Molecular surveillance of carbapenem-resistant Enterobacterales in two nearby tertiary hospitals to identify regional spread of high-risk clones in Germany, 2019-2020.

BACKGROUND: Understanding the transmission dynamics of carbapenem-resistant Enterobacterales (CRE) is critical to addressing the escalating global threat of antimicrobial resistance (AMR). Although hospital transmission of CRE has been extensively studied, information on community transmission is lacking. This study aims to identify genomic clusters of CRE from two nearby institutions that may be indicative of community or inter-facility transmission. METHODS: CRE isolates between 1st of January 2019 and 31st of December 2020 from two tertiary hospitals, detected in the respective routine microbiology laboratories, were collected and characterized by short-read whole genome sequencing. RESULTS: A total of 272 CRE were collected, with Enterobacter cloacae complex (71/192, 37%) predominant in Heidelberg and Escherichia coli (19/80, 24%) in Mannheim. The most common carbapenem resistance gene, blaOXA-48, was detected in 38% of CRE from both centres. Several putative transmission clusters were found, including 6 clusters of E. cloacae complex, 5 clusters of Klebsiella pneumoniae, 4 clusters of Citrobacter freundii, and 2 clusters each of Escherichia coli and K. aerogenes. No clusters involved isolates from both study centres, except for an ST22 C. freundii cluster. Globally circulating clones were identified between the two centres for ST131 E. coli, ST66 E. hormaechei and ST22 C. freundii. CONCLUSION: This study found no widespread transmission clusters among isolates from both centres, suggesting a hospital-specific clonal structure. This suggests that CRE clusters involving both institutions may indicate emerging or circulating clones in the community, highlighting the need for intersectoral surveillance and data sharing.

Genomic epidemiology of Streptococcus pyogenes from pharyngeal and skin swabs in Gabon.

The disease burden of Streptococcus pyogenes is particularly high in low- and middle-income countries. However, data on the molecular epidemiology of S. pyogenes in such regions, especially sub-Saharan Africa, are scarce. To address this, whole-genome sequencing (WGS) of S. pyogenes from Gabon was performed to identify transmission clusters and provide valuable genomic data for public repositories. A total of 76 S. pyogenes isolates from 73 patients, collected between September 2012 and January 2013, were characterized by short-read whole-genome sequencing. The predominant emm types were emm58.0, emm81.2 and emm223.0 with 9.2% (7 of 76), 7.9% (6 of 76), and 6.6% (5 of 76), respectively. Single-nucleotide polymorphism analysis revealed 16 putative transmission clusters. Four of these were household transmissions. Four antimicrobial genes (lmrP, tetM, tetL, and thfT) were found in the S. pyogenes isolates from this study. All strains carried lmrP. Of the 76 isolates, 64 (84.2%) carried at least one tetracycline resistance gene (tetM or tetL). Comparisons with other publicly available African genomic data revealed a significant correlation between geographical location and genetic diversity of S. pyogenes, with Gabonese strains showing similarities to those from Kenya and certain Oceanian regions. Our study showed that transmission of S. pyogenes can occur at the community/household level and that high-resolution molecular typing is needed to monitor changes in circulating clones and to detect community outbreaks. Advocacy for the adoption of WGS for comprehensive molecular characterization of S. pyogenes and data sharing through public repositories should be encouraged to understand the molecular epidemiology and evolutionary trajectory of S. pyogenes in sub-Saharan Africa. IMPORTANCE: The study conducted in Gabon underscores the critical importance of addressing the limited knowledge of the molecular epidemiology of Streptococcus pyogenes in low- and middle-income countries, particularly sub-Saharan Africa. Our molecular analysis identified predominant emm types and unveiled 16 putative transmission clusters, four involving household transmissions. Furthermore, the study revealed a correlation between geographical location and genetic diversity, emphasizing the necessity for a comprehensive understanding of the molecular epidemiology and evolutionary trajectory of S. pyogenes in various regions. The call for advocacy in adopting whole-genome sequencing for molecular characterization and data sharing through public repositories is crucial for advancing our knowledge and implementing effective strategies to combat the spread of S. pyogenes in sub-Saharan Africa.

Airway commensal bacteria in cystic fibrosis inhibit the growth of P. aeruginosa via a released metabolite.

In cystic fibrosis (CF), Pseudomonas aeruginosa infection plays a critical role in disease progression. Although multiple studies suggest that airway commensals might be able to interfere with pathogenic bacteria, the role of the distinct commensals in the polymicrobial lung infections is largely unknown. In this study, we aimed to identify airway commensal bacteria that may inhibit the growth of P. aeruginosa. Through a screening study with more than 80 CF commensal strains across 21 species, more than 30 commensal strains from various species have been identified to be able to inhibit the growth of P. aeruginosa. The underlying mechanisms were investigated via genomic, metabolic and functional analysis, revealing that the inhibitory commensals may affect the growth of P. aeruginosa by releasing a large amount of acetic acid. The data provide information about the distinct roles of airway commensals and provide insights into novel strategies for controlling airway infections.

Nasal lavage microbiome, but not nasal swab microbiome, correlates with sinonasal inflammation in children with cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) is characterized by highly viscous mucus obstructing the lower and upper airways, chronic neutrophil inflammation and infection resulting not only in lung destruction but also in paranasal sinus involvement. The pathogenesis of CF-associated chronic rhinosinusitis (CRS) is still not well understood, and it remains unclear how the microbiome in the upper airways (UAW) influences paranasal sinus inflammation. METHODS: In a cross-sectional study in pediatric patients with CF under stable disease conditions, we examined the microbiome in relation to inflammation by comparing nasal swabs (NS) and nasal lavage (NL) as two UAW sampling methods. The microbiota structure of both NS and NL was determined by 16S rRNA gene amplicon sequencing. In addition, pro-inflammatory cytokines (IL-1ß, IL-6, IL-8, TNF-α) and proteases (SLPI, TIMP-1, NE/A1-AT complex) as well as neutrophil elastase activity were measured in NL. RESULTS: Simultaneous NS and NL samples were collected from 36 patients with CF (age range: 7 - 19 years). The microbiome of NS samples was shown to be significantly lower in α-diversity and evenness compared to NL samples. NS samples were particularly found to be colonized with Staphylococcus species. NL microbiome was shown to correlate much better with the sinonasal inflammation status than NS microbiome. Especially the detection of Moraxella in NL was associated with increased inflammatory response. CONCLUSION: Our results show that the NL microbiome reflects sinonasal inflammation better than NS and support NL as a promising tool for simultaneous assessment of the UAW microbiome and inflammation in children with CF.

Monocentric observational cohort study to investigate the transmission of third-generation cephalosporin-resistant Enterobacterales in a neonatal intensive care unit in Heidelberg, Germany.

Third-generation cephalosporin-resistant Enterobacterales is a major threat for newborns in neonatal intensive care units (NICUs). The route of acquisition in a non-outbreak setting should be investigated to implement adequate infection prevention measures. To identify risk factors for colonization with and to investigate the transmission pattern of third-generation cephalosporin-resistant Enterobacterales in a NICU setting. This monocentric observational cohort study in a tertiary NICU in Heidelberg, Germany, enrolled all hospitalized neonates screened for cephalosporin-resistant Enterobacterales. Data were collected from 1 January 2018 to 31 December 2021. Weekly screening by rectal swabs for colonization with third-generation cephalosporin-resistant Enterobacterales was performed for all newborns until discharge. Whole-genome sequencing was performed for molecular characterization and transmission analysis. In total, 1,287 newborns were enrolled. The median length of stay was 20 (range 1-250) days. Eighy-eight infants (6.8%) were colonized with third-generation cephalosporin-resistant Enterobacterales. Low birth weight [<1500 g (adjusted odds ratio, 5.1; 95% CI 2.2-11.5; P < 0.001)] and longer hospitalization [per 30 days (adjusted odds ratio, 1.7; 95% CI 1.5-2.0; P < 0.001)] were associated with colonization or infection with drug-resistant Enterobacterales in a multivariate analysis. Enterobacter cloacae complex was the most prevalent third-generation cephalosporin-resistant Enterobacterales detected, 64.8% (59 of 91). Whole-genome sequencing, performed for the available 85 of 91 isolates, indicated 12 transmission clusters involving 37 patients. This cohort study suggests that transmissions of third-generation cephalosporin-resistant Enterobacterales in newborns occur frequently in a non-outbreak NICU setting, highlighting the importance of surveillance and preventive measures in this vulnerable patient group. IMPORTANCE Preterm newborns are prone to infections. Therefore, infection prevention should be prioritized in this vulnerable patient group. However, outbreaks involving drug-resistant bacteria, such as third-generation resistant Enterobacterales, are often reported. Our study aims to investigate transmission and risk factors for acquiring third-generation cephalosporin-resistant Enterobacterales in a non-outbreak NICU setting. Our data indicated that premature birth and low birth weight are significant risk factors for colonization/infection with third-generation cephalosporin-resistant Enterobacterales. Furthermore, we could identify putative transmission clusters by whole-genome sequencing, highlighting the importance of preemptive measures to prevent infections in this patient collective.

Longitudinal effects of elexacaftor/tezacaftor/ivacaftor on sputum viscoelastic properties, airway infection and inflammation in patients with cystic fibrosis.

BACKGROUND: Recent studies demonstrated that the triple combination cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy elexacaftor/tezacaftor/ivacaftor (ETI) improves lung function and reduces pulmonary exacerbations in cystic fibrosis (CF) patients with at least one F508del allele. However, effects of ETI on downstream consequences of CFTR dysfunction, i.e. abnormal viscoelastic properties of airway mucus, chronic airway infection and inflammation have not been studied. The aim of this study was to determine the longitudinal effects of ETI on airway mucus rheology, microbiome and inflammation in CF patients with one or two F508del alleles aged ≥12 years throughout the first 12 months of therapy. METHODS: In this prospective observational study, we assessed sputum rheology, the microbiome, inflammation markers and proteome before and 1, 3 and 12 months after initiation of ETI. RESULTS: In total, 79 patients with CF and at least one F508del allele and 10 healthy controls were enrolled in this study. ETI improved the elastic modulus and viscous modulus of CF sputum at 3 and 12 months after initiation (all p<0.01). Furthermore, ETI decreased the relative abundance of Pseudomonas aeruginosa in CF sputum at 3 months and increased the microbiome α-diversity at all time points. In addition, ETI reduced interleukin-8 at 3 months (p<0.05) and free neutrophil elastase activity at all time points (all p<0.001), and shifted the CF sputum proteome towards healthy. CONCLUSIONS: Our data demonstrate that restoration of CFTR function by ETI improves sputum viscoelastic properties, chronic airway infection and inflammation in CF patients with at least one F508del allele over the first 12 months of therapy; however, levels close to healthy were not reached.

Genome-scale metabolic modeling of Aspergillus fumigatus strains reveals growth dependencies on the lung microbiome.

Aspergillus fumigatus, an opportunistic human pathogen, frequently infects the lungs of people with cystic fibrosis and is one of the most common causes of infectious-disease death in immunocompromised patients. Here, we construct 252 strain-specific, genome-scale metabolic models of this important fungal pathogen to study and better understand the metabolic component of its pathogenic versatility. The models show that 23.1% of A. fumigatus metabolic reactions are not conserved across strains and are mainly associated with amino acid, nucleotide, and nitrogen metabolism. Profiles of non-conserved reactions and growth-supporting reaction fluxes are sufficient to differentiate strains, for example by environmental or clinical origin. In addition, shotgun metagenomics analysis of sputum from 40 cystic fibrosis patients (15 females, 25 males) before and after diagnosis with an A. fumigatus colonization suggests that the fungus shapes the lung microbiome towards a more beneficial fungal growth environment associated with aromatic amino acid availability and the shikimate pathway. Our findings are starting points for the development of drugs or microbiome intervention strategies targeting fungal metabolic needs for survival and colonization in the non-native environment of the human lung.

Immune Response in Cystic Fibrosis: Interplay between the Host and Microbes.

Cystic fibrosis (CF) is a rare genetic disease caused by genetic variants of the cystic fibrosis transmembrane conductance regulator (CFTR) [...].

Genomic Modification of TonB and Emergence of Small-Colony Phenotype in VIM- and NDM-Producing Escherichia coli following Cefiderocol Exposure In Vitro.

Knowledge on resistance mechanisms toward cefiderocol, a novel siderophore-conjugated cephalosporin antibiotic, is still limited. Although the presence of New-Delhi metallo-ß-lactamase has been demonstrated to facilitate the resistance development toward cefiderocol via siderophore receptor mutations in Enterobacter cloacae and Klebsiella pneumoniae, the impact of metallo-ß-lactamases on facilitating such mutations in Escherichia coli is not yet elucidated. Our study aimed to study the effect of the presence of various ß-lactamases, such as NDM-5, VIM-1, KPC-2, and OXA-48, on the development of cefiderocol resistance in E. coli. To this end, we performed liquid mating to transfer these ß-lactamases onto a defined K-12 E. coli background (J53) and exposed these transconjugants to increasing cefiderocol concentrations in a serial passage experiment. Cefiderocol-resistant isolates were genotyped by whole-genome sequencing to investigate the underlying resistance mechanism. Cefiderocol-resistant isolates emerged only in isolates producing VIM-1 and NDM-5 metallo-ß-lactamase, but not in those producing the serine ß-lactamases KPC-2 and OXA-48. We observed two distinct morphological changes of the J53 E. coli strain exhibiting reduced colony size after insertions of transposable elements in the tonB gene leading to alterations in the TonB binding site and morphological changes consistent with the small-colony variant (SCV) phenotype due to mutations in the hemB and hemH genes. Passaging experiments suggested that these phenotypes were highly plastic. The SCV phenotype is attributed to immune evasion and decreased susceptibility toward antibiotics. The emergence of SCV following cefiderocol exposure may have clinical implications for bacterial clearance and warrants further investigation.

Gain a Baby Lose a Tooth-Is There an Association between Periodontitis and Preterm Birth?

Preterm birth serves as one of the leading causes of neonatal mortality worldwide. The underlying mechanisms that contribute to preterm birth are not yet fully understood. However, an association between periodontitis and preterm birth has been proposed. The periodontal status and presence of periodontal pathogens in women with different birth outcomes have been previously examined. However, varying definitions of periodontitis and different microbiological methods make their interpretation challenging. The aim of this case-control study on women with and without preterm birth was to investigate their periodontal status using the current classification system for periodontal diseases. Moreover, differences in the periodontal microbiome of the study participants were investigated. Therefore, we collected data on oral and periodontal parameters in 77 puerperal women divided into two groups based on gestational age at delivery: 33 patients with preterm birth (PTB, <37 weeks) and 44 patients with term birth (TB, >37 weeks). These data included pocket probing depth (PPD), clinical attachment loss (CAL), bleeding on probing (BOP), gingival-bleeding index, DMFT index, and gynecologic and dental history. In addition, their oral microbiome was explored. Median CAL and percentage PPD ≥ 4 mm were significantly higher in the PTB group than in the TB group (p = 0.0128 and p = 0.047, respectively). Birth weight was significantly higher in periodontally healthy women than in those with gingivitis (p = 0.0078) or periodontitis (p = 0.0127). The periodontal microbiome differed significantly between groups. Our results are underlining the possible association between periodontitis and preterm delivery. Women with periodontitis had babies with significantly lower birth weights. The microbiome varied between the groups.

The acquisition of transferable extrachromosomal fec operon is associated with a cefiderocol MIC increase in Enterobacterales.

BACKGROUND: Cefiderocol is a novel siderophore cephalosporin active against MDR Gram-negative bacilli, including MBL-harbouring Enterobacterales. The detection of multiple cefiderocol-resistant blaVIM-carrying Enterobacterales isolates (MIC = 4 mg/L) from a single patient suggested an additional, potentially transferable, resistance determinant as blaVIM typically does not elevate cefiderocol MIC above the resistance threshold. METHODS: Transfer of a mobile genetic element was performed in liquid mating experiments. All donor isolates and transconjugants were characterized by short-read WGS to identify potential resistance determinants. mRNA expression of siderophore receptors was determined by quantitative RT-PCR. Validation was performed by transformation. Antibiotic susceptibility was determined by broth microdilution. RESULTS: Liquid mating experiments indicated the presence of transferable resistance determinants. Comparative genomic analysis of the clinical isolates and their respective transconjugants revealed the transfer of an accessory fec operon (fecABCDEIR). Transformation of the fec operon-containing vector into a TOP10 Escherichia coli led to an elevation of the cefiderocol MIC by at least 16-fold. Higher expression of fecA as a proxy for the fec operon mRNA expression was associated with phenotypic cefiderocol resistance. Both VIM and the accessory fec operon contribute to the elevation of cefiderocol MIC beyond the resistance threshold. The acquisition of an accessory fec operon via liquid mating confers phenotypic cefiderocol resistance in both E. coli J53 and Pseudomonas aeruginosa PAO1, indicating a broad-host-range nature of this mobile resistance determinant. CONCLUSIONS: The emergence of a transferable cefiderocol resistance determinant without prior exposure to the substance is worrisome and should be monitored closely.

Diagnosis of pathogens causing bacterial meningitis using Nanopore sequencing in a resource-limited setting.

AIM: The aim of the present study is to compare the performance of 16S rRNA Nanopore sequencing and conventional culture in detecting infectious pathogens in patients with suspected meningitis in a resource-limited setting without extensive bioinformatics expertise. METHODS: DNA was isolated from the cerebrospinal fluid (CSF) of 30 patients with suspected bacterial meningitis. The isolated DNA was subjected to 16S sequencing using MinION™. The data were analysed in real time via the EPI2ME cloud platform. The Nanopore sequencing was done in parallel to routine microbiological diagnostics. RESULTS: Nanopore sequencing detected bacterial pathogens to species level in 13 of 30 (43%) samples. CSF culture showed 40% (12/30) positivity. In 21 of 30 patients (70%) with suspected bacterial meningitis, both methods yielded concordant results. About nine of 30 samples showed discordant results, of these five were false positive and four were false negative. In five of the culture negative results, nanopore sequencing was able to detect pathogen genome, due to the higher sensitivity of the molecular diagnostics. In two other samples, the CSF culture revealed Cryptococcus neoformans and Streptococcus pneumoniae, which were not detected by Nanopore sequencing. Overall, using both the cultures and 16S Nanopore sequencing, positivity rate increased from 40% (12/30) to 57% (17/30). CONCLUSION: Next-generation sequencing could detect pathogens within six hours and could become an important tool for both pathogen screening and surveillance in low- and middle-income countries (LMICs) that do not have direct access to extensive bioinformatics expertise.

Changes in Microbiome Dominance Are Associated With Declining Lung Function and Fluctuating Inflammation in People With Cystic Fibrosis.

Airway inflammation and microbiome dysbiosis are hallmarks of cystic fibrosis (CF) lung disease. However, longitudinal studies are needed to decipher which factors contribute to the long-term evolution of these key features of CF. We therefore evaluated the relationship between fluctuation in microbiome and inflammatory parameters in a longitudinal study including a short- (1-year) and a long-term (3+ years) period. We collected 118 sputum samples from 26 CF adult patients and analyzed them by 16S rRNA gene sequencing. We measured the levels of inflammatory cytokines, neutrophil elastase, and anti-proteinases; lung function (FEV1% predicted); and BMI. The longitudinal evolution was analyzed based on (i) the rates of changes; (ii) the intra-patient stability of the variables; and (iii) the dependency of the rates of changes on the baseline values. We observed that the diversity of the microbiome was highly variable over a 1-year period, while the inflammatory markers showed a slower evolution, with significant changes only observed in the 3+ year cohort. Further, the degree of fluctuation of the biomass and the dominance of the microbiome were associated with changes in inflammatory markers, especially IL-1ß and IL-8. This longitudinal study demonstrates for the first time that the long-term establishment and periodical variation of the abundance of a dominant pathogen is associated with a more severe increase in inflammation. This result indicates that a single time point or 1-year study might fail to reveal the correlation between microbial evolution and clinical degradation in cystic fibrosis.

Maintaining oral health for a hundred years and more? - An analysis of microbial and salivary factors in a cohort of centenarians.

Aim: To investigate associations between oral health-related conditions and the oral microbiome in a representative study sample of centenarians. Materials and methods: Clinical and microbial parameters from 54 centenarians were assessed in the Heidelberg Dental Centenarian Study. Plaque and salivary samples were collected, and the microbiota was characterized by 16S rRNA gene sequencing. Results: Diversity and structure of the oral microbiome were mainly influenced by the presence of natural teeth and the number of decayed, missing, and filled teeth (0.028 ≤ p ≤ 0.001 in plaque and salivary samples). Centenarians with less caries experience possessed a more diverse oral microbiome. Moreover, the number of dental visits also showed a significant influence on the microbial composition. Most centenarians presented with hyposalivation (mean stimulated flow rate = 0.84 ± 0.55 ml/min), a low buffering capacity, and an acidic pH. The latter was between 5.0 and 5.8 in 46.3% of cases, and we observed that an increased salivary pH correlated with higher alpha-diversity in both salivary and plaque samples. Conclusion: The microbiome diversity correlated significantly with successful oral aging. In addition, regular dental visits were a beneficial factor. However, diversity can be negatively influenced by hyposalivation, associated with pH changes due to aging effects.

Comparative Genomic Reveals Clonal Heterogeneity in Persistent Staphylococcus aureus Infection.

Persistent infections caused by Staphylococcus aureus remain a clinical challenge. Adaptational mechanisms of the pathogen influencing infection persistence, treatment success, and clinical outcome in these types of infections by S. aureus have not been fully elucidated so far. We applied a whole-genome sequencing approach on fifteen isolates retrieved from a persistent S. aureus infection to determine their genetic relatedness, virulome, and resistome. The analysis of the genomic data indicates that all isolates shared a common clonal origin but displayed a heterogenous composition of virulence factors and antimicrobial resistance. This heterogeneity was reflected by different mutations in the rpoB gene that were related to the phenotypic antimicrobial resistance towards rifampicin and different minimal inhibitory concentrations of oxacillin. In addition, one group of isolates had acquired the genes encoding for staphylokinase (sak) and staphylococcal complement inhibitor (scn), leading to the truncation of the hemolysin b (hlb) gene. These features are characteristic for temperate phages of S. aureus that carry genes of the immune evasion cluster and confer triple conversion by integration into the hlb gene. Modulation of immune evasion mechanisms was demonstrated by significant differences in biofilm formation capacity, while invasion and intracellular survival in neutrophils were not uniformly altered by the presence of the immune evasion cluster. Virulence factors carried by temperate phages of S. aureus may contribute to the course of infection at different stages and affect immune evasion and pathogen persistence. In conclusion, the application of comparative genomic demonstrated clonal heterogeneity in persistent S. aureus infection.

Commensal Bacteria in the Cystic Fibrosis Airway Microbiome Reduce P. aeruginosa Induced Inflammation.

Chronic Pseudomonas aeruginosa infections play an important role in the progress of lung disease in patients suffering from cystic fibrosis (CF). Recent studies indicate that polymicrobial microbiome profiles in the airway are associated with less inflammation. Thus, the hypothesis was raised that certain commensal bacteria might protect the host from inflammation. We therefore performed a screening study with commensals isolated from CF airway microbiome samples to identify potential beneficial commensals. We isolated more than 80 aerobic or facultative anaerobic commensal strains, including strains from genera Streptococcus, Neisseria, Actinomyces, Corynebacterium, Dermabacter, Micrococcus and Rothia. Through a screening experiment of co-infection in human epithelial cell lines, we identified multiple commensal strains, especially strains belonging to Streptococcus mitis, that reduced P. aeruginosa triggered inflammatory responses. The results were confirmed by co-infection experiments in ex-vivo precision cut lung slices (PCLS) from mice. The underlying mechanisms of the complex host-pathogen-commensal crosstalk were investigated from both the host and the bacterial sides with a focus on S. mitis. Transcriptome changes in the host in response to co-infection and mono-infection were evaluated, and the results indicated that several signalling pathways mediating inflammatory responses were downregulated by co-infection with S. mitis and P. aeruginosa compared to P. aeruginosa mono-infection, such as neutrophil extracellular trap formation. The genomic differences among S. mitis strains with and without protective effects were investigated by whole genome sequencing, revealing genes only present in the S. mitis strains showing protective effects. In summary, through both in vitro and ex vivo studies, we could identify a variety of commensal strains that may reduce host inflammatory responses induced by P. aeruginosa infection. These findings support the hypothesis that CF airway commensals may protect the host from inflammation.

Can the Acid-formation Potential of Saliva Detect a Caries-related Shift in the Oral Microbiome?

PURPOSE: To determine acid-formation potential of saliva and evaluate whether this method corresponds with microbiome composition of individuals with and without caries. MATERIALS AND METHODS: A clinical, controlled pilot study was performed with two groups: individuals without caries (n = 25; DMFT = 0) and individuals with at least one active carious lesion (n = 25; DMFT>0). A detailed intraoral examination was performed, and the gingival bleeding index (GBI) and plaque index (PI) were recorded. The acid-formation potential was measured (ΔpH) after 1 h. Streptococcus mutans (SM) and lactobacilli (LB) were also quantified. Intergroup comparisons were made using the Mann-Whitney U-test. The diagnostic value was evaluated using the receiver operating characteristics (ROC) method and area under the curve (AUC) values were calculated. The saliva microbiome was analysed by 16S rDNA next-generation sequencing. RESULTS: A statistically significant difference was found in ΔpH, with the 'caries' group showing a higher mean value after 1 h ('healthy' = 1.1,'caries' = 1.4; p = 0.035). The AUC values were moderate to good (ΔpH = 0.67; SM = 0.83; LB = 0.83;1 = ideal). Streptococcus mutans and Lactobacilli were more frequently detected in the 'caries' group (p < 0.001), as were statistically significantly higher GBI (p = 0.006) and PI (p = 0.001). The saliva microbiome had a higher α-diversity and greater richness in individuals with active caries. The incidence of the genera Alloprevotella, Prevotella, Campylobacter and Veillonella was statistically significantly higher in the 'healthy' group. The incidence of the genera Fretibacterium, Lactobacillus, and Leptotrichia, as well as the phyla Spirochaetes and Synergistetes, was statistically significantly higher in the 'caries' group. CONCLUSION: Further studies must be carried out to determine the extent to which both tests are suitable for predicting future caries development.