Potent antimicrobial formula -
The antimicrobial effects of alone or in combination with conventional antibiotics were assessed by scanning electron microscopy, transmission electron microscopy, drug combination assay, and growth inhibition assay. In addition, we investigated the antimicrobial efficacy in subcutaneous infection model in vivo.
Results: showed significant synergistic antimicrobial effects in combination with SPR, a polymyxin B derivative, against E. coli standard strain and extensively drug-resistant XDR clinical isolates, and the combination exhibited good safety property in vitro.
In addition, we demonstrated that the combinational treatment of and SPR exhibited a considerable antibacterial activity in vivo, and no tissue damage or other toxicity was observed after the therapeutic dose treatment.
Discussion: To confront the issue of the infectious diseases related to E. coli and its multidrug resistant strains, potential approaches, such as new antibacterial agents with different structures from conventional antibiotics and drug combinations, are urgently needed. In this study, we have determined the in vitro and in vivo antimicrobial potential of alone or in combination with SPR, which might be used as a treatment option for E.
coli related infections. Escherichia coli E. coli is a Gram-negative, facultative anaerobic short bacillus, which was first found and isolated by Theodor Escherich in Escherich, Escherichia coli is known as both harmless commensal of the gastrointestinal tract in warm-blooded animals and one of the most important pathogens in humans, which is the most frequent cause of urinary tract infections Kaper et al.
These infections are common, but also associated with high morbidity and high mortality Russo and Johnson, ; Lefort et al.
However, misuse and overuse of antibiotics are the main reasons for the emergence and spread of severe antimicrobial resistance and multidrug resistance MDR; Llor and Bjerrum, ; Harkins et al. At Global Burden of Diseases, Injuries, and Risk Factors Study GBD , antimicrobial resistance was the third leading GBD Level 3 cause of death Antimicrobial Resistance Collaborators, Antibiotic-resistant bacteria are a threat to public health care and have triggered the development of global action plans World Health Organization, The World Health Organization has included extended-spectrum β-lactamase ESBL -producing E.
In , six pathogens, E. coli, S. aureus, K. pneumoniae, S. pneumoniae, A. baumannii, and P. aeruginosa , led to more than , deaths related to antimicrobial resistance, and E. coli was ranked first for the amount of deaths Antimicrobial Resistance Collaborators, It is urgent to develop new antibacterial agents to combat diseases related to E.
coli and its resistant strains. Confronting with the issue of antibiotic resistance, drug combination is also one of the potential approaches. Antibiotic adjuvants are compounds with weak antibacterial activity when they are used solely, but they can restore or potentiate the activity of antibiotic, such as SPR Liu et al.
SPR NAB is a cationic peptide derived from polymyxin B and retains the ability to permeabilize the outer membrane of Gram-negative bacteria Vaara et al.
Polymyxins polymyxin B and colistin are often the only alternative for some pan-resistant Gram-negative bacteria, although the usage of polymyxins is shadowed by their toxicity, which is mainly referred to nephrotoxicity Arias and Murray, Compared with polymyxin B, SPR exhibits minimal intrinsic Gram-negative antibacterial activity, but an excellent safety profile in vivo.
Eckburg et al. SPR is reported to combat Gram-negative bacteria in combination with traditional antibiotics, such as azithromycin and temocillin Mendes et al. However, the in vitro and in vivo effects of new identified antimicrobials in combination with SPR have not yet been reported.
Here, to identify novel antibacterial compounds with different structures from the conventional antibiotics, we screened MINI Scaffold Library TopScience, L containing 5, bioactive small-molecules and determined a novel small molecular compound, , which was found effective alone or in combination with SPR to combat E.
coli , including its standard strain and drug-resistant clinical isolates. In addition, the synergistic antimicrobial effects are observed in the subcutaneous abscess model, which was established by E. coli infection, while the combination treatment showed good safety property to the host.
In this study, we conducted a series of in vitro and in vivo assays to provide an alternative regimen to treat E. The strains used in this study are listed in Supplementary Table S1. XDR E. coli Y, Y, Y, and Y were kindly provided by Min Li Shanghai Jiaotong University, Shanghai, China.
Gram-positive cocci Staphylococcus was grown in tryptic soy broth TSB; Solarbio, Shanghai, China , and Enterococcus was grown in brain-heart infusion BHI; Solarbio, Shanghai, China. Gram-negative species were grown in Lysogeny Broth LB; Solarbio, Shanghai, China. All drugs used in this study were dissolved in deionized water or dimethyl sulfoxide DMSO.
To identify the antibacterial effects, 5, unique MINI Scaffold molecules, the molecules in MINI Scaffold Library TopScience, L , were used to combat against standard strains E. coli ATCC Figure 1. Antimicrobial effects of against Escherichia coli.
A Workflow of the in vitro high-throughput screening assay of the MINI Scaffold library, containing 5, MINI molecules.
The OD value was determined by a microplate spectrophotometer. The structural formula and characteristics of a hit hit compound , — were shown.
LogP: oil- water partition coefficient. B Growth of E. The experiment was repeated three times. The in vitro antibacterial effects of drugs were evaluated by a standard microdilution broth sensitivity assay according to the guidelines of the Clinical and Laboratory Standard Institute CLSI, MIC was the lowest concentration which cannot be observed visible bacterial growth.
The fixed bacteria were washed with 0. The SEM images of the samples were obtained by SEM HITACHI, Tokyo, Japan; Tan et al. For TEM observation, the bacteria were treated as described above. The samples were sputter-coated with a layer of gold and observed by TEM HITACHI, Tokyo, Japan; Tan et al.
The aberrant cells were manually and qualitatively distinguished from the normal cells in each image. The percentage of aberrant cells was calculated by ImageJ software. The combinational antimicrobial effects between and antibiotics were evaluated through drug combination assays.
FICI, the fractional inhibitory concentration index, is a calculated index employed to evaluate the interaction of drugs combination. Based on the formula of FICI, this index can reflect the MIC changes of drugs before and after drugs combination. FICI was calculated with the formula:.
The viable colonies were quantified by CFU counting. Equal volume of RBC suspension and indicated concentrations of were mixed. The hemolysis rate was calculated with the formula Tan et al.
CCK-8, cell counting kit-8, was used to evaluate cell viability, referring to cell proliferation and cytotoxicity. Cell viability assay using CCK-8 Tongren, Japan was carried out to detect the cytotoxicity of , and four different types of cell lines were used to investigate.
Ten microliters of CCK-8 reagent were plated to each well. The transformation of calcein-AM to calcein contributes to green fluorescence in living cells, and the presence of PI leads to red fluorescence in dead cells. All animal procedures were performed according to the requirements of the Ethic Committee of the Third Xiangya Hospital, Central South University NO: sydw Seven-week-old female ICR mice Hunan SJA Laboratory Animal Co.
The fur on the back was removed by shaving and chemical depilatories. Treatments were directly injected subcutaneously into the infected area. After tissues were excised and homogenized in saline, viable cells were quantified by CFU counting. The abscess tissues were processed with a series of ethanol and embedded in paraffin blocks.
All of the primary antibodies were from Servicebio, Wuhan, China. And the dilution ratios were 1: 1, and 1: , respectively. Color was developed using a DAB substrate kit.
Samples were counterstained with Hematoxylin and dehydrated. The images of samples were observed under an upright epifluorescence microscope Nikon E, Japan. Experiments were independently performed at least in triplicate.
Data were analyzed using the GraphPad Prism 8. To develop the potential antibacterial molecules against E. coli , we performed high-throughput screening with 5, molecules in the MINI Scaffold Library established by TopScience. coli ATCC Supplementary Figure S1.
The chemical structure of was shown in Figure 1A. The dose-dependent growth inhibition effect of was shown in Figure 1B. Furthermore, we conducted primary antimicrobial susceptibility tests of to other pathogens, including P. aeruginosa, A. baumannii, K. pneumoniae, E. faecalis, and S.
Thus, an electron microscope was performed to observe the morphology and microstructure of treated E. For SEM, the surfaces of rodlike bacteria were smooth and structure-intact in the control group Figure 2A.
coli Figures 2A , B ; Supplementary Figure S3. Figure 2. To assess the combinational antimicrobial effects, the interactive activities between and other drugs against E.
coli ATCC were detected by drug combination assays. The FICIs of combination therapies against E. coli ATCC between and most polypeptide antibiotics, including polymyxin B PB , polymyxin E PE , polymyxin B nonapeptide PMBN , and SPR, or other conventional antibiotics, such as erythromycin ERY , gentamicin GEN , Ceftriaxone sodium CRO , tetracycline TET , and as levofloxacin LEV , indicated no interaction Supplementary Figure S4.
Besides, the FICIs of the antimicrobial combination between SPR and other conventional antibiotics against ATCC indicated no or only moderate synergistic effects Table 1. In addition, no interaction or antagonism between and SPR was observed when we changed the objective to other strains, such as K.
pneumoniae, P. baumannii, S. aureus, and S. epidermidis Supplementary Figure S3. Thus, the combinational activity between SPR and was species-dependent. In order to verify the synergy between and SPR against E. Consistently, SYTO9 and PI staining by CLSM observation revealed that the combined treatment of and SPR destroyed the bacterial viability and resulted in reduced viable cell counts compared with the control or mono-treatment group Figures 3E , F.
Further, we evaluated the synergistic antimicrobial activity of SPR and against drug-resistant clinical isolates of E. As shown in Figure 4A , the results of the drug combination assays also indicated the synergistic antimicrobial activity against E.
Consistently, the growth inhibition assays suggested that the combination treatment of and SPR possessed a significant synergistic antimicrobial activity compared with single-usage groups Figure 4C.
Figure 3. Combinational bacteriostatic effects between and SPR against Escherichia coli ATCC A Synergistic antimicrobial effects between and SPR by using drug combination assays.
B Calculation of the fractional inhibitory concentration index FICI. The red circle indicates the optimal FICI. C The time-growth inhibition curves and D time-killing curves of E.
Green indicates live cells and red indicates dead cells. F Fluorescence intensity analysis of PI-stained cells was carried out by ImageJ software. Figure 4.
Synergistic antimicrobial activity of and SPR combination against Escherichia coli XDR strains. A Results of the drug combination assays of four E. coli XDR strains indicate synergism between and SPR B Red circles indicate the optimal FICIs.
The related image of the hemolysis assays was shown in Figure 5B. Further, we performed CCK-8 assays to assess the cytotoxicity of on HepG2, LO2, HK-2, and HSF. Due to the consideration o f potential nephrotoxicity of SPR Vaara, , we further detected the cytotoxicity of and SPR in combination to the renal derived cell line HK Similarly, by comparing the percentages of early apoptotic, late apoptotic, and necrotic cells between groups, the flow cytometry analysis indicated that the combination treatment did not induce apoptosis of the HK-2 cells Figure 5E.
To sum up, in combination with SPR displayed extremely low toxicity in vitro. Figure 5. Hemolytic activity and cytotoxicity of alone or in combination with SPR Human RBCs hemolysis rates A and representative images B in the presence of a series concentrations of , 0.
C The cytotoxicity of and CERI positive control to HepG2 human hepatocarcinoma cell line , LO2 human normal hepatocyte cell line , HK-2 human kidney tubule epithelial cell line , and HSF human skin fibroblast cell line were evaluated by CCK-8 assays.
We performed the subcutaneous abscess models to evaluate the in vivo antimicrobial activity, as described in Figure 6A. coli as compared with the vehicle group, the combination treatment showed a significant reduction of the abscess area Figure 6B , which can be confirmed by visual observation Figure 6D.
As we expected, and SPR in combination also reduced the viable bacteria counts by 2. As shown in Figure 6E , the mono-therapy or vehicle group showed significant granulocyte infiltration and pro-inflammatory cytokines aggregation.
However, there were no significant pathological morphological changes observed in the combination treatment group, a large number of healthy epithelial cells instead were observed in the abscess. Figure 6.
In vivo synergistic antimicrobial efficacy of and SPR in combination in a subcutaneous infection model. Vehicle, 0. B Areas, C viable cell counts, and D representative images of abscess were detected. Vehicle group is used for comparison. Apparently, there was no statistically significant difference in the biomarkers of ALT Figure 7B , UREA Figure 7C , and CK Figure 7D among the control, mono-therapy, and combinational therapy groups.
Additionally, the hematological biomarkers analysis including white blood cell counting, red blood cell counting, hemoglobin amount, platelet counting, and neutrophil classification showed no statistically significant difference among these groups Figure 7E.
Accordingly, these findings suggested that the treatment combination demonstrated good safety in vivo. Figure 7. Acceptable in vivo tolerance of and SPR in combination.
A Whole body pattern diagram of mice, which is labeled with several biomarkers that reflect corresponding organic functions, blood routine analysis. B Serum alanine transaminase ALT quantification for liver function assessment.
C Serum urea UREA quantification for renal function assessment. D Serum creatine kinase CK quantification for myocardial function assessment. One-way ANOVA indicted no statistically significant difference among groups.
Nowadays, the prevalence of drug-resistant bacteria and lack of novel antibiotics have posed critical issues that result in threats to human health. Antibiotics and bioactive molecules screening are according to specific phenotypic traits, rather than traditional target-based procedures, which can give us ideas that we have never thought of or discovered before.
Though it can be a very hard and time-consuming process, several studies have proved satisfactory results in the discovery of antimicrobial compounds with novel mechanisms Watamoto et al. The MINI Scaffold library contains a total of 5, molecules with its own scaffold based on more than 16,, drug-like small molecules from ChemDiv.
A molecule in the MINI Scaffold Library represents a unique scaffold of a class of compounds, giving rise to the great variety of structures among different molecules, which refers to high diversity. Scaffold is the essence of a molecule and different compounds with the same scaffold generally have higher relevance to the target of action.
Thus, discovering a new scaffold can lead us to find a branch of new and effective drugs Mok and Brenk, Besides, all molecules in the MINI Scaffold Library were screened and filtered through their pharmaceutical and chemical properties, and adverse structures, such as toxicity and PAINS, were eliminated by TopScience, Shanghai, China.
To the best of our knowledge, our study is the first one to report the screening of the MINI Scaffold library against Gram-negative bacterial strains.
Due to the existence of outer membrane OM , Gram-negative bacillus is naturally drug-resistant to many antibiotics, leading to less amount of hits than the Gram-positive bacteria during the screening Pengfei et al.
However, we found , the only one hit among these molecules with antimicrobial activity specifically against E. Additional studies to assess the role of these groups are warranted. The varied susceptibilities of may be due to the different cell wall components, structure, and genetic backgrounds among species.
For example, different from the Gram-positive pathogens, the Gram-negative bacteria had lipopolysaccharide LPS structure on the outer membranes as well as cytoplasmic membranes, which could explain their different susceptibilities to polymyxin antibiotics Falagas and Kasiakou, ; Sabnis et al.
Combination therapy is a hopeful solution to the crisis of antimicrobial resistance. Combination therapies possess several key features, such as greater antibacterial effects than mono-therapy; reduced toxicity companioned with lower doses of drugs in combination; reduced incidence of resistance, etc.
Bollenbach, ; Zhou et al. In the present study, although exhibits antibacterial activity against E. Thus, we decide to further investigate the combination therapy between with other antibiotics.
Given that polymyxins like PB, PE, SPR, and SPR can effectively combat multidrug-resistant MDR Gram-negative bacillus, and become the only available drugs for MDR Gram-negative bacteria Phe et al.
However, the toxicity of polymyxins has significantly limited their clinical applications Akhoundsadegh et al. Thus, antibiotics in combination with could be a resort to alleviate the nephrotoxicity of polymyxins.
SPR, as a derivate of polymyxin B, obtains the ability to disrupt the outer membrane of Gram-negative bacteria with higher safety than polymyxin B Zurawski et al. These characters contribute SPR to acting as a potential adjutant, in combination with other antimicrobials that can synergistically improve the antimicrobial effects against Gram-negative strains French et al.
For example, several studies have reported that SPR potentiates the activity of conventional antibiotics like fusidic acid, vancomycin, rifampin, minocycline, or beta-lactam against Gram-negative bacteria Zurawski et al.
According to the advantageous features of SPR, we assume that there are synergistic effects between and SPR In this study, as we expected, we discovered the synergistic antimicrobial activities between and SPR against E.
coli and its drug-resistant clinical strains. However, in combination with other classes of drug indicates no interaction. Different classes of antibiotics show antibacterial effects through different model of actions. And the cell wall or membrane disruptors, like SPR, prone to show synergistic antimicrobial activities with antibiotics targeting inner cellular components.
Similarly, Zurawski et al. reported that SPR could be synergy with rifampin against XDR A. baumannii Zurawski et al. Corbett et al. shown that SPR demonstrated synergistic activities with azithromycin to combat E. coli , K. pneumoniae , and A. baumannii Corbett et al. Thus, based on SPR targeting the outer membrane of Gram-negative strains, we assume that is the reason why the synergistic antimicrobial effects are observed.
Sodium hypochlorite at the concentration used in household bleach 5. The microbicidal activity of chlorine is attributed largely to undissociated hypochlorous acid HOCl.
A potential hazard is production of the carcinogen bis chloromethyl ether when hypochlorite solutions contact formaldehyde and the production of the animal carcinogen trihalomethane when hot water is hyperchlorinated After reviewing environmental fate and ecologic data, EPA has determined the currently registered uses of hypochlorites will not result in unreasonable adverse effects to the environment Alternative compounds that release chlorine and are used in the health-care setting include demand-release chlorine dioxide, sodium dichloroisocyanurate, and chloramine-T.
The advantage of these compounds over the hypochlorites is that they retain chlorine longer and so exert a more prolonged bactericidal effect. Sodium dichloroisocyanurate tablets are stable, and for two reasons, the microbicidal activity of solutions prepared from sodium dichloroisocyanurate tablets might be greater than that of sodium hypochlorite solutions containing the same total available chlorine.
Second, solutions of sodium dichloroisocyanurate are acidic, whereas sodium hypochlorite solutions are alkaline, and the more microbicidal type of chlorine HOCl is believed to predominate Chlorine dioxide-based disinfectants are prepared fresh as required by mixing the two components base solution [citric acid with preservatives and corrosion inhibitors] and the activator solution [sodium chlorite].
In vitro suspension tests showed that solutions containing about ppm chlorine dioxide achieved a reduction factor exceeding 10 6 of S.
aureus in 1 minute and of Bacillus atrophaeus spores in 2. The potential for damaging equipment requires consideration because long-term use can damage the outer plastic coat of the insertion tube In another study, chlorine dioxide solutions at either ppm or 30 ppm killed Mycobacterium avium-intracellulare within 60 seconds after contact but contamination by organic material significantly affected the microbicidal properties The main products of this water are hypochlorous acid e.
As with any germicide, the antimicrobial activity of superoxidized water is strongly affected by the concentration of the active ingredient available free chlorine One manufacturer generates the disinfectant at the point of use by passing a saline solution over coated titanium electrodes at 9 amps.
The product generated has a pH of 5. Although superoxidized water is intended to be generated fresh at the point of use, when tested under clean conditions the disinfectant was effective within 5 minutes when 48 hours old Unfortunately, the equipment required to produce the product can be expensive because parameters such as pH, current, and redox potential must be closely monitored.
The solution is nontoxic to biologic tissues. Although the United Kingdom manufacturer claims the solution is noncorrosive and nondamaging to endoscopes and processing equipment, one flexible endoscope manufacturer Olympus Key-Med, United Kingdom has voided the warranty on the endoscopes if superoxidized water is used to disinfect them As with any germicide formulation, the user should check with the device manufacturer for compatibility with the germicide.
Additional studies are needed to determine whether this solution could be used as an alternative to other disinfectants or antiseptics for hand washing, skin antisepsis, room cleaning, or equipment disinfection e.
In October , the FDA cleared superoxidized water as a high-level disinfectant FDA, personal communication, September 18, The exact mechanism by which free chlorine destroys microorganisms has not been elucidated. Inactivation by chlorine can result from a number of factors: oxidation of sulfhydryl enzymes and amino acids; ring chlorination of amino acids; loss of intracellular contents; decreased uptake of nutrients; inhibition of protein synthesis; decreased oxygen uptake; oxidation of respiratory components; decreased adenosine triphosphate production; breaks in DNA; and depressed DNA synthesis , The actual microbicidal mechanism of chlorine might involve a combination of these factors or the effect of chlorine on critical sites Low concentrations of free available chlorine e.
Higher concentrations 1, ppm of chlorine are required to kill M. tuberculosis using the Association of Official Analytical Chemists AOAC tuberculocidal test One study reported that 25 different viruses were inactivated in 10 minutes with ppm available chlorine Several studies have demonstrated the effectiveness of diluted sodium hypochlorite and other disinfectants to inactivate HIV Chlorine ppm showed inhibition of Candida after 30 seconds of exposure In experiments using the AOAC Use-Dilution Method, ppm of free chlorine killed 10 6 —10 7 S.
aureus , Salmonella choleraesuis , and P. Because household bleach contains 5. A chlorine dioxide generator has been shown effective for decontaminating flexible endoscopes but it is not currently FDA-cleared for use as a high-level disinfectant Chlorine dioxide can be produced by mixing solutions, such as a solution of chlorine with a solution of sodium chlorite In , a chlorine dioxide product was voluntarily removed from the market when its use caused leakage of cellulose-based dialyzer membranes, which allowed bacteria to migrate from the dialysis fluid side of the dialyzer to the blood side tuberculosis , M.
chelonae , poliovirus, HIV, multidrug-resistant S. aureus , E. coli, Candida albicans , Enterococcus faecalis, P. aeruginosa in the absence of organic loading. However, the biocidal activity of this disinfectant decreased substantially in the presence of organic material e.
No bacteria or viruses were detected on artificially contaminated endoscopes after a 5-minute exposure to superoxidized water and HBV-DNA was not detected from any endoscope experimentally contaminated with HBV-positive mixed sera after a disinfectant exposure time of 7 minutes Hypochlorites are widely used in healthcare facilities in a variety of settings.
A — dilution of 5. For small spills of blood i. Because hypochlorites and other germicides are substantially inactivated in the presence of blood 63, , , , large spills of blood require that the surface be cleaned before an EPA-registered disinfectant or a final concentration solution of household bleach is applied If a sharps injury is possible, the surface initially should be decontaminated 69, , then cleaned and disinfected final concentration Extreme care always should be taken to prevent percutaneous injury.
At least ppm available chlorine for 10 minutes is recommended for decontaminating CPR training manikins Full-strength bleach has been recommended for self-disinfection of needles and syringes used for illicit-drug injection when needle-exchange programs are not available. The difference in the recommended concentrations of bleach reflects the difficulty of cleaning the interior of needles and syringes and the use of needles and syringes for parenteral injection Clinicians should not alter their use of chlorine on environmental surfaces on the basis of testing methodologies that do not simulate actual disinfection practices , Other uses in healthcare include as an irrigating agent in endodontic treatment and as a disinfectant for manikins, laundry, dental appliances, hydrotherapy tanks 23, 41 , regulated medical waste before disposal , and the water distribution system in hemodialysis centers and hemodialysis machines Chlorine long has been used as the disinfectant in water treatment.
Water disinfection with monochloramine by municipal water-treatment plants substantially reduced the risk for healthcare—associated Legionnaires disease , Chlorine dioxide also has been used to control Legionella in a hospital water supply. Thus, if a user wished to have a solution containing ppm of available chlorine at day 30, he or she should prepare a solution containing 1, ppm of chlorine at time 0.
Sodium hypochlorite solution does not decompose after 30 days when stored in a closed brown bottle The use of powders, composed of a mixture of a chlorine-releasing agent with highly absorbent resin, for disinfecting spills of body fluids has been evaluated by laboratory tests and hospital ward trials.
The inclusion of acrylic resin particles in formulations markedly increases the volume of fluid that can be soaked up because the resin can absorb — times its own weight of fluid, depending on the fluid consistency. One problem with chlorine-releasing granules is that they can generate chlorine fumes when applied to urine Formaldehyde is used as a disinfectant and sterilant in both its liquid and gaseous states.
Liquid formaldehyde will be considered briefly in this section, and the gaseous form is reviewed elsewhere The aqueous solution is a bactericide, tuberculocide, fungicide, virucide and sporicide 72, 82, OSHA indicated that formaldehyde should be handled in the workplace as a potential carcinogen and set an employee exposure standard for formaldehyde that limits an 8-hour time-weighted average exposure concentration of 0.
The standard includes a second permissible exposure limit in the form of a short-term exposure limit STEL of 2 ppm that is the maximum exposure allowed during a minute period Ingestion of formaldehyde can be fatal, and long-term exposure to low levels in the air or on the skin can cause asthma-like respiratory problems and skin irritation, such as dermatitis and itching.
For these reasons, employees should have limited direct contact with formaldehyde, and these considerations limit its role in sterilization and disinfection processes. Key provisions of the OSHA standard that protects workers from exposure to formaldehyde appear in Title 29 of the Code of Federal Regulations CFR Part Formaldehyde inactivates microorganisms by alkylating the amino and sulfhydral groups of proteins and ring nitrogen atoms of purine bases Varying concentrations of aqueous formaldehyde solutions destroy a wide range of microorganisms.
Four percent formaldehyde is a tuberculocidal agent, inactivating 10 4 M. tuberculosis in 2 minutes 82 , and 2. anthracis The formaldehyde solution required 2 hours of contact to achieve an inactivation factor of 10 4 , whereas glutaraldehyde required only 15 minutes.
For these reasons and others—such as its role as a suspected human carcinogen linked to nasal cancer and lung cancer , this germicide is excluded from Table 1.
When it is used, , direct exposure to employees generally is limited; however, excessive exposures to formaldehyde have been documented for employees of renal transplant units , , and students in a gross anatomy laboratory Formaldehyde is used in the health-care setting to prepare viral vaccines e.
To minimize a potential health hazard to dialysis patients, the dialysis equipment must be thoroughly rinsed and tested for residual formaldehyde before use.
Paraformaldehyde, a solid polymer of formaldehyde, can be vaporized by heat for the gaseous decontamination of laminar flow biologic safety cabinets when maintenance work or filter changes require access to the sealed portion of the cabinet.
Glutaraldehyde is a saturated dialdehyde that has gained wide acceptance as a high-level disinfectant and chemical sterilant Aqueous solutions of glutaraldehyde are acidic and generally in this state are not sporicidal. Once activated, these solutions have a shelf-life of minimally 14 days because of the polymerization of the glutaraldehyde molecules at alkaline pH levels.
This polymerization blocks the active sites aldehyde groups of the glutaraldehyde molecules that are responsible for its biocidal activity. Novel glutaraldehyde formulations e. However, antimicrobial activity depends not only on age but also on use conditions, such as dilution and organic stress.
However, two studies found no difference in the microbicidal activity of alkaline and acid glutaraldehydes 73, The biocidal activity of glutaraldehyde results from its alkylation of sulfhydryl, hydroxyl, carboxyl, and amino groups of microorganisms, which alters RNA, DNA, and protein synthesis.
The mechanism of action of glutaraldehydes are reviewed extensively elsewhere , The in vitro inactivation of microorganisms by glutaraldehydes has been extensively investigated and reviewed , Spores of C. Microorganisms with substantial resistance to glutaraldehyde have been reported, including some mycobacteria M.
chelonae , Mycobacterium avium-intracellulare, M. xenopi , Methylobacterium mesophilicum , Trichosporon , fungal ascospores e. chelonae persisted in a 0. Two percent alkaline glutaraldehyde solution inactivated 10 5 M.
tuberculosis cells on the surface of penicylinders within 5 minutes at 18°C However, subsequent studies 82 questioned the mycobactericidal prowess of glutaraldehydes.
tuberculosis and compares unfavorably with alcohols, formaldehydes, iodine, and phenol Suspensions of M. avium, M. intracellulare, and M. tuberculosis estimated time to complete inactivation ~25 minutes The rate of kill was directly proportional to the temperature, and a standardized suspension of M.
tuberculosis could not be sterilized within 10 minutes An FDA-cleared chemical sterilant containing 2. tuberculosis per membrane Several investigators 55, 57, 73, 76, 80, 81, 84, have demonstrated that glutaraldehyde solutions inactivate 2. tuberculosis in 10 minutes including multidrug-resistant M.
tuberculosis and 4. tuberculosis in 20 minutes. Glutaraldehyde is commonly diluted during use, and studies showed a glutaraldehyde concentration decline after a few days of use in an automatic endoscope washer , This emphasizes the need to ensure that semicritical equipment is disinfected with an acceptable concentration of glutaraldehyde.
Data suggest that 1. Chemical test strips or liquid chemical monitors , are available for determining whether an effective concentration of glutaraldehyde is present despite repeated use and dilution. The frequency of testing should be based on how frequently the solutions are used e.
The bottle of test strips should be dated when opened and used for the period of time indicated on the bottle e. The results of test strip monitoring should be documented.
The glutaraldehyde test kits have been preliminarily evaluated for accuracy and range but the reliability has been questioned To ensure the presence of minimum effective concentration of the high-level disinfectant, manufacturers of some chemical test strips recommend the use of quality-control procedures to ensure the strips perform properly.
In December , EPA issued an order to stop the sale of all batches of this product because of efficacy data showing the product is not effective against spores and possibly other microorganisms or inanimate objects as claimed on the label Other FDA cleared glutaraldehyde sterilants that contain 2.
Glutaraldehyde is used most commonly as a high-level disinfectant for medical equipment such as endoscopes 69, , , spirometry tubing, dialyzers , transducers, anesthesia and respiratory therapy equipment , hemodialysis proportioning and dialysate delivery systems , , and reuse of laparoscopic disposable plastic trocars Glutaraldehyde is noncorrosive to metal and does not damage lensed instruments, rubber.
or plastics. Glutaraldehyde should not be used for cleaning noncritical surfaces because it is too toxic and expensive. Colitis believed caused by glutaraldehyde exposure from residual disinfecting solution in endoscope solution channels has been reported and is preventable by careful endoscope rinsing , Healthcare personnel can be exposed to elevated levels of glutaraldehyde vapor when equipment is processed in poorly ventilated rooms, when spills occur, when glutaraldehyde solutions are activated or changed, , or when open immersion baths are used.
Acute or chronic exposure can result in skin irritation or dermatitis, mucous membrane irritation eye, nose, mouth , or pulmonary symptoms , Epistaxis, allergic contact dermatitis, asthma, and rhinitis also have been reported in healthcare workers exposed to glutaraldehyde , Glutaraldehyde exposure should be monitored to ensure a safe work environment.
The silica gel tube and the DNPH-impregnated cassette are suitable for monitoring the 0. The passive badge, with a 0. ACGIH does not require a specific monitoring schedule for glutaraldehyde; however, a monitoring schedule is needed to ensure the level is less than the ceiling limit. For example, monitoring should be done initially to determine glutaraldehyde levels, after procedural or equipment changes, and in response to worker complaints In the absence of an OSHA permissible exposure limit, if the glutaraldehyde level is higher than the ACGIH ceiling limit of 0.
Engineering and work-practice controls that can be used to resolve these problems include ducted exhaust hoods, air systems that provide 7—15 air exchanges per hour, ductless fume hoods with absorbents for the glutaraldehyde vapor, tight-fitting lids on immersion baths, personal protection e.
If engineering controls fail to maintain levels below the ceiling limit, institutions can consider the use of respirators e. In general, engineering controls are preferred over work-practice and administrative controls because they do not require active participation by the health-care worker. Even though enforcement of the OSHA ceiling limit was suspended in by the U.
Court of Appeals , limiting employee exposure to 0. If glutaraldehyde disposal through the sanitary sewer system is restricted, sodium bisulfate can be used to neutralize the glutaraldehyde and make it safe for disposal.
The literature contains several accounts of the properties, germicidal effectiveness, and potential uses for stabilized hydrogen peroxide in the health-care setting. Published reports ascribe good germicidal activity to hydrogen peroxide and attest to its bactericidal, virucidal, sporicidal, and fungicidal properties Tables 4 and 5 The FDA website lists cleared liquid chemical sterilants and high-level disinfectants containing hydrogen peroxide and their cleared contact conditions.
Hydrogen peroxide works by producing destructive hydroxyl free radicals that can attack membrane lipids, DNA, and other essential cell components. Catalase, produced by aerobic organisms and facultative anaerobes that possess cytochrome systems, can protect cells from metabolically produced hydrogen peroxide by degrading hydrogen peroxide to water and oxygen.
This defense is overwhelmed by the concentrations used for disinfection , Hydrogen peroxide is active against a wide range of microorganisms, including bacteria, yeasts, fungi, viruses, and spores 78, Bactericidal effectiveness and stability of hydrogen peroxide in urine has been demonstrated against a variety of health-care—associated pathogens; organisms with high cellular catalase activity e.
aureus , S. marcescens , and Proteus mirabilis required 30—60 minutes of exposure to 0. Synergistic sporicidal effects were observed when spores were exposed to a combination of hydrogen peroxide 5.
Other studies demonstrated the antiviral activity of hydrogen peroxide against rhinovirus The product marketed as a sterilant is a premixed, ready-to-use chemical that contains 7.
The mycobactericidal activity of 7. tuberculosis after a minute exposure When the effectiveness of 7. No complaints were received from the nursing or medical staff regarding odor or toxicity.
A new, rapid-acting Manufacturer data demonstrate that this solution sterilizes in 30 minutes and provides high-level disinfection in 5 minutes This product has not been used long enough to evaluate material compatibility to endoscopes and other semicritical devices, and further assessment by instrument manufacturers is needed.
Under normal conditions, hydrogen peroxide is extremely stable when properly stored e. Corneal damage from a hydrogen peroxide-soaked tonometer tip that was not properly rinsed has been reported Hydrogen peroxide also has been instilled into urinary drainage bags in an attempt to eliminate the bag as a source of bladder bacteriuria and environmental contamination Although the instillation of hydrogen peroxide into the bag reduced microbial contamination of the bag, this procedure did not reduce the incidence of catheter-associated bacteriuria As with other chemical sterilants, dilution of the hydrogen peroxide must be monitored by regularly testing the minimum effective concentration i.
Compatibility testing by Olympus America of the 7. Iodine solutions or tinctures long have been used by health professionals primarily as antiseptics on skin or tissue. Iodophors, on the other hand, have been used both as antiseptics and disinfectants.
FDA has not cleared any liquid chemical sterilant or high-level disinfectants with iodophors as the main active ingredient. An iodophor is a combination of iodine and a solubilizing agent or carrier; the resulting complex provides a sustained-release reservoir of iodine and releases small amounts of free iodine in aqueous solution.
The best-known and most widely used iodophor is povidone-iodine, a compound of polyvinylpyrrolidone with iodine. This product and other iodophors retain the germicidal efficacy of iodine but unlike iodine generally are nonstaining and relatively free of toxicity and irritancy ,
FDA has antimidrobial cleared Potent antimicrobial formula liquid chemical sterilant or high-level Pootent with Potent antimicrobial formula as the main active ingredient. These alcohols are rapidly bactericidal Raspberry ketones and fat oxidation than bacteriostatic against vegetative forms of Potnt they also are tuberculocidal, fungicidal, and virucidal but do not destroy bacterial spores. Top of Page. The most feasible explanation for the antimicrobial action of alcohol is denaturation of proteins. This mechanism is supported by the observation that absolute ethyl alcohol, a dehydrating agent, is less bactericidal than mixtures of alcohol and water because proteins are denatured more quickly in the presence of water , Introduction: Antibiotic resistance ofrmula posed a serious challenge to global public health. Atnimicrobial the increasing Potent antimicrobial formula antimicroblal of Aromatherapy. coli and mortality caused by drug-resistant E. coli infections, it is urgent to develop novel antibiotics. Methods: By high-throughput screening assay, we found a bioactive molecule, —which demonstrated antimicrobial effects against E. The antimicrobial effects of alone or in combination with conventional antibiotics were assessed by scanning electron microscopy, transmission electron microscopy, drug combination assay, and growth inhibition assay.
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