Clonal Complexes Research Articles

Candida krusei (Pichia kudriavzevii) multilocus sequence typing and antifungal susceptibility profile in Cameroon.

Among the Candida species commonly involved in superficial and more significant life-threatening infections, C. krusei exhibits the most worrisome resistance profile to antifungals. This study aimed to analyse the population structure using multilocus sequence typing (MLST), and to evaluate the antifungal susceptibility profile of C. krusei isolated from patients living with human immunodeficiency virus (HIV) in Cameroon. C. krusei isolated from stool, urine, mouth and vaginal samples were identified using routine laboratory techniques and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). The C. krusei isolates were further analysed by MLST. In vitro antifungal susceptibility testing was performed using the Sensititre Yeast One™ microdilution technique. Forty three (43) C. krusei isolates were included in the study. The MLST identified 32 Diploid sequence type (DST), of which 31 were new that were not included in the current database. New alleles were not observed. Different DSTs were observed in isolates from the same geographical area, from different anatomical sites in the same patient. An eBURST analysis clustered all identified DSTs of former isolates in clonal complex 1. Heterogeneous antifungal MICs were observed in isolates of the same DST and/or from the same geographical area. 32.6% of the isolates displayed a resistant or non-wild-type phenotype to at least 3 distinct antifungal agent classes. The achieved results support the setting up of molecular epidemiology and antifungal resistance surveillance of C. krusei.

Phenotypic and Genotypic Correlates of the NaHCO3-Responsive Phenotype Among Methicillin-Resistant Staphylococcus aureus (MRSA) Isolates from Skin and Soft Tissue Infections (SSTIs)

ObjectivesTo assess the frequency of the novel NaHCO3-responsive phenotype, wherein clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates are rendered susceptible to standard-of-care β-lactams in the presence of NaHCO3, in a collection of 103 clinical US MRSA skin and soft tissue infection (SSTI) isolates and 22 clinical European SSTI isolates. Determined the correlation between specific phenotypic and genotypic metrics and the NaHCO3-responsive phenotype among US SSTI isolates. MethodsAntimicrobial susceptibility testing (AST) was performed to determine susceptibility phenotypes. Targeted and whole genome sequencing (WGS) with a genome-wide sequence analysis (GWAS) were conducted to identify specific and novel genotypes of interest that may be associated with the NaHCO3-responsive phenotype. Gene expression analysis and targeted gene deletion were performed to assess the role of a specific novel genetic locus in the NaHCO3-responsive phenotype. ResultsThe NaHCO3-responsive phenotype was identified in 78/103 US isolates and 4/22 UK isolates to cefazolin (CFZ), and in 17/103 US isolates and 1/22 UK isolates to oxacillin (OXA). In US isolates, a significant association was identified between NaHCO3-responsiveness to CFZ and: i) susceptibility to amoxicillin-clavulanate; ii) a specific mecA genotype; iii) clonal complex (CC) type 8; and iv) spa type t008. GWAS identified SNPs in an AraC family regulator (SAUSA300_RS00540) to be exclusively found in NaHCO3-non-responsive SSTI strains. In vitro HCO3 exposures of NaHCO3-responsive strains, but not -non-responsive strains, caused >2-fold up-regulated expression of this gene. Deletion of this gene rendered NaHCO3-responsive strain MRSA 11/11 no longer NaHCO3-responisve to CFZ; we have termed this gene the staphylococcal AraC bicarbonate-response regulator, sabR. ConclusionsNaHCO3-responsiveness is highly associated with CC8/spa t008, a commonly circulating genetic background in North America. The AraC bicarbonate-response regulator, sabR, appears to be associated with the mechanism of NaHCO3-responisveness, more work is needed to verify. Word count 288.

Eco-Evolutionary Drivers of Vibrio parahaemolyticus Sequence Type 3 Expansion: Retrospective Machine Learning Approach.

Environmentally sensitive pathogens exhibit ecological and evolutionary responses to climate change that result in the emergence and global expansion of well-adapted variants. It is imperative to understand the mechanisms that facilitate pathogen emergence and expansion, as well as the drivers behind the mechanisms, to understand and prepare for future pandemic expansions. The unique, rapid, global expansion of a clonal complex of Vibrio parahaemolyticus (a marine bacterium causing gastroenteritis infections) named Vibrio parahaemolyticus sequence type 3 (VpST3) provides an opportunity to explore the eco-evolutionary drivers of pathogen expansion. The global expansion of VpST3 was reconstructed using VpST3 genomes, which were then classified into metrics characterizing the stages of this expansion process, indicative of the stages of emergence and establishment. We used machine learning, specifically a random forest classifier, to test a range of ecological and evolutionary drivers for their potential in predicting VpST3 expansion dynamics. We identified a range of evolutionary features, including mutations in the core genome and accessory gene presence, associated with expansion dynamics. A range of random forest classifier approaches were tested to predict expansion classification metrics for each genome. The highest predictive accuracies (ranging from 0.722 to 0.967) were achieved for models using a combined eco-evolutionary approach. While population structure and the difference between introduced and established isolates could be predicted to a high accuracy, our model reported multiple false positives when predicting the success of an introduced isolate, suggesting potential limiting factors not represented in our eco-evolutionary features. Regional models produced for 2 countries reporting the most VpST3 genomes had varying success, reflecting the impacts of class imbalance. These novel insights into evolutionary features and ecological conditions related to the stages of VpST3 expansion showcase the potential of machine learning models using genomic data and will contribute to the future understanding of the eco-evolutionary pathways of climate-sensitive pathogens.

Methicillin-resistant and methicillin-susceptible Staphylococcus aureus in French hedgehogs admitted to a wildlife health center

The mecC gene conferring methicillin-resistance has always been found on a SCCmec type XI element and is largely restricted to the few clonal complexes CC130, CC1943, CC425, CC49 and CC599. The occurrence of the mecC gene in many different hosts highlighted its One Health importance, even though European hedgehogs (Erinaceus europaeus) are considered its natural reservoir, most probably because of the selective pressure imposed by beta-lactam-producing dermatophytes (Trichophyton erinacei) that colonize the skin of these mammals. Surprisingly, while the presence of T. erinacei on the French territory has been proven, no mecC-positive methicillin-resistant Staphylococcus aureus (MRSA) isolate has been reported yet from hedgehogs. We thus sampled 139 hedgehogs brought to a wildlife center; 128 were S. aureus carriers and 25 (18.0 %) presented a MRSA isolate, of which 21 (15.1 %) displayed the mecC gene. All 161 S. aureus collected were whole-genome sequenced. The mecC-MRSA belonged to the classical CCs, i.e. CC130, CC1943 and CC49. The majority (98/139, 70.5 %) of the methicillin-susceptible Staphylococcus aureus (MSSA) isolates also belonged to these three CCs. A phylogenetic comparison with mecC-MRSA isolates from all over Europe and New-Zealand showed local adaptations, despite the fact that they all belonged to the same CCs. The acquisition of the SCCmec type XI element by a concomitant MSSA could not be observed in the same animal, but such a transfer might be suggested since identical clones were identified, one MSSA and one MRSA, though in different animals. In parallel, we conducted a detailed analysis of the SCCmec type XI element as well as specific virulence factors (a tst variant and the vwbSaPI gene). Results led us to hypothesize that the mecC gene might be acquired through selective pressure of T. erinacei on MSSA, some of which were acquired a long time ago from ruminants and are now colonizing the skin of the hedgehogs.

Epidemiology of invasive meningococcal disease, Japan, 2013 to 2023.

BackgroundThe National Surveillance for Invasive Meningococcal Disease (IMD) initiative started in Japan in April 2013. Multiple international mass gathering events have since been held in Japan, and the COVID-19 pandemic has occurred.AimWe summarised 10 years of national surveillance data for IMD in Japan to describe epidemiological characteristics of IMD and evaluate the influence of mass gatherings and the COVID-19 pandemic on IMD.MethodsUpon diagnosis of IMD, patient information and specimens were collected and reported to local health centres. We analysed the epidemiology of IMD cases reported between 1 April 2013 and 31 March 2023.ResultsAmong 274 cases reported (median age: 55 years; 55% male), no outbreaks related to mass gathering events were identified. The annual reported incidence of IMD was 0.001-0.039 cases per 100,000 individuals between 2014 and 2022, with a notable decrease after 2020. The overall case fatality rate was 12% (33/274). The most frequent serogroups were Y and B (46 and 17%). Multilocus sequence typing revealed a predominance of clonal complex (cc) 23, followed by cc2057, while cc11 was detected in eight cases.ConclusionThe reported incidence of IMD in Japan is low compared with high-endemic countries and decreased further during the COVID-19 pandemic. This unique epidemiology of IMD in Japan lacks a clear explanation. However, distribution of meningococcal strains, such as predominance of serogroup Y, could be a contributing factor. Maintaining high-quality surveillance, including of serogroups and sequence types, is crucial to manage and prevent future IMD cases in Japan effectively.

Exploring the sequence diversity and surface expression of Factor H-Binding Protein among invasive serogroup B meningococcal strains from selected European countries

ABSTRACT Factor H-Binding Protein (fHbp) is a key component of meningococcal vaccines such as MenB-fHbp, licensed in the EU, UK, and other countries. Sufficient expression of fHbp on the bacterial surface is necessary for vaccine-induced antibodies to bind and exert bactericidal activity. The flow cytometric MEASURE assay quantifies fHbp expression in vitro, and previous studies have shown that strains with a Mean Fluorescence Intensity (MFI) >1000 are likely to be killed by MenB-fHbp-induced antibodies. This study assessed fHbp peptide distribution and expression among 451 invasive group B strains collected in 2016 across England, Wales, and Northern Ireland (EW&NI), Germany, Italy, and Spain. We found that 92% of the strains expressed fHbp above the MFI 1000 threshold. The strain distribution across EW&NI, Germany, and Italy was similar, with coverage ranging from 92.1% to 94.6%, dominated by a small number of clonal complexes and fHbp peptides. Although, the Spanish subset had a higher proportion of lower-expressing strains, particularly clonal complex 213, resulting in a lower predicted coverage for Spain (84%). These results, along with other published MEASURE data, can provide a basis for genotypic MenB-fHbp coverage predictions, however, inclusion of the upstream intergenic sequence of the fHbp gene in the prediction improved its accuracy by distinguishing between low- and high-expressing strains. Future MEASURE analyses of strains with less common fHbp variants would serve to further refine vaccine coverage predictions.

Whole-genome analysis of stress resistance-related genes in Listeria monocytogenes

Listeria monocytogenes is a crucial foodborne pathogen with significant public health implications. This study analyzed whole-genome sequences (WGS) of L. monocytogenes strains from public databases, examining associations between resistance genes, lineage, strain type, isolation source, and geography. Results revealed that after eliminating duplicates and strains with incomplete WGS, a total of 316 strains were deemed suitable for subsequent analyses. Within these strains, lineages I and II were extensively distributed, predominantly isolated from clinical and food sources. 56.65% of these strains fell into seven major Clonal Complexes (CC), identified by Multilocus Sequence Typing (MLST), correlating significantly with isolation information. Analysis of 46 resistance-related genes showed a high consistency of resistance genes in the same type of strains, hinting at a potential causal chain of ‘food-food environment-evolution of L. monocytogenes’. Moreover, the standard strains exhibit similar gene carriage rates as the sample strains, with multiple variations observed in acid-resistance genes. In conclusion, through a comprehensive analysis of the L. monocytogenes genome sequences, this study deepens our understanding of the differences and associations between its lineage, strain typing, isolation sources, geographical distribution, and resistance genes, thereby providing a scientific basis for formulating more effective prevention and control strategies.

Genotypic analysis of Shiga toxin-producing Escherichia coli clonal complex 17 in England and Wales, 2014-2022.

Introduction. Shiga toxin-producing Escherichia coli (STEC) are zoonotic, gastrointestinal pathogens characterized by the presence of the Shiga toxin (stx) gene. Historically, STEC O157:H7 clonal complex (CC) 11 has been the most clinically significant serotype; however, recently there has been an increase in non-O157 STEC serotypes, including STEC O103:H2 belonging to CC17.Gap statement. STEC O103:H2 is an STEC serotype frequently isolated in England, although little is known about the epidemiology, clinical significance, associated public health burden or evolutionary context of this strain.Aim. Surveillance data and whole-genome sequencing data were analysed to determine the microbiological characteristics and public health burden of CC17, including the clinically significant serotype O103:H2, in England and Wales.Methodology. Isolates of E. coli belonging to CC17 (n=425) submitted to the Gastrointestinal Bacteria Reference Unit from 2014 to 2022 were whole genome sequenced, integrated with enhanced surveillance questionnaire data and analysed retrospectively.Results. Overall, diagnoses of CC17 infection increased every year since 2014. Most cases were female (58.5%), with the highest proportion of cases belonging to the 0-4 age group (n=83/424, 19.6%). Clinical presentation data identified diarrhoea (92.1%), abdominal pain (72.4%) and blood in stool (55.3%) as the most frequent symptoms, while 20.4% cases were admitted to hospital and 1.3% developed haemolytic uraemic syndrome. The five most common established serotypes were O103:H2 (64.5%), O123:H2 (11.1%), O151:H2 (6.6%), O71:H2 (3.3%) and O4:H2 (2.6%). The majority of CC17 isolates (78.6%) had the stx1a/eae virulence gene combination. Nine outbreak clusters of STEC infections that were mainly geographically dispersed and temporally related were identified and associated with foodborne transmission.Conclusions. Nationwide implementation of PCR to detect non-O157 STEC and improvements to algorithms for the follow-up of PCR-positive faecal specimens is recommended. Enhanced surveillance is necessary to assess the incidence of CC17 infection and overall burden of this CC within the UK population.

Genetic characteristics and antimicrobial resistance of Staphylococcus aureus isolates from pig farms in Korea: emergence of cfr-positive CC398 lineage

BackgroundLivestock-associated Staphylococcus aureus (LA-SA) has gained global attention because of its ability to colonize farm animals and transmit to the environment and humans, leading to symptomatic infections and the spread of antimicrobial resistance (AMR). In the last decade, numerous studies have reported a high prevalence of S. aureus clonal complex (CC) 398 in pig farms.ResultsIn this study, 163 S. aureus isolates were collected from healthy pigs (n = 110), farm environments (n = 42), and farm workers (n = 11), and their AMR profiles and epidemiological characteristics were analyzed. We identified 51 (31.3%) methicillin-resistant S. aureus (MRSA) and 112 (68.7%) methicillin-susceptible S. aureus (MSSA), with 161 (98.8%) isolates belonging to the CC398 lineage. The highest prevalence of spa type t571 was observed among the CC398 isolates. All 47 sequence type (ST) 398 MRSA isolates carried staphylococcal cassette chromosome mec (SCCmec) V, while four ST541 isolates carried SCCmec IV. High levels of resistance to commonly used antibiotics, including phenicols, quinolones, lincosamides, macrolides, aminoglycosides, and tetracyclines, have been observed on Korean pig farms. Notably, 21 cfr-positive CC398 isolates (four ST541-SCCmec IV MRSA and 17 ST398 MSSA) displaying increased resistance to linezolid were identified in healthy pigs.ConclusionsIn summary, these findings suggest that the multidrug-resistant CC398 S. aureus lineage predominantly colonizes healthy pigs and farm environments in Korea. The emergence of cfr-positive S. aureus at human-animal interfaces presents a significant threat to food safety and public health.

First insights into the prevalence, genetic characteristics, and pathogenicity of Bacillus cereus from generations worldwide.

Bacillus cereus, a global threat, is one of the major causes of toxin-induced foodborne diseases. However, a comprehensive assessment of the prevalence and characteristics of B. cereus worldwide is still lacking. Here, we applied whole-genome sequence analysis to 191 B. cereus collected in Africa, America, Asia, Europe, and Oceania from the 1900s to 2022, finding that CC142 dominated the global B. cereus clonal complex. The results provided direct evidence that B. cereus could spread through the food chain and intercontinentally. B. cereus from different generations worldwide showed coherence in the antibiotic-resistant gene and virulence and biofilm gene profiles, although with high genomic heterogeneity. The BCI-BCII-vanZF-fosB profiles and virulence and biofilm genes were detected at high rates, and we emphasized that B. cereus would pose a serious challenge to global public health and clinical treatment.IMPORTANCEThis study first emphasized the prevalence, genetic characteristics, and pathogenicity of Bacillus cereus worldwide from the 1900s to 2022 using whole-genome sequence analysis. The CC142 dominated the global Bacillus cereus clonal complex. Moreover, we revealed a close evolutionary relationship between the isolates from different sources. B. cereus isolates from different generations worldwide showed coherence in potential pathogenicity, although with high genomic heterogeneity. The BCI-BCII-vanZF-fosB profiles and virulence and biofilm genes were detected at high rates, and we emphasized that B. cereus would pose a serious challenge to global public health and clinical treatment.

An Acinetobacter baumannii nasal carriage isolate recovered from an asymptomatic patient in Vietnam is extensively antibiotic resistant and produces a rare K71 type capsule.

The majority of Acinetobacter baumannii genomes sequenced and analyzed to develop an understanding of extensively drug-resistant (XDR) isolates belong to the globally disseminated CC2 clonal complex. While XDR isolates belonging to rarer lineages are often unexplored, detailed analyses could provide novel insights into the spread of resistance, as well as cell surface features such as the CPS that determine the specificity of non-antibiotic therapeutics required to treat XDR infections that resist antimicrobial chemotherapy. Here, we describe the properties of an XDR asymptomatic nasal carriage isolate recovered in Vietnam that belongs to ST142, a rarely encountered sequence type. We report the resistance profile and correlate this with detected resistance determinants. We also solve the structure of the CPS and reveal its relationship with CPS produced by other A. baumannii isolates.

Exogenous Methicillin-Resistant Staphylococcus aureus Endophthalmitis is Caused by Multidrug-Resistant Lineages that are Associated with Poor Outcomes

ABSTRACT Purpose To investigate the genomic epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) endophthalmitis and correlate it with the presenting clinical features and outcomes. Methods Nine patients presenting with MRSA endophthalmitis from 2014 to 2022 were included. Phenotypic and genomic tests were used for strain characterization. Demographics, clinical presentation, treatment and outcomes were reviewed. Results The MRSA population was dominated by multidrug-resistant (MDR) strains within the clonal complex 5 (CC5) carrying an SCCmec type II genetic element (USA100-like strains). These strains carried genes that confer resistance to five antibiotic classes, in addition to mutations in topoisomerase genes (gyrA and parC) that resulted in resistance to all fluoroquinolones tested. Patients were mostly male (56%), with a median age of 82.7 years, and most had no recent history of extensive healthcare exposure. All cases were exogenous following ocular surgery (67%) or intravitreal injection (33%). The main exam findings were visual acuity ≤ hand motion, hypopyon (89%), and vitreous opacity (89%). Five patients (56%) showed improvement in visual acuity at 1 month following presentation, three (33%) at 3 months, and two (22%) at 6 months. Complications included evisceration (n = 1) and phthisis (n = 1). Patients who had pars plana vitrectomy within 48 hours of presentation had better clinical outcomes compared to those who did not. Conclusion Exogenous MRSA endophthalmitis is caused by MDR strains that resemble the hospital-acquired lineage USA100. These strains cause severe endophthalmitis in patients with no recent hospital/healthcare exposure.

Whole-genome comparative analysis of the genetic, virulence and antimicrobial resistance diversity of Campylobacter spp. from Spain

Whole-Genome Sequencing has the potential to be an effective method for surveillance of foodborne diseases. This study aims to determine the genetic relatedness and prevalence of virulence-associated genes and antimicrobial resistance determinants in 135 Campylobacter jejuni, seven Campylobacter coli and three Campylobacter lari isolates from the poultry supply chain and a hospital in Spain. The isolates showed a wide genetic diversity between and within species with Clonal Complex 21 the most frequent lineage found. Among species, C. jejuni showed the highest prevalence of virulence genes (287/333) in which a high occurring variability was observed in the capsule biosynthesis and transport, O-linked flagellar glycosylation and lipooligosaccharide biosynthesis loci, with a great impact of phase-variation that led to 72 different virulence gene patterns among all isolates. High prevalence (> 90 %) of blaOXA-type β-lactamase genes and mutations in DNA gyrase gene associated with fluoroquinolones resistance were observed, and at a frequency similar to the tet(O) gene in C. jejuni (93 %) and C. coli (86 %), both of which also harboured resistance determinants to aminoglycosides with a higher occurrence rate in C. coli (43 %), that was the only species in which mutations in the 23S ribosomal subunit conferring resistance to erythromycin were identified (43 %). The present study constitutes the largest genomic survey of Campylobacter isolates in Spain providing insight into the prevalence and diversity of the pathogen along the poultry supply chain in the country.

Molecular characteristics, risk factors, and clinical outcomes of methicillin-resistant Staphylococcus aureus infections among critically ill pediatric patients in Shanghai, 2016-2021.

Methicillin-resistant Staphylococcus aureus (MRSA) infection in children has been on the rise, which poses a serious threat to their health and life in China. The purpose of this study was to determine the molecular characteristics, risk factors, and clinical outcomes of MRSA infections among critically ill pediatric patients. A retrospective case-control study was performed in the pediatric intensive care unit (PICU) of a tertiary university teaching hospital. All children infected with culture-positive S. aureus in the PICU between January 2016 and December 2021 were included. Univariate and multivariable logistic regression analyses were used to identify potential risk factors for MRSA infection and clinical outcomes of S. aureus infection. All S. aureus isolates were characterized based on antimicrobial resistance, multilocus sequence typing (MLST) and Staphylococcal protein A (spa) typing. Of 3,974 patients admitted to the PICU, 280 were diagnosed with a S. aureus infection during the 6-year study period. Among them, 43.2% (121/280) were MRSA. All MRSA isolates showed significantly higher rates of resistance to penicillin, erythromycin, clindamycin and tetracycline than MSSA strains. The MRSA strains consisted of 45 spa types and 20 sequence types (STs) (20 clonal complexes), among which the most frequently represented were ST59-t437and ST398-t034. Multivariable logistic regression revealed vaginal delivery, respiratory failure, co-infection with a virus, C-reactive protein (CRP) > 8 mg/L as significant risk factors for MRSA infection. There was no significant difference in all-cause mortality during hospitalization between the MRSA group and the MSSA group. Furthermore, independent predictors for mortality in patients with S. aureus infections were the presence of hypoproteinemia, hematopathy, septic shock, respiratory failure, fever, and white blood cell (WBC) > 15.0 × 109/L. The study revealed a high proportion of MRSA infections among critically ill pediatric patients, and found significant risk factors for MRSA infection and poor prognosis of S. aureus infection. Methicillin resistance did not contribute to the mortality in the current study. These findings will provide evidence-based practices to make the strategies of prevention and rational use of antibiotics for pediatric patients with S. aureus infection in the ICU.

Molecular evidence that GBS early neonatal sepsis results from ascending infection: comparative hybrid genomics analyses show that microorganisms in the vaginal ecosystem, amniotic fluid, chorioamniotic membranes, and neonatal blood are the same.

Streptococcus agalactiae, or Group B Streptococcus (GBS), is a leading cause of neonatal sepsis. Materno-fetal transmission of the microorganisms present in the lower genital tract/perineum is considered to be the most frequent mode for acquisition of infection. It has also been proposed that, in a subset of cases, GBS causes acute chorioamnionitis, intraamniotic infection, and fetal/neonatal sepsis. However, the evidence to support this ascending pathway is derived from microbiologic studies that rely on cultivation methods, which do not have the resolution to determine if the microorganisms causing neonatal sepsis are the same as those found in the amniotic fluid and the vaginal ecosystem. We used whole genome sequencing of the microorganisms isolated from the vagina, amniotic fluid, chorioamniotic membranes, and neonatal blood (four isolates) in a case of early neonatal sepsis. Using hybrid genome assembly, we characterized the genomic features including virulence factors and antimicrobial resistance in four isolates from the same mother, placenta, and newborn. Whole genome sequencing revealed that the microorganisms in the four clinical isolates corresponded to S. agalactiae sequence type 1, clonal complexes 1, and serotype Ib. Comparative genomic analysis illustrated similar DNA sequences of the four genomes. This study presents the first evidence of the genomic similarity of microorganisms in the vaginal ecosystem, the space between the chorioamniotic membranes of the placenta, amniotic fluid, and neonatal blood.

Molecular and Seroepidemiologic Characteristics ofStaphylococcus aureus in Children with Cystic Fibrosis

Abstract Background Cystic fibrosis (CF), through altered sodium and chloride transport across the CF transmembrane receptor (CFTR), is associated with poor mucus clearance, bacterial colonization of the lower airway, chronic pulmonary disease punctuated by acute exacerbations, and chronic inflammation. With increased use of CFTR modulators, changes in the CF pulmonary microbiome have been observed, including decreased prevalence of Pseudomonas colonization; however, overall S. aureus prevalence remains largely unchanged. An important remaining question is whether changes in the CF pulmonary microbiome, while not affecting the overall prevalence of S. aureus colonization, has led to changes in S. aureus diversity or the strain types that are most frequently recovered from children with CF. It is also unknown whether the humoral immune response to S. aureus is altered in the presence of CFTR modulator therapy. Methods Genomic DNA was extracted from S. aureus isolates obtained from two cohorts: a historic cohort, comprised of children not receiving CFTR modulators, and a modern cohort, comprised of children receiving CFTR modulator therapy. Multilocus sequence typing (MLST) and whole genome sequencing (WGS) were used to identify clonotype and characterize virulence factors of interest. Serum was obtained from participants and antibody titers to the extracellular toxins LukAB and LukED were measured. Results One hundred participants were enrolled: 50 in cohort 1 (historic) and 50 in cohort 2 (modern). Compared to cohort 1, isolates identified in cohort 2 were less likely to contain ACME and mecA; similarly, cohort 2 isolates were more likely to contain PVL, sek, and sak. The frequency of specific clonotypes changed over time, with increased prevalence of CC8 and decreased prevalence of CC1, CC5, CC15, and CC30 in cohort 2 compared to cohort 1 (Figure 1A). Serum antibody concentrations to LukAB, a conserved toxin across S. aureus, were similar across cohorts (Figure 1B). Conclusion Over time, the clonotypes responsible for S. aureus colonization in patients receiving CFTR modulators have changed. Despite changes in clonotypes and receipt of CFTR modulators, serum concentrations of LukAB-specific antibodies did not differ. These data suggest that staphylococcal colonization in patients with CF is dynamic over time and may be modified based on the receipt of CFTR modulator therapy. Figure 1. (A) S. aureus clonal complexes, over time, demonstrating a relative increase in CC8 between 2012 and 2022; (B) serum concentrations of LukAB antibodies from 2012 (historic) and 2022 (modern) cohorts. No significant difference was observed in median anti-LukAB concentrations.

Current methodologies available to evaluate the virulence potential among Listeria monocytogenes clonal complexes.

Listeria monocytogenes is a foodborne pathogen that causes listeriosis in humans, the severity of which depends on multiple factors, including intrinsic characteristics of the affected individuals and the pathogen itself. Additionally, emerging evidence suggests that epigenetic modifications may also modulate host susceptibility to infection. Therefore, different clinical outcomes can be expected, ranging from self-limiting gastroenteritis to severe central nervous system and maternal-neonatal infections, and bacteremia. Furthermore, L. monocytogenes is a genetically and phenotypically diverse species, resulting in a large variation in virulence potential between strains. Multilocus sequence typing (MLST) has been widely used to categorize the clonal structure of bacterial species and to define clonal complexes (CCs) of genetically related isolates. The combination of MLST and epidemiological data allows to distinguish hypervirulent CCs, which are notably more prevalent in clinical cases and typically associated with severe forms of the disease. Conversely, other CCs, termed hypovirulent, are predominantly isolated from food and food processing environments and are associated with the occurrence of listeriosis in immunosuppressed individuals. Reports of genetic traits associated with this diversity have been described. The Food and Agriculture Organization (FAO) is encouraging the search for virulence biomarkers to rapidly identify the main strains of concern to reduce food waste and economical losses. The aim of this review is to comprehensively collect, describe and discuss the methodologies used to discriminate the virulence potential of L. monocytogenes CCs. From the exploration of in vitro and in vivo models to the study of expression of virulence genes, each approach is critically explored to better understand its applicability and efficiency in distinguishing the virulence potential of the pathogen.

Construction of core genome multi-locus sequence typing schemes for population structure analyses of Nocardia species

Nocardia, a member of the Actinobacteria phylum, populates diverse habitats globally, with certain species being the cause of various clinical infections in humans. There is paucity of data regarding the population structure of this genus and of established genomic-based phylogenetic methods. We examined the whole genome sequences of 193 isolates spanning five major pathogenic Nocardia species sourced from public databases, encompassing diverse geographic regions. Using the chewBBACA pipeline, a species-specific core genome multilocus sequence typing (cgMLST) schema was created for N. cyriacigeorgica, N. farcinica, N. brasiliensis, N. wallacei, and N. abscessus. Additional genomic features that were examined included virulence factor (VF) profile, total length and open-reading frame count, the core genome length and core gene count, and GC content. Our findings indicated that: (i) N. brasiliensis diverges significantly from the other four species, underscoring its distinct evolutionary trajectory; (ii) the population structures of all species were polyclonal, with phylogenetic clustering occurring in the minority of isolates; (iii) clonal complexes were largely restricted to specific geographical locations, rather than demonstrating a global distribution, and (iv) initial evidence suggests no direct common-source transmission amongst the studied strains. Our study establishes a comprehensive genome-based phylogenetic methodology for population structure of Nocardia species.

Genus-targeted markers for the taxonomic identification and monitoring of coagulase-positive and coagulase-negative Staphylococcus species

The Staphylococcus genus comprises multiple pathogenic and opportunistic species that represent a risk to public health. Epidemiological studies require accurate taxonomic classification of isolates with enough resolution to distinguish clonal complexes. Unfortunately, 16 S rRNA molecular analysis and phenotypic characterization cannot distinguish all species and do not offer enough resolution to assess intraspecific diversity. Other approaches, such as Multilocus Sequence Tagging, provide higher resolution; however, they have been developed for Staphylococcus aureus and a few other species. Here, we developed a set of genus-targeted primers using five orthologous genes (pta, tuf, tpi, groEs, and sarA) to identify all Staphylococcus species within the genus. The primers were initially evaluated using 20 strains from the Collection of Microorganisms of Interest in Animal Health from AGROSAVIA (CMISA), and their amplified sequences were compared to a set of 33 Staphylococcus species. This allowed the taxonomic identification of the strains even on close species and the establishment of intraspecies diversity. To enhance the scope and cost-effectiveness of the proposed strategy, we customized the primer sets for an Illumina paired-end amplicon protocol, enabling gene multiplexing. We assessed five genes across 177 strains, generating 880 paired-end libraries from the CMISA. This approach significantly reduced sequencing costs, as all libraries can be efficiently sequenced in a single MiSeq run at a fraction (one-fourth or less) of the cost associated with Sanger sequencing. In summary, this method can be used for precise identification and diversity analysis of Staphylococcus species, offering an advancement over traditional techniques in both resolution and cost-effectiveness.Graphical

Campylobacter coli of porcine origin exhibits an open pan-genome within a single clonal complex: insights from comparative genomic analysis.

Although Campylobacter spp., including Campylobacter coli, have emerged as important zoonotic foodborne pathogens globally, the understanding of the genomic epidemiology of C. coli of porcine origin is limited. As pigs are an important reservoir of C. coli, we analyzed C. coli genomes that were isolated (n= 3) from pigs and sequenced (this study) them along with all other C. coli genomes for which pig intestines, pig feces, and pigs were mentioned as sources in the NCBI database up to January 6, 2023. In this paper, we report the pan-genomic features, the multi-locus sequence types, the resistome, virulome, and mobilome, and the phylogenomic analysis of these organisms that were obtained from pigs. Our analysis revealed that, in addition to having an open pan-genome, majority (63%) of the typeable isolates of C. coli of pig origin belonged to a single clonal complex, ST-828. The resistome of these C. coli isolates was predominated by the genes tetO (53%), blaOXA-193 (49%), and APH (3')-IIIa (21%); however, the virulome analysis revealed a core set of 37 virulence genes. Analysis of the mobile genetic elements in the genomes revealed wide diversity of the plasmids and bacteriophages, while 30 transposons were common to all genomes of C. coli of porcine origin. Phylogenomic analysis showed two discernible clusters comprising isolates originating from Japan and another set of isolates comprising mostly copies of a type strain stored in three different culture collections.