Breaking down antibiotic resistance in methicillin-resistant Staphylococcus aureus: Combining antimicrobial photodynamic and antibiotic treatments.

The widespread use of antibiotics drives the evolution of antimicrobial-resistant bacteria (ARB), threatening patients and healthcare professionals. Therefore, the development of novel strategies to combat resistance is recognized as a global healthcare priority. The two methods to combat ARB are development of new antibiotics or reduction in existing resistances. Development of novel antibiotics is a laborious and slow-progressing task that is no longer a safe reserve against looming risks. In this research, we suggest a method for reducing resistance to extend the efficacious lifetime of current antibiotics. Antimicrobial photodynamic therapy (aPDT) is used to generate reactive oxygen species (ROS) via the photoactivation of a photosensitizer. ROS then nonspecifically damage cellular components, leading to general impairment and cell death. Here, we test the hypothesis that concurrent treatment of bacteria with antibiotics and aPDT achieves an additive effect in the elimination of ARB. Performing aPDT with the photosensitizer methylene blue in combination with antibiotics chloramphenicol and tetracycline results in significant reductions in resistance for two methicillin-resistant Staphylococcus aureus (MRSA) strains, USA300 and RN4220. Additional resistant S. aureus strain and antibiotic combinations reveal similar results. Taken together, these results suggest that concurrent aPDT consistently decreases S. aureus resistance by improving susceptibility to antibiotic treatment. In turn, this development exhibits an alternative to overcome some of the growing MRSA challenge.

Moving from the traditional paradigm of pathogen inactivation to controlling antibiotic resistance in water - Role of ultraviolet irradiation.

Ultraviolet (UV) irradiation has proven an effective tool for inactivating microorganisms in water. There is, however, a need to look at disinfection from a different perspective because microbial inactivation alone may not be sufficient to ensure the microbiological safety of the treated water since pathogenic genes may still be present, even after disinfection. Antibiotic resistance genes (ARGs) are of a particular concern since they enable microorganisms to become resistant to antibiotics. UV irradiation has been widely used for disinfection and more recently for destroying ARGs. While UV lamps remain the principal technology to achieve this objective, UV light emitting diodes (UV-LEDs) are novel sources of UV irradiation and have increasingly been reported in lab-scale investigations as a potential alternative. This review discusses the current state of the applications of UV technology for controlling antibiotic resistance during water and wastewater treatment. Since UV-LEDs possess several attractive advantages over conventional UV lamps, the impact of UV-LED characteristics (single vs combined wavelengths, and operational parameters such as periodic or pulsed and continuous irradiation, pulse repetition frequencies, duty cycle), type of organism, and fluence response, are critically reviewed with a view to highlighting the research needs for addressing future disinfection challenges. The energy efficiency of the reported UV processes is also evaluated with a focus on relating the findings to disinfection efficacy. The greater experience with UV lamps could be useful for investigating UV-LEDs for similar applications (i.e., antibiotic resistance control), and hence identification of future research directions.

Effectiveness of an ultraviolet-C disinfection system for reduction of healthcare-associated pathogens.

BACKGROUND: Healthcare-associated infections caused by multidrug-resistant (MDR) pathogens are significantly associated with increased mortality and morbidity. Environmental cleaning can reduce transmission of these pathogens but is often inadequate. Adjunctive methods are warranted to enhance the effectiveness of disinfection particularly in hospital settings where healthcare-associated infections are of major concern. METHODS: We conducted a study to examine the effectiveness of a mobile, automatic device, Hyper Light Disinfection Robot (model: Hyper Light P3), which utilized ultraviolet-C (UV-C) to kill MDR-Pseudomonas aeruginosa, MDR- Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), Mycobacterium abscessus and Aspergillus fumigatus. The performance of this device in disinfecting hospital rooms previously admitted by patients harboring MRSA and VRE was also assessed. RESULTS: Except for VRE and M. abscessus, more than 3 log10 reduction of vegetative bacteria colonies was observed after UV-C irradiation of 5 min at a distance of 3 m from the device. At the distance of 1 m, substantial and comparable reduction of colonies was observed across all tested microorganisms regardless of exposure time. The killing effect was less pronounced for A. fumigatus particularly at the distance of 2-3 m. In uncleaned hospital rooms, there was significant reduction in the number of bacteria colonies sampled from different surfaces after UV-C irradiation for 15 min. CONCLUSIONS: UV-C disinfection system was effective in killing MDR pathogens. Further study is warranted to confirm its effectiveness as an adjunctive method in disinfecting hospital environment.

Chlorination caused a shift in marine biofilm niches on microfiltration/ultrafiltration and reverse osmosis membranes and UV irradiation effectively inactivated a chlorine-resistant bacterium.

The effect of chlorine disinfection on marine biofilm populations and communities formed on membrane surfaces was investigated under two feedwater conditions: raw seawater and deep bed filtration-treated seawater. As a result of chlorination, the structure of the biofilm community on the microfiltration/ultrafiltration and reverse osmosis membrane coupons shifted significantly at the genus level. However, the total bacterial population was not reduced under the two feedwater conditions. This failure to control the biofilm was attributed to the adaptation and survival of selected bacteria under chlorine stress. Phaeobacter caeruleus, isolated from the biofilm, was examined as a representative chlorine-resistant biofilm-forming bacterium. The number of viable P. caeruleus was significantly reduced (as much as 99.8%) after ultraviolet (UV) disinfection. The results indicated that additional disinfection by UV irradiation can inactivate chlorine-resistant bacteria. Therefore, tandem chlorination-UV disinfection may enhance the efficiency of biofouling control in seawater reverse osmosis processes. The synergistic effects of tandem chlorination-UV irradiation on the marine biofilm community should be investigated in future studies.

Ozonation and UV254nm radiation for the removal of microorganisms and antibiotic resistance genes from urban wastewater.

Conventional wastewater treatment has a limited capacity to reduce antibiotic resistant bacteria and genes (ARB&ARG). Tertiary treatment processes are promising solutions, although the transitory inactivation of bacteria may select ARB&ARG. This study aimed at assessing the potential of ozonation and UV254nm radiation to inactivate cultivable fungal and bacterial populations, and the selected genes 16S rRNA (common to all bacteria), intI1 (common in Gram-negative bacteria) and the ARG vanA, blaTEM, sul1 and qnrS. The abundance of the different microbiological parameters per volume of wastewater was reduced by ∼2 log units for cultivable fungi and 16S rRNA and intI1 genes, by∼3-4 log units, for total heterotrophs, enterobacteria and enterococci, and to values close or below the limits of quantification for ARG, for both processes, after a contact time of 30min. Yet, most of the cultivable populations, the 16S rRNA and intI1 genes as well as the ARG, except qnrS after ozonation, reached pre-treatment levels after 3days storage, suggesting a transitory rather than permanent microbial inactivation. Noticeably, normalization per 16S rRNA gene evidenced an increase of the ARG and intI1 prevalence, mainly after UV254nm treatment. The results suggest that these tertiary treatments may be selecting for ARB&ARG populations.

Cross-Contamination of Residual Emerging Contaminants and Antibiotic Resistant Bacteria in Lettuce Crops and Soil Irrigated with Wastewater Treated by Sunlight/H2O2.

The sunlight/H2O2 process has recently been considered as a sustainable alternative option compared to other solar driven advanced oxidation processes (AOPs) in advanced treatment of municipal wastewater (WW) to be reused for crop irrigation. Accordingly, in this study sunlight/H2O2 was used as disinfection/oxidation treatment for urban WW treatment plant effluent in a compound parabolic collector photoreactor to assess subsequent cross-contamination of lettuce and soil by contaminants of emerging concern (CECs) (determined by QuEChERS extraction and LC-QqLIT-MS/MS analysis) and antibiotic resistant (AR) bacteria after irrigation with treated WW. Three CECs (carbamazepine (CBZ), flumequine (FLU), and thiabendazole (TBZ) at 100 µg L(-1)) and two AR bacterial strains (E. coli and E. faecalis, at 10(5) CFU mL(-1)) were spiked in real WW. A detection limit (DL) of 2 CFU mL(-1) was reached after 120 min of solar exposure for AR E. coli, while AR E. faecalis was more resistant to the disinfection process (240 min to reach DL). CBZ and TBZ were poorly removed after 90 min (12% and 50%, respectively) compared to FLU (94%). Lettuce was irrigated with treated WW for 5 weeks. CBZ and TBZ were accumulated in soil up to 472 ng g(-1) and 256 ng g(-1) and up-taken by lettuce up to 109 and 18 ng g(-1), respectively, when 90 min treated WW was used for irrigation; whereas no bacteria contamination was observed when the bacterial density in treated WW was below the DL. A proper treatment time (>90 min) should be guaranteed in order to avoid the transfer of pathogens from disinfected WW to irrigated crops and soil.

Distinguishing effects of ultraviolet exposure and chlorination on the horizontal transfer of antibiotic resistance genes in municipal wastewater.

Growing attention has been paid to the dissemination of antibiotic resistance genes (ARGs) in wastewater microbial communities; however, the disinfection processes, as microbial control technologies, have not been evaluated for their impacts on ARGs transfer. In this study, the effects of ultraviolet (UV) disinfection and chlorination on the frequency of ARGs transfer have been explored based on the conjugative transfer model between Gram-negative strains of E. coli. The results indicated that UV disinfection and chlorination exhibit distinct influences on the conjugative transfer. Low UV doses (up to 8 mJ/cm2) had little influence on the frequency of conjugative transfer, and UV exposure only decreased the bacterial number but did not change the cell permeability. By comparison, low chlorine doses (up to 40 mg Cl min/L) significantly promoted the frequency of conjugative transfer by 2-5-fold. The generated chloramine stimulated the bacteria and improved the cell permeability. More pilus were induced on the surface of conjugative cells, which acted as pathways for ARGs transfer. The frequency of ARG transfers was greatly suppressed by high doses of UV (>10 mJ/cm2) or chlorine (>80 mg Cl min/L).

Survival of Escherichia coli in two sewage treatment plants using UV irradiation and chlorination for disinfection.

We investigated the survival of Escherichia coli in two STPs utilising UV irradiation (STP-A) or chlorination (STP-B) for disinfection. In all, 370 E. coli strains isolated from raw influent sewage (IS), secondary treated effluent (STE) and effluent after the disinfection processes of both STPs were typed using a high resolution biochemical fingerprinting method and were grouped into common (C-) and single (S-) biochemical phenotypes (BPTs). In STP-A, 83 BPTs comprising 123 isolates were found in IS and STE, of which 7 BPTs survived UV irradiation. Isolates tested from the same sites of STP-B (n = 220) comprised 122 BPTs, however, only two BPTs were found post-chlorination. A representative isolate from each BPT from both STPs was tested for the presence of 11 virulence genes (VGs) associated with uropathogenic (UPEC) or intestinal pathogenic (IPEC) E. coli strains. Strains surviving UV irradiation were distributed among seven phylogenetic groups with five BPTs carrying VGs associated with either UPEC (4 BPTs) or IPEC (1 BPT). In contrast, E. coli strains found in STP-B carried no VGs. Strains from both STPs were resistant to up to 12 out of the 21 antibiotics tested but there was no significant difference between the numbers of antibiotics to which surviving strains were resistant to in these STPs. Our data suggests that some E. coli strains have a better ability to survive STPs utilising chlorination and UV irradiation for disinfection. However, strains that survive UV irradiation are more diverse and may carry more VGs than those surviving SPTs using chlorination.

Microbial selectivity of UV treatment on antibiotic-resistant heterotrophic bacteria in secondary effluents of a municipal wastewater treatment plant.

Little is known about the microbial selectivity of UV treatment for antibiotic resistant bacteria, and the results of limited studies are conflicting. To understand the effect of UV disinfection on antibiotic resistant bacteria, both total heterotrophic bacteria and antibiotic resistant bacteria (including cephalexin-, ciprofloxacin-, erythromycin-, gentamicin-, vancomycin-, sulfadiazine-, rifampicin-, tetracycline- and chloramphenicol-resistant bacteria) were examined in secondary effluent samples from a municipal wastewater treatment plant. Bacteria resistant to both erythromycin and tetracycline were chosen as the representative of multiple-antibiotic-resistant bacteria and their characteristics after UV treatment were also investigated. UV disinfection results in effective inactivation for total heterotrophic bacteria, as well as all antibiotic resistant bacteria. After UV treatment at a fluence of 5 mJ/cm(2), the log reductions of nine types of antibiotic resistant bacteria varied from 1.0 ± 0.1 to 2.4 ± 0.1. Bacteria resistant to both erythromycin and tetracycline had a similar fluence response as did total heterotrophic bacteria. The findings suggest that UV disinfection could eliminate antibiotic resistance in wastewater treatment effluents and thus ensure public health security. Our experimental results indicated that UV disinfection led to enrichment of bacteria with resistance to sulfadiazine, vancomycin, rifampicin, tetracycline and chloramphenicol, while the proportions of cephalexin-, erythromycin-, gentamicin- and ciprofloxacin-resistant bacteria in the wastewater decreased. This reveals the microbial selectivity of UV disinfection for antibiotic resistant bacteria.

Helicobacter pylori inactivation and virulence gene damage using a supported sensitiser for photodynamic therapy.

About half of the world's population is currently infected with Helicobacter pylori, which is involved in the development of several gastro-duodenal pathologies. The increasing number of antibiotic resistance reduces the effectiveness of the first-line therapy, so new strategies to improve the H. pylori eradication rates are needed. Antimicrobial Photodynamic Therapy (APDT) benefits from photogenerated reactive oxygen species, such as singlet oxygen, which inactivate microorganisms by means of photosensitising dyes and visible light. Therefore, it could be a suitable alternative for H. pylori eradication in the gastro-duodenal tract, particularly in patients infected with antibiotic resistant strains. We evaluated APDT against H. pylori, in vitro, using a new photosensitising material (PSM) based on a ruthenium(II) complex covalently bound to micrometric glass beads. Five H. pylori isolates (classified according to cagA genotype, and metronidazole-clarithromycin resistance) were used. Bacteria were mixed with the PSM and incubated in the dark or illuminated by blue light. Aliquots (min 1', 2', 5', 15' and 30') were cultured and colonies were counted after 2-3 days. A 99.99999% decrease was detected in the number of colonies in the irradiated wells where the bacterium was mixed with the PSM, compared to non-illuminated wells or with irradiated wells without PSM. It was also confirmed that DNA is a molecular target for oxidant species released during APDT (evaluated by alkaline gel electrophoresis after endonuclease III incubation, ureC and cagA RT-PCR, and bacterial fingerprint). Results were independent of cagA gene and antibiotic resistances.

Inactivation/reactivation of antibiotic-resistant bacteria by a novel UVA/LED/TiO2 system.

In this study, an effective photocatalytic disinfection system was established using the newly emerged high power UVA/LED lamp. Crystallizing dish coated with TiO2 was prepared by 32-times impregnation-drying processes, and served as the supporting container for water samples. This study focused on the application of this UVA/LED system for the photocatalytic disinfection of selected antibiotic-resistant bacteria, Escherichia coli ATCC 700891. The disinfection performances were studied under various light intensities and illumination modes. Results show that higher light intensity could reach more significant inactivation of E. coli ATCC 700891. With the same UV dose, log-removal of antibiotic-resistant bacteria decreased with circle time in the studied range, while increased with duty circle. A "residual disinfecting effect" was found in the following dark period for bacteria collected at different phases of photocatalytic process. Residual disinfecting effect was found not significant for bacteria with 30 min periodic illumination. While residual disinfecting effect could kill almost all bacteria after 90 min UV periodic illumination within the following 240 min dark period.

Mecanismo de resistencia a antibióticos ß-lactámicos en Staphylococcus aureus / Mechanisms of resistance to ß-lactam antibiotics in Staphylococcus aureus

S. aureus ha demostrado un gran poder de adaptación a los agentes antimicrobianos, adquiriendo paso a paso resistencia a todos los antibióticos disponibles para el tratamiento de las infecciones que ocasiona. Existen tres mecanismos de resistencia a los antibióticos ß-lactámicos en S. aureus: resistencia mediada por enzimas (penicilinasas o ß-lactamasas) las cuales desactivan al antibiótico; resistencia intrínseca, que no es debida a la inactivación de drogas y es responsable dela resistencia a meticilina; y la modificación de las proteínas de unión a penicilinas (PBPs). Además, S. aureus puede expresar el fenómeno de tolerancia, en el que ocurre una disociación de las acciones inhibitoria y bactericida de los antibióticos ß-lactámicos. De éstos, el mecanismo más importante, es la resistencia intrínseca que es probablemente más compleja, debido a que varios factores pueden afectar también su expresión

S. aureus has shown a great power of adaptation to antimicrobial agents, acquiring, step-bystep, resistance to all available antibiotics for treatment of the infections it causes. S. aureus has three major mechanisms of resistance to ß-lactam antibiotics: enzyme mediated (penicillinase or ß-lactamase) by which the antibiotic is inactivated; intrinsic resistance, which is not due to drug inactivation and accounts for methicillin resistance; and modifications of penicillin-binding proteins (PBPs). Additionally, S. aureus can express the tolerance phenomenon, in which there is a dissociation of the inhibitory and killing actions of ß-lactam antibiotics. Of these, the most important mechanism is intrinsic resistance, which is probably more complex because several factors can affect its expression

â - lactam susceptibility of Escherichia coli isolates from urinary tract infections exhibiting different resistance phenotypes

Susceptibility to â-lactams was determined in 203 recentSpanish E. coli isolates from urinary tract infectionsexhibiting different resistance phenotypes: a) susceptible(n = 60); b) quinolone-resistant (n = 45); c) penicillinase(n=64); d) hyperproduction of penicillinase (n=8); e) inhibitorresistant TEM (IRT) (n=18), and f) extended spectrumbetalactamase (ESBL) (n=8). Minimum inhibitory concentration(MIC) determination by agar dilution and susceptibilitytests for ESBL detection by macrodilution were performedfollowing CLSI recommendations. All the â-lactamstested showed high activity against susceptible and penicillinasephenotypes, with close to 100 % susceptibility.Hyperproduction of penicillinase increased MIC90 values forall antibiotics except for meropenem, with 100% resistanceto cefuroxime and amoxicillin/clavulanic acid, and 100%susceptibility to cefotaxime, piperacillin/tazobactam andmeropenem. All the antibiotics, except for amoxicillin/clavulanicacid, exhibited high activity against IRT. Meropenem,cefminox and piperacillin/tazobactam exhibited thehighest activity against ESBL, followed by amoxicillin/clavulanicacid. The most active compound among the parenteralantibiotics was meropenem, regardless of the resistancephenotype. Among the oral antibiotics, the most activecompound was cefditoren with the exception of ESBL whereamoxicillin/clavulanic acid where the MIC90 value wasone dilution lower (AU)

Se determinó la susceptibilidad a betalactámicos de203 aislados recientes de E. coli procedentes de infeccionesdel tracto urinario en España y que presentaban distintosfenotipos de resistencia: a) susceptible (n = 60);b) resistente a quinolonas (n=45); c) productor de penicilinasa(n=64); d) hiperproductor de penicilinasa (n=8);e) resistente a inhibidores de TEM (IRT) (n=18), y f) productorde betalactamasas de espectro extendido (BLEE)(n=8). La determinación de la concentración mínima inhibitoria(CMI) por dilución en agar y los tests de susceptibilidadpara la detección de BLEE se realizaron siguiendolas recomendaciones del Clinical and Laboratory StandardsInstitute (CLSI). Frente a los fenotipos susceptible yproductor de penicilinasa, todos los betalactámicos ensayadosexhibieron gran actividad, con una sensibilidadcercana al 100% de los aislados. La hiperproducción depenicilinasa incrementó los valores de CMI90 de todos losantibióticos, excepto de meropenem, con un 100% de resistenciaa cefuroxima y amoxicilina/clavulánico y un 100% desensibilidad a cefotaxime, piperacilina/tazobactam y meropenem.Todos los antibióticos, excepto amoxicilina/clavulánico,presentaron gran actividad frente a las cepas IRT.Meropenem, cefminox y piperacilina/tazobactam presentaronla mayor actividad frente a BLEE, seguidas de amoxicilina/clavulánico. Entre los antibióticos parenterales, elcompuesto más activo fue meropenem, con independenciadel fenotipo de resistencia. Entre los antibióticos oralesel compuesto más activo fue cefditoren, excepto frentea las cepas BLEE, donde amoxicilina/clavulánico presentóen un valor de CMI90 una dilución menor (AU)

A genetic selection for Neurospora crassa mutants altered in their light regulation of transcription.

Transcription of the Neurospora crassa gene con-10 is induced during conidiation and following exposure of vegetative mycelia to light, but light activation is transient due to photoadaptation. We describe mutational analyses of photoadaptation using a N. crassa strain bearing a translational fusion of con-10, including its regulatory region, to a selectable bacterial gene conferring hygromycin resistance (hph). Growth of this strain was sensitive to hygromycin, upon continuous culture in the light. Five mutants were isolated that were resistant to hygromycin when cultured under constant light. Three mutant strains displayed elevated, sustained accumulation of con-10::hph mRNA during continued light exposure, suggesting that they bear mutations that reduce or eliminate the presumed light-dependent repression mechanism that blocks con-10 transcription upon prolonged illumination. These mutations altered photoadaptation for only a specific group of genes (con-10 and con-6), suggesting that regulation of photoadaptation is relatively gene specific. The mutations increased light-dependent mRNA accumulation for genes al-1, al-2, and al-3, each required for carotenoid biosynthesis, resulting in a threefold increase in carotenoid accumulation following continuous light exposure. Identification of the altered gene or genes in these mutants may reveal novel proteins that participate in light regulation of gene transcription in fungi.

[Inhibitory effects of electric UHF field and magnetic infrared laser irradiation on Mycobacterium tuberculosis]. / Ingibiruiushchee deistvie élektricheskogo polia UVCh i magnito-infrakrasno-lazernogo izlucheniia na mikobakterii tuberkuleza.

Microbiological studies indicate that stimulating the multiplication of Mycobacteria tuberculosis, electric UHF fields at 40 and 70 W make them available for drug exposure despite drug resistance. Unlike the electric UHF fields, MILI has a direct inhibitory effect on Mycobacteria tuberculosis that are sensitive and resistant to antibiotic drugs, by potentiating their action. In the clinical setting, chemotherapy in combination with electric UHF fields and MILI therapy reduces the time of disappearance of clinical signs of the disease, infiltration resolution, bacterial isolation, and decay cavity closure.

Induction of unique tandem-base change mutations in bacterial spores exposed to extreme dryness.

Despite the remarkable resistance to desiccation, Bacillus subtilis spores manifest indications of DNA damage when being kept in an extremely dry environment made by high vacuum. Spores of strain TKJ3422 (uvrA10 spl-1 recA4) with triple repair defects lost colony-forming capacity dependent on the duration and strength of the exposure. Mutations to rifampicin resistance were induced in the spores of the strain HA101 with wild-type repair capability and the strain TKJ6312 (uvrA10 spl-1) with double repair defects. The majority of nalidixic acid-resistant mutations induced by the exposure to high vacuum belonged to one particular allele gyrA12 carrying a tandem-base change, 5'-CA to 5'-TT, at codon 84 of the gyrA gene coding for DNA gyrase subunit A. This allele has never been found among more than 500 mutants obtained by various treatments other than vacuum exposure. These results indicate forced dehydration of DNA in the microenvironment of the spore core causes unique damage leading to lethal and mutagenic consequences.

[The effect of millimeter-band radiation of nonthermal intensity on sensitivity of Staphylococcus to various antibiotics]. / Vliianie millimetrovogo izlucheniia neteplovoi intensivnosti na chuvstvitel'nost' stafilokokka k razlichnym antibiotikam.

The affect of preliminary irradiation of staphylococcus culture by electromagnetic radiation of extremely high frequency (42, 54, 66 + 78 GHz) of nonthermal intensity on the bacteria growth on the media containing various antibiotics is studied. The reliable change in bacteria sensitivity toward 5 antibiotics, mainly having membranotropic properties is observed in the experiments using 14 antibiotics with various mechanisms of action. It has been established that in the presence of subbactericide concentrations of active antibiotics the irradiation could result in both further suppression of bacteria growth and its stimulation. As shown, the development of these effects takes place even in a matter of minutes of preliminary irradiation, and weak changes are observed at further increase of this period up to 60 min.

A more potent bystander cytocidal effect elicited by tumor cells expressing the herpes simplex virus-thymidine kinase gene than by fibroblast virus-producer cells in vitro.

Retrovirus-mediated herpes simplex virus-thymidine kinase (HSV-tk) gene therapy is a promising approach in the treatment of brain tumors. Previous in vitro and in vivo studies have demonstrated a bystander effect in which nonmodified tumor cells in proximity to HSV-tk-modified tumor cells are killed with the modified cells in the presence of ganciclovir. In the present study the authors assessed the contribution of infectious HSV-tk retrovirus made by producer cells to the bystander cytocidal effect in tissue culture using Walker 256 rat breast carcinosarcoma cells, which represent an established model for carcinomatous meningitis. The authors observed ganciclovir-dependent growth inhibition even when only one HSV-tk-positive Walker cell was mixed with 1000 HSV-tk-negative Walker cells and showed that the bystander cytocidal effect is not mediated by toxic cell lysis products. Walker cells engineered to produce HSV-tk retrovirus with titers ranging from 10(3) to 10(5) colony-forming units/ml exert no greater cytocidal effect than nonviral producer HSV-tk-positive Walker cells in vitro. Murine fibroblast-producer cells with viral titers ranging from 10(6) to 10(7) colony-forming units/ml exerted a stronger cytocidal effect than nonviral producer HSV-tk-positive murine fibroblasts. Despite the high viral titers of fibroblast producer cells, HSV-tk-modified Walker cells performed better than fibroblast producer cells in their cytotoxic effect on wild-type Walker tumor cells. Given that HSV-tk-modified tumor cells can become ganciclovir resistant, we tested gamma-irradiation as a means to overcome resistance. Lethal gamma-irradiation of the HSV-tk-positive Walker cells did not abolish their bystander effect on Walker HSV-tk-negative cells. One can infer from these results that HSV-tk-modified tumor cells, irradiated or not, may be a better alternative to murine fibroblast producer cells in the treatment of central nervous system neoplasia.

Cyclophilin-dependent stimulation of transcription by cyclosporin A.

Exposure to cyclosporin A (CspA) increased laccase (lac-1) transcript accumulation in the chestnut blight fungus Cryphonectria parasitica. This response was suppressed by compounds that interfere with calcium-dependent signal transduction and by the presence of a virulence-attenuating mycovirus. CspA stimulated the accumulation of mRNA from a nonhomologous reporter fused to the lac-1 promoter, indicating that the increased transcript levels resulted from an increase in promoter activity. Based on the current model for the regulation of lac-1 transcription, these results suggest that CspA interferes with a negative regulatory pathway that normally constrains lac-1 promoter activity. Significantly, CspA did not stimulate lac-1 transcription in mutant strains deficient in CspA binding activity, directly demonstrating a requirement for the interaction of CspA and cyclophilin in the modulation of lac-1 transcription. Our results establish that CspA treatment can stimulate gene transcription and that cyclophilin is the cellular receptor that mediates this activity.