Role of gene sequencing for the diagnosis, tracking and prevention of bacterial infections

Renu Kumari; Benu Dhawan

doi:10.4103/jacm.jacm_22_22

REVIEW ARTICLE

Year : 2022 | Volume : 24 | Issue : 3 | Page : 8-14

Role of gene sequencing for the diagnosis, tracking and prevention of bacterial infections

Renu Kumari, Benu Dhawan
Department of Microbiology, AIIMS, New Delhi, India

Date of Submission	13-Sep-2022
Date of Acceptance	19-Oct-2022
Date of Web Publication	11-Nov-2022

Correspondence Address:
Benu Dhawan
Department of Microbiology, AIIMS, New Delhi
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/jacm.jacm_22_22

Abstract

Gene sequencing is the inevitable future of diagnostic microbiology. Of the various molecular assays available, sequencing is the promising technique for detecting culture-negative infections due to uncultivable bacteria namely culture-negative endocarditis, meningitis, brain abscess, keratitis, urinary tract infections, empyema, septic arthritis and septicaemia. Sequencing also helps to predict full resistance profile of bacteria and its virulence traits. Sequencing is an emerging and powerful technique to perform the epidemiological studies in an outbreak situation. This review focuses on the common applications of sequencing in clinical bacteriology including isolate characterisation, antimicrobial resistance and virulence factor profiling, establishing the source of infections and tracking the disease transmission.

Keywords: Bacterial infection, diagnosis, gene sequencing, prevention, tracking

How to cite this article:
Kumari R, Dhawan B. Role of gene sequencing for the diagnosis, tracking and prevention of bacterial infections. J Acad Clin Microbiol 2022;24, Suppl S1:8-14

How to cite this URL:
Kumari R, Dhawan B. Role of gene sequencing for the diagnosis, tracking and prevention of bacterial infections. J Acad Clin Microbiol [serial online] 2022 [cited 2023 Sep 25];24, Suppl S1:8-14. Available from: https://www.jacmjournal.org/text.asp?2022/24/3/8/360981

Introduction

The incidence of hard-to-treat bacterial infections is increasing nowadays. Gene sequencing can help in both limiting these infections and in providing optimum treatment to the patient by timely strain identification as well as virulence and drug-resistance profiling. Sequencing is also helpful in source identification and tracing the transmission of infection. It could thus help to improve the prophylactic measures to be implemented and ultimately reduce the incidence of nosocomial infections.

In this review, we look for the common applications of sequencing in diagnostic bacteriology including isolate characterisation, antimicrobial resistance (AMR) and virulence factor profiling, establishing the source of recurrent infections and tracking the disease transmission.

The Lure of Sequencing in Clinical Microbiology

As a result of the progress in sequencing technologies, gene sequencing is considered as the obvious and inevitable future of diagnostic microbiology.^{[1],[2],[3],[4]} Sequencing is useful for isolate characterization, AMR profiling and establishing the sources of recurrent infections and between-patient transmissions. The comprehensive information so obtained will have an obvious clinical relevance and also provide additional information on disease dynamics. Hence, sequencing can replace the knowledge obtained through standard clinical microbiology techniques. This review reiterates the potential of sequencing in clinical bacteriology in addition the limitations of gene sequencing are also addressed.

Sequencing in Diagnostic Bacteriology

Strain characterization

At the most basic level, sequencing can be used to characterise a clinical isolate. It will not only give information on the likely species and/or subtype but also allow phylogenetic placement of a given sequence relative to an existing set of isolates. Sequencing-based strain characterisation gives a better resolution as compared to high-resolution genotyping methods such as multilocus sequence typing (MLST). Sequencing helps in strain identification, when standard techniques such as pulsed-field gel electrophoresis, variable-number tandem repeat profiling, multiple loci variable number of tandem repeats and MALDI-TOF are unable to accurately distinguish lineages.^[5]

Sequencing could also be of particular significance for bacteria with large accessory genomes, as these are clinically most problematic bacteria, where much of the relevant genetic diversity is driven by differences in the accessory genome on the chromosome and/or plasmid carriage.

Detection of uncultivable bacteria and diagnosis of culture-negative infections

Bacterial culture has been the most important technique for diagnosing bacterial infections in microbiology. Indeed, Koch's original postulates required that the pathogen be grown in a pure culture. However, the requirement has since been updated, and the clinical importance of 'uncultivable' bacteria, e.g., Bartonella, Treponema pallidum, Tropheryma whipplei, Ehrlichiaspp and anaplasmaspp, is now well recognised. The diagnosis of culture-negative infections and those caused by uncultivable bacteria remains a somewhat arduous task. Traditional methods for the diagnosis of these organisms include direct microscopy and immunology based assays. The sensitivity and specificity of these methods vary greatly and depend on the implementation of technical expertise and the organisms concerned.

The introduction of molecular diagnostics significantly enhanced the ability to diagnose culture-negative infections. Among the various molecular assays available, sequencing stands out as a useful technique for detecting uncultivable bacteria. Sequencing has been used for diagnosing culture-negative infections, the best example being culture-negative endocarditis. Amongst all cases of infective endocarditis up to one-third are culture negative, and diagnosis has relied mainly upon clinical and ultrasonographic findings.^[6]

Polymerase chain reaction (PCR) amplification and sequencing of DNA extracted from infected valves is the ultimate solution for culture-negative endocarditis cases as reported by Goldenberger et al.^[7] Another study conducted in France also showed the utility of PCR amplification and sequencing in diagnosing culture-negative endocarditis. Subsequently, many studies confirmed the usefulness of this method in diagnostic microbiology.^{[8],[9],[10],[11],[12],[13],[14],[15],[16],[17]} The result of these studies showed that many culture-negative cases were caused by cultivable Gram-positive bacteria, yet the blood culture results might have been negative due to previous antibiotic usage. Other factors that may contribute to the false-negative blood culture results include inadequate microbiological techniques, for example, use of an insufficient volume of blood or infection by a fastidious organism.^[18]

Sequencing helpful in the diagnosis of culture-negative prosthetic joint infection (PJI). In a study conducted at our institute, of the 22 musculoskeletal infection society criteria confirmed PJIs cases, which were culture-negative, an etiological diagnosis could be established in seven using sequencing. The organisms included Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Escherichia coli and Lysobacter thermophilus.^[19] Sequencing can also help detect rarely described human pathogens and previously identified bacteria never reported in human infection.^[20] The first case of PJI caused by Lysobacterthermophiles a non-cultivable bacteria was diagnosed by 16S rRNA sequencing.^[21] There are the several reports of other culture-negative infections, including meningitis,^[22],[23] brain abscess,^[24] keratitis,^[25] urinary tract infections,^[26] empyema,^[27],[28] septic arthritis^[29],[30] and septicaemia,^[31],[32] where an etiological diagnosis required the use of sequencing. Summary of studies utilising sequencing for the diagnosis of bacterial infections as reported in the literature is given in [Table 1].

Table 1: Summary of studies utilizing sequencing for diagnosis of bacterial infections as reported in the literature

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Limitations of sequencing in diagnostics

Sequencing data, although provides extremely rich information, generates problems of its own.^[33],[34] Since every genome is unique, phylogenetically based classification is possible for only those species, which undergo no horizontal gene transfer (HGT), such as Mycobacterium tuberculosis.^[35] Unfortunately, organisms with a significant accessory genome and undergoing regular HGT, do not fall clearly into existing classification schemes. It is even questionable whether a completely satisfactory classification scheme could be devised for such organisms, as classifications based on the core genome, genotypic markers, accessory genome, housekeeping genes (MLST), plasmid sequence, virulence factors or AMR profile may all produce incompatible categories.

Phenotype and antimicrobial resistance prediction using sequencing

Beyond species identification and characterisation, sequencing also helps in the prediction of phenotypes by providing a rich resource of genome sequences. AMR and virulence are the main microbial traits of clinical relevance. Sequencing-based drug profiling is important to predict the AMR in bacterial pathogens.

Possibly, the first real attempt to predict the full resistance profile to multiple drugs by using sequencing data was done by Holden et al. in 2013, showing that, for a large dataset of Staphylococcus aureus ST22 isolates, 98.8% of all phenotypic resistances could be explained by at least one previously documented AMR element or mutation in the sequence data.^[36] Since then, several tools have been developed for the prediction of resistance profiles from sequencing.

It has been proposed that whole-genome sequencing (WGS)-based phenotyping might, be equal, if not more, accurate than traditional phenotyping.^{[37],[38],[39],[40]} However, the most successful applications to date have primarily been on M. tuberculosis and S. aureus, which are characterized by essentially no, or very limited, accessory genomes, respectively. In streptococcal pathogens, sequence-based predictions and measured phenotypic resistance show good agreement even in the large and diverse samples of isolates.^[41],[42]

Linezolid has emerged as a novel alternative to vancomycin and other second-generation drugs for the treatment of infections from Gram-positive cocci. The first clinical isolates of linezolid-resistant staphylococci and enterococci were reported in 2001.^[43] Since then, linezolid-resistant strains have become an increasing problem worldwide. The most frequently reported mechanisms of linezolid resistance include the mutation in 23S ribosomal RNA (23S rRNA) and presence of cfr gene.

In a study conducted at our institute, linezolid resistance was observed in 13 strain of S. haemolyticus recovered from the pus specimen. Sequencing results revealed G2576T mutations in eight, G2447U in four and C2534U in one isolate of S. haemolyticus. One isolate of S. haemolyticus showed two simultaneous mutations (G2576T and G2447U) in the domain V region of 23S rRNA gene.^[44] All the isolates demonstrated a dual mechanism of resistance with both the mutation in 23S ribosomal and the presence of cfr gene.

Apart from multidrug-resistant bacteria, sequencing is also useful to characterise highly-virulent bacteria, such as shiga toxin-producing E. coli (STEC) O104:H4. This bacterium has also been responsible for large outbreaks. In addition to that core-genome phylogenetic analysis of STEC (EAEC Stx2a+) O104:H4 showed different clustering and different resistance and virulence patterns depending on the time of isolation.^[45] Phylogenetic analysis of outbreak and non-outbreak related isolates using sequencing revealed lineage-specific markers, indicative for selective pressure and niche adaptation.^[46]

In a hospital setting, resistance patterns and virulence of isolates are important in patient management and formulation of infection control measures.

Limitation of sequencing for antimicrobial resistance prediction

On the whole, predicting comprehensive AMR profiles in organisms is challenging and requires extremely extensive and well curated reference databases. To put the problem in context, there are over 2000 described beta-lactamase gene sequences responsible for multiresistance to beta-lactam antibiotics such as penicillins, cephalosporins and carbapenems.^[47]

At this stage, many of the AMR reference databases are not well integrated and often have varying predictive ranges and biases and produce fairly inaccessible output files with little guidance on how to interpret or utilise this information for clinical intervention.

AMR predictions from sequencing data are qualitative, it simply predict whether an isolate is expected to be resistant or susceptible against a compound despite AMR generally being a continuous and often complex trait. The level of resistance of a strain to a drug can be affected by multiple elements or mutations,^[48] the copy number variation of these elements,^[49] the function of the genetic background of the strain^{[50],[51],[52]} and modulating effects by the environment.^[53] The level of resistance is generally well captured by the phenotypic measurement of minimum inhibitory concentration. Quantitative resistance predictions are not just of academic interest but it also a guide to the clinicians, as low-level resistance strains can still be treated with a given antibiotic but the standard dose should be increased, which can be the best option at hand, especially for drugs with low toxicity.

Perhaps, because of these limitations, although of obvious benefit as part of a diagnostics platform, both awareness of sequencing for AMR prediction and its utility in clinical settings is still limited.

Sequencing in Tracking Infection and Prevention

Tracking outbreaks and identifying sources of recurrent infections

Sequencing can help to reveal which isolates is the part of an outbreak lineage. It can detect direct probable transmission events by integrating epidemiological data with phylogenetic information.^{[54],[55],[56],[57]} Advancements in sequencing technologies have shifted the traditional culture-based microbial source tracking approaches towards culture independent technologies.^[58] WGS is an emerging and powerful technique to perform epidemiological studies in outbreak situations. In a study by Yang et al.^[59] WGS was used to identify and evaluate an outbreak of OXA-232–expressing carbapenem-resistant Klebsiella pneumoniae transmitted to 16 patients via endoscopic retrograde cholangiopancreatography procedures. Another study conducted in USA also showed sequencing to be a promising technique for strain typing and outbreak investigations. This study demonstrated sequencing to be superior to conventional typing techniques for A. baumannii strain typing and outbreak analysis.^[60] The findings of this study supported the incorporation of sequencing into healthcare infection prevention efforts. Various studies are there on the use of sequencing in tracking outbreaks and source identification ranging from MRSA^[61],[62] to Streptococcal disease^[63] and Neisseria gonorrhea.^[64]

Hence, beside phenotype prediction for individual isolates, sequencing has also allowed reconstructing outbreaks within hospitals and the community. Genomic data are increasingly being used to understand infectious disease epidemiology. Unfortunately, the phylogenetic trees, made up from sequencing the isolates from a given outbreak are not directly informative, since a phylogenetic tree is not a transmission tree. However, a transmission tree can be inferred from a phylogeny and approaches based on transmission chains can also be used to identify the sources of recurrent infections.^[65] In this way, WGS-based inference can elucidate the patterns of infection which are impossible to recapitulate from standard sequence typing alone.^[66]

Prevention of infection by using sequencing

One of the major advantages of sequencing is for epidemiological studies and public health investigations. It is especially important in outbreak detection, monitoring the evolution of multi-drug resistant pathogens and prevention of infection.^[67] Several studies have illustrated the usefulness of sequencing based methods in disclosing and tracing the dissemination of emerging pathogens. In a study from Netherland, Zhou et al.^[46] characterise a newly emerging CTX-M-15 producing K. pneumoniae clone with sequence type 1427 isolated from their hospital settings. In addition, they also demonstrated the transmission of a CTX-M-15-producing ST15 K. pneumoniae between patients treated in a single centre and the subsequent inter-institutional spread by patient referral was traced by genomic phylogenetic analysis. The investigation allowed the early detection of a K. pneumoniae high-risk-clone with prolonged circulation in the regional patient population.^[68] In addition to outbreak tracing and characterisation, sequencing also allows or helps in the implementation of control measures to avoid the spread of resistant bacterial clones. A study conducted by Weterings et al.^[69] demonstrated how an outbreak of a colistin-resistant carbapenemase-producing K. pneumoniae in the Netherlands, was controlled by transferring all positive residents to a separate location outside the Institution and hence sequencing plays an important role in prevention of the infection as well. Sequencing is a high discriminatory power tool that can differentiate between clones with specific properties and to use the obtained knowledge for patient management, infection prevention and evolutionary studies. Summary of studies using sequencing in tracking outbreak and prevention of infection are given in [Table 2].

Table 2: Summary of studies using sequencing in tracking outbreak and prevention of infection

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Concluding Remarks

Sequencing has an immense role in the diagnosis of infectious diseases including culture negative organisms and fastidious bacteria. In the near future sequencing will completely supersede current conventional diagnostic technique in clinical microbiology. The role of sequencing in diagnostic microbiology is a well-known fact. Other than this, its role in prevention of infections and guiding treatment protocols on the basis of resistance pattern of the organisms is also commendable. In the field of source identification and tracking outbreaks, sequencing has shown promising results based on several studies.

Despite all these successful applications, sequencing has several major limitations in its implementation as a routine approach to diagnose and characterise microbial infections. These include, the current costs of sequencing; a lack of trained personnel; limited necessary computational infrastructure in most hospitals; the inadequacy of existing reference microbial genomics databases necessary for reliable AMR and virulence profiling; and the difficulty of setting up effective, standardised and accredited bioinformatics protocols. There is also a genuine risk in implementing sequencing in routine diagnosis since there is a potential threat of loss of precious knowledge of basic microbiology following transition to sequencing.

In spite of all these problems we cannot ignore the fact that in near future sequencing applications shall fulfill unmet diagnostic needs in clinical microbiology and demonstrate clear benefits to patients.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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