KEY POINTS:Single-molecule real-time sequencing with the PacMUC1 script resolved exact MUC1 variable tandem repeat structure and full allelic variation. In 300 individuals, the protocol identified 215 distinct MUC1 tandem repeat alleles with 80 repeat units and nine frameshift mutation types. Probe extension assay identified 90% of families with frameshift mutations, detection of frameshifted mucin-1 aided genetically unresolved cases. BACKGROUND:ADTKD- MUC1 is caused by frameshift mutations in the MUC1 gene, producing a frameshifted neoprotein (MUC1fs) toxic to kidney cells. The gene's variable number of tandem repeats (VNTR), with approximately 80% guanine/cytosine content, has made it largely inaccessible to standard short-read sequencing, leaving the reference sequence and natural variation poorly defined and complicating mutation detection. METHODS:Using single-molecule real-time (SMRT) sequencing, we characterized MUC1 VNTR in 300 individuals, including 279 from 143 families suspected of having ADTKD- MUC1 , assessing VNTR length, repeat structure, and frameshift mutations. Results were compared with the Clinical Laboratory Improvement Amendments-approved probe-extension assay, detecting the prevalent 59dupC mutation, and with MUC1fs immunohistochemistry, which detects the pathogenic protein independent of the underlying genomic change. RESULTS:We identified 215 unique VNTR alleles composed of 80 distinct repeat units, 46 (58%) of which were novel, and nine distinct frameshift mutations present on 52 mutated alleles. Overall, MUC1 frameshift mutations were identified in 71 of 143 families (50%) with suspected ADTKD- MUC1 , comprising 135 affected individuals (48%). The SMRT assay outperformed the probe-extension assay by identifying frameshift mutations in two families with previously inconclusive results and in eight additional families whose mutations were undetectable by the probe-extension design. When successful, SMRT assay showed 100% concordance with probe-extension assay at the family level and 98% at the individual level, with discordance attributable to allelic dropout inherent to both long-range PCR amplification and long-read sequencing. Analysis of the mutational spectrum confirmed 59dupC as the most prevalent mutation, affecting approximately 90% of families, while the other eight mutation types occurred at most twice. CONCLUSIONS:The SMRT assay outperformed the Clinical Laboratory Improvement Amendments-approved probe-extension assay by detecting essentially all VNTR-associated frameshift mutations. The probe-extension assay identified approximately 90% of affected families. MUC1fs immunohistochemistry added diagnostic value in genetically unresolved cases by detecting the pathogenic protein independent of the underlying mutation.

Background The incidence of Candida tropicalis isolation is increasing in hospital settings. High azole resistance and mortality rates make it a pathogen that requires further analysis.Methods Fourteen azole resistant Candida glabrata clinical isolates were collected from a Lebanese hospital and analysed through whole genome sequencing for single nucleotide polymorphisms in key resistance and virulence genes, and for phylogenetic relatedness. Isolates were then characterised for pathogenicity related attributes.Results All isolates had Lys314Glu mutation in ERG20 with multiple isolates displaying numerous shared mutations, such as Glu291Lys in CDR2 and Ala16Thr in CDR3. With the exception of two isolates that clustered together, most isolates were over 99.6% identical based on a genomic heatmap, implying high relatedness consistent with localised clonal expansion, although SNP differences appeared too high to support this. However, the isolates exhibited increased ergosterol and chitin content, as well as upregulation of drug efflux pumps resulting in drug resistance.Conclusion Our hospital isolates showed convergent resistant pathways, with many isolates having both shared and unique mutations and a high degree of genomic similarities.

The global rise of antimicrobial resistance has renewed interest in fosfomycin (FOS), an old antibiotic with activity against multidrug-resistant Enterobacterales. However, resistance to FOS is increasing, driven by impaired drug uptake, target modification, and by fosA-encoded enzymatic inactivation. This study assessed the prevalence and molecular basis of FOS resistance among Enterobacterales collected in Czech tertiary care hospitals in 2024. A total of 211 preliminary FOS-resistant isolates were obtained from nine hospitals across the Czech Republic, predominantly Proteus mirabilis (n=149) and Escherichia coli (n=57). All isolates showed elevated FOS MICs, and the PPF test identified FosA activity in 34/211 isolates. Carbon-source growth testing demonstrated widespread impairment of GlpT and UhpT transporters (93.8% affecting both). PCR confirmed fosA genes in 14 isolates (9 P. mirabilis, 5 E. coli). WGS revealed fosA3 as the dominant variant (85.7%), followed by fosA4 (14.3%). P. mirabilis isolates primarily belonged to ST185 and ST135, forming two Czech-specific fosA3 clusters with limited relatedness to international genomes. FosA-producing E. coli displayed broader diversity (ST69, ST58, ST550, ST1308). FosA4 was detected exclusively in E. coli. Most fosA-positive strains co-harbored ESBL genes, predominantly blaCTX-M-65. SNP-based phylogenies indicated local clonal circulation of fosA3-positive P. mirabilis ST185, whereas E. coli isolates showed heterogeneous international linkages. Analysis of GlpT/UhpT/MurA identified numerous amino acid substitutions, though only a minority were predicted to affect protein function. This study documents the first broader emergence of plasmid-mediated FOS resistance in Czech Enterobacterales and underscores the importance of continuous genomic surveillance of fosA-mediated resistance.

Wilson disease (WD) is an autosomal recessive disorder of copper transport caused by bi-allelic pathogenic variants in the ATPase copper transporting beta gene (ATP7B). Results of standard genetic diagnostics remain inconclusive in 3%-20% of WD patients in part due to problematic assessment of variants of unknown or conflicting pathogenicity (synonymous variants included). Correct interpretation of potential effects of such variants can be substantially enhanced by RNA analyses. This strategy is, however, of limited utility in WD patients because of predominant liver expression of ATP7B. To avoid invasive bioptic liver collection and increase WD diagnostic yields, we searched for a surrogate tissue sample and identified profiles of ATP7B transcripts in nasopharyngeal swabs that were comparable to liver. Amplicons spanning ATP7B Exons 3-21 were prepared from the swab material and analysed by long-read nanopore sequencing to enable the detection of splicing changes and variant phasing. Diagnostic utility of this novel in vivo methodology was demonstrated by characterization of mRNA splicing abnormalities caused by synonymous ATP7B variants c.1488C>T (p.(Gly496=)), c.2241C>T (p.(Ile747=)), c.2292C>T (p.(Phe764=)), and a nonsense variant c.2336G>A (p.(Trp779Ter)) in four WD patients, who were not genetically resolved by standard techniques. Nasopharyngeal swab sampling is minimally invasive and allows effective analyses of mRNA to detect and/or validate effects of ATP7B variants in WD patients. Conclusive genetic diagnosis attained by this novel technique may facilitate family counselling and substantiate initiation of copper-chelation therapy in presymptomatic individuals.

This study aimed to compare the microbial profiles between hospital wastewater and river water to assess the dissemination of clinical isolates into the environment. Two types of water samples were collected from sampling sites which were geographically close (wastewater from the University Hospital of Larissa and river water from the Pineios River). Gram-negative bacteria isolated from both sample types were identified using MALDI-TOF. Furthermore, the minimum inhibitory concentration (MIC) of antibiotics were evaluated. A total of 54 Gram-negative isolates, belonging to diverse species, were collected from wastewater sample and river sample. All isolates were classified as MDR, exhibiting resistance to at least one agent from more than three different antibiotic classes. Based on species identification and susceptibility profiles, 27 isolates (19 from wastewater and 8 from river-water) were selected to be further characterized by whole-genome sequencing (WGS). Analysis of WGS data, revealed the presence of different STs, even in isolates belonging to the same bacterial species. Additionally, WGS data showed that carbapenemase-encoding genes were identified in the majority of isolates. PlasmidFinder identified a huge variety of plasmid replicons among the isolates studied. In conclusion, both hospital wastewater and river water contained isolates carrying clinically relevant resistance determinants, such as carbapenemase-encoding genes. The presence of these pathogenic bacteria in the river poses a significant public health concern. Although we could not identify the origin of MDR bacteria in the river sample, these findings highlight the growing threat of antimicrobial resistance in the environment and underscore the urgent need for improved treatment methods and stricter surveillance to control its spread.

Wolbachia pipientis is an obligate intracellular bacterium, associated with several arthropods and filarial nematodes. Wolbachia establishes a variety of symbiotic relationships with its hosts, with consequent genomic rearrangements, variation in gene content, and loss of regulatory regions. Despite this, experimental studies show that Wolbachia gene expression is coordinated with host developmental stages, but the mechanism is still unknown. In this work, we analyzed published RNA-seq data of four Wolbachia strains, finding a correlation between gene nucleotide composition and gene expression. The strength and direction of this phenomenon changed with the expression of the S-adenosyl-methionine-dependent methyltransferase midA. Specifically, when midA is overexpressed, there is a negative relationship between gene adenine content and gene expression, while downregulation of midA reverses this trend. MidA is known to methylate protein arginine, with potential effect on protein affinity for substrates, including nucleic acids. To expand our understanding of this poorly characterized enzyme, we investigated its ability to methylate DNA expressing it in Escherichia coli. The experiment revealed that the Wolbachia MidA can methylate both adenine and cytosine. Lastly, we found upstream the midA gene, a conserved binding site for the Ccka/CtrA signaling transduction system, and we hypothesize that this mechanism could be involved in the communication between the host and the bacterium. Overall, these findings suggest a cascade mechanism in which the host activates the bacterium Ccka/CtrA signaling system, thus inducing the expression of the midA gene, with subsequent effect on the expression of several Wolbachia genes on the basis of their nucleotide composition. IMPORTANCE:Wolbachia pipientis is one of the most common intracellular bacteria in insects, and it is currently utilized as a tool for the control of vector-borne diseases. As for many other endosymbiont bacteria, Wolbachia experienced important genome rearrangements, gene content changes, and the loss of several regulatory sequences, affecting the integrity of operons and promoters. Nevertheless, experimental studies have shown that Wolbachia gene expression is coordinated with the host physiology (e.g., developmental stages), although the underlying mechanism remains unclear. In this work, based on in silico analyses and an experimental study on wOo methyltransferase, we propose that bacterial DNA methylation could be a key mechanism regulating Wolbachia gene expression. Additionally, we found evidence suggesting that the DNA methylation process in Wolbachia can be activated by the host.

The aim of this study was to examine the genetic characteristics that could be associated with the virulence characteristics of Escherichia coli collected from clinical samples. A collection of 100 non-repetitive E. coli isolates was analyzed. All isolates were typed by MLST. String production, biofilm formation and serum resistance were examined for all isolates. Twenty E. coli isolates were completely sequenced Illumina platform. The results showed that the majority of E. coli isolates (87%) produced significant levels of biofilm, while none of the isolates were positive for string test and resistance to serum. Additionally, the presence of CRISPR/Cas systems (type I-E or I-F) was found in 18% of the isolates. Analysis of WGS data found that all sequenced isolates harbored a variety of virulence genes that could be implicated in adherence, invasion, iron uptake. Also, WGS data confirmed the presence of a wide variety of resistance genes, including ESBL- and carbapenemase-encoding genes. In conclusion, an important percentage (87%) of the E. coli isolates had a significant ability to form biofilm. Biofilms, due to their heterogeneous nature and ability to make microorganisms tolerant to multiple antimicrobials, complicate treatment strategies. Thus, in combination with the presence of multidrug resistance, expression of virulence factors could challenge antimicrobial therapy of infections caused by such bacteria.

The objective of this study was to characterize the virulence characteristics of a collection of Klebsiella pneumoniae isolates collected from different clinical sources. A collection of 60 non-repetitive K. pneumoniae isolates, was studied. In vitro, virulence was analyzed by testing the survival of bacteria in pooled human serum. Isolates were typed by MLST. The genomes of 23 K. pneumoniae isolates, representatives of different STs and virulence profiles, were completely sequenced using the Illumina platform. Of note, 26/60 of K. pneumoniae isolates were resistant to killing by complement. Serum-resistant isolates belonged to distinct STs. Analysis of WGS data with VFDB showed the presence of several virulence genes related various virulence functions. Specifically, serum-resistant isolates carried a higher number of ORFs, which were associated with serum resistance, compared to serum-sensitive isolates. Additionally, analysis of WGS data showed the presence of multiple plasmid replicons that could be involved with the spread and acquisition of resistance and virulence genes. In conclusion, analysis of virulence characteristics showed that an important percentage (31.6%) of K. pneumoniae isolates were in vitro virulent by exhibiting resistance to serum. Thus, the presence of several virulence factors, in combination with the presence of multidrug resistance, could challenge antimicrobial therapy of infections caused by such bacteria.

Background:ADTKD-MUC1 is caused by frameshift mutations in MUC1 gene that produce a frameshifted protein (MUC1fs) toxic to kidney cells. The gene's variable number of tandem repeats (VNTR), with high GC content, makes it largely inaccessible to standard sequencing. As a result, both the reference sequence and natural variation in this region remain poorly defined, complicating mutation detection and data interpretation. Standard methods also fail to pinpoint the exact VNTR unit affected, limiting insight into mutation mechanisms and genotype-phenotype correlations. Methods:We employed Single Molecule, Real-Time (SMRT) sequencing and characterized the genomic sequence of MUC1 in 300 individuals including 279 individuals from 143 families suspected of having ADTKD-MUC1. We compared these results to those obtained using the CLIA-approved mass spectrometry-based probe extension (PE) assay, which specifically detect the most prevalent 59dupC mutation. We correlated the structural features of the MUC1 VNTR with the rate of kidney function decline in affected individuals. Results:We identified MUC1 consensus sequences for 205 unique VNTR alleles, with 9 distinct types of frameshift mutations present on 52 distinct mutated VNTR alleles. MUC1 frameshift mutations were identified in 71 of 143 families (50%) with suspected ADTKD, comprising 135 genetically affected individuals (48%). The SMRT assay exhibited complete concordance and revealed that the PE assay is capable of detecting frameshift mutations in approximately 85% of affected families. The constellation of VNTR structures supports a genotype-progression model, in which fast progressors exhibit a significantly lower number of repeat units on the wild-type allele and a higher number of repeats on the mutation-bearing allele, including an increased number of frameshifted repeat units. Conclusions:SMRT sequencing outperforms current diagnostic methods for ADTKD-MUC1 and reveals the prognostic value of VNTR structures. Although their contribution to disease progression is modest (~6% variance explained), it remains biologically and clinically meaningful.

FosA10-producing Enterobacterales have an extremely low incidence in Europe. In March 2024, an 83-year-old woman, hospitalized in the Modena Province, developed an infection with fosfomycin-resistant Escherichia coli. The patient was treated with piperacillin/tazobactam and, after 10 days, the clinical picture was resolved. Fosfomycin MIC was evaluated with the reference agar dilution method and the production of FosA enzymes by phenotypic testing. Genomic characterization was assessed using long-read sequencing technology on the Sequel I platform. An E. coli isolate (FO_2) was collected from both blood and urine samples and showed high-level resistance to fosfomycin (MIC > 128 mg/L). The resistance to fosfomycin was ascribed to the production of FosA-like enzymes by phenotypic testing. The genomic analysis pointed to a FosA10-producing E. coli ST69. The fosA10 gene was carried by a highly conjugative IncB/O/K/Z plasmid that showed relevant similarities with other globally circulating plasmids. The acquisition of rare fosA-like genes in clinically relevant clones is concerning and the dissemination of FosA-producing E. coli should be continuously monitored.

Background:FosA10-producing Enterobacterales have an extremely low incidence in Europe. Patients and methods:In March 2024, an 83-year-old woman, hospitalized in the Modena Province, developed an infection with fosfomycin-resistant Escherichia coli. The patient was treated with piperacillin/tazobactam and, after 10 days, the clinical picture was resolved. Fosfomycin MIC was evaluated with the reference agar dilution method and the production of FosA enzymes by phenotypic testing. Genomic characterization was assessed using long-read sequencing technology on the Sequel I platform. Results:An E. coli isolate (FO_2) was collected from both blood and urine samples and showed high-level resistance to fosfomycin (MIC > 128 mg/L). The resistance to fosfomycin was ascribed to the production of FosA-like enzymes by phenotypic testing. The genomic analysis pointed to a FosA10-producing E. coli ST69. The fosA10 gene was carried by a highly conjugative IncB/O/K/Z plasmid that showed relevant similarities with other globally circulating plasmids. Conclusions:The acquisition of rare fosA-like genes in clinically relevant clones is concerning and the dissemination of FosA-producing E. coli should be continuously monitored.

A clonal outbreak of 18 ST773 NDM-1 producing Pseudomonas aeruginosa strains has been detected for the first time in the Czech Republic. The strains were extremely drug-resistant (XDR) and resistant to serum killing. SNP-based phylogeny and CRISPR assay typing showed minimal genomic variations among the isolates. The results suggest a high-risk, persistent, virulent clone causing the hospital outbreak, with the possibility of a nationwide outbreak.IMPORTANCEOur research on the novel detection of the NDM-1 gene in carbapenem-resistant Pseudomonas aeruginosa ST773 in the Czech Republic is of great significance for public health and infection control. Until now, the emergence of this gene in P. aeruginosa strains was uncommon in this region, as carbapenem resistance was primarily associated with IMP and VIM types of MBLs. This nosocomial outbreak was triggered by an index case patient repatriated from areas with reported NDM-1 producing P. aeruginosa, illustrating how international travel contributes to the spread of such resistant pathogens. The results obtained in this study show that it is necessary to focus on tracing the source of infections to control and prevent nosocomial infections, helping to protect public health in the Czech Republic.

The opportunistic pathogen Candida parapsilosis is a major causative agent of candidiasis leading to death in immunocompromised individuals. Azoles are the first line of defense in their treatment. The purpose of this study was to characterize eight fluconazole-resistant and sensitive C. parapsilosis hospital isolates through a battery of phenotypic tests that target pathogenicity attributes such as virulence, biofilm formation, stress resistance, and ergosterol content. Whole genome sequencing was carried out to identify mutations in key pathogenicity and resistance genes. Phylogenetic comparison was performed to determine strain relatedness and clonality. Genomic data and phylogenetic analysis revealed that two isolates were C. orthopsilosis and C. metapsilosis misidentified as C. parapsilosis. Whole genome sequencing analysis revealed known and novel mutations in key drug resistance and pathogenicity genes such as ALS6, ALS7, SAPP3, SAP7, SAP9, CDR1, ERG6, ERG11 and UPC2. Phylogenetic analysis revealed a high degree of relatedness and clonality within our C. parapsilosis isolates. Our results showed that resistant isolates exhibited an increase in biofilm content compared to the sensitive isolates. In conclusion, our study is the first of its kind in Lebanon to describe phenotypic and genotypic characteristics of nosocomial C. parapsilosis complex isolates having a remarkable ability to form biofilms.

Wolbachia pipientis is an endosymbiont bacterium commonly found in association with arthropods and filarial nematodes. Several studies disclosed the main role of Wolbachia in host physiology. Reasonably, the maintenance of a strict symbiosis requires coordination between Wolbachia and host gene expressions. Experimental studies show Wolbachia gene expression varies during host development or after environmental stresses. Despite this, the mechanism behind the regulation of Wolbachia gene expression is still not clear, since their genomes have lost most promoters during genome reduction. The first published RNA-Seq study on Wolbachia showed the differential expression of a DNA methyltransferase, an enzyme able to methylate adenine or cytosine on specific patterns and often involved in the regulation of bacteria gene expression. In this work, we tested the hypothesis that DNA methylation can affect gene expression in Wolbachia. We experimentally characterised the Wolbachia DNA methyltransferase and re-analysed RNA-Seq data from six studies on four hosts. We surprisingly found that the Wolbachia DNA methyltransferase is the only so far described able to methylate both adenine and cytosine without highly specific patterns. We also found that the nucleotide content of the genes correlates with their expressions, with a pattern compatible to be a consequence of DNA methylation. Lastly, we found a conserved eukaryotic-like promoter upstream of the Wolbachia DNA methyltransferase gene. Overall, these results may suggest a cascade mechanism in which the host induces the Wolbachia DNA methyltransferase expression, which in turn affects the bacterium gene expression.### Competing Interest StatementThe authors have declared no competing interest.

Background: The pathogenic fungus Candida albicans is a leading agent of death in immunocompromised individuals with a growing trend of antifungal resistance. Methods: The purpose is to induce resistance to drugs in a sensitive C. albicans strain followed by whole-genome sequencing to determine mechanisms of resistance. Strains will be assayed for pathogenicity attributes such as ergosterol and chitin content, growth rate, virulence, and biofilm formation. Results: We observed sequential increases in ergosterol and chitin content in fluconazole-resistant isolates by 78% and 44%. Surface thickening prevents the entry of the drug, resulting in resistance. Resistance imposed a fitness trade-off that led to reduced growth rates, biofilm formation, and virulence in our isolates. Sequencing revealed mutations in genes involved in resistance and pathogenicity such as ERG11, CHS3, GSC2, CDR2, CRZ2, and MSH2. We observed an increase in the number of mutations in key genes with a sequential increase in drug-selective pressures as the organism increased its odds of adapting to inhospitable environments. In ALS4, we observed two mutations in the susceptible strain and five mutations in the resistant strain. Conclusion: This is the first study to induce resistance followed by genotypic and phenotypic analysis of isolates to determine mechanisms of drug resistance.

This report describes mutations in genes responsible for cell deformities in haemophili under beta-lactam pressure in vitro. Light and transmission electron microscopy confirmed a hypothesis regarding changes in the shape of haemophili that had become more filamentous in the presence of ampicillin (2 mg/L) and cefuroxime (8 mg/L) after 30 days of serial passage. Short-axis size increased by 28% (from 0.767 to 1.06 µm) and long-axis length increased by 54% (from 1 to 2.175 µm). Additionally, whole-genome sequencing analysis (Illumina platform, software PROKKA) revealed a variety of mutations in genes responsible for cell morphology in isolates examined in this study: ftsI (A1576 → C; G1154 → C; T986 → C; G1684 → C), mreB (C476 → T), mreC (A5 → G), mrdA (A1148 → G; C179 → T; G1613 → T), mrdB (T668 → G), mltC (C1016 → T) and rodA (T668 → G). The results of this study indicate that shifts in bacterial shape could play a role in the adaptation of haemophili to a new niche created by beta-lactams as a strategy of antibiotic therapy survival.

Paenibacillus larvae and Melissococcus plutonius represent the most threatening bacterial diseases of honeybee ( Apis mellifera )—American and European foulbrood, respectively. For efficient control of those diseases, rapid and accurate detection of the pathogens is crucial. Therefore, we developed a novel multiplex PCR method simultaneously detecting both pathogens. To design and optimize multiplex PCR reaction, four strains of P. larvae representing four ERIC genotypes I–IV (strain DSM 7030—ERIC I, DSM 25430—ERIC II, LMG 16252—ERIC III, DSM 3615—ERIC IV) were selected. Those strains were fully sequenced using long-read sequencing (Sequel I, Pacific Biosciences). For P. larvae , the multicopy insertion sequence IS 256 identified in all genotypes of P. larvae was selected to provide high sensitivity. M. plutonius was detected by plasmid pMP1 sequence and the virulence verified by following detection of ETX/MTX2 toxin responsible for pore formation in the cell membrane. As an internal control, a gene encoding for major royal jelly protein 1 specific for honeybees was selected. The method was validated on 36 clinical specimens collected from the colonies suffering from American and European foulbrood in the Czech Republic. Based on the results, sensitivity of PCR was calculated to 93.75% and specificity to 100% for P. larvae diagnosed from hive debris and 100% sensitivity and specificity for honeybee workers and larval scales as well as for diseased brood infected by M. plutonius .

Background: Aminopenicillins are recommended agents for non-invasive Haemophilus influenzae infections. One of the mechanisms of resistance to β-lactams is the alteration of the transpeptidase region of penicillin binding protein 3 (PBP3) which is caused by mutations in the ftsI gene. It was shown that exposure to beta-lactams has a stimulating effect on increase of prevalence of H. influenzae strains with the non-enzymatic mechanism of resistance. Objectives: The aim of our study was to compare the mutational potential of ampicillin and cefuroxime in H. influenzae strains, determination of minimum inhibitory concentration and the evolution of mutations over time, focusing on amino acid substitutions in PBP3. Methods: 30 days of serial passaging of strains in liquid broth containing increasing concentrations of ampicillin or cefuroxime was followed by whole-genome sequencing. Results: On average, cefuroxime increased the minimum inhibitory concentration more than ampicillin. The minimum inhibitory concentration was increased by a maximum of 32 fold. Substitutions in the PBP3 started to appear after 15 days of passaging. In PBP3, cefuroxime caused different substitutions than ampicillin. Conclusions: Our experiment observed differences in mutation selection by ampicillin and cefuroxime. Selection pressure of antibiotics in vitro generated substitutions that do not occur in clinical strains in the Czech Republic.