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Abstract Vast amounts of pathogen genomic, demographic and spatial data are transforming our understanding of SARS-CoV-2 emergence and spread. We examined the drivers of molecular evolution and spread of 291,791 SARS-CoV-2 genomes from Denmark in 2021. With a sequencing rate consistently exceeding 60%, and up to 80% of PCR-positive samples between March and November, the viral genome set is broadly whole-epidemic representative. We identify a consistent rise in viral diversity over time, with notable spikes upon the importation of novel variants (e.g., Delta and Omicron). By linking genomic data with rich individual-level demographic data from national registers, we find that individuals aged 75 years had a lower contribution to molecular change (i.e., branch lengths) compared to other age groups, but similar molecular evolutionary rates, suggesting a lower likelihood of introducing novel variants. Similarly, we find greater molecular change among vaccinated individuals, suggestive of immune evasion. We also observe evidence of transmission in rural areas to follow predictable diffusion processes. Conversely, urban areas are expectedly more complex due to their high mobility, emphasising the role of population structure in driving virus spread. Our analyses highlight the added value of integrating genomic data with detailed demographic and spatial information, particularly in the absence of structured infection surveys.

ABSTRACT Phage therapy appears to be a promising approach to tackle multidrug-resistant bacteria, including staphylococci. However, most anti-staphylococcal phages have been characterized in Staphylococcus aureus, while a limited number of studies investigated phage activity against S. epidermidis. We studied the potential of phage training to extend the host range of two types of anti-S. aureus phages against S. epidermidis isolates. The Appelmans protocol was applied to a mixture of Kayvirus and a mixture of Silviavirus phages repeatedly exposed to seven S. epidermidis strains representative of nosocomial-associated sequence types (ST), including the world-wide disseminated ST2. We observed increased activity only for the Kayvirus mixture against two of these strains (ST2 or ST35). Phage subpopulations isolated from the training mixture using these two strains (five/strain) exhibited different evolved phenotypes, active only against their isolation strain or strains of the same ST. Of note, 16/47 ST2 strains were susceptible to one of the groups of trained phages. A comparative genomic analysis of ancestral and trained phage genomes, conducted to identify potential bacterial determinants of such specific activity, found numerous recombination events between two of the three ancestors. However, a small number of trained phage genes had nucleotide sequence modifications impacting the corresponding protein compared to ancestral phages, two to four of them per phage genome being specific of each group of phage subpopulations exhibiting different host range. The results suggest that anti-S. aureus phages can be adapted to S. epidermidis isolates but with inter- and intra-ST specificity.ImportanceS. epidermidis is increasingly recognized as a threat for public health. Its clinical importance is notably related to multidrug resistance. Phage therapy is one of the most promising alternative therapeutic strategies to antibiotics. Nonetheless, only very few phages active against this bacterial species have been described. In the present study, we showed that phage training can be used to extend the host range of polyvalent Kayvirus phages within the Staphylococcus genera to include S. epidermidis species. In the context of rapid development of phage therapy, in vitro forced adaptation of previously characterized phages could be an appealing alternative to fastidious repeated isolation of new phages to improve the therapeutic potential of a phage collection.

ABSTRACT: Objectives: This study characterized the resistome, mobilome and phylogenomic relatedness of Staphylococcus aureus strains previously obtained from healthy nestling storks (HNS), pigs (HP) and pig farmers (HPF) to analyse possible transmission pathways of S. aureus with implications for the spread of antimicrobial resistance. Methods: The genomic contents of 52 S. aureus strains obtained from the nasal cavity of HNS, HP and HPF in Spain were sequenced using the Illumina NextSeq platform to characterize their resistome, virulome and mobile genetic elements. The relatedness of strains was assessed by core-genome single nucleotide polymorphisms (SNPs). Results: The frequencies of multidrug-resistance phenotype and transposons were significantly lower in strains from HNS than in those from HP and HPF (P < 0.005). However, the presence of human immune evasion cluster genes in S. aureus strains from HNS was significantly higher than in those from HP and HPF (P < 0.005). Interestingly, the frequencies of plasmids and phages were not significantly associated with the host (P > 0.05). The phylogenetic analysis identified a cluster of all the MSSA-CC398 strains carrying φSa3 and ermT on rep13 separately from the two MRSA-CC398 strains (carrying ermT on repUS18). Highly related MRSA-CC398 strains were detected in some pigs and related farmers (

Patients with atopic dermatitis (AD) are more likely than healthy individuals to harbour Staphylococcus aureus on their skin. Superantigens (SAgs) produced by specific S. aureus strains may contribute to AD-associated skin inflammation. The present study compared the prevalence and types of SAg-encoding genes between S. aureus isolated from patients with AD and from controls, and within the AD group between isolates from different sampling sites (lesional skin, non-lesional skin, and nares). This retrospective case-control study extracted data from 2 previous studies that examined S. aureus using whole-genome sequencing. The 138 S. aureus isolates obtained from 71 AD patients contained 349 SAg-encoding genes; 22 (6.3%) were found in isolates from nares (0.4 ± 0.6 genes per isolate), 99 (28.4%) in isolates from non-lesional skin (3.7 ± 3.9), and 228 (65.3%) in isolates from lesional skin (4.2 ± 4.5). S. aureus (n = 101) from the control group contained 594 SAg-encoding genes (5.9 ± 4.2). Of the S. aureus isolated from lesional AD skin, 69% carried at least 1 gene encoding SAg compared with 33% of AD nasal isolates. SAg could be a factor in the pathogenesis of a subset of AD patients.

Wastewater sequencing has become a powerful supplement to clinical testing in monitoring SARS-CoV-2 infections in the post-COVID-19 pandemic era. While its applications in measuring the viral burden and main circulating lineages in the community have proved their efficacy, the variations in sequencing quality and coverage across the different regions of the SARS-CoV-2 genome are not well understood. Furthermore, it is unclear how different sample origins, viral extraction and concentration methods and environmental factors impact the reads sequenced from wastewater. Using high-coverage, amplicon-based, paired-end read sequencing of viral RNA extracted from wastewater collected directly from aircraft, pooled from different aircraft and airport buildings or from regular wastewater plants, we assessed the genome coverage across the sample groups with a focus on the 5′-end region covering the leader sequence and investigated whether it was possible to detect subgenomic RNA from viral material recovered from wastewater. We identified distinct patterns in the persistence of the different genomic regions across the different types of wastewaters and the existence of chimeric reads mapping to non-amplified regions. Our findings suggest that preservation of the 5′-end of the genome and the ability to detect subgenomic RNA reads, though highly susceptible to environment and sample processing conditions, may be indicative of the quality and amount of the viral RNA present in wastewater.

Abstract Dolosigranulum pigrum—a lactic acid bacterium that is increasingly recognized as an important member of the nasal microbiome. Currently, there are limited rapid and low-cost options for confirming D. pigrum isolates and detecting D. pigrum in clinical specimens. Here we describe the design and validation of a novel PCR assay targeting D. pigrum that is both sensitive and specific. We designed a PCR assay targeting murJ, a single-copy core species gene identified through the analysis of 21 D. pigrum whole genome sequences. The assay achieved 100% sensitivity and 100% specificity against D. pigrum and diverse bacterial isolates and an overall 91.1% sensitivity and 100% specificity using nasal swabs, detecting D. pigrum at a threshold of 1.0 × 104 D. pigrum 16S rRNA gene copies per swab. This assay adds a reliable and rapid D. pigrum detection tool to the microbiome researcher toolkit investigating the role of generalist and specialist bacteria in the nasal environment.

IntroductionThis study aimed to investigate the epidemiology, serotype distribution, phenotypical antibiogram, and molecular resistance gene characteristics of invasive Haemophilus influenzae infections in Denmark from 2014 to 2022. Additionally, the potential impact of outdoor temperature and COVID-19 restrictions on the epidemiology of H. influenzae was assessed.Materials and methodsInvasive H. influenzae isolates were received from patients with positive culture results from cerebrospinal fluid, blood, or other sterile sites. Sample data were obtained from the Danish laboratory surveillance system/MiBa database, and whole-genome sequencing (WGS) was performed on the isolates. The incidence rates and distribution of H. influenzae cases were analyzed, and antibiotic susceptibility were assessed.ResultsA total of 1,007 invasive H. influenzae cases were identified, with serotyping conducted for 752 (74.7%) isolates. The median incidence per year of H. influenzae was 2.0 cases per 100,000, with the highest incidence in 2014 and the lowest in 2020. The majority of H. influenzae isolates were non-typeable H. influenzae (NTHi), while the most prominent serotypes were serotype f followed by serotype b. Bacteremia cases accounted for the majority (88.6%) of occurrences, although meningitis cases showed an increasing trend during the time period. The age group 85+ exhibited the highest incidence. The implementation of COVID-19 preventive interventions in 2020 resulted in a significant reduction in H. influenzae incidence, which returned to pre-COVID levels in 2021. A negative correlation was observed between monthly H. influenzae cases and outdoor temperature. An overall level of genetic beta-lactamase resistance of 26.3% was observed divided into 10.6% beta-lactamase-positive ampicillin-resistant (gBLPAR), 13.6% beta-lactamase-negative ampicillin-resistant (gBLNAR) and 2.1% beta-lactamase-positive amoxicillin clavulanate-resistant (gBLPACR). Other non-beta-lactam resistance traits were detected in 7.6% of isolates (primarily aminoglycoside-modifying enzymes).ConclusionThe overall incidence of H. influenzae in Denmark returned to stable levels after the COVID-19 epidemic, with NTHi strains dominating. The COVID-19 preventive interventions led to a major reduction in incidence. A significant negative correlation between the incidence of H. influenzae and temperature was observed. The study revealed an overall genetic beta-lactam resistance rate of 26.3%, and the concordance between genotypic and phenotypic beta-lactam resistance was high (98.2%).

In this study, the authors compare the transmission dynamics of the Delta and Omicron SARS-CoV-2 variants using household data from Denmark. They find that Omicron has a higher secondary attack rate, and that the odds of infection with Omicron was higher than with Delta, particularly for vaccinated individuals.

In this study, the authors use household data from Denmark to investigate the transmissibility of the BA.1 and BA.2 Omicron SARS-CoV-2 subvariants. They find that the secondary attack rate was higher for BA.2, but that it had higher infectiousness only when cases were not vaccinated.

Introduction A feared complication after total hip arthroplasty (THA) is prosthetic joint infection (PJI), associated with high morbidity and mortality. Prophylactic antibiotics can reduce the risk of PJI. However, there is no consensus on the dosages and current recommendations are based on a low evidence level. The objective is to compare the effect of a single versus multiple doses of prophylactic antibiotics administered within 24 hours on PJI.Methods and analysis The study is designed as a cross-over, cluster randomised, non-inferiority trial. All clinical centres use both antibiotic practices (1 year of each intervention). All Danish orthopaedic surgery departments will be involved: Based on quality databases, 2-year cohorts of approximately 20 000 primary THAs conducted at 39 public and private hospitals, will be included. Inclusion criteria: age ≥18 years, all indications for THA except patients operated due to acute or sequelae from proximal femoral or pelvic fractures or bone tumour or metastasis. The primary outcome is PJI within 90 days after primary THA. Secondary outcomes include (1) serious adverse events, (2) potential PJI, (3) length of hospitalisation stay, (4) cardiovascular events, (5) hospital-treated infections, (6) community-based antibiotic use, (7) opioid use and (8) use of acetaminophen and non-steroidal anti-inflammatory drugs. All outcome measures will be extracted from national databases. Analyses will be based on the intention-to-treat population. Non-inferiority will be shown if the upper limit of the two-sided 95% CI for the OR is less than 1.32 for the single dose as compared with multiple doses. The results will establish best practice on antibiotic prophylaxis dosages in the future.Ethics and dissemination This study has been approved by Committees on Health Research Ethics for The Capital Region of Denmark (21069108) and The Danish Medicines Agency (2021091723). All results will be presented in peer-reviewed medical journals and international conferences.Trial registration number NCT05530551.

Summary: Background: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has rapidly spread worldwide in the population since it was first detected in late 2019. The transcription and replication of coronaviruses, although not fully understood, is characterised by the production of genomic length RNA and shorter subgenomic RNAs to make viral proteins and ultimately progeny virions. Observed levels of subgenomic RNAs differ between sub-lineages and open reading frames but their biological significance is presently unclear. Methods: Using a large and diverse panel of virus sequencing data produced as part of the Danish COVID-19 routine surveillance together with information in electronic health registries, we assessed the association of subgenomic RNA levels with demographic and clinical variables of the infected individuals. Findings: Our findings suggest no significant statistical relationship between levels of subgenomic RNAs and host-related factors. Interpretation: Differences between lineages and subgenomic ORFs may be related to differences in target cell tropism, early virus replication/transcription kinetics or sequence features. Funding: The author(s) received no specific funding for this work.

A one-health perspective may provide new and actionable information about Escherichia coli transmission. E. coli colonizes a broad range of vertebrates, including humans and food-production animals, and is a leading cause of bladder, kidney, and bloodstream infections in humans. Substantial evidence supports foodborne transmission of pathogenic E. coli strains from food animals to humans. However, the relative contribution of foodborne zoonotic E. coli (FZEC) to the human extraintestinal disease burden and the distinguishing characteristics of such strains remain undefined. Using a comparative genomic analysis of a large collection of contemporaneous, geographically-matched clinical and meat-source E. coli isolates (n = 3111), we identified 17 source-associated mobile genetic elements – predominantly plasmids and bacteriophages – and integrated them into a novel Bayesian latent class model to predict the origins of clinical E. coli isolates. We estimated that approximately 8 % of human extraintestinal E. coli infections (mostly urinary tract infections) in our study population were caused by FZEC. FZEC strains were equally likely to cause symptomatic disease as non-FZEC strains. Two FZEC lineages, ST131-H22 and ST58, appeared to have particularly high virulence potential. Our findings imply that FZEC strains collectively cause more urinary tract infections than does any single non-E. coli uropathogenic species (e.g., Klebsiella pneumoniae). Our novel approach can be applied in other settings to identify the highest-risk FZEC strains, determine their sources, and inform new one-health strategies to decrease the heavy public health burden imposed by extraintestinal E. coli infections.

Abstract In broiler breeders, background mortality is rarely addressed, however, it represents the death of a vast number of birds, a constant productivity loss, welfare concerns and it might affect chick quality. The study aimed to unveil lesions leading to mortality in a study population perceived as healthy, combined with whole-genome sequencing (WGS) of Escherichia coli, a well-known contributor to disease problems in poultry. Broiler breeders (n = 340) originating from three distinct, putative healthy flocks and their progeny (n = 154) were subjected to a comprehensive post-mortem examination, bacteriological sampling, and sequencing of 77 E. coli isolates. Productivity data confirmed an exemplary health status of the enrolled flocks, and post-mortem examination further verified the absence of general disease problems. Among the submitted broiler breeders, exudative peritonitis (31.2%) was the most frequent lesion linked to infectious disease, whereas airsacculitis, pericarditis, perihepatitis, and salpingitis occurred in 18.5%, 3.5%, 3.8% and 17%, respectively. Yolksacculitis occurred in 15.6% of the broilers, whilst pericarditis, perihepatitis and peritonitis were diagnosed in 9.7%, 7.1% and 9.1%, respectively. WGS revealed a diverse population where ST95 dominated the population retrieved from broiler breeders, whereas ST10 was highly prevalent among broilers. Both lineages could be isolated from extraintestinal sites of birds without lesions indicative of infection. In general, the genetic diversity within flocks was comparable to the diversity between farms, and the overall occurrence of resistance markers was low. In conclusion, a comprehensive insight into lesions associated with background mortality is presented, together with a vast diversity of E. coli isolated from extraintestinal sites during a non-outbreak situation.

Abstract Whole genome sequencing (WGS) of methicillin-resistant Staphylococcus aureus (MRSA) provides high-resolution typing, facilitating surveillance and outbreak investigations. The aim of this study was to evaluate the genomic variation rate in MRSA, by comparing commonly used core genome multilocus sequencing (cgMLST) against single nucleotide polymorphism (SNP) analyses. WGS was performed on 95 MRSA isolates, collected from 20 carriers during years 2003–2019. To assess variation and methodological-related differences, two different cgMLST schemes were obtained using Ridom SeqSphere+ and the cloud-based 1928 platform. In addition, two SNP methods, 1928 platform and Northern Arizona SNP Pipeline (NASP) were used. The cgMLST using Ridom SeqSphere+ and 1928 showed a median of 5.0 and 2.0 allele variants/year, respectively. In the SNP analysis, performed with two reference genomes COL and Newman, 1928 showed a median of 13 and 24 SNPs (including presumed recombination) and 3.8 respectively 4.0 SNPs (without recombination) per individual/year. Accordantly, NASP showed a median of 5.5 and 5.8 SNPs per individual/year. In conclusion, an estimated genomic variation rate of 2.0–5.8 genetic events per year (without recombination), is suggested as a general guideline to be used at clinical laboratories for surveillance and outbreak investigations independently of analysis approach used.

COVID-19 vaccines may reduce the susceptibility of an individual to infection and/or the infectiousness of breakthrough infections. Here, the authors use data from Denmark and estimate that vaccine effectiveness was 61% for susceptibility and 31% for infectiousness during a period of Delta variant dominance.

IntroductionNasal carriage of coagulase-positive staphylococci (CoPS) in healthy dogs could indicate increased risks of colonization for in-contact people or vice versa. This study determined the nasal carriage rate of CoPS among healthy dogs and in-contact people, their genotypic characteristics and phylogenetic relatedness.MethodsNasal samples were collected from 27 households (34 dogs and 41 humans) in Spain. Staphylococci were identified by MALDI-TOF-MS, their antimicrobial resistance (AMR) genes and spa-types were tested by PCR/sequencing. The relatedness of CoPS from the same households was assessed by core genome single nucleotide polymorphisms (SNPs) analyses.ResultsStaphylococcus aureus carriage was found in 34.1% of humans (including one methicillin-resistant S. aureus MRSA-CC5-t2220-SCCmec type-IV2B) and 5.9% of dogs; Staphylococcus pseudintermedius in 2.4% of humans and 32.4% of dogs; while Staphylococcus coagulans was only detected in dogs (5.4%). Remarkably, one human co-carried S. aureus/S. pseudintermedius, while a dog co-carried the three CoPS species. Household density was significantly associated with S. pseudintermedius carriage in households with > than 1 dog and >than 1 human (OR = 18.10, 95% CI: 1.24–260.93, p = 0.034). Closely related (

Abstract Background In early 2021, the SARS-CoV-2 lineage B.1.1.7 (Alpha variant) became dominant across large parts of the world. In Denmark, comprehensive and real-time test, contact-tracing, and sequencing efforts were applied to sustain epidemic control. Here, we use these data to investigate the transmissibility, introduction, and onward transmission of B.1.1.7 in Denmark. Methods We analyzed a comprehensive set of 60,178 SARS-CoV-2 genomes generated from high-throughput sequencing by the Danish COVID-19 Genome Consortium, representing 34% of all positive cases in the period 14 November 2020 to 7 February 2021. We calculated the transmissibility of B.1.1.7 relative to other lineages using Poisson regression. Including all 1976 high-quality B.1.1.7 genomes collected in the study period, we constructed a time-scaled phylogeny, which was coupled with detailed travel history and register data to outline the introduction and onward transmission of B.1.1.7 in Denmark. Results In a period with unchanged restrictions, we estimated an increased B.1.1.7 transmissibility of 58% (95% CI: [56%, 60%]) relative to other lineages. Epidemiological and phylogenetic analyses revealed that 37% of B.1.1.7 cases were related to the initial introduction in November 2020. The relative number of cases directly linked to introductions varied between 10 and 50% throughout the study period. Conclusions Our findings corroborate early estimates of increased transmissibility of B.1.1.7. Both substantial early expansion when B.1.1.7 was still unmonitored and continuous foreign introductions contributed considerably to case numbers. Finally, our study highlights the benefit of balanced travel restrictions and self-isolation procedures coupled with comprehensive surveillance efforts, to sustain epidemic control in the face of emerging variants.

ABSTRACT Staphylococcus haemolyticus is a major cause of late-onset sepsis in neonates, and endemic clones are often multidrug-resistant. The bacteria can also act as a genetic reservoir for more pathogenic bacteria. Molecular epidemiology is important in understanding bacterial pathogenicity and preventing infection. To describe the molecular epidemiology of S. haemolyticus isolated from neonatal blood cultures at a Swedish neonatal intensive care unit (NICU) over 4 decades, including antibiotic resistance genes (ARGs), virulence factors, and comparison to international isolates. Isolates were whole-genome sequenced, and single nucleotide polymorphisms in the core genome were used to map the relatedness. The occurrence of previously described ARGs and virulence genes were investigated. Disc diffusion and gradient tests were used to determine phenotypic resistance. The results revealed a clonal outbreak of S. haemolyticus at this NICU during the 1990s. Multidrug resistance was present in 28 (82%) of all isolates and concomitant resistance to aminoglycoside and methicillin occurred in 27 (79%). No isolates were vancomycin resistant. Genes encoding ARGs and virulence factors occurred frequently. The isolates in the outbreak were more homogenous in their genotypic and phenotypic patterns. Genotypic and phenotypic resistance combinations were consistent. Pathogenic traits previously described in S. haemolyticus occurred frequently in the present isolates, perhaps due to the hospital selection pressure resulting in epidemiological success. The clonal outbreak revealed by this study emphasizes the importance of adhering to hygiene procedures in order to prevent future endemic outbreaks. IMPORTANCE This study investigated the relatedness of Staphylococcus haemolyticus isolated from neonatal blood and revealed a clonal outbreak in the 1990s at a Swedish neonatal intensive care unit. The outbreak clone has earlier been isolated in Japan and Norway. Virulence and antibiotic resistance genes previously associated with clinical S. haemolyticus were frequently occuring in the present study as well. The majority of the isolates were multidrug-resistant. These traits should be considered important for S. haemolyticus epidemiological success and are probably caused by the hospital selection pressure. Thus, this study emphasizes the importance of restrictive antibiotic use and following the hygiene procedures, to prevent further antibiotic resistance spread and future endemic outbreaks.

Haemophilus influenzae is a gram-negative coccobacillus known to cause respiratory and invasive infections. It can possess a polysaccharide capsule that can be categorized into six different serotypes (i.e., Hia, Hib, Hic, Hid, Hie, and Hif) and non-encapsulated strains that are defined as non-typeable. Furthermore, H. influenzae can be characterized into eight biotypes (I–VIII). Traditionally, isolates have been serotyped and biotyped using phenotypic methods; however, these methods are not always reliable. In this study, we evaluate the use of whole-genome sequencing (WGS) for national surveillance and characterization of clinical Danish H. influenzae isolates. In Denmark, all clinical invasive isolates between 2014 and 2021 have been serotyped using a traditional phenotypic latex agglutination test as well as in silico serotyped using the in silico programs “hinfluenzae_capsule_characterization” and “hicap” to compare the subsequent serotypes. Moreover, isolates were also biotyped using a phenotypic enzyme test and the genomic data for the detection of the genes encoding ornithine, tryptophan, and urease. The results showed a 99–100% concordance between the two genotypic approaches and the phenotypic serotyping, respectively. The biotyping showed a 95% concordance between genotyping and phenotyping. In conclusion, our results show that in a clinical surveillance setting, in silico serotyping and WGS-based biotyping are a robust and reliable approach for typing clinical H. influenzae isolates.

After the eradication of polio infection, the plan is to phase-out the live-attenuated oral polio vaccine (OPV). Considering the protective non-specific effects (NSE) of OPV on unrelated pathogens, the withdrawal may impact child health negatively. Within a cluster-randomized trial, we carried out 16S rRNA deep sequencing analysis of fecal and nasopharyngeal microbial content of Bissau–Guinean infants aged 4–8 months, before and after 2 months of OPV revaccination (revaccinated infants = 47) vs. no OPV revaccination (control infants = 47). The aim was to address changes in the gut and upper respiratory bacterial microbiotas due to revaccination. Alpha-diversity for both microbiotas increased similarly over time in OPV-revaccinated infants and controls, whereas greater changes over time in the bacterial composition of gut (padjusted < 0.001) and upper respiratory microbiotas (padjusted = 0.018) were observed in the former. Taxonomic analysis of gut bacterial microbiota revealed a decrease over time in the median proportion of Bifidobacterium longum for all infants (25–14.3%, p = 0.0006 in OPV-revaccinated infants and 25.3–11.6%, p = 0.01 in controls), compatible with the reported weaning. Also, it showed a restricted increase in the median proportion of Prevotella_9 genus in controls (1.4–7.1%, p = 0.02), whereas in OPV revaccinated infants an increase over time in Prevotellaceae family (7.2–17.4%, p = 0.005) together with a reduction in median proportion of potentially pathogenic/opportunistic genera such as Escherichia/Shigella (5.8–3.4%, p = 0.01) were observed. Taxonomic analysis of upper respiratory bacterial microbiota revealed an increase over time in median proportions of potentially pathogenic/opportunistic genera in controls, such as Streptococcus (2.9–11.8%, p = 0.001 and Hemophilus (11.3–20.5%, p = 0.03), not observed in OPV revaccinated infants. In conclusion, OPV revaccination was associated with a healthier microbiome composition 2 months after revaccination, based on a more abundant and diversified bacterial community of Prevotellaceae and fewer pathogenic/opportunistic organisms. Further information on species-level differentiation and functional analysis of microbiome content are warranted to elucidate the impact of OPV-associated changes in bacterial microbiota on child health.

BackgroundThe continued occurrence of more contagious Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants and waning immunity over time require ongoing reevaluation of the vaccine effectiveness (VE). This study aimed to estimate the effectiveness in 2 age groups (12 to 59 and 60 years or above) of 2 or 3 vaccine doses (BNT162b2 mRNA or mRNA-1273) by time since vaccination against SARS-CoV-2 infection and Coronavirus Disease 2019 (COVID-19) hospitalization in an Alpha-, Delta-, or Omicron-dominated period.Methods and findingsA Danish nationwide cohort study design was used to estimate VE against SARS-CoV-2 infection and COVID-19 hospitalization with the Alpha, Delta, or Omicron variant. Information was obtained from nationwide registries and linked using a unique personal identification number. The study included all previously uninfected residents in Denmark aged 12 years or above (18 years or above for the analysis of 3 doses) in the Alpha (February 20 to June 15, 2021), Delta (July 4 to November 20, 2021), and Omicron (December 21, 2021 to January 31, 2022) dominated periods. VE estimates including 95% confidence intervals (CIs) were calculated (1-hazard ratio∙100) using Cox proportional hazard regression models with underlying calendar time and adjustments for age, sex, comorbidity, and geographical region. Vaccination status was included as a time-varying exposure. In the oldest age group, VE against infection after 2 doses was 90.7% (95% CI: 88.2; 92.7) for the Alpha variant, 82.3% (95% CI: 75.5; 87.2) for the Delta variant, and 39.9% (95% CI: 26.3; 50.9) for the Omicron variant 14 to 30 days since vaccination. The VE waned over time and was 73.2% (Alpha, 95% CI: 57.1; 83.3), 50.0% (Delta, 95% CI: 46.7; 53.0), and 4.4% (Omicron, 95% CI: -0.1; 8.7) >120 days since vaccination. Higher estimates were observed after the third dose with VE estimates against infection of 86.1% (Delta, 95% CI: 83.3; 88.4) and 57.7% (Omicron, 95% CI: 55.9; 59.5) 14 to 30 days since vaccination. Among both age groups, VE against COVID-19 hospitalization 14 to 30 days since vaccination with 2 or 3 doses was 98.1% or above for the Alpha and Delta variants. Among both age groups, VE against COVID-19 hospitalization 14 to 30 days since vaccination with 2 or 3 doses was 95.5% or above for the Omicron variant. The main limitation of this study is the nonrandomized study design including potential differences between the unvaccinated (reference group) and vaccinated individuals.ConclusionsTwo vaccine doses provided high protection against SARS-CoV-2 infection and COVID-19 hospitalization with the Alpha and Delta variants with protection, notably against infection, waning over time. Two vaccine doses provided only limited and short-lived protection against SARS-CoV-2 infection with Omicron. However, the protection against COVID-19 hospitalization following Omicron SARS-CoV-2 infection was higher. The third vaccine dose substantially increased the level and duration of protection against infection with the Omicron variant and provided a high level of sustained protection against COVID-19 hospitalization among the +60-year-olds.

Staphylococcus pseudintermedius is an opportunistic pathogen that is frequently isolated from canines. It is of escalating interest because of its increasing antimicrobial resistance and zoonotic potential. Although many published articles are available that describe isolates obtained from diseased dogs and humans, this study focused on isolates obtained from healthy dogs and their owners who presented at clinics for routine veterinary care and utilized whole genome sequencing-based analyses for strain comparisons. A total of 25 humans and 27 canines were sampled at multiple sites, yielding 47 and 45 isolates, respectively. Whole genome sequence analysis was performed. We detected mostly new sequence types (STs) and a high diversity. Strains carried few antimicrobial resistance genes and plasmids, albeit three MRSP strains were found that belonged to two internationally distributed STs. The virulence content did not provide insights toward a tendency to colonization of humans but supported that there may be differences in the surface proteins between carrier strains and those causing pyoderma. We identified 13 cases in which humans were infected with strains from the dog they owned.

Abstract Background The global emergence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has seen the dominance of specific clones in different regions around the world with the PVL-positive ST93-IV as the predominant CA-MRSA clone in Australia. In this study we applied a genome-wide association study (GWAS) approach on a collection of Australian ST93-IV MRSA genomes to screen for genetic traits that might have assisted the ongoing transmission of ST93-IV in Australia. We also compared the genomes of ST93-IV bacteraemia and non-bacteraemia isolates to search for potential virulence genes associated with bacteraemia. Results Based on single nucleotide polymorphism phylogenetics we revealed two distinct ST93-IV clades circulating concurrently in Australia. One of the clades contained isolates primarily isolated in the northern regions of Australia whilst isolates in the second clade were distributed across the country. Analyses of the ST93-IV genome plasticity over a 15-year period (2002–2017) revealed an observed gain in accessory genes amongst the clone’s population. GWAS analysis on the bacteraemia isolates identified two gene candidates that have previously been associated to this kind of infection. Conclusions Although this hypothesis was not tested here, it is possible that the emergence of a ST93-IV clade containing additional virulence genes might be related to the high prevalence of ST93-IV infections amongst the indigenous population living in the northern regions of Australia. More importantly, our data also demonstrated that GWAS can reveal candidate genes for further investigations on the pathogenesis and evolution of MRSA strains within a same lineage.

Dromedary camels are an important source of food and income in many countries. However, it has been largely overlooked that they can also transmit antibiotic-resistant bacteria. The aim of this study was to identify the Staphylococcaceae bacteria composition of the nasal flora in dromedary camels and evaluate the presence of methicillin-resistant Mammaliicoccus (MRM) and methicillin-resistant Staphylococcus (MRS) in dromedary camels in Algeria. Nasal swabs were collected from 46 camels from seven farms located in two different regions of Algeria (M’sila and Ouargla). We used non-selective media to determine the nasal flora, and antibiotic-supplemented media to isolate MRS and MRM. The staphylococcal isolates were identified using an Autoflex Biotyper Mass Spectrometer (MALDI-TOF MS). The mecA and mecC genes were detected by PCR. Methicillin-resistant strains were further analysed by long-read whole genome sequencing (WGS). Thirteen known Staphylococcus and Mammaliicoccus species were identified in the nasal flora, of which half (49.2%) were coagulase-positive staphylococci. The results showed that four out of seven farms were positive for MRS and/or MRM, with a total of 16 isolates from 13 dromedary camels. The predominant species were M. lentus, S. epidermidis, and S. aureus. Three methicillin-resistant S. aureus (MRSA) were found to be ST6 and spa type t304. Among methicillin-resistant S. epidermidis (MRSE), ST61 was the predominant ST identified. Phylogenetic analysis showed clonal relatedness among M. lentus strains, while S. epidermidis strains were not closely related. Resistance genes were detected, including mecA, mecC, ermB, tet(K), and blaZ. An SCCmec type VIII element was found in a methicillin-resistant S. hominis (MRSH) belonging to the ST1 strain. An SCCmec-mecC hybrid element was detected in M. lentus, similar to what was previously detected in M. sciuri. This study highlights that dromedary camels may be a reservoir for MRS and MRM, and that they contain a specific set of SCCmec elements. This emphasizes the need for further research in this ecological niche from a One Health perspective.

Abstract Staphylococcus capitis is a coagulase-negative staphylococcus that has been described primarily as causing bloodstream infections in neonatal intensive care units (NICUs), but has also recently been described in prosthetic joint infections (PJIs). The multidrug-resistant S. capitis subsp. urealyticus clone NRCS-A, comprising three sublineages, is prevalent in NICUs across the world, but its impact on other patient groups such as those suffering from PJIs or among adults planned for arthroplasty is unknown. Genome sequencing and subsequent analysis were performed on a Swedish collection of PJI isolates (n = 21), nasal commensals from patients planned to undergo arthroplasty (n = 20), NICU blood isolates (n = 9), operating theatre air isolates (n = 4), and reference strains (n = 2), in conjunction with an international strain collection (n = 248). The NRCS-A Outbreak sublineage containing the composite type V SCCmec-SCCcad/ars/cop element was present in PJIs across three Swedish hospitals. However, it was not found among nasal carrier strains, where the less virulent S. capitis subsp. capitis was most prevalent. The presence of the NRCS-A Outbreak clone in adult patients with PJIs demonstrates that dissemination occurs beyond NICUs. As this clone has several properties which facilitate invasive infections in patients with medical implants or immunosuppression, such as biofilm forming ability and multidrug resistance including heterogeneous glycopeptide-intermediate susceptibility, further research is needed to understand the reservoirs and distribution of this hospital-associated pathogen.

Abstract Background Although generally known as a human commensal, Staphylococcus epidermidis is also an opportunistic pathogen that can cause nosocomial infections related to foreign body materials and immunocompromized patients. Infections are often caused by multidrug-resistant (MDR) lineages that are difficult and costly to treat, and can have a major adverse impact on patients’ quality of life. Heterogeneity is a common phenomenon in both carriage and infection, but present methodology for detection of this is laborious or expensive. In this study, we present a culture-independent method, labelled Epidome, based on an amplicon sequencing-approach to deliver information beyond species level on primary samples and to elucidate clonality, population structure and temporal stability or niche selection of S. epidermidis communities. Results Based on an assessment of > 800 genes from the S. epidermidis core genome, we identified genes with variable regions, which in combination facilitated the differentiation of phylogenetic clusters observed in silico, and allowed classification down to lineage level. A duplex PCR, combined with an amplicon sequencing protocol, and a downstream analysis pipeline were designed to provide subspecies information from primary samples. Additionally, a probe-based qPCR was designed to provide valuable absolute abundance quantification of S. epidermidis. The approach was validated on isolates representing skin commensals and on genomic mock communities with a sensitivity of

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) can acquire phage-encoded immune modulators, such as the immune evasion cluster (IEC), which protects bacteria from components of the human innate immune system, and the enzyme TarP, which protects against antibody-mediated immune recognition. We used whole-genome sequencing and epidemiologic investigations to study the effects of IEC- and tarP-harboring phages on household transmission of LA-MRSA in North Denmark Region during 2004–2011. We reviewed information about all patients throughout Denmark who experienced LA-MRSA infection during 2007–2018 to determine whether IEC is associated with increased spread into the general population. Horizontal acquisition of IEC in the human host was associated with increased household transmission of LA-MRSA and spillover into the community and healthcare settings, whereas we found no evidence to suggest that IEC-positive LA-MRSA isolates have become self-sustainable in the general population. By contrast, TarP did not seem to influence household transmission of LA-MRSA.

Objective: Epidemic methicillin-resistant Staphylococcus aureus (MRSA) clones have been described in Ghana, but so far, no typical livestock-associated MRSA isolates (CC398) have been found. In this study we provide baseline information on antimicrobial resistance, population structure, and virulence gene content of S. aureus isolates from livestock and farm attendants. Methods: Nasal samples were collected from cattle, pigs, goats, sheep, and farm attendants from three farms. Staphylococcus aureus was identified by matrix-assisted laser desorption/ionisation time-of-flight and antimicrobial susceptibility testing was performed using VITEK II (Biomerieux, Marcy l'Etoile, France) and interpreted according to EUCAST guidelines. Whole-genome sequencing was performed using the Illumina, San Diego, CA, USA MiSeq Platform. Results: In total, 401 nasal swab samples were obtained from 57 farm attendants, 208 pigs, 30 goats, 26 sheep, and 80 cattle. The S. aureus isolates (n = 25) recovered (farm attendants: n = 10; pigs: n = 8; and goats: n = 7) were frequently resistant to penicillin (68%), tetracycline (44%), and ciprofloxacin (32%); two human isolates were MRSA. Twelve isolates (48%) were multidrug resistant (MDR) (>3 classes). Genome sequencing of the isolates revealed ST152-t355, ST9-t1430, and ST133-t8662 as dominant clones among farm attendants, pigs, and goats, respectively. The two MRSA isolates detected belonged to ST8-t334 and ST152-t355. The scn and sak genes associated with human-adaption were detected in 10 isolates; 9 from humans and 1 from a goat. Typing results provided evidence of a single potential transmission event (t861, PVL-, scn+). Conclusion: No MRSA was detected among livestock, perhaps because of low intensive farming; however, the relatively high prevalence of MDR isolates may be a result of inappropriate antibiotic usage in Ghanaian livestock production.

Escherichia coli sequence type (ST) 131 is of concern because it can acquire antimicrobial resistance and cause extraintestinal infections. E. coli ST131-H22 sublineage appears capable of being transmitted to humans through poultry. We report on multidrug-resistant ST131-H22 poultry isolates in Brazil closely related to international human and poultry isolates.

Arthroplasty surgery is a common procedure that significantly improves quality of life. The most feared complication is prosthetic joint infection (PJI), which occurs more often following revision surgery. Staphylococci are the most prevalent bacteria in PJIs, although many other pathogens have been reported. We describe a case of PJI in a 75-year-old farmer following revision surgery caused by Corynebacterium striatum, an unusual agent which normally occurs in the normal human skin microbiota with perceived low pathogenicity. Following a cemented right-sided total hip arthroplasty in 2006, a one-stage revision due to an osteolytic process in the right femur took place in 2020 with negative intraoperative tissue cultures. Three weeks later, the patient presented a fulminant infection which was treated with debridement, antibiotics, and implant retention (DAIR). Tissue biopsies showed C. striatum in 6/6 samples including small colony variants. Genome sequencing showed that all isolates differed by ≤6 SNPs with the same gene content related to resistance (tet(W) and erm(X)). The patient was sequentially treated with vancomycin, linezolid, and daptomycin, but due to side effects, treatment was changed to 12 weeks of dalbavancin as a 1000 mg loading dose followed by 500 mg intravenously/week. Impaired renal function during vancomycin treatment was normalized, and >1 year after finishing antibiotic treatment the outcome was still favourable. In conclusion, a case of a fulminant early post-interventional PJI due to C. striatum was successfully treated with DAIR and long-term dalbavancin therapy without any adverse reactions.

Polymyxins are considered as last–resort antibiotics to treat infections caused by Acinetobacter baumannii. However, there are increasing reports of resistance in A. baumannii to polymyxins. In this study, inhalable combinational dry powders consisting of ciprofloxacin (CIP) and polymyxin B (PMB) were prepared by spray–drying. The obtained powders were characterized with respect to the particle properties, solid state, in vitro dissolution and in vitro aerosol performance. The antibacterial effect of the combination dry powders against multidrug–resistant A. baumannii was assessed in a time–kill study. Mutants from the time–kill study were further investigated by population analysis profiling, minimum inhibitory concentration testing, and genomic comparisons. Inhalable dry powders consisting of CIP, PMB and their combination showed a fine particle fraction above 30%, an index of robust aerosol performance of inhaled dry powder formulations in the literature. The combination of CIP and PMB exhibited a synergistic antibacterial effect against A. baumannii and suppressed the development of CIP and PMB resistance. Genome analyses revealed only a few genetic differences of 3–6 SNPs between mutants and the progenitor isolate. This study suggests that inhalable spray–dried powders composed of the combination of CIP and PMB is promising for the treatment of respiratory infections caused by A. baumannii, and this combination can enhance the killing efficiency and suppress the development of drug resistance.

Avian pathogenic Escherichia coli (APEC) is one of the most important bacterial pathogens affecting poultry worldwide. The emergence of multidrug-resistant pathogens has renewed the interest in the therapeutic use of bacteriophages (phages). However, a major concern for the successful implementation of phage therapy is the emergence of phage-resistant mutants. The understanding of the phage-host interactions, as well as underlying mechanisms of resistance, have shown to be essential for the development of a successful phage therapy. Here, we demonstrate that the strictly lytic Escherichia phage vB_EcoM-P10 rapidly selected for resistance in the APEC ST95 O1 strain AM621. Whole-genome sequence analysis of 109 spontaneous phage-resistant mutant strains revealed 41 mutants with single-nucleotide polymorphisms (SNPs) in their core genome. In 32 of these, a single SNP was detected while two SNPs were identified in a total of nine strains. In total, 34 unique SNPs were detected. In 42 strains, including 18 strains with SNP(s), gene losses spanning 17 different genes were detected. Affected by genetic changes were genes known to be involved in phage resistance (outer membrane protein A, lipopolysaccharide-, O- antigen-, or cell wall-related genes) as well as genes not previously linked to phage resistance, including two hypothetical genes. In several strains, we did not detect any genetic changes. Infecting phages were not able to overcome the phage resistance in host strains. However, interestingly the initial infection was shown to have a great fitness cost for several mutant strains, with up to ∼65% decrease in overall growth. In conclusion, this study provides valuable insights into the phage-host interaction and phage resistance in APEC. Although acquired resistance to phages is frequently observed in pathogenic E. coli, it may be associated with loss of fitness, which could be exploited in phage therapy.

ABSTRACT Bacterial pathogens commonly carry prophages that express virulence factors, and human strains of Staphylococcus aureus carry Sa3int phages, which promote immune evasion. Recently, however, these phages have been found in livestock-associated, methicillin-resistant S. aureus (LA-MRSA). This is surprising, as LA-MRSA strains contain a mutated primary bacterial integration site, which likely explains why the rare integration events that do occur mostly happen at alternative locations. Using deep sequencing, we show that after initial integration at secondary sites, Sa3int phages adapt through nucleotide changes in their attachment sequences to increase homology with alternative bacterial attachment sites. Importantly, this homology significantly enhances integrations in new rounds of infections. We propose that promiscuity of the phage-encoded tyrosine recombinase is responsible for establishment of Sa3int phages in LA-MRSA. Our results demonstrate that phages can adopt extensive population heterogeneity, leading to establishment in strains lacking bona fide integration sites. Ultimately, their presence may increase virulence and zoonotic potential of pathogens with major implications for human health. IMPORTANCE A growing number of humans are being infected by antibiotic resistant Staphylococcus aureus originating from livestock. The preference of S. aureus for humans or animals is in part determined by factors encoded by viruses (phages) that reside in the bacterial genome. Here, we reveal a process by which phages adapt to and become integrated in new strains of S. aureus lacking the preferred phage integration site. We propose that this is due to the relaxed specificity of a phage-encoded enzyme called recombinase. As this recombinase is used by many other phages, our results might have implications for a broader range of phages. Importantly, the adaptation described here enables S. aureus to jump between host organisms and increases its zoonotic threat.

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a disease of major impact on pig health, welfare, and productivity globally. Serovar 8 (APP) is the predominant clinical serovar in Norway and the United Kingdom (UK), and has been isolated from clinical cases in Denmark. The primary objective of this study was to characterize the genetic variability of isolates of A. pleuropneumoniae APP8 in the Norwegian population. The secondary objectives were to determine the within-host variability of APP8; to compare the APP8 bacterial populations in Norway, Denmark, and the UK, including antimicrobial resistance (AMR) gene profiles and to assess the effect of national differences in antimicrobial drug use and restricted animal movement on the occurrence of resistance. Isolates of APP8 from the UK (n=67), Denmark (n=22), and Norway (n=123) collected between 1983 and 2020 were compared using whole genome sequencing. To investigate genetic variability within individual hosts, an additional 104 APP8 isolates from the lungs of six Norwegian pigs were compared. Very low within-host variation was observed (≤ 2 single nucleotide polymorphisms). The phylogeny of 123 Norwegian APP8 isolates from 76 herds revealed some within-herd genetic variation, but substantial geographical clustering. When inferring the relatedness of the three international APP8 collections, the topology highlighted the existence of two distinct monophyletic branches characterized by the Norwegian and UK isolates, respectively. Three Danish isolates were scattered across the UK branch, whereas the remaining 19 Danish isolates clustered in two monophyletic groups nested in the Norwegian branch. Coalescence analysis, performed to estimate the divergences from a common ancestor, indicated a last common ancestor several centuries ago. The phylogenetic analyses also revealed striking differences in occurrence of AMR genes, as these were 23-times more prevalent among the UK isolates than among the Norwegian isolates. An increased understanding of the effects of population strategies is helpful in surveillance and control of infectious diseases.

Objectives: To report the complete genome sequence of a methicillin-resistant Staphylococcus vitulinus from ground beef to allow comparison with other available S. vitulinus genomes and to investigate its SCCmec element. Methods: Meat samples from grocery stores in Denmark were examined for the presence of staphylococcal species by plating on selective plates. One colony isolated from beef was identified as S. vitulinus by MALDI-TOF and genome sequenced using a combination of Illumina and Oxford Nanopore technologies. Phylogenetic and in silico resistome analyses were performed for all available S. vitulinus genomes. Results: The closed genome of S. vitulinus Tienloo1 isolate had a chromosome size of 2,628,028 bp and contained a single novel 2,380 bp plasmid based on a hybrid assembly. It carried mecA as the only resistance marker. The isolate was found not to carry any immune evasion cluster genes, which have been putatively associated to human origin. Comparison with all publicly available S. vitulinus draft genomes showed a diverse population and revealed that only the Danish beef isolate contained a mec gene in addition to a ccr gene complex. Additionally, the single ccrC gene within the isolate was novel and distant from the mecA2 gene. Conclusion: This isolate, Tienloo1, from a ground beef meat sample represents the first complete genome of S. vitulinus found to carry a mecA2 gene and a novel ccr allotype in its SCCmec element that is distinct from all publicly available draft S. vitulinus genomes.

We report a fatal aspergillosis case in which STRAf typing and whole-genome sequencing substantiated in vivo emergence of an azole-resistant Aspergillus fumigatus with a 120-bp tandem repeat in the promoter region of cyp51A. This event, previously restricted to the environment, challenges current understanding of azole resistance development in A. fumigatus.

Background: Defined by the emergence of antibiotic resistant strains, Staphylococcus aureus is a priority bacterial species with high antibiotic resistance. However, a rise in the prevalence of penicillin-susceptible S. aureus (PSSA) bloodstream infections has recently been observed worldwide, including in Australia, where the proportion of methicillin-susceptible S. aureus causing bacteremia identified phenotypically as penicillin-susceptible has increased by over 35%, from 17.5% in 2013 to 23.7% in 2020. Objectives: To determine the population structure of PSSA causing community- and hospital-onset bacteremia in Australia and to evaluate routine phenotypic antimicrobial susceptibility methods to reliably confirm penicillin resistance on blaZ-positive S. aureus initially classified as penicillin-susceptible by the Vitek® 2 automated microbiology system. Results: Whole genome sequencing on 470 PSSA collected in the 2020 Australian Group on Antimicrobial Resistance Australian Staphylococcus aureus Sepsis Outcome Programme identified 84 multilocus sequence types (STs), of which 79 (463 isolates) were grouped into 22 clonal complexes (CCs). The dominant CCs included CC5 (31.9%), CC97 (10.2%), CC45 (10.0%), CC15 (8.7%), and CC188 (4.9%). Many of the CCs had multiple STs and spa types and, based on the immune evasion cluster type, isolates within a CC could be classified into different strains harboring a range of virulence and resistance genes. Phylogenetic analyses of the isolates showed most CCs were represented by one clade. The blaZ gene was identified in 45 (9.6%) PSSA. Although multiclonal, approximately 50% of blaZ-positive PSSA were from CC15 and were found to be genetically distant from the blaZ-negative CC15 PSSA. The broth microdilution, Etest® and cefinase, performed poorly; however, when the appearance of the zone edge was considered; as per the EUCAST and CLSI criteria, disc diffusion detected 100% of blaZ-positive PSSA. Conclusions: In Australia, PSSA bacteremia is not caused by the expansion of a single clone. Approximately 10% of S. aureus classified as penicillin-susceptible by the Vitek® 2 harbored blaZ. Consequently, we recommend that confirmation of Vitek® 2 PSSA be performed using an alternative method, such as disc diffusion with careful interpretation of the zone edge.

S. epidermidis is a substantial component of the human skin microbiota, but also one of the major causes of nosocomial infection in the context of implanted medical devices. We here aimed to advance the understanding of S. epidermidis genotypes and phenotypes conducive to infection establishment. Furthermore, we investigate the adaptation of individual clonal lines to the infection lifestyle based on the detailed analysis of individual S. epidermidis populations of 23 patients suffering from prosthetic joint infection. Analysis of invasive and colonizing S. epidermidis provided evidence that invasive S. epidermidis are characterized by infection-supporting phenotypes (e.g. increased biofilm formation, growth in nutrient poor media and antibiotic resistance), as well as specific genetic traits. The discriminating gene loci were almost exclusively assigned to the mobilome. Here, in addition to IS256 and SCCmec, chromosomally integrated phages was identified for the first time. These phenotypic and genotypic features were more likely present in isolates belonging to sequence type (ST) 2. By comparing seven patient-matched nasal and invasive S. epidermidis isolates belonging to identical genetic lineages, infection-associated phenotypic and genotypic changes were documented. Besides increased biofilm production, the invasive isolates were characterized by better growth in nutrient-poor media and reduced hemolysis. By examining several colonies grown in parallel from each infection, evidence for genetic within-host population heterogeneity was obtained. Importantly, subpopulations carrying IS insertions in agrC, mutations in the acetate kinase (AckA) and deletions in the SCCmec element emerged in several infections. In summary, these results shed light on the multifactorial processes of infection adaptation and demonstrate how S. epidermidis is able to flexibly repurpose and edit factors important for colonization to facilitate survival in hostile infection environments.

Whole-genome sequencing is essential to many facets of infectious disease research. However, technical limitations such as bias in coverage and tagmentation, and difficulties characterising genomic regions with extreme GC content have created significant obstacles in its use. Illumina has claimed that the recently released DNA Prep library preparation kit, formerly known as Nextera Flex, overcomes some of these limitations. This study aimed to assess bias in coverage, tagmentation, GC content, average fragment size distribution, and de novo assembly quality using both the Nextera XT and DNA Prep kits from Illumina. When performing whole-genome sequencing on Escherichia coli and where coverage bias is the main concern, the DNA Prep kit may provide higher quality results; though de novo assembly quality, tagmentation bias and GC content related bias are unlikely to improve. Based on these results, laboratories with existing workflows based on Nextera XT would see minor benefits in transitioning to the DNA Prep kit if they were primarily studying organisms with neutral GC content.

ABSTRACT Since the late 1990s, changes in the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) were recognized with the emergence of community-associated MRSA (CA-MRSA). CA-MRSA belonging to clonal complex 152 (CC152), carrying the small staphylococcal cassette chromosome mec (SCCmec) type V and encoding the Panton-Valentine leukocidin (PVL), has been observed in Europe. The aim of this study was to investigate its origin, evolution, and dissemination. Whole-genome sequencing was performed on a global collection of 149 CC152 isolates spanning 20 years (93 methicillin-susceptible S. aureus [MSSA] and 56 MRSA isolates). Core genome phylogeny, Bayesian inference, in silico resistance analyses, and genomic characterization were applied. Phylogenetic analysis revealed two major distinct clades, one dominated by MSSA and the other populated only by MRSA. The MSSA isolates were predominately from sub-Saharan Africa, whereas MRSA was almost exclusively from Europe. The European MRSA isolates all harbored an SCCmec type V (5C2&5) element, whereas other SCCmec elements were sporadically detected in MRSA from the otherwise MSSA-dominated clade, including SCCmec types IV (2B), V (5C2), and XIII (9A). In total, 93% of the studied CC152 isolates were PVL positive. Bayesian coalescent inference suggests an emergence of the European CC152-MRSA in the 1990s, while the CC152 lineage dates back to the 1970s. The CA-MRSA CC152 clone mimics the European CC80 CA-MRSA lineage by its emergence from a PVL-positive MSSA ancestor from North Africa or Europe. The CC152 lineage has acquired SCCmec several times, but acquisition of SCCmec type V (5C2&5) seems associated with expansion of MRSA CC152 in Europe. IMPORTANCE Understanding the evolution of CA-MRSA is important in light of the increasing importance of this reservoir in the dissemination of MRSA. Here, we highlight the story of the CA-MRSA CC152 lineage using whole-genome sequencing on an international collection of CC152. We show that the evolution of this lineage is novel and that antibiotic usage may have the potential to select for the phage-encoded Panton-Valentine leukocidin. The diversity of the strains correlated highly to geography, with higher level of resistance observed among the European MRSA isolates. The mobility of the SCCmec element is mandatory for the emergence of novel MRSA lineages, and we show here distinct acquisitions, one of which is linked to the successful clone found throughout Europe today.

ABSTRACT Escherichia coli sequence type 131 (ST131) is a major cause of urinary and bloodstream infections. Its association with extended-spectrum β-lactamases (ESBLs) significantly complicates treatment. Its best-described component is the rapidly expanding H30Rx clade, containing allele 30 of the type 1 fimbrial adhesin gene fimH. This lineage appears to have emerged in the United States and spread around the world in part due to the acquisition of the ESBL-encoding blaCTX-M-15 gene and resistance to fluoroquinolones. However, non-H30 ST131 sublineages with other acquired CTX-M-type resistance genes are also emerging. Based on whole-genome analyses, we describe here the presence of an (fimH) H27 E. coli ST131 sublineage that has recently caused an outbreak of community-acquired bacteremia and recurrent urinary tract infections (UTIs) in Denmark. This sublineage has acquired both a virulence plasmid (pAA) that defines the enteroaggregative E. coli (EAEC) diarrheagenic pathotype and multiple genes associated with extraintestinal E. coli (ExPEC); combined, these traits have made this particular ST131 sublineage successful at colonizing its human host and causing recurrent UTI. Moreover, using a historic World Health Organization (WHO) E. coli collection and publicly available genome sequences, we identified a global H27 EAEC ST131 sublineage that dates back as far as 1998. Most H27 EAEC ST131 isolates harbor pAA or pAA-like plasmids, and our analysis strongly implies a single ancestral acquisition among these isolates. These findings illustrate both the profound plasticity of this important pathogenic E. coli ST131 H27 sublineage and genetic acquisitions of EAEC-specific virulence traits that likely confer an enhanced ability to cause intestinal colonization. IMPORTANCE E. coli ST131 is an important extraintestinal pathogenic lineage. A signature characteristic of ST131 is its ability to asymptomatically colonize the gastrointestinal tract and then opportunistically cause extraintestinal infections, such as cystitis, pyelonephritis, and urosepsis. In this study, we identified an ST131 H27 sublineage that has acquired the enteroaggregative diarrheagenic phenotype, spread across multiple continents, and caused multiple outbreaks of community-acquired ESBL-associated bloodstream infections in Denmark. The strain’s ability to both cause diarrhea and innocuously colonize the human gastrointestinal tract may facilitate its dissemination and establishment in the community.

Whole-genome sequencing has enabled detailed studies on bacterial evolution during infection, but there is limited knowledge on intraclonal variation. In this study, we sought to provide a snapshot of the intraclonal diversity of Escherichia coli as both commensal in the faecal environment and pathogen during urinary tract infection, respectively. This was performed by whole-genome sequencing and analyses of single nucleotide polymorphisms (SNPs) and gene-content variation in ten isolates belonging to the same clone and isolated from rectal swabs or urine samples. We identified only one clone in eight of the nine urines sampled (89 %). In both the commensal and pathogenic state, the within-host diversity was limited with intraclonal SNP diversity of 0–2 non-synonymous SNPs for each clone. The genetic diversity showed variation in gene content in a range of 2–15 genes in total for all clones, including genes positioned on plasmids, and in the K- and O-antigen cluster. The observed SNP- and gene variation shows that sampling of one colony would be enough for surveillance, outbreak investigations and clonal evolution. However, for studies of adaptation during or between colonization and infection, this variation is relevant to consider.

BACKGROUND:Over the last three decades, hospital adapted clonal complex (CC) 17 strains of Enterococcus faecium have acquired and exchanged antimicrobial resistance genes leading to the widespread resistance to clinically important antimicrobials globally. In Australia, a high prevalence of vancomycin resistance has been reported in E. faecium in the last decade. METHODS:In this study, we determined the phylogenetic relationship and genetic characteristics of E. faecium collected from hospitalized patients with blood stream infections throughout Australia from 2015 to 2017 using high throughput molecular techniques. RESULTS:Using single nucleotide polymorphism based phylogenetic inference, three distinct clusters of isolates were observed with additional sub-clustering. One cluster harboured mostly non-CC17 isolates while two clusters were dominant for the vanA and vanB operons. CONCLUSION:The gradual increase in dominance of the respective van operon was observed in both the vanA and vanB dominant clusters suggesting a strain-van operon affinity. The high prevalence of the van operon within isolates of a particular sub-cluster was linked to an increased number of isolates and 30-day all-cause mortality. Different dominant sub-clusters were observed in each region of Australia. Findings from this study can be used to put future surveillance data into a broader perspective including the detection of novel E. faecium strains in Australia as well as the dissemination and evolution of each strain.

Foods may potentially serve as vehicles for the transmission of antimicrobial-resistant variants of Staphylococcus aureus that are important in a human clinical context. Further, retail food products can be a cause of staphylococcal food poisoning. For these reasons and to account for source attribution and risk assessment, detailed information on the population structure, resistance, and virulence profiles of S. aureus originating from retail food products is necessary. In the current study, whole-genome sequences from 88 S. aureus isolates were subjected to bioinformatics analyses in relation to sequence types, antimicrobial resistance, and virulence profiles. The sequence types (ST) identified belonged to 13 clonal complexes (CC) with CC5 and CC398 being the most common. CC398 was identified as the dominant clone (n = 31). CC5 was identified as of avian origin, with the presence of φAVβ prophage genes (n = 13). In total, 39.8% of the isolates contained multiple resistance genes, and methicillin-resistant Staphylococcus aureus (MRSA) isolates were found in CC8, CC9, and CC398. Genes conferring resistance to the antimicrobial classes of β-lactams, tetracycline, and erythromycin were detected in this study, all of which are commonly used in Danish livestock production. The tst gene encoding the toxic shock syndrome toxin was for the first time identified in ST398 isolates, probably as a result of a single acquisition of a SaPI-like element. The sushi-CC398 isolates carrying the scn gene likely originated from a human reservoir, while the other isolates originated from livestock. Taken together, our results show that both human and animal reservoirs contribute to contamination in food products and that retail foods may serve as a vehicle of S. aureus between livestock and humans.

Klebsiella pneumoniae causes a variety of infections in both humans and animals. In this study, we characterised the genomes of human and animal isolates from two diagnostic laboratories on St. Kitts, a small Caribbean island inhabited by a large population of vervet monkeys. In view of the increased chances of direct or indirect contact with humans and other animal species, we used the One Health approach to assess transmission of K. pneumoniae across host species by sequencing 82 presumptive K. pneumoniae clinical isolates from humans (n = 51), vervets (n = 21), horses (n = 5), dogs (n = 4) and a cat (n = 1). Whole genome sequencing (WGS) was carried out using Illumina technology. De novo assembly was performed in CLC Genomics Workbench v.11.0. Single nucleotide polymorphisms were detected using NASP followed by phylogenetic analysis using IQ-TREE. Virulence and antimicrobial resistance gene contents were analysed using the Kleborate and CGE pipelines. WGS-based analysis showed that 72 isolates were K. pneumoniae sensu stricto and five K. quasipneumoniae and five K. variicola. K. pneumoniae isolates belonged to 35 sequence types (ST), three of which were occasionally shared between humans and animals: ST23, ST37 and ST307. The ST23 strains from vervets formed a separate cluster amongst publicly available sequenced ST23 strains, indicating the presence of a specific vervet sublineage. Animal strains harbored fewer resistance genes and displayed distinct virulence traits that appeared to be host-specific in vervet isolates. Our results show that K. pneumoniae infections on this Caribbean island are usually caused by host-specific lineages.

Recurrent urinary tract infection (rUTI) remains a major problem for many women and therefore the pursuit for genomic and phenotypic traits which could define rUTI has been ongoing. The present study applied a genomic approach to investigate recurrent urinary tract infections by comparative analyses of recurrent and non-recurrent Escherichia coli isolates from general practice. From whole-genome sequencing data, phylogenetic clustering and genomic traits were studied on a collection of isolates which caused recurrent infection compared to non-recurrent isolates. In addition, genomic variation between the 1st and following infection was studied on a subset of the isolates. Evidence of limited adaptation between the recurrent infections based on single nucleotide polymorphism analyses with a range of 0–13 non-synonymous single nucleotide polymorphisms (SNPs) between the paired isolates. This included an overrepresentation of SNPs in metabolism genes. We identified several genes which were more common in rUTI isolates, including nine fimbrial genes, however, not significantly after false-discovery rate. Finally, the results show that recurrent isolates of the present dataset are not distinctive by variation in the core genome, and thus, did not cluster distinct from non-rUTI isolates in a SNP phylogeny.

Abstract Objective Clostridium perfringens causes gastrointestinal diseases in both humans and domestic animals. Type A strains expressing the NetB toxin are the main cause of necrotic enteritis (NE) in chickens, which has remarkable impact on animal welfare and production economy in the international poultry industry. Three pathogenicity loci NELoc-1, -2 and -3 and a collagen adhesion gene cnaA have been found to be associated with NE in chickens, whereas the presence of these has not been investigated in diseased turkeys. The purpose was to investigate the virulence associated genome content and the genetic relationship among 30 C. perfringens isolates from both healthy and NE infected chickens and turkeys, applying whole-genome sequencing. Results NELoc-1, -3, netB and cnaA were significantly associated with NE isolates from chickens, whereas only NELoc-2 was commonly observed in both diseased turkeys and chickens. A putative collagen adhesion gene that encodes a von Willebrand Factor (vWF) domain was identified in all diseased turkeys and designated as cnaD. The phylogenetic analysis based on single nucleotide polymorphisms showed that the isolates generally were not closely related. These results indicate that virulence factors and pathogenicity loci associated with NE in chickens are not important to the same extent in diseased turkeys except for NELoc-2. A putative collagen adhesion gene which potentially could be of importance in regard to the NE pathogenesis in turkeys was identified and need to be further investigated. Thus, the pathogenesis of NE in turkeys appears to be different from that of broiler chickens.

Abstract Norovirus (NoV) is the most common cause of non-bacterial gastroenteritis and is a major agent associated with outbreaks of gastroenteritis. Conventional molecular genotyping analysis of NoV, used for the identification of transmission routes, relies on standard typing methods (STM) by Sanger-sequencing of only a limited part of the NoV genome, which could lead to wrong conclusions. Here, we combined a NoV capture method with next generation sequencing (NGS), which increased the proportion of norovirus reads by ~40 fold compared to NGS without prior capture. Of 15 NoV samples from 6 single-genotype outbreaks, near full-genome coverage (>90%) was obtained from 9 samples. Fourteen polymerase (RdRp) and 15 capsid (cap) genotypes were identified compared to 12 and 13 for the STM, respectively. Analysis of 9 samples from two mixed-genotype outbreaks identified 6 RdRp and 6 cap genotypes (two at >90% NoV genome coverage) compared to 4 and 2 for the STM, respectively. Furthermore, complete or partial sequences from the P2 hypervariable region were obtained from 7 of 8 outbreaks and a new NoV recombinant was identified. This approach could therefore strengthen outbreak investigations and could be applied to other important viruses in stool samples such as hepatitis A and enterovirus.

The possible spillover from pigs into other production animals incites concern for unresolved reservoirs of human exposure. The present investigation was therefore initiated, to elucidate if Danish veal and dairy farms constitute a reservoir of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) CC398 and to potentially identify the source of introduction. We collected nasal swab samples from 17 Danish veal farms, 2 slaughterhouses, and received bulk tank milk samples from 286 dairy farms. All samples were analyzed by culturing and screening on MRSA selective plates and presumed MRSA was verified by MALDI-TOF and PCR. MRSA isolates were subjected to spa typing and whole-genome sequencing. LA-MRSA was found on two veal farms in one and three calves, respectively, with subsequent follow-up samples found negative. Eight of 286 dairy farms (2.8%) were found LA-MRSA positive and follow-up samples, from five farms showed intermittent detection of LA-MRSA. The spa types, t034 and t011, were the most common while a single isolate from a dairy farm belonged to spa type t843 associated to mecC-MRSA CC130 and is the first report of mecC-MRSA in the Danish dairy production. A phylogenetic analysis showed that some of the isolates grouped within or close to the dominant Danish pig clusters, suggesting spillover into cattle farms. Other isolates clustered outside the dominant pig clusters suggesting that other routes of introduction cannot be excluded. Results of the investigation indicated a contamination of veal farms while some dairy farms seemed to be a permanent reservoir. Thus, Danish cattle represent a low prevalence reservoir of LA-MRSA CC398, which at present, is not of major human health concern.

Limited information is available on antimicrobial susceptibility and clonal distribution of Staphylococcus aureus in the Caribbean region. The aims of this study were to determine the prevalence of antimicrobial resistance among S. aureus isolates and to reveal the frequency and population structure of methicillin-resistant S. aureus (MRSA) in St. Kitts and Nevis, a small island country in the West Indies. A total of 152 S. aureus isolates were collected from consecutive samples submitted to the clinical microbiology laboratory of the main referral hospital from March 2017 to January 2018. Samples came from all units in the hospital and a small number came from external submissions, and comprised a total of 119 clinical specimens and 33 nasal swabs collected from staff and patients. All S. aureus isolates were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Minimal Inhibitory Concentrations (MICs) of clinically relevant antimicrobials were determined by broth microdilution, and diversity of MRSA isolates was assessed by whole genome sequencing (WGS) analysis. MRSA accounted for 45% (69/152) of the isolates. The highest rates of resistance to non-β-lactam agents were observed for erythromycin (55%), moxifloxacin (41%), and levofloxacin (40%), whereas resistance to the other drugs tested was ≤6%. All isolates were susceptible to ceftaroline, linezolid, teicoplanin, telavancin, and vancomycin. WGS-based multilocus sequence typing (MLST) showed that approximately 88% of the MRSA isolates belonged to ST8. Phylogenetic analysis on 801 single-nucleotide polymorphisms (SNPs) identified among the MRSA ST8 isolates indicates a large degree of genetic diversity. However, all ST8 strains clustered within the distinct clade that defines the USA300 North American Epidemic lineage (Panton-Valentine Leukocidin (PVL) +, arginine catabolic mobile element (ACME) +, Staphylococcal cassettes chromosome mec IVa (SCCmec IVa)). Our data show high levels of methicillin, macrolide, and fluoroquinolone resistance among S. aureus on St. Kitts and Nevis. The USA300 North American epidemic lineage is responsible for the vast majority of MRSA infections on this Caribbean island.