methicillin-resistant Staphylococcus

.. Background: Methicillin resistant Staphylococcus aureus (MRSA) is one of the notorious bugs to cause nosocomial human infections worldwide. However its prevalence and susceptibility pattern quite varies in hospitals among different countries or within same country. Objectives: To study the prevalence and antibiotic susceptibility pattern of MRSA isolated from pus sample. Study Design: Cross sectional study. Period: 6 months. Setting: Tertiary care hospital of district Rahim Yar Khan -Punjab, Pakistan at its microbiology laboratory. Materials and Methods: A total 100 clinical specimen of pus and wounds were collected from various indoor and outdoor department of this institute. .The Staphylococcus aureus was identified on the basis of colony morphology, Gram’s stain, and biochemical tests. Antibiotic susceptibility pattern was done according to CLSI guidelines. Results: Out of 100 clinical sample of pus and wounds, 21 coagulase positive staph. aureus were isolated, 14 (66.7%) strains were found to be methicillin resistant and 07(33.3%) were found to be methicillin sensitive. Higher percentage of MRSA was isolated from surgical unit (66.7%), indicating its nosocomial source of spread. Maximum resistance to MRSA isolate were developed to Amoxicillin/ clavulinic acid (80%) followed by ofloxacin (71%), cefoxitin (66.7%), tetracycline (58%), Ciprofloxacin (48%), Erthromycin (46%) and Amikacin (40%). However, all strains were sensitive to vancomycin. Conclusion: There is high frequency of resistance among coagulase positive Methicillin resistant staph. aureus. Regular surveillance of hospital-associated infection and frequent monitoring of antibiotic sensitivity pattern is required to reduce MRSA prevalence.


InTroDuCTIon
As the incidence of bacterial strains resistant to antimicrobial agents rises, the development of new antibacterial strategies becomes increasingly important.In the past decade, the first clinical isolate of a methicillin resistant Staphylococcus aureus (MRSA) strain with reduced susceptibility to vancomycin was reported in Japan (Hiramatsu et al., 1997) and later also in the United States and France (Ploy et al., 1998;Smith et al., 1999).In 2002 the first documented case of an infection caused by vancomycin-resistant S. aureus was reported (Sievert, 2002).Additionally, MRSA are important etiological factors responsible for health care-associated life-threatening infections (Grinholc et al., 2007a;Kurlenda et al., 2007).Particularly burn wound infections remain a potentially serious problem and are an important cause of death.In some countries, e.g.Portugal, MRSA strains can constitute up to 65% of all S. aureus isolates in hospitals and produce many therapeutic problems including MRSA outbreaks (Aires de Sousa et al., 1998;Leski et al., 1998;Kurlenda et al., 2007).In Poland the average prevalence of MRSA in hospitals varies from 2.3% to 59.9% (Hryniewicz et al., 1993;Piechowicz et al., 1993).Many human pathogens are now multiresistant to antimicrobial drugs and skin infections with such organisms may be particularly difficult to treat.
An alternative therapeutic approach may be to use photodynamic inactivation (PDI), which employs visible light in the presence of a photosensitizing agent.An activated sensitizer may react with molecules from its direct environment by electron or hydrogen transfer.The sensitizer, in its photoactive triple state, can react with a local substrate to form cytotoxic radicals (type 1 reaction) or transfer its energy directly to oxygen generating the reactive singlet oxygen (type 2 reaction) (Wainwright, 1998).Both pathways can lead to or induce cell death.These highly reactive oxygen species initiate further oxidative reactions in the direct environment.Free radicals and peroxides, which are produced as a result of PDI photooxidation reactions, may damage a number of cellular structures, like the bacterial cell wall, lipid membranes, enzymes, or nucleic acids (Halliwell & Gutteridge, 1984;Baumler et al., 1999).
Different chemical compounds with photoactive properties have already been tested against Gram-positive bacteria.Various photosensitizers such as haematoporphyrin (Bertoloni et al., 2000), porphyrin derivatives (Lasocki et al., 1999;Lambrechts et al., 2005a;2005b), phenothiazinium salts (Bisland et al., 2006;Tegos & Hamblin, 2006), chlorin (Embleton et al., 2002;Gad et al., 2004) and 5-aminolaevulinic acid-induced porphyrin sensitizers (Nitzan et al., 2004;Bisland et al., 2006) have been studied and found to demonstrate high bactericidal effect against S. aureus strains after illumination with visible light.However, in evaluating the potential of PDI for the clinical treatment of, particularly, skin infections, it is important to assess not only the bactericidal efficacy, but also the cytotoxic effects against healthy dermal cells.The cytotoxicity of many sensitizers described above toward keratinocytes and fibroblasts has been studied (Haddad et al., 1999;Ramaiah et al., 2002;Zeina et al., 2002;2003;Chiu et al., 2005;Lambrechts et al., 2005a;2005b;Maisch et al., 2005).Zeina et al. (2002) reported that antimicrobial photodynamic therapy sufficient to reduce microbes by seven log cycles would have little cytotoxic and no genotoxic effect on keratinocytes.Haylett et al. (2003) showed that following photoinactivation, some DNA damage was detected in fibroblasts, but it was fully repaired within 24 h of treatment.Soukos et al. (1996) claimed that photosensitization of keratinocytes and fibroblasts did not reduce cell viability in given experimental conditions, whereas an effective bactericidal activity was obtained.Previously published data confirm the high bactericidal activity of protoporphyrin diarginate (PPArg 2 ) against an S. aureus strain (Grinholc et al., 2007b).However, its toxicity towards fibroblasts has not been investigated yet.
The use of visible light in conjunction with an appropriate photosensitizer may be a useful alternative to antibiotics for microbial skin infections.How-ever, it is important to determine the effect of PDI on skin cells using the same conditions that are known to be effective against microbes.Therefore, the aim of the present in vitro studies was to investigate the photodynamic inactivation of a multiresistant S. aureus strain and healthy human dermal fibroblasts, to determine if microbes could be effectively killed without damaging adjacent fibroblasts.

MATerIAls AnD MeThoDs
Chemicals.All cell culture material was purchased from Gibco-Invitrogen (Paisley, UK).All other chemicals were purchased from Sigma-Aldrich (Germany).
MrsA isolate.The investigated clinical strain of MRSA (methicillin-resistant Staphylococcus aureus) was isolated from the Provincial Hospital in Gdańsk, Poland.The isolate was characterized by Gram staining and ability to produce coagulase and clumping factor using Slidex Staph Plus (BioMerieux, France).Additionally, the species was identified using the biochemical identification system ID 32 Staph (BioMerieux, France).
Photosensitizer.The stock solution of the photosensitizer protoporphyrin diarginate (PPArg 2 ) (Institute of Optoelectronics, Military University of Technology, Warszawa, Poland) was prepared in distilled water at a concentration of 10 mM and stored at -20°C in darkness until use.light source.The illumination was performed with a BioStimul Lamp (Biotherapy, Czech Republic).Delivered light energy was determined with the use of a light power meter (model LM1, CARL Zeiss, Germany) and was approx.0.2 J cm -2 per minute.The BioStimul Lamp emits polarized (96% level of polarization) monochromatic light (624 nm ± 18 nm).
Phototoxicity assay of bacteria.The bacterial culture was grown overnight at 37°C in nutrient trypcase soy broth (BioMerieux, France) and then diluted with fresh broth to an appropriate density (10 7 ml -1 bacterial cells).Such S. aureus culture was incubated with different concentrations of the protoporphyrin diarginate sensitizer (0 to 25 μM) for 30 min in the dark.After the incubation, the cells were transferred into a 96-well microtiter plate (100 μl per well) and illuminated for 30 min (6 J cm -2 ).Control wells were neither sensitized with the photosensi-PPArg 2 -based PDI against MRSA and human fibroblasts tizer nor exposed to the light source or were incubated with the photosensitizer only.After illumination the survival of the bacteria was determined by counting the numbers of colony forming units (cfu).Serially diluted aliquots of treated and untreated (no sensitizer, no light) cells were plated on trypcase soy agar (BioMerieux, France) and the number of cfu ml -1 was counted after 18 to 24 h of incubation at 37°C.Each experiment was done three times.Survival fractions were expressed as ratios of cfu of bacteria treated with light and photosensitizer to cfu of untreated bacteria.
Phototoxicity assay with eukaryotic cells.Cells (5 × 10 4 ) were seeded into 96-well plates and allowed to adhere overnight.Protoporphyrin diarginate was then added to the medium in the concentration range of 0 to 20 μM and cells were incubated for 30 min at 37 o C in the dark prior to irradiation.As the EC 50 value was found within the studied PPArg 2 concentration range, no further concentrations were analyzed.Irradiation was performed with red light using a BioStimul lamp for 30 min (6 J cm -2 ).After illumination, cell survival was determined 24 h later by a standard 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay.MTT is used as an indicator of metabolically active cells, in which a color reaction dependent on enzyme activity takes place in mitochondria, and this activity can be measured.Following treatment, MTT (0.5 mg ml -1 ) was added and cells were incubated for 2 h at 37 o C. Cells were lysed with DMSO and the absorbance of the formazan solution was measured at 550 nm with a plate reader (Victor, 1420 multilabel counter).
Data analysis and statistics.Each experiment was performed in triplicate.All primary data are presented as means with standard deviations of the mean.Statistical analysis was performed with twoway analysis of variance (ANOVA) with Bonferroni post-test.A P value of < 0.05 was considered as statistically significant in each experiment.

Phototoxicity against methicillin resistant Staphylococcus aureus
A clinical S. aureus strain was used to determine the antibacterial toxicities of protoporphyrin diarginate.Illumination of MRSA following incubation with different concentrations (0 to 25 µM) of PPArg 2 caused a significant decrease in viability, as determined by the survival percentage (Fig. 1).PPArg 2 at 10 µM exhibited significant antibacterial activity after illumination for 30 min (6 J cm -2 ) (killing efficacy, 3.6 log 10 -unit reduction) (Table 1).No toxicity of PPArg 2 at concentrations up to 25 μM was observed without illumination (Fig. 1) (Table 1).

Phototoxicity to eukaryotic cells
To determine whether protoporphyrin diarginate induces cytotoxic and phototoxic activity in eukaryotic cells, the viability of cells treated with the sensitizer was assessed with the MTT assay.The toxicity of different concentrations of protoporphyrin diarginate to human skin cells was tested by using healthy dermal fibroblasts.As shown in Fig. 2, incubation of fibroblasts with PPArg 2 yielded reduced cell viability only upon illumination.The corresponding 50% effective concentration (EC 50 ) after 30 min of incubation and a light dose of 6 J cm -2 was evaluated at 17 µM.Incubation of fibroblasts with protoporphyrin diarginate at concentrations up to 20 µM without illumination did not influence cell viability (Fig. 2).
The concentrations of PPArg 2 used in the bacterial and eukaryotic cell toxicity experiments showed concentration-dependent differences upon illumination.Protoporphyrin diarginate at a concentration of 10 μM exerted 3.6 log 10 -unit reduction in viable count after illumination, whereas at this con-  centration fibroblasts were still viable with or without illumination (Figs. 1 and 2) (Table 1).

DIsCussIon
The challenge in antimicrobial photoinactivation (PDI) is to find a therapeutic window in which bacteria are effectively eradicated without harming the surrounding tissue.It is important not only to assess the antimicrobial activity, but also the cytotoxic effects towards healthy dermal cells.In this study, we analyzed the in vitro toxicity of PDI towards human dermal fibroblasts using protoporphyrin diarginate as the photosensitizer.The results of the present study show that the photosensitization with PPArg 2 at the low concentration of 10 μM was effective in killing a methicillin-resistant Staphylococcus aureus strain, against which it achieved 3.6 log 10unit reduction.At the same concentration and with the same incubation and a dose of light of only 6 J cm -2 , PPArg 2 had little effect on skin cells, with 77.5% of fibroblasts still viable in vitro.Interestingly, previously published data (Grinholc et al., 2007b) reveal that at the same PPArg 2 concentration but different incubation time (15 min), 2.1 log 10 reduction could be achieved even though a 12 J cm -2 light dose was applied.In the present study, with a prolonged incubation time (30 min) and reduced light dose (6 J cm -2 ), 3.1 log 10 unit reduction was obtained.It is in accordance with bacterial uptake studies of PPArg 2 showing that after 30 min of incubation, the highest amount of photosensitizer is accumulated inside the bacterial cell (not shown).Lambrechts et al. (2005b) claim that a 90% reduction in cell viability is very substantial for fibroblasts, and, for bacteria, a 4 log (99.99%) reduction in viability is often used as an acceptable indicator.Comparing our data, we conclude that fibroblasts are substantially less sensitive to PDI with the use of PPArg 2 than are S. aureus.
In regard to the previously published data (Lambrechts et al., 2005b), the cell viability assay (MTT), in our study, was performed 24 h after photodynamic treatment.Lambrechts and coworkers indicated that an increased survival value was observed when the MTT assay was performed immediately following PDI.The authors claim that this can be explained by the fact that the MTT assay is unable to distinguish between early apoptotic and living, healthy cells (Lambrechts et al., 2005b).In consequence, it means that still-alive early apoptotic cells are recognized in MTT assay as healthy, even though apoptosis has already been induced by the photodynamic action.To avoid underestimation of cytotoxicity, we decided to perform the viability test after 24 h of post-treatment incubation.The studies of Lambrechts et al. (2005b) involved photoinactivation with the use of another porphyrin-based sensitizer, 5-phenyl-10,15,20-tris(N-methyl-4-pyridyl)porphyrin chloride (TriP[4]).In those studies, a significant reduction in viability of fibroblasts was obtained with 0.78 μM photosensitizer, and no survival was observed with concentrations of 12.5 μM and greater.EC 50 for TriP[4] was estimated at 2 µM.When the bactericidal effect of TriP[4] was analyzed against S. aureus, a 5 log 10 -unit reduction could be obtained at the concentration of 3.1 μM.Other porphyrin-based sensitizers were analyzed by Maisch et al. (2005), who studied novel XF porphyrin derivatives.The EC 50 s of those novel sensitizers ranged from 0.047 to 0.47 μM.With those sensitizers Maisch et al. (2005) reached an approx.3 log 10 -unit reduction at the very low concentration of 0.005 μM.In the case of PPArg 2 , the EC 50 value for fibroblasts was evaluated at 17 μM and a 3.6 log 10 -unit reduction could be reached at the concentration of 10 μM.Moreover, the applied light dose was only 6 J cm -2 , and not 27 J cm -2 or 13.7 J cm -2 as in the studies mentioned above, respectively.
Not only exogenous but also endogenous, naturally occurring porphyrins within target cells are used as photosensitizing agents.The production of these endogenous porphyrins is stimulated by the administration of 5-amino levulinic acid (ALA).Previously published studies demonstrate that S. aureus is able to produce high amounts of porphyrins upon induction by ALA, which may be used as an ideal stimulator of production of endogenous sensitizers in photodynamic therapy (Nitzan et al., 2004;Bisland et al., 2006).Nitzan et al. (2004) reported that staphylococcal strains produced high amounts of porphyrins when incubated with 0.38 mM ALA for 4 h.Upon illumination of the ALA-induced strains with 407-420 nm blue light, a decrease of five orders of magnitude was demonstrated with a light dose of 50 J cm -2 .Total eradication could be achieved with a 100 J cm -2 dose.Chiu et al. (2005) analyzed the phototoxicity of ALA-induced porphyrins against normal adult and neonatal fibroblasts.The cells were incubated for 3 h with ALA at the concentration of 1 mg ml -1 .At 10 or 20 J cm -2 , near total cell death was observed, while at 5 J cm -2 the cell viability was comparable to controls.Also in the studies of Haddad et al. (1999), the effect of ALA-based photodynamic therapy on the viability of normal fibroblasts was evaluated.Fibroblasts were incubated with ALA at the low concentration of 2.5 μg ml -1 for 48 h.Cells were then illuminated with light doses of 50, 100 and 200 J cm -2 .It was reported that the photodynamic therapy caused no significant change in fibroblast viability at all light doses.In the previous report, Grinholc et al. (2007b) present a more detailed comparison of the light source used with the recently published data obtained using other light sources.
To ensure that photoinactivation (PDI) has the potential for clinical use in antimicrobial treatment, it is necessary to assess the cytotoxicity of the studied sensitizers towards healthy dermal cells, in particular the cells that are involved in the wound healing process, such as fibroblasts and keratinocytes.Such research is still being performed (Zeina et al., 2002;2003;Lambrechts et al., 2005b;Maisch et al., 2005).Our results are in accordance with the above studies and are very promising.On the basis of the presented results we can conclude that antibacterial PDI could be an alternative to standard topical antibiotic treatment.However, despite these promising results, only assessment of the technique against a much wider number of clinical S. aureus isolates can prove the efficacy of PDI for the inactivation of bacteria in vivo.Observations against one strain take no account of the clonal/genotypic variance that undoubtedly is present in the population, and deserves further investigation.

Figure 1 .
Figure 1.survival of S. aureus exposed to different concentrations of PPArg 2 and a light dose of 6 J cm -2 .Samples exposed to PPArg 2 and light (filled squares); samples exposed to sensitizer without illumination (filled triangles).Each point is the mean of three experiments ± S.D. Statistical analysis was performed with two-way ANOVA with Bonferroni post-test to compare the two corresponding data points at each concentration of the two curves.***P < 0.001.

Figure 2 .
Figure 2. PPArg 2 dose-dependent PDI of fibroblasts.Illumination parameters: 30 min, 6 J cm -2 (filled squares).Toxicity of PPArg 2 after 30 min incubation at 37°C in the dark (filled triangles).Each point is the mean of three experiments ± S.D. Statistical analysis was performed with two-way ANOVA with Bonferroni post-test to compare the two corresponding data points at each concentration of the two curves.**P < 0.01; ***P < 0.001.