Critical assessment and comparative analysis of phenotypic

Abstract

The present essay, has conducted a comparative analysis on the different methods of bacterial strain identification, taking in consideration the molecular methods of PCR and gene sequencing, the conventional phenotypic methods and the MALDI-TOF-MS method. The advantages and disadvantages of these methods have been thoroughly delved into, to demonstrate which method stands good in the modern times, as development in bacteriology based researches, genetic sequencing and MS have come up in the present times. While the efficiency of the modern methods namely like - faster detection time, high rate of accuracy, economical cost, have been explained, their potential negative side and limitations like similarity in genetic sequences leading to barriers in species-specific accuracy, have also been explained in details. Hence, the conventional methods of Gram-staining, catalase-based identification have been compared amongst themselves and with the modern molecular identification methods of PCR and 16S-rRNA. The MS technique has also been discussed in details and tallied with the rest of the other methods, to evaluate which methods should be considered as the primary method in laboratories and clinics for bacterial strain identification meeting the cost-accuracy, species-specificity, and time arenas.

Introduction

In the present study, a comparative analysis and detailed assessment of several bacterial identification techniques have been conducted, considering the molecular techniques, the conventional phenotypic techniques of culture identification, and the modern MALDI-TOF (Matrix-assisted-laser-desorption-ionization–time-of-flight-mass-spectrometry) technique. The different types of these methods alongside their disadvantages and advantages have thus been delved into details, to draw out a critical assessment on their limitations and their beneficiary aspects, in terms of speed of assays, turnaround times, technique specificity, sensitivity, labor intensiveness, technical training requirements, assay limitations and cost based complications. Hence apart from the MALDI-TOF technique, other techniques namely - API, catalase method, gram test, oxidase method, PCR, 16s rRNA, RFLP, RAPF and DAF and AFLP have been delved into details in the present essay. The essay has also been formulated taking in consideration the data obtained from the esteemed research of Cherkaoui, et al., (2010), in their study entitled "Comparison of two matrix-assisted laser desorption ionization-time of flight mass spectrometry methods with conventional phenotypic identification for routine identification of bacteria to the species level". Finally after a critical analysis of the conventional techniques with modern day techniques have been conducted in the present essay, the key points of the study have been well summarized in the conclusion section of the study.

Main Body

Molecular methods types

Molecular methods have developed as major tools for analyzing microbes in the present day from biological substances and food, providing innovative ways of screening across a wider agent range within single tests (Tuttle, et al., 2011). The field of molecular detection of bacteria, at species level holds great application and scope of development in the future within multiple industries like - food factories, in water processing, in laboratory analytics and research for faster strain identification, species differentiation and strain relatedness with infected samples. However, there are several types of genetic techniques or molecular methods, with each varying from the other in terms of, power of discrimination, reproducibility, interpretation simplicity or ease in usage (Fraher, et al., 2012). Some such molecular techniques have been discussed and critically analyzed under: The PCR or Polymerase-Chain-Reaction, is a widely practiced bacterial strain detection technique and the technique and its experimental data was first published by Saiki et al. (1985), following which the method was widely accepted and used by researchers the world over to understand microorganisms in a more specific way (Hospodsky, et al., 2010). The method's advantages lies in the way of making unlimited number of DNA fragment copies, helping to detect a bacteria of interest within a much shorter span of time over conventional methods (Sasaki, et al., 2010). PCR also helps in detecting disease causing bacteria faster and more efficiently, namely the Campylobacter, causing bacterial gastroenteritis, found in meat (Braga, et al., 2013). Also good bacteria used in the food industry for probiotics, like the Lactobacillus species have all been well identified due to the PCR method (Matamoros, et al., 2010). Furthermore, Dahlenborg et al. (2001) through the help of PCR could successfully investigate upon Clostridium botulinum prevalence, in the primary production, as well as for gene expression monitoring of the botulinum-neurotoxin-gene (Carlin, 2011). However the major drawbacks of PCR remains in the fact where only amount in genetic material can be amplified through this technique, thus stating the obvious fact that low numbers of species within samples can be detected. Other drawbacks of the technique consist of lacking anti-microbial-sensitivity-data, assay procedure complexity, high price range of the PCR kits and equipments required. The 16S rRNA PCR method is a yet novel molecular tool which is used for detection of bacterial species due to the presence of 16S rRNA gene in all the species of bacteria (Motoshima, et al., 2012). This gene comprises of conserved sequences of nucleotides which are interspersed by variable regions in a species specific or a gene specific way. This molecular method remains highly appropriate in situations where pathogen range is wider and in those scenarios wherein organism-specific type PCR's remain inapplicable. Also this method is used for detection of bacterial species which are difficult in culturing. The method also remains applicable to the culturing of post-antibiotic-treatment based samples (Jenkins, et al., 2012). Several researches have also rampantly used the technique in medical studies where results suggest that the technique holds high potential in detecting bacterial pathogenic presence within the clinical samples which are culture-negative. Such results suggest that the 16S rRNA assay sensitivity is affected by the amplified 16S rRNA gene's fragment size (Jenkins, et al., 2012). The disadvantages of this technique though lies in the fact that during bacterial identification, the completeness and availability of the databases severely affect and limit the rate of identification accuracy in the method. From previous researches, results have suggested that MicroSeq-500-16s-rRNA-sequence library remains outdated and incomplete for identifying clinical isolated of the bacterial species Mycobacterium as well as Nocardia (Cloud, et al., 2010). However with increase in affordability in sequencing technologies, more laboratories have started using sequencing techniques with freely-accessible and publicly available databases for identification of bacterial species. One such prominent database is that of GenBank, which has upon proper evaluation, been reported to contain certain sequence errors in relation to those sequences submitted before the year 1995, signifying the limitations and potential drawbacks of the molecular methods of bacterial identification (Lee, et al., 2014).

MALDI-TOF-MS

Yet another state-of-the-art-technique is the MALDI-TOF-MS or the Matrix-assisted-laser-desorption-ionization–time-of-flight-mass-spectrometry, method which is a highly efficient yet emerging technology in the application of bacterial strain identification. According to the research by Cherkaoui, et al., (2010), two such systems were tested wherein 720 bacterial colonies were isolated within routine laboratory conditions. After that the researchers went forward with analyzing the isolates so obtained in parallel, on both the devices. Further on a comparison was drawn out between the conventional phenotypic-biochemical methods and the MS technique, where the discordant results that were obtained were resolved by 16S rRNA "gold-standard" gene sequencing technique. The results depicted that Bruker (1st MS-system) delivered high-confidence in isolate identification corresponding to 680 isolates, from which 99.1% appeared correct, while for Shimadzu (2nd MS) out of 639 isolate identified, a high-confidence rate of 99.4% comprising of 635 isolated came correct. The high-confidence identification of bacterial strains through the MS method, thus signifies that using that technique alone would have lead to a cost-saving of ~ $5 USD, for every isolate. Also the technique successfully reduces turnaround time by 8-h shift or even more, without any loss in the level of strain identification accuracy. These results led the researchers to suggest that instead of conventional biochemical-tests which get routinely performed in laboratories as a first-strategy for strain identification, the MS technique being more accurate, cost and time effective must be the first-test-strategy to be adopted for the one-step detection procedures, in the field of clinical bacteriology and other research arenas. The technique however also comes with limitations in the way. One of the greatest disadvantages of this technique is its analytical sensitivity which is low without any prior culture. The method also discriminates on phylogenetically related microbes namely on E.coli and Shigella. Yet another major limitation lies in the fact that there remains limited database spectra and inherent microbial species similarity, which may result in poor discrimination amongst the species or misidentification of bacterial strains. Though these are low-frequency errors in occurrence, they can be overcome typically via supplemental testing (Rychert, 2019).

Conventional-Phenotypic methods

For appropriate and accurate bacterial species diagnosis and detection, an effective species-specification method of strain identification is highly crucial. The phenotypic method of bacterial identification remains the most used and conventional methods involving numerous techniques and procedures, inclusive of bacterial growth, colony morphology, biochemical reaction analysis, as well as the usage of non-automated or automated biochemical panels, that are commercially available (Cherkaoui, et al., 2010). However, a major drawback of the conventional phenotypic techniques involving gram, oxidase, catalase methods or API techniques, remain in the fact that the commercial databases remain outdated often and with the lacuna of current taxonomy, proper and accurate identification of bacterial strains is often hindered (Law, et al., 2015). Furthermore, the phenotypic methods also cannot account for characteristic diversity and variability as observed in the members of similar species, which ultimately results in poor identification and faulty precision after repeat testing. A commonly used method, developed in 1884 by "Hans-Christian-Gram", and commonly known as the Gram stain, separates bacteria into the two major categories as bacteria which are gram-negative and those that are gram-positive (Menard, et al., 2010). This conventional phenotypic method has several advantages like faster result detection during examination of infections, cost-effectiveness, simplicity and ease of use, appropriate infection treatment and it allows tests to be conducted wherein different sample collection methods can be used. But the method also contains disadvantages like its limitations in detection of bacterial strains at species-level, or limitations in form of misinterpretations due to over-colorization. The catalase-test is yet another test for detecting catalase enzyme's presence or absence in bacterial species, for differentiating streptococcus (catalase-negative) from staphylococci (catalase-positive). But false-negative-results may be obtained if the bacterial cultures are more than 24 hr old, as catalase remains present within viable cultures (Reiner, 2010). Bacterial strain isolation using the 16S rRNA sequencing technique via the usage of the system MicroSeq 500, remains far more accurate over the biochemical tests and conventional phenotypic techniques, or even over other types of commercial systems that involve carbon-utilization and fatty-acid utilization profiles (Cloud, et al., 2010). Even then, limitations also exist for the gene sequencing 16S rRNA technique, as alike other methods. With the ever changing bacterial nomenclature and taxonomy every day, as the bacterial genotypes and genetic features are delved and researched upon in greater details, there increases the risk where accuracy in identification may get hampered. Also through the in-depth analyses of bacterial genotypic characteristics and with development in modern day research and information gathering, it has been made evident that that through these analyses unique strains of bacteria have been identified, possessing distinctive genetic profiles as well as biochemical profiles. This fact suggests that definitive identification of bacterial strain can hence not be made possible and is becoming more difficult, unless the new strains are further accepted and considered as some new bacterial species (Wang, et al., 2014). Even when relatively complete database sequences remain present the 16S sequences remain identical and highly close even from different strains, showing a similarity percentage of more than 99.5%. This leaves expert judgement, in such scenarios, the sole requirement for bacterial identification (Lee, et al., 2014).

Conclusion

The conventional phenotypic techniques though widely used in most laboratories and research studies around the world, remains inaccurate to the species-level of strain detection, and consumes a fairly large amount of time during conduction of biochemical tests. The PCR techniques, which show higher result accuracy, and can be conducted in shorter time span. But the PCR technique involves higher cost and low number of species within samples can be detected. The 16S rRNA PCR method is a yet another novel molecular tool which is used for detection of bacterial species, the limitations of which lies in the fact that during bacterial identification, the completeness and availability of the databases severely affect and limit the rate of identification accuracy in the method (Motoshima, et al., 2012). The most recently adopted method the MALDI-TOF-MS, provides high-confidence identification of strains amounting at 99.4%, a cost-saving of ~ $5 USD, for every isolate, and reduced turnaround time (Carbonnelle, et al., 2011). However analytical sensitivity of the method is low, but although the errors are low-frequency in occurrence, they can be overcome typically via supplemental testing, thus being approved by most researchers at the present time Cherkaoui, et al., (2010).

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