Clinical Isolates Of Staphylococci Research Articles

Biofilm Eradication and Inhibition of Methicillin-resistant Staphylococcus Clinical Isolates by Curcumin-Chitosan Magnetic Nanoparticles.

Biofilm-producing methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative Staphylococci (MR-CoNS) are a clinical challenge for the treatment of healthcare-associated infections. As alternative antimicrobial options are needed, we aimed to determine the effect of curcumin-chitosan magnetic nanoparticles on the biofilm of staphylococcal clinical isolates. MRSA and CoNS clinical isolates were identified by MALDI-TOF mass spectrometry. Antimicrobial susceptibility testing was performed by broth microdilution. Nanoparticles were synthesized by co-precipitation of magnetic nanoparticles (MNP) and encapsulation by ionotropic gelation of curcumin (Cur) and chitosan (Chi). Biofilm inhibition and eradication by nanoparticles with and without the addition of oxacillin was assessed on staphylococcal strains. Cur-Chi-MNP showed antimicrobial activity on planktonic cells of MRSA and MR-CoNS strains and inhibited biofilm of MRSA. The addition of OXA to Cur-Chi-MNP increased biofilm inhibition and eradication activity against all Staphylococci strains (p=0.0007); higher biofilm activity was observed in early biofilm stages. Cur-Chi-MNP showed antimicrobial and biofilm inhibition activity against S. aureus. The addition of OXA increased biofilm inhibition and eradication activity against all Staphylococci strains. A combination treatment of Cur-Chi-MNP and OXA could be potentially used to treat staphylococcal biofilm-associated infections in its early stages before the establishment of biofilm bacterial cells.

Isolation and Characterization of New Bacteriophages against Staphylococcal Clinical Isolates from Diabetic Foot Ulcers.

Staphylococcus sp. is the most common bacterial genus in infections related to diabetic foot ulcers (DFUs). The emergence of multidrug-resistant bacteria places a serious burden on public health systems. Phage therapy is an alternative treatment to antibiotics, overcoming the issue of antibiotic resistance. In this study, six phages (SAVM01 to SAVM06) were isolated from effluents and were used against a panel of staphylococcal clinical samples isolated from DFUs. A genomic analysis revealed that the phages belonged to the Herelleviridae family, with sequences similar to those of the Kayvirus genus. No lysogeny-associated genes, known virulence or drug resistance genes were identified in the phage genomes. The phages displayed a strong lytic and antibiofilm activity against DFU clinical isolates, as well as against opportunistic pathogenic coagulase-negative staphylococci. The results presented here suggest that these phages could be effective biocontrol agents against staphylococcal clinical isolates from DFUs.

Prevalence of Coagulase Negative Strains of Staphylococcus aureus in Clinical Specimens at the University of Benin Teaching Hospital, Benin City, Nigeria

The test for coagulase activity is a simple and non-expensive test for the identification of S. aureus strains from clinical samples in resource-poor settings. The results of these tests have been reported to vary with the source of plasma and with some atypical strains that test negative with the coagulase test. The study was carried out to determine if misidentification of strains of S. aureus exists on account of the reliance on the coagulase tests. Clinical isolates of Staphylococci that tested negative by the coagulase tests from wounds, pus, aspirates, blood cultures and urogenital samples were collected and re-tested by the slide and tube coagulase tests and confirmed to be negative. Each isolate was inoculated onto mannitol sat agar and DNAse agar plates and incubated at 37oC for 18 h. Isolates that fermented mannitol and showed a positive DNAse test were 25/366 (6.8%). The highest number of coagulase-negative Staphylococcus aureus (CNSA) occurred within blood culture samples 25.7% and in wound, pus and aspirates, 6.9% and the least proportion, 2.9% from urogenital samples. The generation of CNSA strains is strongly associated with severely-ill patients and the potential for the administration of cell-wall inhibiting antibacterial agents may have important roles to play in the emergence CNSA. The inclusion of DNAse test in the routine identification of staphylococci is advocated especially when an isolate has tested coagulase-negative.

In-vitro assessment of staphylococci biofilms formed under biologically-relevant conditions and correlation to the biofilm genotype

Staphylococci have been implicated in chronic device-related infections due to their ability to form resistant biofilms on implanted medical devices. For a long time, two different mechanisms of biofilm formation were known in Staphylococcus spp., the ica-dependent biofilms in MSSA and CoNS and the ica-independent biofilms in MRSA. Recently, a new fibrin-based biofilm phenotype was identified when S. aureus isolates were allowed to construct biofilms in biologically-relevant conditions using plasma-coated surfaces and RPMI-1640 for biofilm development (RPMI-1640/Pl). In this study, 140 staphylococci clinical isolates (91 MRSA, 27 MSSA and 22 CoNS) were tested for biofilm formation, biofilm formers were selected and used to scrutinize the ability of RPMI-1640/Pl to support staphylococci biofilm formation. Results showed that, in RPMI-1640/Pl, the biofilm formation abilities of MRSA and MSSA isolates were non-significantly different compared to those formed in TSB and BHI, (Kruskal Wallis test, P = 0.3275 and 0.466 for MRSA and MSSA isolates, respectively). However, a significantly different biofilm formation ability was observed regarding the tested CoNS isolates (ANOVA test, P = 0.0006). Furthermore, biofilm formation in RPMI-1640/Pl under different incubation conditions was tested, and among the tested conditions, 48h of static incubation showed significantly elevated biofilm for both MRSA and MSSA. Finally, PCR was used to detect genes implicated in biofilm formation, and the genotypes were correlated to the biofilm formation ability in different tested conditions. In contrast to ordinary media, biofilm formation by staphylococci in RPMI-1640/Pl was positively correlated to coa, fnbA, fnbB and clfB.

Phenotypic and Genotypic Characterization of Methicillin Resistance in Staphylococci Isolated from an Egyptian University Hospital.

Methicillin-resistant in Staphylococci is a serious public health issue. It is mostly encoded by the mecA gene. The mecC gene is a new mecA analog responsible for resistance to methicillin in some Staphylococcal clinical isolates. This mecC gene is still underestimated in Egypt. The aim of the current study was to detect mecA and mecC genes in clinical Staphylococci isolates from a tertiary care university hospital in Egypt compared to the different phenotypic methods. A total of 118 Staphylococcus aureus (S. aureus) and 43 coagulase-negative Staphylococci (CoNS) were identified from various hospital-acquired infections. Methicillin resistance was identified genotypically using the PCR technique and phenotypically using the cefoxitin disc diffusion test, oxacillin broth microdilution and the VITEK2 system in all Staphylococcal isolates. The mecA gene was detected in 82.2% of S. aureus and 95.3% of CoNS isolates, while all of the isolates tested negative for the mecC gene. Interestingly, 30.2% of CoNS isolates showed the unique character of inducible oxacillin resistance, being mecA-positive but oxacillin-susceptible (OS-CoNS). The dual use of genotypic and phenotypic methods is highly recommended to avoid missing any genetically divergent strains.

High level of persister frequency in clinical staphylococcal isolates

BackgroundStaphylococcus aureus is a notorious human pathogen that causes often lethal systemic conditions that are mostly medical device associated biofilm infections. Similarly, coagulase negative staphylococci are emerging as leading pathogen for nosocomial infections owing to their ability to form biofilm on implanted medical equipment. Chronic in nature, these infections are difficult to treat. Such recalcitrance of these infections is caused mainly due to the presence of persister cells, which exhibit transient yet extreme tolerance to antibiotics. Despite tremendous clinical significance, there is lack of studies on persister cells formation among clinical bacterial isolates. Considering the importance of factors influencing persister formation, in this study, we evaluate the association of antibiotic tolerance with biofilm production, antibiotic stress, growth phase, specimen type, and dependency on staphylococcal species. Biofilm formation was detected among 375 clinical staphylococcal isolates by quantitative tissue culture plate method (TCP) and icaAD genes by genotypic method. The antibiotic susceptibility was determined by Kirby Bauer disc diffusion method while minimum inhibitory concentration values were obtained by agar dilution method. Persister cells were measured in the susceptible staphylococcal isolates in the presence of clinically relevant antibiotics.ResultsIn the study, 161 (43%) S. aureus and 214 (57%) coagulase negative staphylococci (CNS) were isolated from different clinical samples. TCP method detected biofilm production in 84 (52.2%) S. aureus and 90 (42.1%) CNS isolates. The genotypic method detected icaAD genes in 86 (22.9%) isolates. Majority (> 90%) of both the biofilm producers and non-producers were sensitive to chloramphenicol and tetracycline but were resistant to penicillin. Interestingly, all isolates were sensitive to vancomycin irrespective of biofilm production. While high persister frequency was observed among all staphylococci isolates in the stationary growth phase, the persister frequency in exponential growth phase was statistically high among isolates possessing icaAD genes compared to icaAD negative isolates.ConclusionThe research findings provide strong evidence that the clinical staphylococcal isolates exhibit extreme antibiotic tolerance suggesting their causal link with treatment failures. Understanding the factors influencing the formation and maintenance of persister cells are of utmost important aspect to design therapeutics and control recalcitrant bacterial infections.

INVESTIGATING SENSITIVITY OF TYPICAL STRAINS AND CLINICAL ISOLATES OF STAPHYLOCOCCUS SPP. TO DECASAN, CHLORHEXIDINE AND IODOPHERE BY DISCA-DIFFUSION METHOD EUCAST

Antibiotic resistance is a global challenge, whose emergence is associated with the irrational and uncontrolled use of antimicrobial drugs, or the use of antibiotics to prevent local and systemic complications during the surgical operations. Therefore, the number of failed outcomes of the planned treatment of infectious and inflammatory processes with antibiotics has increased. Given this fact, it is important to study the potential of antiseptic drugs, which usually do not cause the microorganism resistance and thus are more advantageous over antibiotics. Objective of this study is to compare the effects of antiseptics Decasan, Chlorhexidine and Iodoform on clinical and typical strains of microorganisms using the disco-diffusion method according to the EUCAST. Materials and methods: Typical strains of Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 14990 were used as the studied cultures of microorganisms. Clinical isolates of the microorganisms Staphylococcus aureus, Staphylococcus epidermidis were collected from patients with postextraction complications, who took treatment at surgical departments of Poltava. Identification of isolated cultures was performed using an automatic bacteriological analyzer Vitec-2 compact bioMarieux (France). The antibacterial effect of antiseptic drugs Dekasan, Chlorhexidine, Iodoform was studied. Given that iodoform is poorly soluble in water, we used its 5% alcohol solution, which was prepared ex tempore. The susceptibility of the studied strains to antiseptics was determined by disco-diffusion method according to the EUCAST committee. The determination was performed by using Mueller-Hinton medium (HIMEDIA, India) with standard sterile disks, which were not impregnated (HIMEDIA, India). Then we impregnated them with solutions of Decasan, Chlorhexidine and 5% alcohol solution of iodoform. We used a microbial inoculum equivalent to according to the McFarland turbidity standard. Prepared Petri dishes containing impregnated paper disks were incubated overnight at 35-36 °C, after which the results were determined. Results and discussion: The study has shown that the antiseptic Decasan has a greater antistaphylococcal effect on typical strains and clinical isolates of staphylococci compared to other antiseptics, which are often used in dentistry. The lowest antistaphylococcal effect on the studied microorganisms has been found by applying 5% alcohol solution of iodoform, Chlorhexidine has demonstrated the moderate effect on the strains under the study.

Assessment of Staphylococcal Clinical Isolates from Periprosthetic Joint Infections for Potential Bacteriophage Therapy.

Bacteriophage therapy is a potential adjunctive treatment for periprosthetic joint infections (PJIs) given the capabilities of bacteriophages to degrade biofilms, self-replicate, and lyse bacteria. However, many aspects of this therapeutic are ill-defined, and the narrow spectrum of bacteriophage activity along with limited available bacteriophage strains curb potential use for specific bacteria such as Staphylococcus aureus at the present time. Therefore, the aim of this study was to determine the feasibility of using bacteriophages for PJI by (1) categorizing the causative organisms in hip and knee PJI at a tertiary academic center and (2) evaluating in vitro activity of a group of bacteriophages against clinical S. aureus PJI isolates. Patients with chronic hip or knee PJI after undergoing the first stage of a 2-stage revision protocol from 2017 to 2020 were identified retrospectively by a query of the hospital billing database. The causative pathogens in 129 cases were reviewed and categorized. From this cohort, preserved S. aureus isolates were tested against a library of 15 staphylococcal bacteriophages to evaluate for bacterial growth inhibition over 48 hours. S. aureus was the most common pathogen causing PJI (26% [33] of 129 cases). Of 29 S. aureus samples that were analyzed for bacteriophage activity, 97% showed adequate growth inhibition of the predominant planktonic colonies by at least 1 bacteriophage strain. However, 24% of the 29 samples demonstrated additional smaller, slower-growing S. aureus colonies, none of which had adequate growth inhibition by any of the initial 14 bacteriophages. Of 5 secondary colonies that underwent subsequent testing with another bacteriophage with enhanced biofilm activity, 4 showed adequate growth inhibition. Effective bacteriophage therapeutics are potentially available for S. aureus PJI isolates. The differences in bacteriophage activity against the presumed small-colony variants compared with the planktonic isolates have important clinical implications. This finding suggests that bacteriophage attachment receptors differ between the different bacterial morphologic states, and supports future in vitro testing of bacteriophage therapeutics against both planktonic and stationary states of PJI clinical isolates to ensure activity.

The SCCmec Types and Antimicrobial Resistance among Methicillin-Resistant Staphylococcus Species Isolated from Dogs with Superficial Pyoderma.

This study characterizes clinical methicillin-resistant staphylococcal (MRS) isolates obtained from superficial pyoderma infections in dogs. Our interest was to determine the staphylococcal cassette chromosome mec (SCCmec) type and the antimicrobial susceptibility among MRS isolates from clinical cases. Skin swabs were collected and cultured. Staphylococcus species were identified and characterized with biochemical tests and MALDI-TOF-MS and antimicrobial susceptibility testing by disk diffusion. mecA detection and staphylococcal cassette chromosome mec (SCCmec) typing were achieved by PCR. Of the 65 clinical samples, 56 (86.2%) staphylococcal infections were identified. Twelve (21%) of 56 isolates were MRS infections. All MRS isolates were multidrug resistant. The ccrC and class-C2 mec, which were SCCmec type V, were the most prevalent (66.7%) among the 12 MRS isolates. The predominant SCCmec type V was found in S. aureus, S. intermedius group, S. lentus, S. xylosus, and S. arlettae. Treatment failure is a concern with the emergence of highly resistant MRS in dogs associated with superficial pyoderma. The detection of type V SCCmec MRS has previously been reported among veterinarians and dog owners but not in Northern Thailand. These infections serve as a reminder to improve infection prevention and control measures including reducing environmental contamination and potential zoonotic exposures to MRS. In addition, educational awareness of these risks in small animal hospitals needs to be increased among veterinary hospital staff, clients, and patients.

The Antibacterial Properties of 4, 8, 4', 8'-Tetramethoxy (1,1'-biphenanthrene) -2,7,2',7'-Tetrol from Fibrous Roots of Bletilla striata.

Biphenanthrene compound, 4, 8, 4', 8'-tetramethoxy (1, 1'-biphenanthrene)-2, 7, 2', 7'-tetrol (LF05), recently isolated from fibrous roots of Bletilla striata, exhibits antibacterial activity against several Gram-positive bacteria. In this study, we investigated the antibacterial properties, potential mode of action and cytotoxicity. Minimum inhibitory concentrations (MICs) tests showed LF05 was active against all tested Gram-positive strains, including methicillin-resistant Staphylococcus aureus (MRSA) and staphylococcal clinical isolates. Minimum bactericidal concentration (MBC) tests demonstrated LF05 was bactericidal against S. aureus ATCC 29213 and Bacillus subtilis 168 whereas bacteriostatic against S. aureus ATCC 43300, WX 0002, and other strains of S. aureus. Time-kill assays further confirmed these observations. The flow cytometric assay indicated that LF05 damaged the cell membrane of S. aureus ATCC 29213 and B. subtilis 168. Consistent with this finding, 4 × MIC of LF05 caused release of ATP in B. subtilis 168 within 10min. Checkerboard test demonstrated LF05 exhibited additive effect when combined with vancomycin, erythromycin and berberine. The addition of rat plasma or bovine serum albumin to bacterial cultures caused significantly loss in antibacterial activity of LF05. Interestingly, LF05 was highly toxic to several tumor cells. Results of these studies indicate that LF05 is bactericidal against some Gram-positive bacteria and acts as a membrane structure disruptor. The application of biphenanthrene in the treatment of S. aureus infection, especially local infection, deserves further study.

Emergence of resistance to last-resort antibiotics in clinical isolates of staphylococci with special reference to daptomycin and linezolid

Background: Treatment of staphylococcal infections has been complicated by the continuous emergence of antibiotic-resistant strains. Objective: In this study, we investigated the resistance pattern of clinical isolates of both coagulase-positive and coagulase-negative staphylococci to antibiotics recently introduced to treat staphylococcal infections. Methodology: Minimum inhibitory concentrations of antibiotics were determined by agar dilution or broth microdilution method. Identification of daptomycin- and linezolid-resistant isolates was performed by matrixassisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Moreover, the mechanism of resistance to daptomycin and linezolid was investigated on the molecular basis using PCR and amplicon sequencing. Results: Out of 104 staphylococci, 9 were resistant to ciprofloxacin (8.7%), 68 to clindamycin (65.4%), 2 to daptomycin (1.9%), 54 to erythromycin (51.9%), 6 to linezolid (5.8%), 3 to nitrofurantoin (2.9%), 75 to oxacillin (72.1%), 37 to teichoplanin (35.6%), 69 to tetracycline (66.3%), 51 to tigecycline (49%), and 41 to vancomycin (39.4%). Identification of daptomycin- and linezolid-resistant isolates revealed that they belong to Staphylococcus aureus, S. hominis, S. capitis, and S. epidermidis. In addition, sequencing of the mprF gene conferring daptomycin resistance revealed L431F and S829L point mutations in the two resistant isolates identified as S. aureus and S. hominis, respectively. Furthermore, linezolid resistance was due to optrA gene in two, and cfr in three of the resistant isolates. Conclusion: Resistance to the last-resort antibiotics used to treat staphylococcal infection has emerged. Therefore, the use of daptomycin and linezolid should be restricted to critical cases not susceptible to other available agents.

Prevalence and mechanisms of linezolid resistance among staphylococcal clinical isolates from Egypt

The emergence of methicillin-resistant staphylococci necessitated the search for alternative agents as linezolid, introduced to treat infections due to multidrug-resistant bacteria. Linezolid resistance has since emerged, yet its global prevalence remains low. In Egypt, little is known about the situation. We investigated the prevalence and mechanisms of resistance among Egyptian staphylococcal clinical isolates. Linezolid resistance among 232 staphylococcal isolates obtained from Alexandria Main Hospitals between 2011 and 2016 was assessed using disc diffusion and minimum inhibitory concentration. Resistant isolates were checked for cfr presence using polymerase chain reaction. The V domain of different alleles of 23S rRNA gene was investigated for mutations. Selection for linezolid-resistant mutants was performed in vitro through serial passages in linezolid sub-inhibitory concentrations. Combinations of linezolid with imipenem or anti-inflammatory agents were investigated using time-kill and modified checkerboard assays. Three Staphylococcus haemolyticus isolates (1.3%) from 2015 to 2016 were linezolid-resistant. One isolate carried cfr which was plasmid-borne, and together with another isolate which had a G2603T point mutation in the V domain of 23S rRNA gene. Successive exposure to linezolid sub-inhibitory concentrations was selected for three resistant Staphylococcus aureus mutants out of ten susceptible isolates. These mutants were more resistant towards different antibiotic classes than their susceptible parents. Linezolid combinations with imipenem, ibuprofen, or aspirin were synergistic against the isolates and mutants. Despite unregulated use of linezolid, resistance remains fairly low among the Egyptian isolates. Strict antimicrobial stewardship guidelines are needed in hospitals and the community to guard against further evolution of resistant mutants.

Phenotypic Detection of Inducible Clindamycin Resistance among Clinical Isolates of Staphylococcus aureus in Bharatpur Hospital

Background: Clindamycin is regarded as a reserve drug in the treatment of staphylococcal infections. Among few therapeutic alternatives available for treatment of erythromycin-resistant Staphylococcus aureus infections, clindamycin has several advantages but major limitation in its use is the development of resistance resulting in treatment failure. Routine clindamycin susceptibility test may fail to detect such inducible resistance which can be detected by Double disc diffusion test (D-test). The present study was undertaken to determine the incidence of inducible clindamycin resistance among clinical isolates of S. aureus in a tertiary care hospital in central Nepal. Methods: A cross-sectional study was carried out among the patients visiting Bharatpur Hospital from September to November 2019. A total of 1279 clinical samples were examined for the identification of S. aureus by standard microbiological procedures. Antibiotic susceptibility testing of the isolates was done by Kirby-Bauer disc diffusion method and all the erythromycin-resistant isolates were subjected to D-test for the phenotypic detection of inducible clindamycin resistance according to CLSI guidelines (2016). Results: S. aureus was recovered from 4.5% (58/1279) samples of which 35 isolates were Methicillin-Resistant Staphylococcus aureus (MRSA) and 23 were multi-drug resistant (MDR). Tetracycline was found to be the most effective antibiotic whereas erythromycin was the least effective. D-test revealed that 39.7% isolates showed iMLSB phenotype, 3.5% showed cMLSB phenotype and 56.8% showed MS phenotype. The percentage of inducible and constitutive resistance was seen higher amongst MRSA isolates compared to Methicillin-Sensitive Staphylococcus aureus (MSSA) isolates. Incidence of S. aureus was found higher among females and in the age group 20-30 years and in pus samples (p<0.01). Conclusions: Routine testing of inducible clindamycin resistance is suggested among the clinical isolates of erythromycin-resistant Staphylococci to avoid treatment failure.

Phenotypic and Molecular Detection of Macrolide Lincosamide Streptogramin B Resistance in Clinical Isolates of Staphylococci

Phenotypic and Molecular Detection of Macrolide Lincosamide Streptogramin B Resistance in Clinical Isolates of Staphylococci

A potent enzybiotic against methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus is one of the most dreadful infectious agents, responsible for high mortality and morbidity in both humans and animals. The increased prevalence of multidrug-resistant (MDR) Staphylococcus aureus strains has limited the number of available treatment options, which calls for the development of alternative and effective modalities against MDR S. aureus. Endolysins are bacteriophage-derived antibacterials, which attack essential conserved elements of peptidoglycan that are vital for bacterial survival, making them promising alternatives or complements to existing antibiotics for tackling such infections. For developing endolysin lysin-methicillin-resistant-5 (LysMR-5) as an effective antimicrobial agent, we evaluated its physical and chemical characteristics, and its intrinsic antibacterial activity against staphylococcal strains, including methicillin-resistant Staphylococcus aureus (MRSA). In this study, we cloned, expressed, and purified LysMR-5 from S. aureus phage MR-5. In silico analysis revealed that LysMR-5 harbors two catalytic and one cell wall-binding domain. Biochemical characterization and LC-MS analysis showed that both catalytic domains were active and had no dependence on divalent ions for their action, Zn2+ exerted a negative effect. The optimal lytic activity of the endolysin was at 37°C/pH 7.0 and in the presence of ≥ 300mM concentration of NaCl. Circular dichroism (CD) demonstrated a loss in secondary structure with an increase in temperature confirming the thermosensitive nature of endolysin. Antibacterial assays revealed that LysMR-5 was active against diverse clinical isolates of staphylococci. It showed high lytic efficacy against S. aureus ATCC 43300, as an endolysin concentration as low as 15µg/ml was sufficient to achieve maximum lytic activity within 30min and it was further confirmed by scanning electron microscopy. Our results indicate that rapid and strong bactericidal activity of LysMR-5 makes it a valuable candidate for eradicating multidrug-resistant S. aureus.

The emergence of plasmid-borne cfr-mediated linezolid resistant-staphylococci in Vietnam

ObjectivesLinezolid is one of the last resort antibiotics effectively used in the treatment of infections caused by multidrug-resistant Gram-positive bacteria. Recent outbreaks of Linezolid resistance have been the great concern worldwide, while many countries have not experienced it. In this work, we aimed to evaluate the existence of linezolid resistance and further clarify potential resistance mechanism(s) in staphylococcal isolates obtained from the hospital in Vietnam, a country in which linezolid resistance had not been previously detected. MethodsSeventy staphylococcal clinical isolates including MRSA (n=63) and methicillin-resistant coagulase-negative staphylococci (MRCNS, n=7) were collected and analyzed for linezolid resistance. Linezolid-resistant isolates were submitted for whole genome sequencing to search for the resistance determinants. ResultsWe identified two coagulase-negative staphylococcal isolates that were resistant to linezolid. Whole genome sequencing revealed several alterations in the 23S rRNA and L3, L17, L22, L24, L30 ribosomal proteins. Importantly, both isolates harbour the chloramphenicol/florfenicol resistance (cfr) gene on a plasmid. The plasmid was closely identical to the pLRSA417 plasmid that was originally reported in China. ConclusionsTo the best of our knowledge, this is the first report of cfr-mediated linezolid resistance in clinically isolated staphylococci in Vietnam. We suggest that adequate surveillance is necessary to monitor the dissemination of linezolid resistance among staphylococcal species and other important pathogens.

Assessment of inducible clindamycin resistance and Hyper Variable Region (HVR) of mecA gene in clinical staphylococci.

Objective:To study the prevalence of inducible clindamycin along with vancomycin and methicillin resistance and assessment of hyper variable region (HVR) of mecA gene among different clinical isolates of Staphylococcus spp.Methods:A total of 176 clinical isolates of Staphylococci were collected from Pakistan Institute of Medical Sciences (PIMS), Islamabad during 2014-2015. The sample sources were pus, blood, urine, sputum, tracheal secretions and tissue fluids. Bacterial identification was done by colony morphology and biochemical tests. Kirby-Bauer disc-diffusion method was carried out to assess the susceptibility against different antibiotics. Minimal inhibitory concentrations (MICs) were done for vancomycin resistance. Double Disk Diffusion test (D-test) was used to detect the clindamycin inducible resistance. PCR was performed to detect erm(C), mecA and HVR genes.Results:Clindamycin inducible resistance among Staphylococcal isolates was found to be 7%, whereas in S. aureus it was 4%, and in coagulase negative Staphylococci (CoNS) it was 11%. The highest resistance was observed against fosfomycin, fusidic acid and cefoxitin. Vancomycin resistance was observed in 23 isolates (13%) of Staphylococci. erm(C), mecA and HVR genes were found in 18%, 50% and 42% respectively.Conclusions:D-test must be performed routinely to avoid clindamycin failure. A high level of resistance against vancomycin in Staphylococcal isolates is a concern for public health.

Assessing efficiency of synthetic peptide-containing spray in combination therapy of chronic generalized periodontitis

Antibacterial drugs are routinely used in therapy of periodontal diseases. However, increasing incidence of antibiotics resistance necessitates development of novel therapeutic approaches for oral diseases. Currently, newly designed antibacterial agents based on natural, semi-synthetic and synthetic peptides is the most promising approach in dentistry. Among them is a product containing synthetic peptide (ZP2) replicating active site of granulocyte-macrophage colonystimulating factor (GM-CSF) as the main active ingredient in Atsegram-spray (manufactured by Academic Innovation Research Center, Chelyabinsk). Our study was aimed at assessing efficacy of a combination therapy of inflammatory periodontal diseases by introducing Atsegram-spray as well as examining potential relationship between peptide ZP2-related biological properties, physicochemical properties of the spray and mechanism of antibacterial and immunotropic action for substantiating its application on oral mucosa. During the first stage experiments, it was found that the peptide ZP2 was able to trigger lymphocyte blast transformation in vitro indicating that it might influence cell proliferation and exhibit marked immunotropic activity. Next, we assessed potential effects of the peptide ZP2 on biofilm formation mediated by staphylococcal clinical isolates. It was shown that peptide ZP2 inhibited biofilm formation in 75.0±9.0% of S. aureus and 50.0±15.1% of S. epidermidis strains, with mean inhibition index of biofilm formation reaching 25.1±3.8 and 50.4±6.0%, respectively. However, peptide ZP2 in 8.3–25.0% of staphylococcal clinical isolates was found to stimulate/lack effect on biofilm formation by 14.9–48.5 and 16.7–25.0% cultures, respectively. Thus, the synthetic peptide ZP2 exerts divergent, but mainly inhibitory effects on biofilm formation with staphylococcal clinical strains, which are characterized by inter- and intraspecific (strain) variability. Use of a synthetic peptide-based spray in antibacterial therapy of mild chronic generalized periodontitis (main group) one month after the onset was found to improve oral hygiene by 28.5% as well as decrease PMA index and gum bleeding index by 82.8 and 100%, respectively. In contrast, such parameters in patients receiving basic therapy (comparison group) were lower on average by 2-fold. While analyzing physicochemical properties of the spray such as pH, buffer capacity and solution osmotic pressure, it was found that they were related to the antibacterial mechanism of drug activity and efficacy in treatment of inflammatory periodontal diseases. Thus, assessing peptide ZP2-related biological properties and physicochemical parameters of the spray allows to evaluate their role in mechanism of previously unknown antibacterial and immunotropic activity. These findings confirm feasibility and efficacy of using Atsegram-spray in dentistry as an alternative means to antimicrobial agents, such as antibacterial drugs.

2125. Staphylococcus Species Identification by Fourier Transform Infrared (FTIR) Spectroscopic Techniques: A Cross-Lab Study

Background Staphylococcus aureus is well known to be associated with atopic dermatitis. Recent studies also report S. aureus presence in lesional skin of squamous cell carcinoma (SCC) and its precursor lesion, actinic keratosis (AK). Therefore, it is of potential clinical interest to monitor skin S. aureus colonization on AK lesions. Fourier transform infrared (FTIR) spectroscopy is a cost-effective, nondestructive, and reagent-free technique for rapid microbial identification. It is based on the use of spectral databases developed with well-characterized strains in conjunction with the application of multivariate statistical analysis to elaborate classification models. In the present cross-lab study, spectral databases containing FTIR spectra of over 1000 staphylococcal isolates obtained from reference and clinical microbiology laboratories across Canada were employed in the FTIR spectroscopic identification of Staphylococcus spp. isolated from AK, SCC and perilesional skin of patients at the Princess Alexandra Hospital Dermatology Clinic in Brisbane, Australia.MethodsFTIR spectra of 51 staphylococcal isolates from AK, SCC and perilesional skin were acquired by both attenuated total reflectance (ATR)-FTIR and transflection-FTIR spectroscopy. All isolates had been previously characterized by 16S rRNA sequencing. ATR- and transflection-FTIR spectra were recorded in triplicate from isolated colonies taken from the same agar plate. Identification of the bacteria was based on the similarities of their spectra with those in ATR- and transflection-FTIR spectral databases originating from the Canadian lab.ResultsAmong the 51 staphylococcal isolates included in this study, identification of S. aureus (n = 24) with 100% specificity and 100% sensitivity was achieved by both ATR- and transflection-FTIR spectroscopy. Overall, FTIR-based species identification was in 90.2% concordance with 16S rRNA sequencing.ConclusionThis cross-lab study demonstrates the applicability of Canadian isolate-based ATR- and transflection-FTIR spectral databases for the identification of clinical staphylococcal isolates obtained in Australia. The results support the potential utility of FTIR spectroscopic techniques to monitor skin S. aureus colonization on AK lesions.Disclosures All authors: No reported disclosures.

Incidence of clindamycin resistance among clinical isolates of Staphylococci in a tertiary care centre of Manipur, India

Incidence of clindamycin resistance among clinical isolates of Staphylococci in a tertiary care centre of Manipur, India