A program to identify microbial organisms is integral to the pharmaceutical microbiology laboratory. Whether it is used to identify contamination in clean rooms, on personnel, or confirming the identification of testing organisms, it is important to be able to successfully and correctly identify [1].
Two basic categories of identification methods are routinely used for microbial identification, phenotypic methods and genotypic methods. Sutton describes phenotypic methods as those which typically incorporate reactions to different chemicals or different biochemical markers [1]. Wenning, et al., (2010) describes these methods as those which analyze the metabolic characteristics like the assimilation of different carbon or nitrogen sources, or the expression of specific enzymes [2]. Genotypic methods utilize the cells genetic material to determine the identity, e.g., sequencing some or all of the genetic material, or polymerase chain reaction. Genotypic methods have been considered the “gold standard” for identification due to their high level of accuracy. However, there have been concerns regarding the cost of the test and the need for highly skilled analysts [2]. Both phenotypic methods and genotypic methods have been widely used.
Today, several methods for identification have been developed using equipment commonly used by our friends in the chemistry laboratory, like Fourier-Transform (FTIR) spectroscopy, Matrix-Assisted Laser Desorption Ionization- Time of Flight (MALDI-TOF) mass spectroscopy, and Raman Spectroscopy. While many clinical studies have been performed using these technologies, few pharmaceutical quality control microbiology laboratories have implemented these methods. All of these methods generate spectral analysis that can be compared to a database of spectra to determine a match. The referenced spectrum is associated with a specific identification of a microorganism. Collectively, these methods are being considered proteotypic methods, as much of the data generated is based upon the bonding taking place in the cell. For FT-IR many of the features in the spectra that allow differentiation of species and strains are known to originate from proteins. For MALDITOF, it has little to do with bonding but rather a direct measure of the proteins. In the mass range used, the proteins are the most abundant and most easily ionized components of the cell, so that is what is seen and analyzed.
Fourier-Transform (FTIR) Spectroscopy
FTIR is a method that can be used to obtain an infrared spectrum of the absorption, emission, photoconductivity, or Raman scattering of a solid, liquid, or gas. The infrared (IR) range of the electromagnetic spectrum is between that of microwaves and visible light. The spectrometer collects the data across a wide spectral range. It is a type of vibrational spectroscopy. When molecules are exposed to the infrared radiation, the bonds vibrate as a result of the energy uptake. The amount of energy required to “lift” the bonds to a higher level differs, the light is absorbed in specific wave numbers. The absorption can be correlated to the concentration of the specific components in the sample and thus the spectrum reflects the overall composition of the sample (microorganism). As such, it provides a biochemical fingerprint of the entire microbial cell [2].
Chemists have used this technology for many years in the identification of chemical samples.
FTIR was suggested as a way to identify microbial isolates by Nauman and his associates in the early 1900’s. The intact microbial cells are analyzed using the FTIR spectra and provide a type of biochemical fingerprint that can be compared to a reference database of known microbial isolates. Some of the benefits of this technology include: the small sample size utilized, little sample preparation is required, few consumables are required resulting in low cost for testing per sample, and high levels of sample throughput can be obtained [2].
Numerous studies have been conducted to demonstrate that this technology is appropriate for use in characterizing microorganisms, e.g., with coagulase-positive and negative Staphylococcus microbes, enterococci, Listeria, Brucella, and Candida species [2].
The actual procedure for identification is quite simple. The cells from a pure culture are inoculated directly onto a solid media and incubated (24 hours). The cells are harvested and suspended in water. They are then applied to the sample carrier in an array. After the sample has dried on the carrier, the FTIR analysis can be conducted. It takes about 1 minute to obtain the spectrum [2].
In most cases, samples identified in the database, have been analyzed using a genotypic method to ensure the accuracy of the identification. Some organisms which are hard to identify using phenotypic and genotypic methods, like distinguishing between species groups of B. cereus, can be differentiated using FTIR. [2] provides a description of the methods used to develop a database of microbial identifications as well as discussing the strengths and weakness with using existing databases.
Extensive work has been performed using this technology to identify pharmaceutical environmental isolates [2]. Some companies have implemented this technology for microbial identification at their sites.
Matrix-Assisted Laser Desorption Ionization- Time of Flight (MALDITOF) Mass Spectroscopy
Maldi-TOF is an acronym for a series of words that describes how it works. Matrix-assisted refers to the use of an organic solution of a matrix compound, which is placed on the sample and in the case of bacterial analysis, to lyse the cell walls and extract the proteins and other intracellular material. When this matrix solution dries it create a crystalline matrix. The dried sample is placed in the source region of a Time Of Flight mass spectrometer and irradiated with focused laser light. The matrix absorbs the laser light causing the energetic ejection of a small volume of the matrix into the gas phase above the sample. The proteins are not damaged during this process. Desorption and ionization refers to the process where the proteins are released and become charged in a gaseous state. Time of flight mass separation is where the charged proteins are accelerated by high electric fields and they drift up the vacuum tube towards the detector. The spectral profiles are a result of the proteins which are separated based upon molecular weight. It utilizes the average of many laser pulses data [3].
One of the methods that can be used called the “direct smear” is very simple. It allows for use of a single colony to generate the identification. A portion of the colony is placed upon the stainless steel plate which is approximately ½ the size of a 96-well microtiter plate, but it still has 96 wells in an array of hydrophobic circles delineating individual reaction wells [3].
The analyst smears the sample in the circle and adds the matrix solution, which is allowed to dry. After drying, the plate is loaded into the Maldi- TOF equipment. After several minutes, a high vacuum is established. The plate is positioned under the pulsed laser lens. The laser is pulsed repeatedly in different positions within the sample circle. The spectrum is generated, predominantly based upon the ribosomal proteins [3]. Like the other systems described, the spectrum is compared to the database of spectra with known microbial identifications to determine whether a match can be made. There are commercial software systems available to aid in the process [3].
Some of the benefits of Maldi-TOF when performing identifications are: a very small sample size is needed for identification, minimal analyst preparation is required, the cost for processing the sample is minimal, the identification takes very little time, and a large number of samples can be processed.
Raman Spectroscopy
Sir C.V. Raman is credited with the discovery of the inelastic scattering of light by matter. This phenomenon is called Raman scattering. It represents the small changes in the frequency of scattered light that are a result of the interaction of the incident light with the molecular bonds of the illuminated material. This technology has become more popular as technology has improved the narrow frequency lasers and improvements in optical detection [4].
Raman spectroscopy is similar to FTIR in many ways. It is another vibrational spectroscopy method. However, they differ in the light-matter interaction. Raman spectroscopy changes the polarizability of the electrons shared between atoms. FTIR necessitates a change in the dipole moment for the bond to be active. As such, both methods provide complementary information. Most microbial cells are predominantly water and for some Raman is a preferred method to use since the technology is blind to the water content when using Raman scattering [4].
Ronninger and Bartko (2009) indicate that there are several advantages to using Raman for microbial identifications over the methods currently used for environmental isolates [4]. Some of these advantages include: no growth stage is required; identification can be performed on single cells; the test is non-destructive allowing for other testing on the sample; the time frame for identification is a few minutes for environmental isolates; minimal sample preparation is required; and the sample analysis may be automated.
Discussion
While these technologies are not well known in the realm of pharmaceutical microbiology, they provide opportunities for rapid and accurate identifications. The data published and presented for pharmaceutical applications indicates a high level of accuracy for these methods. One such presentation discussing Maldi-TOF indicated that the level of accuracy in a study conducted at a contract testing laboratory was comparable to genotypic methods. Before being able to claim this for all applications, however more data is needed. One of the biggest limitations to date for these types of systems is the size of the database incorporating the known identifications. For FTIR systems in commercial use at pharmaceutical companies, the early adopters were very involved in the development of the database of known microorganisms. Most systems have the capability to enter spectra for unknown organisms into the database, without knowing the correct identification. The organism could be subsequently subcultured and identified using genetic sequencing allowing for the database to be updated with the correct identification. The very low cost and the short time period required for identification makes these units attractive for the identification of microbial isolates from your environmental monitoring program. Most of the identifications can be performed for a few pennies to a cost of about a dollar. The time to obtain results is also low. This short turn-around time allows one to identify the contamination and hopefully respond to the contaminant quickly reducing the risk of the product becoming contaminated.
References
1. Sutton, S. (2006) How Do You Decide Which Microbial Identification System is Best? PMF Newsletter. Downloaded from http://www.microbiol.org/resources/monographswhitepapers/how-do-you-decide-which-microbial-identification-system-is-best? on April 4, 2011.
2. Wenning, M. Rieser, G., Scherer, S., von Brehmer, S., and Schuffenhauer, G. (2010) Rapid, simple, and cost efficient environmental monitoring of microorganisms by Fourier-Transform Infrared Spectroscopy. In. Environmental Monitoring: A Comprehensive Handbook Volume 4. Ed. by Moldenhauer, J. PDA/DHI Publishers. Bethesda, MD. 203- 221.
3. Shelep, D. (2011) Matrix-Assisted Laser Desorption Ionization – Time of Flight Mass Spectrometry for Identification of Microbial Isolates in Environmental Monitoring. Currently in Publication for Environmental Monitoring: A Comprehensive Handbook Volume 5. Ed. By Moldenhauer, J. PDA/DHI Publishers. Bethesda, MD.
4. Ronningen, T.J. and Bartko, A.P. (2009) Microbial Detection, Identification, and Enumeration Based Upon Raman Spectroscopy. In Environmental Monitoring: A Comprehensive Handbook Volume 3. Ed. by Moldenhauer, J. PDA/DHI Publishers. Bethesda, MD. 183 – 197.
Author Biography
Jeanne Moldenhauer is the VP of Excellent Pharma Consulting. She has numerous years in the pharmaceutical industry and is a strong proponent of emerging technologies for microbiology. She has authored many books and publications in the field of pharmaceutical microbiology.