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.

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 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.

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.

Cryptic Species Diversity and Phylogenetic Relationship in the Rust Genus Chrysomyxa from China.

Chrysomyxa rusts are fungal pathogens widely distributed in the Northern hemisphere, causing spruce needle and cone rust diseases, and they are responsible for significant economic losses in China. Taxonomic delimitation and precise species identification are difficult within this genus because some characters often overlap in several species. Adequate species delimitation, enhanced by the use of DNA-based methodologies, will help to establish well-supported species boundaries and enable the identification of cryptic species. Here, we explore the cryptic species diversity in the rust genus Chrysomyxa from China. Species delimitation analyses are conducted using a distance-based method (ABGD) and three tree-based methods (GMYC, bPTP, and mPTP) based on combined LSU and ITS sequences of over 60 specimens. Although there is some incongruence among species delimitation methods, two new species and three putative cryptic species are identified. The key to 20 Chrysomyxa species distributed in China is presented. These results suggest that a significant level of undiscovered cryptic diversity is likely to be found in Chrysomyxa from China. Future studies should consider multiple analytical methods when dealing with multi-locus datasets.

Phylogenetic Relationships, Speciation, and Origin of Armillaria in the Northern Hemisphere: A Lesson Based on rRNA and Elongation Factor 1-Alpha.

Armillaria species have a global distribution and play various roles in the natural ecosystems, e.g., pathogens, decomposers, and mycorrhizal associates. However, their taxonomic boundaries, speciation processes, and origin are poorly understood. Here, we used a phylogenetic approach with 358 samplings from Europe, East Asia, and North America to delimit the species boundaries and to discern the evolutionary forces underpinning divergence and evolution. Three species delimitation methods indicated multiple unrecognized phylogenetic species, and biological species recognition did not reflect the natural evolutionary relationships within Armillaria; for instance, biological species of A. mellea and D. tabescens are divergent and cryptic species/lineages exist associated with their geographic distributions in Europe, North America, and East Asia. While the species-rich and divergent Gallica superclade might represent three phylogenetic species (PS I, PS II, and A. nabsnona) that undergo speciation. The PS II contained four lineages with cryptic diversity associated with the geographic distribution. The genus Armillaria likely originated from East Asia around 21.8 Mya in early Miocene when Boreotropical flora (56-33.9 Mya) and the Bering land bridge might have facilitated transcontinental dispersal of Armillaria species. The Gallica superclade arose at 9.1 Mya and the concurrent vicariance events of Bering Strait opening and the uplift of the northern Tibetan plateau might be important factors in driving the lineage divergence.

Draft Genome Sequence of Rhodotorula mucilaginosa from an Adult Patient in Qatar.

Rhodotorula mucilaginosa is an opportunistic fungal pathogen of public health importance. We present the draft genome sequence of an isolate (Rhodo3571) cultured from an immunocompetent patient. The isolate is similar to other R. mucilaginosa genomes in the NCBI database. Presented here are the genome assembly and its comparison to other reference genomes.

Disease-induced changes in plant microbiome assembly and functional adaptation.

BACKGROUND: The plant microbiome is an integral part of the host and increasingly recognized as playing fundamental roles in plant growth and health. Increasing evidence indicates that plant rhizosphere recruits beneficial microbes to the plant to suppress soil-borne pathogens. However, the ecological processes that govern plant microbiome assembly and functions in the below- and aboveground compartments under pathogen invasion are not fully understood. Here, we studied the bacterial and fungal communities associated with 12 compartments (e.g., soils, roots, stems, and fruits) of chili pepper (Capsicum annuum L.) using amplicons (16S and ITS) and metagenomics approaches at the main pepper production sites in China and investigated how Fusarium wilt disease (FWD) affects the assembly, co-occurrence patterns, and ecological functions of plant-associated microbiomes. RESULTS: The amplicon data analyses revealed that FWD affected less on the microbiome of pepper reproductive organs (fruit) than vegetative organs (root and stem), with the strongest impact on the upper stem epidermis. Fungal intra-kingdom networks were less stable and their communities were more sensitive to FWD than the bacterial communities. The analysis of microbial interkingdom network further indicated that FWD destabilized the network and induced the ecological importance of fungal taxa. Although the diseased plants were more susceptible to colonization by other pathogenic fungi, their below- and aboveground compartments can also recruit potential beneficial bacteria. Some of the beneficial bacterial taxa enriched in the diseased plants were also identified as core taxa for plant microbiomes and hub taxa in networks. On the other hand, metagenomic analysis revealed significant enrichment of several functional genes involved in detoxification, biofilm formation, and plant-microbiome signaling pathways (i.e., chemotaxis) in the diseased plants. CONCLUSIONS: Together, we demonstrate that a diseased plant could recruit beneficial bacteria and mitigate the changes in reproductive organ microbiome to facilitate host or its offspring survival. The host plants may attract the beneficial microbes through the modulation of plant-microbiome signaling pathways. These findings significantly advance our understanding on plant-microbiome interactions and could provide fundamental and important data for harnessing the plant microbiome in sustainable agriculture. Video abstract.

Whole-Genome Sequencing for Molecular Characterization of Carbapenem-Resistant Enterobacteriaceae Causing Lower Urinary Tract Infection among Pediatric Patients.

Antibiotic resistance is a growing public health problem globally, incurring health and cost burdens. The occurrence of antibiotic-resistant bacterial infections has increased significantly over the years. Gram-negative bacteria display the broadest resistance range, with bacterial species expressing extended-spectrum ß-lactamases (ESBLs), AmpC, and carbapenemases. All carbapenem-resistant Enterobacteriaceae (CRE) isolates from pediatric urinary tract infections (UTIs) between October 2015 and November 2019 (n = 30). All isolates underwent antimicrobial resistance phenotypic testing using the Phoenix NMIC/ID-5 panel, and carbapenemase production was confirmed using the NG-Test CARBA 5 assay. Whole-genome sequencing was performed on the CREs. The sequence type was identified using the Achtman multi-locus sequence typing scheme, and antimicrobial resistance markers were identified using ResFinder and the CARD database. The most common pathogens causing CRE UTIs were E. coli (63.3%) and K. pneumoniae (30%). The most common carbapenemases produced were OXA-48-like enzymes (46.6%) and NDM enzymes (40%). Additionally, one E. coli harbored IMP-26, and two K. pneumoniae possessed mutations in ompK37 and/or ompK36. Lastly, one E. coli had a mutation in the marA porin and efflux pump regulator. The findings highlight the difference in CRE epidemiology in the pediatric population compared to Qatar's adult population, where NDM carbapenemases are more common.