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.

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.

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).

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.

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.

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.

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.

UV sensitivity of several genetic markers of Haemophilus influenzae DNA.

Studies on inactivation by UV of several genetic markers of the streptomycin and novobiocin resistance regions of H. influenzae DNA have shown that two kinds of markers may be found with respect to frequency of integration, UV sensitivity and reactivability. One class of markers has high integration frequency and low UV sensitivity, which can be partly explained by the reactivability of the irradiated markers. The second class of markers has low integration frequency and high UV sensitivity. Markers of each class have been found in the novobiocin region.-In one recipient strain with decreased transformability the difference in integration frequency between the two classes is enhanced.

Increased spontaneous mitotic segregation in MMS-sensitive mutants of Saccharomyces cerevisiae.

Methyl methanesulfonate (MMS)-sensitive mutants of Saccharomyces cerevisiae belonging to four different complementation groups, when homozygous, increase the rate of spontaneous mitotic segregation to canavanine resistance from heterozygous sensitive (canr/+) diploids by 13-to 170-fold. The mms8-1 mutant is MMS and X-ray sensitive and increases the rate of spontaneous mitotic segregation 170-fold. The mms9-1 and mms13-1 mutants are sensitive to X rays and UV, respectively, in addition of MMS, and increase the rate of spontaneous mitotic segregation by 13-fold and 85-fold, respectively. The mutant mms21-1 is sensitive to MMS, X rays and UV and increases the rate of spontaneous mitotic segregation 23-fold.

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.

Induction of petite "mutants" in an ethidium-resistant strain of Saccharomyces cerevisiae by photoaffinity labeling. Distinction between early and late steps.

A strain of Saccharomyces cerevisiae (MH41-7B/011) was resistant to petite induction by ethidium bromide at 30 degrees, but was sensitive to induction by photolabeling with ethidium monoazide. These results suggested a defect in the mutant in metabolic activation of ethidium to account for its resistance. Synchronized cultures of both the mutant and the normal parent strains showed a substantial reduction in petite response to photolabeling in stationary phase cells which could not be accounted for by changes in cell penetration of the drug. The use of photolabeling with normal and mutant cells suggested that petite induction can be divided into early and late steps.

Genetic analysis of DNA repair in Aspergillus: evidence for different types of MMS-sensitive hyperrec mutants.

To identify genes which affect DNA repair and possibly recombination in Aspergillus nidulans, mutants hypersensitive to methyl methanesulphonate (MMS) were induced with ultraviolet light (UV) or gamma-rays. About half of them contained associated translocations and many were hypersensitive to UV and/or defective in meiosis. Two are alleles of the known uvsB gene while most others define new genes. In addition, among available uvs mutants many were found to be MMS-sensitive. Some of the various uncharacterized ones were identified as alleles of known uvs, but 5 of them were mapped in 2 new genes, uvsH and uvsJ. To identify functional and epistatic groups, mutants from each uvs gene were tested for effects on recombination and mutation, and double mutant uvs strains were compared for UV survival to their component single mutant strains. 3 epistatic pairs were identified, (1) uvsF and H, (2) uvsB and D, and (3) uvsC and E. Conclusive interpair tests were difficult, because such double mutant combinations were frequently lethal or nearly so. The first pair, uvsF and H, shared some of the properties of excision-defective mutants, both uvs being very highly sensitive to UV for mutation as well as survival. But unlike such mutants, uvsH was also sensitive to gamma-rays and defective in meiosis. Both uvs showed normal levels of meiotic recombination, but greatly increased spontaneous mitotic crossing-over, being the most "hyperrec" types among all uvs. The second pair, uvsB and uvsC, which was similarly hyperrec showed only slight increases of UV-induced mutation (less than 2-fold). As a main effect, these uvs caused very high frequencies of unbalanced, unstable segregants from diploid conidia (30 X), but few of these were recognizable aneuploids. The third pair, uvsC and E, which are known to be rec- for gene conversion, caused reduced mitotic crossing-over in diploids and increased levels of haploid segregants. These mutants are spontaneous mutators, but showed less UV-induced mutation than wild-type controls.

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.

[Plasmid DNA transduction in Bacillus subtilis]. / Transduktsiia plazmidnoi DNK u Bacillus subtilis.

Transduction of Bacillus subtilis pUB110 plasmid by AR9 phage is described. Some aspects of this process are studied. Plasmid transduction depended on multiplicity of infection similar to cases of chromosomal markers transduction, though optimal multiplicity of infection was achieved using low number of phage particles. No cotransduction of plasmid and chromosomal markers was demonstrated. The transduction frequencies of plasmid and chromosomal markers increased after UV irradiation of phage suspensions within the range of definite doses.

[Characteristics of the range of mutations in nystatin resistance induced in yeasts by 6-N-hydroxylaminopurine]. / Osobennosti spektra mutatsii nistatinustoichivosti, indutsirovannykh u drozhzhei 6-N-gidroksilaminopurinom.

A simple procedure for induction of nistatin-resistant mutants in yeasts under the action of 6-N-hydroxylaminopurine (HAP) is proposed. Some differences between the spectra of mutations induced by HAP and UV-light are observed: HAP induces dominant and double recessive mutants more frequently.

[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.

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.

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.

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.