Quantitative real-time PCR assay for the rapid identification of the multidrug-resistant bacterial pathogen Stenotrophomonas maltophilia

Stenotrophomonas maltophilia is emerging as an important cause of disease in nosocomial and community-acquired settings, including bloodstream, wound and catheter-associated infections. Cystic fibrosis airways also provide optimal growth conditions for various opportunistic pathogens with high antibiotic tolerance, including S. maltophilia. Currently, there is no rapid, cost-effective, and accurate molecular method for detecting this potentially life-threatening pathogen, particularly in polymicrobial specimens, suggesting that its true prevalence may be underestimated. Here, we used large-scale comparative genomics to identify a specific genetic target for S. maltophilia, with subsequent development and validation of a real-time PCR assay for its detection. Analysis of 165 Stenotrophomonas spp. genomes identified a 4kb region specific to S. maltophilia, which was targeted for Black Hole Quencher assay design. Our assay yielded the positive detection of 89 of 89 (100%) clinical S. maltophilia strains, and no amplification of 23 non-S. maltophilia clinical isolates. S. maltophilia was detected in 10/16 CF sputa, demonstrating the utility for direct detection in respiratory specimens. The assay demonstrated good sensitivity, with limits of detection and quantitation on pure culture of ~10 and ~100 genome equivalents, respectively. Our assay provides a highly specific, sensitive, and cost-effective method for the accurate identification of S. maltophilia, and will improve the diagnosis and treatment of this under-recognized pathogen by enabling its accurate and rapid detection from polymicrobial clinical and environmental samples.


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Stenotrophomonas maltophilia is a Gram-negative, intrinsically multidrug-resistant bacterium 43 that is ubiquitous in aqueous environments such as soils, plant roots, water treatments and 44 distribution systems (1). Whilst conventionally overlooked as a laboratory contaminant, or as 45 a common commensal in hospitalized patients, S. maltophilia is increasingly being 46 recognized as an important nosocomial pathogen in its own right due to its ability to cause 47 life-threatening disease in immunocompromised individuals (2). This opportunistic pathogen 48 has been isolated from a variety of hospital settings including faucets, sinks, central venous 49 catheters, ice machines, tap water, and water fountains, reinforcing its nosocomial importance 50 (1, 3, 4). S. maltophilia most commonly infects people with meningitis, cancer, chronic 51 obstructive pulmonary disease (COPD), or cystic fibrosis (CF), with pneumonia, bacteraemia, 52 and wound and urinary infections being the most frequent clinical manifestations (5, 6). Risk 53 factors for S. maltophilia infection include prolonged hospitalization, neutropenia, 54 catheterization, and previous use of broad-spectrum antibiotics (7). The recommended 55 antibiotic treatment for S. maltophilia infections is co-trimoxazole; however, resistance 56 towards this antibiotic combination has been documented (2,8,9). Indeed, treatment options 57 are limited for S. maltophilia, with this pathogen also exhibiting resistance towards several 58 antibiotic classes including fluoroquinolones, macrolides, β-lactams, aminoglycosides, 59 carbapenems, tetracyclines, polymyxins, chloramphenicol and cephalosporins (1, 2). With a 60 mortality rate approaching 70%, the importance of timely identification and effective 61 treatment of S. maltophilia infections is paramount (10).

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S. maltophilia is a common pathogen in CF airways due to its ability to evade many 63 antipseudomonal antibiotics, with chronic S. maltophilia infection associated with an 64 increased risk of respiratory disease and mortality (11,12). CF is an autosomal recessive 65 genetic disorder effecting multiple organs; however, its pathogenesis is most prominent in 66 4 airways, with ~90% of CF deaths associated with respiratory failure (13). The excessive 67 production of mucus in CF airways provides optimal growth conditions for opportunistic 68 pathogens, which drives most CF morbidity and mortality. Molecular methods have 69 confirmed that CF lower airways harbour diverse microbial communities, with Pseudomonas 70 aeruginosa and Burkholderia cepacia complex species of greatest concern due to frequent 71 rapid respiratory decline in people infected with these pathogens (14,15  in the phylogenomic analysis to confirm that they were in fact S. maltophilia. Sequence data 120 from assembled genomes were converted to simulated 100bp paired-end Illumina reads at 121 85x coverage using ART version MountRainier (21). SRA data were quality-filtered using 122 Trimmomatic v0.33 (22) using previously described filtering parameters (23) prior to 123 analysis.

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This phylogenomic analysis was used to delineate S. maltophilia from other species, and to 201 modify incorrect species designations for 43 taxa, including reclassification of all four "S.  (Table S1).   There was an 84.6% congruence between the two methods, with two PCR-positive sputa 250 (SCHI0020 Day 1 and SCHI0021 Day 1) being negative by culture (Table 1)

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There is currently a lack of a rapid, cost-effective, accessible, and accurate diagnostic method 280 for S. maltophilia detection, particularly from polymicrobial clinical specimens such as CF 281 sputa. As S. maltophilia is thought to be the only Stenotrophomonas species to cause human 282 disease, mass spectrometry-based systems such as VITEK 2 and VITEK MS are a common 283 diagnostic method in large, centralized pathology laboratories. However, the accuracy of 284 species determination using mass spectrometry is heavily dependent on the quality of the 285 associated databases, and it is currently unknown whether other Stenotrophomonas spp. can 286 be accurately differentiated from S. maltophilia on these systems. In addition, access to this 287 instrument is limited to well-resourced laboratories owing to a large barrier-to-entry cost 288 (~USD$200,000) (34-36). From a genotyping standpoint, 16S rDNA PCR has been used to 289 identify S. maltophilia in blood samples for patients undergoing chemotherapy for leukemia 290 (37), and a multiplex PCR to detect P. aeruginosa, S. maltophilia and B. cepacia successfully 291 identified S. maltophilia in 85% of cases (38). However, these assays have either not been 292 optimized to avoid non-specific amplification in other Stenotrophomonas spp. and members 293 of the closely related Xanthomonas genus, or they require downstream processing (e.g. gel 294 electrophoresis, Sanger sequencing) to confirm results, which is laborious, time-consuming, 295 and raises potential laboratory contamination issues.

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Therefore, the purpose of this study was to use large-scale comparative genomics to identify 297 a S. maltophilia-specific genetic target, and to subsequently design a highly-specific and 298 accurate real-time PCR-based assay for identifying S. maltophilia. Using this approach, we 299 identified a genetic region specific to S. maltophilia, which was subsequently targeted for 300 assay development. We found that our newly developed assay correctly identified 89 S. 301 maltophilia isolates with 100% accuracy. The accuracy and specificity of this assay is both analysis showed that there were two SNPs in the probe-binding region, including a SNP at 320 the 5' ultimate base of the BHQ probe, which would likely result in poor or no amplification.

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Taken together, we show that our assay is highly specific for S. maltophilia, particularly in 322 clinical samples, but it also has applicability for testing environmental samples, such as Day 1). This finding demonstrates that our assay has a higher sensitivity for detecting S. 336 maltophilia in CF clinical specimens than culture methods. Of four longitudinally collected 337 sputa, one patient (SCHI0019) had S. maltophilia at all time points (Days 1, 11, and 46; Table   338 1) despite intravenous antibiotic therapy during this time, and another patient, SCHI0002, 339 was positive for S. maltophilia in samples that were collected nearly 12 months apart (Days 1 340 and 320), indicating either long-term airway persistence or reinfection with this organism. In 341 one sputum sample from SCHI0019 (Day 11), the ∆CT value between the 16S rDNA and S. 342 maltophilia PCRs was identical to that of pure S. maltophilia culture (Table 1), indicating that 343 S. maltophilia had become the dominant, and potentially sole bacterial species, in this 344 specimen. Although outside the scope of this study, this finding demonstrates the potential 345 for S. maltophilia to persist and dominate in CF airways following antibiotic-driven 346 microbiome perturbations, which may have implications for rapid re-infection with more 347 formidable pathogens such as P. aeruginosa. A further two patients, SCHI0020 and 348 SCHI0021, had S. maltophilia at Day 1, but subsequent sampling (up to Day 31 and 13, 349 respectively) were PCR-negative during the intravenous antibiotic treatment phase, indicating 350 successful eradication of S. maltophilia in these cases. Future work will entail testing across 351 larger CF sputum panels, including longitudinal samples, to further examine potential 352 mutualistic relationships between S. maltophilia and other pathogens such as P. aeruginosa, 353 and on assessing assay performance directly on clinical specimens to further reduce sample 354 processing timeframes.

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In conclusion, the ability to accurately, rapidly, and cheaply detect S. maltophilia is critical 356 for understanding the prevalence of this underappreciated opportunistic pathogen and for 357 reducing its burden of disease. The implementation of this assay in the clinical setting will 358 enable researchers, clinicians and pathologists to more accurately identify this multidrug-359 resistant bacterium, particularly in isolates that have been ruled out as other multidrug-360 resistant Gram-negative pathogens, such as P. aeruginosa or Burkholderia spp. Finally, the 361 correct and rapid identification of S. maltophilia will improve antibiotic stewardship 362 measures by enabling more targeted eradication of this pathogen, and in polymicrobial 363 infections such as those commonly found in CF airways, S. maltophilia eradication may 364 reduce the prevalence and persistence of more serious pathogens such as P. aeruginosa, 365 leading to improved quality of life and lifespans for people with CF.