-being and life. Due to their significant antibiofilm activities, antimicrobial peptides are being intensively investigated in this regard. A promising approach is a combination therapy that aims to increase the efficacy and broaden the spectrum of antibiotics. The main goal of this study was to evaluate the antimicrobial efficacy of temporin A and the short lipopeptides (C10)2-KKKK-NH2 and (C12)2-KKKK-NH2 in combination with gentamicin against biofilm formed by Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA). Peptides were synthesized with solid-phase temperature-assisted synthesis methodology. The minimum inhibitory concentrations (MICs), fractional inhibitory concentrations (FICs), minimum biofilm eradication concentrations (MBECs), and the influence of combinations of compounds with gentamicin on bacterial biofilm were determined for reference strains of SA (ATCC 25923) and PA (ATCC 9027). The peptides exhibited significant potential to enhance the antibacterial activity of gentamicin against SA biofilm, but there was no synergy in activity against planktonic cells. The antibiotic applied alone demonstrated strong activity against planktonic cells and poor effectiveness against SA biofilm. Biofilm formed by PA was much more sensitive to gentamicin, but some positive influences of supplementation with peptides were noticed. The results of the performed experiments suggest that the potential application of peptides as adjuvant agents in the treatment of biofilm-associated infections should be studied further.

Epidemiology, Drug Resistance, and Virulence of Staphylococcus aureus Isolated from Ocular Infections in Polish Patients.

Analysis of the epidemiology of Staphylococcus aureus (SA) ocular infections and virulence factors of the isolates with a special emphasis on their drug resistance, and the ability of biofilm formation. In a period from 2009 to 2013, 83 isolates of SA were prospectively collected and preserved in a multicenter laboratory-based study carried out in southern Poland. Epidemiological, phenotypic, and genotypic analyses were performed. The resistance and virulence genes were analyzed. Screening for the biofilm formation was provided. Among the materials derived from ocular infections from 456 patients, SA was found in 18.2% (n = 83) of cases (one SA isolate per one patient). Most infections were identified in the age group of over 65 years (OR 8.4 95%CI; 1.03-68.49). The majority of patients (73.4%) were hospitalized. Among the virulence and resistance genes, the most frequently detected were the lukE (72.2%, n = 60) and ermA (15.6%, n = 13) genes. A positive result of the CRA test (the ability of biofilm formation) was found in 66.2% (n = 55) of isolates. Among the strains under study, 6.0% (n = 5) had the methicillin-resistant Staphylococcus aureus phenotype, and 26.5% (n = 22) had the macrolide-lincosamide-streptogramin B phenotype. In 48 (57.8%) isolates the neomycin resistance was revealed. All isolates under study were sensitive to vancomycin. The population most susceptible to ocular SA infections consists of hospitalized patients aged 65 and more. The SA strains under study showed the increased ability to biofilm formation. In the strains tested, high susceptibility to chloramphenicol and fluoroquinolones was demonstrated. However, the high level of drug resistance to neomycin detected in this study among SA isolates and the blood-ocular barrier makes it difficult to treat ocular infections.

627. Molecular Epidemiology of Daptomycin-resistant Staphylococcus aureus Causing Bloodstream Infections

BackgroundDaptomycin (Dap) has become increasingly important to combat infections caused by Staphylococcus aureus (SA). However, reports of daptomycin nonsusceptible (DNS) SA strains have emerged over the recent years, specifically with alteration of the MprF gene. Mutations in this gene lead to an increase in lysyl-phosphatidyl glycerol production resulting in a lower susceptibility to Dap and cationic antimicrobial peptides. Point mutations in the cls2 gene have also been associated with DNS SA through similar mechanisms of action. This study describes the gene mutations involved in our patients with SA bloodstream infections (BSIs) that developed Dap resistance.MethodsThis is a retrospective study in a tertiary healthcare system in southeast Michigan. SA blood isolates were sequenced through a gene-by-gene approach using Ridom SeqSphere. Charts were reviewed for clinical and laboratory data. Dap minimum inhibitory concentrations (MICs) were determined by E-test. DNS was defined as an MIC > 1.0 µg/mL.ResultsFrom December 3, 2015 to May 8, 2017, 7 patients were identified with DNS SA BSI. Mean age of the patients was 59 (SD = 13.7) and 86% were male. Mean bacteremia duration was 7 days (SD = 6.3), mean hospital length of stay was 13 days (SD = 9.8), 5 isolates were methicillin-resistant SA, and 2 were methicillin-sensitive SA. When comparing molecular sequencing of all seven patients with their Dap-sensitive strains vs. resistant strains, all patients, except for patient 3, had a point mutation at different allele loci in the MprF gene (Table 1). Additionally, two patients had point mutations in the cls2 gene. When comparing all 27 strains from the seven patients, each cluster was attributed to an individual patient with the exception of cluster one that included patients 5 and 6 suggesting transmission (Figure 1).ConclusionDNS SA is infrequent; however, the present study shows that it can emerge both from methicillin-resistant and -susceptible SA most commonly when developing MprF and cls2 gene mutations as seen in our patients. Patient 3 was the exception in our analysis and likely developed DNS SA through another gene mutation. Further studies are necessary to know the frequency of each mutation with all genes associated with DNS SA, and epidemiological risk factors of transmission. Disclosures All authors: No reported disclosures

Associations of pathogen-specific and host-specific characteristics with disease outcome in patients with Staphylococcus aureus bacteremic pneumonia.

ObjectiveTo understand the relationships of Staphylococcus aureus (SA) bacteremic pneumonia (SABP) outcome with patient‐specific and SA‐specific variables.MethodsWe analysed SA bloodstream isolates and matching sera in SABP patients by sequencing SA isolates (n = 50) and measuring in vitro AT production, haemolytic activity and expression of ClfA and ClfB. Controls were sera from gram‐negative bacteremia patients with or without pneumonia and uninfected subjects. Levels of IgGs, IgMs and neutralizing antibodies (NAbs) against SA antigens were quantified and analysed by one‐way ANOVA. Associations of patient outcomes with patient variables, antibody levels and isolate characteristics were evaluated by univariate and multivariate logistic regression analyses.ResultsSABP patients had higher levels of IgGs against eight virulence factors and anti‐alpha toxin (AT) NAbs than uninfected controls. Levels of IgG against AT and IgMs against ClfA, FnbpA and SdrC were higher in clinically cured SABP patients than in clinical failures. Anti‐LukAB NAb levels were elevated in all cohorts. Increased odds of cure correlated with higher haemolytic activity of SA strains, longer time between surgery and bacteremia (> 30 days), longer duration of antibiotic therapy, lower acute physiology and total APACHE II scores, lack of persistent fever for > 72 h and higher levels of antibodies against AT (IgG), ClfA (IgM), FnbpA (IgM) and SdrC (IgM).DiscussionLimitations included the cross‐sectional observational nature of the study, small sample size and inability to measure antibody levels against all SA virulence factors.ConclusionOur results suggest that SABP patients may benefit from immunotherapy targeting multiple SA antigens.

Detection of β-lactamase and antibiotic susceptibility of clinical isolates of Staphylococcus aureus

Abstract Staphylococcus aureus (SA) is the pathogen of greatest concern in the clinical worldwide because of its intrinsic virulence and ability to cause the diverse array of life-threatening infections. In the last two decades or so, antimicrobial resistance has evolved into one of the most formidable problems of infectious diseases. Bacteria acquire resistance to β-lactam antibiotics, most commonly due to the production of enzymes called β-lactamase. This enzyme hydrolyzes the amide bond of β-lactam antibiotics to make them ineffective. In this study, screening of clinical isolates of SA showed β-lactamase activity in 11 clinical isolates (SA-1745, SA-2071, SA-2940, SA-3151, SA-4423, SA-4620, SA-4627, SA-4693, SA-4696, SA-10760, Methicillin-resistant Staphylococcus aureus (MRSA), and one wild- type strain (SA-96) by iodometric and nitrocefin disk methods. The antibiotic susceptibility profiles of 12 strains of SA were determined for four β-lactam antibiotics viz. penicillin, ampicillin, cefoxitin and oxacillin. All the clinical isolates showed resistant to these antibiotics. Further, minimum inhibitory concentration (MIC) of four β-lactam antibiotics was also determined by micro-broth dilution assay. The MIC of penicillin, ampicillin and oxacillin against wild-type strain of SA was 0.078 µg/ml, whereas the MIC of cefoxitin against wild-type strain was recorded as 1.25 µg/ml. The MIC of these antibiotics against 11 clinical isolates of SA including methicillin-resistant Staphylococcus aureus (MRSA) ranged between 1.95 µg/ml to 1000 µg/ml. All clinical isolates were highly resistant to penicillin and ampicillin followed by cefoxitin and oxacillin. The outcomes of the present study can be useful for the screening of other natural bioactive compounds against these virulent strains.

Genetic typing and susceptibility testing of strains from Staphylococcus aureus keratitis or conjunctivitis patients

Objective: To study the relationship between genetic typing and the antibiotic susceptibility of staphylococcus aureus (SA) isolated from keratitis or conjunctivitis patients. Methods: Experimental study. Thirty-four (34) strains of Staphylococcus aureus were isolated from 34 cases of keratitis or conjunctivitis. The genomic DNA was extracted and amplified with PCR. With the method of multi locus sequences typing (MLST), gene fragments from 7 house-keeping genes were amplified and the products were sequenced. The results were submitted to the MLST website (www.pubmlst.org). In comparison with the allele of the corresponding gene, the allele spectrums of the strain were obtained with 7 housekeeping genes. At last, the MLST genotypes of the isolated strains were determined. With the START software, the evolutionary tree was established with UPGMA method. With the microdilution method, the MIC(90) of 13 antimicrobial agents was determined. The MIC(90) value of antimicrobial agents among different genotypes of Staphylococcus aureus was comparatively analyzed. Results: Ten (10) genotypes were obtained from 34 strains of Staphylococcus aureus. The dominant types were ST239, ST2592 and ST188. The clustering of genotyping was relatively concentrated, mainly in group Ⅰ (25 strains of SA, 83.3% of the total), and followed by group Ⅱ (5 strains of SA, 16.7%). The conjunctival isolates were distributed in the subgroup A of group Ⅰ. The cornea isolates were concentrated in subgroup B and group Ⅱ. With the exact probability method, the R×C chi square tests were used as statistic analysis method. The difference between the bacterial genotyping of two sources was statistically significant (P=0.011). Twenty-four strains of SA in group Ⅰ was sensitive to Vancomycin, Rifampicin and Amikacin (sensitivity ratio was 24/24, 20/24 and 20/24, respectively), and was generally resistant to other antibiotics. The values of MIC(90) of ciprofloxacin, ofloxacin, gatifloxacin and moxifloxacin to Staphylococcus aureus in subgroup A (0.16±0.07, 0.51±0.42, 0.31±0.14, 0.22±0.33) were significantly lower than the values in subgroup B(0.74±0.11, 0.84±0.45, 0.67±0.03, 0.68±0.26). The difference was statistically significant (P=0.004, 0.026, 0.034, 0.001). There was no significant difference between the MIC(90) values of the other 9 kinds of antibiotics in the subgroup A and in the subgroup B of Staphylococcus aureus (P value 0.047-0.561). Conclusion: The genotype of Staphylococcus aureus of corneal isolations and conjunctival isolations were different. The conjunctival isolates were distributed in the subgroup A of group Ⅰ and the corneal isolates were concentrated in subgroup B and group Ⅱ. There is a significant correlation between the MLST genotypes and antibiotic sensitivity. (Chin J Ophthalmol, 2018, 54:767-774).