Abstract
A microbial identification method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF) is an innovative dimension in proteomic analysis. MALDI-ToF mass spectrometry allows not only determine the species and subspecies bacteria, but also determined by proteomic analysis and the corresponding software degree of kinship analyzed strains, which allows this method to be used in epidemiological studies and in comparing strains isolated from patients with chronic infection. The aim of this study was to evaluate the possibility of using protein spectra of microorganisms obtained by MALDI-ToF mass spectrometry as additional microbiological criteria in assessing the course of the infection process caused by Burkholderia cepacia complex among patients with cystic fibrosis. The analysis of protein profiles, which were obtained by using MALDI-ToF mass spectrometry (Bruker Daltonik GmbH, Germany), was performed by using flexAnalisis 3.0 software (Bruker Daltonik GmbH, Germany). Differences in protein profiles of Burkholderia spp. isolates were found depended on the stage of infection of a cystic fibrosis patient with prolonged colonisation of the lower respiratory tract. The protein profiles of Burkholderia spp. isolates that formed a heterogeneous population containing both NCV and SCV morphotypes were also studied. A regular dynamic monitoring and comparison of protein profiles of microbial strains can be useful in forecasting effort of the clinical course of the disease, as well as in assessing of risks of severe infectious complications development.
Keywords: MALDI-ToF mass spectrometry, Lower respiratory tract infection, Cystic fibrosis, Burkholderia cepacia strains
Introduction
An innovative dimension in proteomic analysis is a microbial identification method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF). This method made possible to extend quite a bit the boundaries and to reduce the limitations in the possibilities of species-level identification of microorganisms, including mycobacteria [1, 2]. This fact provides an essential advantage to mass spectrometry technology compared with other microbial identification methods. Among functions of MALDI-ToF mass spectrometry, aside from identification of bacterial species, it should be noted that it is possible to carry out more detailed exploratory development of microorganisms. Additional software is capable of biomarkers definition in which subtyping can be carried out [3–6].
By identification of some or other microorganisms, a mass spectrum, which is specific to a particular isolated strain, is formed. Moreover, during epidemiological analysis or linking between isolated microorganisms, mass spectra, available from identification, can be subjected to a bioinformatic analysis [7–9].
One way to solve problems of the comparison of mass spectra’ wealth is using the MALDI Biotyper 3.0 Offline Classification software (Bruker Daltonik GmbH, Germany). Pseudogel imaging is possible for the analyzed strains. The picture formed is similar to the results of one-dimensional electrophoresis of proteins in GelView polyacrylamide gel, which simplifies the work with mass spectra analysis. Another way of visualization and the following analysis is the creation of mass spectra dendrograms (MSP-dendrograms)—a hierarchical structure of mass spectra, where the position of the specific strain and the correlation between all interrogated strains are formed according to characteristics of the mass spectra elution bands of each strain [8, 9].
Thus, MALDI-ToF mass spectrometry technologies allow not only to carry out species and subspecies identification of bacteria, but also to determine the degree of relationship of the analyzed strains from proteomic analysis and applicable software, this lets, in some ways, to use mass spectrometry in epidemiological studies and in studies of strains, which are isolated from patients who have chronic infection with any microorganism.
Cystic fibrosis (CF) is an autosomal recessive monogenic disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). The CFTR protein is an important regulator of fluid and electrolyte homeostasis in many mucosal tissues. Persistent respiratory tract colonisation and infection with bacteria can enhance the inflammatory response by causing neutrophils to release large amounts of DNA and matrix proteins into the respiratory tract. In combination with CF-induced impaired respiratory tract’s clearance and chronic inflammation, it increases respiratory tract’s mucus viscosity. In addition, CFTR dysfunction in the bronchial epithelium also leads to an increased inflammatory response and impaired immune response. This contributes to acute and recurrent infections in the patient, associated with the accumulation of inflammatory cells and the release of their contents. This leads to damage of the bronchial walls, loss of cartilaginous support and muscle tone and, consequently, chronic bacterial colonisation of the lungs [2, 9].
Approximately one thousand new cases of CF are diagnosed each year worldwide, with more than 75% of CF patients diagnosed before the age of 2 years, and the average age of diagnosis is about 3 years.
Increased viscosity of mucus on the surface of the epithelium provokes the development of bacterial infection, with Staphylococcus aureus and Pseudomonas aeruginosa most commonly involved. However, disease progression is associated with the emergence of unusual pathogens, such as Stenotrophomonas maltophilia, Achromobacter xylosoxidans, nontuberculous mycobacteria, and Burkholderia cepacia complex. Bacteria of the Burkholderia cepacia complex species are the group of pathogens considered to be the main cause of death in CF patients [9].
Burkholderia cepacia complex can cause the so-called “cepacia” syndrome, which is a necrotising pneumonia with septicaemia, often resulting in death. Infection of CF patients with Burkholderia cepacia complex bacteria is particularly dangerous, as this microorganism is naturally resistant to a wide range of antimicrobial agents and rapidly acquires resistance to new antibiotics. Such properties make it difficult to carry out eradication in the process of treatment of the patient, contributing to the long-term persistence of the pathogen and leading to a rapid transition of acute lower respiratory tract infection into a chronic form, thereby causing significant impairment of lung function [10].
In this article are reported clinical case studies that illustrate the possibility of exploitation of microbial protein spectra, which are obtained by MALDI-ToF mass spectrometry as additional microbiological criteria in the assessment of the course of the infection process, that is caused by Burkholderia cepacia complex in patients with cystic fibrosis.
Materials and Methods
Biomaterial Collection and Its Transportation
CF patients from the Samara region colonised with Burkholderia cepacia strain ST208 were included in the study from 2014 to 2020. 40 patients were examined, and a total of 500 strains of the genus Burkholderia were isolated and sampled in different years.
The process of collecting and transporting samples from CF patients was standardised, and all samples were examined under identical conditions. A uniform protocol for isolation of proteins that depended on the stage of disease exacerbation was used. None of the examined patients had chronic diseases in decompensation stage. The material was collected on an out-patient basis, before routine hospitalisation, except for the patient with cepacia syndrome, in whom the last isolate was taken during hospitalisation, taking into account the severity of the condition.
Collection and transportation of respiratory samples from patients with cystic fibrosis, as well as from their family members, were implemented as the biomaterial was collected in sterile tubes, without transport medium, and then was transported to the laboratory under isothermal conditions within 2 h after collection. Material, that was placed in tubes with Ames transport medium was transported under isothermal conditions within 24–72 h [11].
Microbial Isolation Methods
Primary seedings of nasal lavage fluid, which was isolated from patients with cystic fibrosis, swabs from environmental medium, as well as respiratory samples of homogenized material, which was isolated from the lower respiratory tract of patients with cystic fibrosis as well as their family members, were carried out according to the following scheme: 200 µl of the collected material was sown using a sterile swab on 5 culture media, including 5% blood agar, chocolate agar, universal chromogenic medium, selective medium for Burkholderia cepacia (OFPBL agar), and Sabouraud agar. For swabs from environmental medium and for respiratory samples of homogenized material inoculation technique based on the «streak» was used, while for nasal lavage fluid inoculation technique based on the «lawn» was used. After that Petri dishes seeded with 5% blood agar as well as with universal chromogenic medium were incubated at a temperature of 37 °C within 24–48 h with cultures daily checked. Petri dishes with chocolate agar were incubated at a temperature of 35 °C under 5–7% CO2 within 24–48 h with cultures daily checked. For swabs from environmental medium and for respiratory samples of homogenized material Petri dishes seeded with selective medium for Burkholderia cepacia (OFPBL agar) were incubated within 24–48 h at a temperature of 37 °C, then they were incubated up to 14 days at a temperature of 28 °C with daily checking of the cultures. While for nasal lavage fluid Petri dishes with selective medium for Burkholderia cepacia (OFPBL agar) were incubated within 24–48 h at a temperature of 37 °C and then were incubated for up to 14 days in ambient conditions with cultures daily checked, their subsequent identification and recalculation of colony-forming units (CFU) per 1 ml of lavage fluid [12].
Microbial Identification
In this work the MALDI-ToF mass spectrometry method was used, as a matrix the α-cyano-4-hydroxycinnamic acid (HCAA) (Bruker Daltonik GmbH, Germany) was used. For identification the standard spectra library of Bruker Daltonik GmbH company was used. The result of identification was estimated automatically by using MALDI Biotyper RTC software, by the level of coefficient of coincidence (Score) from 0 to 3, at the same time the certainty of identification at the level of 0.000–1.699 was considered as low, identification to genus, at the level of 1.700–1.999, as well as identification to species at the level of 2.000–2.999, were considered as high-quality, according to recommendations of the equipment manufacturer.
Proteomic Analysis of Protein Spectra
The analysis of protein profiles, which were obtained by using MALDI-ToF mass spectrometry, was performed by using flexAnalisis 3.0 software (Bruker Daltonik GmbH, Germany). While analyzing mass spectra, the elution bands detection algorithm (Centroid) was used.
Results
Assessing of Dynamic Changes in the Protein Profiles of Strains of the Genus Burkholderia Bacteria, Which were Isolated from Patients with Cystic Fibrosis with Long-Term Colonization of the Lower Respiratory Tract at Different Times of Infection
As an example of potential applications of protein mass spectra in prognosis of the deterioration of the patient's condition, the analysis of protein profiles of Burkholderia cenocepacia strains, which were isolated from Patient A, birth year—2003 (13 years old at the baseline), was treated. The patient was observing at the Centre for treatment of cystic fibrosis since 2004. Cystic fibrosis in mixed form and severe disease course were diagnosed. F508del/CFTRdele2,3 mutations. Chronic infection of the respiratory tract with Pseudomonas aeruginosa, B. cenocepacia (first inoculation in 2012), 4 years of colonization of the lower respiratory tract with B. cenocepacia strain. The patient died affected by progression of “cepacia” syndrome. Protein spectra of three B. cenocepacia isolates, which were obtained from the patient at different stages of infection—isolate No. 13 (13.02.2015), isolate No. 36 (29.09.2015) and isolate No. 83 (11.04.2016) were examined. It is important to note, that isolate No. 83 (11.04.2016) was obtained in a matter of weeks before the patient's death. We paid attention to the fact that during the specified time some differences in protein profiles of isolates were identified. These differences are tabulated in Table 1.
Table 1.
Differences in protein profiles of examined isolates of patient A
| Protein (mass, Da) | Isolate No. 13 | Isolate No. 36 | Isolate No. 83 |
|---|---|---|---|
| 2083 | + | − | − |
| 2090 | + | − | − |
| 3767 | + | − | − |
| 3780 | + | − | − |
| 4651 | + | − | − |
| 5023 | + | − | − |
| 5345 | − | + | + |
| 5444 | + | − | − |
| 6318 | + | + | − |
| 6340 | − | − | + |
| 7575 | + | − | − |
Proteins with masses of 2083, 2090, 3767, 3780, 4651, 5023, 5445, 7575 Da presented only in the first isolate (No. 13), but were lost in the later isolates (Fig. 1).
Fig. 1.
Protein spectra of patient’s A. isolates No. 13, No. 36 and No. 83. X-direction the mass-to-charge ratio is presented, Y-direction the elution bands intensity is presented. Proteins with masses of 2083, 2090, 3767, 3780, 4651, 5023, 5445, 7575 Da were present only in the first isolate (No. 13), but were lost in the later isolates
In a time of obtaining of isolate No. 13, the patient's condition was stable, there were no clinical or radiographic signs of deterioration in the condition.
Since 2015, the patient's condition began to deteriorate, there multiple exacerbations of the underlying condition with signs of pneumonia were noted. He was hospitalized four times in a given period. Throughout the year, the body temperature rises from subfebrile to febrile values was noted. When assessing protein profiles of the strain, that was obtained during this period (isolate No. 36), changes in protein spectra were identified. They were characterized by losing of considerable amount of proteins. At the same time, the protein with a mass of 6318 Da was remained in the first and in the second isolates. The protein with a mass of 5345 Da appeared, that was also identified in the last isolate. From the beginning of 2016, the patient's condition continued to get worse and clinical and radiographic signs of “cepacia” syndrome appeared from the beginning of April. Despite massive antibiotic therapy, the patient's death occurred in May 2016. While analyzing protein profiles of the strain, that was isolated at the end-stage of the disease (isolate No. 83), alongside earlier lost proteins, it can be observed the absence of the protein with a mass of 6318 Da, the maintenance of the protein with a mass of 5345 Da and the appearance of a protein with a mass of 6340 Da, that was absent in earlier isolates (Fig. 2).
Fig. 2.
Protein spectra of patient’s A. isolates No. 13, No. 36 and No. 83. X-direction the mass-to-charge ratio is presented, Y-direction the elution bands intensity is presented. While analyzing protein profiles of the strain, that was isolated at the end-stage of the disease (isolate No. 83), alongside earlier lost proteins, it can be observed the absence of the protein with a mass of 6318 Da, the maintenance of the protein with a mass of 5345 Da and the appearance of a protein with a mass of 6340 Da, that was absent in earlier isolates
Possibility of Application MALDI-ToF Mass Spectrometry to Estimate Protein Profiles of Heterogeneous Isolates of Burkholderia spp.
We examined protein profiles of isolates of patient P., 26 years old, that was observed at the Centre for treatment of cystic fibrosis with the diagnosis: cystic fibrosis, in mixed form, severe disease course. F508del/F508del mutations. Chronic infection of the respiratory tract with P. aeruginosa, B. cenocepacia (first inoculation in 2013).
A heterogeneous B. cenocepacia population from the sputum sample obtained was received, and both normal colony variant (NCV) and small colony variant (SCV) presented in the sample (Fig. 3). At the same time, the growth of the SCV of Burkholderia was 24–48 h days later than the growth of the NCV. Meanwhile, SCV were more resistant to antimicrobial.
Fig. 3.
Normal and small colony variants of Burkholderia spp. × 4 magnification, direct microscopy from Petri dish, own observations. A heterogeneous B. cenocepacia population from the obtained sputum sample was received, and both normal colony variant (NCV) and small colony variant (SCV) were presented in the sample
As opposed to the forequoted example, in examining differences in the protein spectra of NCV and SCV strains, we did not find out differences in a large number of proteins. Nonetheless, it was noted that proteins with masses of 3768 Da and 6146 Da presented in the normal colony morphotype, while in the small colony morphotype they were absent (Figs. 4, 5).
Fig. 4.
Protein spectra of normal and small colonies morphotypes of patient P. X-direction the mass-to-charge ratio is presented, Y-direction the elution bands intensity is presented. As opposed to the forequoted example, in examining differences in the protein spectra of NCV and SCV strains, we did not find out differences in a large number of proteins. Nonetheless, it was noted that proteins with masses of 3768 Da and 6146 Da were present in the normal colony morphotype, while in the small colony morphotype they were absent
Fig. 5.
Protein spectra of normal and small colonies morphotypes of patient P. X-direction the mass-to-charge ratio is presented, Y-direction the elution bands intensity is presented. As opposed to the forequoted example, in examining differences in the protein spectra of NCV and SCV strains, we did not find out differences in a large number of proteins. Nonetheless, it was noted that proteins with masses of 3768 Da and 6146 Da were present in the normal colony morphotype, while in the small colony morphotype they were absent
Probably, the loss of these proteins reflects in changes of the morphology and the growth rate of colonies. It should be noticed that proteins with given masses, which were detected in the normal colonies morphotype, also presented in the early isolate of patient A., that is described above.
Discussion
Over the last few years, MALDI-ToF mass spectrometry technology plays an increasingly important role in the description of new species of microorganisms, as well as in subtyping. Subtyping is capable for not only identify a wide range of bacteria and fungi, but also carry out the bioinformatic analysis of their protein spectra due to the low cost of reagents. It can be used for epidemiological and clinical goals.
From year to year a growing number of publications appear, with a description of new microorganisms. They are involved in various infectious processes in the human body and Burkholderia cepacia complex is one of their representatives [13, 14]. By representing different types of saprophitic bacteria, that mostly inhabit the environment, they are increasingly act as the cause of the development of infectious processes under varying localizations, especially in patients from vulnerable groups, including patients with cystic fibrosis [15].
In cases of chronic lung infection, opportunistic microorganisms in the respiratory tract of patients with cystic fibrosis develop in response to stress-selective pressure. It is generated by the host environment in response to the action of immune system factors, antimicrobial therapy, reducing of nutrient availability, and oxygen limitations. This results in occurrence of phenotypic variants of the main clonal population that exhibit different patterns of antimicrobial susceptibility. They are also differ in size of formed biofilms, etc. [16–18].
As known, a microbial population, especially in culture in vivo, can’t remain feature-wise homogenous, even if the whole population was from the outset a clonal descendent of a single cell. After a certain number of divisions, genetically heterogeneous individuals appear in each bacterial population. This occurs as a result of the exchange of genetic information both within the given population and with other microorganisms presented in the lungs in cases of cystic fibrosis. The exchange of information in this case is caused by various phenomena: transformation, transduction, conjugation, as well as in attendance with transposable genetic elements, besides other R plasmids. These formations are not only capable of leading to the spread of drug resistance, but also increase the survival time of pathogens in the environment, that is an additional factor that increases the possibility of infection in medical settings and in everyday life [17, 19].
The results obtained in the study of Burkholderia spp. isolated from cystic fibrosis patients with long-term colonisation of the lower respiratory tract at different periods of infection demonstrate that changes in the proteomic characteristics of Burkholderia spp. strains in conditions of chronic infection are noted several months before the clinical manifestation of the ‘cepacia’ syndrome, which is expressed in the loss of proteins with masses of 2083, 2090, 3767, 3780, 4651, 5023, 5444, 6318 and 7575 Da, and in the acquisition of proteins with masses of 5345 and 6340 Da, which may be related to the clinical condition of the patient. Similar studies have been conducted to describe the strain specificity of protein profiles in members of the genus Bifidobacterium [20].
The study also demonstrates that patients with chronic infection have a heterogeneous population of Burkholderia spp., which is also reflected in the protein profiles obtained. It was shown that strains giving growth as SCV morphotype were lack of proteins with masses of 3768 and 6146 Da, while they were present in NCV morphotype. Similar studies describing the occurrence of heterogeneity of formed colonies of Burkholderia cepacia complex bacteria are also described in world studies [21–24].
Conclusion
A regular dynamic monitoring and comparison of protein profiles of microbial strains, which were isolated from patients with chronic lung infections with a long term history of lower respiratory tract colonization, can be useful in forecasting effort of the clinical course of the disease, as well as in assessing of risks of severe infectious complications development.
Funding
This work received no funding from any source.
Declarations
Ethical Approval
This study was approved by the Bioethics Committee of the Samara State Medical University with the Approval Number 151; November 26, 2014 so as approved by the Bioethics Committee of the Samara State Medical University with the Approval Number 204; December 11, 2019. All procedures performed were in accordance with the ethical standards of the university Bioethics Committee and with the principles of the 1964 Declaration of Helsinki and its later amendments.
Informed Consent
All the patients signed informed consent statement to participate in the research. For the patient who was under 18 years old and mentioned in our research, informed consent was obtained from the parents.
Conflict of interest
The authors declare that they have no conflict of interest.
Footnotes
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