The evolution of antimicrobial-resistant pathogens poses a global health threat, and it is unclear if evolutionary trajectories to resistance lead to predictable phenotypes. We analyzed antimicrobial resistance (AMR) evolution in the human pathogen Haemophilus influenzae in controlled evolution experiments with increasing concentrations of either ampicillin, cefotaxime, or ceftriaxone. We isolated 315 clones from different time points of six independent experiments and characterized changes in genome sequences, bacterial fitness, and minimum inhibitory concentrations (MICs) to 14 antibiotics. Resistance evolution under ampicillin and cefotaxime was mainly driven by mutations in the ftsI gene, encoding the penicillin-binding protein 3. However, ceftriaxone exposure repeatedly selected for amino acid substitutions in the outer membrane protein P2 (OmpP2). Some OmpP2 mutants reproducibly showed phenotypic heterogeneity not only for ceftriaxone but also for fluoroquinolones, rifampicin, and tetracycline. Bacterial fitness assessments revealed trade-offs between resistance-associated mutations and growth, though no systematic correlation of MIC increase and growth deficit was detected. Over 50% of the selected clones became more susceptible to aminoglycosides, clarithromycin, and colistin, while some mutation patterns resulted in cross-resistance to meropenem and fluoroquinolones. Overall, beta-lactam antibiotics reproducibly selected mutants with increased MICs, but evolutionary pathways and resulting phenotypes remain unpredictable. These findings highlight the complexity of resistance evolution and suggest that future AMR treatment strategies may need to consider strain-specific and collateral effects more closely.

The increasing prevalence of antimicrobial resistance (AMR) poses significant challenges in clinical settings. In particular, early screening and detection of colonization by multidrug-resistant organisms (MDROs) in patients at admission is crucial. In this context, the clinical use of metagenomics (mNGS) holds promise for fast and untargeted diagnostic methods. Here, we aimed to evaluate the long-term stability of the rectal microbiome and the diagnostic accuracy of mNGS in comparison to culture and whole-genome sequencing (WGS) of MDROs. We analyzed rectal swabs from 26 patients with two consecutive admissions over a four-year period. The detected antimicrobial resistance genes and assembled metagenomes were compared to those obtained via classical culture-based antimicrobial susceptibility testing and WGS of isolated MDROs. Our results showed that the rectal microbiome is variable during the two timepoints, highlighting the variability in the niche. Nevertheless, we also observed strong co-occurrence of taxa, suggesting that the rectal swab microbiome is also a regulated environment with cooperative biotic interactions. In total, we isolated and sequenced 6 MDROs from 6 patients at individual timepoints. Almost all AMR genes from the genomes of the isolates (median: 100%, range: 84.6-100%) could be detected by mNGS of the rectal swabs at the time of isolation of the MDRO but not at the time of culture negativity. In addition, we detected AMR genes and potentially pathogenic species in patients with negative cultures. In conclusion, our study showed that, in principle, mNGS of rectal swabs can detect clinically relevant AMR profiles. However, the cooccurrence of AMR genes and potentially-pathogenic species does not always correlate with culture-based diagnostic results but rather indicates a potential risk of horizontal AMR gene transfer. However, it is unclear whether the observed discrepancies are due to transient or locally confined colonization of MDROs, limits of detection, or variability of the sampling method and specimens.

Background Pathogenic mycobacteria, such as Mycobacterium tuberculosis complex (Mtbc), and non-tuberculous mycobacteria (NTMs) can cause severe chronic pulmonary infections. However, not all infected patients develop active disease, and it remains unclear whether key lung microbiome taxa play a role in the pathogenesis of tuberculosis (TB) and NTM lung diseases (LD). Here, we aim to further define the lung microbiome composition in TB, and NTM-LD prior to the initiation of therapy. Study design We employed 16S rRNA amplicon sequencing to characterize the baseline microbiome in bronchoalveolar lavage fluid (BALF) from patients diagnosed with TB (n = 23), NTM-LD (n = 19), or non-infectious inflammatory disease (n = 4). We applied depletion of human cells, removal of extracellular DNA, implementation of a decontamination strategy, and exploratory whole-metagenome sequencing (WMS) of selected specimens. Results Genera Serratia and unclassified Yersiniaceae dominated the lung microbiome of most patients with a mean relative abundance of >15% and >70%, respectively. However, at the sub-genus level, as determined by amplicon sequence variants (ASVs), TB-patients exhibited increased community diversity, and distinct signatures of ASV_7, ASV_21 abundances which resulted in a significant association with disease state. Exploratory WMS, and ASV similarity analyses suggested the presence of Serratia liquefaciens , Serratia grimesii , Serratia myotis and/or Serratia quinivorans in TB and NTM-LD patients. Conclusions The lung microbiome of TB-patients harbored a distinct, and heterogenous structure, with specific occurrences of certain Serratia traits. Some of these traits may play a role in understanding the microbial interactions in the lung microbiome of patients infected with Mtbc.

Non-tuberculous mycobacteria (NTM) are a diverse group of environmental bacteria that are increasingly associated with human infections and difficult to treat. Plasmids, which might carry resistance and virulence factors, remain largely unexplored in NTM. We used publicly available complete genome sequence data of 328 NTM isolates belonging to 125 species to study gene content, genomic diversity, and clusters of 196 annotated NTM plasmids. Furthermore, we analyzed 3755 draft genome assemblies from over 200 NTM species and 5415 short-read sequence datasets from six clinically relevant NTM species or complexes including M. abscessus, M. avium complex, M. ulcerans complex and M. kansasii complex, for the presence of these plasmids. Between one and five plasmids were present in approximately one-third of the complete NTM genomes. The annotated plasmids varied widely in length (most between 10 and 400 kbp) and gene content, with many genes having an unknown function. Predicted gene functions primarily involved plasmid replication, segregation, maintenance, and mobility. Only a few plasmids contained predicted genes that are known to confer resistance to antibiotics commonly used to treat NTM infections. Out of 196 annotated plasmid sequences, 116 could be grouped into 31 clusters of closely related sequences, and about one-third were found across multiple NTM species. Among clinically relevant NTM, the presence of NTM plasmids showed significant variation between species, within (sub)species, and even among strains within (sub)lineages, such as dominant circulating clones of Mycobacterium abscessus. Our analysis demonstrates that plasmids are a diverse and heterogeneously distributed feature in NTM bacteria. The frequent occurrence of closely related putative plasmid sequences across different NTM species suggests they may play a significant role in NTM evolution through horizontal gene transfer at least in some groups of NTM. However, further in vitro investigations and access to more complete genomes are necessary to validate our findings, elucidate gene functions, identify novel plasmids, and comprehensively assess the role of plasmids in NTM.

The gut microbiome (GM) is increasingly recognized as a key modulator of neurodevelopment via the microbiome-gut-brain axis. Fragile X syndrome (FXS), the most common inherited monogenic cause of intellectual disability, shares behavioural and molecular features with other neurodevelopmental disorders (NDDs), yet the role of the GM in FXS remains largely unexplored. In this open-label, single-arm trial, 15 children with genetically confirmed FXS received a daily probiotic formulation containing Lactobacillus casei, Lactobacillus salivarius, and Bifidobacterium breve for 12 weeks. Behavioural analysis and metagenomic sequencing with network and pathway analyses were performed before and after probiotic supplementation. Significant improvements were observed in irritability (-3.9, SD: ± 5.2; p = 0.027), communication (+ 1.7, SD: ± 2.5; p = 0.022), socialization (+ 1.4, SD: ± 2.1; p = 0.033), and adaptive behaviour (+ 1.3, SD: ± 1.4; p = 0.004). While overall microbial diversity remained stable, SparCC network analysis revealed increases in connectivity measures such as edge count and clustering coefficient, indicating denser microbial interactions and greater community coordination after probiotic supplementation. Functional profiling showed trends toward increased microbial activity in fatty acid biosynthesis, NAD salvage, and starch degradation pathways. This pilot study provides initial evidence that probiotics may modulate structural and functional properties of the GM, with potential links to improved behavioural outcomes in children with FXS. Larger, controlled trials are needed to validate the therapeutic potential of GM-targeted interventions in FXS and related NDDs.

Mycobacterium tuberculosis is well adapted to survive and persist in the infected host, escaping the host's immune response. Since polyamines such as spermine, which are synthesized by infected macrophages, are able to inhibit the growth of M. tuberculosis, the pathogen needs strategies to cope with these toxic metabolites. The actinomycete Streptomyces coelicolor, a close relative of M. tuberculosis, makes use of a gamma-glutamylation pathway to functionally neutralize spermine. We therefore considered whether a similar pathway would be functional in M. tuberculosis. In the current study, we demonstrated that M. tuberculosis growth was inhibited by the polyamine spermine. Using in vitro enzymatic assays we determined that GlnA3Mt (Rv1878) possesses genuine gamma-glutamylspermine synthetase catalytic activity. We further showed that purified His-Strep-GlnA3Mt, as well as native GlnA3Mt, prefer spermine as a substrate over putrescine, cadaverine, spermidine, or other monoamines and amino acids, suggesting that GlnA3Mt may play a specific role in the detoxification of the polyamine spermine. However, the deletion of the glnA3 gene in M. tuberculosis did not result in growth inhibition or enhanced sensitivity of M. tuberculosis in the presence of high spermine concentrations. Gene expression analysis of spermine-treated M. tuberculosis revealed no difference in the level of glnA3Mt expression relative to untreated cells, whereas a gene encoding a previously characterized efflux pump (Mmr; rv3065) was significantly upregulated. This suggests that bacterial survival under elevated spermine concentrations can not only be achieved by detoxification of spermine itself but also by mechanisms resulting in decreased spermine levels in the bacteria. IMPORTANCE:Upon Mycobacterium tuberculosis infection macrophages synthesize the polyamine spermine, which at elevated concentrations is toxic for M. tuberculosis. Based on our investigations of spermine resistance in the closely related actinomycete Streptomyces coelicolor, we hypothesized that the glutamylspermine synthetase GlnA3 may be responsible for the resistance of M. tuberculosis against toxic spermine. Here we show that GlnA3Mt can indeed covalently modify spermine via glutamylation. However, GlnA3Mt is probably not the only resistance mechanism since a glnA3 null mutant of M. tuberculosis can survive under spermine stress. Gene expression studies suggest that an efflux pump may participate in resistance. Thus a combination of GlnA3Mt and specific efflux pumps acting as putative spermine transporters may constitute an active spermine-detoxification system in M. tuberculosis.

Global health is threatened by the rise of antibiotic resistance. Bacteria of the Mycobacterium tuberculosis complex (Mtbc) are a major contributor to this antibiotic crisis, with about 450,000 new multidrug-resistant tuberculosis (MDR-TB) cases per year. This study investigates resistance evolution by defining the resistance mutant selection window (MSW) for the important MDR-TB treatment drugs moxifloxacin and bedaquiline. We employed a combination of long-term in vitro experiments supplemented with mathematical modeling that combined pharmacodynamics with population genetics. We assessed resistance selection at concentrations below the minimum inhibitory concentration (MIC), the MSW and fitness cost of eight mutant clones with different resistance-associated variants. Both computational and experimental results show that mutant clone populations are selected far below the MIC, leading to a major growth advantage of resistant populations under weak selection pressure. An eighth of the MIC was enough to enrich mutant clone populations in the short term (five bacterial passages or 20 generations), even in mutant clones with a major competitive fitness loss. In fact, gyrA, gyrB and most Rv0678 mutations have virtually no effect on the bacteria's competitive fitness in vitro. This work highlights the risk that ineffective drug delivery and dosing can lead to the emergence of resistance.

OBJECTIVES:Heteroresistance, the presence of a resistant subpopulation within a predominantly susceptible bacterial isolate, is usually difficult to detect and can cause treatment failure. Mechanisms driving unstable heteroresistant phenotypes, which rapidly revert in the absence of antibiotics, remain poorly understood. METHODS:To investigate the mechanisms underlying heteroresistance in Haemophilus influenzae, we isolated four heteroresistant clones and characterized them through experimental evolution, gradient diffusion assays, population analysis profiling (PAP), and whole genome sequencing. RESULTS:All clones initially exhibited increased ceftriaxone minimum inhibitory concentrations (MICs) but rapidly reverted to wild-type susceptibility within 4 days in drug-free conditions. The main mechanism was reversible structural changes of the ompP2 gene. Three clones exhibiting a 168 bp genomic inversion affecting ompP2 reverted to wild-type sequence. A fourth clone with an ompP2 frameshift insertion reproducibly acquired compensatory deletions restoring the open reading frame. PAP analysis still detected minority subpopulations with 4x-8x increased MICs at day 4. We identified site-specific DNA breakpoints with a palindrome and a homologous sequence facilitating rapid structural genome changes and phenotypic switching. CONCLUSION:We discovered a novel mechanism of unstable heteroresistance in H. influenzae, mediated by reversible structural changes in the gene ompP2. Rapid phenotypic switching was induced by ceftriaxone exposure and likely reflects the mutational plasticity of ompP2.

BACKGROUND:Several European countries have reported a rise in invasive Group A Streptococcus (GAS) infections, particularly linked to the toxigenic emm1 sublineage M1UK. In Germany, historical molecular data are limited due to the absence of systematic molecular surveillance. METHODS:We performed whole-genome sequencing (WGS) on 189 invasive Streptococcus pyogenes isolates collected between January 1, 2015, and May 31, 2023, at University Medical Center Carl Gustav Carus, TU Dresden. Clinical data were extracted from patient records. M1UK sublineage identification was based on 27 characteristic single nucleotide polymorphisms (SNPs). A Bayesian coalescent analysis estimated the evolutionary timescales of the M1UK clade in Germany. RESULTS:The most common emm type was emm1 (34%, 64/189), followed by emm12, emm4, and emm89. Of the 64 emm1 isolates, 31 (48%) were M1UK. No significant associations were found between clinical outcomes and M1UK or M1global genotypes. Although a post-pandemic shift favouring M1UK was observed, our analysis indicates that M1UK had already been circulating in Germany by 2017. The estimated most recent common ancestor dates to 2012 (95% highest posterior density: 2009-2015), with a stable effective population size over time. CONCLUSION:Our findings confirm the pre-pandemic circulation of M1UK in Germany. While the clinical impact of M1UK remains unclear, integrating clinical data with high-resolution molecular surveillance may improve early detection of emerging high-risk clones.

ABSTRACTPrevious work reported unprecedented differences in the intrinsic in vitro susceptibility of the Mycobacterium tuberculosis complex (MTBC) to pretomanid (Pa) using the Mycobacteria Growth Indicator Tube (MGIT) system. We tested 125 phylogenetically diverse strains from all known MTBC lineages (1–9) without known Pa resistance mutations and four strains with known resistance mutations as controls. This confirmed that MTBC, unlike most bacteria-antimicrobial combinations, displayed substantial differences in the intrinsic susceptibility relative to the technical variation of Pa MIC testing. This was also the case for the Middlebrook 7H11 (7H11) medium, demonstrating that these differences were not specific to MGIT. Notably, lineage 1 was confirmed to have intrinsically elevated MICs compared with lineages 2, 3, 4, and 7 (L2–4/7), underlining the urgent need for WHO to publish its decision of whether lineage 1 should be deemed treatable by BPaL(M), the now preferred all-oral regimen for treating rifampin-resistant tuberculosis. Lineages 5 and 6, which are most frequent in West Africa, responded differently to Pa, with lineage 5 being more similar to L2–4/7 and lineage 6 being more susceptible. More data are needed to determine whether 7H11 MICs are systematically lower than those in MGIT.IMPORTANCEThis study confirmed that the Mycobacterium tuberculosis complex lineage 1, responsible for 28% of global tuberculosis cases, is less susceptible to pretomanid (Pa). It also refined the understanding of the intrinsic susceptibilities of lineages 5 and 6, most frequent in West Africa, and lineages 8 and 9. Regulators must review whether these in vitro differences affect the clinical efficacy of the WHO-recommended BPaL(M) regimen and set breakpoints for antimicrobial susceptibility testing accordingly. Notably, regulators should provide detailed justifications for their decisions to facilitate public scrutiny.

Whole genome sequencing of Mycobacterium tuberculosis complex (MTBC) isolates has been shown to provide accurate predictions for resistance and susceptibility for many first- and second-line anti-tuberculosis drugs. However, bioinformatic pipelines and mutation catalogs to predict antimicrobial resistances in MTBC isolates are often customized and detailed protocols are difficult to access. Here, we provide a step-by-step workflow for the processing and interpretation of short-read sequencing data and give an overview of available analysis pipelines.

Abstract Background Haemophilus influenzae is an opportunistic bacterial pathogen that can cause severe respiratory tract and invasive infections. The emergence of β-lactamase-negative ampicillin-resistant (BLNAR) strains and unclear correlations between genotypic (i.e., gBLNAR) and phenotypic resistance are challenging empirical treatments and patient management. Thus, we sought to revisit molecular resistance mechanisms and to identify new resistance determinants of H. influenzae. Methods We performed a systematic meta-analysis of H. influenzae isolates (n = 291) to quantify the association of phenotypic ampicillin and cefotaxime resistance with previously defined resistance groups, i.e., specific substitution patterns of the penicillin binding protein PBP3, encoded by ftsI. Using phylogenomics and a genome-wide association study (GWAS), we investigated evolutionary trajectories and novel resistance determinants in a public global cohort (n = 555) and a new clinical cohort from three European centers (n = 298), respectively. Results Our meta-analysis confirmed that PBP3 group II- and group III-related isolates were significantly associated with phenotypic resistance to ampicillin (p < 0.001), while only group III-related isolates were associated with resistance to cefotaxime (p = 0.02). The vast majority of H. influenzae isolates not classified into a PBP3 resistance group were ampicillin and cefotaxime susceptible. However, particularly group II isolates had low specificities (< 16%) to rule in ampicillin resistance due to clinical breakpoints classifying many of them as phenotypically susceptible. We found indications for positive selection of multiple PBP3 substitutions, which evolved independently and often step-wise in different phylogenetic clades. Beyond ftsI, other possible candidate genes (e.g., oppA, ridA, and ompP2) were moderately associated with ampicillin resistance in the GWAS. The PBP3 substitutions M377I, A502V, N526K, V547I, and N569S were most strongly related to ampicillin resistance and occurred in combination in the most prevalent resistant haplotype H1 in our clinical cohort. Conclusions Gradient agar diffusion strips and broth microdilution assays do not consistently classify isolates from PBP3 groups as phenotypically resistant. Consequently, when the minimum inhibitory concentration is close to the clinical breakpoints, and genotypic data is available, PBP3 resistance groups should be prioritized over susceptible phenotypic results for ampicillin. The implications on treatment outcome and bacterial fitness of other extended PBP3 substitution patterns and novel candidate genes need to be determined.

Applying whole-genome-sequencing, we aimed to detect transmission events of multidrug-resistant/rifampin-resistant strains of Mycobacterium tuberculosis complex at a tuberculosis hospital in Chisinau, Moldova. We recorded ward, room, and bed information for each patient and monitored in-hospital transfers over 1 year. Detailed molecular and patient surveillance revealed only 2 nosocomial transmission events.

•Moxifloxacin daily doses of 2400 mg were tolerated over several months.•M. tuberculosis drug resistance could be overcome by a high-dose treatment regimen.•No occurrence of cardiac AEs despite high doses of QTc interval prolonging drugs.•No eradication of M. tuberculosis despite longstanding culture conversion.•Development of pretomanid resistance on an insufficient treatment regimen.

Globally, tuberculosis (TB) remains the deadliest bacterial infectious disease, and spreading antibiotic resistances is the biggest challenge for combatting the disease. Rapid and comprehensive diagnostics including drug susceptibility testing (DST) would assure early treatment, reduction of morbidity and the interruption of transmission chains. To date, rapid genetic resistance testing addresses only one to four drug groups while complete DST is done phenotypically and takes several weeks. To overcome these limitations, we developed a two-stage workflow for rapid TB diagnostics including DST from a single sputum sample that can be completed within three days. The first stage is qPCR detection of M. tuberculosis complex (MTBC) including antibiotic resistance testing against the first-line antibiotics, isoniazid (Inh) and rifampicin (Rif). The test is automated by centrifugal microfluidics and designed for point of care (PoC). Furthermore, enriched MTBC DNA is provided in a detachable sample tube to enable the second stage: if the PCR detects MTBC and resistance to either Inh or Rif, the MTBC DNA is shipped to specialized facilities and analyzed by targeted next generation sequencing (tNGS) to assess the complete resistance profile. Proof-of-concept testing of the PoC test revealed an analytical sensitivity of 44.2 CFU ml-1, a diagnostic sensitivity of 96%, and a diagnostic specificity of 100% for MTBC detection. Coupled tNGS successfully provided resistance profiles, demonstrated for samples from 17 patients. To the best of our knowledge, the presented combination of PoC qPCR with tNGS allows for the fastest comprehensive TB diagnostics comprising decentralized pathogen detection with subsequent resistance profiling in a facility specialized in tNGS.

As meropenem-clavulanic acid is recommended for the treatment of drug-resistant tuberculosis, the repurposing of new carbapenem combinations may provide new treatment options, including oral alternatives. Therefore, we studied the in vitro activities of meropenem-vaborbactam, meropenem-clavulanic acid, and tebipenem-clavulanic acid. One hundred nine Mycobacterium tuberculosis complex (MTBC) clinical isolates were tested, of which 69 were pan-susceptible and the remaining pyrazinamide- or multidrug-resistant. Broth microdilution MICs were determined using the EUCAST reference method. Meropenem and tebipenem were tested individually and in combination with vaborbactam 8 mg/L and clavulanic-acid 2 and 4 mg/L, respectively. Whole-genome sequencing was performed to explore resistance mechanisms. Clavulanic acid lowered the modal tebipenem MIC approximately 16-fold (from 16 to 1 mg/L). The modal meropenem MIC was reduced twofold by vaborbactam compared with an approximately eightfold decrease by clavulanic acid. The only previously described high-confidence carbapenem resistance mutation, crfA T62A, was shared by a subgroup of lineage 4.3.4.1 isolates and did not correlate with elevated MICs. The presence of a beta-lactamase inhibitor reduced the MTBC MICs of tebipenem and meropenem. The resulting MIC distribution was lowest for the orally available drugs tebipenem-clavulanic acid. Whether this in vitro activity translates to similar or greater clinical efficacy of tebipenem-clavulanic acid compared with the currently WHO-endorsed meropenem-clavulanic acid requires clinical studies. IMPORTANCE Repurposing of already approved antibiotics, such as beta-lactams in combination with beta-lactamase inhibitors, may provide new treatment alternatives for drug-resistant tuberculosis. Meropenem-clavulanic acid was more active in vitro compared to meropenem-vaborbactam. Notably, tebipenem-clavulanic acid showed even better activity, raising the potential of an all-oral treatment option. Clinical data are needed to investigate whether the better in vitro activity of tebipenem-clavulanic acid correlates with greater clinical efficacy compared with the currently WHO-endorsed meropenem-clavulanic acid.

The Colombian Amazon around Puerto Nariño has a high tuberculosis burden with a prevalence of 1,267/100,000 people in 2016. Recently, an outbreak of Mycobacterium tuberculosis complex (MTBC) bacteria among the indigenous populations was identified with classical MTBC genotyping methods.

Drug-resistant tuberculosis is a substantial health-care concern worldwide. Despite culture-based methods being considered the gold standard for drug susceptibility testing, molecular methods provide rapid information about the Mycobacterium tuberculosis mutations associated with resistance to anti-tuberculosis drugs. This consensus document was developed on the basis of a comprehensive literature search, by the TBnet and RESIST-TB networks, about reporting standards for the clinical use of molecular drug susceptibility testing. Review and the search for evidence included hand-searching journals and searching electronic databases. The panel identified studies that linked mutations in genomic regions of M tuberculosis with treatment outcome data. Implementation of molecular testing for the prediction of drug resistance in M tuberculosis is key. Detection of mutations in clinical isolates has implications for the clinical management of patients with multidrug-resistant or rifampicin-resistant tuberculosis, especially in situations when phenotypic drug susceptibility testing is not available. A multidisciplinary team including clinicians, microbiologists, and laboratory scientists reached a consensus on key questions relevant to molecular prediction of drug susceptibility or resistance to M tuberculosis, and their implications for clinical practice. This consensus document should help clinicians in the management of patients with tuberculosis, providing guidance for the design of treatment regimens and optimising outcomes.