Microbiological hazards can occur when foodstuffs come into contact with contaminated surfaces or infectious agents dispersed by air currents in the manufacturing environment. An environmental moni-toring program (EMP) is a critical aspect of sustainable and safe food manufacturing used to evaluate the effectiveness of the microbial controls in place. An effective EMP should be based on risk analy-sis, taking into account previous sampling history to determine the selection of the sampling points, the scope of the test, and the frequency of analysis. This study involved evaluation of the environmen-tal monitoring regime and microbiological status of a medium-sized dairy plant manufacturing food ingredients, e.g., proteins, milk powders, and dairy fats. The data specific to microbial tests (n¼3,468), recorded across 124 fixed sampling locations over a 2-year period (2014 to 2015) from air (n¼1,787) and surfaces (n¼1,681) were analyzed. The aim of this study was to highlight the strengths and weak-nesses of the EMP in a select dairy processing plant. The results of this study outline the selection of sampling locations, the scope of the test, and the frequency of analysis. An analysis of variance re-vealed subsections of the manufacturing areas with high risk factors, especially the packaging subsec-tion specified for bulk packaging, the atomizer, and the fluidized bed. The temporal and spatial analy-sis showed the potential to reduce or relocate the monitoring effort, most notably related to total coli-forms and Staphylococcus aureus, across the dairy plant due to homogeneity across the sampling sub-sections with little or no deviations. The results suggest a need to reevaluate the current EMP and the corrective action plan, especially with regard to detection of pathogens. Recommendations for optimi-zation of the EMP are presented to assist the dairy industry with reviewing and revising the control measures and hazard assessment with regard to existing contamination issues.
In situ Study for Incubation Conditions for Contact Plates from Microbiological Environmental Moni-toring The current study investigates different incubation conditions for agar plates from the microbio-logical environmental monitoring on the growth of aerobic mesophilic microorganisms and molds. The study was performed using naturally contaminated surfaces. Four different incubation conditions were tested. Additionally, the effect of a hold time step of the used agar plates for 3 days at room tem-perature before incubation was investigated. Neither the incubation condition nor the hold time at room temperature had a significant effect on the total aerobic microbial count. However, the hold time significantly increased the numbers of molds found, at least in 3 out of 4 incubations conditions. This effect probably relied on the increased incubation time due to the hold time step. In conclusion, the current study shows the minimal effect of the different investigated incubation conditions on the mi-crobial counts of agar plates from the environmental monitoring.
Environmental monitoring and aseptic process simulations represent an integral part of the microbio-logical quality control system of sterile pharmaceutical products manufacturing operations. However, guidance documents and manufacturers practices differ regarding recommendations for incubation time and incubation temperature, and, consequently, the environmental monitoring and aseptic process simulation incubation strategy should be supported by validation data. To avoid any bias coming from in vitro studies or from single-site manufacturing in situ studies, we performed a collaborative study at four manufacturing sites with four samples at each location. The environmental monitoring study was performed with tryptic soy agar settle plates and contact plates, and the aseptic process simulation study was performed with tryptic soy broth and thioglycolate broth. The highest recovery rate was obtained with settle plates (97.7%) followed by contact plates (65.4%) and was less than 20% for liq-uid media (tryptic soy broth 19% and thioglycolate broth 17%). Gram-positive cocci and non-spore-forming Gram-positive rods were largely predominant with more than 95% of growth and recovered best at 32.5 °C. The highest recovery of molds was obtained at 22.5 °C alone or as the first incubation temperature. Strict anaerobes were not recovered. At the end of the five days of incubation no signifi-cant statistical difference was obtained between the four conditions. Based on these data a single incu-bation temperature at 32.5 °C could be recommended for these four manufacturing sites for both envi-ronmental monitoring and aseptic process simulation, and a second plate could be used, periodically incubated at 22.5 °C. Similar studies should be considered for all manufacturing facilities in order to determine the optimal incubation temperature regime for both viable environmental monitoring and aseptic process simulation.
This study compared the recovery of naturally occurring micro-organisms on tryptone soya agar (TSA) and Sabouraud dextrose agar (SDA), when incubated under three different temperature condi-tions. The micro-organisms were sourced directly from a factory environment. The incubation condi-tions employed were 20–25°C, 30–35°C and a combination of each, termed dual temperature incuba-tion, for a period of 5 days. The results demonstrated that TSA was the most effective medium for the primary isolation of Environmental Monitoring both bacterial and fungal/yeast micro-organisms. Bac-teria were recovered best at 30–35°C with human commensals providing the largest numbers, while fungi and yeasts showed best recovery at 20–25°C. The use of dual temperature incubation at 20–25°C for 3 days followed by 2 days at 30–35°C gave reduced recovery for both types of micro-organisms. The authors recommend that similar studies should be considered for all manufacturing facilities in order to determine the optimal incubation temperature regime for the recovery of local, naturally oc-curring species of bacteria and fungi which may present a threat to aseptic manufacturing areas. This process should be undertaken as part of an overall risk assessment for the establishment and mainte-nance of a viable environmental monitoring programme and may also be relevant to the incubation conditions employed in process simulation studies.
Statistical tools are required to organize and present microbial environmental monitoring data for the purpose of evaluating it against regulatory action limits and of determining if the microbial moni-toring process is in a state of control. This paper applies a known methodology of a simple and straightforward construction of control XmR (X data and moving range) charts of individual microbial counts as they are or of contamination rates derived from them, irrespective of the type of the par-ent data distribution and without the need to transform the data into a normal distribution. Plotting of monthly and cumulative sample contamination rates, as newly suggested by USP <1116>, is also shown. Both types of the control charts and plots allow an evaluation of the behavior of the microbi-al monitoring process. After addressing the magnitude of microbial counts expected in environmen-tal monitoring samples, this paper presents the rationale behind the use of XmR charts. Employing data taken from environmental monitoring programs of pharmaceuticals manufacturing facilities, this paper analyzes examples of (1) microbial counts from passive or active air sampling in area Grade D or B or Class 100,000 in XmR charts, (2) contamination recovery rates as suggested by USP <1116> from active air samples in area Grade B and contact plates in area Grade C, and (3) instantaneous contamination rates with calculations illustrated on microbial counts of contact plates in area Grade D.
Environmental monitoring represents an integral part of the microbiological quality control system of a pharmaceutical manufacturing operation. However, guidance documents differ regarding recom-mendation of a procedure, particularly regarding incubation time, incubation temperature, or nutri-ent media. Because of these discrepancies, many manufacturers decide for a particular environmen-tal monitoring sample incubation strategy and support this decision with validation data. Such valida-tions are typically laboratory-based in vitro studies, meaning that these are based on comparing in-cubation conditions and nutrient media through use of cultured microorganisms. An informal survey of the results of these in vitro studies performed at Novartis or European manufacturing sites of dif-ferent pharmaceutical companies highlighted that no consensus regarding the optimal incubation conditions for microbial recovery existed. To address this question differently, we collected a signifi-cant amount of samples directly from air, inanimate surfaces, and personnel in pharmaceutical pro-duction and packaging rooms during manufacturing operation (in situ study). Samples were incubat-ed under different conditions suggested in regulatory guidelines, and recovery of total aerobic mi-croorganisms as well as moulds was assessed. We found the highest recovery of total aerobic count from areas with personnel flow using a general microbiological growth medium incubated at 30-35 °C. The highest recovery of moulds was obtained with mycological medium incubated at 20-25 °C. Single-plate strategies (two-temperature incubation or an intermediate incubation temperature of 25-30 °C) also yielded reasonable recovery of total aerobic count and moulds. However, recovery of moulds was found to be highly inefficient at 30-35 °C compared to lower incubation temperatures. This deficiency could not be rectified by subsequent incubation at 20-25 °C. A laboratory-based in vitro study performed in parallel was inconclusive. We consider our results potentially conferrable to other pharmaceutical manufacturing sites in moderate climate zones and believe that these should represent a valuable reference for definition of the incubation strategy of microbiological environ-mental monitoring samples.
This paper introduces an initiative based on microbiological risk profiling and a proactive response to bio-contamination risk escalation in classified and controlled environments. The concept is included in the PHSS Bio-contamination monograph 201. The monograph considers four principle require-ments of a systematic approach to bio-contamination risk management and a microbial control strategy with guidance on best practice (see Figure 1); risk profiling is one key area where a system-atic approach could yield significant benefits.
Bioburden testing is an important part of pharmaceutical microbiology and provides data in relation to the quality of pharmaceutical products during manufacture. Little guidance is provided in relation to test methodology, culture media and incubation parameters. The quality control laboratory, therefore, needs to establish the most appropriate method. This paper outlines a case study for the selection of incubation parameters for the bioburden assessment of in-process samples using the Total Viable Count technique and pour plate method. While the outcome of the experiment contained within the paper relates to a specific set of processes, the approach taken can be used by other laboratories to compare or to develop their test methods and techniques for bioburden determinations.
Background: Since air can play a central role as a reservoir for microorganisms, in controlled envi-ronments such as operating theatres regular microbial monitoring is useful to measure air quality and identify critical situations. The aim of this study is to assess microbial contamination levels in operat-ing theatres using both an active and a passive sampling method and then to assess if there is a correla-tion between the results of the two different sampling methods.Methods: The study was performed in 32 turbulent air flow operating theatres of a University Hospital in Southern Italy. Active sampling was carried out using the Surface Air System and passive sampling with settle plates, in accordance with ISO 14698. The Total Viable Count (TVC) was evaluated at rest (in the morning before the beginning of surgical activity) and in operational (during surgery).Results: The mean TVC at rest was 12.4 CFU/m3 and 722.5 CFU/m2/h for active and passive sam-plings respectively. The mean in operational TVC was 93.8 CFU/m3 (SD = 52.69; range = 22-256) and 10496.5 CFU/m2/h (SD = 7460.5; range = 1415.5-25479.7) for active and passive samplings re-spectively. Statistical analysis confirmed that the two methods correlate in a comparable way with the quality of air.Conclusion: It is possible to conclude that both methods can be used for general monitoring of air contamination, such as routine surveillance programs. However, the choice must be made between one or the other to obtain specific information.Keywords: Bioaerosol, Air sampling, Operating theatres, Surveillance
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 environ-ment (Gram-positive rods) and water (Gram-negative rods) are also detected, although in lower numbers.
Inanimate surfaces have often been described as the source for outbreaks of noso-comial infections. The aim of this review is to summarize data on the persistence of different nosocomial pathogens on inanimate surfaces.
The literature was systematically reviewed in MedLine without language restrictions. In addition, cited articles in a report were assessed and standard textbooks on the topic were reviewed. All reports with experimental evidence on the duration of per-sistence of a nosocomial pathogen on any type of surface were included.
Most gram-positive bacteria, such as Enterococcus spp. (including VRE), Staphylococ-cus aureus (including MRSA), or Streptococcus pyogenes, survive for months on dry surfaces. Many gram-negative species, such as Acinetobacter spp., Escherichia coli, Klebsiella spp., Pseudomonas aeruginosa, Serratia marcescens, or Shigella spp., can also survive for months. A few others, such as Bordetella pertussis, Haemophilus in-fluenzae, Proteus vulgaris, or Vibrio cholerae, however, persist only for days. Myco-bacteria, including Mycobacterium tuberculosis, and spore-forming bacteria, includ-ing Clostridium difficile, can also survive for months on surfaces. Candida albicans as the most important nosocomial fungal pathogen can survive up to 4 months on sur-faces. Persistence of other yeasts, such as Torulopsis glabrata, was described to be similar (5 months) or shorter (Candida parapsilosis, 14 days). Most viruses from the respiratory tract, such as corona, coxsackie, influenza, SARS or rhino virus, can persist on surfaces for a few days. Viruses from the gastrointestinal tract, such as astrovirus, HAV, polio- or rota virus, persist for approximately 2 months. Blood-borne viruses, such as HBV or HIV, can persist for more than one week. Herpes viruses, such as CMV or HSV type 1 and 2, have been shown to persist from only a few hours up to 7 days.
The most common nosocomial pathogens may well survive or persist on surfaces for months and can thereby be a continuous source of transmission if no regular preven-tive surface disinfection is performed.
This article describes an environmental monitoring data study for clean rooms. Two parameters werecompared in order to investigate the possibility of correlation between them: (1) total airborne nonvia-ble (0.5 and 5_m) and viable (CFU) particulates, and (2) surface contamination (Rodac_). The investi-gation took into account A, B, and C classified areas, in operational moment or status, which involved “at rest” (just equipment working) and “dynamic” (equipment and operators working) conditions. Available data in the literature indicates that the parameters are not particularly dependent upon the layout or classification of areas, but rather on the use of the areas and operator behaviour. Our results showed a correlation around 0.6 between different sampling places for nonviable 0.5- and 5-_m parti-cles. There was no correlation between nonviable 0.5- and 5-_m particles in places with laminar flow.
Microbial stress due to the impaction of microorganisms onto an agar collection surface was studied experimentally. The relative recovery rates of aerosolized Pseudomonas fluorescens and Micrococcus luteus were determined as a function of the impaction velocity by using a moving agar slide impactor operating over a flow rate range from 3.8 to 40 liters/min yielding impaction velocities from 24 to 250 m/s. As a reference, the sixth stage of the Andersen Six-Stage Viable Particle Sizing Sampler was used at its operating flow rate of 28.3 liters/min (24 m/s). At a collection efficiency of close to 100% for the agar slide impactor, an increase in sampling flow rate and, therefore, in impaction velocity produced a significant decline in the percentage of microorganisms recovered. Conversely, when the collection efficiency was less than 100%, greater recovery and lower injury rates occurred. The high-est relative rate of recovery (approximately 51% for P. fluorescens and approximately 62% for M. luteus) was obtained on the complete (Trypticase soy agar) medium at 40 and 24 m/s (6.4 and 3.8 li-ters/min), respectively. M. luteus demonstrated less damage than P. fluorescens, suggesting the hardy nature of the gram-positive strain versus that of the gram-negative microorganism. Comparison of results from the agar slide and Andersen impactors at the same sampling velocity showed that recov-ery and injury due to collection depends not only on the magnitude of the impaction velocity but also on the degree to which the microorganisms may be embedded in the collection medium. Impaction velocity, characterized by the sampler’s operating flow rate and inlet design, is unique for each sam-pling device. The resulting impaction stress influences the recovery and injury of collected microor-ganisms and ultimately affects the measurement data for colony enumeration. This can be one of the most important reasons for variations that occur when using different sampling devices to measure bioaerosols from the same environment.