Diagnostic clinical bacteriology – recent developments in the application of molecular biology tools
Diagnostic clinical bacteriology has been relatively slow in picking up on the molecular biology tools now available for identifying genes and classifying living matter. The demands for well-validated procedures in the diagnostic laboratory mean that the analytic as well as diagnostic performance of new techniques is a major issue when exciting new tools become available. A common finding among those clinical bacteriology specialists trying to move clinical bacteriology into the new era of molecular biology has been that the introduction of molecular biology in the diagnostic laboratory provides not only a wealth of new insights but also procedural problems and logistic issues that need to be addressed. Revision of bacterial taxonomy based on molecular genetic properties in addition to more conventional phenotyping has in many cases proven to be clinically and/or epidemiologically relevant. Procedures for extraction of DNA from human specimens in a consistent manner are not sufficiently standardized. In all probability this is due to the dearth of systematic information on the principle polymerase inhibitors that occur in various body fluids and biopsies when processed in vitro for DNA extraction. The setting up of rational cost-effective high-throughput analytical platforms is only in its infancy and considerable development can be expected with respect to the use of sophisticated integrated sequencing, and analysis, as well as interpretive software platforms that explore all properties – be they phenotypic or genotypic – for relevant bacterial taxonomy. In the present twilight zone between traditional procedural approaches in clinical bacteriology and the new rapidly evolving molecular biology-based techniques, we considered it pertinent to highlight the present state of the art of molecular diagnostics in clinical bacteriology by asking colleagues who are in the forefront of this development to review their respective areas of research critically. We hope that the present volume will meet those expectations and be useful in charting the future direction of diagnostic clinical bacteriology.
David R Murdoch presents the topic molecular genetic methods in the diagnosis of lower respiratory tract infections. He evaluates the development of nucleic acid amplification technologies (NAATs) for the detection of the many respiratory pathogens. Commercial assays are available for some of these. The need for standardized protocols is stated. Bacteria that are isolated only rarely or with difficulty, such as Mycoplasma pneumoniae, Legionella spp. and Chlamydia pneumoniae, together with tuberculosis are most suitable for non-culture diagnoses. Also Bordetella pertussis is readily diagnosed with these methods. Further work is required the better to characterize the role of molecular diagnostic tests for diagnosing lower respiratory tract infections and to develop a standard assay that can be readily adopted by routine diagnostic laboratories.
Shamputa et al. review in detail the application of all the molecular methods commercially available, as well as those not yet available, for the direct detection of mycobacteria in clinical specimens. They also cover the methods used for the detection of mutations causing drug resistance in M. tuberculosis. Several informative tables are included.
The contribution of Muhamed-Kheir Taha and Per Olcén takes the form of an overview entitled “Molecular genetic methods in diagnosis and direct characterization of acute bacterial central nervous system infections”. Acute infections of the central nervous system require rapid and adequate management. Molecular techniques can be employed for enhanced identification and characterization of etiologic agents, especially for non-culture diagnoses of bacterial infections. After an introduction covering general issues with respect to acute bacterial infections in the central nervous system, and a description of conventional identification/characterization of etiologic agents, the limitations are outlined. Methods are described for molecular identification of bacteria as such and for species, as well as directly in cerebrospinal fluid without culture. It is concluded that the molecular approach has provided powerful tools for diagnosis, epidemiological surveillance and tracking of the microevolution of bacterial populations. The development of systems that can reveal the aetiology and characterize most agents causing central nervous system infections, even including selected genetic analyses of host DNA, can be foreseen.
Fredlund at al. provide an outline of the sexually transmitted infections in “Molecular genetic methods for diagnosis and characterisation of Chlamydia trachomatis and Neisseria gonorrhoeae: impact on epidemiological surveillance and interventions”. The two major sexually transmitted bacteria C. trachomatis and N. gonorrhoeae are presented. The benefits and shortcomings of NAATs for these two disease-causing bacteria are described in the perspective of prevention and diagnosis/treatment. It is a problem when an old technique (culture) is considered the standard when in reality a new technique for evaluation has a higher diagnostic sensitivity. Characterisation of bacterial isolates by classical methods and by non-culture techniques is discussed.
In their review Fenollar & Raoult examine the molecular biology methods used for the diagnosis of difficult-to-culture bacteria, such as Rickettsia spp., Ehrlichia spp., Coxiella burnetii, Bartonella sp., Tropheryma whipplei and Yersinia pestis. The authors make the case that whilst molecular biology methods may allow quick diagnosis of infections caused by fastidious bacteria, several pitfalls, such as false positives, have been observed with PCR, underlining the necessity of interpreting the results obtained with caution. The examples given are DNA sequences from aquatic bacteria (Pseudomonas species) that are retrieved from human samples such as cerebrospinal fluid from aseptic meningitis post craniotomy, and the claim that nanobacteria exist based on 16S rRNA sequencing. The case of nanobacteria shows that without proper validation this area of molecular diagnosis of fastidious bacteria can be troublesome and too esoteric for the routine clinical bacteriology laboratory with the present state of our technology. Procedural stringency can help the diagnostic laboratory to avoid these pitfalls, but clearly more contained instrumentations are also needed.
Sample preparation and validation in PCR-based bacterial diagnostics is thus one of the most challenging tasks for workers crossing the threshold between the research laboratory and the routine clinical microbiology laboratory. Hoorfar et al. have made an excellent contribution by reviewing recent progress.
DNA-based methods in the detection of antibiotic resistance genes in bacteria are dealt with in two reviews in this issue.
Arnfinn Sundsfjord and colleagues present molecular genetic methods for the determination of antibiotic susceptibility and resistance. They point out that accurate and rapid methods are needed to guide antimicrobial therapy in infection control interventions. The development of the user-friendly PCR technique has increased the use of genetic assays as part of a larger strategy to minimize the spread of antimicrobial resistance bacteria. The principle features of genetic assays in determination of antimicrobial resistance are outlined in the review along with their advantages and limitations. The overview focuses on methicillin-resistant Staphylococcus aureus, glycopeptide-resistant enterococci, aminoglycoside resistance in staphylococci and enterococci, broad-spectrum resistance to β-lactam antibiotics in Gram-negative bacteria as well as genetic elements involved in the assembly and spread of antimicrobial resistance.
Jalava & Marttila review the current state of research into macrolide, lincosamide and streptogramin resistance diagnostics. The clear and concise text is further corroborated by four excellent tables listing the primers currently in use for the detection of the relevant resistance genes. In their review they also introduce the genetic basis of drug resistance in M. tuberculosis and the molecular methods used to detect the resistance genes and especially the application of pyrosequencing in detection.
In an extensive review clearly pointing out the way ahead for diagnostic bacteriology, Schweickert et al. describe the application of MALDI-TOF mass spectroscopy and FISH (fluorescence in situ hybridization) to bacteriology. First they review the use of MALDI-TOF in the analysis of bacterial nucleíc acids. MALDI-TOF has been used especially in determining the masses of enzymatically cleaved plasmids or PCR products and RNA, in the analysis of single nucleotide polymorphisms (SNPs) by primer extension analysis, and in sequence validation of nucleic acid fragments generated by base-specific cleavage. Then the authors review different applications where MALDI-TOF has so far been used and also consider how nucleic acid analysis by MALDI-TOF has developed rapidly and become a candidate method for high-throughput genetic testing. It is fast, accurate and cost-effective. FISH is the tool of choice when studying microbial communities, especially in environmental microbiology. Its application to clinical bacteriology, especially using rRNA-targeted probes, has become widespread lately. The review dissects in great detail the benefits and limitations of FISH when applied to diagnostics.
Progress respecting the molecular biology of some selected areas of intense research with a clear focus on typing of bacteria, transmission of bacteria and virulence forms the last part of this special issue.
Simala-Grant & Taylor review molecular biology methods for the characterization of Helicobacter pylori infections and their diagnosis. They emphasize that it is likely that antibiotic resistance will increase in the future. Thus, genotypic characterization of antibiotic susceptibility and virulence genes of H. pylori and patient populations prior to selecting patient therapy must be carried out. This quest for suitable diagnosis and treatment strategies of H. pylori infections should be population-based since in geographic areas where H. pylori infection is common, targeting intervention towards individuals possessing genotypes predisposing them to cancer of the stomach and infected with more virulent strains of H. pylori might be an attainable goal. Simala-Grant & Taylor point out that molecular methods are often limited at present to comparing bacterial genotypes with disease outcome and eradication rates; however, studies of quantitative expression of genes should be done to correlate gene expression with clinical findings and outcome of interventions. As pointed out in the review, the selection of suitable molecular methods to do this will require a balance between quick results, simplicity of technique, and the use of sophisticated instrumentation and trained individuals. The case of H. pylori and its relation to ventricular pathology illustrates the present state of the art in developing and introducing molecular biology techniques in clinical bacteriology in as much as the actual development of suitable techniques parallels the exploration of the disease concepts. During this exciting stage of the development great emphasis must be placed on technical details in DNA extraction, validation of findings in relation to known and possible unknown disease entities, and logistics strategies in the laboratory for wide-large-scale introduction of H. pylori molecular diagnostics in patient care.
The development of molecular techniques for diagnosis and also typing of Francisella tularensis provides a fascinating insight into `a necessity turned into a virtue' because of the well-known safety concern in the laboratories growing these bacteria. Johansson et al., in their review of this field, point out that elucidation of the epidemiology and epizootology of the disease has been hampered by the lack of suitable and safe methods, and that PCR will most probably be the preferred method for detection of F. tularensis in clinical samples for the foreseeable future. Johansson et al. also describe strategies for high-resolution typing of bacteria with a high degree of genome conservation, such as F. tularensis. Once extensive genome data are procured this will allow not only the development of methods for subspecies differentiation but also a search for targets suitable for distinction of strains based on even more characteristics as exemplified by investigating variable number tandem repeats in the bacterial genomes. This should be applicable to virtually any human bacterial pathogen. As the various parts of a bacterial genome differ in their rate of accumulating genetic variability and DNA repeats often account for higher degrees of variability, the review authors argue that it seems that such repeats could be suitable targets for discriminating closely related bacterial isolates.
The article by Lukinmaa et al. reviews the use of molecular genetic methods in the diagnostics and epidemiology of food-borne bacterial pathogens. The authors focus on the most commonly used genetic molecular typing methods, such as PFGE and a number of PCR-based methods, in the diagnosis of enteric and environmental food-borne pathogenic bacteria. As modern food production has become more and more centralized and food delivery routes are getting longer, single food-borne outbreaks have expanded to wider and wider areas. This has presented a challenge when endeavouring to solve these outbreaks. Molecular typing methods used together with relevant analytical epidemiology enable better identification of epidemics. Several examples of epidemiological investigations are provided, and laboratory-based surveillance using different DNA-based methods is described. Furthermore, the importance of national and international co-operation for tracing the food-borne infections is emphasized. This paper thus touches upon an important issue and provides a clear insight into genetic typing methodologies and the current situation respecting food-borne bacterial diseases.
Oelschlaeger & Hacker have written a concise review on bacterial pathogenicity islands, which are today attracting more and more attention in the light of the application of the accumulating data in clinical microbiology. The article describes how PAIs were first identified from Gram-negative bacteria as well as giving information on those few PAIs identified in Gram-positive bacteria.
As is evident from the papers in this APMIS special issue the application of molecular biology tools in diagnostic bacteriology is at a crucial stage of its development. It can be anticipated that our perception of diagnostic bacteriology will change from being a relatively slow but useful and necessary activity in health care to one that is at the forefront, where not only new rapid diagnostic procedures will be developed but also where we are given new insights into the pathogenesis of bacterial infections. Furthermore, better validated categorization of bacteria and their ways of sharing genetic material in ways sometimes useful for mankind but often troublesome can be foreseen. Clearly, applying this new-found knowledge to the development of strategies for antimicrobial drug development holds great promise for the future.
