group leade: Prof. Höfle

Recent Submissions

  • Comparison of in situ sequence type analysis of Legionella pneumophila in respiratory tract secretions and environmental samples of a hospital in East Jerusalem.

    Jaber, Lina; Amro, Mahmod; Tair, Hadeel Abu; Bahader, Shereen A; Alalam, Hanna; Butmeh, Suha; Hilal, Dalia Abu; Brettar, Ingrid; Höfle, Manfred G; Bitar, Dina M; et al. (Cambridge University Press, 2018-01-01)
    Legionella pneumophila genotyping is important for epidemiological investigation of nosocomial and community-acquired outbreaks of legionellosis. The prevalence of legionellosis in pneumonia patients in the West Bank was monitored for the first time, and the sequence types (STs) from respiratory samples were compared with STs of environmental samples from different wards of the hospital. Sputum (n = 121) and bronchoalveolar lavage (BAL) (n = 74) specimens were cultured for L. pneumophila; genomic DNA was tested by 16S rRNA polymerase chain reaction (PCR) amplification. Nested PCR sequence-based typing (NPSBT) was implemented on DNA of the respiratory and environmental PCR-positive samples. Only one respiratory specimen was positive for L. pneumophila by culture. BAL gave a higher percentage of L. pneumophila-positive samples, 35% (26/74) than sputum, 15% (18/121) by PCR. NPSBT revealed the following STs: ST 1 (29%, 7/24), ST 461 (21%, 5/24), ST 1037 (4%, 1/24) from respiratory samples, STs from environmental samples: ST 1 (28.5%, 4/14), ST 187 (21.4%, 3/14) and ST 2070, ST 461, ST 1482 (7.1%, 1/14) each. This study emphasises the advantage of PCR over culture for the detection of L. pneumophila in countries where antibiotics are indiscriminately used prior to hospital admission. ST 1 was the predominant ST in both respiratory and environmental samples.
  • Genomic Variations Underlying Speciation and Niche Specialization of Shewanella baltica.

    Deng, Jie; Auchtung, Jennifer M; Konstantinidis, Konstantinos T; Brettar, Ingrid; Höfle, Manfred G; Tiedje, James M; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (American Society of Microbiology, 2019-10-15)
    Shewanella baltica was the dominant culturable nitrate-reducing bacterium in the eutrophic and strongly stratified Baltic Sea in the 1980s, where it primarily inhabited the oxic-anoxic transition zone. The genomic structures of 46 of these isolates were investigated through comparative genomic hybridization (CGH), which revealed a gradient of genomic similarity, ranging from 65% to as high as 99%. The core genome of the S. baltica species was enriched in anaerobic respiration-associated genes. Auxiliary genes, most of which locate within a few genomic islands (GIs), were nonuniformly distributed among the isolates. Specifically, hypothetical and mobile genetic element (MGE)-associated genes dominated intraclade gene content differences, whereas gain/loss of functional genes drove gene content differences among less related strains. Among the major S. baltica clades, gene signatures related to specific redox-driven and spatial niches within the water column were identified. For instance, genes involved in anaerobic respiration of sulfur compounds may provide key adaptive advantages for clade A strains in anoxic waters where sulfur-containing electron acceptors are present. Genes involved in cell motility, in particular, a secondary flagellar biosynthesis system, may be associated with the free-living lifestyle by clade E strains. Collectively, this study revealed characteristics of genome variations present in the water column and active speciation of S. baltica strains, driven by niche partitioning and horizontal gene transfer (HGT).IMPORTANCE Speciation in nature is a fundamental process driving the formation of the vast microbial diversity on Earth. In the central Baltic Sea, the long-term stratification of water led to formation of a large-scale vertical redoxcline that provided a gradient of environmental niches with respect to the availability of electron acceptors and donors. The region was home to Shewanella baltica populations, which composed the dominant culturable nitrate-reducing bacteria, particularly in the oxic-anoxic transition zone. Using the collection of S. baltica isolates as a model system, genomic variations showed contrasting gene-sharing patterns within versus among S. baltica clades and revealed genomic signatures of S. baltica clades related to redox niche specialization as well as particle association. This study provides important insights into genomic mechanisms underlying bacterial speciation within this unique natural redoxcline.
  • Antimicrobial agent susceptibilities of Legionella pneumophila MLVA-8 genotypes.

    Sharaby, Yehonatan; Nitzan, Orna; Brettar, Ingrid; Höfle, Manfred G; Peretz, Avi; Halpern, Malka; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Nature publishing group, 2019-04-16)
    Legionella pneumophila causes human lung infections resulting in severe pneumonia. High-resolution genotyping of L. pneumophila isolates can be achieved by multiple-locus variable-number tandem-repeat analysis (MLVA-8). Legionella infections in humans occur as a result of inhalation of bacteria-containing aerosols, thus, our aim was to study the antimicrobial susceptibilities of different MLVA-8 genotypes to ten commonly used antimicrobial agents in legionellosis therapy. Epidemiological cut-off values were determined for all antibiotics. Significant differences were found between the antimicrobial agents’ susceptibilities of the three studied environmental genotypes (Gt4, Gt6, and Gt15). Each genotype exhibited a significantly different susceptibility profile, with Gt4 strains (Sequence Type 1) significantly more resistant towards most studied antimicrobial agents. In contrast, Gt6 strains (also Sequence Type 1) were more susceptible to six of the ten studied antimicrobial agents compared to the other genotypes. Our findings show that environmental strains isolated from adjacent points of the same water system, exhibit distinct antimicrobial resistance profiles. These differences highlight the importance of susceptibility testing of Legionella strains. In Israel, the most extensively used macrolide for pneumonia is azithromycin. Our results point at the fact that clarithromycin (another macrolide) and trimethoprim with sulfamethoxazole (SXT) were the most effective antimicrobial agents towards L. pneumophila strains. Moreover, legionellosis can be caused by multiple L. pneumophila genotypes, thus, the treatment approach should be the use of combined antibiotic therapy. Further studies are needed to evaluate specific antimicrobial combinations for legionellosis therapy.
  • Pseudomonas-Specific NGS Assay Provides Insight Into Abundance and Dynamics of Species Including P. aeruginosa in a Cooling Tower.

    Pereira, Rui P A; Peplies, Jörg; Mushi, Douglas; Brettar, Ingrid; Höfle, Manfred G; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2018-01-01)
    Pseudomonas species are frequent inhabitants of freshwater environments and colonizers of water supply networks via bioadhesion and biofilm formation. P. aeruginosa is the species most commonly associated with human disease, causing a wide variety of infections with links to its presence in freshwater systems. Though several other Pseudomonas species are of ecological and public health importance, little knowledge exists regarding environmental abundances of these species. In the present study, an Illumina-based next-generation sequencing (NGS) approach using Pseudomonas-specific primers targeting the 16S rRNA gene was evaluated and applied to a set of freshwater samples from different environments including a cooling tower sampled monthly during 2 years. Our approach showed high in situ specificity and accuracy. NGS read counts revealed a precise quantification of P. aeruginosa and a good correlation with the absolute number of Pseudomonas genome copies in a validated genus-specific qPCR assay, demonstrating the ability of the NGS approach to determine both relative and absolute abundances of Pseudomonas species and P. aeruginosa. The characterization of Pseudomonas communities in cooling tower water allowed us to identify 43 phylotypes, with P. aeruginosa being the most abundant. A shift existed within each year from a community dominated by phylotypes belonging to P. fluorescens and P. oleovorans phylogenetic groups to a community where P. aeruginosa was highly abundant. Co-occurrence was observed between P. aeruginosa and other phylotypes of P. aeruginosa group as well as the potentially pathogenic species P. stutzeri, but not with phylotypes of the P. fluorescens group, indicating the need to further investigate the metabolic networks and ecological traits of Pseudomonas species. This study demonstrates the potential of deep sequencing as a valuable tool in environmental diagnostics and surveillance of health-related pathogens in freshwater environments
  • Virulence traits of environmental and clinicalLegionella pneumophilaMLVA genotypes.

    Sharaby, Yehonatan; Rodríguez-Martínez, Sarah; Pecellin, Marina; Sela, Rotem; Peretz, Avi; Höfle, Manfred; Halpern, Malka; Brettar, Ingrid; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-03-09)
    Legionella pneumophila causes water-based infections resulting in severe pneumonia. Recently, we showed that different variable numbers of tandem repeat analysis (MLVA-8) genotypes dominated different sites of a drinking-water distribution system, with a genotype-related temperature and abundance regime. Here we compare the pathogenicity potential of different MLVA-8 genotypes of environmental and clinical strains. The virulence traits studied were hemolytic activity and cytotoxicity towards amoebae and macrophages. Clinical strains were significantly more hemolytic than environmental strains, while their cytotoxicity towards amoebae was significantly lower at 30°C. No significant differences were detected between clinical and environmental strains in cytotoxicity towards macrophages. Significant differences in virulence were observed between the environmental genotypes (Gt). Gt15 strain showed a significantly higher hemolytic activity. In contrast, Gt4 and Gt6 strains were more infective towardsAcanthamoeba castellaniiMoreover, Gt4 strain exhibited increased cytotoxicity towards macrophages and demonstrated a broader temperature range of amoebal lysis compared to Gt6 and Gt15. Understanding the virulence traits ofLegionellagenotypes may improve the assessment of public health risks ofLegionellain drinking water.ImportanceLegionella pneumophilais the causative agent of a severe form of pneumonia. Here we demonstrate that clinical strains were significantly more cytotoxic towards red blood cells compared to environmental strains, while their cytotoxicity towards macrophages was similar. Genotype 4 (Gt4) strains were highly cytotoxic towards amoebae, macrophages, and lysed amoebae in a broader temperature range, compared to the other studied genotypes. The results can explain the Gt4 relatively high success in the environment and in clinical samples; thus Gt4 strains should be considered as a main factor for the assessment of public health risks ofLegionellain drinking water. Our findings shed light on the ecology, virulence, and pathogenicity potential of differentL. pneumophilagenotypes that can be a valuable parameter for future modelling and Quantitative Microbial Risk Assessment ofLegionellain drinking water systems.
  • Divergence in gene regulation contributes to sympatric speciation of Shewanella baltica strains.

    Deng, Jie; Auchtung, Jennifer; Konstantinidis, Konstantinos T; Caro-Quintero, Alejandro; Brettar, Ingrid; Höfle, Manfred; Tiedje, James M; Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017-12-08)
    Niche partitioning and sequence evolution drive genomic and phenotypic divergence, which ultimately leads to bacterial diversification. This study investigated genomic composition of two Shewanella baltica clades previously identified through multi-locus sequencing typing and recovered from the redox transition zone in the central Baltic Sea. Comparative genomic analysis revealed significantly higher inter- than intra-clade genomic dissimilarity, and that a subset of genes present in Clade A were associated with potential adaptation to respiration of sulfur compounds present in the redox-transition zone. Transcriptomic divergence between two representative strains of Clades A and D, OS185 and OS195, was also characterized and revealed marked regulatory differences. We found that both transcriptional divergence of shared genes and expression of strain-specific genes lead to differences in regulatory patterns between strains that correlate with environmental redox niches. For instance, under anoxic conditions of respiratory nitrate ammonification, OS185 - the strain isolated from nitrate-rich environment, upregulated nearly twice the number of shared genes compared to OS195 - the strain isolated from H2S-containing anoxic environment. Conversely, OS195 showed stronger induction of strain-specific genes, especially those associated with sulfur compound respiration, under thiosulfate-reducing conditions. A positive association between the level of transcriptional divergence and sequence divergence for shared genes was also noted. Our results provide further support for the hypothesis that genomic changes impacting transcriptional regulation play an important role in the diversification of ecologically distinct populations.Importance This study examined potential mechanisms through which co-occurring Shewanella baltica strains diversified to form ecologically distinct populations. At the time of isolation, the strains studied composed the major fraction of culturable nitrate-reducing communities in the Baltica Sea. Analysis of genomic content of 13 S. baltica strains from two clades representing different ecotypes demonstrated that one clade specifically possesses a number of genes that could favor successful adaptation to respire sulfur compounds in the portion of the water column from which these strains were isolated. In addition, transcriptional profiling of fully-sequenced strains representative of these two clades, OS185 and OS195, under oxygen-, nitrate- and thiosulfate-respiring conditions demonstrated that the strains exhibit relatively similar transcriptional responses during aerobic growth, but more distinct transcriptional responses under nitrate and thiosulfate-respiring conditions. Results from this study provide insights into how genomic and gene regulatory diversification together impacted the redox specialization of the S. baltica strains.
  • Comparison of sputum microbiome of legionellosis-associated patients and other pneumonia patients: indications for polybacterial infections.

    Mizrahi, Hila; Peretz, Avi; Lesnik, René; Aizenberg-Gershtein, Yana; Rodríguez-Martínez, Sara; Sharaby, Yehonatan; Pastukh, Nina; Brettar, Ingrid; Höfle, Manfred G.; Halpern, Malka; et al. (2017-01-06)
    Bacteria of the genus Legionella cause water-based infections resulting in severe pneumonia. Here we analyze and compare the bacterial microbiome of sputum samples from pneumonia patients in relation to the presence and abundance of the genus Legionella. The prevalence of Legionella species was determined by culture, PCR, and Next Generation Sequencing (NGS). Nine sputum samples out of the 133 analyzed were PCR-positive using Legionella genus-specific primers. Only one sample was positive by culture. Illumina MiSeq 16S rRNA gene sequencing analyses of Legionella-positive and Legionella-negative sputum samples, confirmed that indeed, Legionella was present in the PCR-positive sputum samples. This approach allowed the identification of the sputum microbiome at the genus level, and for Legionella genus at the species and sub-species level. 42% of the sputum samples were dominated by Streptococcus. Legionella was never the dominating genus and was always accompanied by other respiratory pathogens. Interestingly, sputum samples that were Legionella positive were inhabited by aquatic bacteria that have been observed in an association with amoeba, indicating that amoeba might have transferred Legionella from the drinking water together with its microbiome. This is the first study that demonstrates the sputum major bacterial commensals and pathogens profiles with regard to Legionella presence.
  • Shewanella baltica Ecotypes Have Wide Transcriptional Variation under the Same Growth Conditions.

    Hambright, W S; Deng, Jie; Tiedje, James M; Brettar, Ingrid; Rodrigues, Jorge L M; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-05-09)
    In bacterial populations, subtle expressional differences may promote ecological specialization through the formation of distinct ecotypes. In a barrier-free habitat, this process most likely precedes population divergence and may predict speciation events. To examine this, we used four sequenced strains of the bacterium Shewanella baltica, OS155, OS185, OS195, and OS223, as models to assess transcriptional variation and ecotype formation within a prokaryotic population. All strains were isolated from different depths throughout a water column of the Baltic Sea, occupying different ecological niches characterized by various abiotic parameters. Although the genome sequences are nearly 100% conserved, when grown in the laboratory under standardized conditions, all strains exhibited different growth rates, suggesting significant expressional variation. Using the Ecotype Simulation algorithm, all strains were considered to be discrete ecotypes when compared to 32 other S. baltica strains isolated from the same water column, suggesting ecological divergence. Next, we employed custom microarray slides containing oligonucleotide probes representing the core genome of OS155, OS185, OS195, and OS223 to detect natural transcriptional variation among strains grown under identical conditions. Significant transcriptional variation was noticed among all four strains. Differentially expressed gene profiles seemed to coincide with the metabolic signatures of the environment at the original isolation depth. Transcriptional pattern variations such as the ones highlighted here may be used as indicators of short-term evolution emerging from the formation of bacterial ecotypes. IMPORTANCE Eukaryotic studies have shown considerable transcriptional variation among individuals from the same population. It has been suggested that natural variation in eukaryotic gene expression may have significant evolutionary consequences and may explain large-scale phenotypic divergence of closely related species, such as humans and chimpanzees (M.-C. King and A. C. Wilson, Science 188:107-116, 1975,; M. F. Oleksiak, G. A. Churchill, and D. L. Crawford, Nat Genet 32:261-266, 2002, However, natural variation in gene expression is much less well understood in prokaryotic organisms. In this study, we used four sequenced strains of the marine bacterium Shewanella baltica to better understand the natural transcriptional divergence of a stratified prokaryotic population. We found substantial low-magnitude expressional variation among the four S. baltica strains cultivated under identical laboratory conditions. Collectively, our results indicate that transcriptional variation is an important factor for ecological speciation.
  • Development of a genus-specific next generation sequencing approach for sensitive and quantitative determination of the Legionella microbiome in freshwater systems.

    Pereira, Rui P A; Peplies, Jörg; Brettar, Ingrid; Höfle, Manfred G; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-03-31)
    Next Generation Sequencing (NGS) has revolutionized the analysis of natural and man-made microbial communities by using universal primers for bacteria in a PCR based approach targeting the 16S rRNA gene. In our study we narrowed primer specificity to a single, monophyletic genus because for many questions in microbiology only a specific part of the whole microbiome is of interest. We have chosen the genus Legionella, comprising more than 20 pathogenic species, due to its high relevance for water-based respiratory infections.
  • Temperature-dependent growth modeling of environmental and clinical Legionella pneumophila MLVA-genotypes.

    Sharaby, Yehonatan; Rodríguez-Martínez, Sarah; Oks, Olga; Pecellin, Marina; Mizrahi, Hila; Peretz, Avi; Brettar, Ingrid; Höfle, Manfred; Halpern, Malka; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-02-03)
    Legionella pneumophila cause waterborne infections resulting in severe pneumonia. High resolution genotyping of L. pneumophila isolates can be achieved by Multiple-Locus Variable number of tandem repeat Analysis (MLVA). Recently, we found that different MLVA genotypes of L. pneumophila, dominated different sites in a small drinking-water network, with a genotype-related temperature and abundance regime. The current study focuses on understanding the temperature-dependent growth kinetics of the genotypes that dominated the water network. Our aim was to model mathematically the influence of temperature on the growth kinetics of different environmental and clinical L. pneumophila genotypes and compare it with their ecological niches. Environmental strains showed a distinct temperature preference with significant differences among the growth kinetics of the three studied genotypes (Gt4, Gt6 and Gt15). Gt4 strains exhibited superior growth at lower temperatures (25-30 °C) while Gt15 strains appeared to be best adapted to relatively higher temperatures (42-45 °C). The temperature-dependent growth traits of the environmental genotypes were consistent with their distribution and temperature preferences in the water network. Clinical isolates exhibited significantly higher growth rates and reached higher maximal cell densities at 37°C and 42°C than the environmental strains. Further research on the growth preferences of L. pneumophila clinical and environmental genotypes will result in better understanding of their ecological niches in drinking water systems as well as in the human body.
  • Cohort Study of Airway Mycobiome in Adult Cystic Fibrosis Patients: Differences in Community Structure between Fungi and Bacteria Reveal Predominance of Transient Fungal Elements.

    Kramer, Rolf; Sauer-Heilborn, Annette; Welte, Tobias; Guzman, Carlos A; Abraham, Wolf-Rainer; Höfle, Manfred G; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2015-09)
    The respiratory mycobiome is an important but understudied component of the human microbiota. Like bacteria, fungi can cause severe lung diseases, but their infection rates are much lower. This study compared the bacterial and fungal communities of sputum samples from a large cohort of 56 adult patients with cystic fibrosis (CF) during nonexacerbation periods and under continuous antibiotic treatment. Molecular fingerprinting based on single-strand conformation polymorphism (SSCP) analysis revealed fundamental differences between bacterial and fungal communities. Both groups of microorganisms were taxonomically classified by identification of gene sequences (16S rRNA and internal transcript spacer), and prevalences of single taxa were determined for the entire cohort. Major bacterial pathogens were frequently observed, whereas fungi of known pathogenicity in CF were detected only in low numbers. Fungal species richness increased without reaching a constant level (saturation), whereas bacterial richness showed saturation after 50 patients were analyzed. In contrast to bacteria, a large number of fungal species were observed together with high fluctuations over time and among patients. These findings demonstrated that the mycobiome was dominated by transient species, which strongly suggested that the main driving force was their presence in inhaled air rather than colonization. Considering the high exposure of human airways to fungal spores, we concluded that fungi have low colonization abilities in CF, and colonization by pathogenic fungal species may be considered a rare event. A comprehensive understanding of the conditions promoting fungal colonization may offer the opportunity to prevent colonization and substantially reduce or even eliminate fungus-related disease progression in CF.
  • Effects of Global Warming on Vibrio Ecology.

    Vezzulli, Luigi; Pezzati, Elisabetta; Brettar, Ingrid; Höfle, Manfred; Pruzzo, Carla; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2015-06)
    Vibrio-related infections are increasing worldwide both in humans and aquatic animals. Rise in global sea surface temperature (SST), which is approximately 1 °C higher now than 140 years ago and is one of the primary physical impacts of global warming, has been linked to such increases. In this chapter, major known effects of increasing SST on the biology and ecology of vibrios are described. They include the effects on bacterial growth rate, both in the field and in laboratory, culturability, expression of pathogenicity traits, and interactions with aquatic organisms and abiotic surfaces. Special emphasis is given to the effect of ocean warming on Vibrio interactions with zooplankters, which represent one of the most important aquatic reservoirs for these bacteria. The reported findings highlight the biocomplexity of the interactions between vibrios and their natural environment in a climate change scenario, posing the need for interdisciplinary studies to properly understand the connection between ocean warming and persistence and spread of vibrios in sea waters and the epidemiology of the diseases they cause.
  • Spatial distribution of Legionella pneumophila MLVA-genotypes in a drinking water system.

    Rodríguez-Martínez, Sarah; Sharaby, Yehonatan; Pecellín, Marina; Brettar, Ingrid; Höfle, Manfred; Halpern, Malka; Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany. (2015-06-15)
    Bacteria of the genus Legionella cause water-based infections, resulting in severe pneumonia. To improve our knowledge about Legionella spp. ecology, its prevalence and its relationships with environmental factors were studied. Seasonal samples were taken from both water and biofilm at seven sampling points of a small drinking water distribution system in Israel. Representative isolates were obtained from each sample and identified to the species level. Legionella pneumophila was further determined to the serotype and genotype level. High resolution genotyping of L. pneumophila isolates was achieved by Multiple-Locus Variable number of tandem repeat Analysis (MLVA). Within the studied water system, Legionella plate counts were higher in summer and highly variable even between adjacent sampling points. Legionella was present in six out of the seven selected sampling points, with counts ranging from 1.0 × 10(1) to 5.8 × 10(3) cfu/l. Water counts were significantly higher in points where Legionella was present in biofilms. The main fraction of the isolated Legionella was L. pneumophila serogroup 1. Serogroup 3 and Legionella sainthelensis were also isolated. Legionella counts were positively correlated with heterotrophic plate counts at 37 °C and negatively correlated with chlorine. Five MLVA-genotypes of L. pneumophila were identified at different buildings of the sampled area. The presence of a specific genotype, "MLVA-genotype 4", consistently co-occurred with high Legionella counts and seemed to "trigger" high Legionella counts in cold water. Our hypothesis is that both the presence of L. pneumophila in biofilm and the presence of specific genotypes, may indicate and/or even lead to high Legionella concentration in water. This observation deserves further studies in a broad range of drinking water systems to assess its potential for general use in drinking water monitoring and management.
  • High Individuality of Respiratory Bacterial Communities in a Large Cohort of Adult Cystic Fibrosis Patients under Continuous Antibiotic Treatment.

    Kramer, Rolf; Sauer-Heilborn, Annette; Welte, Tobias; Jauregui, Ruy; Brettar, Ingrid; Guzman, Carlos A; Höfle, Manfred G (2015)
    Routine clinical diagnostics of CF patients focus only on a restricted set of well-known pathogenic species. Recent molecular studies suggest that infections could be polymicrobial with many bacteria not detected by culture-based diagnostics.