Trends in Molecular Diagnostics No. 1/19: 1-9

Whitepaper

Rare Pathogens – Culture-Independent Eubacterial

16S rRNA Gene Diagnosis

Michael G. Lorenz – Molzym GmbH & Co. KG, Bremen, Germany

Keywords: Sepsis, septicaemia, bacteraemia, diversity, Gram-positive, Gram-negative pathogens, direct diagnosis, ribosomal genes, broad-range PCR, unbiased Real-Time PCR, Sanger sequencing, routine diagnosis, fluid and tissue biopsies, one sample processing protocol, contamination-free products, auto- mated extraction

Summary classes targeting the most probable, i.e., common pathogens. Rare pathogens are associated with critical conditions of patients in many cases demanding Common pathogens timely identification for adjustment of the antibiotic regime. Cultural diagnosis of rare In clinical practice in the Western and Eastern pathogens, however, can be laborious and time- hemisphere, Escherichia coli, Staphylococcus consuming, which lowers the prognosis of aureus and coagulase-negative staphylococci successful treatment. A prominent reason for have been shown in clinical studies to make up this is the enormous diversity of rare pathogens 43.1 to 65% of cases of bacteraemia in sepsis that may be identifiable only with delay or even (Table 1). In large studies from Germany (4380 not identifiable by usual phenotyping methods. A patients), France (842), USA (1585), Saudi review of clinical studies conducted in five Arabia (1626) and Pakistan (521), further countries from Europe, Asia and the USA common pathogens or representatives of the reveals that 90.7 to 98.2% of systemic infections genus were identified with incidences of sepsis were caused by strains belonging to only 7 cases between 1 and 10%, including strepto- bacterial classes and one yeast genus. With less cocci, Salmonella typhi/paratyphi, Pseudomonas than 1% incidence in the majority of the spp., Acinetobacter spp., Klebsiella spp., countries, the taxonomic diversity of rare enterococci, Candida spp., Enterobacter spp., pathogens markedly increased adding another Proteus spp., Serratia spp., Bacteroides spp., 13 bacterial classes to the list of aetiologies of Clostridium spp., and, in USA, sepsis. Even more, a survey of 54 case studies spp. (Table 1). Depending on the country, these uncovered further unusual species that 16 pathogens/groups cause sepsis in 32.9 to belonged to 27 classes. In order to break 47.6% of cases. Together, common pathogens through the obstacles of poor cultural diagnosis, account for 90.7 to 98.2% of systemic infections. the use of culture-independent, pan-bacterial 16S rDNA PCR and sequencing analysis has Rare pathogens entered routine as a helpful method of precise Other pathogenic were associated with strain identification within hours instead of days. infections at lower than 1% incidence. This arbitrarily defined group of rare pathogens Introduction accounted for 1.4 to 13.2% of bacteraemias in Latest 2016 recommendations of the Surviving the studies of the 5 countries (Table 1). The Sepsis Campaign guide to the immediate (within pathogens represent 13 taxonomic classes, one hour) administration of empiric broad- including Enterobacterales (Citrobacter spp., spectrum antimicrobials after diagnosis of sepsis Yersinia enterocolitica), Pasteurellales (Hae- or septic shock. The problem with gold standard mophilus influenzae), Vibrionales (Vibrio spp.), blood culture is that cultures are often negative Neisseriales (Neisseria meningitidis), Clostrid- with blood draws after initiation of antibiotic iales (Peptostreptococcus spp., Peptococcus treatment. Also, as demonstrated in a recent spp.), Rhizobiales (Brucella spp.), Burkhold- study (1), cultures grow positive with bacteria eriales (Burkholderia spp.), Propionibacteriales and yeasts only after 12 to 61 hours which ( spp.), Bacillales (Listeria delays targeted antibiotic treatment and monocytogenes, Bacillus spp.), Fusobacteriales increases the risk of poor prognosis of the (Fusobacterium spp.), Campylobacterales patients. According to the recommendations, (Campylobacter fetus), Corynebacteriales (Cory- empiric antimicrobials should include combina- nebacterium spp.) and Veillonellales (Veillonella tions of two antimicrobials of different antibiotic spp.). For comparison, the 16 common patho- Direct 16S rRNA Gene Diagnosis

Table 1: Pathogens identified in blood cultures of septic patients.

gens (Table 1) fall into only 7 classes (Bacillales, reports emphasise the notion that unusual Enterobacterales, Lactobacillales, Pseudomona- bacteria stem from taxonomically highly diverse dales, Bacteroidales, Clostridiales and Coryne- groups. bacteriales). This diversity of can pose There is a long record of further bacteria problems to the cultivation and the identification involved in rare cases of sepsis. A Google of strains in clinical routine, because standard Scholar search under the term “rare pathogens media not always meet their growth conditions in sepsis” resulted in 54 case reports uncovering and phenotypic tests may not represent their an enormous diversity of unusual aetiologies of characters for identification purposes. In fact, the bacteraemia. Most of the identified bacteria diagnostic procedure of unexpected aetiologies normally live in the environment, on plants and of sepsis can be laborious and time-consuming in animals, and can give rise to sepsis in and standard phenotypic identification practices patients with neutropenia, meningitis, may fail or lead to misidentification. For endocarditis, diabetic foot ulcers or catheterised, instance, in their report Malkan et al. (15) in the course of transplantations and surgeries, described a case of an anaemic patient in neonates, after cat and dog bites and in other supported with iron by a venous infusion port incidences. Table 2 sorts the pathogens into 40 catheter. The patient developed sepsis by a families and 27 classes to underline the methicillin-sensitive S. aureus which was treated enormous diversity of bacterial species involved with daptomycin. At day 3, the catheter was in septicaemia. This list does not claim to be removed. Cultures of the device were positive comprehensive. Nonetheless, these case with Gram-positive cocci and Gram-negative

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Table 2: Cultured rare strains causing sepsis and detectability by 16S rRNA gene PCR and sequencing.

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Table 2: Continued.

rods. Same day blood cultures grew only Gram- (BLAST®) leBIBI and Molzym’s Sep- negative rods and treatment was changed to siTest™BLAST. gentamicin. Subculturing was performed on various media resulting in very small colonies, Culture-independent diagnosis and Vitek 2 (bioMérieux) compact Gram- by rRNA gene sequencing negative identification resulted in Cupriavidus pauculus. The bioMérieux API 20E system could rRNA gene sequencing approaches, including not assign the organism to a certain species. At Sanger and NGS sequencing of amplified and the end, definitive identification of the strain as cloned 16S rRNA gene sequences, respectively, Acidovorax avenae was reached by sequencing have been used for the direct, i.e., culture- analysis of a 485 bp PCR amplicon of the 16S independent analysis of bacterial strains and rRNA gene. multiple infections for quite a while. Many of the reported methods are used for research purpos- Ribosomal RNA gene sequenc- es to answer specific scientific questions rather ing in clinical practice than for use of surveillance and in daily routine diagnostic practice. Ribosomal RNA gene sequencing is used in rou- rRNA gene sequencing promises to add benefit tine laboratories as a reliable method of strain to culture diagnosis in two clinical situations: di- identification. Mahlen and Clarridge (58), for in- agnosis of i) fastidious and anaerobic bacteria stance, elaborated a strategy of broad-range with unusual growth requirements and ii) growth- 16S rRNA gene sequencing analysis of Gram- inhibited, but not cleared pathogens as a result negative rods and coccobacilli as aid for routine of antimicrobial treatment of patients. Reported identification methods. For strain identification, applications of the use of rRNA gene sequenc- the rRNA genes or parts thereof are amplified in ing comprise of diseases like pneumonia, men- a PCR reaction. The reaction includes PCR pri- ingitis, thrombosis, pleuritis, septic arthritis, sep- mers that bind to sequences highly conserved tic coxitis, intra-abdominal infection, intra- among the of bacteria, while the se- amniotic infection, brain abscess, recurrent quences in-between vary among species. Identi- cholecystitis, spondylitis, osteomyelitis, septi- fication of an unknown strain is achieved by caemia, sepsis, subcutaneous abscess, spinal comparison of its determined sequence to a li- abscess, empyema (69), endophthalmitis (70), brary of sequences of known strains. Online cystic fibrosis (71), infective endocarditis (72), tools are available that perform the assignment urinary tract infections (73), airway inflammation by an elaborated algorithm. Examples are the (74), chronic venous leg ulcer (75), prosthetic NCBI Basic Local Alignment Search Tool joint infections (76) and others. Unlike other as-

Trends in Molecular Diagnostics No. 1/19 Page 4 of 9 Direct 16S rRNA Gene Diagnosis says for direct analysis of a limited number of cluding rare pathogens directly in fluid and tis- selected common pathogens, the unbiased na- sue biopsies (59-68). ture of broad-range 16S rRNA gene PCR and All of the common and rare species found in the sequencing follows a universal approach of the studies discussed above (Table 1) have been identification of common, rare and unusual shown to be detectable in other evaluations. pathogens. Among the 54 unusual species listed in Table 2, 31 have been identified by Molzym’s direct 16S Standardisation rRNA gene sequencing approaches in studies The majority of methods for direct rRNA gene employing other specimens. In 19 of the cases, sequencing analysis of pathogens in clinical 16S rRNA gene sequencing evaluations have specimens is in-house developed tests and thus not yet identified the same strains, but other are not comparable among laboratories. Such species of the same genus, family or class of the tests differ in their pre-analytical and analytical cultured strains. No relatives of Chlamydophila procedures. A variety of commercial kits used in abortus, Chromobacterium violaceum, Ignatz- the laboratories are usually for DNA extraction schineria indica and Wohlfahrtiimonas chitini- from clinical samples and amplification. The clastica have been found at the family or class problem is that chemicals, reagents and con- level so far (Table 2). However, except for C. sumables are generally not produced under con- abortus, in silico testing of primer binding to the trol of the absence of microbial DNA (77), be- 16S rRNA gene targets was positive, indicating cause they are designed for applications where that the strains are detectable by the assay. DNA contamination is irrelevant. Under these Thus, 16S rRNA gene sequencing as represent- conditions the avoidance of false positive results ed by Molzym’s molecular diagnostic kits have in the diagnosis of low bacterial load materials is the potential to identify essentially all pathogens difficult to reach if not impossible. Moreover, the directly found in samples. The kits are CE- sensitivity and specificity of tests can be lowered marked for the in-vitro diagnosis of pathogens. by the vast load of human DNA in many clinical specimens (78). Another consequence is that Conclusions Sanger and NGS sequencing analysis can be Ribosomal RNA gene amplification and se- disturbed by human sequences from unspecific quencing analysis is a frequently used method amplification (78). for the unbiased identification of rare and unu- The only unbiased 16S rRNA gene PCR and sual pathogenic bacteria in clinical routine. In the sequencing tests, which are CE-approved for past years, the method has been adapted to the the in-vitro diagnostics of hundreds of pathogens culture-independent diagnosis of infected clinical are provided by Molzym. The products are com- materials at low loads of pathogens. With this plete solutions for the whole process, including approach, however, a variety of problems have enrichment (depletion of human DNA) and ex- been faced that disturb the precise diagnosis. traction of DNA from bacteria present in samples Factors negatively influencing sensitivity, speci- at low loads (<100 cfu/ml), detection by Real- ficity and sequencing identification include, Time PCR amplification and sequencing analy- among others contamination of extraction and sis of the amplified hypervariable V3/V4 region amplification reagents and consumables with of the 16S rRNA gene. By employing highly ac- microbial DNA and a great excess of human tive and contamination-free PCR reagents, am- DNA. Molzym addressed these problems by the plification runs of 40 cycles are performed with- supply of contamination-free kits and reagents out signals in negative PCR controls. Solutions that enable the processing of human DNA- are available for manual extraction, Sep- depleted preparations from a variety of clinical siTest™-UMD, semi-automated extraction, samples by just one protocol and the amplifica- UMD-SelectNA™, and fully automated extrac- tion over 40 cycles without background in nega- tion, Micro-Dx™ in the SelectNA™plus robot. tive PCR controls. By this solution hundreds of Other, contamination-free research-use-only rare and unusual pathogens can be identified products provide options for the extraction of within hours without the need of cultivation. bacterial DNA from fluid (MolYsis™ kits) and tissue samples (Ultra-Deep Microbiome kit) and amplification of custom targets as well as de- fined parts of the 16S rRNA gene for qualitative, References quantitative Real-Time PCR (Mastermix 16S/18S kits) and library preparation purposes 1. Ning Y, Hu R, Yao G, Bo S (2016) Time to (NGSeq 16S V3/V4). Validations in clinical stud- positivity of blood culture and its prognostic ies and routine use have proven that unbiased value in bloodstream infection. Eur J Clin 16S rRNA gene analysis can reliably detect and Microbiol Inf Dis 35, 619–624. identify a great variety of microorganisms, in-

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