Current trends of antibiotic resistance in clinical isolates of Staphylococcus aureus

Staphyloccus aureus （S. aureus） is a well known human pathogen known to causes a verity of infections in humans. In recent years S. aureus is reported to show drug resistant toward commonly known drugs. Therefore, this study was designed to study the pattern of antibiotic resistance in 50 clinical isolates ofS. aureus isolated at Dhanwantri Hospital and Research Centre, Jaipur, Rajasthan, India. S. aureus cultures were isolated from different clinical samples, pus, throat swabs and urine on Blood agar and MacConkey agar and Chrom agar plats and characterized by an array of microscopic and biochemical tests. Antibiotic sensitivity test was performed by standard disc diffusion method （Kirby bayer＇s method） on Muller Hinton agar plates. During this study, among 50 S. aureus isolates 48 （96%） were found to be resistance toward Aztreonam and Doxicycline followed by Ciprofloxacin （n = 45, 90%）, Cefpodoxime and Ceftazidime （n = 44, 88%）, Cefuroxime （n = 40, 80%）, Pipracillin ＋ Tazobactum （n = 38, 76%）, Cefoparazone （n = 36, 72%）, Amoxicillin ＋ Clavulanic acid and Ceftriaxone （n = 33, 66%）, Levofloxacin （n = 32, 64%）, Moxifloxacin （n = 31, 62%）, Ofloaxacin （n = 25, 50%）, Cloxacillin （n = 22, 44%）, Azithromycin （n = 21, 42%）, Clindamycin （n = 19, 38%）, Meropenem （n = 18, 36%）, Clarithromycin （n = 16, 32%）, Ampicillin ＋ sulbactam （n = 13, 26%）, Amikacin （n = 12, 24%）, Impipenem （n = 8, 16%）, Linezolid and Methicillin （n = 7, 14%） and Teicoplanin （n = 3, 6%）. In conclusion, the isolated S. aureus found to be resistant toward common antibiotics, however all isolates were found to be susceptible to Vancomycin.     

长沙地区临床分离金黄色葡萄球菌的耐药监测

目的了解长沙地区临床分离金黄色葡萄球菌（以下简称金葡菌）对常用抗菌药物的耐药现状,探讨金黄色葡萄球菌对甲氧西林的耐药水平。方法收集长沙地区11家医院2009年11月至2010年11月临床分离的非重复金葡菌279株,应用Vitek-2全自动微生物分析系统进行鉴定,K-B法检测金葡菌对24种药物的敏感性,产色头孢菌素试验检测β-内酰胺酶以及D试验检测诱导型克林霉素耐药。应用头孢西丁和苯唑西林纸片扩散法筛查耐甲氧西林的金葡菌（MRSA）,琼脂稀释法检测头孢西丁和苯唑西林的最低抑菌浓度（MIC）。结果在被检测的24种药物中,敏感率〉50%的药物为9种,未发现对万古霉素、替考拉宁和利奈唑胺耐药菌株;耐药率〉50%的抗菌药物有11种,其中以青霉素和氨苄西林的耐药率最高（均为97.1%）。MRSA的分离率达54.5%,且对常用的16种抗菌药物的耐药率均显著高于甲氧西林敏感金黄色葡萄球菌（MSSA）。279株金葡菌中,β-内酰胺酶阳性250株（89.6%）;红霉素耐药而克林霉素敏感或中介的30株中,D试验阳性22株（73.3%）。苯唑西林（OXA）和头孢西丁（FOX）MIC范围分别为0.125～〉256μg/mL和2～〉256μg/mL,苯唑西林的MIC50和MIC90分别为128μg/mL和256μg/mL,头孢西丁的MIC50和MIC90分别为64μg/mL和256μg/mL。结论长沙地区临床分离金葡菌对常用抗菌药物呈多重耐药;MRSA不仅分离率高,而且对甲氧西林呈高水平耐药。     

Antibacterial potential of Malaysian ethnomedicinal plants against methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA)

This study aimed to evaluate the antibacterial activities of 61 plant extracts from 49 Malaysian ethnomedicinal plants and to investigate the interaction of the active plant extracts in combination with synthetic antibiotics against the MSSA and MRSA strains. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the plant extracts were determined using a microdilution method against MSSA and MRSA strains. The interaction between active plant extracts and the antibiotics was assessed using the checkerboard method. The total fractional inhibitory concentration (∑FIC) indices from the combination were calculated to determine the nature of the interaction. Out of the 61 plant extracts tested against the MSSA strain, 7 plant extracts (̴ 11%) showed MIC values of less than 200 μg/mL, 17 extracts (̴ 28%) showed MIC between 200 and 800 µg/mL and seed extracts of Areca catechu showed MBC values of 400 μg/mL. The seed extract of A. catechu showed MIC and MBC of 400 μg/mL against the MRSA strains while leaf extract of Cocos nucifera showed MIC of 400 μg/mL against MRSA NCTC 12493. When the active plant extracts (MIC ≤ 200 µg/mL for MSSA, and ≤ 400 µg/mL for MRSA) were tested in combination with vancomycin and ciprofloxacin, they showed no interaction against both MSSA and MRSA with ∑FIC between 1.06 and 2.03. These findings provide a preliminary overview of the anti-MSSA and anti-MRSA properties of Malaysian ethnobotanical plants to combat Staphylococcal infections. Further research is needed to establish an antibacterial profile of the tested plant extracts.     

Detection of Staphylococcus aureus (MRSA/MSSA) in surfaces of dental medicine equipment

Methicillin-Resistant Staphylococcus aureus (MRSA) represents one of the major causes of nosocomial infections, leading to high mortality. Surfaces in clinics, as well as the attending uniform and the hands of the dental doctor can be MRSA reservoirs. Having this in mind, the purpose of this study was to evaluate the presence of Methicillin-Sensitive Staphylococcus aureus (MSSA) and MRSA on dental medicine equipment surfaces. 354 Samples were collected from six equipment surfaces in six attendance areas before and after patient consultation and cultured in a selective medium. Polymerase Chain Reaction (PCR) was used to confirm the identity of bacterial strains as MRSA or MSSA. Data analysis was performed with chi-square tests with Bonferroni correction. It was observed 55.6% of uncontaminated samples. Contamination was: 17.5% MRSA (5.9% of samples collected before patient attendance and 11.6% after); 39.3% MSSA (14.1% collected before and 25.2% after). The prevalence of MRSA and MSSA was significantly higher after patient care. Integrated Clinic represented the most contaminated attendance area (MRSA − 41.7%, MSSA − 51.2%), the chair arm rest was the most contaminated surface for MRSA (29.7%) and the dental spittoon the most contaminated surface for MSSA (23.5%). Although a low level of contamination was observed, dental clinics, through patients possibly carrying bacteria, may be reservoirs for MRSA and MSSA transmission, and might contribute to potential nosocomial infections.     

Macrolide resistance genotypes and phenotypes among erythromycin-resistant clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci, Italy

One hundred macrolide-resistant staphylococcal isolates from clinically relevant infections in Italy during a 19-month period were studied. Four distinct resistance phenotypes were observed using the triple-disk induction test (erythromycin, clindamycin, telithromycin): the cMLS_B phenotype (24 isolates); the iMLS_B phenotype (41 isolates); the MS phenotype (three isolates); and the iMTS phenotype (erythromycin-induced telithromycin resistance) (32 isolates). ermC and ermA genes predominated within erythromycin-resistant Staphylococcus aureus isolates with iMLS_B phenotype and cMLS_B phenotype, respectively. Among erythromycin-resistant CoNS isolates, half of the strains showed the iMTS or MS/ msrA association, and ermC gene predominated among isolates with MLS_B phenotype. By pulsed-field gel electrophoresis, high genetic heterogeneity was observed among the isolates studied. Both independent acquisition of macrolide resistance genes and spread of specific resistant clones were observed. Association between certain clonal types and specific types of infection could be detected. To our knowledge, this is the first report on characterization of erythromycin-resistant staphylococci in Italy.     

Detection and quantification of methicillin‐resistant Staphylococcus aureus (MRSA) clones in retail meat products

Aims: The objective of the study was to determine the prevalence of methicillin‐resistant Staphylococcus aureus (MRSA) contamination of retail meat and to determine the level of contamination. Methods and Results: Pork (pork chops and ground pork), ground beef and chicken (legs, wings and thighs) were purchased at retail outlets in four Canadian provinces and tested for the presence of methicillin‐resistant Staph. aureus using qualitative and quantitative methods. MRSA was isolated from 9·6% of pork, 5·6% of beef and 1·2% of chicken samples (P = 0·0002). Low levels of MRSA were typically present, with 37% below the detection threshold for quantification and <100 CFU g^?1 present in most quantifiable samples. All isolates were classified as Canadian epidemic MRSA‐2 (CMRSA‐2) by pulsed field gel electrophoresis (PFGE), with two different PFGE subtypes, and were spa type 24/t242. Conclusions: MRSA contamination of retail meat is not uncommon. While CMRSA‐2, a human epidemic clone, has been found in pigs in Canada, the lack of isolation of livestock‐associated ST398 was surprising. Significance and Impact of the Study: The relevance of MRSA contamination of meat is unclear but investigation is required because of the potential for exposure from food handling. Sources of contamination require investigation because these results suggest that human or animal sources could be involved.     

Increased tolerance of Staphylococcus aureus to vancomycin in viscous media

The increased viscosity observed in biofilms, adherent communities of bacterial cells embedded in a polymeric matrix, was hypothesized to induce increased tolerance of bacteria to antibiotics. To test this concept, planktonic Staphylococcus aureus cells were grown and exposed to vancomycin in media brought to specific viscosities in order to mimic the biofilm extracellular polymeric matrix. A viscous environment was observed to decrease the vancomycin susceptibility of planktonic S. aureus to levels seen for biofilms. Both planktonic S. aureus at a viscosity of 100 mPa s and staphylococcal biofilms were able to survive at >500 times the levels of the antibiotic effective against planktonic populations in standard medium. Time-dependent and dose-dependent viability curves revealed that more than one mechanism was involved in high S. aureus tolerance to vancomycin in viscous media. Increased viscosity affects antibiotic susceptibility by reducing diffusion and the mass transfer rate; this mechanism alone, however, cannot explain the increased tolerance demonstrated by S. aureus in viscous media, suggesting that viscosity may also alter the phenotype of the planktonic bacteria to one more resistant to antimicrobials, as seen in biofilms. However, these latter changes are not yet understood and will require further study.     

Dynamics of Biofilm Formation and the Interaction between Candida albicans and Methicillin-Susceptible (MSSA) and -Resistant Staphylococcus aureus (MRSA)

Polymicrobial biofilms are an understudied and a clinically relevant problem. This study evaluates the interaction between C. albicans, and methicillin- susceptible (MSSA) and resistant (MRSA) S. aureus growing in single- and dual-species biofilms. Single and dual species adhesion (90 min) and biofilms (12, 24, and 48 h) were evaluated by complementary methods: counting colony-forming units (CFU mL^-1), XTT-reduction, and crystal violet staining (CV). The secretion of hydrolytic enzymes by the 48 h biofilms was also evaluated using fluorimetric kits. Scanning electron microscopy (SEM) was used to assess biofilm structure. The results from quantification assays were compared using two-way ANOVAs with Tukey post-hoc tests, while data from enzymatic activities were analyzed by one-way Welch-ANOVA followed by Games-Howell post hoc test (α = 0.05). C. albicans, MSSA and MRSA were able to adhere and to form biofilm in both single or mixed cultures. In general, all microorganisms in both growth conditions showed a gradual increase in the number of cells and metabolic activity over time, reaching peak values between 12 h and 48 h (ρ<0.05). C. albicans single- and dual-biofilms had significantly higher total biomass values (ρ<0.05) than single biofilms of bacteria. Except for single MRSA biofilms, all microorganisms in both growth conditions secreted proteinase and phospholipase-C. SEM images revealed extensive adherence of bacteria to hyphal elements of C. albicans. C. albicans, MSSA, and MRSA can co-exist in biofilms without antagonism and in an apparent synergistic effect, with bacteria cells preferentially associated to C. albicans hyphal forms.     

Molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) isolates from major hospitals in Riyadh, Saudi Arabia

The few studies that have reported the incidence of methicillin-resistant Staphylococcus aureus (MRSA) in Saudi Arabia have indicated that a diverse number of circulating MRSA strains have been detected in several major hospitals. Thus, this study was designed to track the presence of MRSA strains in major hospitals in Riyadh, Saudi Arabia, and perform comparative chromosomal DNA analysis of MRSA strains for epidemiological investigation using pulsed-field gel electrophoresis (PFGE). Correlation of the PFGE types generated with microbiological and clinical data of the isolates was attempted. Screening for decreased susceptibility to vancomycin among the isolates was also done. A dendogram was generated using PFGE macrorestriction fragments and 6 types were identified (M1-M6) with M1 being predominant and widespread. A clear link between PFGE types and some clinical and microbiological data available for the strains was found. For example, M1 was statistically associated with male patients, whereas the unique types were associated with female patients, M2 was associated with isolates from wounds and age group <5 years, and M4 was associated with isolates from patients admitted to intensive care units. M5 was highly correlated with low sensitivity to linezolid. No vancomycin-resistant isolates were detected.     

Overexpression of genes of the cell wall stimulon in clinical isolates of Staphylococcus aureus exhibiting vancomycin-intermediate- S. aureus-type resistance to vancomycin

Custom-designed gene chips (Affymetrix) were used to determine genetic relatedness and gene expression profiles in Staphylococcus aureus isolates with increasing MICs of vancomycin that were recovered over a period of several weeks from the blood and heart valve of a patient undergoing extensive vancomycin therapy. The isolates were found to be isogenic as determined by the GeneChip based genotyping approach and thus represented a unique opportunity to study changes in gene expression that may contribute to the vancomycin resistance phenotype. No differences in gene expression were detected between the parent strain, JH1, and JH15, isolated from the nares of a patient contact. Few expression changes were observed between blood and heart valve isolates with identical vancomycin MICs. A large number of genes had altered expression in the late stage JH9 isolate (MIC = 8 microg/ml) compared to JH1 (MIC = 1 microg/ml). Most genes with altered expression were involved in housekeeping functions or cell wall biosynthesis and regulation. The sortase-encoding genes, srtA and srtB, as well as several surface protein-encoding genes were downregulated in JH9. Two hypothetical protein-encoding genes, SAS016 and SA2343, were dramatically overexpressed in JH9. Interestingly, 27 of the genes with altered expression in JH9 grown in drug-free medium were found to be also overexpressed when the parental strain JH1 was briefly exposed to inhibitory concentrations of vancomycin, and more than half (17 of 27) of the genes with altered expression belonged to determinants that were proposed to form part of a general cell wall stress stimulon (S. Utaida et al., Microbiology 149:2719-2732, 2003).     

A CD-1 mouse model of infection with Staphylococcus aureus: influence of gender on infection with MRSA and MSSA isolates

Staphylococcus aureus is an important pathogen of humans and other animals, causing bacteremia, abscessation, toxemia, and other infectious diseases. An animal model using CD-1 mice was developed to study the pathogenesis of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA). When inoculated into the CD-1 mouse model, it was shown that both MSSA isolates, (HR 78 and CSA-1) and MRSA isolates (MRSA 456 and MRSA 457) led to chronic infection of the kidney. Female CD-1 mice inoculated with MRSA 456 proved to be more susceptible to infection and mortality than their male counterparts. Castrated mice became more susceptible to infection than intact male mice, suggesting a hormonal involvement in the infection process.     

Functional group characterization of lactic bacterial biosurfactants and evaluation of antagonistic actions against clinical isolates of methicillin-resistant Staphylococcus aureus

The present study investigated the antimicrobial and antibiofilm potential of biosurfactants derived from Lactobacillus fermentum Lf1, L. fermentum LbS4 and Lactobacillus plantarum A5 against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA). The cell wall-bound and intracellular biosurfactants were extracted by solvent extraction method. Fourier-transform infrared spectroscopy-based characterization of biosurfactants revealed the heterogeneous chemical composition involving proteins, fatty acids and carbohydrate moieties in LbS4 and A5, while only the sugar and lipid fractions in Lf1. Fatty acid profiling using Gas chromatography-mass spectrometry indicated hexadecanoic acid and stearic acid as the predominant fatty acids in the biosurfactants of all these strains. Biosurfactants demonstrated dose-dependent antibacterial action against MRSA isolates with the highest inhibition zone diameter (30·0 ± 0·0 to 35·0 ± 0·0 mm) recorded at 400 mg ml⁻¹. Biosurfactants showed an excellent staphylococcal antibiofilm activity by preventing the biofilm formation and disrupting the preformed biofilms. Visual inspection through scanning electron microscopy witnessed the biosurfactants-induced alteration in the cell membrane integrity and subsequent membrane pore formation on staphylococcal cells. Taken together, our findings emphasize the prospects of biomedical applications of biosurfactants as bactericidal and biofilm controlling agents to confront staphylococcal nosocomial infections.     

Impedimetric detection of single-stranded PCR products derived from methicillin resistant Staphylococcus aureus (MRSA) isolates

Using electrochemical impedance spectroscopy (EIS) the sensitive and specific detection of the antibiotic resistance gene mecA has been demonstrated. The gene sequence was obtained from clinical Staphylococcus aureus isolates. Initially a mecA specific probe was selected from hybridisation tests with a 3' and 5' version of a previously published probe sequence. When immobilised on a gold electrode in PNA form it was possible to detect hybridisation of mecA PCR product electrochemically at concentrations as low as 10nM. By incorporating an undecane-thiol and 1.8 nm glycol spacer into the PNA probe it was possible to extend the limit of detection for mecA to 10 pM. Most published studies on EIS and nucleic acid detection report the use of short artificial DNA sequences or novel signal amplification schemes which improve sensitivity whereas this study reports the successful detection of long DNA fragments produced by PCR following extraction from clinical isolates. Finally, using screen printed electrodes the paper demonstrates hybridisation monitoring of mecA in an "on-line" assay format under ambient conditions which paves the way for rapid mecA detection in point of care scenarios.     

Potent in vitro synergism of fusidic acid (FA) and berberine chloride (BBR) against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA)

It was found in the present study that combined use of fusidic acid (FA) and berberine chloride (BBR) offered an in vitro synergistic action against 7 of the 30 clinical methicillin-resistant Staphylococcus aureus (MRSA) strains, with a fractional inhibitory concentration (FIC) index ranging from 0.5 to 0.19. This synergistic effect was most pronounced on MRSA 4806, an FA-resistant isolate, with a minimum inhibitory concentration (MIC) value of 1,024 μg/ml. The time-kill curve experiment showed that FA plus BBR yielded a 4.2 log₁₀ c.f.u./ml reduction in the number of MRSA 4806 bacteria after 24-h incubation as compared with BBR alone. Viable count analysis showed that FA plus BBR produced a 3.0 log₁₀ c.f.u./ml decrease in biofilm formation and a 1.5 log₁₀ c.f.u./ml decrease in mature biofilm in viable cell density as compared with BBR alone. In addition, phase contrast micrographs confirmed that biofilm formation was significantly inhibited and mature biofilm was obviously destructed when FA was used in combination with BBR. These results provide evidence that combined use of FA and BBR may prove to be a promising clinical therapeutic strategy against MRSA.     

体外诱导耐万古霉素金黄色葡萄球菌及分子机制

【目的】通过前期体外诱导获得耐万古霉素金黄色葡萄球菌,从基因突变方面对万古霉素耐药性菌株进行研究。【方法】通过低浓度万古霉素逐步诱导13株敏感性金黄色葡萄球菌,用琼脂稀释法和E-test法检测所有菌株对万古霉素的耐药性(最低抑菌浓度,MIC),PCR扩增与万古霉素耐药性密切相关的4个重要基因:rpo B、vra S、gra R和gra S,并测序分析,比较诱导前后不同菌株的基因序列。【结果】通过60 d的体外诱导实验,13株对万古霉素敏感性金黄色葡萄球菌中有6株被诱导为中介耐药金黄色葡萄球菌(Vancomycin intermediate Staphylococcus aureus,VISA),7株菌被诱导之后对万古霉素仍处于敏感状态,MIC4 mg/L。检测诱导前后所有菌株的rpo B、vra S、gra R和gra S基因发现:有3株VISA的rpo B基因同时有L466S和H481N的突变,gra S基因同时有R232K的突变。【结论】对万古霉素敏感的金黄色葡萄球菌经过较长时间的体外诱导可发展为VISA。在已检测的重要基因中,rpo B和gra S的突变对耐药性的发展很可能起关键作用,而vra S和gra R对这一过程没有显著影响。     

Aminoglycosides resistance in clinical isolates of Staphylococcus aureus from a University Hospital in Bialystok, Poland

Staphylococcus aureus obtained from a University Hospital in Poland were characterized in relation to resistance to aminoglycoside antibiotics and the distribution of the genes encoding the most clinically relevant aminoglycoside modifying enzymes (AMEs). Of a total of 118 S. aureus, 45 (38.1%) isolates were found to be resistant to at least one of the tested antibiotics. All aminoglycoside resistant isolates except one 44 (97.8%) were resistant to kanamycin. The majority of strains 37 (82.2%) and 32 (71.1%) expressed resistance to neomycin and tobramycin, respectively. Eleven strains (24.4%) were resistant to gentamicin or amikacin. All S. aureus strains were sensitive to netilmicin. The most prevalent resistance gene was aac(6')-Ie+aph(2') found in 13 (28.9%) strains and 12 (26.7%) isolates carried ant(4')-Ia gene, whilst aph(3')-IIIa gene was detected in only 7 (15.6%) isolates. Additionally, the ant(6)-Ia and str genes were detected in 14 (31.1%) and 2 (4.4%) strains, respectively. Ten (22.2%) strains resistant to amikacin, tobramycin, kanamycin or neomycin did not harbor any of the above-noted genes.