Microbiological Evaluation of Clean Rooms in Pharmaceutical Industries and Molecular Characterization of Microbial Isolates
Gunaseelan Ramaiyan1*, Viswanathan Thirumoorthy2
1Research and Development Centre, Bharathiar University, Coimbatore 641046, India.
2Department of Microbiology, L.R.G Govt. Arts College for Women, Tirupur-641 604.
*Corresponding Author E-mail: cvramguns@gmail.com
ABSTRACT:
Environmental monitoring in pharmaceutical industry was studied by using settle plate, air sampling, surface monitoring and personnel monitoring. These methods are commonly employed to collect the samples in pharmaceutical industry. The collected samples were incubated and predominant bacteria were isolated based on the colony morphology and identified by molecular characterisation. The PCR sequenced bi-directionally for seven isolates i.e, Micrococcus luteus, Bacillus sp, Bacillus Sp. Strain S3Sr84, Kocuria sp. LWYT2000(2016), Staphylococcus sp. Bca53(2016), Brevibacterium sanguinis strain CF52, Staphylococcus hominis strain HB14. Environmental monitoring statistical trend data will provide the process control and environmental isolate must be identified to understand their disinfectant susceptibility.
KEYWORDS: Environmental monitoring, Molecular Characterization, 16S rDNA, Phylogenetic tree, Disinfectant Validation.
INTRODUCTION:
The design and construction of clean rooms and controlled environments are demonstrated in USFDA GMP (United States food and drug association, Good manufacturing practice) guideline, adequately separating areas of operation is an important for contamination prevention in the pharmaceutical clean rooms and should maintain at least 10-15 Pascal’s of positive pressure differential between adjacent clean room[4,5,6]. USFDA recommends and classified aseptic processing area to meet minimum, Class 10,000 (ISO 7) standards under dynamic conditions. Manufacturers can also classify this area as Class 1,000 (ISO 6) or maintain the entire aseptic filling room at Class 100 (ISO 5). An area classified at a Class 100,000 (ISO 8) air cleanliness level is appropriate for less critical activities (e.g., equipment cleaning)[4].
Aseptic processing facility should be appropriately controlled to achieve different degrees of air quality depending on the nature of the operation[7,8]. As the level of airborne contaminants in the environment may have impact on the level of product quality, hence microbiological assessment of aseptic processes is crucial[7,8,9,10].
The presence of high numbers of microorganisms and pathogens represents a serious health threat to consumers as the products are consumed by humans. The microbiological quality is necessary for their efficacy and patient safety, because microbial contamination of drug cause immediate adverse effects on patient’s health in morbidity and mortality[4]. The disease can be based upon microbial infection or metabolic disorders. Therefore, minimizing the numbers or preventing the introduction of significant numbers of microorganism into pharmaceutical clean rooms is necessary[11].
Hence to have a controlled environment pharmaceutical industry should have an effective cleaning and sanitization programme[12]. Pharmaceutical facilities must be kept clean and microbial count under control in order to protect the quality of the products and ultimately the safety of the patients[10,13].
Microbiological testing alone does not provide completed or absolute assurance of absence of microbial contamination. However, such testing combined with robust environmental monitoring program and the use of validated manufacturing processes provides a high degree of assurance of the microbial safety of drugs[14]. The suitability, efficacy, and limitations of disinfecting agents and procedures should be assessed[4]. A comprehensive cleaning and sanitization program is needed for controlled environments used in the manufacture of Pharmacopeial articles to prevent the microbial contamination[15].
The development of recent technologies to enumerate microbial population have greater resolution and sensitivity to describe the presence of microbial distribution on Earth. All microbes in nature do not grow on plate media. Similar results have been observed in pharmaceutical environments. The available information from environmental monitoring programme support on optimizing process control and controlling microbial contamination[16].
Environmental monitoring programs for sterile and non-sterile pharmaceutical facilities comprise the analysis of personnel, processes, raw materials, and finished products. Critical areas during pharmaceutical manufacturing must always be in control to minimize the distribution, viability, and proliferation of microorganisms. When an environmental monitoring program is in place, environmental monitoring data are evaluated to determine whether or not the series of environmental controls continue to operate as intended. Statistical analysis is used to evaluate an environmental monitoring program. A gradual increase or decrease in microbial counts over time, or a change in Microbial flora or counts on several plates of a particular area on a given day, would constitute a trend. Environmental fluctuations are intrinsic of an environmental monitoring system. This is because clean rooms and controlled environments are not supposed to be sterile, and constant intervention by personnel and materials represents continuous challenge to process control and cGMP. Optimization of pharmaceutical manufacturing relies on the integration of different systems and processes to minimize microbial insult resulting in safe and efficacious products[11,16].
The presence of microorganisms in air can impact the quality of the processes and products manufactured in pharmaceutical environments. Although Quantitation of the air borne microbial flora depends upon the sensitivity and accuracy of the methods used, several methods are recommended for air monitoring[2,3]. The most common viable monitoring methods used in pharmaceutical industry such as Settle plate, active air sampling, surface monitoring and personal monitoring[16].
The purpose of the study was to identify the predominant bacteria from pharmaceutical clean rooms by molecular characterization and to minimize and control the microbial proliferation in the clean room by using appropriate disinfectant[17,18,19]. 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 [6,20].
MATERIALS AND METHODS:
Chemicals and reagents:
Instrumentation:
Air sampler Make: VWR PBI Model: SAS super ISO, Incubators Make: Thermolab: 800L, Autoclave make machine fabric Model: 180, Laminar airflow unit Make: Esco, Microscope Make: Zeiss Mode; Primo star. Sequencing Machine: ABI 3500 Genetic Analyzer.
Pre-incubated Soyabean casein digest agar plates were exposed at predetermined sampling location at working level from the floor on petri plate stand, lifted and slide open the lid of the media plate and kept aside. Exposed individual media plates for 4 hours[21,22]. After the exposure period, closed the media plate with the lid. Collected the exposed media plates in Petri plate’s carrier and brought back to the microbiology department. Incubated the media plates at 20 - 25ºC for 72 hours; recorded the observations after 72 hours for fungal count. Incubated the same Media plates, further at 30 - 35ºC for 48 hours and recorded the observation after 48 hours for bacterial count.
Active air sampling:
Air sampling was performed by using 90mm Pre-incubated Soyabean casein digest agar by using active air sampler SAS PBI. Collected 1000L of air hours[21]. After air sampling, collected the plates in petriplates carrier and brought back to the microbiology department. Incubated the media plates at 20º to 25ºC for 72 hours; recorded the observations after 72 hours for fungal count. Incubated the same Media plates, further at 30º - 35ºC for 48 hours and recorded the observation after 48 hours for bacterial count.
Surface monitoring:
Surface monitoring was performed by using contact plate method. The plates contain Tryptic Soy Agar with Lecithin and Polysorbate 80 added to inactivate residual disinfectants and are used for enumeration of microorganisms on environmental[3,15]. and personnel gowning surfaces.
Identification of microbial isolate from environmental monitoring:
Primary Screening:
The predominant bacterial colonies identified were (named EI-121, EI-122, EI-129, EI-130, EI-133, EI-134 and EI-135) isolated and performed grams staining by using microscope. Grams staining will provide information’s i.e cell arrangement (single cell, cluster and clumps), shape (rod or cocci) and grams-Staining characteristics[23]. Transferred a loopful of the liquid culture to the surface of a clean glass slide and allowed to spread over a small area, set aside the film to air dry. Dried film fixed by passing it briefly through the Bunsen flame two or three times without exposing the dried film directly to the flame. The slide should not be so hot as to be uncomfortable to the touch. Deluged slide with crystal violet solution for up to one minute. Wash off briefly with tap water (not over 5 seconds) and drain. Flooded slide with Gram's Iodine solution, and allowed to act (as a mordant) for about one minute. Wash off with tap water and drain. Removed excess water from slide and blot, so that alcohol used for decolorization is not diluted. Flood slide with 95% alcohol for 10 seconds and wash off with tap water. (Smears that are excessively thick may require longer decolonization. This is the most sensitive and variable step of the procedure, and requires experience to know just how much to decolorize) and drained the slide. Flooded slide with safranin solution and allowed to counterstain for 30 seconds. Wash off with tap water. Drain and blot dry with bibulous paper. The slides of bacteria were examined under the oil immersion lens.
Molecular Characterization:
Genomic DNA was extracted from 7 isolates following DNAzol-based cell lysis protocol and the lysates were purified on DNA-binding columns. Polymerase chain reaction (PCR) amplification performed in ABI-2720 thermal cyclers using 341F and 907R as primers (2). The PCR amplification combination include 0.2mmol/L (each) dNTP, 400nmol/L (each) primer, 5mmol/L MgCl, and 1 U Taq polymerase in a final volume of 50μl. After an initial denaturation step at 94°C for 3 min, 30 cycles of PCR reaction were run as follow: denaturation at 94 °C for 1 min, annealing at 55°C for 45 s, and extension at 72°C for 1 min. In addition, a final extension at 72°C for 10 min was added. The resulting products were analysed by electrophoresis in 1.0% agarose gel and purified with PCR Clean-up Kit Refer Fig. 1 and 2. Sequences were determined in an ABI-3500 XL Genetic Analyser using 341F as a sequencing primer, and their closest matches were found by blasting against the short and nearly exact matches from NCBI (National Center for Biotechnology Information) databases (http://www.ncbi.nlm.nih.gov). Sequences were aligned and the phylogenetic tree was generated using DNAMAN package (Lynnon Biosoft, Canada) with evolutionary distances method (boot-strapping 100-times)[24] Refer Fig.3.
Figure 1: Genomic DNA from Bacterial sample using the Bacterial Genomic DNA Isolation Kit.
Figure -3: Represented phylogenetic tree of Sample – EI-135.
Disinfectant Efficacy Validation:
Prepared the desired volume of Dey-Engley agar medium, Normal saline, prepared inoculum for the test Bacillus subtilis ATCC-6633, Staphylococcus aureus ATCC-6538, Pseudomonas aeruginosa ATCC-9027, Escherichia coli ATCC-8739, Aspergillus brasilliensis ATCC-16404, Candida albicans ATCC-10231, and isolate obtained from environment i.e., Micrococcus luteus strain CL10 16S (EI-121), Bacillus sp. H1-115 (EI-122), Kocuria sp. LWYT2000 (EI-129), Staphylococcus sp. Bca53 (EI-130), Brevibacterium sanguinis (EI-133), Staphylococcus hominis strain HB14 (EI-134), Bacterium Sanya 2013001(EI-135). Culture suspension prepared for the above mentioned cultures & environmental isolates in predetermined the inoculum count to achieve 106-107 cells/ml bacteria and 105-106 fungi cells/ml by dilution method. 10-100 cfu culture suspension is prepared by diluting the stock culture appropriately.
Selection of disinfectants:
Selected based on the chemical composition and characteristics of isolates i.e. broad spectrum antimicrobial activity i.e., Vesphene IISe (2-phenylphenol- 9.09%, p-tertiary amylphenol- 7.66%, Potassium hydroxide-5.00%, Sodium hydroxide -<2.00%) LpH Se (7.7%.), -7.7%, Isopropanol- ~10%, Phorsphoric acid- ~14.0%, Dodecylbenzene sulfonic acid-5.0%), Bacillocid extra (Dimethanol-14.0%, Glutraldehyde-5.0%), Microbac Forte (Benzyl-C12-18-alkyldimethylammonium chlorides 199mg/g, N-3aminopropyl)-N-dodecylpropane-1,3-diamine 50mg/g). Collected the required disinfectant and prepared the disinfectants concentrations in a sterilized container by using sterile water. Above disinfectant solution was filtered by using 0.2micron, 47mm diameter membrane filter. Collected the filtered disinfectant solution in a sterilized container and labeled the container which shall represent the name of disinfectant, concentration and date of preparation.
Verification of Filtration technique:
10mL of selected higher concentration of diluted disinfectant was filtered through 0.45µ membrane filter. Rinsed the membrane with 100mL Normal saline solution three times and added the 10-100cfu culture in the final rinse and placed the membrane in to pre-incubated Dey-Engley agar. Incubated at respective temperature to verify the suitability of the test method.
Disinfectant Efficacy Test:
Transferred
10ml of prepared disinfectant solution into different sterilized test tubes and
marked with different time intervals, Inoculated 0.1ml (106-107
cells/ml bacteria and 105-106 fungi cells/ml) of any
one of the challenge microorganisms in to each test tube, inoculum should not exceed
1% of the test solution volume. Mixed well and allowed it to the pre-determined
contact time such as 0 minutes, 15 minutes, 30 minutes for Vesphene IISe,
LpH Se, Bacillocid extra, Bacillocid extra, Pre-sterile for each organism. At the
end of contact time, serially diluted the solution and filtered the contents of
each tube through sterilized 0.45 micron, 47mm diameter membrane filter. Rinsed
the membrane filter with 100ml of sterilized normal saline three times. Aseptically
transferred the membrane filter on to the surface of pre- incubated Dey-Engley agar
plate repeated the steps for the other disinfectant solution with all microorganisms.
Taken separately, 10ml of disinfectant used in the study for all the disinfectants
and filter through a sterilized 0.45-micron 47mm diameter membrane filter. Rinsed
the membrane filter with 100ml of normal saline three times, inoculated with 10-100
cells of one of the microorganisms. Aseptically transferred the membrane filter
on to the surface of pre-incubated Dey-Engley agar plate for positive control.
Filtered 10ml of disinfectant solution through a sterilized 0.45micron, 47mm
diameter membrane filter. Rinsed the membrane filter with 100ml quantity of Normal
saline. Aseptically transferred the membrane filter on to the surface of pre-incubated
Dey-Engley agar plate for negative control. Incubated the bacterial challenged
plates at 30-35°C for 72 hours and fungal challenged plates at 20-20ºC for 5 days.
Observed the plate at the end of incubation period and recorded. Required achieve
the acceptance criteria of Not less than 3 log reduction for vegetative bacteria
and 2 log reductions for spores and Fungi[3].
Surface Test:
Took sterilized 2 x 2 inches Epoxy template and added 0.1mL of test culture suspension containing 106 to107 bacterial cells and for fungi 105 to106 cells using micropipette and spread using a sterile spreader. Allowed to dry all the culture inoculated Epoxy templates inside the laminar air flow cabinet. Flooded 1.0mL selected disinfectant listed in table - 5 of test concentration and allowed the surface as it is with disinfectant until contact time 10 minutes. After the specified contact time, discarded the flooded disinfectant by tilting the template and swabbed the total area with the help of sterile swab Collected swab sample from the entire template surface adequately and immediately inoculated the swab into 10mL of normal saline vortexed the test solution vigorously and serially diluted using 10mL normal saline to get 10-100 CFU/mL for bacteria and fungi after dilution, content filtered through sterile 0.45micron, 47mm diameter membrane filter. Rinsed the membrane filter with 100ml of sterilized normal saline three times. Aseptically transferred the membrane filter on the surface of pre-incubated Dey-Engley agar plate and incubated. Performed the test for all the organisms listed in Table -5 and repeated the procedure for all other disinfectant solution.
RESULTS:
The samples were collected from different location of pharmaceutical industry by using settle plate, air sampling, surface monitoring and personnel monitoring. Total numbers of samples by settle plate was 53663 while 44004 samples (82%) were showed positive and 9659 (18%) samples showed no growth, by air sampling 39312 while 34987 samples (89%) were showed positive and 4325 (11%) samples showed no growth, by surface monitoring 25584 were tested among that 23793 samples (93%) were showed positive and 1791 (7%) samples showed no growth and personnel monitoring 14976 samples among which 898 samples (6%) showed positive growth while 14077 samples (94%) showed no growth Refer Table No 1,2,3, and 4.
Settle plate (Passive Air Sampling):
Samples were taken from microbiology laboratory and production area in facility using settle plate technique. Results were shown in table-1.
Table 1: Environmental Monitoring by Settle Plate in pharmaceutical industry
|
No. samples |
Positive |
Negative |
||
|
No. |
% |
No. |
% |
|
|
53663 |
44004 |
82 |
9659 |
18% |
Active air sampling:
Samples were taken from microbiology laboratory and production area in facility using air sampling apparatus (SAS PBI Air sampler) for quantitative determination of microorganisms. Results were shown in Table-2.
Table 2: Environmental Monitoring by Air sampling in Pharmaceutical Industry
|
No. samples |
Positive |
Negative |
||
|
No. |
% |
No. |
% |
|
|
39312 |
34987 |
89 |
4325 |
11% |
Surface Monitoring:
Samples were taken using contact plates from production area and microbiology laboratory in facility results were shown in Table-3.
Table 3: Environmental Monitoring by Surface Monitoring in Pharmaceutical Industry
|
No. samples |
Positive |
Negative |
||
|
No. |
% |
No. |
% |
|
|
25584 |
23793 |
93 |
1791 |
7% |
Personnel Monitoring:
Samples were taken using contact plates from production area and microbiology laboratory in facility Results were shown in Table-4
Table 4: Environmental Monitoring by Personnel Monitoring in Pharmaceutical Industry
|
No. samples |
Positive |
Negative |
||
|
No. |
% |
No. |
% |
|
|
14976 |
898 |
6 |
14077 |
94% |
Molecular characterization:
Seven bacterial species were isolated from different locations in the pharmaceutical facility. Primary screened microbial isolates were further molecular characterization performed for the microbial isolates. Genomic DNA was isolated from the sample and further ~1.3 kb/1.5kb, 16s-rDNA fragment was amplified using high–fidelity PCR polymerase. The PCR product was sequenced bi-directionally. Represented Gel photo of Sample EI- 133 and EI-134. Refer Figure – 1 and 2.
The results of molecular characterization of bacterial isolate are as below:
Sample: EI-121:
Sample: EI-122:
The Microbe was found to be most similar to Bacillus sp. H1-115 16S ribosomal RNA gene, partial sequence. The next closest homologue was found to be Geobacillus stearothermophilus strain NB3-8 16S ribosomal RNA gene, partial sequence.
Sample: EI-129:
The Microbe was found to be most similar to Kocuria sp. LWYT2000 16S ribosomal
RNA gene, partial sequence. The next closest homologue was found to be Kocuria palustris partial 16S rRNA gene, strain Marseille-P699.
Sample: EI-130:
The Microbe was found to be most similar to Staphylococcus sp. Bca53 16S ribosomal RNA gene, partial sequence. The next closest homologue was found to be Staphylococcus cohnii strain PC-05 16S ribosomal RNA gene, partial sequence.
Sample: EI-133:
The Microbe was found to be most similar to Brevibacterium sanguinis partial 16S rRNA gene, strain CF 52. The next closest homologue was found to be Brevibacterium sanguinis strain T124 16S ribosomal RNA gene, partial sequence.
Sample: EI-134:
The Microbe was found to be most similar to Staphylococcus hominis strain HB14 16S ribosomal RNA gene, partial sequence. The next closest homologue was found to be Staphylococcus hominis strain 77 (BC26) 16S ribosomal RNA gene, partial sequence.
Sample: EI-135:
The Microbe was found to be most similar Bacterium Sanya 2013001 16S ribosomal RNA gene, partial sequence. The next closest homologue was found to be Ralstonia mannitolilytica strain OS8.6 16S ribosomal RNA gene, partial sequence.
Disinfectant validation:
|
Disinfectant |
Microorganism |
Efficacy test |
Surface test |
||
|
0 min |
15 min |
30 min |
Epoxy |
||
|
Vesphene IISe 0.8% |
Escherichia coli ATCC-8739 |
6.69 |
6.69 |
6.69 |
6.94 |
|
Pseudomonas aeruginosa ATCC-9027 |
6.67 |
6.67 |
6.67 |
6.67 |
|
|
Staphylococcus aureus ATCC-6538 |
6.69 |
6.86 |
6.86 |
6.82 |
|
|
Bacillus subtilis ATCC-6633 |
2.69 |
4.63 |
5.15 |
4.59 |
|
|
Candida albicans ATCC-10231 |
6.06 |
6.06 |
6.06 |
6.00 |
|
|
Aspergillus brasiliensis ATCC-16404 |
3.18 |
4.43 |
5.43 |
5.43 |
|
|
Micrococcus luteus strain CL10 16S (EI-121) |
5.60 |
7.55 |
7.55 |
5.04 |
|
|
Bacillus sp. H1-115 (EI-122) |
4.04 |
5.48 |
7.60 |
5.43 |
|
|
Kocuria sp. LWYT2000 (EI-129) |
5.83 |
6.83 |
6.83 |
6.46 |
|
|
Staphylococcus sp. Bca53 (EI-130) |
3.76 |
5.06 |
5.67 |
6.67 |
|
|
Brevibacterium sanguinis (EI133) |
4.76 |
7.60 |
7.60 |
6.51 |
|
|
Staphylococcus hominis strain HB14 (EI-134) |
2.84 |
7.61 |
7.61 |
6.46 |
|
|
Bacterium Sanya 2013001(EI-135) |
4.64 |
6.02 |
7.54 |
5.37 |
|
|
LpH Se 0.4% |
Escherichia coli ATCC-8739 |
6.66 |
6.66 |
6.66 |
6.92 |
|
Pseudomonas aeruginosa ATCC-9027 |
6.74 |
6.74 |
6.74 |
6.69 |
|
|
Staphylococcus aureus ATCC-6538 |
7.01 |
7.01 |
7.01 |
6.86 |
|
|
Bacillus subtilis ATCC-6633 |
2.69 |
7.02 |
7.02 |
6.85 |
|
|
Candida albicans ATCC-10231 |
6.08 |
6.08 |
6.08 |
5.99 |
|
|
Aspergillus brasiliensis ATCC-16404 |
2.10 |
3.47 |
4.47 |
5.38 |
|
|
Micrococcus luteus strain CL10 16S (EI-121) |
4.64 |
7.54 |
7.54 |
6.82 |
|
|
Bacillus sp. H1-115 (EI-122) |
4.69 |
6.72 |
7.59 |
6.94 |
|
|
Kocuria sp. LWYT2000 (EI-129) |
6.87 |
6.87 |
6.87 |
6.43 |
|
|
Staphylococcus sp. Bca53 (EI-130) |
4.87 |
7.56 |
7.56 |
6.56 |
|
|
Brevibacterium sanguinis (EI133) |
7.00 |
7.00 |
7.00 |
6.67 |
|
|
Staphylococcus hominis strain HB14 (EI-134) |
4.73 |
7.57 |
7.57 |
6.79 |
|
|
Bacterium Sanya 2013001(EI-135) |
4.43 |
7.73 |
7.73 |
6.51 |
|
|
Bacillocid extra 1% |
Escherichia coli ATCC-8739 |
6.69 |
6.69 |
6.69 |
6.94 |
|
Pseudomonas aeruginosa ATCC-9027 |
6.67 |
6.67 |
6.67 |
6.67 |
|
|
Staphylococcus aureus ATCC-6538 |
6.86 |
6.86 |
6.86 |
6.82 |
|
|
Bacillus subtilis ATCC-6633 |
4.74 |
5.09 |
6.99 |
5.19 |
|
|
Candida albicans ATCC-10231 |
6.06 |
6.06 |
6.06 |
6.00 |
|
|
Aspergillus brasiliensis ATCC-16404 |
3.26 |
4.43 |
5.43 |
5.43 |
|
|
Micrococcus luteus strain CL10 16S (EI-121) |
6.97 |
6.97 |
6.97 |
6.51 |
|
|
Bacillus sp. H1-115 (EI-122) |
4.25 |
7.84 |
7.84 |
5.38 |
|
|
Kocuria sp. LWYT2000 (EI-129) |
6.83 |
6.83 |
6.83 |
6.46 |
|
|
Staphylococcus sp. Bca53 (EI-130) |
4.32 |
7.55 |
7.55 |
6.60 |
|
|
Brevibacterium sanguinis (EI133) |
3.39 |
7.71 |
7.71 |
6.07 |
|
|
Staphylococcus hominis strain HB14 (EI-134) |
2.20 |
7.43 |
7.43 |
5.83 |
|
|
Bacterium Sanya 2013001(EI-135) |
4.64 |
7.54 |
7.54 |
6.77 |
|
|
Microbac Forte 2% |
Escherichia coli ATCC-8739 |
6.66 |
6.66 |
6.66 |
6.94 |
|
Pseudomonas aeruginosa ATCC-9027 |
6.74 |
6.74 |
6.74 |
6.67 |
|
|
Staphylococcus aureus ATCC-6538 |
5.71 |
7.01 |
7.01 |
6.82 |
|
|
Bacillus subtilis ATCC-6633 |
3.44 |
4.34 |
7.02 |
4.28 |
|
|
Candida albicans ATCC-10231 |
6.08 |
6.08 |
6.08 |
6.00 |
|
|
Aspergillus brasiliensis ATCC-16404 |
2.15 |
5.47 |
5.47 |
5.43 |
|
|
Micrococcus luteus strain CL10 16S (EI-121) |
7.00 |
7.00 |
7.00 |
6.51 |
|
|
Bacillus sp. H1-115 (EI-122) |
3.56 |
5.22 |
6.82 |
5.44 |
|
|
Kocuria sp. LWYT2000 (EI-129) |
7.02 |
7.02 |
7.02 |
6.46 |
|
|
Staphylococcus sp. Bca53 (EI-130) |
6.87 |
6.87 |
6.87 |
6.82 |
|
|
Brevibacterium sanguinis (EI133) |
5.88 |
6.84 |
6.84 |
5.68 |
|
|
Staphylococcus hominis strain HB14 (EI-134) |
6.84 |
7.28 |
7.28 |
6.04 |
|
|
Bacterium Sanya 2013001(EI-135) |
7.42 |
7.42 |
7.42 |
5.66 |
|
DISCUSSION:
Predominant bacterial isolates from the environmental monitoring were identified by molecular characterization. The identified isolates were submitted in NCBI are Micrococcus luteus (EI-121) Accession No. KX082816, Bacillus sp (EI-122) Accession No KX082817, Bacillus sp. strain S3Sr84 (EI-135) Accession No KY678781, Kocuria sp. LWYT2000 (2016)( EI-129) Accession No. KX845571, Staphylococcus sp. Bca53 (2016) (EI-130) Accession No KX845572, Brevibacterium sanguinis strain CF 52 (EI-133) Accession No KX953856, Staphylococcus hominis strain HB14 (EI-134) Accession No KX953857. Statistical trend data will provide the process control and further available isolate must be identified to understand the existing flora in the clean room and must be challenged with disinfectant periodically to confirm their susceptibility.
The authors would like to express the gratitude to the management of Bharathiar University and Sequent Research limited for all the support.
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
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Received on 09.01.2020 Modified on 03.04.2020
Accepted on 05.06.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2021; 14(1):337-343.
DOI: 10.5958/0974-360X.2021.00062.7