Low Levels Of Microbial Contamination Research Articles

Assessment of Performance of Two Rapid Methods for On-Site Control of Microbial and Biofilm Contamination

(1) Background: The validation of hygiene procedures in food industries is paramount to ensure that food contact surfaces are properly decontaminated before production. Rapid, sensitive and reliable tools are needed for routine hygiene validation in order to increase food safety levels. Two novel tools for biofilm detection (TBF 300) and detection of low levels of microbial contamination (FreshCheck) have been assessed. (2) Methods: Biofilms of relevant food pathogens: Listeria monocytogenes and Salmonella spp. were grown for 3 and 10 days to assess the performance of the biofilm detection product. Surfaces were inoculated with different levels of L. monocytogenes to determine the limit of detection of FreshCheck. (3) Results: TBF 300 visibly stained 3 days-old biofilms of both pathogens, containing 5.0–5.4 log CFU/cm2. FreshCheck showed a positive reaction with contamination levels as low as 10 CFU/cm2 for L. monocytogenes. (4) Conclusions: Assessment of the hygienic status of food contact surfaces before production can be greatly improved with the use of the two novel tools evaluated in this study. The detection of microorganisms’ presence at very low levels of contamination as well as identification of biofilm growth spots is available in a rapid and easy way, with a big potential contribution to food safety.

Measuring environmental contamination in critical care using dilute hydrogen peroxide (DHP) technology: An observational cross-over study.

The environment has an important role in the transmission of healthcare associated infections. This has encouraged interest in novel methods to improve hygiene in hospitals. One such technology is the use of hydrogen peroxide to decontaminate rooms and equipment; there are, however, few studies that have investigated the effect of continuous dilute hydrogen peroxide (DHP) in the clinical environment. The aim of this study was to examine the use of dilute hydrogen peroxide (DHP) in a critical care unit and measure the microbiological impact on surface contamination. We conducted a prospective observational cross-over study in a ten-bed critical care unit in one rural Australian hospital. Selected high-touch sites were screened using dipslides across three study phases: baseline; continuous DHP; and no DHP (control). Quantitative aerobic colony counts (ACC) were assessed against a benchmark standard of ACC >2.5cfu/cm2 to indicate hygiene failure. There were low levels of microbial contamination in the unit for baseline; DHP; and no DHP phases: 2.2% (95% CI 0.7-5.4%) vs 7.7% (95% CI 4.3-13.0%) vs 6% (95% CI 3.2-10.4%) hygiene failures, respectively. Significant reduction in ACCs did not occur when the DHP was operating compared with baseline and control phases. Further work is needed to determine whether continuous DHP technology has a role in decontamination for healthcare settings.

Antibiotics inhibit sphere-forming ability in suspension culture.

BackgroundThis last decade, a lot of emphasis has been placed on developing new cancer cell culture models, closer to in vivo condition, in order to test new drugs and therapies. In the case of colorectal cancer, the use of patient biopsies to seed 3D primary cultures and mimic tumor initiation necessitates the use of antibiotics to prevent microbial intestinal contamination. However, not only long term use of antibiotics may mask the presence of low levels of microbial contamination, it may also impact cancer cell phenotype.MethodsIn this study we tested the impact of penicillin-streptomycin cocktail addition in both monolayer and suspension culture. To ensure the reliability of our observations we used six different cell lines and each experiment was performed in triplicate. Results were analyzed with Student’s t test.ResultsWe show that penicillin–streptomycin cocktail inhibits the sphere-forming ability of six cancer cell lines in suspension culture though it has no impact in monolayer culture. We correlate this effect with a significant decrease of cancer stem cells pool which holds self-renewal potential.ConclusionsOverall, this study warns against systematic addition of antibiotics in growth medium and raises the interesting possibility of using antibiotics to target cancer stem cells.

How reliable are ATP bioluminescence meters in assessing decontamination of environmental surfaces in healthcare settings?

BackgroundMeters based on adenosine triphosphate (ATP) bioluminescence measurements in relative light units (RLU) are often used to rapidly assess the level of cleanliness of environmental surfaces in healthcare and other settings. Can such ATP measurements be adversely affected by factors such as soil and cleaner-disinfectant chemistry?ObjectiveThis study tested a number of leading ATP meters for their sensitivity, linearity of the measurements, correlation of the readings to the actual microbial contamination, and the potential disinfectant chemicals’ interference in their readings.MethodsFirst, solutions of pure ATP in various concentrations were used to construct a standard curve and determine linearity and sensitivity. Serial dilutions of a broth culture of Staphylococcus aureus, as a representative nosocomial pathogen, were then used to determine if a given meter’s ATP readings correlated with the actual CFUs. Next, various types of disinfectant chemistries were tested for their potential to interfere with the standard ATP readings.ResultsAll four ATP meters tested herein demonstrated acceptable linearity and repeatability in their readings. However, there were significant differences in their sensitivity to detect the levels of viable microorganisms on experimentally contaminated surfaces. Further, most disinfectant chemistries tested here quenched the ATP readings variably in different ATP meters evaluated.ConclusionsApart from their limited sensitivity in detecting low levels of microbial contamination, the ATP meters tested were also prone to interference by different disinfectant chemistries.

Estimating the correlation between concentrations of two species of bacteria with censored microbial testing data

Indicator organisms, such as generic Escherichia coli (GEC) and coliforms, can be used to measure changes in microbial contamination during the production of food products. Large and consistent reductions in the concentration of these organisms demonstrates an effective and well-controlled production process. Nevertheless, it is unclear to what degree concentrations of indicator organisms are related to pathogenic organisms such as Campylobacter and Salmonella on a sample-by-sample basis. If a strong correlation exists between the concentrations of different organisms, then the monitoring of indicator organisms would be a cost-effective surrogate for the measurement of pathogenic organisms. Calculating the correlation between the concentrations of an indicator and pathogenic organism is complicated because microbial testing datasets typically contain a large proportion of censored observations (i.e., samples where the true concentration is not observable, with nondetects and samples that are only screen-test positive being examples). This study proposes a maximum likelihood estimator that can be used to estimate the correlation between the concentrations of indicator and pathogenic organisms. An example based on broiler chicken rinse samples demonstrates modest, but significant positive correlations between the concentration of the indicator organism GEC when compared to the concentration of both Campylobacter and Salmonella. A weak positive correlation was also observed between concentrations of Campylobacter and Salmonella, but it was not statistically significant.

Bioactive amines and internal quality of commercial eggs

The physicochemical and microbiological qualities of commercial eggs produced by layer hens of different ages (approximately 30 and 60 wk old) were submitted to storage under room temperature or refrigeration for 28 d. A total of 600 eggs were subjected to microbiological analyses of their inner contents and another 600 to a determination of Haugh units (HU) and bioactive amine content. A decrease in the quality of the inner contents of the eggs was observed during the experiment, mainly in the eggs from the 60-wk-old layers, which presented the worst HU values when stored at room temperature (P < 0.05). Microbiological analyses showed an absence of Salmonella spp., Staphylococcus aureus, and coliforms, either total or thermal-tolerant; however, low counts of other Staphylococcus species, Enterobacter spp., Pseudomonas spp., mesophilic aerobic bacteria, and fungi were also recorded. The chromatographic analysis of bioactive amines detected the presence of phenylethylamine in all albumens (38.0 mg/kg) and spermidine in the yolks (1.02 mg/kg). It was concluded that the age of the hens and the time and temperature of storage influenced the quality parameters of the eggs (P < 0.05). Furthermore, despite the low levels of microbial contamination found, phenylethylamine was detected in the albumen. It was not possible to establish index of quality of eggs using bioactive amines present in the yolk and albumen of eggs.

Ward assessment of SmartIdeas Project: bringing source isolation to the patient

Most UK hospitals lack enough single rooms to provide source isolation for all infected patients. The aim of this study was to test prototype isolation systems on general wards together with specifically designed portable sink units and toilets. Questionnaires were offered to staff, patients and visitors covering ease of use and acceptability. A total of 53 patients were isolated, with concurrent collection of environmental samples and staff hand hygiene audit. Blocking of beds next to infected patients was avoided but patients and staff were concerned about limited space and communication. Hand hygiene compliance on entry or exit to/from an isolated bed space significantly improved [43/76 (56.6%) to 107/147 (72.8%), P<0.05]. Although popular, the toilets were mechanically unreliable. Low levels of microbial contamination (<1-3.4cfu/cm(2)) were present within all isolated bed spaces. The highest colony counts were obtained from high contact sites (e.g. remote controls). Meticillin-resistant Staphylococcus aureus (MRSA) was present at similar levels inside all systems. Although one system was designed to provide airborne as well as contact isolation, MRSA was isolated from air inside and outside the system suggesting poor efficiency of the air door. The finding was confirmed by aerobiology tests at the Health Protection Agency Laboratory, Porton Down, UK. A trial of redesigned units is required to establish efficacy (Trial Identifier: ISRCTN02681602).

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.

ASSESSMENT OF CONVENTIONAL DETECTION METHOD, IMMUNOANALYSIS AND POLYMERASE CHAIN REACTION FOR SALMONELLA SPP. MONITORING IN CHICKEN

ABSTRACT Three Salmonella spp. detection methodologies were evaluated in superficial chicken carcasses samples collected in a poultry slaughter. One hundred thirty‐five were tested by conventional isolation procedure, 70 by immunoanalysis and 90 by polymerase chain reaction (PCR). The frequency of Salmonella spp. isolation among the methodologies was evaluated considering their applicability as monitoring tools for quality control programs, like the hazard analysis critical control point (HACCP). The immunoanalysis and PCR methods showed significant differences from the conventional isolation procedure (qui‐square = 18.51; gl = 2; P &lt; 0.0001), and they presented higher frequencies of positive results. Besides being more sensitive and specific, immunoanalysis and PCR were faster and more practical in Salmonella spp. monitoring. These characteristics make these alternative techniques more suitable as monitoring tools for quality control programs as HACCP.PRACTICAL APPLICATIONS Salmonella spp. detection in chicken during the slaughter is fundamental to assure microbiological safety, a worldwide concern. The chicken slaughter process must be conducted in order to avoid the contamination of the carcasses, resulting in food with low levels of microbial contamination, including pathogens as Salmonella spp. This paper shows the adequacy of three methodologies for this purpose (the conventional isolation and two alternative methodologies, VIDAS and polymerase chain reaction [PCR]), so as to identify the advantages and disadvantages of each one. Considering the results, it is possible to identify which one is more convenient for proper Salmonella spp. monitoring in distinct slaughterhouses. In addition, the obtained results allow the observation of the most efficient tested methodologies which showed more sensitivity and specificity, leading their application in food industries, scientific researches and official inspection services.

The appropriateness of swab cultures for the release of human allograft tissue

Surgeries utilizing human allograft tissues have increased dramatically in recent years. With this increase has come a greater reliance on the use of swab culturing to assess allograft tissues for microbial contamination prior to distribution. In contrast to the typical industrial microbiological uses for swabs, the tissue banking industry has relied on swab cultures as a sterility release method for allograft tissues. It has been reported in the literature that swabs have limitations, both in sensitivity and reproducibility, so their suitability as a final sterility release method was evaluated in this study. Two different swab-culturing systems were evaluated (COPAN, EZ Culturette) using human allograft tissues spiked with low levels of multiple bacterial and fungal microorganisms. The average microbial recoveries for all challenge microorganisms for each tissue type and each swab system were calculated. Percent recoveries for each challenge microorganism were also calculated and reported. The results indicated that both swab systems exhibited low and highly variable recoveries from the seeded allograft tissues. Further analysis indicated there was no statistical difference ( proportional, variant=0.05) between the two swab systems. It is the recommendation of the authors that swab culturing not be used to assess relatively low levels of microbial contamination on allografts. Instead, alternative validated microbial detection methods with improved sensitivity and reproducibility should be employed and validated for this critical task.

Temporal variations in the depth-specific hydrochemistry and sewage-related microbiology of an urban sandstone aquifer, Nottingham, United Kingdom

The temporal and spatial characteristics of groundwater recharge in urban environments remain poorly understood. Depth-specific monitoring of groundwater quality in the Triassic Sandstone underlying the city of Nottingham, UK, indicates that contamination results primarily from sewage and atmospheric sources. The temporal and depth-specific characteristics of microbial and inorganic (e.g. nitrate, chloride, sulphate) contamination over the investigation period differ significantly and reflect the contrasting transport characteristics of surface-loaded solutes and particulate microbial species (bacteria and viruses) in the Triassic Sandstone. Differences result from a variety of factors, which include microbial die-off, dilution, and the contaminant-source characteristics. Observations in this study show that low levels of microbial contamination should be expected at depth in fissured sandstone due to aquifer heterogeneities such as fissuring and the occurrence of mudstone bands, though the magnitude of this contamination will vary over time. Furthermore, urban groundwater-protection measures based on solute-transport estimates may not be applicable to microbial contamination.

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-μl 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-μl 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.

Levels of naturally occurring microorganisms on surgical instruments after clinical use and after washing

Surgical instruments exposed to sterile body sites should be contaminated with relatively low levels of microbial contamination or bioburden; however, few studies in the literature have determined the quantitative level and types of contamination. A study was conducted at 2 clinical sites to determine the level of microbial contamination of surgical instruments after clinical use and after washing. Quantitative assays showed that bioburden levels were in the range of 0 to 4415 colony forming units per instrument after clinical use, and 88% of the instruments had bioburden levels lower than 1000. As expected, a reduction in counts occurred after washing; however, in some cases, higher counts were found on the instruments after the washing process. Although the washing procedure is effective in reducing the microbial levels deposited on the surgical instruments during use, a recon-tamination process occurs that results in increased counts after washing. The low bioburden level after washing consists of predominantly vegetative microorganisms that present a relatively low challenge to sterilization and disinfection systems. (AJIC Am J Infect Control 1999;27:315-9)

The Measurement of Chlorine Activity in Biofilm Contaminated Dental Unit Water Lines

Potentially pathogenic microorganisms may participate in the formation of microbial biofilms on the interior walls of the plastic tubing used to supply coolant and irrigating water in dental units. A program was designed to test the effect of diluted commercial bleach (5.25% sodium hypochlorite) on bacteria colonizing dental unit water lines. A baseline bacteriological assay was performed on 24 dental units located at four separate institutional dental clinics located in the same metropolitan area. Twelve units at three clinics (designated Clinics I, II, and III) had been treated once weekly with 1:10 NaOCl for periods ranging from 6 mo to 6 yr. Twelve units at Clinic IV, which had never been treated with hypochlorite, were selected for a 28-day study of the effectiveness of two dilutions (1:10, 1:100) of 5.25% sodium hypochlorite for control of biofilm growth in dental water lines. Eight units were treated weekly with either 1:10 or 1:100 solutions of commercial bleach. The four control units were treated only with sterile deionized water. The eight units at Dental Clinics I and II had relatively low levels of planktonic bacterial colony forming units/ml (CFU/ml) ranging from 0 to 8.8 × 10 3CFU/ml with a mean of 1.1 × 10 3CFU/ml (5 of 8 units had 0 CFU/ml and two had only 30 CFU/ml). Characteristic profile curves were produced when chlorine concentration (ppm) was plotted versus a cumulative volume of solution flushed through the units. The chlorine concentration showed very little initial change, then rapidly fell to zero as the solutions were flushed from the units with sterile deionized water. The total chlorine mass recovered at these clinics varied from 94 to 101.7%, with the average being approximately 96%. When chlorine concentration was plotted against the total volume of effluent recovered (including diluent), a characteristic chlorine profile curves resulted which was dubbed a Type I Curve. Planktonic bacteria counts at Clinics III and IV were much higher (Clinic III range: 1 × 10 2CFU/ml to 4.8 × 10 5CFU/ml, median: 1.0 × 10 4CFU/ml; Clinic IV range: 5.6 × 10 4CFU/ml to 1.1 × 10 6CFU/ml, mean: 4.7 × 10 5CFU/ml), suggesting high levels of biofilm contamination. The chlorine recovered from Clinics III and IV varied from 40 to 88% with the average being 80%. The chlorine profile curves for the units at Clinics III and IV initially fell very rapidly, then more slowly as sterile distilled water was used to flush residual hypochlorite from the lines throughout the dilution flush phase of the treatment. These curves were called Type II Curves. The difference in the two profile curves may be due to absorption and reaction of the available chlorine by adherent biofilms. At the conclusion of the 28-day treatment of Clinic IV units, the average percent chlorine recovered for the 1:10 NaOCl group increased to 99.7% and the profile curves yielded the same characteristics as those observed for Clinics I and II, suggesting low levels of microbial contamination. Although each subsequent treatment of units in the 1:100 NaOCl group consumed less chlorine than the previous treatment, planktonic bacteria were still recovered at the end of the 28 day study (range: 30 CFU/ml to 5 × 10 6CFU/ml, median: 130 CFU/ml). A linear regression of all samples ( n= 52) using the percentage of active chlorine recovered versus log CFU/ml yielded an inverse relationship, indicating that there is an indirect relationship between the amount of chlorine consumed when treating a dental unit and the number of planktonic bacteria in the water lines.

Microbiological Hazards from Red Meat and Their Control

Contamination of red meat with foodborne pathogens is associated with symptom‐less carriage of the organisms in the live animal. In the United Kingdom, meat is an acknowledged source of human food poisoning from Salmonella and Clostridium perfringens, and other pathogens that are sometimes present may also be important in this respect. Within the EU, much attention has been given to improving the design and structure of abattoirs, although, in themselves, such changes do not ensure low levels of microbial contamination. However, it has been suggested that, with due care in slaughtering and meat handling, microbial counts from carcasses can be reduced from around 103‐104 to 102‐103/cm2 and that contamination with any foodborne pathogens can also be reduced. Discusses those stages in meat handling which have the greatest effect on carcass contamination and the importance of using a system of quality assurance that incorporates the hazard analysis critical control point concept (HACCP). As an adjunct to good abattoir hygiene, possible processes for decontamination of finished carcasses, especially with hot water or lactic acid, are considered.

The detection and inhibition of proteolytic enzyme activity in concentrated preparations of inactivated foot-and-mouth disease virus

Proteolytic enzyme activity was detected in a large number of concentrated preparations of inactivated foot-and-mouth disease virus. Several lines of evidence indicated that at least some of this activity could be attributed to BHK cells, although low levels of microbial contamination in many of our preparations could not be discounted and would certainly enhance the cellular proteolytic activity. From an experiment with different concentrations of trypsin, it was concluded that the proteolytic activities of virus concentrates were sufficient to cause significant degradation of VP1. It was also shown that Trasylol and ox serum were effective inhibitors of proteolytic enzymes in concentrated virus preparations stored at 4 degrees C for 8 months.

Microbiologic Quality Assurance for Intravenous Admixtures in a Small Hospital

A simple, inexpensive method for end-product testing of intravenous admixtures for microbial contamination was developed and tested by challenging the system with low levels of microbial contamination. The 16-step procedure for testing i.v. admixtures for microbial contamination used total-sample membrane filtration A 0.2-micrometers Nalgene filter unit was used; the entire contents of randomly selected admixtures were to be filtered and discarded under the procedure. Filters were incubated on sheep-blood agar plates for 48 hours at 35 degrees C. Low concentrations (Klebsiella pneumoniae and Pseudomonas aeruginosa were used to contaminate admixtures deliberately to challenge the system. Seventy-two solutions were contaminated with each microbe; 72 other solutions were inoculated with sterile 0.9% sodium chloride; and 72 uninoculated solutions served as controls. Filtration was performed on a laboratory bench to prevent contamination of the laminar-flow hood. In deliberately contaminated solutions, a mean of 82% of inoculated organisms was isolated by membrane filtration. Five instances of adventitious contamination were noted among the 288 samples; these occurred across all experimental groups. Cost per sample was $4-5. This system can be used by hospital pharmacists to produce documentation of quality assurance that will be acceptable in terms of cost, simplicity, and accuracy.