Microbial contamination of water-soaked cotton gauze and its cause

Seven in-use cotton gauze samples and three cotton balls soaked in sterile distilled water in canisters were investigated 7 days after they were prepared in hospital. All samples were contaminated with bacteria including 10(6) to 10(7) colony forming units/ml of Pseudomonas aeruginosa. In vitro viability tests using cotton gauze and cotton balls soaked in sterile distilled water revealed rapid proliferation of P. aeruginosa, Serratia marcescens and Candida albicans. Since the cotton gauze and the cotton balls were soaked in water containing nutrients, such as protein and glucose, these materials may be readily contaminated with bacteria including P. aeruginosa. Thus, when using cotton gauze and cotton balls containing water, microbial contamination should be expected.     

Multiparametric analysis of waterline contamination in dental units.

Microbial contamination of dental unit waterlines is thought to be the result of biofilm formation within the small-bore tubing used for these conduits. Systematic sampling of 121 dental units located at the dental school of Université de Montréal showed that none of the waterlines was spared from bacterial contamination. Multilevel statistical analyses showed significant differences between samples taken at the beginning of the day and samples taken after a 2-min purge. Differences were also found between water from the turbine and the air/water syringe. Random variation occurred mainly between measurements (80%) and to a lesser extent between dental units (20%). In other analyses, it was observed to take less than 5 days before initial bacterial counts reached a plateau of 2 x 10(5) CFU/ml in newly installed waterlines. Sphyngomonas paucimobilis, Acinetobacter calcoaceticus, Methylobacterium mesophilicum, and Pseudomonas aeruginosa were the predominant isolates. P. aeruginosa showed a nonrandom distribution in dental unit waterlines, since 89.5% of the all the isolates were located in only three of the nine clinics tested. Dental units contaminated by P. aeruginosa showed significantly higher total bacterial counts than the others. By comparison, P. aeruginosa was never isolated in tap water remote from or near the contaminated dental unit waterlines. In conclusion, dental unit waterlines should be considered an aquatic ecosystem in which opportunistic pathogens successfully colonize synthetic surfaces, increasing the concentration of the pathogens in water to potentially dangerous levels. The clinical significance of these findings in relation to routine dental procedures is discussed.     

Detection of indicator pathogens from pharmaceutical finished products and raw materials using multiplex PCR and comparison with conventional microbiological methods

A multiplex PCR assay was devised and compared with standard conventional methods for quality evaluation of pharmaceutical raw materials and finished products with low levels of microbial contamination. Samples which were artificially contaminated with <10 colony forming units of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella species and possibly contaminated samples were incubated for 16 h with different enrichment media. Primers that deduce 559 bp fragment of the 16S rRNA gene was employed in amplifying E. coli species, similarly invasion protein gene with 275 bp fragment size was used as target for detecting Salmonella spp., in case of S. aureus a 461 bp amplicon from m-RNA nuclease gene, and an 709 bp fragment from oprL gene was used for amplifying P. aeruginosa. The detection limits for artificially contaminants by multiplex PCR was 1 CFU/g, where as in case of conventional method the detection limit was >2 CFU/g. Similarly, when tested with possibly contaminated samples, 35% were detected for E. coli, Salmonella spp., S. aureus and P. aeruginosa species with multiplex PCR, while only 21% were detected with standard conventional microbial methods. Multiplex PCR assay provides sensitive and reliable results and allows for the cost-effective detection of all four bacterial pathogens in single reaction tube.     

Bacteria of public health significance in blackcurrant juice

A total of 192 samples of blackcurrant juice were investigated for hygienic quality and drug resistance. The average aerobic bacteria in 1 ml of the blackcurrant juice was 4.8 x 10(8) and the average MPN was 8.4 x 10(2) coliform/100 ml. The percentage of the samples contaminated with coliform bacteria was 23.4. The incidence of Escherichia coli, Klebsiella, Pseudomonas aeruginosa, Staphylococcus aureus and faecal streptococci were 19.8, 9.9, 29.2, 14.0 and 18.2%, respectively. Biochemical and serological studies showed that 10.5% of total Escherichia coli were enteropathogenic E. coli. Of sixty strains tested against six antibacterial drugs, 57% were resistant to one or more of the drugs. The incidence of resistance to ampicillin, chloramphenicol, sulphonamide, streptomycin, tetracyline and gentamycin was 40.0, 25.0, 43.3, 18.3, 20.0 and 8.3%, respectively.     

C-390 as sole selective agent for isolation of Pseudomonas aeruginosa from hospital waste water

Pseudomonas aeruginosa was recovered (in numbers ranging from 10(2) to 10(5) colony-forming units per millilitre) from heavily contaminated hospital waste water when grown at 41.5 degrees C on a differential medium agar containing 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan (C-390) at a final concentration of 30 micrograms/mL. The medium appeared to be highly selective for P. aeruginosa with 95-100% of all colonies isolated from four different hospital waste waters being identified as P. aeruginosa. Many strains of P. aeruginosa isolated from hospital waste waters failed to hydrolyse casein when grown on skim milk agar and this medium appeared to restrict pigment production to only pyoverdin (detectable only under ultraviolet light). However, most strains were capable of casein hydrolysis when grown on a modified skim milk medium.     

Use of PCR analysis for detecting low levels of bacteria and mold contamination in pharmaceutical samples

PCR assays were developed and compared to standard methods for quality evaluation of pharmaceutical raw materials and finished products with low levels of microbial contamination. Samples were artificially contaminated with less than 10 CFU of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Aspergillus niger. Bacterial DNA was extracted from each enrichment broth by mild lysis in Tris-EDTA-Tween 20 buffer containing proteinase K while mold DNA was extracted by boiling samples in Tris-EDTA-SDS buffer for 1 h. A 10-microl aliquot of extracted DNA was added to Ready-To-Go PCR beads and specific primers for E. coli, S. aureus, and P. aeruginosa. However, 50-microl aliquots of extracted mold DNA were used for amplification of specific A. niger DNA sequences. Standard methods required 6-8 days while PCR detection of all microorganisms was completed within 27 h. Low levels of microbial contamination were detected in all raw materials and products using PCR assays. Rapid quality evaluation of pharmaceutical samples resulted in optimization of product manufacturing, quality control, and release of finished products.     

Dynamics of the contamination of mice during the treatment of burn sepsis due to Pseudomonas aeruginosa using tobramycin alone or combined with active and passive immunization

An experimental study was made with a view to finding out the possible bacteriological advantages of the combined use of tobramycin (Tb) and P. aeruginosa corpuscular polyvalent vaccine (PaCPV) or P. aeruginosa hyperimmune plasma (PaHIP) in burn sepsis caused by P. aeruginosa. The use of the median therapeutic dose of Tb (2.5 mg/kg body weight per day), alone or in combination with immunopreparations, ensured the survival rate of the animals equal to 100%. The contamination of the body with P. aeruginosa after treatment with Tb and PaHIP or PaCPV was lower than after the administration of Tb alone, this phenomenon becoming manifest starting from day 5 of observation in the first case and from day 10 in the second case. The combined use of Tb and immunopreparations (PaCPV or PaHIP) in acute P. aeruginosa infection proved to be more effective than treatment with Tb alone.     

Mortality in adult tsetse, Glossina morsitans morsitans, caused by entomopathogenic bacteria

Mortality in adult tsetse, Glossina morsitans morsitans, caused by Pseudomonas aeruginosa, Serratia marcescens, Bacillus sphaericus, Bacillus cereus, Bacillus thuringiensis H-14, B. thuringiensis 1, B. thuringiensis 5, B. thuringiensis var. insraelensis, and Providentia rettgeri was determined. When bacteria were smeared on rabbit skin and tsetse allowed to feed only once on the contaminated area, mortality 8 days postingestion was significantly higher (P less than 0.01) in tsetse fed on P. aeruginosa, S. marcescens, B. thuringiensis 1, and P. rettgeri and increased when tsetse were allowed to feed for the second time on the contaminated skin. With this smear technique, however, mortalities were generally not remarkable. In artificial membrane feeding experiments using low concentrations of bacteria (-10(6)/ml of blood), the B. thuringiensis strains caused low mortality, except B. thuringiensis H-14, which caused 59% mortality. However, at this concentration, P. aeruginosa, S. marcescens, B. cereus, and P. rettgeri caused highly significant (P less than 0.01) mortality (64-96%). When higher concentrations of bacteria (10(7)/ml) were used, all the bacteria tested, except B. sphaericus, caused high mortality ranging from 70 to 98%. Thus, mortality depended on the species of bacteria, the dose ingested, and time postingestion.     

Investigations into the survival of Pseudomonas aeruginosa in poloxamer-iodine

Laboratory investigations were conducted to study potential mechanisms for prolonged survival of Pseudomonas aeruginosa in poloxamer-iodine (PxI). P. aeruginosa organisms isolated from PxI and adapted for growth in distilled water or found as part of a mixed microbial population from water in a manufacturing plant did not survive more than 15 s after challenge in stock PxI solution. Batches of PxI were compounded in the laboratory to determine the survival and growth of P. aeruginosa during the various stages of preparation. No P. aeruginosa organisms were recovered from the finished product at 1 min after the addition of iodine-iodide. However, we found P. aeruginosa in PxI 48 h after adding sterile PxI to the inside of a naturally contaminated polyvinyl chloride water distribution pipe. These organisms (10(4) CFU/ml) survived for as long as 98 days in contaminated stock PxI after it was removed from the polyvinyl chloride pipe. Both decreasing the free iodine level through addition of potassium iodide and increasing the free iodine level through dilution of the product resulted in an increased length of survival of P. aeruginosa in contaminated PxI solution. Comparative survival studies with pipes of different composition revealed that other materials may exert an effect similar to polyvinyl chloride. We concluded that polyvinyl chloride and perhaps other materials may play an important role in the survival of P. aeruginosa in iodophors and may be one source of resistant microbial populations when used in manufacturing plants which produce these antimicrobial solutions.     

Investigations into the survival of Pseudomonas aeruginosa in poloxamer-iodine.

Laboratory investigations were conducted to study potential mechanisms for prolonged survival of Pseudomonas aeruginosa in poloxamer-iodine (PxI). P. aeruginosa organisms isolated from PxI and adapted for growth in distilled water or found as part of a mixed microbial population from water in a manufacturing plant did not survive more than 15 s after challenge in stock PxI solution. Batches of PxI were compounded in the laboratory to determine the survival and growth of P. aeruginosa during the various stages of preparation. No P. aeruginosa organisms were recovered from the finished product at 1 min after the addition of iodine-iodide. However, we found P. aeruginosa in PxI 48 h after adding sterile PxI to the inside of a naturally contaminated polyvinyl chloride water distribution pipe. These organisms (10(4) CFU/ml) survived for as long as 98 days in contaminated stock PxI after it was removed from the polyvinyl chloride pipe. Both decreasing the free iodine level through addition of potassium iodide and increasing the free iodine level through dilution of the product resulted in an increased length of survival of P. aeruginosa in contaminated PxI solution. Comparative survival studies with pipes of different composition revealed that other materials may exert an effect similar to polyvinyl chloride. We concluded that polyvinyl chloride and perhaps other materials may play an important role in the survival of P. aeruginosa in iodophors and may be one source of resistant microbial populations when used in manufacturing plants which produce these antimicrobial solutions.     

Bacterial hand contamination and transfer after use of contaminated bulk-soap-refillable dispensers

Bulk-soap-refillable dispensers are prone to extrinsic bacterial contamination, and recent studies demonstrated that approximately one in four dispensers in public restrooms are contaminated. The purpose of this study was to quantify bacterial hand contamination and transfer after use of contaminated soap under controlled laboratory and in-use conditions in a community setting. Under laboratory conditions using liquid soap experimentally contaminated with 7.51 log(10) CFU/ml of Serratia marcescens, an average of 5.28 log(10) CFU remained on each hand after washing, and 2.23 log(10) CFU was transferred to an agar surface. In an elementary-school-based field study, Gram-negative bacteria on the hands of students and staff increased by 1.42 log(10) CFU per hand (26-fold) after washing with soap from contaminated bulk-soap-refillable dispensers. In contrast, washing with soap from dispensers with sealed refills significantly reduced bacteria on hands by 0.30 log(10) CFU per hand (2-fold). Additionally, the mean number of Gram-negative bacteria transferred to surfaces after washing with soap from dispensers with sealed-soap refills (0.06 log(10) CFU) was significantly lower than the mean number after washing with contaminated bulk-soap-refillable dispensers (0.74 log(10) CFU; P < 0.01). Finally, significantly higher levels of Gram-negative bacteria were recovered from students (2.82 log(10) CFU per hand) than were recovered from staff (2.22 log(10) CFU per hand) after washing with contaminated bulk soap (P < 0.01). These results demonstrate that washing with contaminated soap from bulk-soap-refillable dispensers can increase the number of opportunistic pathogens on the hands and may play a role in the transmission of bacteria in public settings.     

Phytoremediation of Abandoned Crude Oil Contaminated Drill Sites of Assam with the Aid of a Hydrocarbon-Degrading Bacterial Formulation

Environmental deterioration due to crude oil contamination and abandoned drill sites is an ecological concern in Assam. To revive such contaminated sites, a field study was conducted to phytoremediate four crude oil abandoned drill sites of Assam (Gelakey, Amguri, Lakwa, and Borholla) with the aid of two hydrocarbon-degrading Pseudomonas strains designated N3 and N4. All the drill sites were contaminated with 15.1 to 32.8% crude oil, and the soil was alkaline in nature (pH8.0–8.7) with low moisture content, low soil conductivity and low activities of the soil enzymes phosphatase, dehydrogenase and urease. In addition, N, P, K, and C contents were below threshold limits, and the soil contained high levels of heavy metals. Bio-augmentation was achieved by applying Pseudomonas aeruginosa strains N3 and N4 followed by the introduction of screened plant species Tectona grandis, Gmelina arborea, Azadirachta indica, and Michelia champaca. The findings established the feasibility of the phytoremediation of abandoned crude oil-contaminated drill sites in Assam using microbes and native plants.     

Ten years-long survey on pathogen status of mouse and rat breeding colonies

Eleven pathogens including P. aeruginosa, Salmonella spp., E. coli O115a, c: K(B), P. pneumotropica, B. bronchiseptica, C. kutscheri, Tyzzer's organism, M. pulmonis, Sendai virus, MHV and Syphacia spp. were surveyed in 217 mouse and rat breeding colonies during 1972-1981. In conventional animals, P. pneumotropica and/or Syphacia spp. were detected in nearly 90% of 89 mouse and 64 rat colonies. Sendai virus, M. pulmonis, P. aeruginosa and MHV were positive in 51.7 to 23.6% of the colonies, and Tyzzer's organism, B. bronchiseptica and probably SDA virus were also detected in more than 10% of the rat colonies. Salmonella spp., E. coli O115a, c: K(B) and C. kutscheri were found in a few colonies. In SPF animals, P. aeruginosa was isolated from about one third of 33 mouse and 31 rat colonies, and P. aeruginosa was isolated from about one third of 33 mouse and 31 rat colonies, and P. pneumotropica was also positive in 3 rat colonies. Infection rates of P. pneumotropica, M. pulmonis, Sendai virus and Syphacia spp. were usually higher than 40% of animals sampled from colonies contaminated with them. Accidental contaminations of SPF colonies were usually caused by P. pneumotropica and Syphacia spp.     

On the methods of disinfecting bronchofibroscopes

An apparatus is proposed for the wet disinfection of bronchofibroscopes which enables to wash with disinfectant solution the external surfaces of the tube and instrument channel while keeping dry the control mechanism. Several disinfecting regimens have been tested using chlorohexidine (Gibitan), benzalkonium chloride (Roccal), diocide and ethanol solutions. On experimental contamination of bronchofibroscopes with a P. aeruginosa culture effective disinfection was achieved using 0.5% aqueous and ethanol solution of chlorohexidine and 1% benzalkonium chloride solution (5-min, exposure in the circulation mode of the apparatus), 0.1% solution of diocide only produced a bactericidal effect after a 30-min, exposure. Adequate disinfection was not feasible when the endoscope was soaked in disinfectant. Similar results were obtained when bronchofibroscopes were disinfected in a clinical setup where they became contaminated with the most common pathogenic and potentially pathogenic microflora from the airways of patients suffering from purulent pulmonary diseases.     

Survival of selected bacterial species in sterilized activated carbon filters and biological activated carbon filters.

The survival of selected hygienically relevant bacterial species in activated carbon (AC) filters on a bench scale was investigated. The results revealed that after inoculation of the test strains the previously sterilized AC absorbed all bacteria (10(6) to 10(7)). After a period of 6 to 13 days without countable bacteria in the effluent, the numbers of Escherichia coli, Pseudomonas aeruginosa, and Pseudomonas putida increased up to 10(4) to 10(5) CFU/ml of effluent and 10(6) to 10(7) CFU/g of AC. When Klebsiella pneumoniae and Streptococcus faecalis were used, no growth in filters could be observed. The numbers of E. coli, P. aeruginosa, and P. putida, however, decreased immediately and showed no regrowth in nonsterile AC from a filter which had been continuously connected to running tap water for 2 months. Under these conditions an autochthonous microflora developed on the carbon surface which could be demonstrated by scanning electron microscopy and culturing methods (heterotrophic plate count). These bacteria reduced E. coli, P. aeruginosa, and P. putida densities in the effluent by a factor of more than 10(5) within 1 to 5 days. The hypothesis that antagonistic substances of the autochthonous microflora were responsible for the elimination of the artificial contamination could not be confirmed because less than 1% of the isolates of the autochthonous microflora were able to produce such substances as indicated by in vitro tests. Competition for limiting nutrients was thought to be the reason for the observed effects.     

Evaluation of disinfective potential of reactivated free chlorine in pooled tap water by electrolysis

Tap water is one of the causative factors of hospital infections. We examined the disinfective potential of electrolysis and mechanism of disinfection, and clarified the disinfective effect of electrolysis on tap water contaminated with bacteria, and discussed its clinical applications. Tap waters artificially contaminated with Pseudomonas aeruginosa, Escherichia coli, Legionella pneumophila, and Staphylococcus aureus could be sterilized by electrolysis at 20-30 mA for 5 min. A high-density suspension (10(6) CFU/ml) of a spore forming bacterium, Bacillus subtilis was not completely sterilized by electrolysis at 50 mA up to 30 min, but a low-density suspension (10(5) CFU/ml) was totally sterilized by electrolysis at 50 mA for 5 min. Electrolyzed P. aeruginosa changed morphologically, that is, there was bleb formation on the cell wall and irregular aggregation of cytoplasmic small granules. Moreover, cytoplasmic enzyme, nitrate reductase, was inactivated by the electrolysis. On the other hand, genomic DNA of the electrolyzed bacteria was not degenerated, therefore, their DNA polymerase activity was not completely inactivated. Consequently, the major agent in electrolysis for bactericidal action was considered to be free chlorine, and the possible bactericidal mechanism was by destruction of bacterial membranes, followed by the aggregation of peripheral cytoplasmic proteins. Electrolysis of tap water for both disinfecting contaminating bacteria and increasing the disinfectant capacity was considered effective with some limitations, particularly against high-density contamination by spore-forming bacteria. In clinical settings, electrolysis of tap water is considered effective to disinfect water for hand washing in operation theatres, and bathing water for immunocompromised hosts.     

Siderophore production by using free and immobilized cells of two pseudomonads cultivated in a medium enriched with Fe and/or toxic metals (Cr, Hg, Pb)

Pseudomonads are serious candidates for siderophore production applied to toxic metal (TM) solubilization. The bioaugmentation of contaminated soils by these TM-solubilizing bacteria combined with phytoextraction is an emerging clean-up technology. Unfortunately, siderophore synthesis may be drastically reduced by soluble iron in soils and bacteria can suffer from TM toxicity. In this study, we compared siderophore production by Pseudomonas aeruginosa and Pseudomonas fluorescens by using free and immobilized cells in Ca-alginate beads incubated in a medium containing Fe and/or TM (mixture of Cr, Hg, and Pb in concentrations which represented the soluble fraction of a contaminated agricultural soil). Free cell growth was stimulated by Fe, whatever the microorganism, the inoculum size and the presence or not of TM might have been. P. aeruginosa was less sensitive to TM than P. fluorescens. By comparison with free cells, immobilization with the high inoculum size showed less sensitivity to TM most probably because of lower metal diffusion in beads. Indeed, a maximum of 99.1% of Cr, 57.4% of Hg, and 99.6% of Pb were adsorbed onto beads. The addition of iron in the culture medium reduced significantly siderophore production of free cells while it led only to a low decrease with their immobilized counterparts, in particular with P. aeruginosa. In culture medium enriched with Fe and/or TM, siderophore-specific production of immobilized cells was higher than for free cells.     

Survival of selected bacterial species in sterilized activated carbon filters and biological activated carbon filters

The survival of selected hygienically relevant bacterial species in activated carbon (AC) filters on a bench scale was investigated. The results revealed that after inoculation of the test strains the previously sterilized AC absorbed all bacteria (10(6) to 10(7)). After a period of 6 to 13 days without countable bacteria in the effluent, the numbers of Escherichia coli, Pseudomonas aeruginosa, and Pseudomonas putida increased up to 10(4) to 10(5) CFU/ml of effluent and 10(6) to 10(7) CFU/g of AC. When Klebsiella pneumoniae and Streptococcus faecalis were used, no growth in filters could be observed. The numbers of E. coli, P. aeruginosa, and P. putida, however, decreased immediately and showed no regrowth in nonsterile AC from a filter which had been continuously connected to running tap water for 2 months. Under these conditions an autochthonous microflora developed on the carbon surface which could be demonstrated by scanning electron microscopy and culturing methods (heterotrophic plate count). These bacteria reduced E. coli, P. aeruginosa, and P. putida densities in the effluent by a factor of more than 10(5) within 1 to 5 days. The hypothesis that antagonistic substances of the autochthonous microflora were responsible for the elimination of the artificial contamination could not be confirmed because less than 1% of the isolates of the autochthonous microflora were able to produce such substances as indicated by in vitro tests. Competition for limiting nutrients was thought to be the reason for the observed effects.     

Routes of the transmission of Pseudomonas aeruginosa infection in resuscitation and intensive therapy departments

To find out if the transfer of P. aeruginosa infection by droplet route is possible in resuscitation and intensive care units, the bacteriological study of air samples taken in different rooms of resuscitation units (altogether 234 air samples) was carried out with the subsequent identification and typing of isolated P. aeruginosa strains. In most cases (70.5%) the microbial contamination of the air in the main rooms of resuscitation units was found not to exceed 500 microbial cells per cu. m, and no P. aeruginosa strains were isolated. The identification and typing of six P. aeruginosa strains isolated from the air of an isolation ward for patients with infectious complications made it possible to find out intraspecific differences of these microorganisms, as all of them belonged to strains of different sero- and pyocinotypes. Thus, the results of these investigations indicate that the droplet route of the transfer of P. aeruginosa hospital infection is not characteristic of resuscitation and intensive care units, as no P. aeruginosa strains are isolated from the main rooms of such units; likewise, no circulation of this microorganism was observed in the air of an isolation ward for patients with infectious complications.     

Microbiological study of cosmetic products during their use by consumers: health risk and efficacy of preservative systems

AIM: To evaluate the microbial contamination of 91 cosmetics (23 o/w emulsions, 47 tensiolytes, 21 aqueous pastes) in three different states of use (intact, in-use, ending product) and the protection efficacy of the preservative systems most frequently used in the analysed cosmetic formulations. METHODS AND RESULTS: Total bacterial count, isolation and identification of pathogenic isolates were performed on the collected cosmetics. About 10.6% of tensiolytes (13.5% bath foam, 6.7% shampoo, 10% liquid soaps) were contaminated by Staphylococcus warneri, Staphylococcus epidermidis and Pseudomonas putida. The efficacy of the preservative systems of two cosmetic products, tested against standard micro-organisms (Staphylococcus aureus ATCC 4338 and Pseudomonas aeruginosa ATCC 9027) and two isolates from cosmetics in this study (S. epidermidis and P. putida), satisfied the Cosmetics, Toiletries, and Fragrance Association and Official Italian Pharmacopeia criteria, while only one tested cosmetic respected the Rapid Challenge Test criterion. CONCLUSIONS: Contaminated cosmetic products are relatively uncommon, but some products, unable to suppress the growth of several micro-organisms, represent a potential health hazard. SIGNIFICANCE AND IMPACT OF THE STUDY: The challenge test may be performed not only during the preparation of the preservative system in the intact cosmetics, but also be used to evaluate the protection efficacy during their use.