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]