Deoxyribonucleic acid (DNA) contains the genetic information that controls the development of a microbial cell. DNA determines the genotypic and phenotypic potential of a microbial cell. With the latest advances in genomics, where more than 25 microbial genomes have been sequenced, the potential to use genetic information for the detection and discrimination of micro- organisms is endless. Genetic technologies can increase the resolution and specificity of microbial detection and identification in pharmaceutical envi- ronments. DNA-based technologies are used in clinical, food, and environ- mental samples providing valuable information on the survival, distribution, and function of microorganisms in those habitats [36,37]. One of the tech- nologies based on DNA analysis is the polymerase chain reaction.
Polymerase chain reaction (PCR) amplifies specific DNA sequences along the microbial genome. For example, a set of DNA primers is used to target the specific sequence to be amplified (Table 7). The PCR reaction takes place in three different steps. First, the target sequence is denatured by
heating. Second, the primers anneal to complementary sequences on the target DNA strands. Third, the primers are extended by the DNA polymerase enzyme resulting in two different strands. The three steps are repeated again for a given number of cycles, e.g., 30–35. As soon as the target is amplified, the products are detected by gel electrophoresis. However, new systems have been developed that rely on fluorescence detection of amplified products. PCR based assays are used routinely in the food industry and clinical laboratories to detect and identify bacteria, yeast, and mold [36,37].
TABLE 7 PCR Assay Reaction Steps (1) Double helix denatured by heating
5V 3V A T C G C A G G G A T C 95jC 5V 3V T A G C G T C C C T A G A T C G C A G G G A T C 3V 5V
!
T A G C G T C C C T A G 3V 5V(2) Primers are bound to complementary sequences on template strands Template Strand 5V 3V A T C G C A G G G A T C T A G j > > > > j Target Region j j < < < A T C T A G C G T C C C T A G 3V 5V Template Strand
(3) Primers are extended by DNA polymerase resulting in two DNA strands 5V 3V A T C G C A G G G A T C T A G C G T C C C T A G 3V 5V 5V 3V A T C G C A G G G A T C T A G C G T C C C T A G 3V 5V
In pharmaceutical laboratories, PCR-based assays have been shown to be capable of detecting S. typhimurium, E. coli, P. aeruginosa, S. aureus, B. cepacia, A. niger, and eubacterial sequences after an incubation period [38– 43]. Analysts, raw materials, equipment, or water contamination introduces some of these microorganisms into pharmaceutical environments. Further- more, when analysts do not follow good laboratory practices, they become major sources of microbial contamination in clean rooms and aseptic man- ufacturing. Rapid detection of objectionable microorganisms results in faster implementation of corrective actions. Detection times using PCR range from 24 to 27 hr (Table 8). This is a significant reduction when compared to the standard 5–7 days detection time. Furthermore, high-throughput screening of samples is possible by using a 96-well format.
The simplification of PCR analysis for pharmaceutical quality control is achieved by using a tablet and PCR bead formats. The PCR reagents, in- cluding DNA primers, are combined in a tablet form, while the beads provide the necessary reagents for the PCR reaction but without the DNA primers. Time-consuming preparations and handling of individual PCR reagents are not required due to the tablet and bead formats incorporated in the assay. During assay development, different experiments are performed to determine
TABLE8 Pharmaceutical Samples Analyzed by PCR [21,23–27]
Inhibitory reaction Detection Dilution Time (hr)
Neobee Oil No Yes 1/10 24–27
Simethicone No Yes 1/10 24–27
CMC No Yes 1/10 24–27
Sodium alginate No Yes 1/10 24–27
Rasberry flavor No Yes 1/10 24–27
Hydroxymethylcellulose No Yes 1/10 24–27
Xantham gum No Yes 1/10 24–27
Silica calcinated No Yes 1/10 24–27
Guar gum No Yes 1/10 24–27
Starch No Yes 1/10 24–27
Lactose monohydrate No Yes 1/10 24–27
Diatomaceous earth No Yes 1/10 24–27
Tablets No Yes 1/10 24–27
Medicated skin cream No Yes 1/10 24–27
Ointment No Yes 1/10 24–27
Antiflatulent drops No Yes 1/10 24–27
Medical device No Yes 1/10 24–27
the optimal numbers of beads. Optimal DNA amplification is found to be obtained with two or three beads (Table 9).
DNA extraction from sample enrichments is performed in single-step assays. For bacteria and yeast, a sample preparation using Tris–EDTA– Tween 20 buffer with proteinase K at 35jC resulted in high-quality DNA, while boiling the samples in sodium dodecyl sulfate (SDS) for 1 hr is required for efficient mold DNA extraction. None of the product suspensions shows PCR inhibition allowing rapid determination of sample quality (Table 8). The amount of DNA needed for detecting the different target sequences ranged from 10 to 50 Al of lysate (Table 9). Higher concentrations of the lysate are found to be inhibitory for successful PCR amplification.
TABLE 9 Optimization of PCR Reactions for Objectionable Microorganisms in Pharmaceutical Products
Microorganism Beads Aliquot PCR band
S. aureus 3 10 + S. aureus 2 10 S. aureus 1 10 S. aureus 3 25 P. aeruginosa 2 10 + P. aeruginosa 1 10 P. aeruginosa 2 25 P. aeruginosa 1 25 E. coli 2 10 + E. coli 1 10 E. coli 2 25 E. coli 1 25 S. typhimuriuma 1 50 + B. cepacia 2 10 + B. cepacia 1 10 B. cepacia 2 25 B. cepacia 1 25 A. niger 2 50 + A. niger 2 25 A. niger 2 10 A. niger 1 50 C. albicans 2 50 + C. albicans 2 25 C. albicans 2 10 C. albicans 1 50 aCommercial system.
The development of new PCR formats allows for the simplification of PCR protocols where only sample addition and primers are needed to per- form the assay. With the latest advances in microbial genomics, the avail- ability of DNA primer sequences are limitless allowing the development of universal primers for bacteria, yeast, and mold. A recent study has shown the applicability of detecting bacterial contamination for sterility testing by using a simple PCR assay. The study is based upon the universal and inclusivity nature of the DNA sequences coding for bacterial ribosomal genes. DNA primers targeting these common bacterial sequences are capable of rapidly screening samples for bacteria contamination.
All the previously discussed studies have been performed using a single PCR amplification format where a specific microorganism DNA sequence was targeted. However, simultaneous detection of bacteria and mold DNA sequences in pharmaceutical samples using a gradient thermocycler has been recently reported [44]. The gradient thermocycler allows the use of primers with annealing temperatures ranging from 54 to 65jC leading to the detection of different microorganisms in a single PCR run. This allows the immediate screening of a pharmaceutical sample for bacteria, yeast, and mold.
PCR has also been used for the monitoring of pharmaceutical water samples in manufacturing processes [8]. Ribosomal DNA sequences are amplified with universal bacterial primers. After amplification, the samples are loaded onto polyacrylamide gels [denaturing gradient gel electrophoresis (DGEE)] to detect the amplified products. This will allow the separation of DNA fragments of the same length but different pair sequences. After sepa- ration, the gels are scanned to generate a densitometric profile. The se- quencing of the amplified fragments has revealed that the dominant bacteria in the water samples are not culturable on standard media. Most of the cul- turable bacterial species have been found to be related to Bradyrhizobium spp., Xanthomonas spp., and Stenotrophomonas spp., while the dominant unculturable bacterial species have not been characterized. These studies further showed the limit capacity of standard methods to determine and characterize the community structure of pharmaceutical environments. Similar results have been found in other environmental conditions.
Similar results are found in pharmaceutical clean room environments [11]. DNA extracted from selected samples have been analyzed by using 16S rDNA sequencing. Results indicate that bacterial isolates do not grow on plate media but are major components of the microbial populations. 8. GENETIC IDENTIFICATION
When microbial contamination is detected in a given pharmaceutical sample, characterization of the types of microorganisms by genera and species is an
important criterion to determine the source of the contamination. The first step in the phenotypic identification of microorganisms in pharmaceutical laboratories is performed using the gram strain method [7]. This method is based upon the chemical and structural differences between the membranes and cell walls of gram-negative and gram-positive bacteria.
For bacteria, once the results of the gram reaction have been determined and other simple biochemical tests are completed, e.g., catalase and oxidase test, a standardized pure culture suspension of the isolate is inoculated into strips, cards, or microtiter plates [7]. These systems are based upon the de- tection of enzymatic activity by different types of enzymes such as oxidases and carbon utilization profiles. However, new genetic tests provide a greater resolution and discrimination for microbial identification. Table 10 shows a comparison of phenotypic and genotypic identification of bacterial species by biochemical, lipids, and genetic methods. The genetic method demonstrated a higher accuracy and reproducibility than lipid and biochemical analysis. Similar results were obtained with environmental isolates from different pharmaceutical environments (Table 11). DNA sequencing analysis provided
TABLE10 Microbial Characterization of Bacteria Using Different Identification Systems
Species Vitek Biolog Lipids Genetic
Bacillus cereus Unidentified Yes Yes Yes
Burkholderia cepacia Yes Unidentified Yes Yes
Enterobacter cloacae Yes Yes Unidentified Yes
Escherichia coli Yes Yes Yes Yes
Micrococcus luteus Unidentified Yes Unidentified Yes
Pseudomonas aeruginosa Yes Yes Yes Yes
Shigella flexneri Unidentified Yes Unidentified Yes Staphylococcus aureus Unidentified Unidentified Unidentified Yes Staphylococcus epidermidis Yes Unidentified Unidentified Yes Acinetobacter radioresistens Unidentified Yes Yes Yes Macrococcus caseolyticus Unidentified Yes Yes Yes Methylobacterium
radiotolerans
Unidentified Unidentified Unidentified Yes
Ochrobactrum anithropi Yes Yes Yes Yes
Ralstonia pickettii Unidentified Yes Unidentified Yes Streptococcus salivarius Unidentified Unidentified Unidentified Yes Corynebacterium xerosis Unidentified Unidentified Unidentified Yes Kokuria rosea Unidentified Unidentified Unidentified Yes Paenibacillus glucanolyticus Unidentified Unidentified Unidentified Yes
TABLE11 Microbial Identification of Common Microbial Contaminants in Pharmaceutical Environments Using Lipid Analysis and DNA-Based Tests
Species Lipid analysis Genetic fingerprinting Genetic sequencing
Ralstonia spp. R. pickettii R. pickettii Ralstonia spp. Kokuria rosea Unidentified Unidentified K. rosea Bacillus pumilus B. pumilus B. pumilus B. pumilus Bacillus pumilus B. pumilus Unidentified B. pumilus Bacillus pumilus B. pumilus B. pumilus B. pumilus Bacillus pumilus Unidentified B. pumilus B. pumilus Bacillus pumilus B. pumilus B. pumilus B. pumilus Bacillus pumilus B. pumilus B. pumilus B. pumilus Bacillus pumilus B. pumilus B. pumilus B. pumilus Ralstonia pickettii R. pickettii R. pickettii R. pickettii Staphylococcus
hominis
S. hominis S. epidermidis S. hominis
Ralstonia pickettii Unidentified R. pickettii R. pickettii Corynebacterium spp. Unidentified C. amycolatum Corynebacterium
spp. Stenotrophomonas
maltophila
S. maltophila S. maltophila S. maltophila
Enterobacter cancerogenous
E. cancerogenous E. cloacae E. cancerogenous
Aeromonas hydrophila Unidentified Unidentified A. hydrophila Pantoea spp. Cedecea lapagei Unidentified Pantoea spp. Moraxella osloensis M. osloensis Unidentified M. osloensis Staphyloccus warneri S. warneri S. aureus S. warneri
Stenotrophomonas spp. S. maltophila S. maltophila Stenotrophomonas spp.
Staphyloccus aureus Unidentified S. aureus S. aureus
Microbacterium sp. Unidentified Unidentified Microbacterium sp. Bacillus circulans Cellulomonas
turbata
Unidentified B. circulans
Bacillus megaterium B. megaterium B. megaterium B. megaterium Bacillus
amyloliquefaciens
B. subtilis B. subtilis B. amyloliquefaciens
Bacillus sp. Bacillus sp. Unidentified Bacillus sp. Staphylococcus
epidermidis
Unidentified S. epidermidis S. epidermidis
Burkholderia cepacia Unidentified B. cepacia B. cepacia Micrococcus luteus Unidentified M. lylae M. luteus Paenibacillus
glucanolyticus
P. polymyxa P. glucanolyticus P. glucanolyticus
Stenotrophomonas maltophila
S. maltophila S. maltophila S. maltophila
a higher level of accuracy resolution, and identification than lipid analysis or DNA fingerprinting. Several studies demonstrate that unidentified environ- mental isolates not characterized by phenotypic analysis are correctly char- acterized by 16SrRNA and 16SrDNA sequencing [45]. This provides accurate information for the tracking of the contamination source in phar- maceutical environments and microbial community characterization allow- ing faster corrections actions to be implemented.