Whole Genome Sequencing Analysis Research Articles

Clinical Course, Antifungal Susceptibility, and Genomic Sequencing of Trichophyton indotineae

Trichophyton indotineae is an emerging dermatophyte causing outbreaks of extensive tinea infections often unresponsive to terbinafine. This species has been detected worldwide and in multiple US states, yet detailed US data on infections with T indotineae are sparse and could improve treatment practices and medical understanding of transmission. To correlate clinical features of T indotineae infections with in vitro antifungal susceptibility testing results, squalene epoxidase gene sequence variations, and isolate relatedness using whole-genome sequencing. This retrospective cohort study of patients with T indotineae infections in New York City spanned May 2022 to May 2023. Patients with confirmed T indotineae infections were recruited from 6 New York City medical centers. Improvement or resolution at the last follow-up assessment. Among 11 patients with T indotineae (6 male and 5 female patients; median [range] age, 39 [10-65] years), 2 were pregnant; 1 had lymphoma; and the remainder were immunocompetent. Nine patients reported previous travel to Bangladesh. All had widespread lesions with variable scale and inflammation, topical antifungal monotherapy failure, and diagnostic delays (range, 3-42 months). Terbinafine treatment failed in 7 patients at standard doses (250 mg daily) for prolonged duration; these patients also had isolates with amino acid substitutions at positions 393 (L393S) or 397 (F397L) in squalene epoxidase that correlated with elevated terbinafine minimum inhibitory concentrations of 0.5 μg/mL or higher. Patients who were treated with fluconazole and griseofulvin improved in 2 of 4 and 2 of 5 instances, respectively, without correlation between outcomes and antifungal minimum inhibitory concentrations. Furthermore, 5 of 7 patients treated with itraconazole cleared or had improvement at the last follow-up, and 2 of 7 were lost to follow-up or stopped treatment. Based on whole-genome sequencing analysis, US isolates formed a cluster distinct from Indian isolates. The results of this case series suggest that disease severity, diagnostic delays, and lack of response to typically used doses and durations of antifungals for tinea were common in this primarily immunocompetent patient cohort with T indotineae, consistent with published data. Itraconazole was generally effective, and the acquisition of infection was likely in Bangladesh.

Marine Pediococcus pentosaceus E3 Probiotic Properties, Whole-Genome Sequence Analysis, and Safety Assessment.

Probiotics play a significant role in enhancing health, and they are well known for bacteriocins production. Evaluating probiotics' whole-genome sequence provides insights into their consumption outcomes. Thus, genomic studies have a significant role in assessing the safety of probiotics more in-depth and offer valuable information regarding probiotics' functional diversity, metabolic pathways, and health-promoting mechanisms. Marine Pediococcus pentosaceus E3, isolated from shrimp gut, exhibited beneficial properties, indicating its potential as a probiotic candidate. Phenotypically, E3 strain was susceptible to most antibiotics assessed, tolerant to low pH and high bile salt conditions, and revealed no hemolysin activity. Interestingly, E3-neutralized CFS revealed significant antibacterial activity against pathogens under investigation. Therefore, the concentrated CFS was prepared and evaluated as a natural biopreservative and showed outstanding antimicrobial activity. Furthermore, integrated-based genome assessment has provided insight into probiotic characteristics at the genomic level. Whole-genome sequencing analysis revealed that the E3 genome possesses 1805 protein-coding genes, and the genome size was about 1.8Mb with a G + C content of 37.28%. Moreover, the genome revealed the absence of virulence factors and clinically related antibiotic genes. Moreover, several genes consistent with probiotic microorganisms' features were estimated in the genome, including stress response, carbohydrate metabolism, and vitamin biosynthesis. In addition, several genes associated with survival and colonization within the gastrointestinal tract were also detected across the E3 genome. Therefore, the findings suggest that insights into the genetic characteristics of E3 guarantee the safety of the strain and facilitate future development of E3 isolate as a health-promoting probiotic and source of biopreservative.

Unveiling antibiofilm potential: proteins from Priestia sp. targeting Staphylococcus aureus biofilm formation.

Staphylococcus aureus is the etiologic agent of many nosocomial infections, and its biofilm is frequently isolated from medical devices. Moreover, the dissemination of multidrug-resistant (MDR) strains from this pathogen, such as methicillin-resistant S. aureus (MRSA) strains, is a worldwide public health issue. The inhibition of biofilm formation can be used as a strategy to weaken bacterial resistance. Taking that into account, we analysed the ability of marine sponge-associated bacteria to produce antibiofilm molecules, and we found that marine Priestia sp., isolated from marine sponge Scopalina sp. collected on the Brazilian coast, secretes proteins that impair biofilm development from S. aureus. Partially purified proteins (PPP) secreted after 24 hours of bacterial growth promoted a 92% biofilm mass reduction and 4.0 µg/dL was the minimum concentration to significantly inhibit biofilm formation. This reduction was visually confirmed by light microscopy and Scanning Electron Microscopy (SEM). Furthermore, biochemical assays showed that the antibiofilm activity of PPP was reduced by ethylenediaminetetraacetic acid (EDTA) and 1,10 phenanthroline (PHEN), while it was stimulated by zinc ions, suggesting an active metallopeptidase in PPP. This result agrees with mass spectrometry (MS) identification, which indicated the presence of a metallopeptidase from the M28 family. Additionally, whole-genome sequencing analysis of Priestia sp. shows that gene ywad, a metallopeptidase-encoding gene, was present. Therefore, the results presented herein indicate that PPP secreted by the marine Priestia sp. can be explored as a potential antibiofilm agent and help to treat chronic infections.

Prevalence and genetic diversity of azole-resistant Malassezia pachydermatis isolates from canine otitis and dermatitis: a two-year study.

Despite previous reports on the emergence of Malassezia pachydermatis strains with decreased susceptibility to azoles, there is limited information on the actual prevalence and genetic diversity of azole-resistant isolates of this yeast species. We assessed the prevalence of azole resistance in M. pachydermatis isolates from cases of dog otitis or skin disease attended in a veterinary teaching hospital during a 2-year period and analyzed the ERG11 (encoding a lanosterol 14-α demethylase, the primary target of azoles) and whole genome sequence diversity of a group of isolates that displayed reduced azole susceptibility. Susceptibility testing of 89M. pachydermatis isolates from 54 clinical episodes (1-6 isolates/episode) revealed low minimum inhibitory concentrations (MICs) to most azoles and other antifungals, but 11 isolates from six different episodes (i.e., 12.4% of isolates and 11.1% of episodes) had decreased susceptibility to multiple azoles (fluconazole, itraconazole, ketoconazole, posaconazole, ravuconazole, and/or voriconazole). ERG11 sequencing of these 11 azole-resistant isolates identified eight DNA sequence profiles, most of which contained amino acid substitutions also found in some azole-susceptible isolates. Analysis of whole genome sequencing (WGS) results revealed that the azole-resistant isolates from a same episode of otitis, or even different episodes affecting a same animal, were more genetically related to each other than to isolates from other dogs. In conclusion, our results confirmed the remarkable ERG11 sequence variability in M. pachydermatis isolates of animal origin observed in previous studies and demonstrate the value of WGS for disentangling the epidemiology of this yeast species.

The influence of an innovative antibacterial drug of the thiadiazinone class on the virulence factors of bacteria of the phylum Pseudomonadota, which chronically infect patients with cystic fibrosis

Introduction. Infections of the lower respiratory tract by bacteria of the Pseudomonadota phylum: Pseudomonas aeruginosa, Burkholderia spp., Achromobacter spp. are critical to the quality and life expectancy of patients with cystic fibrosis (CF). When the infection is chronic, eradication of bacteria with existing antibacterial drugs is practically impossible. To explore alternative drugs, trials are needed on bacteria isolated from CF patients and characterized using genomic approaches. The objective of our study was a comparative analysis of virulence factors of 6 isolates of bacteria of the Pseudomonadota phylum and testing the efficacy of the innovative drug Fluorothiazinone (FT) in suppressing the pathogenicity of bacteria in vitro. Materials and methods. Isolates of A. ruhlandii ST36, A. xylosoxidans ST555, B. cepacia ST2140, B. gladioli ST2141, P. aeruginosa ST859 and ST198 were examined using whole-genome sequencing and bioinformatics analysis to search for resistance and virulence determinants. The FT drug was tested for its effect on bacteria in in vitro experiments on cytotoxicity on HeLa cells, motility and biofilm formation. Results. Genomic studies have confirmed the arsenal of resistance determinants, especially the efflux systems of bacteria isolated from patients with CF, and the diversity of virulence factors, among which we identified factors in the categories of motility, signals of quorum-sensing systems, secretion systems, exotoxins, as the most essential for the adaptation of bacteria to conditions of the lower respiratory tract. In vitro tests of the FT drug showed its effectiveness in suppressing cytotoxicity (2.6–4.0 times), motility (2.0–3.6 times) and the process of biofilm formation (2.0–7.7 times). Conclusion. For the first time, the effectiveness of the innovative antibacterial drug Fluorothiazinone has been shown against bacteria of the Pseudomonadota phylum, isolated from chronically infected patients with CF, with the described potential of virulence factors.

Mechanisms governing melon fruit skin pigmentation: Insights from transcriptome sequencing and whole-genome bisulfite sequencing analyses

Fruit skin color is a critical agronomic trait determining fruit quality, but the mechanisms governing melon (Cucumis melo L.) fruit skin pigmentation remain inadequately characterized. We performed cytological, transcriptomic, and whole-genome bisulfite sequencing (WGBS) comparative analyses on two near-isogenic lines (NILs) at different developmental stages: the green-skinned (GS) and white-skinned (WS) melon. The enriched functions among the 536 identified differentially expressed genes (DEGs) were related to chloroplast development, pigment metabolism, and photosynthetic pathway. The genes in these pathways were downregulated in the WS line, potentially contributing to the decreased chlorophyll content and abnormal chloroplast development in WS. WGBS analysis revealed that the levels of genomic DNA methylation in WS progressively exceeded those in GS as the fruits developed. The integrated analysis of the transcriptome and methylome identified DMR-associated DEGs (CHLI, CRD1, PORA, and HCAR) involved in chlorophyll metabolism. Notably, PORA exhibited downregulated expression and was hypermethylated in WS fruits during two developmental stages, showing a coordinated expression with the chlorophyll contents in developing WS and GS fruits. Furthermore, we identified a transcription factor gene, MELO3C011576, that was closely associated with the expression of PORA and chlorophyll levels across three developmental stages in both NILs. We speculate that PORA positively regulates chlorophyll biosynthesis in the two NILs, with its expression may be co-controlled by DNA methylation and transcription factors.

Novel oceanic cyanobacterium isolated from Bangaram island with profound acid neutralizing ability is proposed as Leptolyngbya iicbica sp. nov. strain LK

An acid-neutralizing, filamentous, non-heterocytous, marine cyanobacterium named 'LK' has been isolated from the seashore of Bangaram Island, an atoll of Lakshadweep, India, and is described here as a novel species. LK has been characterized using morphological, ecological, and genomic features. Based on 16S rRNA, whole-genome sequencing, and marker gene-based analysis, LK has been identified as a new species. LK clustered with Leptolyngbya-like strains belonging to the LPP group but diverged from Leptolyngbya sensu stricto, indicating the polyphyletic nature of the Leptolyngbya genus. Leptolyngbya sp. SIOISBB and Halomicronema sp. CCY15110 were identified as LK's two closest phylogenetic neighbors in various phylogenetic studies. The analysis of 16S rRNA, ITS secondary structures, and genome relatedness indices such as AAI, ANI, and gANI strongly support LK as a novel species of the Leptolyngbya genus. The mechanism behind acid neutralization in LK has been delineated, attributing it to a surface phenomenon most likely due to the presence of salts of calcium, magnesium, sodium, and potassium. We name LK as Leptolyngbya iicbica strain LK which is a novel species with prominent acidic pH-neutralizing properties.

In vitro, in planta, and comparative genomic analyses of Pseudomonas syringae pv. syringae strains of pepper (Capsicum annuum var. annuum).

Pseudomonas leaf spot (PLS) caused by Pseudomonas syringae pv. syringae (Pss) causes significant losses to the pepper industry. Highly virulent Pss strains under optimal environmental conditions (cool-moderate temperatures, high moisture) can cause severe necrotic lesions on pepper leaves that consequently can decrease pepper yield if the disease persists. Hence, it is important to understand the virulence mechanisms of Pss to be able to effectively control PLS in peppers. In our study, in vitro, in planta, and whole-genome sequence analyses were conducted to better understand the virulence and pathogenicity characteristics of Pss strains in peppers. Our findings fill a knowledge gap regarding potential virulence and pathogenicity characteristics of Pss in peppers, including virulence and antimicrobial gene content. Our study helps pave a path to further identify the role of specific virulence genes in causing disease in peppers, which can have implications in developing strategies to effectively control PLS in peppers.

Genomic characterization of Staphylococcus aureus isolated from patients admitted to intensive care units of a tertiary care hospital: epidemiological risk of nasal carriage of virulent clone during admission.

We conducted a molecular epidemiological study of Staphylococcus aureus using whole-genome sequence data and clinical data of isolates from nasal swabs of patients admitted to the intensive care unit (ICU) of Hiroshima University hospital. The relationship between isolate genotypes and virulence factors, particularly for isolates that caused infectious diseases during ICU admission was compared with those that did not. The nasal carriage rates of methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) in patients admitted to the ICU were 7.0% and 20.1%, respectively. The carriage rate of community-acquired (CA)-MRSA was 2.3%, accounting for 32.8% of all MRSA isolates. Whole-genome sequencing analysis of the MRSA isolates indicated that most, including CA-MRSA and healthcare-associated (HA)-MRSA, belonged to clonal complex (CC) 8 [sequence type (ST) 8] and SCCmec type IV. Furthermore, results for three disease foci (pneumonia, skin and soft tissue infection, and deep abscess) and the assessment of virulence factor genes associated with disease conditions [bacteremia, acute respiratory distress syndrome (ARDS), disseminated intravascular coagulopathy (DIC), and septic shock] suggested that nasal colonization of S. aureus clones could represent a risk for patients within the ICU. Particularly, MRSA/J and MSSA/J may be more likely to cause deep abscess infection; ST764 may cause ventilation-associated pneumonia, hospital-acquired pneumonia and subsequent bacteremia, and ARDS, and tst-1-positive isolates may cause DIC onset.IMPORTANCENasal colonization of MRSA in patients admitted to the intensive care unit (ICU) may predict the development of MRSA infections. However, no bacteriological data are available to perform risk assessments for Staphylococcus aureus infection onset. In this single-center 2-year genomic surveillance study, we analyzed all S. aureus isolates from nasal swabs of patients admitted to the ICU and those from the blood or lesions of in-patients who developed infectious diseases in the ICU. Furthermore, we identified the virulent clones responsible for causing infectious diseases in the ICU. Herein, we report several virulent clones present in the nares that are predictive of invasive infections. This information may facilitate the design of preemptive strategies to identify and eradicate virulent MRSA strains, reducing nosocomial infections within the ICU.

XA21-mediated resistance to Xanthomonas oryzae pv. oryzae is dose dependent.

The rice receptor kinase XA21 confers broad-spectrum resistance to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of rice bacterial blight disease. To investigate the relationship between the expression level of XA21 and resulting resistance, we generated independent HA-XA21 transgenic rice lines accumulating the XA21 immune receptor fused with an HA epitope tag. Whole-genome sequence analysis identified the T-DNA insertion sites in sixteen independent T0 events. Through quantification of the HA-XA21 protein and assessment of the resistance to Xoo strain PXO99 in six independent transgenic lines, we observed that XA21-mediated resistance is dose dependent. In contrast, based on the four agronomic traits quantified in these experiments, yield is unlikely to be affected by the expression level of HA-XA21. These findings extend our knowledge of XA21-mediated defense and contribute to the growing number of well-defined genomic landing pads in the rice genome that can be targeted for gene insertion without compromising yield.

Genomic epidemiology reveals multiple mechanisms of linezolid resistance in clinical enterococci in China

BackgroundInfections caused by linezolid-resistant enterococci (LRE) are clinically difficult to treat and threaten patient health. However, there is a lack of studies on long time-span LRE strains in China. For this reason, our study comprehensively revealed the resistance mechanisms of LRE strains collected in a Chinese tertiary care hospital from 2011 to 2022.MethodsEnterococcal strains were screened and verified after retrospective analysis of microbial data. Subsequently, 65 LRE strains (61 Enterococcus faecalis and 4 Enterococcus faecium, MIC ≥ 8 µg/ml), 1 linezolid-intermediate Enterococcus faecium (MIC = 4 µg/ml) and 1 linezolid-susceptible Enterococcus faecium (MIC = 1.5 µg/ml) were submitted for whole-genome sequencing (WGS) analysis and bioinformatics analysis.ResultsThe optrA gene was found to be the most common linezolid resistance mechanism in our study. We identified the wild-type OptrA and various OptrA variants in 98.5% of LRE strains (61 Enterococcus faecalis and 3 Enterococcus faecium). We also found one linezolid-resistant Enterococcus faecium strain carried both optrA and cfr(D) gene, while one linezolid-resistant Enterococcus faecium only harbored the poxtA gene. Most optrA genes (55/64) were located on plasmids, with impB-fexA-optrA, impB-fexA-optrA-erm(A), fexA-optrA-erm(A), and fexA-optrA segments. A minority of optrA genes (9/64) were found on chromosomes with the Tn6674-like platform. Besides, other possible linezolid resistance-associated mechanisms (mutations in the rplC and rplD genes) were also found in 26 enterococcal strains.ConclusionsOur study suggested that multiple mechanisms of linezolid resistance exist among clinical LRE strains in China.

Rapid detection of the source of a Listeria monocytogenes outbreak in Switzerland through routine interviewing of patients and whole-genome sequencing.

Listeriosis is a notifiable disease in Switzerland. In summer 2022, the Swiss Federal Office of Public Health noticed an increase in reports of listeriosis cases, indicating a possible ongoing outbreak. Here we present the approaches applied for rapidly confirming the outbreak, detecting the underlying source of infection and the measures put in place to eliminate it and contain the outbreak. For close surveillance and early detection of outbreak situations with their possible sources, listeriosis patients in Switzerland are systematically interviewed about risk behaviours and foods consumed prior to the infection.Listeria monocytogenesisolates derived from patients in medical laboratories are sent to the National Reference Laboratory for Enteropathogenic Bacteria and Listeria, where they routinely undergo whole-genome sequencing. Interview and whole-genome sequencing data are continuously linked for comparison and analysis. In summer 2022, 20 patient-derivedL. monocytogenesserotype 4b sequence type 388 strains were found to belong to an outbreak cluster (≤10 different alleles between neighbouring isolates) based on core genome multilocus sequence typing analysis. Geographically, 18 of 20 outbreak cases occurred in northeastern Switzerland. The median age of patients was 77.4 years (range: 58.1-89.7), with both sexes equally affected. Rolling analysis of the interview data revealed smoked trout from a local producer as a suspected infection source, triggering an on-site investigation of the production facility and sampling of the suspected products by the responsible cantonal food inspection team on 15 July 2022. Seven of ten samples tested positive forL. monocytogenesand the respective cantonal authority ordered a ban on production and distribution as well as a product recall. The Federal Food Safety and Veterinary Office released a nationwide public alert covering the smoked fish products concerned. Whole-genome sequencing analysis confirmed the interrelatedness of theL. monocytogenessmoked trout product isolates and the patient-derived isolates. Following the ban on production and distribution and the product recall, reporting of new outbreak-related cases rapidly dropped to zero. This listeriosis outbreak could be contained within a relatively short time thanks to identification of the source of contamination through the established combined approach of timely interviewing of every listeriosis patient or a representative and continuous molecular analysis of the patient- and food-derivedL. monocytogenesisolates. These findings highlight the effectiveness of this well-established, joint approach involving the federal and cantonal authorities and the research institutions mandated to contain listeriosis outbreaks in Switzerland.

Multidrug resistance in Salmonella isolates of swine origin: mobile genetic elements and plasmids associated with cephalosporin resistance with potential transmission to humans.

The emergence of foodborne Salmonella strains carrying antimicrobial resistance (AMR) in mobile genetic elements (MGE) is a significant public health threat in a One Health context requiring continuous surveillance. Resistance to ciprofloxacin and cephalosporins is of particular concern. Since pigs are a relevant source of foodborne Salmonella for human beings, we studied transmissible AMR genes and MGE in a collection of 83 strains showing 9 different serovars and 15 patterns of multidrug resistant (MDR) previously isolated from pigs raised in the conventional breeding system of Northern Spain. All isolates were susceptible to ciprofloxacin and three isolates carried blaCMY-2 or blaCTX-M-9 genes responsible for cefotaxime resistance. Filter mating experiments showed that the two plasmids carrying blaCTX-M-9 were conjugative while that carrying blaCMY-2 was self-transmissible by transformation. Whole-genome sequencing and comparative analyses were performed on the isolates and plasmids. The IncC plasmid pSB109, carrying blaCMY-2, was similar to one found in S. Reading from cattle, indicating potential horizontal transfer between serovars and animal sources. The IncHI2 plasmids pSH102 in S. Heidelberg and pSTM45 in S. Typhimurium ST34, carrying blaCTX-M-9, shared similar backbones and two novel "complex class 1 integrons" containing different AMR and heavy metal genes. Our findings emphasize the importance of sequencing techniques to identify emerging AMR regions in conjugative and stable plasmids from livestock production. The presence of MGE carrying clinically relevant AMR genes raises public health concerns, requiring monitoring to mitigate the emergence of bacteria carrying AMR genes and subsequent spread through animals and food.IMPORTANCEThe emergence of foodborne Salmonella strains carrying antimicrobial resistance (AMR) in mobile genetic elements (MGE) is a significant public health threat in a One Health context. Since pigs are a relevant source of foodborne Salmonella for humans, in this study, we investigate different aspects of AMR in a collection of 83 Salmonella showing nine different serovars and 15 patterns of multidrug resistant (MDR) isolated from pigs raised in the conventional breeding system. Our findings emphasize the importance of sequencing techniques to identify emerging AMR regions in conjugative and stable plasmids from livestock production. The presence of MGE carrying clinically relevant AMR genes raises public health concerns, requiring monitoring to mitigate the emergence of bacteria carrying AMR genes and subsequent spread through animals and food.

Characterization of MLST-99 Salmonella Typhimurium and the monophasic variant I:4,[5],12:i:- isolated from Canadian Atlantic coast shellfish.

Salmonella enterica subsp. enterica Typhimurium and its monophasic variant I 1;4,[5],12:i:- (MVST) are responsible for thousands of reported cases of salmonellosis each year in Canada, and countries worldwide. We investigated S. Typhimurium and MVST isolates recovered from raw shellfish harvested in Atlantic Canada by the Canadian Food Inspection Agency (CFIA) over the past decade, to assess the potential impact of these isolates on human illness and to explore possible routes of shellfish contamination. Whole-genome sequence analysis was performed on 210 isolates of S. Typhimurium and MVST recovered from various food sources, including shellfish. The objective was to identify genetic markers linked to ST-99, a sequence type specifically associated with shellfish, which could explain their high prevalence in shellfish. We also investigated the genetic similarity amongst CFIA ST-99 isolates recovered in different years and geographical locations. Finally, the study aimed to enhance the molecular serotyping of ST-99 isolates, as they are serologically classified as MVST but are frequently misidentified as S. Typhimurium through sequence analysis. To ensure recovery of ST-99 from shellfish was not due to favourable growth kinetics, we measured the growth rates of these isolates relative to other Salmonella and determined that ST-99 did not have a faster growth rate and/or shorter lag phase than other Salmonella evaluated. The CFIA ST-99 isolates from shellfish were highly clonal, with up to 81 high-quality single nucleotide variants amongst isolates. ST-99 isolates both within the CFIA collection and those isolated globally carried numerous unique deletions, insertions and mutations in genes, including some considered important for virulence, such as gene deletions in the type VI secretion system. Interestingly, several of these genetic characteristics appear to be unique to North America. Most notably was a large genomic region showing a high prevalence in genomes from Canadian isolates compared to those from the USA. Although the functions of the majority of the proteins encoded within this region remain unknown, the genes umuC and umuD, known to be protective against UV light damage, were present. While this study did not specifically examine the effects of mutations and insertions, results indicate that these isolates may be adapted to survive in specific environments, such as ocean water, where wild birds and/or animals serve as the natural hosts. Our hypothesis is reinforced by a global phylogenetic analysis, which indicates that isolates obtained from North American shellfish and wild birds are infrequently connected to isolates from human sources. These findings suggest a distinct ecological niche for ST-99, potentially indicating their specialization and adaptation to non-human hosts and environments, such as oceanic habitats.

Occurrence and characteristics of blaOXA-181-carrying Klebsiella aerogenes from swine in China

Klebsiella aerogenes is a largely understudied opportunistic pathogen that can cause sepsis and lead to high mortality rates. In this study, we reported the occurrence of carbapenem-resistant blaOXA-181-carrying Klebsiella aerogenes from swine in China and elucidate their genomic characteristics. A total of 126 samples, including 109 swine fecal swabs, 14 environmental samples, and 3 feed samples were collected from a pig farm in China. The samples were enriched with LB broth culture and then inoculated into MacConkey agar plates for bacterial isolation. After PCR detection of carbapenemases genes, the blaOXA-181-carrying isolates were subjected to antimicrobial susceptibility testing, and whole-genome sequence analysis. Four Klebsiella aerogenes isolates carrying the blaOXA-181 gene were obtained from swine fecal samples. All the four strains were belonged to ST438. The blaOXA-181 genes were located in IncX3-ColKP3 hybrid plasmids with the core genetic structure of IS26-ΔIS3000-ΔISEcp1-blaOXA-181-ΔlysR-ΔereA-ΔrepA-ISKpn19-tinR-qnrS1-ΔIS2-IS26, which suggests the potential for horizontal transfer and further dissemination of this resistance gene among Enterobacteriaceae and other sources. This study represents the first instance of OXA-181-producing K. aerogenes being identified from swine feces in China. It is crucial to maintain continuous monitoring and ongoing attention to the detection of K. aerogenes carrying blaOXA-181 and other resistance genes in pigs.

Genetic Characterization of Plasmid-Borne blaOXA-58 and blaOXA-72 in Acinetobacter pittii in Shaanxi, China

BackgroundAcinetobacter pittii has emerged as an opportunistic nosocomial pathogen associated with hospital-acquired infections. The purpose of this study was to investigate the genetic structures of plasmids carrying carbapenemase genes blaOXA-58 and blaOXA-72 in A. pittii strains AR3676 and AR3651 isolated from patients. MethodsAntimicrobial susceptibility testing was performed using broth microdilution. Whole-genome sequencing and bioinformatics analysis were performed to characterize the genome of A. pittii AR3676 and AR3651. Conjugation experiments were conducted to evaluate plasmids transferability. Phylogenetic and comparative genomic analysis were performed to explore the characteristics of carbapenem-resistant A. pittii isolates worldwide. ResultsThe AR3676 strain exhibited resistance to imipenem. The 19,700-bp plasmid pAR3676-OXA-58 harbored blaOXA-58 with genetic contexts consisting of a truncated ISAba3-like-blaOXA58-ISAba3. Additionally, the AR3651 strain exhibited resistance to imipenem and meropenem. The genome of AR3651 comprised one 9,837-bp RepA_AB plasmid pAR3651-OXA-72 harboring blaOXA-72. The blaOXA-72 was flanked by XerC/XerD recombination sites. The conjugation of plasmids pAR3676-OXA-58 and pAR3651-OXA-72 from A. pittii to A. baumannii ATCC 17978RIFR failed three independent times. Phylogenetic analysis of A. pittii strains AR3676, AR3651 and other 504 A. pittii strains collected between 1966 and 2022 from various geographic localities, revealed genetic diversity with a heterogeneous distribution of carbapenemase genes. ConclusionA. pittii strains with plasmid carrying blaOXA-58 or blaOXA-72 may serve as an important reservoir of carbepenemase genes. The carbepenemase genes on a single plasmid may facilitate their dissemination and persistence. Additionally, the pdif sites and mobile elements play an important role in the mobilization of resistance genes and plasmid evolution.

Plasmodium ovale spp dhfr mutations associated with reduced susceptibility to pyrimethamine in sub-Saharan Africa: a retrospective genetic epidemiology and functional study

Mutations in the Plasmodium falciparum dhfr gene confer resistance to pyrimethamine, which is widely used for malaria chemoprevention in Africa. We aimed to evaluate the frequency and evolution of dhfr mutations in Plasmodium ovale spp in Africa and their functional consequences, which are incompletely characterised. We analysed dhfr mutations and their frequencies in P ovale spp isolates collected between Feb 1, 2004, and Aug31, 2023, from the French National Malaria Reference Centre collection and from field studies in Benin, Gabon, and Kenya. Genetic patterns of positive selection were investigated. Full-length recombinant wild-type and mutant DHFR enzymes from both P ovale curtisi and P ovale wallikeri were expressed in bacteria to test whether the most common mutations reduced pyrimethamine susceptibility. We included 518 P ovale spp samples (314 P ovale curtisi and 204 P ovale wallikeri). In P ovale curtisi, Ala15Ser-Ser58Arg was the most common dhfr mutation (39%; 124 of 314 samples). In P ovale wallikeri, dhfr mutations were less frequent, with Phe57Leu-Ser58Arg reaching 17% (34 of 204 samples). These two mutants were the most prevalent in central and east Africa and were fixed in Kenyan isolates. We detected six and four other non-synonymous mutations, representing 8% (24 isolates) and 2% (five isolates) of the P ovale curtisi and Povale wallikeri isolates, respectively. Whole-genome sequencing and microsatellite analyses revealed reduced genetic diversity around themutant pocdhfr and powdhfr genes. The mutant DHFR proteins showed structural changes at the pyrimethamine binding site in-silico, confirmed by a 4-times increase in pyrimethamine half-maximal inhibitory concentration in an Escherichia coli growth assay for the Phe57Leu-Ser58Arg mutant and 50-times increase for the Ala15Ser-Ser58Arg mutant, compared with the wild-type counterparts. The widespread use of sulfadoxine-pyrimethamine for malaria chemoprevention might have exerted fortuitous selection pressure for dhfr mutations in P ovale spp. This calls for closer monitoring of dhfr and dhps mutations in P ovale spp. French Ministry of Health, Agence Nationale de la Recherche, and Global Emerging Infections Surveillance branch of the Armed Forces Health Surveillance Division.

Conversion behavior of heterotrophic nitrification - aerobic denitrification bacterium Paracoccus denitrificans HY-1 in nitrogen and phosphorus removal

The heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria harbored remarkable removal capacity in eliminating single nitrogen pollutant, but their comprehensive performance in dealing with mixed nitrogen sources remained unknown. In this study, the denitrification capacity of HN-AD bacteria Paracoccus denitrificans HY-1 was explored through whole-genome sequencing and nitrogen metabolic pathway analysis, and the complex nitrogen sources with gradient concentration were used to validate its nitrogen removal efficiency under both aerobic and anaerobic conditions. The results demonstrated that strain HY-1 harbored an efficient degradation capacity on three nitrogen sources, and the removal rates of NH4+-N, NO2−-N and NO3−-N were 10.67 mg·L−1·h−1, 27.81 mg·L−1·h−1, and 22.05 mg·L−1·h−1, respectively. The denitrification rate of 15.33 mg·L−1·h−1 was obtained under anaerobic condition with mixed nitrogen sources. Also, the simultaneous nitrification denitrification phosphorus removal (SNDPR) trial revealed that strain HY-1 could eliminate PO43−-P with a rate of 0.38 mg·L−1·h−1 under coexistence condition of the three nitrogen sources. Combined with the nitrogen balance analysis, the results indicated that strain HY-1 possesses a great potential for application in complex nitrogen and phosphorus contaminated environments.

Emergence of novel hypervirulent Acinetobacter baumannii strain and herpes simplex type 1 virus in a case of community-acquired pneumonia in China

BackgroundA. baumannii is an important and common clinical pathogen, especially in the intensive care unit (ICU). This study aimed to characterize one hypervirulent A. baumannii strain in a patient with community-acquired pneumonia and herpes simplex type 1 virus infection. MethodsMinimum inhibitory concentrations (MICs) were determined using the Kirby-Bauer (K-B) and broth microdilution methods. Galleria mellonella infection model experiment was conducted. Whole-genome sequencing (WGS) was performed using the Illumina and Nanopore platforms. The resistance and virulence determinants were identified using the ABRicate program with ResFinder and the VFDB database. The capsular polysaccharide locus (K locus) and lipooligosaccharide outer core locus (OC locus) were identified using Kleborate with Kaptive. Phylogenetic analyses were conducted using the BacWGSTdb server. ResultsA. baumannii XH2146 strain belongs to ST10Pas and ST447Oxf. The strain was resistant to cefazolin, ciprofloxacin, and trimethoprim/sulfamethoxazole (TMP-SMX). Bautype and Kaptive analyses showed that XH2146 contains OCL2 and KL49. WGS analysis revealed that the strain harbored blaADC-76, blaOXA-68, ant(3’’)-IIa, tet(B), and sul2. Notably, tet(B) and sul2, both were located within a 114,700-bp plasmid (designated pXH2146-1). Virulence assay revealed A. baumannii XH2146 possessed higher virulence than A. baumannii AB5075 at 12h. Comparative genomic analysis showed that A. baumannii ST447 strains were mainly isolated from the USA and exhibited a relatively close genetic relationship. Importantly, 11 strains were observed to carry blaOXA-58; blaOXA-23 was identified in 11 isolates and three ST447 A. baumannii strains harbored blaNDM-1. ConclusionsEarly detection of community-acquired hypervirulent Acinetobacter baumannii strains is recommended to prevent their extensive spread in hospitals.

First Report of Kiwifruit Root Rot Caused by Globisporangium spinosum in China.

Kiwifruit is widely cultivated for its high vitamin C content and nutritional value. In January 2022, root rot symptoms were found in about 30% of Actinidia chinensis cv. Jinyan plants grafted on A. deliciosa rootstocks in an orchard located in Sanming (26.32°N, 117.23°E), Fujian Province of China. The affected plants appeared stunted, with brown and decaying roots, some of which were covered with white hyphae. To isolate the pathogen, the surfaces of typical symptomatic roots were sterilized for 30 s using 75% ethanol, followed by four rinses in sterile water, placing on potato dextrose agar (PDA), and incubating away from light at 25°C for 7 days. 16 Globisporangium-like isolates were obtained through hyphal tip isolation, displaying a milky-white appearance with irregular protuberances on the surface, and yellow-white backs with radial fold lines. The isolates were then cultured on corn meal agar for 5 days at 25°C in dark for morphological characteristics. Under microscope, the hyphae appeared as long strips without septa and 4.1 to 8.2 µm wide (average 6.7 µm), containing irregularly sized spherical droplets. Both terminal and intercalary hyphae swellings were observed; these appeared either spherical or subspherical, with some having projections. Their dimensions were 12.3 to 27.6 µm (average 17.3 µm). The oospores were mostly spherical, either plerotic or aplerotic, 11.8 to 22.3 µm wide (average 18.9 µm), with occasional projections. The antheridia were rod-shaped and curved, with one end attached to the oogonia. Amplification of the sequences of internal transcribed spacer (ITS) regions and cytochrome c oxidase subunit I (COI) were conducted using the primers ITS1/ITS4 (White et al. 1990) and OomCoxI-Levlo/OomCoxI-Levup (Robideau et al. 2011), respectively. The sequencing results revealed identical ITS and COI sequences in all 16 isolates. BLASTn analysis of the 969-bp ITS sequence ON202808 showed 99.38-99.59% similarity (965/971bp, 967/971bp) with the KJ162353 and AY598701 sequences from Globisporangium spinosum isolates, while the 700-bp COI sequence ON075783 showed 100% and 99.41% identity (680/680bp, 676/680bp) with the GenBank sequences HQ708835 and HQ708832, respectively, from G. spinosum. Phylogenetic analysis also showed that the obtained isolate (termed MA16) clustered with isolates from G. spinosum on the same evolutionary branch. For pathogenicity testing, four-month-old healthy Jinyan (A. chinensis) plants grown in sterilized media were transferred to sterile petri dishes covered with wet filter paper, and their roots were inoculated with a 5-mm-wide disk of MA16 when cultivated on PDA medium for 5 days. Miliang-1 (A. deliciosa) and Hongyang (A. chinensis) plants were treated similarly. The control groups each included three plants that were inoculated with non-colonized PDA. The plants were kept at 25 °C with a 12-/12-h light/dark cycle for 10 days when the inoculated plants exhibited root rot symptoms similar to those seen in the field, together with rotting and browning of the leaves. The control plants appeared healthy with no symptoms. After re-isolated from infected tissues, the pathogen was verified to be G. spinosum according to its ITS sequence, thus fulfilling the Koch's postulates. Recently, Pythium spinosum has been classified as G. spinosum according to whole-genome sequencing and phylogenomic analysis (Nguyen et al. 2022). Based on the morphological features and pathogenicity results, MA16 was identified as G. spinosum (van der Plaats-Niterink 1981; Huo et al. 2023). This report appears to be the first description of kiwifruit root rots caused by G. spinosum in China, and its identification will assist the development of strategies to counteract the disease.