Microbial Contamination in Water Systems.

The microbiological impurities are the most critical ones in a water system and are thus of special concern. In case of contamination of a purification unit or a distribution system, the user must react quickly. The authors have written down their experience and explain details about the typical microbiome of a water system. In addition, advice is given on how to remediate microbial contamination.

Assessment of airborne endotoxin in sandstorm dust and indoor environments using a novel passive sampling device in Al Zulfi city, Saudi Arabia - Establishing threshold exposure levels.

The impact of sandstorm dust events affects local air quality and public health. These issues are becoming of greater concern in Saudi Arabia. There is a significant lack of research on airborne endotoxin exposure and analysis in the Middle East countries and no coherent body of research exists focusing on sandstorm dust in worldwide. In this study, we used a novel design of an aluminum foil plate (AFP) electrostatic dust cloth (EDC) for the passive air sampling of sandstorm dust. A total of 38 sandstorm dust samples were collected during sandstorm episodes occurring between January and April 2020 in both indoor (7 days, n = 20) and outdoor environments (24 h, n = 18). After exposure, and following an extraction procedure, bacterial endotoxin levels were measured using the Limulus Amoebocyte Lysate (LAL) gel clot method. The study highlights that the airborne endotoxin level observed was between 10 and 200 EU/m2 in both indoor and outdoor environments, during a sandstorm event. Agricultural activities and farmhouses observed higher airborne endotoxin levels. In general, increased endotoxin levels were related to the severity of the sandstorms. Given that the observed values were high as per existing guidelines for respiratory health, we recommend the setting an occupational airborne exposure limit for bacterial endotoxin. This is the first report and further studies across various sandstorm-hit regions will need to be undertaken, together with various sampling methods, in order to assess for seasonal and geographic trends.

Virulence profiles of some Pseudomonas aeruginosa clinical isolates and their association with the suppression of Candida growth in polymicrobial infections.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause a variety of diseases especially in the hospital environment. However, this pathogen also exhibits antimicrobial activity against Gram-positive bacteria and fungi. This study aimed to characterize different virulence factors, secreted metabolites and to study their role in the suppression of Candida growth. Fifteen P. aeruginosa isolates were tested for their anticandidal activity against 3 different Candida spp. by the cross-streak method. The effect on hyphae production was tested microscopically using light and scanning electron microscopy (SEM). Polymerase chain reaction was used in the detection of some virulence genes. Lipopolysaccharide profile was performed using SDS-polyacrylamide gel stained with silver. Fatty acids were analyzed by GC-MS as methyl ester derivatives. It was found that 5 P. aeruginosa isolates inhibited all tested Candida spp. (50-100% inhibition), one isolate inhibited C. glabrata only and 3 isolates showed no activity against the tested Candida spp. The P. aeruginosa isolates inhibiting all Candida spp. were positive for all virulence genes. GC-Ms analysis revealed that isolates with high anticandidal activity showed spectra for several compounds, each known for their antifungal activity in comparison to those with low or no anticandidal activity. Hence, clinical isolates of P. aeruginosa showed Candida species-specific interactions by different means, giving rise to the importance of studying microbial interaction in polymicrobial infections and their contribution to causing disease.

Antimicrobial Activity of Silver-Treated Bacteria against other Multi-Drug Resistant Pathogens in Their Environment.

Silver is a potent antimicrobial agent against a variety of microorganisms and once the element has entered the bacterial cell, it accumulates as silver nanoparticles with large surface area causing cell death. At the same time, the bacterial cell becomes a reservoir for silver. This study aims to test the microcidal effect of silver-killed E. coli O104: H4 and its supernatant against fresh viable cells of the same bacterium and some other species, including E. coli O157: H7, Multidrug Resistant (MDR) Pseudomonas aeruginosa and Methicillin Resistant Staphylococcus aureus (MRSA). Silver-killed bacteria were examined by Transmission Electron Microscopy (TEM). Agar well diffusion assay was used to test the antimicrobial efficacy and durability of both pellet suspension and supernatant of silver-killed E. coli O104:H4 against other bacteria. Both silver-killed bacteria and supernatant showed prolonged antimicrobial activity against the tested strains that extended to 40 days. The presence of adsorbed silver nanoparticles on the bacterial cell and inside the cells was verified by TEM. Silver-killed bacteria serve as an efficient sustained release reservoir for exporting the lethal silver cations. This promotes its use as a powerful disinfectant for polluted water and as an effective antibacterial which can be included in wound and burn dressings to overcome the problem of wound contamination.

A novel mechanism of action of ketoconazole: inhibition of the NorA efflux pump system and biofilm formation in multidrug-resistant Staphylococcus aureus.

Background: The rapid emergence of antimicrobial resistance among Gram-positive organisms, especially staphylococci, has become a serious clinical challenge. Efflux machinery and biofilm formation are considered two of the main causes of antimicrobial resistance and therapy failure. Aim: Our study aims to evaluate the antibiofilm and efflux pump inhibitory activity of the antifungal ketoconazole against multidrug-resistant (MDR) Staphylococcus aureus. Methods: Ketoconazole was tested for its effect on the following: minimum inhibitory concentrations (MICs) of ciprofloxacin, norfloxacin, levofloxacin, and ethidium bromide (EtBr) by the broth microdilution method, the efflux of EtBr by NorA-positive MDR S. aureus, and the relative expression of NorA, NorB, and NorC efflux pump genes. Docking studies of ketoconazole were performed using 1PW4 (glycerol-3-phosphate transporter from Escherichia coli which was the representative structure from the major facilitator superfamily). Results: Ketoconazole significantly decreased the MICs of levofloxacin, ciprofloxacin, norfloxacin, and EtBr (a substrate for efflux pump) by 8 to 1024-fold (P<0.01) and decreased the efflux of EtBr. Furthermore, a time-kill assay revealed that combinations of levofloxacin with ketoconazole or carbonyl cyanide m-chlorophenylhydrazone showed no growth for the tested strains after 24 h in comparison to the effect of levofloxacin alone. Docking studies and the ability of ketoconazole to diminish the relative expression of NorA gene in comparison to control (untreated strains) confirmed its action as an efflux pump inhibitor. Conclusion: The findings showed that the antifungal ketoconazole has no antibacterial activity but can potentiate the activity of the fluroquinolones against MDR S. aureus via inhibiting efflux pump and biofilm formation in vitro.

Distribution of biocide resistant genes and biocides susceptibility in multidrug-resistant Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii - A first report from the Kingdom of Saudi Arabia.

PURPOSES: The aim of this study was to determine the frequency of biocide resistant genes, qacA, qacE and cepA in multidrug resistant (MDR) bacteria: Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii and to correlate the presence or absence of resistant genes with biocides susceptibility. MATERIALS AND METHODS: The study included 44 MDR K. pneumoniae, P. aeruginosa and A. baumannii microorganisms. The bacteria were screened for the presence of biocide resistant genes by the polymerase chain reaction (PCR) method. The test organisms were isolated from various clinical specimens in the Qassim region, Saudi Arabia. The in vitro susceptibility tests of the three biocides (benzalkonium chloride, cetrimide and chlorhexidine gluconate) were studied against the test isolates by broth microdilution method following Clinical and Laboratory Standards Institute guidelines. RESULTS: With the distribution of biocide resistant genes in K. pneumoniae, all 9 isolates (100%) possessed cepA; 4 (44.4%) and 1 (11.1%) isolate contained qacA and qacE genes respectively. Among 24 isolates of A. baumannii tested, cepA, qacA and qacE genes were found in 54.2%, 16.7% and 33.3% of isolates respectively. Among 11 P. aeruginosa isolates, 63.6% contained cepA gene, 18.2% contained qacE genes, and none of the isolates harboured qacA gene. There was no significant correlation between presence or absence of biocide resistant genes and high MIC values of the test isolates (p≥0.2). CONCLUSION: Our observations imply that there was no significant correlation between presence or absence of biocide resistant genes and MICs observed in MDR K. pneumoniae, P. aeruginosa and A. baumannii. Further studies are required to find to confirm the trend of reduced susceptibility to biocides of problematic nosocomial pathogens.

Microbiological Test Data-Assuring Data Integrity.

Marketed drugs and devices possess specifications including critical microbiological quality attributes purposed to assure efficacy and patient safety. These attributes are legislated requirements intended to protect the recipient patient. Sampling, microbiological testing, interpretation of data for final products, raw materials, and intermediates all contribute to a cohesive assessment in the assurance of finished product quality. Traditional culture-based microbiological methods possess inherent and unavoidable variability, recognized by the compendia and which might lead to erroneous conclusion pertaining to product quality. Such variability has been associated and intrinsically linked with data integrity issues; manufacturers have subsequently been encouraged by regulatory authorities to introduce multiple microbiologists or checks to prevent such issues. Understanding microbiological variability is essential such that genuine data integrity issues are identified. Furthermore, a range of meaningful preventative strategies are feasible beyond increasing the capacity of the quality control microbiological laboratory. This short review describes the legislative requirements, inherent microbiological variability, and realistic actions and activities that genuinely assure patient safety.LAY ABSTRACT: Marketed drugs and devices possess specifications including critical microbiological quality attributes purposed to assure efficacy and patient safety. These attributes are legislated requirements intended to protect the recipient patient. Sampling, microbiological testing, interpretation of data for final products, raw materials, and intermediates all contribute to a cohesive assessment in the assurance of finished product quality. Traditional culture-based microbiological methods possess inherent and unavoidable variability, recognized by the compendia and which might lead to erroneous conclusion pertaining to product quality. Such variability has been associated and intrinsically linked with data integrity issues; manufacturers have subsequently been encouraged by regulatory authorities to introduce multiple microbiologists or checks to prevent such issues. Understanding microbiological variability is essential such that genuine data integrity issues are identified. Furthermore, a range of meaningful preventative strategies are feasible beyond increasing the capacity of the quality control microbiological laboratory. This short review describes the legislative requirements, inherent microbiological variability, and realistic actions and activities that genuinely assure patient safety.

Examination of the order of incubation for the recovery of bacteria and fungi from pharmaceutical-grade cleanrooms.

A study was undertaken to compare microbial recoveries from pharmaceutical-grade cleanrooms using two different incubation regimes and a general-purpose agar (Tryptone Soy Agar). One temperature regime (A) incubated plates first at 30 degrees C to 35 degrees C, followed by 20 degrees C to 25 degrees C; the second temperature regime (B) began the incubation with plates at 20 degrees C to 25 degrees C, followed by 30 degrees C to 35 degrees C. The experimental outcomes demonstrated that there was no significant difference with the total microbial count when measured using a t-test (0.05 significance level; 95% confidence interval). However, with the recovery of fungi, the second incubation regime (B), which began with the lower 20 degrees C to 25 degrees C temperature, produced higher incidents and numbers of fungi. While this finding might provide the basis for adopting one incubation regime over another, a review of the types of cleanrooms recovering fungi suggests that fungal incidents are low, and they are more often confined to specific areas. Thus, as an alternative, incubation regimes could be varied to suit different cleanroom environments or a selective mycological agar adopted for specific areas.

Comparison of different fungal agar for the environmental monitoring of pharmaceutical-grade cleanrooms.

In relation to a growth in reported incidents of fungal contamination of pharmaceutical products, there has been a developing interest by U.S. and U.K. regulators concerning the risk of fungi. This paper describes a study undertaken to examine the suitability of different commercially available mycological agars for the environmental monitoring of pharmaceutical-grade cleanrooms. Five agars were evaluated in relation to the detection of both numbers and different species of fungi (yeasts and moulds). The objective was to determine if one mycological medium is more suitable than another. Data was collected using different sampling techniques (settle plates, active air samples, and contact plates) from different locations within representative cleanrooms. Samples were taken over a 3 month time period. The study results indicated that fungi are not distributed evenly across cleanrooms and that that the prevalence of fungi partly relates to the room design and operation. In relation to the different agar types, the study indicated that Sabouraud dextrose agar was the most effective at detecting the widest number of different types of isolates, and that Sabouraud dextrose agar and malt extract agar were the most efficient in terms of the numbers of recovered isolates. Other media, notably potato dextrose agar, was relatively less effective. LAY ABSTRACT: There has been an increased regulatory concern about the presence of fungi in cleanrooms. Some environmental monitoring regimes are not especially orientated towards the examination of fungi, and it may be that special agars are required. Given the choice of different agars available, this paper outlines a case study where different fungal agars were evaluated. The study showed that Sabouraud dextrose agar was the optimal agar.

Application of quality risk management to set viable environmental monitoring frequencies in biotechnology processing and support areas.

Environmental monitoring programs are essential for pharmaceutical facilities in order to assess the level of environmental control. For biotechnology facilities there is little advice as to the frequency at which viable environmental monitoring should be conducted. This paper outlines an approach, based on the principles of quality risk management, for the development of a framework from which monitoring frequencies can be determined. This involved the identification of common hazards and the evaluation those hazards in terms of the severity of contamination and the probability of contamination occurring. These elements of risk were evaluated for different cleanrooms and the relative risks ranked. Once the risk scores were calculated, the methods for detecting risks within the cleanrooms were assessed. Risk filtering was then used to group different cleanrooms based on their relative risks and detection methods against predetermined monitoring frequencies. Through use of case study examples, the paper presents the model and describes how appropriate frequencies for the environmental monitoring of cleanrooms can be set. LAY ABSTRACT: Cleanrooms in which biotechnology pharmaceutical processing takes place are subject to environmental monitoring. The frequency at which such monitoring should be performed can be difficult to determine. This paper uses quality risk assessment methods to construct a framework for determining monitoring frequencies and illustrates the suitability of the framework through a case study.

In vitro Antifungal Efficacy of Biguanides and Quaternary Ammonium Compounds against Cleanroom Fungal Isolates.

In vitro antifungal activities of three biocides including one biguanide (chlorhexidine) and two quaternary ammonium compounds (benzalkonium chloride and cetrimide) were studied against eight cleanroom fungal isolates by using a microbiological broth dilution technique as per Clinical and Laboratory Standards Institute (CLSI) M38-A guidelines. No data exists on the activity of biocides on pharmaceutical cleanroom fungal isolates. Minimum inhibitory concentrations (MICs) for all three biocides against species of Aspergillus and Penicillium species ranged between 4 and 16 µg/mL. MICs of Curvularia, Cladosporium, and Alternaria species also showed less than 16 µg/mL. To date, susceptibility breakpoints have not been established for biocides, and this is the first study using the CLSI broth microdilution antifungal susceptibility testing to determine the MIC value of biocides. This study clearly demonstrates that the most frequently isolated micro-organisms from an environmental monitoring program may be periodically subjected to microbroth dilution testing with cleanroom disinfectant agents used in the disinfection program to confirm their sensitivity profile. Further work is needed in this field to increase our understanding of biocides against different fungal isolates and to enable to the design of more efficient disinfection and contamination control programs. LAY ABSTRACT: Increased trend of fungal growth is observed in pharmaceutical and medical device cleanrooms. It is essential to have knowledge of choosing effective disinfectants for minimizing fungal occurrence in cleanrooms. The present study establish minimum cut-offs for specific fungal isolates that are problematic in cleanrooms against commonly used disinfectants (quaternary ammonium compounds and biguanides). Further studies based on this minimum inhibitory concentration of disinfectants, effective time, and cleaning methods could prevent fungal occurrence.

A review of cleanroom microflora: types, trends, and patterns.

Cleanroom microflora are of importance for microbiologists and quality control personnel in order to assess changes in trends. Shifts in the types of microflora may indicate deviations from the "norm" such as resistant strains or problems with cleaning practices. Given the few published studies of the typical microflora, this paper uniquely reviews over 9000 microbial isolates from a range of different grades of cleanroom. The paper concludes that the typical flora are primarily those associated with human skin (Gram-positive cocci), although microorganisms from other sources such as the environment (Gram-positive rods) and water (Gram-negative rods) are also detected, although in lower numbers. LAY ABSTRACT: It is of importance that pharmaceutical manufacturers and healthcare pharmacies review the types and numbers of microorganisms found within their clean areas. Such examination should be carried out over a long period of time so that the complete picture can be revealed. This is important in order to understand if certain species are being recovered pose a product or environmental risk and to check if the cleaning and sanitization practices are effective.

An approach for the reporting of microbiological results from water systems.

Many statistical textbooks lend themselves readily to the analysis of physical and chemical data. This is due to the material gathered from such analyses, following, in most cases, normal distribution. Normally distributed data can be examined over time to allow the obtained profile to be studied for variation. Microbiological data rarely follows such distribution. However, such approaches can be adapted for non-normally distributed (or skewed) microbiological data. This paper applies Shewhart Control Charts to the trend analysis of microbiological data from two different pharmaceutical water systems.