Phenylmercuric Nitrate Research Articles

Integron occurrence is linked to reduced biocide susceptibility in multidrug resistant Pseudomonas aeruginosa

Objective: Integrons are gene acquisition systems commonly found in bacterial genomes that play a major role in the dissemination of resistance to antibiotics. This work aimed to study the relationship between the presence of integrons and the reduced susceptibility of multidrug-resistant (MDR) Pseudomonas aeruginosa isolates towards different groups of biocides.Methods: The antimicrobial susceptibility patterns of 104 clinical isolates were determined against different antibiotics by the disk diffusion method. The isolates were also tested for their susceptibility to six biocides (glutaraldehyde, benzalkonium chloride, cetrimide, chlorhexidine gluconate, chlorocresol and gluconate, and phenyl mercuric nitrate) by agar dilution. The presence of integrons and resistance genes in MDR isolates were detected by polymerase chain reaction.Results: Thirty-six Pseudomonas isolates were MDR, and the majority of these isolates showed reduced susceptibility to biocides. In the MDR isolates, Class I integron was detected in 22 isolates (61.1%), while Class II and III integrons were identified in only four isolates (11.1%), In addition, aacA4 and qacE genes were detected in 22 (61.1%) and 11 (30.5%) isolates, respectively. Integron I-positive isolates showed reduced susceptibility to tested biocides.Conclusions: The current study reveals the presence of different classes of integrons, with class I being predominant. Class I integron may be responsible for generating MDR P. aeruginosa isolates with reduced susceptibility to biocides. This linkage between integrons and biocide resistance in MDR-Pseudomonas isolates is notable and could be clinically important. Strict antibiotic prescription policies and the adequate use of biocides could help in controlling this problem.

A proposed approach to correlate antibiotic and biocide resistance in staphylococcal isolates based on Minimum Inhibitoy Concentration (MIC) data analyses

The study aimed to compare the sensitivity of Staphylococcus isolates from hospital and non hospital sources to some commonly used antibiotics and biocides, and to investigate whether resistance to either types of antimicrobials are correlated. The minimum inhibitory concentrations (MICs) of the antibiotics and biocides were determined for clinical (47), hospital environment (64) and non hospital (33) isolates using agar dilution method. Arithmetic progression in biocides concentration was used instead of the geometric progression for antibiotics. Pearson's correlation coefficients (r) between MIC-MIC, Log MIC-Log MIC, and MIC-Log MIC for individual biocides and antibiotics, respectively, were compared. Also, the frequencies of antibiotic resistance among biocide-susceptible and biocide-resistant isolates were calculated. The antibiotics included: ampicillin, penicillin G, oxacillin, cefepime, streptomycin, tetracycline, gentamicin, azithromycin, ciprofloxacin, vancomycin and chloramphenicol, while biocides included chlorocresol, benzalkonium chloride, cetrimide, chlorhexidine, phenyl mercuric nitrate, povidone-iodine and ethidium bromide. Hospital isolates showed higher resistance rates to antibiotics and biocides and more predominance of methicillin resistance compared to non-hospital isolates. The most likely correlation between antibiotic and biocide resistance was best expressed by comparing log MIC of antibiotic with MIC of biocide. For hospital isolates, positive correlations were found between increased resistance to most of tested antibiotics (except for vancomycin and occasionally ciprofloxacin and tetracycline) and reduced susceptibility to biocides, except for chlorhexidine and povidone-iodine. For non hospital isolates, only resistance to benzalkonium chloride and cetrimide correlated with resistance to most of the tested antibiotics. Except for chlorhexidine and povidone-Iodine higher resistance rates to antibiotics were found among biocide resistant isolates.

Temperature-mediated absorption of phenylmercuric nitrate on polyethylene and polypropylene containers in chloramphenicol eye drops

The aim of this work was to find the effect of temperature and manufacturing source of phenylmercuric nitrate (PMN) on PMN absorption on low-density polyethylene (LDPE) and polypropylene containers in chloramphenicol eye drops. Two factorial experiments were designed to study the effect of temperature on PMN assay in chloramphenicol eye drops stored in LDPE and prepared from two different PMN sources. PMN source had no effect on PMN assay at 2–8 °C, however at stress conditions (30 °C/75%RH) for 3 weeks, the effect of PMN source on PMN assay was found significant (p < 0.05) in formulations stored in LDPE bottles. Temperature was the major contributor to decreased PMN assay. In formulations stored in polypropylene containers, PMN source had significant effect on PMN assay at 2–8 °C and 30 °C/75%RH. Overall, new PMN and polypropylene bottles performed better. The eye drops complied with preservative efficacy test both initially and at the end of shelf life. The concentration exponent of PMN is very low and in spite of its high absorption by container/closure, PMN was still able to protect the eye drops at the end of shelf life. It can be inferred that preservative efficacy test is the better indicator of preservative activity.

In Vitro Permeation Characteristics of Itraconazole from Oil Drops and Ophthalmic Ointment Through Excised Goat And Sheep Corneas

In vitro transcorneal permeation of itraconazole from oil drops and ophthalmic ointments was studied using freshly excised goat and sheep corneas. Permeation of itraconazole from castor oil was found maximum and minimum with kardi oil formulation. The higher permeation of itraconazole from castor oil formulation could be attributed to the lower partition coefficient of drug between oil and aqueous phase. The addition of benzyl alcohol, a preservative, in oil drops, increased the permeation of itraconazole. Partition experiments indicated increased partitioning of itraconazole in the aqueous phase in the presence of benzyl alcohol. The addition of phenyl mercuric acetate, phenyl mercuric nitrate and thiomersal as preservative, in castor oil drop decreased the permeation of itraconazole. Corneal hydration was within the desired limit below 80 %. Permeation of itraconazole from ointment containing dissolved drug was higher than the ointment containing solid drug. In antifungal study, the castor oil formulation showed maximum zone of inhibition against Candida albicans. Stability study conducted at 40 0 C and 70% RH indicated the formulations as quite

Antifungal effect of ophthalmic preservatives phenylmercuric nitrate and benzalkonium chloride on ocular pathogenic filamentous fungi

In the present study, the antifungal effects of phenylmercuric nitrate and benzalkonium chloride versus those of natamycin and ketoconazole were assessed against 216 filamentous fungi isolates from cases of fungal keratitis. They included 112 Fusarium isolates, 94 Aspergillus isolates, and 10 Alternaria alternata isolates. The strains were tested by broth dilution antifungal susceptibility testing of filamentous fungi approved by the Clinical and Laboratory Standards Institute M38-A document. The results showed that the MIC50 values of phenylmercuric nitrate were 0.0156, 0.0156, and 0.0313 μg/mL for Fusarium spp., Aspergillus spp., and A. alternata, respectively. The MIC90 values of phenylmercuric nitrate were 0.0313, 0.0313, and 0.0313 μg/mL for Fusarium spp., Aspergillus spp., and A. alternata, respectively. The MIC50 values of benzalkonium chloride were 16, 32, and 8 μg/mL for Fusarium spp., Aspergillus spp., and A. alternata, respectively. The MIC90 values of benzalkonium chloride were 32, 32, and 16 μg/mL for Fusarium spp., Aspergillus spp., and A. alternata, respectively. The study indicates that phenylmercuric nitrate has considerable antifungal activity and its effect is significantly superior to those of benzalkonium chloride, natamycin, and ketoconazole against ocular pathogenic filamentous fungi in vitro, deserving further investigation for treating fungal keratitis as a main drug.

Discovery of potent inhibitors of pseudomonal quorum sensing via pharmacophore modeling and in silico screening

HipHop-Refine was employed to derive a binding hypothesis for pseudomonal quorum sensing (QS) antagonists. The model was employed as 3D search query to screen the National Cancer Institute (NCI) database. One of the hits illustrated nanomolar QS inhibitory activity. The fact that this compound contained tetravalent lead (Pb) prompted us to evaluate the activities of phenyl mercuric nitrate and thimerosal, both fit the binding pharmacophore. The two mercurials illustrated nanomolar to low micromolar IC 50 inhibitory values against pseudomonal QS. The three compounds represent a new class of QS inhibitors.

Oral lichenoid lesions (OLL) and mercury in amalgam fillings.

84 patients with oral lichenoid lesions (OLL) were seen in the contact dermatitis clinic. All these patients had reticulate, lacy, plaque-like or erosive lichenoid changes adjacent to amalgam fillings. Patch testing to metallic mercury, 0.1% thimerosal, 1% ammoniated mercury, 0.1% mercuric chloride, and in some cases 0.05% phenylmercuric nitrate and amalgam discs was undertaken. 33 (39%) patients had positive patch test findings. 30/33 patch test positive patients had replacement of their amalgam fillings, with 28 (87%) patients experiencing improvement of symptoms and signs within 3 months. This confirms that mercury allergy is a factor in the pathogenesis of OLL in some cases. In cases where patch test negative patients improve with amalgam replacement, mercury may be acting as an irritant in the pathogenesis of OLL.

Patch Testing by Additional Series of Allergens: Results of Further Experiences

Background: Patch testing with additional series (AS) of allergens may be a useful tool in diagnosing allergic contact dermatitis (ACD). Objective: Aim of the study was to verify the usefulness, to check the reliability in clinical practice and to evaluate the economic costs of AS previously built up. Methods: A total of 281 patients with suspicious ACD underwent patch test with the standard series (SS) and with one or more AS (51 among 71 built up). Results: A total of 170 patients (60.5%) showed positive reactions to SS; 116 (41.3%) to AS. Among 582 nonstandard allergens used, 113 (19.4%) elicited 1 or more positive reactions: out of 10,916 patch tests carried out, 260 (2.4%) positive reactions were observed. The correlation between SS and AS indicated that 8.2% patients resulted SS-/AS+, 27.7% SS+/AS-, 32.7% SS+/AS+, 31.3% SS-/AS-. The most frequently used AS showed the following percentages of patients with 1 or more positive reactions: clothes 41.4%, building industry 51.8%, hairdressers 77.3%, textile industry 42.1%, shoes 36.8%. Positive reactions to the most frequently used nonstandard allergens resulted: propylene glycol 0.4%, cobalt chloride 12.6%, phenylmercuric nitrate 2.2%, p-aminophenol 4.5%. The approximate economic cost of patch testing with AS has been evaluated in € 1.3 per single patch test. Conclusion: The cost of patch testing AS is not irrelevant, but it can be compensated by the advantages deriving from the increase of data concerning ACD etiology. A reduction in the number of allergens included in single AS should be performed. Cobalt chloride, taking into account the high percentage of positive reactions observed and its presence in a large number of AS, could be (re)introduced in the standard series.

Quaternary ammoniums and other preservatives’ contribution in oxidative stress and apoptosis on Chang conjunctival cells. Debbasch C, Brignole F, Pisella PJ, Warnet JM, Rat P, Baudouin C. Invest Ophthalmol Vis Sci 2001;42:642–652.

The authors investigated some of the toxicity mechanisms in vitro of ten preservatives currently used in ophthalmic solutions. A continuous human conjunctival cell line was treated with different concentrations of various preservatives for 15 minutes and for 15 minutes followed by 24 hours of cell recovery: three benzalkonium chlorides (BACs) with different hydrocarbon chain length, benzododecinium bromide (BOB), cetrimide (Cet), phenylmercuric nitrate, thimerosal, methyl parahydroxybenzoate, chlorobutanol, and EDTA. An inhibition study was then conducted using a one-hour vitamin E pretreatment followed by a 15-minute BAC treatment. Membrane integrity was assessed using a neutral red test and chromatin condensation with a Hoechst 33342 test. Reactive oxygen species were measured using dichlorofluorescein diacetate test for H2O2 production and hydroethidine test for peroxide anion production. These tests were performed using microplate cold light cytofluorometry. Cell size and DNA content were also analyzed using flow cytometry. Confocal microscopy was used to explore morphologic changes. A significant decrease of membrane integrity with chromatin condensation was observed with all the quaternary ammoniums tested at concentrations of 0.005% and higher. The effect was amplified after 24 hours of cell recovery. The other preservatives tested did not decrease membrane integrity. H2O2 production was observed with all the preservatives, whereas peroxide anion production was significantly higher with the quaternary ammoniums at 0.005% and 0.01%, compared with the other preservatives. Flow cytometry results confirmed the cytotoxicity observed with cold light cytofluorometry. The authors conclude that the quaternary ammoniums tested (BAC, BOB, and Cet) were the most cytotoxic preservatives in the current model. An apoptotic mechanism appeared to be present at low concentrations of quaternary ammoniums, whereas a necrotic process appeared at higher concentrations. Superoxide anions may play an important role in tissue damage induced by preservatives in ocular surface disorders.—Thomas J. Liesegang

Rapid evaluation of biocidal activity using a transposon-encoded catechol 2,3-dioxygenase from Pseudomonas putida.

Pseudomonas putida (UWC1), containing a genetically-engineered plasmid (pQM899), that encodes for the production of catechol 2,3-dioxygenase (C230), was used as a potential means of rapidly estimating bactericidal activity of chlorhexidine diacetate (CHA), phenol, cetylpyridinium chloride (CPC) and phenylmercuric nitrate (PMN). Enzyme C230 converts catechol to 2-hydroxymuconic semialdehyde (2-HMS), which is yellow in colour, via a meta cleavage pathway. Ideal conditions for production and measurement spectrophotometrically of 2-HMS were determined. However, the correlation between this method and viable plate counts was not sufficiently accurate to enable 2-HMS production to provide a sufficiently sensitive determination of biocidal activity. An alternative method, synchronous scanning fluorimetry, in which the decrease in catechol concentration was measured under standardized conditions, provided a good dose-response histogram for all the biocides tested. Although, in comparison with plate counts, there was an underestimation of the bactericidal effects of phenol an PMN, the results of this study suggest that this method has potential in determining the bactericidal efficacy of agents such as CHA and CPC.

The effect of amino acid modifying reagents on the activity of a (1→3)-β-glucan synthase from Italian ryegrass (Lolium multiflorum) endosperm

Abstract A series of amino acid modifying reagents were tested for their effects on the activity of the (1→3)-β-glucan synthase (EC 2.4.1.34) from endosperm of ryegrass ( Lolium multiflorum ). Of these reagents only the carbodiimide EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), DEPC (diethyl pyrocarbonate), iodine, Cu 2+ , Hg 2+ , phenylmercuric nitrate, N -bromosuccinimide and 2-hydroxy-5-nitrobenzylbromide gave significant inhibition. It was concluded that carboxyl and His groups are likely to be essential for enzyme activity but the involvement of Trp residues was not excluded. Significantly, free thiols or disulfides do not appear to be necessary for enzyme activity. The kinetics of the inhibition reaction with diethyl pyrocarbonate were complex. No evidence for methoxyformylation of His was obtained by measurements of absorbance at 242 nm. EDC inhibited in a time and concentration dependent manner, with kinetics suggesting that no reversible complex between the enzyme and the EDC was formed. The apparent bimolecular rate constant was 0.029 M −1 s −1 . The order of the inactivation was 1 indicating that one molecule of EDC is bound per active site. The rate of inhibition is slowed in the presence of UDP–Glc, suggesting that the modified groups may be located in or near the substrate-binding domain. The pH dependence of the rate of inactivation indicates the presence of a critical group with a p K a of 5.7. These results are consistent with the finding that a `D,D,D(35)Q(RQ)XRW' motif is present in a number of repetitive β-glycan synthases and suggest that the critical acidic group may be an aspartate.

The Effect of Organic Ligands on the Rate of Loss of Phenylmercuric Nitrate into Rubber Closures and Plastic Containers

AbstractThe effect of the presence of various organic compounds containing a variety of functional groups on the rate of loss of phenylmericuric (PM) nitrate into plastic containers and rubber closures has been investigated. The presence of organic compounds profoundly influenced the loss of the phenylmercuric nitrate, that loss being dependent upon their chemical structure and concentration. This effect explains the unpredictability of the loss of PM nitrate into polymer matrices from pharmaceutical products.

Oral lichenoid lesions caused by allergy to mercury in amalgam fillings

Oral lichenoid lesions (OLL) or lichen-planus-like lesions are often idiopathic. Our aim was to determine whether OLL can be caused by allergy to mercury in amalgam fillings, and whether resolution of OLL occurs after replacement of amalgam with other dental fillings. Patients with only OLL (except for 1 case with cutaneous lichen planus) referred for patch testing during 1985-1994 to the Contact and Occupational Dermatitis Clinic of the Skin & Cancer Foundation, Darlinghurst, were reviewed. Patch tests were performed with 1% mercury, 1% ammoniated mercury, 0.1% thimerosal, 0.1% mercuric chloride, 0.05% phenylmercuric nitrate and an amalgam disc, using Finn Chambers occluded for 2 days, 19 patients (17 women and 2 men; age range: 28-72 years) had OLL in close contact with amalgam fillings and showed positive patch test reactions to mercury compounds, 16 out of 19 patients had their amalgam fillings replaced. In 13 patients, the OLL healed. 1 patient had marked improvement. 1 patient had no improvement and developed multiple oral squamous cell carcinoma. In conclusion, OLL can be caused by allergy to mercury in amalgam fillings. Replacement of amalgam with other dental fillings usually results in resolution of OLL and is recommended for cases with positive patch test reactions to mercury compounds.

Development of resistance to biocides during sporulation of Bacillus subtilis

From synchronized sporulation and spore mutant studies, the order of development of resistance to biocides during sporulation of Bacillus subtilis strain 168 was toluene, formaldehyde, sodium lauryl sulphate, phenol, phenylmercuric nitrate, m‐cresol, chlorocresol, chlorhexidine gluconate, cetylpyridinium chloride, moist heat, sodium dichlorisocyanurate, sodium hypochlorite, lysozyme and glutaraldehyde. These resistances could be assigned to different stages in spore development.

Effect of plasmids conferring preservative resistance on performance of bacterial strains in compendial preservative efficacy tests

R plasmids, which increased resistance of Staphylococcus aureus and Escherichia coli to a variety of pharmaceutical preservatives, were shown, using the protocol of the Preservative Efficacy Test of the British Pharmacopoeia (1988), to influence the antibacterial efficacy of 0.001% benzalkonium chloride, 0.001% cetrimide and 0.0001% phenyl mercuric nitrate solutions, but not 0.0005% chlorhexidine gluconate. Some strains, which failed the BP (1988) criteria for ophthalmic and parenteral products, satisfied the less stringent A or B criteria of the BP (1993). However, the death rates of all strains tested complied with the criteria of the BP (1988) for 0.003% benzalkonium chloride, 0.003% cetrimide and 0.001% phenylmercuric nitrate solutions. The data illustrate the undoubted effects R plasmids can have on the survival of bacteria contaminating pharmaceutical products but suggest that the levels of resistance conferred are not sufficient to affect, adversely, the activities of in-use concentrations that are required by compendial preservative efficacy tests.

The decomposition of phenylmercuric nitrate in sulphacetamide drops during heat sterilization.

Analytical methods have been developed which enable phenylmercuric nitrate, mercuric ion and diphenylmercury to be measured in sulphacetamide drops. Application of these methods demonstrate that during heat sterilization of both 10 and 30% sulphacetamide drops containing phenylmercuric nitrate, together with disodium edetate and sodium metabisulphite, there is a 20-30% overall loss of phenylmercuric nitrate. This loss occurs mainly due to the phenylmercuric ion establishing an equilibrium with equimolar amounts of mercuric ion and diphenylmercury.

Determination of phenylmercuric nitrate in pharmaceuticals by HPLC.

Phenylmercuric nitrate (PMN) is commonly used in pharmaceutical preparations as an antimicrobial preservative in a concentration of lo-20 mg 1-l. Few methods have been reported for the analysis of organomercurial compounds. Although atomic absorption spectrophotometry has been applied [l], highperformance liquid chromatographic methods with UV detection have been described as more specific; these methods involve the analysis of PMN after chloroform extraction [2] or after chelation of organic mercury with substituted dithiocarbamates 13, 41. In particular, the chelation of PMN by morpholinedithiocarbamate was developed by Parkin [4]; the reaction was shown to be rapid and to result in the formation of a compound with a suitable molar absorptivity in the UV range. The present study was undertaken to validate a rapid and sensitive method for the direct determination of PMN in a nasal pharmaceutical formulation. Since it has been shown that organomercurial compounds may be sorbed into plastics [l, 51 the possible loss of PMN from a nasal solution packed in glass or plastic containers was assessed in this study.

The chemical degradation of phenylmercuric nitrate by disodium edetate during heat sterilization at pH values commonly encountered in ophthalmic products.

The stability following heat sterilization (121 degrees C for 15 min) of phenylmercuric (PM) nitrate in the presence of disodium edetate at pH values 5-8 has been investigated by both high-performance liquid chromatography (HPLC) and atomic absorption spectroscopy (AAS). A stability-indicating HPLC method involving formation of the diethylamine-dithiocarbamate complexes of phenylmercuric and mercuric ions was found to suffer a pH-dependent interference from disodium edetate. A second method was therefore also employed involving selective extraction into diethylether of the phenylmercuric ion followed by HPLC of the piperidinedithiocarbamate complex with concomitant analysis of the unextracted mercuric ion by AAS using the cold-vapour technique. An HPLC method was also developed for benzene in the degraded mixtures. The application of these methods to autoclaved solutions containing PM nitrate and disodium edetate demonstrates that under the conditions of heat sterilization the phenylmercuric ion is degraded to mercuric ion and benzene to the extent of 15% at pH 8, 80% at pH 7 and completely degraded at pH 5 and 6.

Resistogram typing as an epidemiological tool for Escherichia coli isolates of poultry origin

Twenty isolates of Escherichia coli from cases of colibacillosis-affected birds in different farms in and around Madras city of Tamil Nadu State (India) were collected and subjected to resistogram typing. The resistogram types obtained from seven dyes and toxic chemicals were useful in differentiating the isolates. In all 14 different types were obtained with 100% showing resistance to phenyl mercuric nitrate and only 10% to 4-chloresorcinol. The resistance patterns towards other dyes and chemicals were, in descending order, copper sulphate, 95%, sodium arsenate, 80%, acriflavine, 70%, boric acid, 65%, and malachite green, 60%. The usefulness of this test as an epidemiological tool is discussed.

The decomposition of phenylmercuric nitrate caused by disodium edetate in neomycin eye drops during the process of heat sterilization.

High-performance liquid chromatographic (HPLC) assays have been developed for phenylmercuric nitrate and mercuric ions in the presence of disodium edetate. Application of these methods to neomycin eye drops of the Pharmaceutical Codex and Australian Pharmaceutical Formulary, which contain both phenylmercuric nitrate and disodium edetate, show that edetate promotes approximately 20% decomposition of the phenylmercuric nitrate in the drops of the Pharmaceutical Codex but little in the drops of the Australian Pharmaceutical Formulary during sterilization by the recommended method. The reason for the stability of the organomercurial in the Australian product was determined as being due to the presence of sodium chloride in the formulation.