Abstract:
We report on the spectroscopic investigation of common bacteria encountered in biopharmaceutical industries with spectroscopic definition and specificity using mid-infrared laser spectroscopy. This study describes the detection of three different bacteria species using quantum cascade laser spectroscopy coupled to a grazing angle probe (QCL-GAP). Stainless steel material, like surfaces commonly used in biopharmaceutical industries, was used as support media substrates for the bacterial samples. QCL-GAP spectroscopy was assisted by multivariate analysis (MVA) to assemble a powerful spectroscopic technique with classification, identification, and quantification resources. The bacterial species analyzed,Staphylococcus aureus , Staphylococcus epidermidis , andMicrococcus luteus, were used to challenge the technique’s capability to discriminate microorganisms from the same family. Principal component analysis and partial least squares discriminant analysis differentiated between the bacterial species, using (QCL-GAP) as the reference. Spectral differences in the bacterial membrane were used to determine if these microorganisms were present in the samples analyzed. Results herein provided effective discrimination for the bacteria under study with high sensitivity and specificity values.
Keywords: quantum cascade laser spectroscopy (QCLS), infrared spectroscopy (IRS), bacteria, stainless steel substrates (SS), principal component analysis (PCA)
1 Introduction
Microbial contamination happens because of the existence and proliferation of microorganisms in the environment [1]. The ability of microorganisms to grow in food, pharmaceutical and cosmetic products, and medical devices has been the subject of several studies [2]. From the infectious point of view, pathogenic microbes in pharmaceutical and food products make them hazardous and objectionable [2]. Microbial infections can alter active ingredients’ physicochemical and biological properties or even become toxic materials [3]. Some diseases, such as diarrhea, acute gastroenteritis, and abdominal pain, result from microbial toxins. Nevertheless, depending on the individual sensitivity to the toxin, symptoms are different and range from mild distress to the individual’s death [4].
Biological contamination in the drug manufacturing industry is a medical problem that can lead to drug degradation and sub-potency. Patients can be exposed to pathogens or opportunistic microorganisms that can cause serious metabolic harm or lead to death, especially if the patient is immunocompromised. It also represents a hazardous threat in the pharmaceutical and biotechnology clean-room production environment. Regulatory agencies require continuous, routine, or regular environmental monitoring of microorganisms to ensure that the environment during production activities is precisely controlled. Maintaining a controlled environment is vital to avoid contamination and protect the patient from contact with possible extraneous matter or product contamination. Environmental monitoring requirements may include but are not limited to the manufacturing room, surfaces, walls, ceiling roof HEPA filters, equipment, and personnel. Identifying and characterizing bacterial isolates starts by inspecting the colony morphology once the cells have been cultured in solid media, followed by microscopic analysis of Gram-stained preparations [5].
Today, pharmaceutical and biotechnology industries assess their activities, such as daily environmental monitoring for their facilities, bioburden, endotoxins, and sterility for product microorganism contamination. Industries, specifically the parenteral product industries, monitor the water used to manufacture the products. Biotechnology industries often use Polymerase Chain Reaction (PCR) and Micro sequencers. These techniques take long periods to implement because the operator needs to collect the sample in the area and inoculate it in a culture media to allow the microorganism’s growth. Results obtained from these studies will commonly take about 12 to 24 hours, depending on the culture media used. Once the incubation period is completed, the analyst studies the morphology of the recovered microorganism under the microscope and performs the identification using the abovementioned techniques. The industry may select to ship the recovered microorganism to an external laboratory to complete the identification process.
Therefore, in these fields, fast identification of pathogenic and non-pathogenic microorganisms is necessary because the time required for identifying pathogens is essential in determining the contamination source promptly and reducing manufacturing costs. Rapid identification techniques play a critical role in reducing costs associated with contamination to mitigate the event faster and avoid further product processing that may increase product disposition expenses.
Bacteria can be divided and identified into groups based on their morphology (macro and micro), physiological, biochemical, serological, and genetic characteristics. Generally, the tests are combined in a series of solid or liquid media inoculated with bacteria and identified after a certain incubation period. Many different tests are often needed for the definitive identification of the bacteria. These tests require turnaround times from 24 hours to up to 5 days between receipt of material and identification results by the clinician [6].
Thus, empirical treatment with broad-spectrum antibiotics is often started while awaiting further identification of the bacteria. It has been reported that 10-30% of patients suffering from bloodstream infections in intensive care units (ICUs) do not receive the correct antibacterial therapy initially. Mortality rates in this group have been reported to be 30-60% higher than in the group that promptly receives appropriate therapy [7].
The effort has been invested in developing new techniques for identifying microorganisms, including molecular methods, such as mass spectrometry, electrospray ionization, matrix-assisted laser desorption ionization, Fourier Transform Infrared (FT-IR), and Raman spectroscopy. Among these methods, vibrational spectroscopy Quantum Cascade Laser Grazing Angle Probe (QCL-GAP) is a reagent less/solventless, in which there is no need to add chemical dyes or labels for identification. QCLs differ from traditional semiconductor laser diodes, which use p-n junctions for light emission, consisting of diode arrays with an active region where electrons and holes recombine to produce light emission. Instead, QCLs have multiple active regions composed of a multilayered semiconductor material structure specially designed to have the appropriate electronic bands [8].
QCL sources consist of semiconductor lasers based on sub-band transitions in a manifold of quantum-well structure [9].
QCL sources operate at wavelengths in the MIR starting from 3300 to approximately 750 cm-1, which matches well with the fundamental vibrational absorption bands of many biological species, in contrast with typical diode sources where the laser emission generally matches the weaker overtone. The emission wavelengths of these lasers depend on the thickness of the quantum well and the barrier layers of the active region rather than the band gap of diode lasers. They operate near room temperature, produce milliwatts of radiation, and offer the possibility of tailoring the emission wavelengths within a broad range of frequencies [9,12].
Vibrational spectra of bacterial cells consist of signal contributions of all components in the cells and, therefore, reflect their overall molecular composition. FT-IR spectroscopy has proven that a wide range of microorganisms can be identified using the spectral response [13-15]. Gram-positive and Gram-negative sugar-based coating structures are also relevant properties that can contribute to the spectral differences. Other factors that have been used as discrimination in FTIR are the biochemical fingerprints observed on spectra consistently correlated with sugar-based coating structures that, besides reflecting strain variation, are also highly relevant for the specificity in pathogen-host interactions [16].
Thus, the present research aims to detect, identify, and discriminate between three bacteria from the same group: Staphylococcus aureus , Staphylococcus epidermidis, and Micrococcus luteus deposited over a stainless steel (SS) substrate-like material used in the biopharmaceutical industry clean rooms using QCL-GAP. Therefore, the bacteria concentration was not considered for this study since the investigation is associated with detection and discrimination. Studies regarding concentration are in the plan and will be included in a subsequent publication.
2 Materials and Methods
2.1 Materials and Reagents
Three environmental bacteria isolates (Staphylococcus aureus(Sa ), Staphylococcus epidermidis (Se ), andMicrococcus luteus (Ml ) from the manufacturing areas were inoculated from 30 - 35°C. The environmental isolates used in this study were sent to Microbiologics® Laboratory (St. Cloud, MN, 56303 USA) to certify and identify the bacteria using MALDI-TOF with supplemental tests, for example, ID determination with MALDI-TOF confirmation. The method used for concentration verification is the spread plate method, using the materials and incubation conditions required for the bacteria under characterization. After that process, the sample is lyophilized with a 50 EU/0.1mL concentration. The bacteria were reconstituted with a reconstitution buffer provided by Microbiologics company and inoculated in Tryptic Soy Broth (TSB). The bacteria were incubated at 30 - 35°C for 12 hours. A solution of 10 mL in 90 mL of TSB media was prepared. Each solution containing the microorganism was left to grow for 24 hours. The SS substrate (2-in x 2-in) used for the sample deposition of the bacteria was provided by Stainless Supply AJW Metal Product Company (Moroe, NC, 28110 USA). The SS substrate used in this study is a grade material similar to that used in clean rooms in biopharmaceutical industries.
2.2 Instrumentation
A QCL-GAP pre-dispersive spectrometer based on QCL technology (LaserTune™, Block Engineering, LLC, Southborough, MA, 01772 USA) set up for reflection-absorption measurements was used for data acquisition in the MIR: 5.4 – 12.8 μm of the bacterial suspension samples (BSS). The microorganism’s vibrational response was collected using a 4 cm-1 spectral resolution. The spectrometer was equipped with an internal thermoelectrically mercury-cadmium-telluride (MCT) detector and a 2 x 4 mm2 MIR laser beam. The setup has two adjustable mirrors that allow the laser incidence at the grazing angle (~82°). The light is emitted towards the first gold (Au) mirror, which goes to the sample. A second gold mirror reflects the light from the sample, allowing a double-pass reflection-absorption measurement. The QCL-GAP array is mainly useful for detection at low concentration ranges with added sensitivity [18]