Pan-drug resistance and hypervirulence in a human fungal pathogen are enabled by mutagenesis induced by mammalian body temperature.

The continuing emergence of invasive fungal pathogens poses an increasing threat to public health. Here, through the China Hospital Invasive Fungal Surveillance Net programme, we identified two independent cases of human infection with a previously undescribed invasive fungal pathogen, Rhodosporidiobolus fluvialis, from a genus in which many species are highly resistant to fluconazole and caspofungin. We demonstrate that R. fluvialis can undergo yeast-to-pseudohyphal transition and that pseudohyphal growth enhances its virulence, revealed by the development of a mouse model. Furthermore, we show that mouse infection or mammalian body temperature induces its mutagenesis, allowing the emergence of hypervirulent mutants favouring pseudohyphal growth. Temperature-induced mutagenesis can also elicit the development of pan-resistance to three of the most commonly used first-line antifungals (fluconazole, caspofungin and amphotericin B) in different Rhodosporidiobolus species. Furthermore, polymyxin B was found to exhibit potent activity against the pan-resistant Rhodosporidiobolus mutants. Collectively, by identifying and characterizing a fungal pathogen in the drug-resistant genus Rhodosporidiobolus, we provide evidence that temperature-dependent mutagenesis can enable the development of pan-drug resistance and hypervirulence in fungi, and support the idea that global warming can promote the evolution of new fungal pathogens.

Intra-hospital microbiome variability is driven by accessibility and clinical activities.

The hospital environmental microbiome, which can affect patients' and healthcare workers' health, is highly variable and the drivers of this variability are not well understood. In this study, we collected 37 surface samples from the neonatal intensive care unit (NICU) in an inpatient hospital before and after the operation began. Additionally, healthcare workers collected 160 surface samples from five additional areas of the hospital. All samples were analyzed using 16S rRNA gene amplicon sequencing, and the samples collected by healthcare workers were cultured. The NICU samples exhibited similar alpha and beta diversities before and after opening, which indicated that the microbiome there was stable over time. Conversely, the diversities of samples taken after opening varied widely by area. Principal coordinate analysis (PCoA) showed the samples clustered into two distinct groups: high alpha diversity [the pediatric intensive care unit (PICU), pathology lab, and microbiology lab] and low alpha diversity [the NICU, pediatric surgery ward, and infection prevention and control (IPAC) office]. Least absolute shrinkage and selection operator (LASSO) classification models identified 156 informative amplicon sequence variants (ASVs) for predicting the sample's area of origin. The testing accuracy ranged from 86.37% to 100%, which outperformed linear and radial support vector machine (SVM) and random forest models. ASVs of genera that contain emerging pathogens were identified in these models. Culture experiments had identified viable species among the samples, including potential antibiotic-resistant bacteria. Though area type differences were not noted in the culture data, the prevalences and relative abundances of genera detected positively correlated with 16S sequencing data. This study brings to light the microbial community temporal and spatial variation within the hospital and the importance of pathogenic and commensal bacteria to understanding dispersal patterns for infection control. IMPORTANCE: We sampled surface samples from a newly built inpatient hospital in multiple areas, including areas accessed by only healthcare workers. Our analysis of the neonatal intensive care unit (NICU) showed that the microbiome was stable before and after the operation began, possibly due to access restrictions. Of the high-touch samples taken after opening, areas with high diversity had more potential external seeds (long-term patients and clinical samples), and areas with low diversity and had fewer (short-term or newborn patients). Classification models performed at high accuracy and identified biomarkers that could be used for more targeted surveillance and infection control. Though culturing data yielded viability and antibiotic-resistance information, it disproportionately detected the presence of genera relative to 16S data. This difference reinforces the utility of 16S sequencing in profiling hospital microbiomes. By examining the microbiome over time and in multiple areas, we identified potential drivers of the microbial variation within a hospital.

Clinical, phenotypic, and genotypic characteristics of ESBL-producing Salmonella enterica bloodstream infections from Qatar.

Background: Resistant Salmonella infections are a major global public health challenge particularly for multidrug-resistant (MDR) isolates manifesting as bloodstream infections (BSIs). Objectives: To evaluate clinical, phenotypic, and genotypic characteristics of extended-spectrum beta-lactamase (ESBL) producing Salmonella enterica BSIs from Qatar. Methods: Phenotypic ESBL Salmonella enterica from adult patients presenting with positive BSIs were collected between January 2019 to May 2020. Microbiological identification and characterization were performed using standard methods while genetic characteristics were examined through whole genome sequencing studies. Results: Of 151 episodes of Salmonella enterica BSI, 15 (10%) phenotypic ESBL isolates were collected. Recent travel was recorded in most cases (80%) with recent exposure to antimicrobials (27%). High-level resistance to quinolines, aminoglycosides, and cephalosporins was recorded (80-100%) while meropenem, tigecycline and colistin demonstrated universal susceptibility. Genomic evaluation demonstrated dominance of serotype Salmonella Typhi sequence type 1 (93%) while antimicrobial resistance genes revealed dominance of aminoglycoside resistance (100%), qnrS1 quinolones resistance (80%), blaCTX-M-15 ESBLs (86.7%), and paucity of AmpC resistance genes (6.7%). Conclusions: Invasive MDR Salmonella enterica is mainly imported, connected to patients from high prevalent regions with recent travel and antimicrobial use caused by specific resistant clones. In suspected cases of multidrug resistance, carbapenem therapy is recommended.

Streptococcus gordonii-associated infective endocarditis: Case series, literature review, and genetic study.

Key Clinical Message: Streptococcus gordonii-associated endocarditis is a rare occurrence, raising diagnostic challenges, and is often associated with considerable morbidity. However, vigilance can prevent devastating consequences. Abstract: Streptococcus gordonii-associated endocarditis is rarely reported but often associated with considerable morbidity. We describe three cases of infective endocarditis caused by S. gordonii during a four-week period in 2023, and the use of whole-genome sequencing to determine whether these isolates were genetically related. The available literature was reviewed.

Epidemiology, Clinical, and Microbiological Characteristics of Multidrug-Resistant Gram-Negative Bacteremia in Qatar.

Antimicrobial resistance is a global healthcare threat with significant clinical and economic consequences peaking at secondary and tertiary care hospitals where multidrug-resistant Gram-negative bacteria (MDR GNB) lead to poor outcomes. A prospective study was conducted between January and December 2019 for all invasive bloodstream infections (BSIs) secondary to MDR GNB in Qatar identified during routine microbiological service to examine their clinical, microbiological, and genomic characteristics. Out of 3238 episodes of GNB BSIs, the prevalence of MDR GNB was 13% (429/3238). The predominant MDR pathogens were Escherichia coli (62.7%), Klebsiella pneumoniae (20.4%), Salmonella species (6.6%), and Pseudomonas aeruginosa (5.3%), while out of 245 clinically evaluated patients, the majority were adult males, with the elderly constituting almost one-third of the cohort and with highest observed risk for prolonged hospital stays. The risk factors identified included multiple comorbidities, recent healthcare contact, previous antimicrobial therapy, and admission to critical care. The in-hospital mortality rate was recorded at 25.7%, associated with multiple comorbidities, admission to critical care, and the acquisition of MDR Pseudomonas aeruginosa. Resistant pathogens demonstrated high levels of antimicrobial resistance but noticeable susceptibility to amikacin and carbapenems. Genomic analysis revealed that Escherichia coli ST131 and Salmonella enterica ST1 were the predominant clones not observed with other pathogens.

Population genomic analyses reveal high diversity, recombination and nosocomial transmission among Candida glabrata (Nakaseomyces glabrata) isolates causing invasive infections.

Candida glabrata is a commensal yeast of the gastrointestinal tract and skin of humans. However, it causes opportunistic infections in immunocompromised patients, and is the second most common Candida pathogen causing bloodstream infections. Although there are many studies on the epidemiology of C. glabrata infections, the fine- and large-scale geographical nature of C. glabrata remain incompletely understood. Here we investigated both the fine- and large-scale population structure of C. glabrata through genome sequencing of 80 clinical isolates obtained from six tertiary hospitals in Qatar and by comparing with global collections. Our fine-scale analyses revealed high genetic diversity within the Qatari population of C. glabrata and identified signatures of recombination, inbreeding and clonal expansion within and between hospitals, including evidence for nosocomial transmission among coronavirus disease 2019 (COVID-19) patients. In addition to signatures of recombination at the population level, both MATa and MATα alleles were detected in most hospitals, indicating the potential for sexual reproduction in clinical environments. Comparisons with global samples showed that the Qatari C. glabrata population was very similar to those from other parts of the world, consistent with the significant role of recent anthropogenic activities in shaping its population structure. Genome-wide association studies identified both known and novel genomic variants associated with reduced susceptibilities to fluconazole, 5-flucytosine and echinocandins. Together, our genomic analyses revealed the diversity, transmission patterns and antifungal drug resistance mechanisms of C. glabrata in Qatar as well as the relationships between Qatari isolates and those from other parts of the world.

Pathogen invasion increases the abundance of predatory protists and their prey associations in the plant microbiome.

Soil and plant-associated protistan communities play a key role in shaping bacterial and fungal communities, primarily through their function as top-down predators. However, our understanding of how pathogen invasion influences these protistan communities and their relationships with bacterial and fungal communities remains limited. Here, we studied the protistan communities along the soil-plant continuum of healthy chilli peppers and those affected by Fusarium wilt disease (FWD), and integrated bacterial and fungal community data from our previous research. Our research showed that FWD was associated with a significant enrichment of phagotrophic protists in roots, and also increased the proportion and connectivity of these protists (especially Cercozoa and Ciliophora) in both intra- and inter-kingdom networks. Furthermore, the microbiome of diseased plants not only showed a higher relative abundance of functional genes related to bacterial anti-predator responses than healthy plants, but also contained a greater abundance of metagenome-assembled genomes with functional traits involved in this response. The increased microbial inter-kingdom associations between bacteria and protists, coupled with the notable bacterial anti-predator feedback in the microbiome of diseased plants, suggest that FWD may catalyse the associations between protists and their microbial prey. These findings highlight the potential role of predatory protists in influencing microbial assembly and functionality through top-down forces under pathogenic stress.

Genomic Epidemiology of Carbapenem-Resistant Klebsiella in Qatar: Emergence and Dissemination of Hypervirulent Klebsiella pneumoniae Sequence Type 383 Strains.

The emergence of carbapenem-resistant, hypervirulent Klebsiella pneumoniae is a new threat to health care. We studied the molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae isolates in Qatar using whole-genome sequence data. We also characterized the prevalence and genetic basis of hypervirulent phenotypes and established the virulence potential using a Galleria mellonella model. Of 100 Klebsiella isolates studied, NDM and OXA-48 were the most common carbapenemases. Core genome single-nucleotide polymorphism (SNP) analysis indicated the presence of diverse sequence types and clonal lineages; isolates belonging to Klebsiella quasipneumoniae subsp. quasipneumoniae sequence type 196 (ST196) and ST1416 may be disseminated among several health care centers. Ten K. pneumoniae isolates carried rmpA and/or truncated rmpA2, and 2 isolates belonged to KL2, indicating low prevalence of classical hypervirulent isolates. Isolates carrying both carbapenem resistance and hypervirulence genes were confined mainly to ST231 and ST383 isolates. One ST383 isolate was further investigated by MinION sequencing, and the assembled genome indicated that blaNDM was located on an IncHI1B-type plasmid (pFQ61_ST383_NDM-5) which coharbored several virulence factors, including the regulator of the mucoid phenotype (rmpA), the regulator of mucoid phenotype 2 (rmpA2), and aerobactin (iucABCD and iutA), likely resulting from recombination events. Comparative genomics indicated that this hybrid plasmid may be present in two additional Qatari ST383 isolates. Carbapenem-resistant, hypervirulent K. pneumoniae ST383 isolates pose an emerging threat to global health due to their simultaneous hypervirulence and multidrug resistance.

Genomic Identification of Two Phytobacter diazotrophicus Isolates from a Neonatal Intensive Care Unit in Singapore.

We report the draft genome sequences of two Phytobacter diazotrophicus isolates recovered from a swab specimen from the water faucet located in the Neonatal Intensive Care Unit (ICU), National University Hospital, Singapore. The isolates were misidentified as Cronobacter sakazakii and Klebsiella oxytoca using biochemical methods. Whole-genome sequencing (WGS) was performed to determine their identity.

Molecular characterization of Candida auris outbreak isolates in Qatar from patients with COVID-19 reveals the emergence of isolates resistant to three classes of antifungal drugs.

OBJECTIVES: During the COVID-19 pandemic in Qatar, many patients who were severely ill were colonized and infected by Candida auris, an invasive multidrug-resistant yeast pathogen that spreads through nosocomial transmission within healthcare facilities. Here, we investigated the molecular epidemiology of these C. auris isolates and the mechanisms associated with antifungal drug resistance. METHODS: Whole genomes of 76 clinical C. auris isolates, including 65 from patients with COVID-19 collected from March 2020 to June 2021, from nine major hospitals were sequenced on Illumina NextSeq. Single nucleotide polymorphisms were used to determine their epidemiological patterns and mechanisms for antifungal resistance. The data were compared with those published prior to the COVID-19 pandemic from 2018 to 2020 in Qatar. RESULTS: Genomic analysis revealed low genetic variability among the isolates from patients with and without COVID-19, confirming a clonal outbreak and ongoing dissemination of C. auris among various healthcare facilities. Based on antifungal susceptibility profiles, more than 70% (22/28) of isolates were resistant to both fluconazole and amphotericin B. Variant analysis revealed the presence of multi-antifungal resistant isolates with prominent amino acid substitutions: Y132F in ERG11 and V704L in CDR1 linked to reduced azole susceptibility and the emergence of echinocandin resistance samples bearing mutations in FKS1 in comparison with pre-COVID-19 pandemic samples. One sample (CAS109) was resistant to three classes of antifungal drugs with a unique premature stop codon in ERG3 and novel mutations in CDR2, which may be associated with elevated amphotericin B and azole resistance. DISCUSSION: Candida auris isolates from patients with COVID-19 and from most patient samples without COVID-19 in Qatar were highly clonal. The data demonstrated the emergence of multidrug-resistant strains that carry novel mutations linked to enhanced resistance to azoles, echinocandins, and amphotericin B. Understanding the epidemiology and drug resistance will inform the infection control strategy and drug therapy.

High prevalence of fluconazole resistant Candida tropicalis among candiduria samples in China: An ignored matter of concern.

Introduction: The rapid rise of azole resistance in Candida tropicalis causing invasive infections has become a public health concern; however, the prevalence of resistant isolates in urine samples was not well studied, because the clinical significance of candiduria was not unambiguous due to possible host colonization. Methods: We performed a 12-year laboratory-based surveillance study of C. tropicalis causing either invasive infection or candiduria and studied their susceptibility profiles to common antifungal drugs. The complete coding domain sequence of the ERG11 gene was amplified in all fluconazole resistant isolates, and aligned with the wild-type sequence to detect nucleotide mutations. Results: A total of 519 unique C. tropicalis strains isolates, 69.9% of which were isolated from urine samples and remaining 30.1% were invasive strains. Overall, 16.5% isolates were confirmed to be resistant to fluconazole, of which 91.9% were cross-resistant voriconazole. Of note, at the beginning of surveillance (2010-2011), the fluconazole resistance rates were low in both candiduria and invasive groups (6.8% and 5.9%, respectively). However, the resistant rate in the candiduria group significantly increased to 29.5% since 2012-2013 (p = 0.001) and stayed high since then, whilst the resistance rate in the invasive group only showed a gradually increasing trends till 2021 (p > 0.05). Sequence analysis of ERG11 from fluconazole-resistant strains revealed the prevalence of A395T/W mutations were relatively low (16.7%) in the beginning but reached 87.5-100% after 2014. Moreover, the A395W heterozygous mutation isolates became predominant (>60% of resistant strains) after 2016, and indeed isolates carrying corresponding amino acid substitution (Y132F) was highly resistant to fluconazole with MIC50 exceeded 256 µg/ml. Conclusion: Our study revealed high azole resistant rate in candiduria with its increasing trends observed much earlier than stains causing invasive infections. Given antimicrobial resistance as a critical "One Health" issue, the emergence of antifungal resistance in Candida species that are common commensal colonizers in the human body should be concerned.

Potency of olorofim (F901318) compared to contemporary antifungal agents against clinical Aspergillus fumigatus isolates, and review of azole resistance phenotype and genotype epidemiology in China.

Triazole resistance in A. fumigatus is an increasing worldwide problem that causes major challenges in the management of aspergillosis. New antifungal drugs are needed with novel targets, that are effective in triazole-resistant infection. In this study, we retrospectively evaluated potency of the novel drug olorofim compared to contemporary antifungal agents against 111 clinical A. fumigatus isolates collected from Huashan Hospital, Shanghai, China, using EUCAST methodology, and reviewed the literature on triazole resistant A. fumigatus published between 1966 and 2020 in China. Olorofim was active in vitro against all tested A. fumigatus isolates with MIC90 of 0.031mg/L (range 0.008-0.062 mg/L). For 4 triazole-resistant A. fumigatus (TRAF) isolates, the olorofim MIC ranged between 0.016-0.062mg/L. The reported rates of TRAF in China is 2.5% - 5.56% for clinical isolates, and 0-1.4% for environmental isolates.TR34/L98H/S297T/F495I is the predominant resistance mechanism, followed by TR34/L98H. Non TR-mediated TRAF isolates, mostly harboring a cyp51A single point mutation, showed greater genetic diversity than TR-mediated resistant isolates. Resistance due toTR34/L98H and TR34/L98H/S297T/F495I mutations among TRAF isolates might have evolved from separate local isolates in China. Continuous isolation of TRAF in China underscores the need for systematic resistance surveillance as well as the need for novel drug targets such as olorofim.

HIF1-α upregulation induces proinflammatory factors to boost host killing capacity after Aspergillus fumigatus exposure.

Aims: HIF1-α is an important transcription factor in the regulation of the immune response. The protective function of HIF1-α in the host epithelial immune response to Aspergillus fumigatus requires further clarification. Methods: In this study we demonstrated the effect of upregulation of HIF1-α expression in A549 cells and mouse airway cells exposed to A. fumigatus in vivo. Results: The killing capacity was enhanced by boosting proinflammatory factors both in vitro and in vivo. Moreover, airway inflammation was reduced in the HIF1-α-upregulated mice. Conclusion: We identified a protective role for HIF1-α in anti-A. fumigatus immunity. Modulation of HIF1-α might be a target for the development of aspergillosis therapy.

Haplotype-phased and chromosome-level genome assembly of Puccinia polysora, a giga-scale fungal pathogen causing southern corn rust.

Rust fungi are characterized by large genomes with high repeat content and have two haploid nuclei in most life stages, which makes achieving high-quality genome assemblies challenging. Here, we described a pipeline using HiFi reads and Hi-C data to assemble a gigabase-sized fungal pathogen, Puccinia polysora f.sp. zeae, to haplotype-phased and chromosome-scale. The final assembled genome is 1.71 Gbp, with ~850 Mbp and 18 chromosomes in each haplotype, being currently one of the two giga-scale fungi assembled to chromosome level. Transcript-based annotation identified 47,512 genes for the dikaryotic genome with a similar number for each haplotype. A high level of interhaplotype variation was found with 10% haplotype-specific BUSCO genes, 5.8 SNPs/kbp, and structural variation accounting for 3% of the genome size. The P. polysora genome displayed over 85% repeat contents, with genome-size expansion and copy number increasing of species-specific orthogroups. Interestingly, these features did not affect overall synteny with other Puccinia species having smaller genomes. Fine-time-point transcriptomics revealed seven clusters of coexpressed secreted proteins that are conserved between two haplotypes. The fact that candidate effectors interspersed with all genes indicated the absence of a "two-speed genome" evolution in P. polysora. Genome resequencing of 79 additional isolates revealed a clonal population structure of P. polysora in China with low geographic differentiation. Nevertheless, a minor population differentiated from the major population by having mutations on secreted proteins including AvrRppC, indicating the ongoing virulence to evade recognition by RppC, a major resistance gene in Chinese corn cultivars. The high-quality assembly provides valuable genomic resources for future studies on disease management and the evolution of P. polysora.

Cross-kingdom synthetic microbiota supports tomato suppression of Fusarium wilt disease.

The role of rhizosphere microbiota in the resistance of tomato plant against soil-borne Fusarium wilt disease (FWD) remains unclear. Here, we showed that the FWD incidence was significantly negatively correlated with the diversity of both rhizosphere bacterial and fungal communities. Using the microbiological culturomic approach, we selected 205 unique strains to construct different synthetic communities (SynComs), which were inoculated into germ-free tomato seedlings, and their roles in suppressing FWD were monitored using omics approach. Cross-kingdom (fungi and bacteria) SynComs were most effective in suppressing FWD than those of Fungal or Bacterial SynComs alone. This effect was underpinned by a combination of molecular mechanisms related to plant immunity and microbial interactions contributed by the bacterial and fungal communities. This study provides new insight into the dynamics of microbiota in pathogen suppression and host immunity interactions. Also, the formulation and manipulation of SynComs for functional complementation constitute a beneficial strategy in controlling soil-borne disease.

Hit Compounds and Associated Targets in Intracellular Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, is one of the most devastating infectious agents in the world. Chemical-genetic characterization through in vitro evolution combined with whole genome sequencing analysis was used identify novel drug targets and drug resistance genes in Mtb associated with its intracellular growth in human macrophages. We performed a genome analysis of 53 Mtb mutants resistant to 15 different hit compounds. We found nonsynonymous mutations/indels in 30 genes that may be associated with drug resistance acquisitions. Beyond confirming previously identified drug resistance mechanisms such as rpoB and lead targets reported in novel anti-tuberculosis drug screenings such as mmpL3, ethA, and mbtA, we have discovered several unrecognized candidate drug targets including prrB. The exploration of the Mtb chemical mutant genomes could help novel drug discovery and the structural biology of compounds and associated mechanisms of action relevant to tuberculosis treatment.

Persistence of an epidemic cluster of Rhodotorula mucilaginosa in multiple geographic regions in China and the emergence of a 5-flucytosine resistant clone.

Rhodotorula mucilaginosa, an environmental yeast widely used in industry and agriculture, is also an opportunistic pathogen resistant to multi-antifungals. During the national surveillance in China, R. mucilaginosa has been documented from various hospitals and regions. At present, the molecular epidemiology of invasive infections caused by R. mucilaginosa and their resistance profiles to antifungals were unknown. Here we collected 49 strains from four hospitals located in different geographic regions from 2009 to 2019 in China, determined their genotypes using different molecular markers and quantified susceptibilities to various antifungals. Sequencing of ITS and D1/D2 regions in rDNA indicated that 73.5% (36/49) of clinical strains belong to same sequence type (rDNA type 2). Microsatellite (MT) genotyping with 15 (recently developed) tandem repeat loci identified 5 epidemic MT types, which accounted for 44.9% (22/49) of clinical strains, as well as 27 sporadic MT types. Microsatellite data indicated that the presence of an epidemic cluster including 35 strains (71.4%) repeatedly isolated in four hospitals for eight years. Single nucleotide variants (SNVs) from the whole genome sequence data also supported the clustering of these epidemic strains due to low pairwise distance. In addition, phylogenetic analysis of SNVs from these clinical strains, together with environmental and animal strains showed that the closely related epidemic cluster strains may be opportunistic, zoonotic pathogens. Also, molecular data indicated a possible clonal transmission of pan echinocandins-azoles-5-flucytosine resistant R. mucilaginosa strains in hospital H01. Our study demonstrated that R. mucilaginosa is a multi-drug resistant pathogen with the ability to cause nosocomial infection.