The Effects of Salt Concentration and Growth Phase on MRSA Solar and Germicidal Ultraviolet Radiation Resistance

    Antimicrobial drugs are used continuously and extensively all over the world. While much of their use is warranted, considerable employment of these agents is inappropriate. The Centers for Disease Control and Prevention (CDC) estimates in the US that many prescriptions are unnecessary: 30% are prescribed for ear infections, 50% for sore throats, and the majority for the common cold.¹ Drugs are overused in animal and poultry feed and also greatly misused in over-the-counter sales in much of the world. All of this contributes greatly to the development of drug resistance. One organism that is becoming an increasing problem worldwide is methicillin-resistant Staphylococcus aureus (MRSA).²

    With the widespread emergence of organisms like MRSA, emphasis has increased on the need to develop alternative treatments for chronic wounds such as diabetic ulcers and pressure ulcers. One such alternative treatment method employs ultraviolet (UV) radiation. Ultraviolet radiation can be conveniently categorized into germicidal (UV C, with a wavelength of 190 nm to 290 nm) and solar (UV B and UV A, with wavelength ranges of 290 nm to 320 nm and 320 nm to 400 nm, respectively). Although essentially all UV C radiation is screened out by the stratospheric ozone layer, substantial UV A and B radiation still reaches the Earth’s surface.³ A range of in vitro and in vivo studies has been conducted involving germicidal and solar UV radiation as an adjunctive therapy to the use of antimicrobials in the treatment of various skin disorders.^2,4-8 Taken together, these studies reveal the potential of UV radiation as an adjunctive treatment. The authors extended these studies by quantifying UV inactivation rates under differing physicochemical and growth conditions. In addition, the response of MRSA and the Gram-negative bacterium, Pseudomonas aeruginosa, a common cause of wound and burn infections, was compared.

Literature Review

    For the last 20 years or so, outbreaks of MRSA have been associated with healthcare facilities such as hospitals and nursing homes. During this time span, this problem has been become much more prevalent. Recent studies document the situation.^9–16 In hospital neurosurgical populations, MRSA infection is a growing problem; most cases are hospital-acquired, are often associated with extended lengths of stays, and many involve wound infections. Often these MRSA patients are discharged before eradication of the infection was achieved.⁹ The results of one study, conducted after MRSA was observed in a tertiary hospital and a geriatric institution where isolates were recovered from pressure ulcers, suggested nosocomial acquisition and highlight the need for epidemiological analysis to control dissemination of MRSA in such facilities.¹⁰ Another report documented the frequent transmission of MRSA from colonized hospital employees to their households.¹¹

    Many recent investigations of MRSA outbreaks have involved community settings such as individual households, daycare centers, correctional facilities, and athletic teams. A detailed study conducted in Texas dealt with the management and outcome of children with skin and soft tissue abscesses caused by community-acquired (CA) MRSA. Researchers determined that incision and drainage without adjunctive antibiotic therapy was an effective management strategy for CA-MRSA skin and soft tissue abscesses with a diameter of <5 cm in immunocompetent children.¹² A comprehensive study involving children in southern New England was performed to understand the epidemiology of both CA-MRSA and healthcare-associated (HCA) MRSA. Between 1997 and 2001, S. aureus was isolated from 1,063 children (57 with MRSA). A case was classified at time of culture and other strict criteria into either CA-MRSA or HCA-MRSA. Over the course of the study, the absolute number and proportion of MRSA cases rose more than threefold due to increases in both disease types. Community-acquired MRSA patients were more likely to have skin and soft tissue infections than HCA-MRSA patients. Although both disease types were resistant to beta-lactam antimicrobials, the CA-MRSA isolates were more likely to be susceptible to non-beta-lactam antimicrobials. All isolates were vancomycin susceptible.¹³ Because of the resistance of MRSA to most if not all commonly used beta-lactam antimicrobials, other antimicrobials often must be selected by physicians before the conclusive identification of MRSA as the source of many of these skin infections has occurred. Some other studies of CA-MRSA have involved outbreaks in correctional facilities and sports participants.^14-16

    Previous in vitro work has considered the effect of UV radiation on antimicrobial resistant bacteria. Chapple et al¹⁷ proposed that S. aureus is more sensitive to germicidal UV (UV C radiation) than to solar UV (UV B and UV A radiation). They also considered the effect of various bacterial growth phases on the killing efficiency of UV radiation. Akiyama et al¹⁸ isolated strains of S. aureus from atopic dermatitis lesions. The attachment of these cells to coverslips was suppressed in the presence of 10% salts and irradiation with UV A and B. Plasma coagulation of these same cells was suppressed in the presence of 10% salts, irradiation with UV A, and heating. Additional studies also have investigated the effects of solar UV on S. aureus.^19,20 More recently, Conner-Kerr and Sullivan have run a range of in vitro studies on the effectiveness of UV C radiation in killing antibiotic-sensitive and resistant strains of S. aureus. Their work also involved other bacteria and fungi.^4-6

    The in vivo use of UV radiation to treat skin infections was documented in a 1983 report.⁷ Using a combination of solar and germicidal UV radiation (A and B and C), it was determined in a randomized placebo-controlled trial of 18 patients that the radiation played a useful role in the treatment of pressure sores. In 1994, a study involving 20 patients found that ultrasound/UV C treatment decreased skin healing time for spinal cord injury patients with pressure ulcers.⁸ More recently, in 2002, Thai et al² documented in a small sample of patients that UV C radiation proved to be a promising form of treatment for chronic wounds containing MRSA.

Methods

    Bacterial strains. While the focus of this research was MRSA (strain ATCC 33591), two other strains were used for some comparative studies: methicillin-susceptible S. aureus (ATCC 25923) and P. aeruginosa PAO-1 (ATCC BAA-47). The two S. aureus strains were grown in the complex medium Bacto Tryptic Soy Broth (TSB) that contained the following: 17 g pancreatic digest of casein, 3 g enzymatic digest of soybean meal, 2.5 g dextrose, 5 g sodium chloride (NaCl) (0.085M), and 2.5 g dipotassium phosphate. The indicated pH was 7.3. Additional NaCl was added to this medium to make media at concentrations of 1.37 M, 2.0 M, 2.5 M, and 3.0 M. For solid media Bacto Agar was added at 15 g/L. Pseudomonas aeruginosa PAO-1 was grown in Difco LB Broth that contained 10 g pancreatic digest of casein, 5 g yeast extract, and 5g NaCl. For the final medium, an additional 5 g of NaCl was added. The indicated pH was 7.0. For solid medium, Bacto Agar was added at 15g/L.

    All cells for growth curves and UV radiation kill curves were prepared by inoculating 20 mL of the appropriate liquid medium from a 24- to 48-hour slant of a bacterial strain. Cultures were incubated for 12 to 14 hours at 37^o C with shaking at 180 rpm. Cells then were transferred to a new sidearm flask with 20 mL using of the medium. For this step-up growth stage, 0.1 mL was employed at the lower salt concentrations (0.085 M and 1.37 M); whereas, 0.3 mL was used at the higher salt concentrations (2.0 M, 2.5 M, and 3.0 M). Step-up growth is defined as a series of culture transfers to ensure starting with the exact type of cells necessary to initiate an experiment. Time points were selected periodically to construct a growth curve. At each optical density (OD) reading (employing a Genesys 20 Spectrophotometer, Thermo Electron Corp., Madison, Wis.), serial dilutions were made and plate counts determined. For subsequent UV radiation kill curves, cells were grown to mid-log or stationary phase. Mid-log phase was equivalent to an OD of 0.5 to 0.6 and stationary phase to an OD of 1.4 to 1.6. A bacterial growth curve follows the growth of a population of cells over time. Mid-log phase cells are increasing exponentially; whereas, stationary phase cells are not increasing in numbers and remain in equilibrium. Cells were harvested at 8,000 rpm for 15 minutes, the supernatant discarded, and the pellet suspended in a small amount (5 mL to 10 mL) of the appropriate saline solution.

    UV A and B radiation of bacterial cells. A UV radiation kill curve is determined initially using an aliquot of bacterial cells at a level of 10⁸ to 10⁹/mL, applying UV radiation, and measuring the rate of cell death over time. To determine the effect that solar UV (UV A and B) has on S. aureus, a 2.5-mL sample of a given cell type was added to a glass petri dish (32-mm diameter) and the cells were maintained in suspension during radiation by continuous agitation with a flea-sized stir bar. The lid was removed to expose cells to the solar UV radiation generated by a 150-W Oriel Solar Simulator (Model number 66002, Oriel Corp., Stratford, Conn.) at a flux equivalent to 5.5 J.m-2 sec-1. The flux rate is the lamp output determined using a Model Li-1800uw Underwater Spectroradiometer (Li-Cor, Lincoln, Neb.).²¹ Exposure times for the S. aureus strains were 0, 30, 60, 90, 120, and 150 minutes for 0.085 M and 1.37 M NaCl. Cells at the higher salt concentrations (2.0 M, 2.5 M, and 3.0 M) were exposed for 0, 30, 60, and 90 minutes. For each exposure time, appropriate dilutions were made and a set of triplicate plates was prepared to quantify colony forming units (CFU). Each CFU is assumed to have arisen from a single living cell. Dead cells would not produce a CFU using this technique. For a given sample, the saline dilution blanks and medium used for plating contained NaCl at the same concentration used for growing the cells. Surviving cells were determined by quantifying CFU at each exposure time.

    All experimental manipulations were carried out under amber light and plates were wrapped in aluminum foil and incubated under dark conditions to prevent photoreactivation. For specific experiments to determine the level of photoreactivation, duplicate sets of plates were made. One set of plates was incubated in the dark and the other set was placed under visible light from General Electric (GE) cool white bulbs at a range of 4 to 6 microEinsteins.m-2.sec-1. All results are from an average of three or more experiments.

    UV C radiation of bacterial cells. To determine the effect of germicidal UV (UV C) on S. aureus and P. aeruginosa, experiments were conducted in the manner described for the solar UV radiation experiments. Cells were exposed to UV C radiation from a GE 15 W germicidal lamp (G15T8) at a flux of 0.14 J.m-2 sec-1. The flux rate is the lamp radiation output that was determined using a UVX Radiometer (Ultra-Violet Products, San Gabriel, Calif.) equipped with a UV-25 probe. For S. aureus, exposure times were 0, 10, 20, 30, and 40 minutes. For P. aeruginosa, the cells were exposed for 0, 10, 20, 40, 80, 160, and 300 seconds. All results are from an average of three or more experiments.

Results

    The growth curves for MRSA (ATCC 33591) are presented in Figure 1. As expected, optimum growth rates occurred at the lower NaCl concentrations of 0.085 M and 1.37 M with stationary phase being reached at 5 hours and 8 hours, respectively. As the medium NaCl concentration was increased to 2.0 M and 2.5 M, the growth rate slowed; stationary phase was entered in the range of 13 hours to 15 hours. At a medium NaCl concentration of 3.0 M, the growth rate had slowed down immensely with the final OD level of approximately 1.0 achieved in the range of 25 to 30 hours. This growth level was substantially lower than for the other medium NaCl concentrations. It is also noted that although growth occurred on liquid medium at 3.0 M NaCl, growth was marginal and inconsistent on solid medium containing this level of salt. Therefore, kill curves to UV radiation could not be run at 3.0M NaCl. No growth was observed for a medium containing NaCl at a level of 3.5 M. The growth curves for the methicillin-susceptible S. aureus (ATCC 25923) mirrored the results for the MRSA strain (see Figure 1) and are not presented in this paper.

    When exposed to solar UV (UV A and B) radiation, MRSA, as expected, exhibited a decrease in CFU in both log and stationary phases of growth (see Figure 2 a,b). For both growth phases, the kill curves at the four different levels of NaCl were very similar. For the 0.085 M and 1.37 M NaCl kill curves, no decrease occurred in cell viability until after 20.0 KJ.m-2 but by a dose of 40.0 KJ.m-2 a 10,000-fold decrease in cell viability had occurred. For both the 2.0 M and 2.5 M kill curves, a 10,000-fold decrease in viability was noted after solar UV doses of 30.0 and 20.0 KJ.m-2, respectively. Therefore, at both log and stationary phases of growth, cell susceptibility to solar UV radiation increased as the medium NaCl increased.

    Kill curves to germicidal (UV C) radiation were attained for MRSA in both log and stationary phases at a range of NaCl concentrations (see Figure 3 a,b). For all of the samples, some decrease in CFU occurred immediately. As was true for solar UV radiation, as the concentration of NaCl in the medium increased, the general trend was for the cells to become more sensitive to the germicidal UV radiation. For log phase by a dose of 172 J.m-2, cells grown in the medium containing 1.37 M, 2.0 M, and 2.5 M NaCl all had shown a 10,000-fold or greater reduction in cell viability (see Figure 3a). By a dose of 258 J.m-2, the log phase cells from the 0.085 M medium also exhibited a reduction in cell viability of greater than 10,000-fold. For stationary phase cells exposed to germicidal UV radiation, a somewhat different pattern emerged (see Figure 3b). Cells grown in medium containing NaCl at 0.085 M, 1.37 M, and 2.0 M all produced the same general type of kill curve. An immediate decrease was noted in cell number with a 10,000-fold decrease in cell number for 1.37 M and 2.0 M cells with a dose of 172 J.m-2 and for 0.085 M cells with a dose of 258 J.m-2. Cells grown in 2.5 M NaCl medium were the most susceptible to germicidal UV radiation in stationary phase. Once again the methicillin-susceptible S. aureus (ATCC 25923) mirrored the results for the MRSA strain with both solar and germicidal UV radiation (see Figures 2 a,b and 3 a,b) and are not presented in this paper.

    An experiment employing germicidal UV radiation was run on P. aeruginosa PAO-1 and MRSA (see Figure 4 a,b).The dual purpose of this work was to test for photoreactivation in both strains and to compare the UV survival capacities of a Gram-negative bacterium with a Gram-positive one. Log phase cells were employed for both organisms. Pseudomonas aeruginosa was grown as previously described. For MRSA, 0.085 M NaCl cells were used. Under the incubation conditions mentioned previously, substantial photoreactivation was noted for P. aeruginosa with none observed for MRSA. It was also noted that the Gram-negative bacterium was much more sensitive than the Gram-positive MRSA to the germicidal UV radiation.

    Control experiments were performed on cells exposed to the respective experimental conditions without turning on the UV lamps. Staphylococcus aureus retained 100% viability for a few days; whereas, P. aeruginosa was 100% viable for at least a week (data not shown).

Discussion

    The widespread use of antimicrobials throughout the world has led to the increasing appearance of micro-organisms with multiple drug resistance. Methicillin-resistant Staphylococcus aureus is a prime example of this phenomenon and has been widely found in both healthcare facilities^9-12 and community settings.^12-16 The importance of UV radiation as an adjunctive therapy to the use of antimicrobials has been documented.^2,7-8

    In 2002, Thai et al² demonstrated that UV C radiation had the ability to reduce bioburden and improve wound status in patients with chronic ulcers of at least 3 months’ duration that were also infected with MRSA. Using a previously described technique,⁸ “the ulcer was cleansed with sterile saline, a thick layer of petroleum jelly was applied to the surrounding periulcer skin and any healthy granulation tissue, and the wound edges were covered with a drape.”² After a 5-minute warm-up period, a 254 nm cold Quartz generator was applied at a distance of 1 inch for 180 seconds per wound site. This method was based on a killing rate reported in a previous study⁴ using the same protocol with an MRSA inoculum of 10⁸ organisms per mL spread on the surface of a series sheep blood agar (SBA) plates. These researchers selected a concentration of 10⁸ organisms per plate surface on the basis that a clinical infection of a wound bed is at least 10⁵ organisms per gram of tissue and 10⁸ would represent a 1,000-fold increase in the minimum number of bacteria present in a clinically infected wound.⁴ Although this extrapolation may be justified, it is an approximation and arguably somewhat closer to the hypothetical minimum bacterial wound load because the bacteria were spread over the entire surface of a petri plate. Also, it is still difficult to compare bacteria spread on SBA to bacteria intermixed within material from a wound site in which growth conditions may be suboptimum and the invading cells subjected to multiple concurrent stresses. Despite some limitations, the previous studies^4-6 clearly revealed the potential importance of UV radiation and point out the need to conduct laboratory and clinical investigations that may be easily compared to one another. Sullivan et al⁵ further noted that UV C treatment was not as effective when administered through thin film dressings.

    Within 7 to 14 days, each of the following patients received a series of seven UV C wound treatments: Case 1, a 77-year-old man with multiple leg ulcers due to venous and arterial insufficiency; Case 2, a 78-year-old woman with poorly controlled type 2 diabetes; and Case 3, an 81-year-old man with a medical history of Alzheimer’s disease and falls. In all of these three cases, the UV C therapy greatly reduced the bacterial load and facilitated healing.² Because of the medical importance of UV treatment, extending basic knowledge involving MRSA in this scientific/medical area is of value.²

    Comparing the current UV C kill curves with the data of Conner-Kerr et al⁴ is possible but some limitations must be noted due to a variation in experimental conditions. In both cases, the starting bacterial concentration was in the range of 10⁸ cells per mL. For Conner-Kerr et al,⁴ the UV C generator was directed at cells spread on SBA at a distance of 1 inch. For the current work, the UV C lamp was applied to cells suspended in saline at a distance of 42 inches in a specially prepared lightbox. Conner-Kerr’s data designate a point when 99.9% of a sample has been killed; whereas, current data quantify cell survival against a measured UV dose and reveal inactivation rates across the entire exposure regimen. The current method also enables direct quantitative comparisons of cell survival under differing environmental conditions. Both the results from Connor-Kerr and current research show that MRSA (see Figure 3 a,b) and methicillin-susceptible S. aureus are readily killed by UV C radiation. The shorter wavelength UV C has far more potential for the direct damage of DNA than the longer wavelength UV A and B. Examples of direct effects on DNA include photodimerizations between adjacent pyrimidine bases, photohydration of cytosine, and, at lower frequencies, inter- and intra-strand cross-links and rare base adducts.²² Current data also show that as the medium NaCl concentration increases, the bacteria are more susceptible to the UV C radiation. This is an important point because MRSA has the ability to multiply in liquid culture up to a level of 3.0 M NaCl (see Figure 1). Increasing ionic strength media was employed to better model the in vivo situation. Methicillin-resistant Staphylococcus aureus survives well in high osmotic strength environments such as sweaty skin and wounds that could exhibit at least a physiologic concentration of saline. With drying, the osmotic level of saline and other solutes could perhaps be even higher. Applying radiation under the conditions of concurrent stress that may be nominally similar to those observed in vivo is important because combined stresses clearly impact resistance capacity. Current results further demonstrate that both log and stationary cells produce the same pattern of kill curves. Chapple et al¹⁷ found that that S. aureus cells had a comparable sensitivity to UV C in both stationary and exponential phases of growth. Methicillin-resistant Staphylococcus aureus found clinically in chronic wounds probably alternates between active growth (log phase) and periods of inactivity (stationary phase), with the latter predominating.

    The authors’ UV A and B kill curves determine that both MRSA (see Figure 2 a,b) and methicillin-susceptible S. aureus are killed by solar UV radiation. However, a much higher dose of the longer wavelength UV A and B must be used to kill the bacteria. Ultraviolet B is responsible for direct and indirect DNA damage.²² Indirect DNA damage results from photo-oxidative processes and the generation of reactive oxygen species. The majority of damage caused by UV A is from indirect damage of the DNA. Bacteria were once again shown to be more susceptible to the solar UV radiation as the medium NaCl increased. Akiyama et al¹⁸ have previously determined that the presence of 10% salts and irradiation with UV A and B suppress the attachment of S. aureus cells to coverslips. The S. aureus strains had been isolated from atopic dermatitis lesions. Additionally, UV A, 10% salts, and heat suppressed the plasma coagulation of these cells. As was true for UV C radiation, the pattern of current kill curves was similar for both log and stationary phase (see Figure 2 a,b).

    Both light and dark mechanisms are used by some bacteria to repair UV-induced damage. Light repair or photoreactivation is inducible and requires visible light in the violet-to-blue range (approximately 380 nm to 430 nm) to activate the enzyme photolyase. This enzyme directly restores the original DNA structure by repairing UV-induced pyrimidine dimers.²² Photoreactivation data is presented in Figure 4. Under the current experimental conditions using log phase cells, photoreactivation was observed for P. aeruginosa PAO-1 but not for MRSA. This observation could be important for the medical use of UV C because treated MRSA cells at least would be unable to repair DNA damage via light DNA repair and that exposure to visible light during or after irradiation would not act as a confounding factor for this species. Photoreactivation capacity is not universal — previous work done in the authors’ laboratory showed light repair for the moderate halophile Halomonas elongata but not for the extreme halophile Halobacterium salinarum.³
Ultraviolet-damaged bacteria such as MRSA could possibly use at least three types of dark repair mechanisms for DNA (nucleotide-excision repair, SOS-error prone repair, and post-replication recombinational repair). All of these involve the product of the bacterial recA gene.²²

    Figure 4 data further show that the Gram-positive MRSA is substantially more resistant to the UV C radiation than the Gram-negative P. aeruginosa PAO-1. Previous work with P. aeruginosa,²³ the Gram-negative H. elongata,³ and Gram-positive moderate halophiles²⁴ support this observation. Sullivan and Conner-Kerr⁶ also have determined that eukaryotic cells (Candida albicans, Aspergillus fumigatus) are much more resistant than some prokaryotic organisms to UV C radiation (P. aeruginosa and Mycobacterium abscessus).

Conclusion

    The experiments detailed herein clearly reveal that medium composition exerts a substantial effect on S. aureus UV resistance. This finding suggests that in order to develop rational UV irradiation protocols that kill infecting bacteria with minimal collateral damage to the host tissue, careful consideration must be given to the precise physicochemical conditions prevalent in the wound site. Data suggest that effective eradication of infecting S. aureus cells may be accomplished at far lower doses than those applied by Thai et al.²

    An accurate understanding of necessary rates and total doses required for control or eradication would be important information, enabling this technology to be employed over large surface areas as would be necessary to treat extensive wounds such as burns. In principle, useful inactivating doses may be supplied by blacklight or germicidal fluorescent bulbs — UV sources that operate at low temperatures — are readily available and capable of irradiating comparatively large areas.

    Although growth phase has no impact on S. aureus UV resistance, whether P. aeruginosa is growing rapidly or in the stationary phase has an important effect on inactivation rates. Because P. aeruginosa colonizing wounds probably will be in stationary phase, UV treatments will need to account for an enhanced UV resistance of such cells.

    The fact that photoreactivation does not occur in S. aureus (at least not under the current experimental conditions) is of potential importance to the conduct of clinical UV treatment of wounds. Application of germicidal (UV C) radiation will be confounded in the event that wounds are subsequently exposed to any visible light as photoreactivation enzymes reverse lethal DNA damage. For S. aureus, exposure to visible light will probably not increase cell survival, but in wounds with a mixed flora or infected with species such as P. aeruginosa,, excluding visible light during and after irradiation is important to preventing cell recovery. Current experiments and previous studies conducted with several species reveal that not all bacteria will photoreactivate UV C damage, but apart from testing for the ability, assuming most species will lack this capacity is not advised.
In principle, UV radiation therapy could be combined effectively with other future adjunctive therapies such as bacteriophage treatments. Ultraviolet exposure of many lysogens induces prophages to lytic growth, causing the death of the host bacterium.²⁵ Treating wounds with bacteriophage could both kill bacteria due to ordinary infection and, in surviving lysogenized cells exposed to UV radiation, provoke a second round of intense killing due to prophage induction. Possibly, lysogenized cells would be more susceptible to UV radiation, enabling excellent responses to be obtained with lower patient exposure and decreased risk of collateral tissue damage.

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