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Serratia marcescens is an opportunistic pathogen with a propensity to cause nosocomial outbreaks, particularly in neonatal intensive care units (NICUs). We present a sustained outbreak spanning over 18 months (1 January 2022-29 August 2023) in a NICU in Antwerp, Belgium, affecting 61 neonates, identified through samples taken for diagnostic purposes and by rectal screening. Ten neonates were infected: five with lower respiratory tract infection, four with conjunctivitis and one fatal case with sepsis. In a logistic regression analysis, nursing in an incubator was significantly associated with acquisition of S. marcescens (odds ratio (OR): 2.99; 95% confidence interval (CI): 1.14-8.25; p < 0.05). Whole genome sequencing-based multilocus sequence typing (wgMLST) and core genome single nucleotide polymorphism (cgSNP) analysis of isolates from clinical (n = 4), screening (n = 52) and environmental samples (n = 8), identified eight clusters and five singletons not associated with the clusters. Although outbreak measures were successful in containing further spread within the ward during sudden surges when > 4 cases per week were identified (peak events), several peaks with different clonal clusters occurred. The emergence of similar outbreaks in Belgian hospitals underscores the need of continuous surveillance and NICU-specific infection prevention and control (IPC) measures.

Objectives: Escherichia coli can cause infections in the urinary tract and in normally sterile body sites leading to invasive E. coli disease (IED), including bacteraemia and sepsis, with older populations at increased risk. We aimed to estimate the theoretical coverage rate by the ExPEC4V and 9V vaccine candidates. In addition, we aimed at better understanding the diversity of E. coli isolates, including their genetic and phenotypic antimicrobial resistance (AMR), sequence types (STs), O-serotypes and the bacterial population structure., Methods: Blood and urine culture E. coli isolates (n = 304) were collected from hospitalized patients ≥60 years (n = 238) with IED during a multicentric, observational study across three continents. All isolates were tested for antimicrobial susceptibility, O-serotyped, whole-genome sequenced and bioinformatically analysed., Results: A large diversity of STs and of O-serotypes were identified across all centres, with O25b-ST131, O6-ST73 and O1-ST95 being the most prevalent types. A total of 45.4% and 64.7% of all isolates were found to have an O-serotype covered by the ExPEC4V and ExPEC9V vaccine candidates, respectively. The overall frequency of MDR was 37.4% and ST131 was predominant among MDR isolates. Low in-patient genetic variability was observed in cases where multiple isolates were collected from the same patient., Conclusions: Our results highlight the predominance of MDR O25b-ST131 E. coli isolates across diverse geographic areas. These findings provide further baseline data on the theoretical coverage of novel vaccines targeting E. coli associated with IED in older adults and their associated AMR levels., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Introduction. Listerine Ò is a bactericidal mouthwash widely used to prevent oral health problems such as dental plaque and gingivitis. However, whether it promotes or undermines a healthy oral microbiome is unclear. Hypothesis/Gap Statement. We hypothesized that the daily use of Listerine Cool Mint would have a significant impact on the oropharyngeal microbiome. Aim. We aimed to assess if daily usage of Listerine Cool Mint influenced the composition of the pharyngeal microbiome. Methodology. The current microbiome substudy is part of the Preventing Resistance in Gonorrhoea trial. This was a double-blind single-centre, crossover, randomized controlled trial of antibacterial versus placebo mouthwash to reduce the incidence of gonorrhoea/chlamydia/syphilis in men who have sex with men (MSM) taking HIV pre-exposure prophylaxis (PrEP). Fifty-nine MSM taking HIV PrEP were enrolled. In this crossover trial, participants received 3 months of daily Listerine followed by 3 months of placebo mouthwash or vice versa. Oropharyngeal swabs were taken at baseline and after 3 months use of each mouthwash. DNA was extracted for shotgun metagenomic sequencing (Illumina Inc.). Non-host reads were taxonomically classified with MiniKraken and Bracken. The alpha and beta diversity indices were compared between baseline and after each mouthwash use. Differentially abundant bacterial taxa were identified using ANOVA-like differential expression analysis. Results. Streptococcus was the most abundant genus in most samples ( n = 103, 61.7 %) with a median relative abundance of 31.5% (IQR 20.6-44.8), followed by Prevotella [13.5% (IQR 4.8-22.6)] and Veillonella [10.0% (IQR 4.0-16.8)]. Compared to baseline, the composition of the oral microbiome at the genus level (beta diversity) was significantly different after 3 months of Listerine ( P = 0.006, pseudo- F = 2.29) or placebo ( P = 0.003, pseudo- F = 2.49, permutational multivariate analysis of variance) use. Fusobacterium nucleatum and Streptococcus anginosus were significantly more abundant after Listerine use compared to baseline. Conclusion. Listerine use was associated with an increased abundance of common oral opportunistic bacteria previously reported to be enriched in periodontal diseases, oesophageal and colorectal cancer, and systemic diseases. These findings suggest that the regular use of Listerine mouthwash should be carefully considered.

Gram-negative bacteria (GNB) are a major cause of neonatal sepsis in low- and middle-income countries (LMICs). Although the World Health Organization (WHO) reports that over 80% of these sepsis deaths could be prevented through improved treatment, the efficacy of the currently recommended first- and second-line treatment regimens for this condition is increasingly affected by high rates of drug resistance. Here we assess three well known antibiotics, fosfomycin, flomoxef and amikacin, in combination as potential antibiotic treatment regimens by investigating the drug resistance and genetic profiles of commonly isolated GNB causing neonatal sepsis in LMICs. The five most prevalent bacterial isolates in the NeoOBS study (NCT03721302) are Klebsiella pneumoniae, Acinetobacter baumannii, E. coli, Serratia marcescens and Enterobacter cloacae complex. Among these isolates, high levels of ESBL and carbapenemase encoding genes are detected along with resistance to ampicillin, gentamicin and cefotaxime, the current WHO recommended empiric regimens. The three new combinations show excellent in vitro activity against ESBL-producing K. pneumoniae and E. coli isolates. Our data should further inform and support the clinical evaluation of these three antibiotic combinations for the treatment of neonatal sepsis in areas with high rates of multidrug-resistant Gram-negative bacteria., (© 2024. The Author(s).)

Background: Streptococcus pneumoniae is a global cause of community-acquired pneumonia (CAP) and invasive disease in children. The CAP-IT trial (grant No. 13/88/11; https://www.capitstudy.org.uk/ ) collected nasopharyngeal swabs from children discharged from hospitals with clinically diagnosed CAP, and found no differences in pneumococci susceptibility between higher and lower antibiotic doses and shorter and longer durations of oral amoxicillin treatment. Here, we studied in-depth the genomic epidemiology of pneumococcal (vaccine) serotypes and their antibiotic resistance profiles., Methods: Three-hundred and ninety pneumococci cultured from 1132 nasopharyngeal swabs from 718 children were whole-genome sequenced (Illumina) and tested for susceptibility to penicillin and amoxicillin. Genome heterogeneity analysis was performed using long-read sequenced isolates (PacBio, n = 10) and publicly available sequences., Results: Among 390 unique pneumococcal isolates, serotypes 15B/C, 11 A, 15 A and 23B1 were most prevalent (n = 145, 37.2%). PCV13 serotypes 3, 19A, and 19F were also identified (n = 25, 6.4%). STs associated with 19A and 19F demonstrated high genome variability, in contrast to serotype 3 (n = 13, 3.3%) that remained highly stable over a 20-year period. Non-susceptibility to penicillin (n = 61, 15.6%) and amoxicillin (n = 10, 2.6%) was low among the pneumococci analysed here and was independent of treatment dosage and duration. However, all 23B1 isolates (n = 27, 6.9%) were penicillin non-susceptible. This serotype was also identified in ST177, which is historically associated with the PCV13 serotype 19F and penicillin susceptibility, indicating a potential capsule-switch event., Conclusions: Our data suggest that amoxicillin use does not drive pneumococcal serotype prevalence among children in the UK, and prompts consideration of PCVs with additional serotype coverage that are likely to further decrease CAP in this target population. Genotype 23B1 represents the convergence of a non-vaccine genotype with penicillin non-susceptibility and might provide a persistence strategy for ST types historically associated with vaccine serotypes. This highlights the need for continued genomic surveillance., (© 2024. The Author(s).)

Background: Colistin serves as the last line of defense against multidrug resistant Gram-negative bacterial infections in both human and veterinary medicine. This study aimed to investigate the occurrence and spread of colistin-resistant Enterobacterales (ColR-E) using a One Health approach in Belgium and in the Netherlands., Methods: In a transnational research project, a total of 998 hospitalized patients, 1430 long-term care facility (LTCF) residents, 947 children attending day care centres, 1597 pigs and 1691 broilers were sampled for the presence of ColR-E in 2017 and 2018, followed by a second round twelve months later for hospitalized patients and animals. Colistin treatment incidence in livestock farms was used to determine the association between colistin use and resistance. Selective cultures and colistin minimum inhibitory concentrations (MIC) were employed to identify ColR-E. A combination of short-read and long-read sequencing was utilized to investigate the molecular characteristics of 562 colistin-resistant isolates. Core genome multi-locus sequence typing (cgMLST) was applied to examine potential transmission events., Results: The presence of ColR-E was observed in all One Health sectors. In Dutch hospitalized patients, ColR-E proportions (11.3 and 11.8% in both measurements) were higher than in Belgian patients (4.4 and 7.9% in both measurements), while the occurrence of ColR-E in Belgian LTCF residents (10.2%) and children in day care centres (17.6%) was higher than in their Dutch counterparts (5.6% and 12.8%, respectively). Colistin use in pig farms was associated with the occurrence of colistin resistance. The percentage of pigs carrying ColR-E was 21.8 and 23.3% in Belgium and 14.6% and 8.9% in the Netherlands during both measurements. The proportion of broilers carrying ColR-E in the Netherlands (5.3 and 1.5%) was higher compared to Belgium (1.5 and 0.7%) in both measurements. mcr-harboring E. coli were detected in 17.4% (31/178) of the screened pigs from 7 Belgian pig farms. Concurrently, four human-related Enterobacter spp. isolates harbored mcr-9.1 and mcr-10 genes. The majority of colistin-resistant isolates (419/473, 88.6% E. coli; 126/166, 75.9% Klebsiella spp.; 50/75, 66.7% Enterobacter spp.) were susceptible to the critically important antibiotics (extended-spectrum cephalosporins, fluoroquinolones, carbapenems and aminoglycosides). Chromosomal colistin resistance mutations have been identified in globally prevalent high-risk clonal lineages, including E. coli ST131 (n = 17) and ST1193 (n = 4). Clonally related isolates were detected in different patients, healthy individuals and livestock animals of the same site suggesting local transmission. Clonal clustering of E. coli ST10 and K. pneumoniae ST45 was identified in different sites from both countries suggesting that these clones have the potential to spread colistin resistance through the human population or were acquired by exposure to a common (food) source. In pig farms, the continuous circulation of related isolates was observed over time. Inter-host transmission between humans and livestock animals was not detected., Conclusions: The findings of this study contribute to a broader understanding of ColR-E prevalence and the possible pathways of transmission, offering insights valuable to both academic research and public health policy development., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 De Koster et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Klebsiella pneumoniae (Kp), which is associated with hospital-acquired infections, is extensively drug-resistant (XDR), making treatment difficult. Understanding the genetic epidemiology of XDR-Kp can help determine its potential to be hypervirulent (hv) through the presence of siderophores. We characterized the genomes of 18 colistin-resistant XDR-Kp isolated from 14 patients with complicated tract infection at an Indian healthcare facility. The 18 organisms comprised the following sequence types (STs): ST14 (n = 9), ST147 (n = 5), ST231 (n = 2), ST2096 (n = 1), and ST25 (n = 1). Many patients in each ward were infected with the same ST, suggesting a common source of infection. Some patients had recurrent infections with multiple STs circulating in the ward, providing evidence of hospital transmission. β-lactamase genes (bla CTX-M-1 , bla SHV , and bla ampH ) were present in all isolates. bla NDM-1 was present in 15 isolates, bla OXA-1 in 16 isolates, bla TEM-1D in 13 isolates, and bla OXA-48 in 3 isolates. Disruption of mgrB by various insertion sequences was responsible for colistin resistance in 6 isolates. The most common K-type among isolates was K2 (n = 10). One XDR convergent hvKp ST2096 mutation (iuc+ybt+bla OXA-1 +bla OXA-48 ) was associated with prolonged hospitalization. Convergent XDR-hvKp has outbreak potential, warranting effective antimicrobial stewardship and infection control.

Importance: Staphylococcus aureus surgical site infections (SSIs) and bloodstream infections (BSIs) are important complications of surgical procedures for which prevention remains suboptimal. Contemporary data on the incidence of and etiologic factors for these infections are needed to support the development of improved preventive strategies., Objectives: To assess the occurrence of postoperative S aureus SSIs and BSIs and quantify its association with patient-related and contextual factors., Design, Setting, and Participants: This multicenter cohort study assessed surgical patients at 33 hospitals in 10 European countries who were recruited between December 16, 2016, and September 30, 2019 (follow-up through December 30, 2019). Enrolled patients were actively followed up for up to 90 days after surgery to assess the occurrence of S aureus SSIs and BSIs. Data analysis was performed between November 20, 2020, and April 21, 2022. All patients were 18 years or older and had undergone 11 different types of surgical procedures. They were screened for S aureus colonization in the nose, throat, and perineum within 30 days before surgery (source population). Both S aureus carriers and noncarriers were subsequently enrolled in a 2:1 ratio., Exposure: Preoperative S aureus colonization., Main Outcomes and Measures: The main outcome was cumulative incidence of S aureus SSIs and BSIs estimated for the source population, using weighted incidence calculation. The independent association of candidate variables was estimated using multivariable Cox proportional hazards regression models., Results: In total, 5004 patients (median [IQR] age, 66 [56-72] years; 2510 [50.2%] female) were enrolled in the study cohort; 3369 (67.3%) were S aureus carriers. One hundred patients developed S aureus SSIs or BSIs within 90 days after surgery. The weighted cumulative incidence of S aureus SSIs or BSIs was 2.55% (95% CI, 2.05%-3.12%) for carriers and 0.52% (95% CI, 0.22%-0.91%) for noncarriers. Preoperative S aureus colonization (adjusted hazard ratio [AHR], 4.38; 95% CI, 2.19-8.76), having nonremovable implants (AHR, 2.00; 95% CI, 1.15-3.49), undergoing mastectomy (AHR, 5.13; 95% CI, 1.87-14.08) or neurosurgery (AHR, 2.47; 95% CI, 1.09-5.61) (compared with orthopedic surgery), and body mass index (AHR, 1.05; 95% CI, 1.01-1.08 per unit increase) were independently associated with S aureus SSIs and BSIs., Conclusions and Relevance: In this cohort study of surgical patients, S aureus carriage was associated with an increased risk of developing S aureus SSIs and BSIs. Both modifiable and nonmodifiable etiologic factors were associated with this risk and should be addressed in those at increased S aureus SSI and BSI risk.

Background: Four randomized controlled trials have now established that doxycycline post exposure (sex) prophylaxis (PEP) can reduce the incidence of chlamydia and syphilis in men who have sex with men. These studies have concluded that the risk of selecting for antimicrobial resistance is low. We evaluated this risk in vitro and in vivo using a Galleria mellonella infection model., Methods: We evaluated how long it took for doxycycline resistance to emerge during passage on doxycycline containing agar plates in 4 species - Escherichia coli , Klebsiella pneumoniae , Neisseria gonorrhoeae and Neisseria subflava . We then assessed if K. pneumoniae could acquire resistance to doxycycline (and cross resistance to other antimicrobials) during intermittent exposure to doxycycline in a Galleria mellonella model of doxycycline PEP., Results: In our passage experiments, we found that resistance first emerged in K. pneumoniae . By day 7 the K. pneumoniae MIC had increased from 2 mg/L to a median of 96 mg/L (IQR 64-96). Under various simulations of doxycycline PEP in the G. mellonella model, the doxycycline MIC of K. pneumoniae increased from 2 mg/L to 48 mg/L (IQR 48-84). Ceftriaxone and ciprofloxacin MICs increased over ten-fold. Whole genome sequencing revealed acquired mutations in ramR which regulates the expression of the AcrAB-TolC efflux pump., Conclusion: Doxycycline PEP can select for doxycycline, ceftriaxone and ciprofloxacin resistance in K. pneumoniae in a G. mellonella model. The emergent ramR mutations were similar to those seen in circulating strains of K. pneumoniae . These findings suggest that we need to assess the effect of doxycycline PEP on resistance induction on a broader range of bacterial species than has hitherto been the case., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kenyon, Gestels, Vanbaelen, Abdellati, Van Den Bossche, De Baetselier, Xavier and Manoharan-Basil.)

Antibiotic resistance poses a global health threat, but the within-host drivers of resistance remain poorly understood. Pathogen populations are often assumed to be clonal within hosts, and resistance is thought to emerge due to selection for de novo variants. Here we show that mixed strain populations are common in the opportunistic pathogen P. aeruginosa. Crucially, resistance evolves rapidly in patients colonized by multiple strains through selection for pre-existing resistant strains. In contrast, resistance evolves sporadically in patients colonized by single strains due to selection for novel resistance mutations. However, strong trade-offs between resistance and growth rate occur in mixed strain populations, suggesting that within-host diversity can also drive the loss of resistance in the absence of antibiotic treatment. In summary, we show that the within-host diversity of pathogen populations plays a key role in shaping the emergence of resistance in response to treatment., (© 2023. The Author(s).)

Colistin heteroresistance has been identified in several bacterial species, including Escherichia coli and Klebsiella pneumoniae , and may underlie antibiotic therapy failures since it most often goes undetected by conventional antimicrobial susceptibility tests. This study utilizes population analysis profiling (PAP) and time-kill assay for the detection of heteroresistance in K. pneumoniae and for evaluating the association between in vitro regrowth and heteroresistance. The mechanisms of colistin resistance and the ability of combination therapies to suppress resistance selection were also analysed. In total, 3 (18%) of the 16 colistin-susceptible strains (MIC ≤ 2 mg/L) were confirmed to be heteroresistant to colistin by PAP assay. In contrast to the colistin-susceptible control strains, all three heteroresistant strains showed regrowth when exposed to colistin after 24 h following a rapid bactericidal action. Colistin resistance in all the resistant subpopulations was due to the disruption of the mgrB gene by various insertion elements such as IS Kpn14 of the IS 1 family and IS 903B of the IS 5 family. Colistin combined with carbapenems (imipenem, meropenem), aminoglycosides (amikacin, gentamicin) or tigecycline was found to elicit in vitro synergistic effects against these colistin heteroresistant strains. Our experimental results showcase the potential of combination therapies for treatment of K. pneumoniae infections associated with colistin heteroresistance.

Background: The increasing number of infections caused by Escherichia coli resistant to clinically important antibiotics is a global concern for human and animal health. High overall levels of extended-spectrum beta-lactamase (ESBL)-producing and ciprofloxacin-resistant (ciproR) Escherichia coli in livestock are reported in Belgium. This cross-sectional study aimed to genotypically characterize and trace ESBL-and ciproR- E. coli of Belgian food-producing animals., Methods: A total of 798 fecal samples were collected in a stratified-random sampling design from Belgian broilers and sows. Consequently, 77 ESBL- E. coli and 84 ciproR- E. coli were sequenced using Illumina MiSeq. Minimum inhibitory concentration (MIC) for fluoroquinolones and cephalosporins were determined. Molecular in silico typing, resistance and virulence gene determination, and plasmid identification was performed. Scaffolds harboring ESBL or plasmid-mediated quinolone resistance (PMQR) genes were analyzed to detect mobile genetic elements (MGEs) and plasmid origins. Core genome allelic distances were used to determine genetic relationships among isolates., Results: A variety of E. coli sequence types (ST) ( n  = 63), resistance genes and virulence profiles was detected. ST10 was the most frequently encountered ST (8.1%, n  = 13). The pandemic multidrug-resistant clone ST131 was not detected. Most farms harbored more than one ESBL type, with bla CTX-M-1 (41.6% of ESBL- E. coli ) being the most prevalent and bla CTX M-15 ( n  = 3) being the least prevalent. PMQR genes (15.5%, n  = 13) played a limited role in the occurrence of ciproR- E. coli . More importantly, sequential acquisition of mutations in quinolone resistance-determining regions (QRDR) of gyrA and parC led to increasing MICs for fluoroquinolones. GyrA S83L, D87N and ParC S80I mutations were strongly associated with high-level fluoroquinolone resistance. Genetically related isolates identified within the farms or among different farms highlight transmission of resistant E. coli or the presence of a common reservoir. IncI1-I(alpha) replicon type plasmids carried different ESBL genes ( bla CTX-M-1 , bla CTX-M-32 and bla TEM-52C ). In addition, the detection of plasmid replicons with associated insertion sequence (IS) elements and ESBL/PMQR genes in different farms and among several STs (e.g., IncI1-I(alpha)/IncX3) underline that plasmid transmission could be another important contributor to transmission of resistance in these farms., Conclusion: Our findings reveal a multifaceted narrative of transmission pathways. These findings could be relevant in understanding and battling the problem of antibiotic resistance in farms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 De Koster, Ringenier, Xavier, Lammens, De Coninck, De Bruyne, Mensaert, Kluytmans-van den Bergh, Dewulf, Goossens and on behalf of the i-4-1-Health Study Group.)

Background: Phenotypic studies have found high levels of antimicrobial resistance to cephalosporins, macrolides and fluoroquinolones in commensal Neisseria species in the oropharynx of men who have sex with men (MSM) using HIV pre-exposure prophylaxis (PrEP). These species include Neisseria subflava and Neisseria mucosa. This may represent a risk to pathogens like Neisseria gonorrhoeae which tend to take up antibiotic resistance genes (ARGs) from other bacteria. We aimed to explore to what extent the oropharyngeal resistome of MSM using PrEP differed from the general population., Methods: We collected oropharyngeal swabs from 32 individuals of the general population and from 64 MSM using PrEP. Thirty-two MSM had consumed antibiotics in the previous six months, whereas none of the other participants had. Samples underwent shotgun metagenomic sequencing. Sequencing reads were mapped against MEGARes 2.0 to estimate ARG abundance. ARG abundance was compared between groups by zero-inflated negative binomial regression., Findings: ARG abundance was significantly lower in the general population than in MSM (ratio 0.41, 95% CI 0.26-0.65). More specifically, this was the case for fluoroquinolones (0.33, 95% CI 0.15-0.69), macrolides (0.37, 95% CI 0.25-0.56), tetracyclines (0.41, 95% CI 0.25-0.69), and multidrug efflux pumps (0.11, 95% CI 0.03-0.33), but not for beta-lactams (1.38, 95% CI 0.73-2.61). There were no significant differences in ARG abundance between MSM who had used antibiotics and those that had not., Interpretation: The resistome of MSM using PrEP is enriched with ARGs, independent of recent antibiotic use. Stewardship campaigns should aim to reduce antibiotic consumption in populations at high risk for STIs., Competing Interests: Declaration of Competing Interest There are no conflicts of interest to declare., (Copyright © 2023 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

Introduction: International travel has been a major determinant for the introduction of pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) into naïve geographic areas. MRSA clonal complex 239 (CC239) is a highly virulent clone that is predominant in Asia. The objective of this study was to determine the geographic origin of MRSA CC239 isolates recovered from Danish cases with or without a history of international travel during 2004-2016., Materials and Methods: Human MRSA isolates with spa types t030 and t037 ( n  = 60) were obtained from the National Reference Laboratory for Antimicrobial Resistance. For each case, the following data were collected from notification forms: sex, age, isolation year, specimen source (screening swab or clinical sample), infection type, and international travel history. All isolates were whole-genome sequenced, and a comparative genome and phylogenetic analysis was performed., Results: The majority of isolates originated from skin and soft tissue (SST) infections and screening swabs. In 31 out of 60 cases reported international travel to different parts of the world. Fifty-four isolates belonged to CC239, including sequence type 239 (ST239) ( n  = 43), ST241 ( n  = 5), ST4377 ( n  = 2), ST4378 ( n  = 1), ST1465 ( n  = 1), ST343 ( n  = 1), and ST592 ( n  = 1). The majority of the CC239 MRSA isolates (40/54) belonged to well-known geographic clades, including the Asian ( n  = 12), Serbian ( n  = 11), South American ( n  = 2), and Turkish ( n  = 15). Most MRSA ST239 isolates belonging to the highly virulent Asian clade carried sasX and were recovered from individuals who had travelled to Asia, Africa and the Middle East., Conclusion: Our data reveal multiple introductions of MRSA CC239 into Denmark through international travel, which highlights the importance of continued genomic surveillance of MRSA in persons returning from international travel to areas where MRSA is endemic., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Coppens, Xavier, Vlaeminck, Larsen, Lammens, Van Puyvelde, Goossens, Larsen and Malhotra-Kumar.)

Understanding the myriad pathways by which antimicrobial-resistance genes (ARGs) spread across biomes is necessary to counteract the global menace of antimicrobial resistance. We screened 17939 assembled metagenomic samples covering 21 biomes, differing in sequencing quality and depth, unevenly across 46 countries, 6 continents, and 14 years (2005-2019) for clinically crucial ARGs, mobile colistin resistance (mcr), carbapenem resistance (CR), and (extended-spectrum) beta-lactamase (ESBL and BL) genes. These ARGs were most frequent in human gut, oral and skin biomes, followed by anthropogenic (wastewater, bioreactor, compost, food), and natural biomes (freshwater, marine, sediment). Mcr-9 was the most prevalent mcr gene, spatially and temporally; bla OXA-233 and bla TEM-1 were the most prevalent CR and BL/ESBL genes, but bla GES-2 and bla TEM-116 showed the widest distribution. Redundancy analysis and Bayesian analysis showed ARG distribution was non-random and best-explained by potential host genera and biomes, followed by collection year, anthropogenic factors and collection countries. Preferential ARG occurrence, and potential transmission, between characteristically similar biomes indicate strong ecological boundaries. Our results provide a high-resolution global map of ARG distribution and importantly, identify checkpoint biomes wherein interventions aimed at disrupting ARGs dissemination are likely to be most effective in reducing dissemination and in the long term, the ARG global burden., (© 2022. The Author(s).)

Background: Methicillin-resistant Staphylococcus aureus (MRSA), a leading cause of chronic infections, forms prolific biofilms which afford an escape route from antibiotic treatment and host immunity. However, MRSA clones are genetically diverse, and mechanisms underlying biofilm formation remain under-studied. Such studies form the basis for developing targeted therapeutics. Here, we studied the temporal changes in the biofilm transcriptome of three pandemic MRSA clones: USA300, HEMRSA-15, and ST239., Methods: Biofilm formation was assessed using a static model with one representative strain per clone. Total RNA was extracted from biofilm and planktonic cultures after 24, 48, and 72 h of growth, followed by rRNA depletion and sequencing (Illumina Inc., San Diego, CA, United States, NextSeq500, v2, 1 × 75 bp). Differentially expressed gene (DEG) analysis between phenotypes and among early (24 h), intermediate (48 h), and late (72 h) stages of biofilms was performed together with in silico co-expression network construction and compared between clones. To understand the influence of SCC mec and ACME on biofilm formation, isogenic mutants containing deletions of the entire elements or of single genes therein were constructed in USA300., Results: Genes involved in primarily core genome-encoded KEGG pathways (transporters and others) were upregulated in 24-h biofilm culture compared to 24-h planktonic culture. However, the number of affected pathways in the ST239 24 h biofilm ( n = 11) was remarkably lower than that in USA300/EMRSA-15 biofilms (USA300: n = 27, HEMRSA-15: n = 58). The clfA gene, which encodes clumping factor A, was the single common DEG identified across the three clones in 24-h biofilm culture (2.2- to 2.66-fold). In intermediate (48 h) and late (72 h) stages of biofilms, decreased expression of central metabolic and fermentative pathways (glycolysis/gluconeogenesis, fatty acid biosynthesis), indicating a shift to anaerobic conditions, was already evident in USA300 and HEMRSA-15 in 48-h biofilm cultures; ST239 showed a similar profile at 72 h. Last, SCC mec +ACME deletion and opp3D disruption negatively affected USA300 biofilm formation., Conclusion: Our data show striking differences in gene expression during biofilm formation by three of the most important pandemic MRSA clones, USA300, HEMRSA-15, and ST239. The clfA gene was the only significantly upregulated gene across all three strains in 24-h biofilm cultures and exemplifies an important target to disrupt early biofilms. Furthermore, our data indicate a critical role for arginine catabolism pathways in early biofilm formation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Vlaeminck, Lin, Xavier, De Backer, Berkell, De Greve, Hernalsteens, Kumar-Singh, Goossens and Malhotra-Kumar.)

Objectives: To determine the susceptibility profiles and the resistome of Pseudomonas aeruginosa isolates from European ICUs during a prospective cohort study (ASPIRE-ICU)., Methods: 723 isolates from respiratory samples or perianal swabs of 402 patients from 29 sites in 11 countries were studied. MICs of 12 antibiotics were determined by broth microdilution. Horizontally acquired β-lactamases were analysed through phenotypic and genetic assays. The first respiratory isolates from 105 patients providing such samples were analysed through WGS, including the analysis of the resistome and a previously defined genotypic resistance score. Spontaneous mutant frequencies and the genetic basis of hypermutation were assessed., Results: All agents except colistin showed resistance rates above 20%, including ceftolozane/tazobactam and ceftazidime/avibactam. 24.9% of the isolates were XDR, with a wide intercountry variation (0%-62.5%). 13.2% of the isolates were classified as DTR (difficult-to-treat resistance). 21.4% of the isolates produced ESBLs (mostly PER-1) or carbapenemases (mostly NDM-1, VIM-1/2 and GES-5). WGS showed that these determinants were linked to high-risk clones (particularly ST235 and ST654). WGS revealed a wide repertoire of mutation-driven resistance mechanisms, with multiple lineage-specific mutations. The most frequently mutated genes were gyrA, parC, oprD, mexZ, nalD and parS, but only two of the isolates were hypermutable. Finally, a good accuracy of the genotypic score to predict susceptibility (91%-100%) and resistance (94%-100%) was documented., Conclusions: An overall high prevalence of resistance is documented European ICUs, but with a wide intercountry variability determined by the dissemination of XDR high-risk clones, arguing for the need to reinforce infection control measures., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Presence of SARS-CoV-2 was monitored in nasopharyngeal samples from young children aged 6-30 months attending day-care centres (DCCs) in Belgium from May 2020-February 2022. SARS-CoV-2 carriage among DCC children was only detected from November 2021, after emergence of Delta and Omicron variants, in 9 of the 42 DCCs screened. In only one DCC, two children tested positive for SARS-CoV-2 at the same sampling time point, suggesting limited transmission of SARS-CoV-2 in Belgian DCCs among young children during the studied period.

Obesity is a major public health problem. Gut microbiome dysbiosis has been associated with obesity, however, little is known on the effect of the dysbiosis on the microbiotic bio-transformation of xenobiotics. Fecal samples of lean (n = 9) and obese (n = 4) female volunteers were collected and characterized by 16 S rDNA gene sequencing. The microbiotic biotransformation of chlorogenic acid was studied using the collected fecal samples of lean and obese subjects in the colon stage of the gastro-intestinal dialysis model with colon stage (GIDM-Colon). The concentration of anaerobic bacteria was lower for all obese samples in comparison to the samples of the lean volunteers. Differences in gut microbiome composition and bacterial concentration were observed between the two populations. The obese gut microbiome presented a lower metabolic activity in comparison to the lean population. Chlorogenic acid was completely biotransformed after 24 h colonic dialysis in the lean population while it was still present in the obese population. Furthermore, 23 and 13 biotransformation products were identified in the lean and obese population, respectively; 11 unique biotransformation products from the caffeic, feruoylquinic and quinic acid pathways were identified in the lean population. The results confirm that changes in gut microbiota related to obesity are associated with differences in microbiotic biotransformation of xenobiotics and thus possibly influencing the activity, bioavailability and toxicity of orally administered xenobiotics and drugs., (Copyright © 2022. Published by Elsevier B.V.)

The influence of gut microbiota on host immunity is widely studied, and its disturbance has been linked to several immune-mediated disorders. Conversely, whether and how inherently disturbed canonical Th1 (pro-inflammatory) and/or Th2 (anti-inflammatory) immune pathways modify the host microbiome is not sufficiently investigated. Here, we characterized the humoral, cellular, and cytokine immunity, and associated alterations in gut microbiota of naïve wild-type mice (C57BL/6 and BALB/c), and mice with deficiencies in Th2 responses (IL-4Rα and IL-33 knockout mice) or in both Th1 and Th2 responses (NOD scid gamma, NSG mice). A global analysis by de novo clustering of 16S rRNA profiles of the gut microbiota independently grouped wild-type immunocompetent (C57BL/6 and BALB/c), Th2-deficient (IL-4Rα -/- and IL-33 -/- ), and severely immunodeficient (NSG) mice; where wild-type mice, but not Th2 or severely immunodeficient mice, were enriched in gut bacteria that produce short-chain fatty acids. These include members of phyla Firmicutes, Verrucomicrobia, and Bacteroidetes such as Lactobacillus spp., Akkermansia muciniphila , and Odoribacter spp. Further comparison of the two naïve wild-type mouse strains showed higher microbial diversity (Shannon), primarily linked to higher richness (Chao1), as well as a distinct difference in microbial composition (weighted UniFrac) in BALB/c mice compared to C57BL/6. T-cell and blood cytokine analyses demonstrated a Th1-polarization in naïve adaptive immunity in C57BL/6 animals compared to BALB/c mice, and an expected Th2 deficient cellular response in IL-4Rα -/- and IL-33 -/- mice compared to its genetic background BALB/c strain. Together, these data suggest that alterations in the Th1/Th2 balance or a complete ablation of Th1/Th2 responses can lead to major alterations in gut microbiota composition and function. Given the similarities between the human and mouse immune systems and gut microbiota, our finding that immune status is a strong driver of gut microbiota composition has important consequences for human immunodeficiency studies., Competing Interests: ESC is employed by AstraZeneca. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Van averbeke, Berkell, Mysara, Rodriguez-Ruiz, Xavier, De Winter, Jongers, Jairam, Hotterbeekx, Goossens, Cohen, Malhotra-Kumar and Kumar-Singh.)

Background: Klebsiella pneumoniae ST101 is an emerging high-risk clone which exhibits extensive drug resistance. Bacterial strains residing in multiple hosts show unique signatures related to host adaptation. In this study, we assess the genetic relationship of K. pneumoniae ST101 isolated from hospital samples, the environment, community, and livestock using whole genome sequencing (WGS)., Materials and Methods: We selected ten K. pneumoniae ST101 strains from hospitalized patients in Italy ( n = 3) (2014) and Spain ( n = 5) (2015-2016) as well as Belgian livestock animals ( n = 2) (2017-2018). WGS was performed with 2 × 250 bp paired-end sequencing (Nextera XT) sample preparation kit and MiSeq (Illumina Inc.). Long-read sequencing (Pacbio Sequel I) was used to sequence the two livestock strains and three Italian hospital-associated strains. Furthermore, a public ST101 sequence collection of 586 strains (566 hospital-associated strains, 12 environmental strains, six strains from healthy individuals, one food-associated strain and one pig strain) was obtained. BacPipe and Kleborate were used to conduct genome analysis. ISFinder was used to find IS elements, and PHASTER was utilized to identify prophages. A phylogenetic tree was constructed to illustrate genetic relatedness., Results: Hospital-associated K. pneumoniae ST101 showed higher resistance scores than non-clinical isolates from healthy individuals, the environment, food and livestock (1.85 ± 0.72 in hospital-associated isolates vs. 1.14 ± 1.13 in non-clinical isolates, p < 0.01). Importantly, the lack of integrative conjugative elements ICE Kp bearing iron-scavenging yersiniabactin siderophores ( ybt ) in livestock-associated strains suggests a lower pathogenicity potential than hospital-associated strains. Mobile genetic elements (MGE) appear to be an important source of diversity in K. pneumoniae ST101 strains from different origins, with a highly stable genome and few recombination events outside the prophage-containing regions. Core genome MLST based analysis revealed a distinct genetic clustering between human and livestock-associated isolates., Conclusion: The study of K. pneumoniae ST101 hospital-associated and strains from healthy individuals and animals revealed a genetic diversity between these two groups, allowing us to identify the presence of yersiniabactin siderophores in hospital-associated isolates. Resistance and virulence levels in livestock-associated strains were considerably lower than hospital-associated strains, implying that the public health risk remains low. The introduction of an ICE Kp into animal strains, on the other hand, might pose a public threat over time., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 De Koster, Rodriguez Ruiz, Rajakani, Lammens, Glupczynski, Goossens and Xavier.)

Objectives: To evaluate a testing algorithm for the rapid identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that includes the use of PCR-based targeted single nucleotide polymorphism (SNP) detection assays preceded by a multiplex PCR sensitive to S-Gene Target Failure (SGTF)., Methods: PCR SNP assays targeting SARS-CoV-2 S-gene mutations ΔH69-V70, L452R, E484K, N501Y, H655Y and P681R using melting curve analysis were performed on 567 samples in which SARS-CoV-2 viral RNA was detected by a multiplex PCR. Viral whole-genome sequencing (WGS) was performed to confirm the presence of SNPs and to identify the Pangolin lineage. Additionally, 1133 SARS-CoV-2 positive samples with SGTF were further assessed by WGS to determine the presence of ΔH69-V70., Results: The N501Y-specific assay (n = 567) had an overall percentage agreement (OPA) of 98.5%. The ΔH69-V70-specific (n = 178) and E484K-specific (n = 401) assays had OPA of 96.6% and 99.7%, respectively. Assessment of H655Y (n = 139) yielded a 100.0% concordance when applied in the proposed algorithm. The L452R-specific (n = 67) and P681R-specific (n = 62) assays had an OPA of 98.2% and 98.1%, respectively. The proposed algorithm identified six variants of concern/interest (VOC/VOI)-Alpha (n = 149), Beta (n = 65), Gamma (n = 86), Delta (n = 49), Eta (n = 6), Kappa (n = 6)-and 205 non-VOC/VOI strains-including the variants under monitoring B.1.214.2 (n = 43) and B.1.1.318 (n = 18) and Epsilon (n = 1). An excellent concordance was observed for the identification of all SARS-CoV-2 lineages evaluated., Conclusions: We present a flexible testing algorithm for the rapid detection of current and emerging SARS-CoV-2 VOC/VOIs, which can be easily adapted based on the local endemicity of specific variants., (Copyright © 2021 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Objectives: Chlorhexidine digluconate (chlorhexidine) and Listerine ® mouthwashes are being promoted as alternative treatment options to prevent the emergence of antimicrobial resistance in Neisseria gonorrhoeae . We performed in vitro challenge experiments to assess induction and evolution of resistance to these two mouthwashes and potential cross-resistance to other antimicrobials. Methods: A customized morbidostat was used to subject N. gonorrhoeae reference strain WHO-F to dynamically sustained Listerine ® or chlorhexidine pressure for 18 days and 40 days, respectively. Cultures were sampled twice a week and minimal inhibitory concentrations (MICs) of Listerine ® , chlorhexidine, ceftriaxone, ciprofloxacin, cefixime and azithromycin were determined using the agar dilution method. Isolates with an increased MIC for Listerine ® or chlorhexidine were subjected to whole genome sequencing to track the evolution of resistance. Results: We were unable to increase MICs for Listerine ® . Three out of five cultures developed a 10-fold increase in chlorhexidine MIC within 40 days compared to baseline (from 2 to 20 mg/L). Increases in chlorhexidine MIC were positively associated with increases in the MICs of azithromycin and ciprofloxacin. Low-to-higher-level chlorhexidine resistance (2-20 mg/L) was associated with mutations in NorM. Higher-level resistance (20 mg/L) was temporally associated with mutations upstream of the MtrCDE efflux pump repressor ( mtrR ) and the mlaA gene, part of the maintenance of lipid asymmetry (Mla) system. Conclusion: Exposure to sub-lethal chlorhexidine concentrations may not only enhance resistance to chlorhexidine itself but also cross-resistance to other antibiotics in N. gonorrhoeae . This raises concern regarding the widespread use of chlorhexidine as an oral antiseptic, for example in the field of dentistry., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Laumen, Van Dijck, Manoharan-Basil, Abdellati, De Baetselier, Cuylaerts, De Block, Van den Bossche, Xavier, Malhotra-Kumar and Kenyon.)

Objectives: We performed a One Health surveillance in Hanoi-a region with a high-density human population and livestock production, and a recognized hotspot of animal-associated antimicrobial resistance (AMR)-to study the contribution of bla CTX-M -carrying Escherichia coli and plasmids from food-animal sources in causing human community-acquired urinary tract infections (CA-UTIs)., Methods: During 2014-2015, 9090 samples were collected from CA-UTI patients (urine, n = 8564), pigs/chickens from farms and slaughterhouses (faeces, carcasses, n = 448), and from the slaughterhouse environment (surface swabs, water, n = 78). E. coli was identified in 2084 samples. Extended-spectrum β-lactamase (ESBL) production was confirmed in 235 and bla CTX-M in 198 strains by PCR with short-read plasmid sequencing. Fourteen strains were long-read sequenced to enable plasmid reconstruction., Results: The majority of the ESBL-producing E. coli strains harboured bla CTX-M (n = 198/235, 84%). High clonal diversity (48 sequence types, STs) and distinct, dominant STs in human sources (ST1193, n = 38/137; ST131, n = 30/137) and non-human sources (ST155, n = 25/61) indicated lack of clonal transmission between habitats. Eight bla CTX-M variants were identified; five were present in at least two sample sources. Human and food-animal strains did not show similar plasmids carrying shared bla CTX-M genes. However, IS6 elements flanking ISEcp1-bla CTX-M -orf477/IS903B structures were common across habitats., Conclusions: In this study, animal-associated bla CTX-M E. coli strains or bla CTX-M plasmids were not direct sources of CA-UTIs or ESBL resistance in humans, respectively, suggesting evolutionary bottlenecks to their adaptation to a new host species. Presence of common IS6 elements flanking bla CTX-M variants in different plasmid backbones, however, highlighted the potential of these transposable elements for AMR transmission either within or across habitats., (Copyright © 2021 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

We identified a novel van gene cluster in a clinical Enterococcus faecium isolate with vancomycin minimum inhibitory concentration (MIC) of 4 µg/mL. The ligase gene, vanP , was part of a van operon cluster of 4,589 bp on a putative novel integrative conjugative element located in a ca 98 kb genomic region presumed to be acquired by horizontal gene transfer from Clostridium scidens and Roseburia sp. 499. Screening for van genes in E. faecium strains with borderline susceptibility to vancomycin is important.

The antibiotic growth promoters (AGPs) flavophospholipol and virginiamycin have been widely used for decades in food animal production. AGP activity is believed to be partly modulated by gut microbial composition although exact AGP-induced changes remain unclear. In a controlled intervention study, we studied the effect of flavophospholipol and virginiamycin on the broiler chicken ileal microbiota spanning from birth to 39 days. Using 16S rRNA gene profiling and prediction of metabolic activity, we show that both AGPs result in dynamic microbial shifts that potentially increase anti-inflammatory mechanisms and bioavailability of several essential nutrients by decreasing degradation (flavophospholipol) or increasing biosynthesis (virginiamycin). Further, virginiamycin-supplemented broilers showed increased colonization with potentially pathogenic bacteria, Clostridium perfringens, Campylobacter, and Escherichia/ Shigella spp. Overall, we show that both AGPs induce microbial changes potentially beneficial for growth. However, the increase in (foodborne) pathogens shown here with virginiamycin use could impact not only broiler mortality but also human health. IMPORTANCE Antibiotic growth promoters (AGPs) are commonly used within poultry farming to increase muscle growth. Microbial composition in the gut is known to be influenced by AGP use although exact AGP-induced changes remain unclear. Utilizing 16S rRNA gene profiling, this study provides a first head-to-head comparison of the effect of the two most commonly used AGPs, flavophospholipol and virginiamycin, on the broiler chicken ileum microbiota over time. We found that supplementation with both AGPs altered ileal microbial composition, thereby increasing potential bioavailability of essential nutrients and weight gain. Flavophospholipol showed a slight benefit over virginiamycin as the latter resulted in more extensive microbial perturbations including increased colonization by enteropathogens, which could impact broiler mortality.

Introduction: Pseudomonas aeruginosa is a common cause of ventilator-associated pneumonia (VAP). Rapid and accurate detection of lower respiratory tract colonization and/or infection with P. aeruginosa may advise targeted preventive (antibody-based) strategies and antibiotic therapy. To investigate this, we compared semi-quantitative culture results from 80 endotracheal aspirates (ETA) collected from mechanically-ventilated patients, to two culture and two non-culture-based methods for detection of P. aeruginosa., Methods: P. aeruginosa-positive (n = 40) and -negative (n = 40) ETAs from mechanically ventilated patients analyzed initally by (i) routine semi-quantitative culture, were further analyzed with (ii) quantitative culture on chromogenic ChromID P. aeruginosa and blood agar; (iii) enrichment in brain heart infusion broth followed by plating on blood agar and ChromID P. aeruginosa; (iv) O-antigen acetylase gene-based TaqMan qPCR; and (v) GeneXpert PA PCR assay., Results: Of the 80 ETA samples included, one sample that was negative for P. aeruginosa by semi-quantitative culture was found to be positive by the other four methods, and was included in an "extended" gold standard panel. Based on this extended gold standard, both semi-quantitative culture and the GeneXpert PA assay showed 97.6% sensitivity and 100% specificity. The quantitative culture, enrichment culture and O-antigen acetylase gene-based TaqMan qPCR had a sensitivity of 97.6%, 89.5%, 92.7%, and a specificity of 97.4%, 100%, and 71.1%, respectively., Conclusion: This first evaluation of the GeneXpert PA assay with ETA samples found it to be as sensitive and specific as the routine, hospital-based semi-quantitative culture method. Additionally, the GeneXpert PA assay is easy to perform (hands-on time ≈ 5 min) and rapid (≈ 55 min assay time). The combination of the high sensitivity and high specificity together with the rapid acquisition of results makes the GeneXpert PA assay a highly recommended screening technique. Where this equipment is not available, semi-quantitative culture remains the most sensitive of the culture methods evaluated here for P. aeruginosa detection in ETA samples., (© 2021. The Author(s).)

Background: The prevalence of azithromycin resistance in Neisseria gonorrhoeae is increasing in numerous populations worldwide., Objectives: To characterize the genetic pathways leading to high-level azithromycin resistance., Methods: A customized morbidostat was used to subject two N. gonorrhoeae reference strains (WHO-F and WHO-X) to dynamically sustained azithromycin pressure. We tracked stepwise evolution of resistance by whole genome sequencing., Results: Within 26 days, all cultures evolved high-level azithromycin resistance. Typically, the first step towards resistance was found in transitory mutations in genes rplD, rplV and rpmH (encoding the ribosomal proteins L4, L22 and L34 respectively), followed by mutations in the MtrCDE-encoded efflux pump and the 23S rRNA gene. Low- to high-level resistance was associated with mutations in the ribosomal proteins and MtrCDE efflux pump. However, high-level resistance was consistently associated with mutations in the 23S ribosomal RNA, mainly the well-known A2059G and C2611T mutations, but also at position A2058G., Conclusions: This study enabled us to track previously reported mutations and identify novel mutations in ribosomal proteins (L4, L22 and L34) that may play a role in the genesis of azithromycin resistance in N. gonorrhoeae., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Multi-drug-resistant Acinetobacter baumannii isolates are key pathogens that contribute to the global burden of antimicrobial resistance. This study aimed to investigate the phenotypic and molecular characteristics of carbapenem-resistant A. baumannii (CRAB) isolates from the EURECA clinical trial. In total, 228 CRAB clinical strains were recovered from 29 sites in 10 European countries participating in the EURECA study between May 2016 and November 2018. All strains were reconfirmed centrally for identification and antimicrobial susceptibility testing, and were then subjected to DNA isolation and whole-genome sequencing (WGS), with analysis performed using BacPipe v.1.2.6. K and O typing was performed using KAPTIVE. Overall, 226 (99.1%) strains were confirmed as CRAB isolates. The minimum inhibitory concentration (MIC 90 ) results of imipenem and meropenem were >16 mg/L. WGS showed that the isolates mainly harboured bla OXA-23 (n=153, 67.7%) or bla OXA-72 (n=70, 30.1%). Four bla OXA-72 isolates from Serbia co-harboured bla NDM-1. An IS5 transposase family element, ISAba31, was found upstream of the bla OXA-72 gene harboured on a small (~10-kb) pSE41030-EUR plasmid. The majority of isolates (n=178, 79.1%) belonged to international clone II. Strains belonging to the same sequence type but isolated in different countries or within the same country could be delineated in different clusters by core-genome multi-locus sequence typing (MLST). Whole-genome/core-genome MLST showed high diversity among the isolates, and the most common sequence type was ST2 (n=153, 67.7%). The EURECA A. baumannii strain collection represents a unique, diverse repository of carbapenem-resistant isolates that adds to the existing knowledge of A. baumannii epidemiology and resistance genes harboured by these strains., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

It is well established that antibiotic treatment selects for resistance, but the dynamics of this process during infections are poorly understood. Here we map the responses of Pseudomonas aeruginosa to treatment in high definition during a lung infection of a single ICU patient. Host immunity and antibiotic therapy with meropenem suppressed P. aeruginosa, but a second wave of infection emerged due to the growth of oprD and wbpM meropenem resistant mutants that evolved in situ. Selection then led to a loss of resistance by decreasing the prevalence of low fitness oprD mutants, increasing the frequency of high fitness mutants lacking the MexAB-OprM efflux pump, and decreasing the copy number of a multidrug resistance plasmid. Ultimately, host immunity suppressed wbpM mutants with high meropenem resistance and fitness. Our study highlights how natural selection and host immunity interact to drive both the rapid rise, and fall, of resistance during infection.

Antibiotic-induced modulation of the intestinal microbiota can lead to Clostridioides difficile infection (CDI), which is associated with considerable morbidity, mortality, and healthcare-costs globally. Therefore, identification of markers predictive of CDI could substantially contribute to guiding therapy and decreasing the infection burden. Here, we analyze the intestinal microbiota of hospitalized patients at increased CDI risk in a prospective, 90-day cohort-study before and after antibiotic treatment and at diarrhea onset. We show that patients developing CDI already exhibit significantly lower diversity before antibiotic treatment and a distinct microbiota enriched in Enterococcus and depleted of Ruminococcus, Blautia, Prevotella and Bifidobacterium compared to non-CDI patients. We find that antibiotic treatment-induced dysbiosis is class-specific with beta-lactams further increasing enterococcal abundance. Our findings, validated in an independent prospective patient cohort developing CDI, can be exploited to enrich for high-risk patients in prospective clinical trials, and to develop predictive microbiota-based diagnostics for management of patients at risk for CDI.

An amorphous and mesoporous silica/titania (SiTi) material was synthesized by sol-gel method and its surface was modified with gold nanoparticles (AuNP) previously stabilized in a chitosan solution. The presence of small AuNP, with diameter lower than 10 nm was confirmed by transmission electron microscopy (TEM) and UV-Vis spectroscopy. Carbon paste electrodes were prepared to test the electrochemical properties by using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in [Fe(CN) 6 ] 3-/4- solution probe whereby the material silica-titania/gold nanoparticles (SiTi/AuNP) showed a huge improvement in the redox peak current and low charge transfer resistance. This electrode presented a good response for both norepinephrine and dopamine by means of square wave voltammetry (SWV) measurements; great sensitivity for both analytes, in an extensive linear range, was obtained. The limits of detection were 0.35 μmol L -1 and 0.57 μmol L -1 for norepinephrine and dopamine, respectively. Additionally, this electrode showed high selectivity for both analytes and it was applied in the simultaneous determination of norepinephrine and dopamine. The sensor was also tested in simulated biological fluids presenting a good recovery. The SWV electrochemical response of norepinephrine was also investigated in the presence of possible interferers such as uric acid, ascorbic acid and glucose and there was no significant interference. The prepared electrode also exhibits good reproducibility for norepinephrine detection, with relative standard deviation of 5.19%., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objectives: WGS-based antimicrobial susceptibility testing (AST) is as reliable as phenotypic AST for several antimicrobial/bacterial species combinations. However, routine use of WGS-based AST is hindered by the need for bioinformatics skills and knowledge of antimicrobial resistance (AMR) determinants to operate the vast majority of tools developed to date. By leveraging on ResFinder and PointFinder, two freely accessible tools that can also assist users without bioinformatics skills, we aimed at increasing their speed and providing an easily interpretable antibiogram as output., Methods: The ResFinder code was re-written to process raw reads and use Kmer-based alignment. The existing ResFinder and PointFinder databases were revised and expanded. Additional databases were developed including a genotype-to-phenotype key associating each AMR determinant with a phenotype at the antimicrobial compound level, and species-specific panels for in silico antibiograms. ResFinder 4.0 was validated using Escherichia coli (n = 584), Salmonella spp. (n = 1081), Campylobacter jejuni (n = 239), Enterococcus faecium (n = 106), Enterococcus faecalis (n = 50) and Staphylococcus aureus (n = 163) exhibiting different AST profiles, and from different human and animal sources and geographical origins., Results: Genotype-phenotype concordance was ≥95% for 46/51 and 25/32 of the antimicrobial/species combinations evaluated for Gram-negative and Gram-positive bacteria, respectively. When genotype-phenotype concordance was <95%, discrepancies were mainly linked to criteria for interpretation of phenotypic tests and suboptimal sequence quality, and not to ResFinder 4.0 performance., Conclusions: WGS-based AST using ResFinder 4.0 provides in silico antibiograms as reliable as those obtained by phenotypic AST at least for the bacterial species/antimicrobial agents of major public health relevance considered., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

Escherichia coli is the leading cause of urinary tract infection, one of the most common bacterial infections in humans. Despite this, a genomic perspective is lacking regarding the phylogenetic distribution of isolates associated with different clinical syndromes. Here, we present a large-scale phylogenomic analysis of a spatiotemporally and clinically diverse set of 907 E. coli isolates, including 722 uropathogenic E. coli (UPEC) isolates. A genome-wide association approach identifies the (P-fimbriae-encoding) papGII locus as the key feature distinguishing invasive UPEC, defined as isolates associated with severe UTI, i.e., kidney infection (pyelonephritis) or urinary-source bacteremia, from non-invasive UPEC, defined as isolates associated with asymptomatic bacteriuria or bladder infection (cystitis). Within the E. coli population, distinct invasive UPEC lineages emerged through repeated horizontal acquisition of diverse papGII-containing pathogenicity islands. Our findings elucidate the molecular determinants of severe UTI and have implications for the early detection of this pathogen.

This study was performed to investigate the genotypic causes of colistin resistance in 18 colistin-resistant Klebsiella pneumoniae ( n = 13), Escherichia coli ( n = 3) and Pseudomonas aeruginosa ( n = 2) isolates from patients at the Hamad General Hospital, Qatar. MIC testing for colistin was performed using Phoenix (BD Biosciences, Heidelberg, Germany) and then verified with SensiTest Colistin (Liofilchem, Zona Ind. le, Italy). Strains determined to be resistant (MIC > 4-16 μg/mL) were then whole-genome sequenced (MiSeq, Illumina, Inc.). Sequences were processed and analysed using BacPipe v1.2.6, a bacterial whole genome sequencing analysis pipeline. Known chromosomal modifications were determined using CLC Genomics Workbench v.9.5.3 (CLCbio, Denmark). Two K. pneumoniae isolates (KPN-15 and KPN-19) harboured mcr-8.1 on the IncFII(K) plasmids, pqKPN-15 and pqKPN-19, and belonged to ST383 and ST716, respectively. One E. coli isolate harboured mcr-1.1 on the IncI2 plasmid pEC-12. The other 15 isolates harboured known chromosomal mutations linked to colistin resistance in the PhoPQ two-component system. Also, three K. pneumoniae strains (KPN-9, KPN-10 and KPN-15) showed disruptions due to IS elements in mgrB . To our knowledge, this marks the first description of mcr-8.1 in K. pneumoniae of human origin in Qatar. Currently, more research is necessary to trace the source of mcr-8.1 and its variants in humans in this region., (Copyright © 2020 Eltai, Kelly, Al-Mana, Ibrahim, Yassine, Al Thani, Al Maslmani, Lammens, Xavier and Malhotra-Kumar.)

Despite rapid advances in whole genome sequencing (WGS) technologies, their integration into routine microbiological diagnostics has been hampered by the lack of standardized downstream bioinformatics analysis. We developed a comprehensive and computationally low-resource bioinformatics pipeline (BacPipe) enabling direct analyses of bacterial whole-genome sequences (raw reads or contigs) obtained from second- or third-generation sequencing technologies. A graphical user interface was developed to visualize real-time progression of the analysis. The scalability and speed of BacPipe in handling large datasets was demonstrated using 4,139 Illumina paired-end sequence files of publicly available bacterial genomes (2.9-5.4 Mb) from the European Nucleotide Archive. BacPipe is integrated in EBI-SELECTA, a project-specific portal (H2020-COMPARE), and is available as an independent docker image that can be used across Windows- and Unix-based systems. BacPipe offers a fully automated "one-stop" bacterial WGS analysis pipeline to overcome the major hurdle of WGS data analysis in hospitals and public-health and for infection control monitoring., Competing Interests: Declaration of Interests None declared., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Recent data clearly show that the gut microbiota plays a significant role in the biotransformation of many endogenous molecules and xenobiotics, leading to a potential influence of this microbiotic metabolism on activation, inactivation and possible toxicity of these compounds. To study the colonic biotransformation of xenobiotics by the gut microbiome, in vitro models are often used as they allow dynamic and multiple sampling overtime. However, the pre-analytical phase should be carefully optimized to enable biotransformation product identification representative for the in vivo situation. During this study, chlorogenic acid was used as a model compound to optimize a ready-to-use gut microbiome biotransformation platform using an in vitro gastrointestinal dialysis-model with colon phase together with an instrumental platform using liquid chromatography coupled to high resolution mass spectrometry (LC-QTOF-MS). Identification of the biotransformation products of chlorogenic acid was performed using complementary suspect and non-targeted data analysis approaches (MZmine + R and MPP workflow). Concerning the pre-analytical phase, (i) the influence of different incubation media (Wilkins-Chalgren Anaerobic Broth (WCB) and (versus) phosphate buffer) and different incubation times (prior to implementation in the colonic stage of the dialysis model) on fecal bacterial composition and concentration were investigated and (ii) four different sample preparation methods (centrifugation, extraction, sonication and freeze-drying) were evaluated targeting colonic biotransformation of chlorogenic acid. WCB as incubation medium showed to introduce substantial variation in the bacterial composition of the fecal samples, while the sterile phosphate buffer guaranteed a closer resemblance to the in vivo composition. Furthermore, incubation during 24 h in sterile phosphate buffer as medium showed no significant increase or decrease in anaerobic bacterial concentration, concluding that incubation prior to the colonic stage is not needed. Concerning sample preparation, centrifugation, sonication and extraction gave similar results, while freeze-drying appeared to be inferior. The extraction method was selected as an optimal sample preparation method given the quick execution together with a good instrumental sensitivity. This study optimized a ready-to-use platform to investigate colonic biotransformation of xenobiotics by using chlorogenic acid as a model compound. This platform can be used in the future to study differences in colonic biotransformation of xenobiotics using fecal samples of different patient groups., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A 60-year-old woman received meropenem/colistin treatment for bilateral pneumonia caused by a ST15 carbapenemase producing Klebsiella pneumoniae. The patient recovered but re-infection with the same (ST15), but now colistin-resistant K. pneumoniae, occurred. The molecular mechanism of the emerged colistin resistance was identified as mgrB gene modification by insertion element (IS) IS903B., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Lichens, encompassing 20,000 known species, are symbioses between specialized fungi (mycobionts), mostly ascomycetes, and unicellular green algae or cyanobacteria (photobionts). Here we describe the first parallel genomic analysis of the mycobiont Cladonia grayi and of its green algal photobiont Asterochloris glomerata. We focus on genes/predicted proteins of potential symbiotic significance, sought by surveying proteins differentially activated during early stages of mycobiont and photobiont interaction in coculture, expanded or contracted protein families, and proteins with differential rates of evolution., Results: A) In coculture, the fungus upregulated small secreted proteins, membrane transport proteins, signal transduction components, extracellular hydrolases and, notably, a ribitol transporter and an ammonium transporter, and the alga activated DNA metabolism, signal transduction, and expression of flagellar components. B) Expanded fungal protein families include heterokaryon incompatibility proteins, polyketide synthases, and a unique set of G-protein α subunit paralogs. Expanded algal protein families include carbohydrate active enzymes and a specific subclass of cytoplasmic carbonic anhydrases. The alga also appears to have acquired by horizontal gene transfer from prokaryotes novel archaeal ATPases and Desiccation-Related Proteins. Expanded in both symbionts are signal transduction components, ankyrin domain proteins and transcription factors involved in chromatin remodeling and stress responses. The fungal transportome is contracted, as are algal nitrate assimilation genes. C) In the mycobiont, slow-evolving proteins were enriched for components involved in protein translation, translocation and sorting., Conclusions: The surveyed genes affect stress resistance, signaling, genome reprogramming, nutritional and structural interactions. The alga carries many genes likely transferred horizontally through viruses, yet we found no evidence of inter-symbiont gene transfer. The presence in the photobiont of meiosis-specific genes supports the notion that sexual reproduction occurs in Asterochloris while they are free-living, a phenomenon with implications for the adaptability of lichens and the persistent autonomy of the symbionts. The diversity of the genes affecting the symbiosis suggests that lichens evolved by accretion of many scattered regulatory and structural changes rather than through introduction of a few key innovations. This predicts that paths to lichenization were variable in different phyla, which is consistent with the emerging consensus that ascolichens could have had a few independent origins.

During the last two decades, there has been a public health concern of severe invasive infections caused by Group A Streptococcus (GAS) of the emm1 genotype. This study investigated the dynamics of emm1 GAS during 1994-2013 in Belgium. emm1 GAS isolated from blood, tissue, and wounds of patients with invasive infections (n = 23, S1-S23), and from patients with uncomplicated pharyngitis (n = 15, NS1-NS15) were subjected to whole-genome mapping (WGM; kpn) (Opgen). Whole-genome sequencing was performed on 25 strains (WGS; S1-S23 and NS6-NS7) (Illumina Inc.). Belgian GAS belonged to the M1T1 clone typified by the 36-kb chromosomal region encoding extracellular toxins, NAD+-glycohydrolase and streptolysin O. Strains from 1994-1999 clustered together with published strains (MGAS5005 and M1476). From 2001 onward, invasive GAS showed higher genomic divergence in the accessory genome and harbored on average 7% prophage content. Low evolutionary rate (2.49E-008; P > 0.05) was observed in this study, indicating a highly stable genome. The studied invasive and pharyngitis isolates were no genetically distinct populations based on the WGM and core genome phylogeny analyses. Two copies of the speJ superantigen were present in the 1999 and 2010 study strains (n = 3), one being chromosomal and one being truncated and associated with phage remnants. This study showed that emm1 GAS in Belgium, compared with Canada and UK M1 strains, were highly conserved by harboring a remarkable genome stability over a 19-year period with variations observed in the accessory genome., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Objectives: sasX is a colonization-virulence factor that potentially underlies the success of methicillin-resistant Staphylococcus aureus (MRSA) sequence type (ST) 239 in Asia. We aimed to study the spread of sasX and the population structure of MRSA in two geographically distinct regions, Europe and India., Methods: MRSA (n = 128) from screening and clinical samples from tertiary care patients in 12 European countries (n = 119), and from India (n = 9) were multilocus-sequence-typed and screened for sasX and its carrier φSPβ-like prophage by PCR. Whole genome sequencing was performed on sasX-harbouring strains from India (n = 5) and Europe (n = 2) and on a selection non-harbouring sasX (n = 36) (2 × 150 bp, Miseq, Illumina). Reads were mapped to the ST239 reference strain, TW20., Results: sasX and sesI, a sasX homologue native to Staphylococcus epidermidis, were detected in five of the nine Indian MRSA belonging to ST239 and to other sequence types of CC8. In contrast, sasX was restricted to two ST239 strains in Europe. The intact sasX and sesI carrier φSPβ-like prophages were ∼80 kb and ∼118 kb, and integrated in the yeeE gene. We identified 'novel' ST239 clades in India and Serbia that showed significant differences in base substitution frequencies (0.130 and 0.007, respectively, Tamura-Nei model) (p <0.05)., Conclusions: Our data highlight dissemination of sasX to non-ST239 sequence types of CC8. Detection of the S. epidermidis-associated sesI in MRSA provided unquestionable evidence of transfer between the two species. Stark differences in evolutionary rates between the novel Indian and Serbian ST239 clades identified here might be due to inherent clade characteristics or influenced by other environmental differences such as antibiotic use., (Copyright © 2018 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Horizontal transfer of plasmid-mediated antibiotic resistance has rarely been documented in vivo. Utilizing long-read (Oxford Nanopore) and short-read (Illumina) sequencing, we confirmed that a gut-colonizing Escherichia coli and a hypervirulent Klebsiella pneumoniae ST23, isolated from a surgical site culture of a patient receiving cefuroxime therapy, harboured a 100% identical bla CTX-M-14 -harbouring plasmid, indicative of a potential transfer in vivo., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Data sharing enables research communities to exchange findings and build upon the knowledge that arises from their discoveries. Areas of public and animal health as well as food safety would benefit from rapid data sharing when it comes to emergencies. However, ethical, regulatory and institutional challenges, as well as lack of suitable platforms which provide an infrastructure for data sharing in structured formats, often lead to data not being shared or at most shared in form of supplementary materials in journal publications. Here, we describe an informatics platform that includes workflows for structured data storage, managing and pre-publication sharing of pathogen sequencing data and its analysis interpretations with relevant stakeholders., (© The Author(s) 2019. Published by Oxford University Press.)

The initial report of the mcr-1 (mobile colistin resistance) gene has led to many reports of mcr-1 variants and other mcr genes from different bacterial species originating from human, animal and environmental samples in different geographical locations. Resistance gene nomenclature is complex and unfortunately problems such as different names being used for the same gene/protein or the same name being used for different genes/proteins are not uncommon. Registries exist for some families, such as bla (β-lactamase) genes, but there is as yet no agreed nomenclature scheme for mcr genes. The National Center for Biotechnology Information (NCBI) recently took over assigning bla allele numbers from the longstanding Lahey β-lactamase website and has agreed to do the same for mcr genes. Here, we propose a nomenclature scheme that we hope will be acceptable to researchers in this area and that will reduce future confusion.

Susceptibility testing for colistin remains challenging primarily due to its inherent properties. We evaluated colistin stability in agar and reproducibility of colistin MICs obtained by agar dilution, broth macro- and micro-dilution and MIC gradient strips on 3-7 iterations of each method using clinical Klebsiella pneumoniae (susceptible-CS, and resistant-CR, n = 2 each), mcr-harboring Escherichia coli (n = 2), and reference strains E. coli ATCC25922 and Pseudomonas aeruginosa ATCC27853. MICs for reference strains were not in the given range using Etest and broth microdilution (ATCC25922, 0.125 and 4 μg/ml, respectively). MICs of CR-1 and CR-2, and of the mcr-harboring E. coli showed high concordance between agar and broth dilution varying up to one 2-fold dilution. However, remarkable variations were observed on broth dilution with CS-1 and CS-2 (MIC range 0.25-32 and 0.5-64 μg/ml, respectively); whereas for agar dilution the MIC for both CS strains was 0.5 μg/ml in all the runs. MICs obtained by MIC gradient strips were lower than those obtained by dilution methods (1-2 dilutions for CS and mcr strains, and up to five dilutions for CR strains). To confirm uniform distribution of colistin in agar, a single strain was spotted in five different regions of the same plate. All spots showed concordant growth with maximum one dilution difference. No effect on MIC was found due to storage of colistin-containing agar plates for 7 days at 4 °C. In our hands, agar dilution was superior in terms of reproducibility and robustness, compared to broth dilution methods, for colistin MIC determination.