An international consortium found a remarkable global spread of strains of a multi-resistant bacterium that can cause severe infections – Stenotrophomonas maltophilia.
The study, published under the supervision of the Research Center Borstel Leibniz Lung Center (FZB), provides for the first time a systematic understanding of the global phylogeny of S. maltophilia strains and shows ways to efficiently monitor the pathogen using a genomic classification system.
DZIF scientists from Lübeck, Borstel and Braunschweig are involved in the study.
S. maltophilia strains occur in several natural and human associated ecosystems. The bacterium was long regarded as relatively unproblematic but is now considered to be one of the most feared hospital pathogens, as it frequently causes infections and is resistant to a number of antibiotics.
This can be particularly dangerous for immune-compromised patients or for patients with underlying inflammatory lung diseases such as cystic fibrosis. Although almost any organ can be affected, infections of the respiratory tract, bacteraemia or catheter-related infections of the bloodstream are the most common.
In view of the increasing importance of this pathogen and the often-severe clinical consequences of an infection, knowledge about the virulence factors and about the local and global transmission of S. maltophilia bacteria is urgently needed.
Scientists from a total of eight countries initially established a genotyping method that enables the standardised analysis of the different genomes of S. maltophilia strains. The DZIF teams around Prof. Stefan Niemann (FZB), Prof. Jan Rupp, (Clinic of Infectiology and Microbiology, Campus Lübeck) and Prof. Ulrich Nübel from the Leibniz Institute DSMZ (German Collection of Microorganisms and Cell Cultures GmbH ) in Braunschweig were involved.
The scientists found that the S. maltophilia complex can be divided into a total of 23 lineages with different prevalence levels. One particular line of descent appeared worldwide and had the highest rate of human-associated strains.
This “Sm6” strain was also characterised by the presence of key virulence genes and resistance genes. “This suggests that a specific gene configuration may promote the spread of different S. maltophilia subtypes in the hospital setting, i.e. under antimicrobial treatment,” says Matthias Gröschel, lead author of the study.
Transmission analysis also identified several potential outbreak events of genetically closely related strains that were isolated within days or weeks in the same hospitals.
“Combined with studies on other pathogens, our results show how systematic genome-based monitoring of S. maltophilia and other pathogens in hospital settings can help detect transmission pathways and improve infection control,” Thomas Kohl, senior author of the study at FZ Borstel, adds.
Stenotrophomonas maltophilia is a Gram-negative, intrinsically multidrug-resistant bacterium that is ubiquitous in aqueous environments, such as soils, plant roots, and water treatment and distribution systems .
Whilst conventionally overlooked as a laboratory contaminant, or as a common commensal in hospitalized patients, S. maltophilia is increasingly being recognized as an important nosocomial pathogen in its own right, due to its ability to cause life-threatening disease in immunocompromised individuals .
This opportunistic pathogen has been isolated from a variety of hospital settings, including taps, sinks, central venous catheters, ice machines, and water fountains, reinforcing its nosocomial importance [1, 3, 4].
S. maltophilia most commonly infects people with meningitis, cancer, chronic obstructive pulmonary disease or cystic fibrosis (CF), with pneumonia, bacteraemia, and wound and urinary infections being the most frequent clinical manifestations [5, 6].
Risk factors for S. maltophilia infection include prolonged hospitalization, neutropenia, catheterization and previous use of broad-spectrum antibiotics . The recommended antibiotic treatment for S. maltophilia infections is co-trimoxazole; however, resistance towards this antibiotic combination has been documented [2, 8, 9].
Indeed, treatment options are limited for S. maltophilia , with this pathogen also exhibiting resistance towards several antibiotic classes, including fluoroquinolones, macrolides, β-lactams, aminoglycosides, carbapenems, tetracyclines, polymyxins, chloramphenicol and cephalosporins [1, 2].
With a mortality rate approaching 70 %, the importance of timely identification and effective treatment of S. maltophilia infections is paramount .
S. maltophilia is a common pathogen in CF airways due to its ability to evade many antipseudomonal antibiotics, with chronic S. maltophilia infection associated with an increased risk of respiratory disease and mortality [11, 12].
CF is an autosomal recessive genetic disorder effecting multiple organs; however, its pathogenesis is most prominent in airways, with ~90 % of CF deaths associated with respiratory failure .
The excessive production of mucus in CF airways provides optimal growth conditions for opportunistic pathogens, which drives most CF morbidity and mortality. Molecular methods have confirmed that CF lower airways harbour diverse microbial communities, with Pseudomonas aeruginosa and Burkholderia cepacia complex species of greatest concern due to frequent rapid respiratory decline in people infected with these pathogens [14, 15].
However, other co-infecting opportunistic pathogens, such as Achromobacter spp., S. maltophilia , Staphylococcus aureus , Haemophilus influenzae and certain fungal species (e.g. Aspergillus), are also prominent in CF airways, and are known to contribute to pathogenesis .
Indeed, recent studies have shown a mutualistic relationship between S. maltophilia and P. aeruginosa in CF airways, with compounds produced by S. maltophilia under exposure to certain antibiotics, such as imipenem, supporting the survival of otherwise antibiotic-susceptible P. aeruginosa strains .
Furthermore, these S. maltophilia compounds can enhance P. aeruginosa stress tolerance, increasing polymyxin tolerance . These studies highlight the importance of S. maltophilia in CF airway pathogenesis, particularly during antibiotic treatment, and emphasize the need for correct species identification in polymicrobial infections.
Although there are a variety of diagnostic methods available for S. maltophilia detection, such as PCR amplicon sequencing, VITEK MS identification or key morphological characteristics on growth media, these methods suffer from issues such as limited access to equipment with a large capital expenditure [e.g. ~US $200 000 (£162 586, £1=$1.23) for VITEK MS instrumentation], high per-assay cost, the need for highly trained personnel, laboriousness, slow turnaround time, the requirement for purified colonies, and misidentification issues [16, 18, 19].
For example, S. maltophilia and P. aeruginosa exhibit colony colour differences when grown on bromothymol blue-containing media, which reflects their different metabolic processes .
However, the use of media containing bromothymol blue is not routine and, thus, the retrieval of S. maltophilia from polymicrobial specimens requires clinical expertise in identifying appropriate culture media for differentiation of this bacterium from other pathogens.
As a non-exhaustive list, S. maltophilia has been misidentified as several other organisms, including Bordetella bronchiseptica , Alcaligenes faecalis , Burkholderia cepacia and numerous Pseudomonas species, which are common in clinical settings, including CF sputa [1, 20].
Diagnostic inconsistencies in S. maltophilia detection from clinical specimens can lead to inappropriate or even detrimental treatment , particularly for those patients requiring urgent care. Therefore, there is a need to accurately identify this emerging pathogen to improve antibiotic-treatment regimens, stewardship and patient outcomes.
Here, we report the development and validation of a black hole quencher (BHQ) probe-based real-time PCR assay for the specific detection of S. maltophilia . Our results indicate that our real-time PCR assay is more sensitive than routine culture for detecting S. maltophilia , particularly in polymicrobial respiratory specimens.
S. maltophilia is emerging as an important multidrug-resistant nosocomial pathogen, being amongst the top three most common non-fermentative Gram-negative bacilli identified in hospitalized patients [5, 36].
Despite S. maltophilia being well-adapted to many environments, most infections occur in immunocompromised individuals in the nosocomial setting , although community-acquired infections are also on the rise .
Here, we describe what is to the best of our knowledge the first real-time PCR assay to detect S. maltophilia with 100 % accuracy in purified colonies, and demonstrate that this assay is superior to microbiological culture for detecting this multidrug-resistant bacterium in polymicrobial respiratory specimens collected from CF patients.
There is currently a lack of a rapid, cost-effective, accessible and accurate diagnostic method for S. maltophilia detection, particularly from polymicrobial clinical specimens such as CF sputa. As S. maltophilia is thought to be the only Stenotrophomonas species to cause human disease, mass spectrometry (MS)-based systems such as VITEK 2 and VITEK MS are a common diagnostic method in large, centralized pathology laboratories.
However, the accuracy of species determination using MS is heavily dependent on the quality of the associated databases, and it is currently unknown whether other Stenotrophomonas spp. can be accurately differentiated from S. maltophilia on these systems.
In addition, access to this instrument is limited to well-resourced laboratories owing to a large barrier-to-entry cost [~US $200 000 (£162 586)] [37–39]. Furthermore, as CF sputa are polymicrobial, overgrowth of other bacteria on selective culture plates is common. This is particularly problematic for VITEK diagnosis when S. maltophilia is present in low abundance or the patient is co-infected with mucoid P. aeruginosa .
From a genotyping standpoint, 16S rRNA gene PCR has been used to identify S. maltophilia in blood samples for patients undergoing chemotherapy for leukemia , and a multiplex PCR targeting P. aeruginosa , S. maltophilia and Burkholderia cepacia successfully identified S. maltophilia in 85 % of cases .
However, these assays have either not been optimized to avoid non-specific amplification in other Stenotrophomonas spp. and members of the closely related genus Xanthomonas , or they require downstream processing (e.g. gel electrophoresis, Sanger sequencing) to confirm results, which is laborious, time-consuming and raises potential laboratory contamination issues.
Therefore, the purpose of this study was to use large-scale comparative genomics to identify a S. maltophilia-specific genetic target, and to subsequently design a highly specific and accurate real-time PCR-based assay for identifying S. maltophilia . Using this approach, we identified a genetic region with high specificity for S. maltophilia , which was subsequently targeted for assay development.
We found that our newly developed assay correctly identified 89 S . maltophilia isolates with 100 % accuracy. The accuracy and specificity of this assay is both highly sensitive and selective for S. maltophilia , with an LoQ and LoD of ~94 and ~9 GEs, respectively.
We chose the BHQ probe real-time PCR format due to its relatively inexpensive up-front cost [~US $25 000 (£30 750) for real-time instrumentation], low per-reaction cost [~US $0.80 (£0.98) per sample when performed in duplicate], high-throughput capacity, closed-tube format (which eliminates post-PCR contamination concerns), simple set-up and rapid turnaround-time (~1 h).
This format also enables robust identification of target species in polymicrobial specimens. Although not examined in this study, the multi-fluorophore capacity of many real-time PCR instruments also enables multiplexing of probe-based assays for the simultaneous identification of multiple organisms in a single specimen, leading to further cost reductions.
Our in silico and laboratory results indicate that all non- S. maltophilia micro-organisms failed to amplify, with the possible exception of two S. indicatrix strains, RS1 and RS7, the genomes of which only became available subsequent to assay design. S. indicatrix is a newly identified Stenotrophomonas species  that has so far been isolated from dirty dishes in Germany (strain WS40 ), sewage in China (strain G4; unpublished), a rotting apple in Germany (Myb57; unpublished), soil in Lebanon (strains RS1 and RS7; unpublished) and a human respiratory infection in Germany (ICU300 ).
Comparative analysis of RS7 and S. maltophilia indicated recent homologous recombination of the formate dehydrogenase locus targeted by our study; however, blastn analysis showed that there were two SNPs in the probe-binding region, including a SNP at the 5′ ultimate base of the BHQ probe, which would likely result in poor or no amplification.
Taken together, we show that our assay is highly specific for S. maltophilia , particularly in clinical samples, but it also has applicability for testing environmental samples, such as hospital water supplies.
Although the quality of life and life expectancy for people with CF has markedly increased in recent decades due to improvements in antibiotic treatments and clinical management, persistent polymicrobial infections in CF airways remain the primary cause of morbidity and mortality .
A recent longitudinal study of a single CF patient’s airways using a cutting-edge metatranscriptomic approach, which measures only the ‘active’ microbial population through mRNA characterization, revealed that S. maltophilia was the second most prevalent bacterium behind P. aeruginosa in the 6 months prior to death .
Our results also revealed a high prevalence of S. maltophilia in adult CF sputa, with 10/16 samples positive for this bacterium according to our assay. As some of these sputa had concurrent culture results, we demonstrated an improvement in detection with 71.4 % congruence to real-time PCR results, with two culture-negative samples returning as positive by real-time PCR (SCHI0020 day 1 and SCHI0021 day 1).
While our sample size is low, this finding demonstrates that our assay may have a higher sensitivity for detecting S. maltophilia in CF clinical specimens than culture methods, although it cannot be ruled out that S. maltophilia PCR positivity may be due to the presence of DNA in the CF sputa from a recently eradicated S. maltophilia infection.
Of four longitudinally collected sputa, one patient (SCHI0019) had S. maltophilia at all time points (days 1, 11 and 46; Table 1) despite intravenous meropenem and tobramycin antibiotic therapy administered on day 2 of admission, and another patient, SCHI0002, was positive for S. maltophilia in samples that were collected nearly 12 months apart (days 1 and 320), indicating either long-term airway persistence or reinfection with this organism.
In one sputum sample from SCHI0019 (day 11), the ∆C t value between the 16S rRNA gene and S. maltophilia PCRs was identical to that of pure S. maltophilia culture (Table 1), indicating that S. maltophilia had become the dominant, and potentially sole, bacterial species in this specimen.
Although outside the scope of this study, this finding demonstrates the potential for S. maltophilia to persist and dominate in CF airways following antibiotic-driven microbiome perturbations, which may have implications for rapid re-infection with more formidable pathogens such as P. aeruginosa .
A further two patients, SCHI0020 and SCHI0021, had S. maltophilia at day 1, but subsequent samples (up to day 31 and 13, respectively) were PCR-negative following intravenous ceftazidime and tobramycin antibiotic treatment phase administered on day 2 of admission, indicating successful eradication of S. maltophilia in these cases.
Future work will entail testing our newly developed assay across larger CF sputum panels, including longitudinal samples, to further examine the potential mutualistic relationships between S. maltophilia and other pathogens such as P. aeruginosa , and to assess assay performance directly on clinical specimens to further reduce sample processing timeframes.
In conclusion, the ability to accurately, rapidly and cheaply detect S. maltophilia is critical for understanding the prevalence of this underappreciated opportunistic pathogen and for reducing its burden of disease.
The implementation of this assay in the clinical setting will enable researchers, clinicians and pathologists to more accurately identify this multidrug-resistant bacterium, particularly in isolates that have been ruled out as other multidrug-resistant Gram-negative pathogens, such as P. aeruginosa or Burkholderia spp.
Finally, the correct and rapid identification of S. maltophilia will improve antibiotic stewardship measures by enabling more targeted eradication of this pathogen, and in polymicrobial infections such as those commonly found in CF airways, S. maltophilia eradication may reduce the prevalence and persistence of more serious pathogens such as P. aeruginosa , leading to improved quality of life and lifespans for people with CF.
More information: Matthias I. Gröschel et al, The phylogenetic landscape and nosocomial spread of the multidrug-resistant opportunist Stenotrophomonas maltophilia, Nature Communications (2020). DOI: 10.1038/s41467-020-15123-0
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