土井 洋平

BACKGROUND: Stenotrophomonas maltophilia is a carbapenem-resistant Gram-negative pathogen increasingly responsible for difficult-to-treat nosocomial infections. OBJECTIVES: To describe the contemporary clinical characteristics and genome epidemiology of patients colonized or infected by S. maltophilia in a multicentre, prospective cohort. METHODS: All patients with a clinical culture growing S. maltophilia were enrolled at six tertiary hospitals across Japan between April 2019 and March 2022. The clinical characteristics, outcomes, antimicrobial susceptibility and genomic epidemiology of cases with S. maltophilia were investigated. RESULTS: In total, 78 patients were included representing 34 infection and 44 colonization cases. The median age was 72.5 years (IQR, 61-78), and males accounted for 53 cases (68%). The most common comorbidity was localized solid malignancy (39%). Nearly half of the patients (44%) were immunosuppressed, with antineoplastic chemotherapy accounting for 31%. The respiratory tract was the most common site of colonization (86%), whereas bacteraemia accounted for most infection cases (56%). The 30 day all-cause mortality rate was 21%, which was significantly higher in infection cases than colonization cases (35% versus 9%; adjusted HR, 3.81; 95% CI, 1.22-11.96). Susceptibility rates to ceftazidime, levofloxacin, minocycline and sulfamethoxazole/trimethoprim were 14%, 65%, 87% and 100%, respectively. The percentage of infection ranged from 13% in the unclassified group to 86% in genomic group 6A. The percentage of non-susceptibility to ceftazidime ranged from 33% in genomic group C to 100% in genomic groups 6 and 7 and genomic group geniculate. CONCLUSIONS: In this contemporary multicentre cohort, S. maltophilia primarily colonized the respiratory tract, whereas patients with bacteraemia had the highest the mortality from this pathogen. Sulfamethoxazole/trimethoprim remained consistently active, but susceptibility to levofloxacin was relatively low. The proportions of cases representing infection and susceptibility to ceftazidime differed significantly based on genomic groups.

Carbapenemase-producing Enterobacterales (CPEs) are one of the top priority antimicrobial-resistant pathogens. Among CPEs, those producing acquired metallo-β-lactamases (MBLs) are considered particularly problematic as few agents are active against them. Imipenemase (IMP) is the most frequently encountered acquired MBL in Japan, but comprehensive assessment of clinical and microbiological features of IMP-producing Enterobacterales infection remains scarce. Here, we retrospectively evaluated 62 patients who were hospitalized at a university hospital in Japan and had IMP-producing Enterobacterales from a clinical culture. The isolates were either Enterobacter cloacae complex or Klebsiella pneumoniae, and most of them were isolated from sputum. The majority of K. pneumoniae, but not E. cloacae complex isolates, were susceptible to aztreonam. Sequence type (ST) 78 and ST517 were prevalent for E. cloacae complex and K. pneumoniae, respectively, and all isolates carried blaIMP-1. Twenty-four of the patients were deemed infected with IMP-producing Enterobacterales. Among the infected patients, therapy varied and largely consisted of conventional β-lactam agents, fluoroquinolones, or combinations. Three (13%), five (21%), and nine (38%) of them died by days 14, 30, and 90, respectively. While incremental mortality over 90 days was observed in association with underlying comorbidities, active conventional treatment options were available for most patients with IMP-producing Enterobacterales infections, distinguishing them from more multidrug-resistant CPE infections associated with globally common MBLs, such as New Delhi metallo-β-lactamase (NDM) and Verona integron-encoded metallo-β-lactamase (VIM).

Pseudomonas aeruginosa causes chronic lung infection in cystic fibrosis (CF), resulting in structural lung damage and progressive pulmonary decline. P. aeruginosa in the CF lung undergoes numerous changes, adapting to host-specific airway pressures while establishing chronic infection. P. aeruginosa undergoes lipid A structural modification during CF chronic infection, not seen in any other disease state. Lipid A, the membrane anchor of lipopolysaccharide (i.e., endotoxin), comprises the majority of the outer membrane of Gram-negative bacteria and is a potent toll-like receptor (TLR)4 agonist. The structure of P. aeruginosa lipid A is intimately linked with its recognition by TLR4, and subsequent immune response. Prior work has identified P. aeruginosa strains with altered lipid A structures that arise during chronic CF lung infection; however, the impact of P. aeruginosa lipid A structure on airway disease has not been investigated. Here, we show that P. aeruginosa lipid A lacks PagL-mediated deacylation during human airway infection using a direct-from-sample mass spectrometry approach on human bronchoalveolar lavage fluid. This structure triggers increased pro-inflammatory cytokine production by primary human macrophages. Furthermore, alterations in lipid A 2-hydroxylation impact cytokine response in a site-specific manner, independent of CFTR function. Interestingly, there is a CF-specific reduction in IL-8 secretion within the epithelial-cell compartment that only occurs in CF bronchial epithelial cells when infected with CF-adapted P. aeruginosa that lack PagL-mediated lipid A deacylation. Taken together, we show that P. aeruginosa alters its lipid A structure during acute lung infection and that this lipid A structure induces stronger signaling through TLR4.

UNLABELLED: Streptococcus pyogenes causes a variety of human infections, and hospital outbreaks with this pathogen have also been reported. The purpose of this study is to describe the clinical characteristics of an outbreak of S. pyogenes involving 15 patients and four healthcare workers (HCWs), as well as the molecular characteristics of the causative isolates. The course and response to the outbreak were reviewed, and information on the characteristics of the patients was extracted retrospectively from the medical records. Whole-genome sequencing of the 16 causative isolates (14 from patients and two from HCWs) was also performed. All 15 patients were postoperative of head and neck cancer with tracheotomy, and 12 had invasive infections, primarily surgical site infections, all of which resolved without causing serious illness. All but the first case was detected more than 7 days after admission. S. pyogenes was detected in two patients after empiric antimicrobial administration was performed on all inpatients and HCWs, and the outbreak was finally contained in approximately 2 months. All isolates detected in patients and HCWs belonged to emm89/clade 3, a hypervirulent clone that has emerged worldwide and was classified as sequence type 646. These isolates had single nucleotide polymorphism (SNP) differences of zero to one, indicating clonal transmission. This study demonstrated an outbreak of S. pyogenes emm89/clade 3 in a ward of patients with head and neck cancer. The global emergence of hypervirulent isolates may increase the risk of outbreaks among high-risk patients. IMPORTANCE: This study describes an outbreak of Streptococcus pyogenes that occurred in a ward caring for patients with head and neck cancer and tracheostomies. Many cases of invasive infections occurred in a short period, and extensive empiric antimicrobial administration on patients and healthcare workers was performed to control the outbreak. Whole-genome sequencing analysis of the causative strains confirmed that it was a monoclonal transmission of strains belonging to emm89/clade 3. The epidemiology and clinical characteristics of S. pyogenes infections have changed with the replacement of the prevalent clones worldwide. In the 1980s, there was a reemergence of S. pyogenes infections in high-income countries due to the spread of hypervirulent emm1 strains. emm89/clade 3 has recently been spreading worldwide and shares common features with emm1, including increased production of two toxins, NADase, and streptolysin O. The outbreak reported here may reflect the high spreading potential and virulence of emm89/clade 3.

We evaluated the secondary COVID-19 incidence among uninfected hospitalized patients after nosocomial COVID-19 exposure. An exposure source of SARS-CoV-2 was hospitalized patients or healthcare personnel (HCP) newly diagnosed as having COVID-19. Patients exposed to a COVID-19-infected patient in a shared room more frequently developed COVID-19 than those exposed to an infected HCP.

The activity of a novel β-lactamase inhibitor combination, sulbactam-durlobactam (SUL-DUR), was tested against 87 colistin-resistant and/or cefiderocol-non-susceptible carbapenem-resistant Acinetobacter baumannii clinical isolates collected from U.S. hospitals between 2017 and 2019. Among them, 89% and 97% were susceptible to SUL-DUR and imipenem plus SUL-DUR, with MIC50/MIC90 values of 2 µg/mL/8 µg/mL and 1 µg/mL/4 µg/mL, respectively. The presence of amino acid substitutions in penicillin-binding protein 3, including previously reported A515V or T526S, was associated with SUL-DUR non-susceptibility.

With the coronavirus disease 2019 (COVID-19) pandemic, there is growing interest in utilizing adaptive platform clinical trials (APTs), in which multiple drugs are compared with a single common control group, such as a placebo or standard-of-care group. APTs evaluate several drugs for one disease and accept additions or exclusions of drugs as the trials progress; however, little is known about the efficiency of APTs over multiple stand-alone trials. In this study, we simulated the total development period, total sample size, and statistical operating characteristics of APTs and multiple stand-alone trials in drug development settings for hospitalized patients with COVID-19. Simulation studies using selected scenarios reconfirmed several findings regarding the efficiency of APTs. The APTs without staggered addition of drugs showed a shorter total development period than stand-alone trials, but the difference rapidly diminished if patient's enrollment was accelerated during the trials owing to the spread of infection. APTs with staggered addition of drugs still have the possibility of reducing the total development period compared with multiple stand-alone trials in some cases. Our study demonstrated that APTs could improve efficiency relative to multiple stand-alone trials regarding the total development period and total sample size without undermining statistical validity; however, this improvement varies depending on the speed of patient enrollment, sample size, presence/absence of family-wise error rate adjustment, allocation ratio between drug and placebo groups, and interval of staggered addition of drugs. Given the complexity of planning and implementing APT, the decision to implement APT during a pandemic must be made carefully.

The antibiotic cefiderocol hijacks iron transporters to facilitate its uptake and resists β-lactamase degradation. While effective, resistance has been detected clinically with unknown mechanisms. Here, using experimental evolution, we identified cefiderocol resistance mutations in Pseudomonas aeruginosa. Resistance was multifactorial in host-mimicking growth media, led to multidrug resistance and paid fitness costs in cefiderocol-free environments. However, kin selection drove some resistant populations to cross-protect susceptible individuals from killing by increasing pyoverdine secretion via a two-component sensor mutation. While pyochelin sensitized P. aeruginosa to cefiderocol killing, pyoverdine and the enterobacteria siderophore enterobactin displaced iron from cefiderocol, preventing uptake by susceptible cells. Among 113 P. aeruginosa intensive care unit clinical isolates, pyoverdine production directly correlated with cefiderocol tolerance, and high pyoverdine producing isolates cross-protected susceptible P. aeruginosa and other Gram-negative bacteria. These in vitro data show that antibiotic cross-protection can occur via degradation-independent mechanisms and siderophores can serve unexpected protective cooperative roles in polymicrobial communities.

OBJECTIVE: Imipenem-relebactam (IMR), a novel β-lactam/β-lactamase inhibitor combination, is recommended for infections caused by difficult-to-treat Pseudomonas aeruginosa. This study aimed to investigate the evolution trajectory of IMR resistance under the selection of levofloxacin in P. aeruginosa. METHODS: Antimicrobial susceptibility testing, complete genome sequencing and gene manipulation experiments were performed. Quantitative reverse transcription PCR for specific genes and porin levels were detected. Evolution trajectory was simulated in vitro by induction assay. RESULTS: P. aeruginosa HS347 and HS355 were isolated from abdominal drainage of two neighboring patients (S and Z) undergoing surgery of colon carcinoma in Shanghai, China, with the latter patient having received levofloxacin. They were closely related ST16 strains, and both carried blaKPC-2 plasmids highly similar to those of P. aeruginosa endemic clones from Zhejiang province, where patient Z had received enteroscopy before this admission. Acquisition of resistance was observed for both IMR and fluoroquinolones in HS355, likely prompted by treatment with levofloxacin. The T274I substitution in MexS (putative oxidoreductase), upregulated efflux pump operon mexEF-oprN and decreased production of porin OprD leading to cross-resistance to fluoroquinolones and IMR, which was also verified by in vitro mutant selection under levofloxacin selection. CONCLUSION: The emergence of a rare blaKPC-2-plasmid-bearing ST16 clone implies the horizonal spread and inter-regional dissemination of a high-risk plasmid-clone combination, representing a public health challenge. Levofloxacin exposure can select for mexS inactivating mutation, which in turn leads to IMR resistance phenotype, implicating the role of an unrelated, widely used antimicrobial agent in insidiously triggering the development of cross resistance to a latest β-lactam/β-lactamase inhibitor combination.

β-Lactamases can accumulate stepwise mutations that increase their resistance profiles to the latest β-lactam agents. CMY-185 is a CMY-2-like β-lactamase and was identified in an Escherichia coli clinical strain isolated from a patient who underwent treatment with ceftazidime-avibactam. CMY-185, possessing four amino acid substitutions of A114E, Q120K, V211S, and N346Y relative to CMY-2, confers high-level ceftazidime-avibactam resistance, and accumulation of the substitutions incrementally enhances the level of resistance to this agent. However, the functional role of each substitution and their interplay in enabling ceftazidime-avibactam resistance remains unknown. Through biochemical and structural analysis, we present the molecular basis for the enhanced ceftazidime hydrolysis and impaired avibactam inhibition conferred by CMY-185. The substituted Y346 residue is a major driver of the functional evolution as it rejects primary avibactam binding due to the steric hindrance and augments oxyimino-cephalosporin hydrolysis through a drastic structural change, rotating the side chain of Y346 and then disrupting the H-10 helix structure. The other substituted residues E114 and K120 incrementally contribute to rejection of avibactam inhibition, while S211 stimulates the turnover rate of the oxyimino-cephalosporin hydrolysis. These findings indicate that the N346Y substitution is capable of simultaneously expanding the spectrum of activity against some of the latest β-lactam agents with altered bulky side chains and rejecting the binding of β-lactamase inhibitors. However, substitution of additional residues may be required for CMY enzymes to achieve enhanced affinity or turnover rate of the β-lactam agents leading to clinically relevant levels of resistance.IMPORTANCECeftazidime-avibactam has a broad spectrum of activity against multidrug-resistant Gram-negative bacteria including carbapenem-resistant Enterobacterales including strains with or without production of serine carbapenemases. After its launch, emergence of ceftazidime-avibactam-resistant strains that produce mutated β-lactamases capable of efficiently hydrolyzing ceftazidime or impairing avibactam inhibition are increasingly reported. Furthermore, cross-resistance towards cefiderocol, the latest cephalosporin in clinical use, has been observed in some instances. Here, we clearly demonstrate the functional role of the substituted residues in CMY-185, a four amino-acid variant of CMY-2 identified in a patient treated with ceftazidime-avibactam, for high-level resistance to this agent and low-level resistance to cefiderocol. These findings provide structural insights into how β-lactamases may incrementally alter their structures to escape multiple advanced β-lactam agents.

OBJECTIVES: To investigate the genomic diversity and β-lactam susceptibilities of Enterococcus faecalis collected from patients with infective endocarditis (IE). METHODS: We collected 60 contemporary E. faecalis isolates from definite or probable IE cases identified between 2018 and 2021 at the University of Pittsburgh Medical Center. We used whole-genome sequencing to study bacterial genomic diversity and employed antibiotic checkerboard assays and a one-compartment pharmacokinetic-pharmacodynamic (PK/PD) model to investigate bacterial susceptibility to ampicillin and ceftriaxone both alone and in combination. RESULTS: Genetically diverse E. faecalis were collected, however, isolates belonging to two STs, ST6 and ST179, were collected from 21/60 (35%) IE patients. All ST6 isolates encoded a previously described mutation upstream of penicillin-binding protein 4 (pbp4) that is associated with pbp4 overexpression. ST6 isolates had higher ceftriaxone MICs and higher fractional inhibitory concentration index values for ampicillin and ceftriaxone (AC) compared to other isolates, suggesting diminished in vitro AC synergy against this lineage. Introduction of the pbp4 upstream mutation found among ST6 isolates caused increased ceftriaxone resistance in a laboratory E. faecalis isolate. PK/PD testing showed that a representative ST6 isolate exhibited attenuated efficacy of AC combination therapy at humanized antibiotic exposures. CONCLUSIONS: We find evidence for diminished in vitro AC activity among a subset of E. faecalis IE isolates with increased pbp4 expression. These findings suggest that alternate antibiotic combinations against diverse contemporary E. faecalis IE isolates should be evaluated.

IMPORTANCE: Treatment options for COVID-19 are warranted irrespective of the presence of risk factors for severe disease. OBJECTIVE: To assess the efficacy and safety of ensitrelvir in patients with mild to moderate COVID-19. DESIGN, SETTING, AND PARTICIPANTS: This phase 3 part of a phase 2/3, double-blind, placebo-controlled randomized clinical trial was conducted from February 10 to July 10, 2022, with a 28-day follow-up period, at 92 institutions in Japan, Vietnam, and South Korea. Patients (aged 12 to <70 years) with mild to moderate COVID-19 within 120 hours of positive viral test results were studied. INTERVENTIONS: Patients were randomized (1:1:1) to receive 125 mg of once-daily ensitrelvir (375 mg on day 1), 250 mg of once-daily ensitrelvir (750 mg on day 1), or placebo for 5 days. MAIN OUTCOMES AND MEASURES: The primary end point was the time to resolution of the composite of 5 characteristic symptoms of SARS-CoV-2 Omicron infection, assessed using a Peto-Prentice generalized Wilcoxon test stratified by vaccination history. Virologic efficacy and safety were also assessed. RESULTS: A total of 1821 patients were randomized, of whom 1030 (347 in the 125-mg ensitrelvir group, 340 in the 250-mg ensitrelvir group, and 343 in the placebo group) were randomized in less than 72 hours of disease onset (primary analysis population). The mean (SD) age in this population was 35.2 (12.3) years, and 552 (53.6%) were men. A significant difference was observed between the 125-mg ensitrelvir group and the placebo group (P = .04 with a Peto-Prentice generalized Wilcoxon test). The difference in median time was approximately 1 day between the 125-mg ensitrelvir group and the placebo group (167.9 vs 192.2 hours; difference, -24.3 hours; 95% CI, -78.7 to 11.7 hours). Adverse events were observed in 267 of 604 patients (44.2%) in the 125-mg ensitrelvir group, 321 of 599 patients (53.6%) in the 250-mg ensitrelvir group, and 150 of 605 patients (24.8%) in the placebo group, which included a decrease in high-density lipoprotein level (188 [31.1%] in the 125-mg ensitrelvir group, 231 [38.6%] in the 250-mg ensitrelvir group, and 23 [3.8%] in the placebo group). No treatment-related serious adverse events were reported. CONCLUSIONS AND RELEVANCE: In this randomized clinical trial, 125-mg ensitrelvir treatment reduced the time to resolution of the 5 typical COVID-19 symptoms compared with placebo in patients treated in less than 72 hours of disease onset; the absolute difference in median time to resolution was approximately 1 day. Ensitrelvir demonstrated clinical and antiviral efficacy without new safety concerns. Generalizability to populations outside Asia should be confirmed. TRIAL REGISTRATION: Japan Registry of Clinical Trials Identifier: jRCT2031210350.

OBJECTIVES: Multidrug-resistant (MDR) bacterial infections are highly prevalent among long-term care facility (LTCF) residents, and are thus important targets for antimicrobial stewardship. Diagnoses of urinary tract infections (UTIs), which are associated with antimicrobial use in these facilities, are not always made by physicians. Past epidemiologic studies have included asymptomatic bacteriuria together with UTIs. The National Healthcare Safety Network has initiated a surveillance program to identify the causative organisms of UTIs in LTCF residents. In Japan, medical care for these residents is provided through in-person physician visits; however, limited related data are available. Therefore, we investigated the organisms causing UTIs and their drug susceptibility among LTCF residents in central Japan, and examined the prevalence of multidrug resistance, its risk factors, and correlations with clinical outcomes. METHODS: We retrospectively evaluated clinical and urine culture data of LTCF residents with physician-diagnosed UTIs between April 1, 2019, and April 30, 2022. RESULTS: The detection rate of multidrug-resistant organisms was high, with Escherichia coli being the most prevalent. Ceftriaxone was frequently used for initial therapy. The initial antimicrobial agents were significantly less active against MDR pathogens than non-MDR pathogens. Most residents continued to receive the initial agents regardless of culture results. Nonetheless, differences in the therapy duration, relapse and hospitalization rates, and death rate within 28 days between the multidrug-resistant and non-multidrug-resistant groups were not significant. CONCLUSIONS: Antimicrobial stewardship is essential for reducing antimicrobial use and selective pressure in LTCFs in Japan; however, more specific data are needed for its effective implementation.

OBJECTIVES: Taxonomic assignment based on whole-genome sequencing data facilitates clear demarcation of species within a complex genus. Here, we applied a unique pan-genome phylogenetic method, open reading frame (ORF)-based binarized structure network analysis (OSNA), for taxonomic inference of Aeromonas spp., a complex taxonomic group consisting of 30 species. METHODS: Data from 335 publicly available Aeromonas genomes, including the reference genomes of 30 species, were used to build a phylogenetic tree using OSNA. In OSNA, whole-genome structures are expressed as binary sequences based on the presence or absence of ORFs, and a tree is generated using neighbor-net, a distance-based method for constructing phylogenetic networks from binary sequences. The tree built by OSNA was compared to that constructed by a core-genome single-nucleotide polymorphism (SNP)-based analysis. Furthermore, the orthologous average nucleotide identity (OrthoANI) values of the sequences that clustered in a single clade in the OSNA-based tree were calculated. RESULTS: The phylogenetic tree constructed with OSNA successfully delineated the majority of species of the genus Aeromonas forming conspecific clades for individual species, which was corroborated by OrthoANI values. Moreover, the OSNA-based phylogenetic tree demonstrated high compositional similarity to the core-genome SNP-based phylogenetic tree, supported by the Fowlkes-Mallows index. CONCLUSIONS: We propose that OSNA is a useful tool in predicting the taxonomic classification of complex bacterial genera.

BACKGROUND: Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) bloodstream infections are associated with high mortality. We studied clinical bloodstream KPC-Kp isolates to investigate mechanisms of resistance to complement, a key host defense against bloodstream infection. METHODS: We tested growth of KPC-Kp isolates in human serum. In serial isolates from a single patient, we performed whole genome sequencing and tested for complement resistance and binding by mixing study, direct ELISA, flow cytometry, and electron microscopy. We utilized an isogenic deletion mutant in phagocytosis assays and an acute lung infection model. RESULTS: We found serum resistance in 16 of 59 (27%) KPC-Kp clinical bloodstream isolates. In five genetically-related bloodstream isolates from a single patient, we noted a loss-of-function mutation in the capsule biosynthesis gene, wcaJ. Disruption of wcaJ was associated with decreased polysaccharide capsule, resistance to complement-mediated killing, and surprisingly, increased binding of complement proteins. Furthermore, an isogenic wcaJ deletion mutant exhibited increased opsono-phagocytosis in vitro and impaired in vivo control in the lung after airspace macrophage depletion in mice. CONCLUSIONS: Loss of function in wcaJ led to increased complement resistance, complement binding, and opsono-phagocytosis, which may promote KPC-Kp persistence by enabling co-existence of increased bloodstream fitness and reduced tissue virulence.

Rapid diagnostic tests (RDTs) for bloodstream infections have the potential to reduce time to appropriate antimicrobial therapy and improve patient outcomes. Previously, an in-house, lipid-based, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) method, Fast Lipid Analysis Technique (FLAT MS), has shown promise as a rapid pathogen identification method. In this study, FLAT MS for direct from blood culture identification was evaluated and compared to FDA-cleared identification methods using the Benefit-risk Evaluation Framework (BED-FRAME) analysis. FLAT MS was evaluated and compared to Bruker Sepsityper and bioMérieux BioFire FilmArray BCID2 using results from a previous study. For this study, 301 positive blood cultures were collected from the University of Maryland Medical Center. The RDTs were compared by their sensitivities, time-to-results, hands-on time, and BED-FRAME analysis. The overall sensitivity of all platforms compared to culture results from monomicrobial-positive blood cultures was 88.3%. However, the three RDTs differed in their accuracy for identifying Gram-positive bacteria, Gram-negative bacteria, and yeast. Time-to-results for FLAT MS, Sepsityper, and BioFire BCID2 were all approximately one hour. Hands-on times for FLAT MS, Sepsityper, and BioFire BCID2 were 10 (±1.3), 40 (±2.8), and 5 (±0.25) minutes, respectively. BED-FRAME demonstrated that each RDT had utility at different pathogen prevalence and relative importance. BED-FRAME is a useful tool that can used to determine which RDT is best for a healthcare center.

Escherichia coli belonging to sequence type ST131 constitute a globally distributed pandemic lineage that causes multidrug-resistant extra-intestinal infections. ST131 E. coli frequently produce extended-spectrum β-lactamases (ESBLs), which confer resistance to many β-lactam antibiotics and make infections difficult to treat. We sequenced the genomes of 154 ESBL-producing E. coli clinical isolates belonging to the ST131 lineage from patients at the University of Pittsburgh Medical Center (UPMC) between 2004 and 2018. Isolates belonged to the well described ST131 clades A (8%), B (3%), C1 (33%), and C2 (54%). An additional four isolates belonged to another distinct subclade within clade C and encoded genomic characteristics that have not been previously described. Time-dated phylogenetic analysis estimated that the most recent common ancestor (MRCA) for all clade C isolates from UPMC emerged around 1989, consistent with previous studies. We identified multiple genes potentially under selection in clade C, including the cell wall assembly gene ftsI, the LPS biosynthesis gene arnC, and the yersiniabactin uptake receptor fyuA. Diverse ESBL genes belonging to the blaCTX-M, blaSHV, and blaTEM families were identified; these genes were found at varying numbers of loci and in variable numbers of copies across isolates. Analysis of ESBL flanking regions revealed diverse mobile elements that varied by ESBL type. Overall, our findings show that ST131 subclades C1 and C2 dominated and were stably maintained among patients in the same hospital and uncover possible signals of ongoing adaptation within the clade C ST131 lineage.

The emerging and global spread of a novel plasmid-mediated colistin resistance gene, mcr-1, threatens human health. Expression of the MCR-1 protein affects bacterial fitness and this cost correlates with lipid A perturbation. However, the exact molecular mechanism remains unclear. Here, we identified the MCR-1 M6 variant carrying two-point mutations that conferred co-resistance to β-lactam antibiotics. Compared to wild-type (WT) MCR-1, this variant caused severe disturbance in lipid A, resulting in up-regulation of L, D-transpeptidases (LDTs) pathway, which explains co-resistance to β-lactams. Moreover, we show that a lipid A loading pocket is localized at the linker domain of MCR-1 where these 2 mutations are located. This pocket governs colistin resistance and bacterial membrane permeability, and the mutated pocket in M6 enhances the binding affinity towards lipid A. Based on this new information, we also designed synthetic peptides derived from M6 that exhibit broad-spectrum antimicrobial activity, exposing a potential vulnerability that could be exploited for future antimicrobial drug design.

BACKGROUND: The genus Aeromonas is increasingly implicated in human infections, but knowledge of its clinical characteristics and antimicrobial resistance profiles has been limited owing to its complex taxonomy. METHODS: We conducted a multicenter prospective cohort study of patients with Aeromonas infections at hospitals across Japan. Patients were eligible for inclusion if they had an Aeromonas spp. strain in a clinical culture and were considered infected at the culture site. Clinical data were collected, and isolates underwent susceptibility testing and whole-genome sequencing. RESULTS: A total of 144 patients were included. Hepatobiliary infection accounted for a majority of infections (73% [105 of 144]), which mostly occurred in elderly patients with comorbid conditions, including hepatobiliary complications. The all-cause 30-day mortality rate was 10.0% (95% confidence interval, 4.9%-14.8%). By whole-genome sequencing, 141 strains (98%) belonged to 4 Aeromonas species-A caviae, A hydrophila, A veronii, and A dhakensis-with significant intraspecies diversity. A caviae was predominant in all infection sites except skin and soft tissue, for which A hydrophila was the prevailing species. The genes encoding chromosomally mediated class B, C, and D β-lactamases were harbored by 92%-100% of the isolates in a species-specific manner, but they often lacked association with resistance phenotypes. The activity of cefepime was reliable. All isolates of A hydrophila and A dhakensis carried an mcr-3-like colistin resistance gene and showed reduced susceptibility to colistin. CONCLUSIONS: Hepatobiliary tract was the most common infection site of Aeromonas spp., with A caviae being the dominant causative species. The resistance genotype and phenotype were often incongruent for β-lactam agents.

<jats:title>Abstract</jats:title> <jats:sec> <jats:title>Background</jats:title> <jats:p>Carbapenem-resistant bacteria in patients with cancer are concerning due to the high risk of infection and mortality rates; however, the characteristics and prognoses of carbapenem-resistant infections in this population are currently unknown in Japan. We hence investigated the features and outcomes of carbapenem-resistant bacterial infections (CRBI) in patients with cancer in Japan.</jats:p> </jats:sec> <jats:sec> <jats:title>Methods</jats:title> <jats:p>From April 1, 2019, to March 31, 2022, patients with CRBI who either had cancer or no cancer were prospectively enrolled at eight centers as part of the Multi-Drug Resistant Organisms Clinical Research Network (MDRnet). The primary outcome was the 30-day all-cause mortality rates in patients with and without cancer. Two secondary outcomes were evaluated: 1) composite outcomes including mortality, worsening of clinical course after culture collection, intensive care unit stay, intubation, new dialysis from the date of culture collection to the end of antimicrobial therapy, and readmission within 90 days after discharge; and 2) the length of hospital stay after CRBI excluding death.</jats:p> </jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>We included a total of 167 patients, with 66 (39.5%) in the cancer group and 101 (60.5%) in the non-cancer group. The 30-day mortality rates in the cancer and non-cancer groups were 18.2% (12/66) and 14.0% (14/101), respectively (p = 0.45), while the composite outcomes in the cancer and non-cancer groups were 56.1% (37/66) and 43.6% (44/101), respectively (p = 0.12). Average duration of hospitalization was not significantly different between the two groups (cancer group, 44.6 days; non-cancer group, 51.0 days; p = 0.55). Propensity score analysis using inverse probability weighting also showed no significant difference in 30-day mortality and average duration of hospitalization (p = 0.22 and 0.98, respectively); however, the composite outcome was significantly higher in the cancer group than in non-cancer controls (odds ratio, 2.41; 95% confidence interval, 1.11–5.21; p = 0.03).</jats:p> </jats:sec> <jats:sec> <jats:title>Conclusion</jats:title> <jats:p>There was no difference in 30-day mortality rates between the cancer and non-cancer patient groups; however, we found a significant difference in the composite outcome. Patients with cancer who had CRBI experienced a worse clinical course that non-cancer patients.</jats:p> </jats:sec> <jats:sec> <jats:title>Disclosures</jats:title> <jats:p>Masahiro Suzuki, PhD, KANTO Chemical co., inc.: Grant/Research Support Yasufumi Matsumara, MD, PhD, Beckman Coulter: Grant/Research Support|Presicion System Science: Grant/Research Support|Toyobo: Grant/Research Support David van Duin, MD, PhD, Entasis: Advisor/Consultant|Merck: Advisor/Consultant|Merck: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Honoraria|Qpex: Advisor/Consultant|Roche: Advisor/Consultant|Shionogi: Advisor/Consultant|Shionogi: Grant/Research Support|Union: Advisor/Consultant|Utility: Advisor/Consultant Yohei Doi, MD, PhD, bioMerieux: Advisor/Consultant|FujiFilm: Advisor/Consultant|Gilead: Advisor/Consultant|Gilead: Honoraria|GSK: Advisor/Consultant|Meiji Seika Pharma: Advisor/Consultant|Moderna: Advisor/Consultant|Moderna: Honoraria|MSD: Advisor/Consultant|MSD: Honoraria|Shionogi: Advisor/Consultant|Shionogi: Grant/Research Support|Shionogi: Honoraria Sho Saito, MD, PhD, Shionogi &amp; Company, Limited: Grant/Research Support</jats:p> </jats:sec>

<jats:title>Abstract</jats:title> <jats:sec> <jats:title>Background</jats:title> <jats:p>Stenotrophomonas maltophilia has become one of the major gram-negative pathogens causing nosocomial infections. However, comprehensive analysis of the clinical characteristics and molecular epidemiology of patients with S. maltophilia remains limited.</jats:p> </jats:sec> <jats:sec> <jats:title>Methods</jats:title> <jats:p>All patients with a clinical culture growing S. maltophilia were collected from April 2019 to March 2022 through the Multi-Drug Resistant organisms clinical research network (MDRnet), consisting of 12 tertiary care hospitals in Japan. The clinical characteristics, outcomes, antimicrobial susceptibility and molecular epidemiology of cases with S. maltophilia colonization and infection were investigated and compared.</jats:p> </jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>In total, 78 cases, 44 with colonization and 34 with infection, were included. The median age was 72.5 years (IQR: 61–78), and males accounted for 53 cases (67.9%). The most common comorbidity was localized solid malignancy (38.5%). Almost half of the patients (43.6%) were immunosuppressed, and the most common reason was antineoplastic chemotherapy (30.8%). Respiratory tract was the most common site of colonization (86.4%), whereas bacteremia accounted for more than half of infection cases (55.9%). The overall 30-day all-cause mortality rate was 20.5%, which was significantly higher in infection cases than in colonization cases (35.3% vs 9.1%; odds ratio, 5.33; 95% confidence interval, 1.40–25.45). Susceptibility rates to ceftazidime, levofloxacin, minocycline, and sulfamethoxazole-trimethoprim were 14.1%, 65.2%, 87.2%, and 100%, respectively. Multilocus sequence typing of the 78 strains revealed that they belonged to 62 different sequence types (STs), of which 42 were known STs and 36 were novel STs.</jats:p> <jats:p>Kaplan-Meier survival analysis</jats:p> </jats:sec> <jats:sec> <jats:title>Conclusion</jats:title> <jats:p>S. maltophilia frequently colonizes the respiratory tract, and the mortality is significantly higher in infection cases. Sulfamethoxazole-trimethoprim remains active, but susceptibility to levofloxacin appear to be declining. The strains were genetically highly diverse, consistent with the likely environmental origin.</jats:p> </jats:sec> <jats:sec> <jats:title>Disclosures</jats:title> <jats:p>Masahiro Suzuki, PhD, KANTO Chemical co., inc.: Grant/Research Support Sho Saito, MD, PhD, Shionogi &amp; Company, Limited: Grant/Research Support Yasufumi Matsumara, MD, PhD, Beckman Coulter: Grant/Research Support|Presicion System Science: Grant/Research Support|Toyobo: Grant/Research Support David van Duin, MD, PhD, Entasis: Advisor/Consultant|Merck: Advisor/Consultant|Merck: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Honoraria|Qpex: Advisor/Consultant|Roche: Advisor/Consultant|Shionogi: Advisor/Consultant|Shionogi: Grant/Research Support|Union: Advisor/Consultant|Utility: Advisor/Consultant Yohei Doi, MD, PhD, bioMerieux: Advisor/Consultant|FujiFilm: Advisor/Consultant|Gilead: Advisor/Consultant|Gilead: Honoraria|GSK: Advisor/Consultant|Meiji Seika Pharma: Advisor/Consultant|Moderna: Advisor/Consultant|Moderna: Honoraria|MSD: Advisor/Consultant|MSD: Honoraria|Shionogi: Advisor/Consultant|Shionogi: Grant/Research Support|Shionogi: Honoraria</jats:p> </jats:sec>

mRNA vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have played a key role in reducing morbidity and mortality from coronavirus disease 2019 (COVID-19). We conducted a double-blind, placebo-controlled phase I/II trial to evaluate the safety, tolerability, and immunogenicity of EXG-5003, a two-dose, controllable self-replicating RNA vaccine against SARS-CoV-2. EXG-5003 encodes the receptor binding domain (RBD) of SARS-CoV-2 and was administered intradermally without lipid nanoparticles (LNPs). The participants were followed for 12 months. Forty healthy participants were enrolled in Cohort 1 (5 µg per dose, n = 16; placebo, n = 4) and Cohort 2 (25 µg per dose, n = 16; placebo, n = 4). No safety concerns were observed with EXG-5003 administration. SARS-CoV-2 RBD antibody titers and neutralizing antibody titers were not elevated in either cohort. Elicitation of antigen-specific cellular immunity was observed in the EXG-5003 recipients in Cohort 2. At the 12-month follow-up, participants who had received an approved mRNA vaccine (BNT162b2 or mRNA-1273) &gt;1 month after receiving the second dose of EXG-5003 showed higher cellular responses compared with equivalently vaccinated participants in the placebo group. The findings suggest a priming effect of EXG-5003 on the long-term cellular immunity of approved SARS-CoV-2 mRNA vaccines.

Addressing the treatment and prevention of antibacterial-resistant gram-negative bacterial infections is a priority area of the Antibacterial Resistance Leadership Group (ARLG). The ARLG has conducted a series of observational studies to define the clinical and molecular global epidemiology of carbapenem-resistant and ceftriaxone-resistant Enterobacterales, carbapenem-resistant Pseudomonas aeruginosa, and carbapenem-resistant Acinetobacter baumannii, with the goal of optimizing the design and execution of interventional studies. One ongoing ARLG study aims to better understand the impact of fluoroquinolone-resistant gram-negative gut bacteria in neutropenic patients, which threatens to undermine the effectiveness of fluoroquinolone prophylaxis in these vulnerable patients. The ARLG has conducted pharmacokinetic studies to inform the optimal dosing of antibiotics that are important in the treatment of drug-resistant gram-negative bacteria, including oral fosfomycin, intravenous minocycline, and a combination of intravenous ceftazidime-avibactam and aztreonam. In addition, randomized clinical trials have assessed the safety and efficacy of step-down oral fosfomycin for complicated urinary tract infections and single-dose intravenous phage therapy for adult patients with cystic fibrosis who are chronically colonized with P. aeruginosa in their respiratory tract. Thus, the focus of investigation in the ARLG has evolved from improving understanding of drug-resistant gram-negative bacterial infections to positively affecting clinical care for affected patients through a combination of interventional pharmacokinetic and clinical studies, a focus that will be maintained moving forward.

INTRODUCTION: Favipiravir terminates severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. Accordingly, early administration of favipiravir to SARS-CoV-2-infected coronavirus disease 2019 (COVID-19) patients may be expected to suppress disease progression. METHODS: A randomized double-blind placebo-controlled trial was conducted to demonstrate efficacy of favipiravir in reducing disease progression in patients with mild COVID-19. The participants were unvaccinated patients with comorbidities and at risk of progression to severe disease. Patients were enrolled within 72 h of disease onset and randomized to receive either favipiravir (1800 mg/dose on Day 1 followed by 800 mg/dose) or matching placebo twice daily for 10 days. The primary endpoint was the proportion of patients requiring oxygen therapy within 28 days of randomization. RESULTS: The trial was discontinued after enrolling 84 patients due to slower than anticipated enrollment caused by rapid uptake of SARS-CoV-2-vaccines and the emergence of the Omicron variant. Results from the 84 patients demonstrated no significant difference in all clinical outcomes. In post-hoc analyses, favipiravir treatment showed higher efficacy in patients within 48 h of onset. No deaths or severe adverse events were documented in the favipiravir group. Plasma concentrations of favipiravir from Day 2 onward were maintained above 40 μg/mL. CONCLUSIONS: Conducting clinical trials for pathogens like SARS-CoV-2 that rapidly accumulate mutations leading to altered disease characteristics carries significant risks unless it can be done in a short period. Therefore, it would be important to prepare the comprehensive clinical trial platform that can appropriately and promptly evaluate drugs even under a pandemic.

BACKGROUND: Cefiderocol demonstrates excellent activity against MDR Pseudomonas aeruginosa; however, the activity against isolates from patients previously treated with β-lactam agents is unknown. We aimed to determine the activity of cefiderocol against P. aeruginosa collected before and after treatment with traditional β-lactams and new β-lactam/β-lactamase inhibitors. METHODS: Cefiderocol MICs were determined in triplicate in iron-depleted cation-adjusted Mueller-Hinton broth and compared with β-lactam MICs tested by standard methods. All isolates underwent WGS analysis to identify mutations associated with resistance. RESULTS: One hundred and seventy-eight P. aeruginosa isolates were evaluated; 48% (86/178) were non-susceptible to ceftazidime/avibactam, ceftolozane/tazobactam and/or imipenem/relebactam. The cefiderocol MIC50 and MIC90 were 0.12 and 1 mg/L, respectively. Median cefiderocol MICs did not vary against isolates classified as MDR, XDR, or those non-susceptible to ceftazidime/avibactam, ceftolozane/tazobactam and/or imipenem/relebactam when compared with non-MDR isolates. Against isolates collected from patients previously treated with ceftolozane/tazobactam, cefiderocol MICs were increased 4-fold compared with baseline. Cross-resistance to cefiderocol was identified in 21% (3/14) of patients who developed treatment-emergent resistance to ceftolozane/tazobactam. Overall, 6% (11/178) of isolates demonstrated cefiderocol MICs ≥2 mg/L, which were disproportionately collected from patients previously treated with ceftolozane/tazobactam (73%; 8/11). Isolates with reduced cefiderocol susceptibility harboured mutations in ampC, tonB-dependent receptors, the response regulator pirR and ftsI. CONCLUSIONS: Cefiderocol demonstrates excellent in vitro activity against P. aeruginosa isolates exposed to other novel β-lactam agents; however, some exceptions were identified. Cross-resistance between cefiderocol and ceftolozane/tazobactam was evident, but not with ceftazidime/avibactam or imipenem/relebactam. Reduced cefiderocol susceptibility was mediated by mutations in ampC and tonB-dependent receptors.

BACKGROUND: Carbapenem-resistant Acinetobacter baumannii (CRAb) is one of the most problematic antimicrobial-resistant bacteria. We sought to elucidate the international epidemiology and clinical impact of CRAb. METHODS: In a prospective observational cohort study, 842 hospitalized patients with a clinical CRAb culture were enrolled at 46 hospitals in five global regions between 2017 and 2019. The primary outcome was all-cause mortality at 30 days from the index culture. The strains underwent whole-genome analysis. RESULTS: Of 842 cases, 536 (64%) represented infection. By 30 days, 128 (24%) of the infected patients died, ranging from 1 (6%) of 18 in Australia-Singapore to 54 (25%) of 216 in the United States and 24 (49%) of 49 in South-Central America, whereas 42 (14%) of non-infected patients died. Bacteremia was associated with a higher risk of death compared with other types of infection (40 [42%] of 96 vs. 88 [20%] of 440). In a multivariable logistic regression analysis, bloodstream infection and higher age-adjusted Charlson comorbidity index were independently associated with 30-day mortality. Clonal group 2 (CG2) strains predominated except in South-Central America, ranging from 216 (59%) of 369 in the United States to 282 (97%) of 291 in China. Acquired carbapenemase genes were carried by 769 (91%) of the 842 isolates. CG2 strains were significantly associated with higher levels of meropenem resistance, yet non-CG2 cases were over-represented among the deaths compared with CG2 cases. CONCLUSIONS: CRAb infection types and clinical outcomes differed significantly across regions. While CG2 strains remained predominant, non-CG2 strains were associated with higher mortality. CLINICALTRIALS.GOV: #NCT03646227.

Acute otitis media (AOM) is the most common childhood bacterial infectious disease requiring antimicrobial therapy. Most cases of AOM are caused by translocation of Streptococcus pneumoniae or Haemophilus influenzae from the nasopharynx to the middle ear during an upper respiratory tract infection (URI). Ongoing genomic surveillance of these pathogens is important for vaccine design and tracking of emerging variants, as well as for monitoring patterns of antibiotic resistance to inform treatment strategies and stewardship.In this work, we examined the ability of a genomics-based workflow to determine microbiological and clinically relevant information from cultured bacterial isolates obtained from patients with AOM or an URI. We performed whole genome sequencing (WGS) and analysis of 148 bacterial isolates cultured from the nasopharynx (N = 124, 94 AOM and 30 URI) and ear (N = 24, all AOM) of 101 children aged 6-35 months presenting with AOM or an URI. We then performed WGS-based sequence typing and antimicrobial resistance profiling of each strain and compared results to those obtained from traditional microbiological phenotyping.WGS of clinical isolates resulted in 71 S. pneumoniae genomes and 76 H. influenzae genomes. Multilocus sequencing typing (MSLT) identified 33 sequence types for S. pneumoniae and 19 predicted serotypes including the most frequent serotypes 35B and 3. Genome analysis predicted 30% of S. pneumoniae isolates to have complete or intermediate penicillin resistance. AMR predictions for S. pneumoniae isolates had strong agreement with clinical susceptibility testing results for beta-lactam and non beta-lactam antibiotics, with a mean sensitivity of 93% (86-100%) and a mean specificity of 98% (94-100%). MLST identified 29 H. influenzae sequence types. Genome analysis identified beta-lactamase genes in 30% of H. influenzae strains, which was 100% in agreement with clinical beta-lactamase testing. We also identified a divergent highly antibiotic-resistant strain of S. pneumoniae, and found its closest sequenced strains, also isolated from nasopharyngeal samples from over 15 years ago.Ultimately, our work provides the groundwork for clinical WGS-based workflows to aid in detection and analysis of H. influenzae and S. pneumoniae isolates.

Cefmetazole is active against extended-spectrum β-lactamase-producing Escherichia coli (ESBLEC) and is a potential candidate for carbapenem-sparing therapy. This multicenter, observational study included patients hospitalized for invasive urinary tract infection due to ESBLEC between March 2020 and November 2021 at 10 facilities in Japan, for whom either cefmetazole or meropenem was initiated as a definitive therapy within 96 h of culture collection and continued for at least 3 d. Outcomes included clinical and microbiological effectiveness, recurrence within 28 d, and all-cause mortality (14 d, 30 d, in-hospital). Outcomes were adjusted for the inverse probability of propensity scores for receiving cefmetazole or meropenem. Eighty-one and forty-six patients were included in the cefmetazole and meropenem groups, respectively. Bacteremia accounted for 43% of the cefmetazole group, and 59% of the meropenem group. The crude clinical effectiveness, 14 d, 30 d, and in-hospital mortality for patients in the cefmetazole and meropenem groups were 96.1% vs 90.9%, 0% vs 2.3%, 0% vs 12.5%, and 2.6% vs 13.3%, respectively. After propensity score adjustment, clinical effectiveness, the risk of in-hospital mortality, and the risk of recurrence were similar between the two groups (P = 0.54, P = 0.10, and P = 0.79, respectively). In all cases with available data (cefmetazole : n = 61, meropenem : n = 22), both drugs were microbiologically effective. In all isolates, blaCTX-M was detected as the extended-spectrum β-lactamase gene. The predominant CTX-M subtype was CTX-M-27 (47.6%). Cefmetazole showed clinical and bacteriological effectiveness comparable to meropenem against invasive urinary tract infection due to ESBLECs.

Fosfomycin, approved in the United States only for cystitis, is an attractive alternative for oral treatment of outpatient complicated urinary tract infections (cUTIs) as it has antimicrobial activity against most common uropathogens. The study was a multicenter, randomized, open-label pragmatic superiority clinical trial evaluating the efficacy of oral fosfomycin versus oral levofloxacin strategies in cUTIs (FOCUS study). The trial compared two strategies for initial or step-down oral therapy of cUTI without bacteremia after 0-48 hours of parenteral antibiotic therapy. Subjects were assigned to 3 g of fosfomycin or 750 mg (or dose adjusted for kidney function) of levofloxacin daily for 5-7 days. Clinical and microbiological cures were assessed at the end of therapy (EOT) and test of cure (TOC) (approximately 21 days from the start of antibiotics). The trial did not meet accrual goals; thus, the results were descriptive. Only 51 subjects were included in the microbiological intention-to-treat population. The subjects were mainly females (76%), with a mean age of 46.7 years (standard deviation [SD] = 20.8) and acute pyelonephritis (88%). At the end of therapy, clinical cure remained similar (69% and 68% for fosfomycin and levofloxacin strategies, respectively), and microbiological success was 100% for both strategies. At the test of cure, clinical cure was similar (84% and 86% in the fosfomycin and levofloxacin strategies, respectively); however, a numerically lower microbiological success was observed for fosfomycin (69% compared to 84% for levofloxacin). These limited data suggest that fosfomycin could be an oral alternative as a step-down therapy for the treatment of cUTIs (registry number NCT03697993). IMPORTANCE Concerns over resistance and safety have been identified in the current treatment regimen for complicated urinary tract infections. Fosfomycin is a drug that is routinely used for the treatment of uncomplicated cystitis. This study shows that fosfomycin could be an oral alternative as step-down therapy for the treatment of complicated urinary tract infections, with a clinical cure rate comparable to levofloxacin but a lower microbiological success rate 3 weeks from start of antibiotics.

Escherichia coli is the most common cause of urinary tract infections (UTIs) in children, and yet the underlying mechanisms of virulence and antibiotic resistance and the overall population structure of the species is poorly understood within this age group. To investigate whether uropathogenic E. coli (UPEC) from children who developed pyelonephritis carried specific genetic markers, we generated whole-genome sequence data for 96 isolates from children with UTIs. This included 57 isolates from children with either radiologically confirmed pyelonephritis or cystitis and 27 isolates belonging to the well-known multidrug-resistant sequence type ST131, selected to investigate their population structure and antibiotic resistance characteristics. We observed a UPEC population structure that is similar to those reported in adults. In comparison with prior investigations, we found that the full pap operon was more common among UPEC from pediatric cases of pyelonephritis. Further, in contrast with recent reports that the P-fimbriae adhesin-encoding papGII allele is substantially more prevalent in invasive UPEC from adults, we found papGII was common to both invasive and non-invasive UPEC from children. Among the set of ST131 isolates from children with UTIs, we found antibiotic resistance was correlated with known genetic markers of resistance, as in adults. Unexpectedly, we observed that fimH30, an allele of the fimbrial gene fimH often used as a proxy to type ST131 isolates into the most drug-resistant subclade C, was carried by some of the subclade A and subclade B isolates, suggesting that the fimH30 allele could confer a selective advantage for UPEC. IMPORTANCE Urinary tract infections (UTIs), which are most often caused by Escherichia coli, are not well studied in children. Here, we examine genetic characteristics that differentiate UTI-causing bacteria in children that either remain localized to the bladder or are involved in more serious kidney infections. We also examine patterns of antibiotic resistance among strains from children that are part of E. coli sequence type 131, a group of bacteria that commonly cause UTIs and are known to have high levels of drug resistance. This work provides new insight into the virulence and antibiotic resistance characteristics of the bacteria that cause UTIs in children.

Abstract Objectives To characterize a blaCMY variant associated with ceftazidime/avibactam resistance from a serially collected Escherichia coli isolate. Methods A patient with an intra-abdominal infection due to recurrent E. coli was treated with ceftazidime/avibactam. On Day 48 of ceftazidime/avibactam therapy, E. coli with a ceftazidime/avibactam MIC of &amp;gt;256 mg/L was identified from abdominal drainage. Illumina and Oxford Nanopore Technologies WGS was performed on serial isolates to identify potential resistance mechanisms. Site-directed mutants of CMY β-lactamase were constructed to identify amino acid residues responsible for ceftazidime/avibactam resistance. Results WGS revealed that all three isolates were E. coli ST410. The ceftazidime/avibactam-resistant strain uniquely acquired a novel CMY β-lactamase gene, herein called blaCMY-185, harboured on an IncI-γ/K1 conjugative plasmid. The CMY-185 enzyme possessed four amino acid substitutions relative to CMY-2, including A114E, Q120K, V211S and N346Y, and conferred high-level ceftazidime/avibactam resistance with an MIC of 32 mg/L. Single CMY-2 mutants did not confer reduced ceftazidime/avibactam susceptibility. However, double and triple mutants containing N346Y previously associated with ceftazidime/avibactam resistance in other AmpC enzymes, conferred ceftazidime/avibactam MICs ranging between 4 and 32 mg/L as well as reduced susceptibility to the newly developed cephalosporin, cefiderocol. Molecular modelling suggested that the N346Y substitution confers the reduction of avibactam inhibition due to steric hindrance between the side chain of Y346 and the sulphate group of avibactam. Conclusions We identified ceftazidime/avibactam resistance in E. coli associated with a novel CMY variant. Unlike other AmpC enzymes, CMY-185 appears to require an additional substitution on top of N346Y to confer ceftazidime/avibactam resistance.

The emergence of carbapenem-resistant, hypervirulent Klebsiella pneumoniae is a new threat to health care. We studied the molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae isolates in Qatar using whole-genome sequence data. We also characterized the prevalence and genetic basis of hypervirulent phenotypes and established the virulence potential using a Galleria mellonella model. Of 100 Klebsiella isolates studied, NDM and OXA-48 were the most common carbapenemases. Core genome single-nucleotide polymorphism (SNP) analysis indicated the presence of diverse sequence types and clonal lineages; isolates belonging to Klebsiella quasipneumoniae subsp. quasipneumoniae sequence type 196 (ST196) and ST1416 may be disseminated among several health care centers. Ten K. pneumoniae isolates carried rmpA and/or truncated rmpA2, and 2 isolates belonged to KL2, indicating low prevalence of classical hypervirulent isolates. Isolates carrying both carbapenem resistance and hypervirulence genes were confined mainly to ST231 and ST383 isolates. One ST383 isolate was further investigated by MinION sequencing, and the assembled genome indicated that blaNDM was located on an IncHI1B-type plasmid (pFQ61_ST383_NDM-5) which coharbored several virulence factors, including the regulator of the mucoid phenotype (rmpA), the regulator of mucoid phenotype 2 (rmpA2), and aerobactin (iucABCD and iutA), likely resulting from recombination events. Comparative genomics indicated that this hybrid plasmid may be present in two additional Qatari ST383 isolates. Carbapenem-resistant, hypervirulent K. pneumoniae ST383 isolates pose an emerging threat to global health due to their simultaneous hypervirulence and multidrug resistance.

Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) bloodstream infections rarely overwhelm the host but are associated with high mortality. The complement system is a key host defense against bloodstream infection. However, there are varying reports of serum resistance among KPC-Kp isolates. We assessed growth of 59 KPC-Kp clinical isolates in human serum and found increased resistance in 16/59 (27%). We identified five genetically-related bloodstream isolates with varying serum resistance profiles collected from a single patient during an extended hospitalization marked by recurrent KPC-Kp bloodstream infections. We noted a loss-of-function mutation in the capsule biosynthesis gene, wcaJ, that emerged during infection was associated with decreased polysaccharide capsule content, and resistance to complement-mediated killing. Surprisingly, disruption of wcaJ increased deposition of complement proteins on the microbial surface compared to the wild-type strain and led to increased complement-mediated opsono-phagocytosis in human whole blood. Disabling opsono-phagocytosis in the airspaces of mice impaired in vivo control of the wcaJ loss-of-function mutant in an acute lung infection model. These findings describe the rise of a capsular mutation that promotes KPC-Kp persistence within the host by enabling co-existence of increased bloodstream fitness and reduced tissue virulence.

Enterococcus faecalis is a hospital-associated opportunistic pathogen that can cause infections with high mortality, such as infective endocarditis. With an increasing occurrence of multidrug-resistant enterococci, there is a need for alternative strategies to treat enterococcal infections. We isolated a gentamicin-hypersusceptible E. faecalis strain from a patient with infective endocarditis that carried a mutation in the alpha-carbonic anhydrase (α-CA) and investigated how disruption of α-CA sensitized E. faecalis to killing with gentamicin. The gentamicin-hypersusceptible α-CA mutant strain showed increased intracellular gentamicin uptake in comparison to an isogenic strain encoding full-length, wild-type α-CA. We hypothesized that increased gentamicin uptake could be due to increased proton motive force (PMF), increased membrane permeability, or both. We observed increased intracellular ATP production in the α-CA mutant strain, suggesting increased PMF-driven gentamicin uptake contributed to the strain's gentamicin susceptibility. We also analyzed the membrane permeability and fatty acid composition of isogenic wild-type and α-CA mutant strains and found that the mutant displayed a membrane composition that was consistent with increased membrane permeability. Finally, we observed that exposure to the FDA-approved α-CA inhibitor acetazolamide lowered the gentamicin MIC of eight genetically diverse E. faecalis strains with intact α-CA but did not change the MIC of the α-CA mutant strain. These results suggest that α-CA mutation or inhibition increases PMF and alters membrane permeability, leading to increased uptake of gentamicin into E. faecalis. This connection could be exploited clinically to provide new combination therapies for patients with enterococcal infections. IMPORTANCE Enterococcal infections can be difficult to treat, and new therapeutic approaches are needed. In studying an E. faecalis clinical strain from an infected patient, we found that the bacteria were rendered hypersusceptible to aminoglycoside antibiotics through a mutation that disrupted the α-CA. Our follow-on work suggested two different ways that α-CA disruption causes increased gentamicin accumulation in E. faecalis: increased proton motive force-powered uptake and increased membrane permeability. We also found that a mammalian CA inhibitor could sensitize a variety of E. faecalis strains to killing with gentamicin. Given that mammalian CA inhibitors are frequently used to treat conditions such as glaucoma, hypertension, and epilepsy, our findings suggest that these "off-the-shelf" inhibitors could also be useful partner antibiotics for the treatment of E. faecalis infections.

Despite technological advancements in infectious disease rapid diagnostic tests (RDTs) and use to direct therapy at the per-patient level, RDT utilisation in antimicrobial stewardship programmes (ASPs) is variable across low-to-middle income and high-income countries. Key insights from a panel of seven infectious disease experts from Colombia, Japan, Nigeria, Thailand, the UK, and the USA, combined with evidence from a literature review, were used to assess the value of RDTs in ASPs. From this, a value framework is proposed which aims to define the benefits of RDT use in ASPs, separate from per-patient benefits. Expert insights highlight that, to realise the value of RDTs within ASPs, effective implementation is key; actionable advice for choosing an RDT is proposed. Experts advocate the inclusion of RDTs in the World Health Organization Model List of essential in vitro diagnostics and in iterative development of national action plans.

Bartonella elizabethae is a rat-borne zoonotic bacterium that causes human infectious endocarditis or neuroretinitis. Recently, a case of bacillary angiomatosis (BA) resulting from this organism was reported, leading to speculation that B. elizabethae may also trigger vasoproliferation. However, there are no reports of B. elizabethae promoting human vascular endothelial cell (EC) proliferation or angiogenesis, and to date, the effects of this bacterium on ECs are unknown. We recently identified a proangiogenic autotransporter, BafA, secreted from B. henselae and B. quintana, which are recognized as Bartonella spp. responsible for BA in humans. Here, we hypothesized that B. elizabethae also harbored a functional bafA gene and examined the proangiogenic activity of recombinant B. elizabethae-derived BafA. The bafA gene of B. elizabethae, which was found to share a 51.1% amino acid sequence identity with BafA of B. henselae and 52.5% with that of B. quintana in the passenger domain, was located in a syntenic region of the genome. The recombinant protein of the N-terminal passenger domain of B. elizabethae-BafA facilitated EC proliferation and capillary structure formation. Furthermore, it upregulated the receptor signaling pathway of vascular endothelial growth factor, as observed in B. henselae-BafA. Taken together, B. elizabethae-derived BafA stimulates human EC proliferation and may contribute to the proangiogenic potential of this bacterium. So far, functional bafA genes have been found in all BA-causing Bartonella spp., supporting the key role BafA may play in BA pathogenesis.

We evaluated the impact of discontinuing universal preadmission screening for severe acute respiratory coronavirus virus 2 (SARS-CoV-2) on the occurrence of nosocomial clusters of coronavirus disease 2019 (COVID-19) during the SARS-CoV-2 o (omicron) variant period. No increasing trend in nosocomial clusters was observed during community-based surges before and after discontinuation. This finding supports the safety of the practice change.

OBJECTIVES: We evaluated the clinical characteristics and outcomes of patients with COVID-19 who received three-drug combination regimens for treatment of carbapenem-resistant Acinetobacter baumannii (CRAB) infections during a single-centre outbreak. Our objective was to describe the clinical outcomes and molecular characteristics and in vitro synergy of antibiotics against CRAB isolates. MATERIALS AND METHODS: Patients with severe COVID-19 admitted between April and July 2020 with CRAB infections were retrospectively evaluated. Clinical success was defined as resolution of signs/symptoms of infection without need for additional antibiotics. Representative isolates underwent whole-genome sequencing (WGS) and in vitro synergy of two- or three-drug combinations was assessed by checkerboard and time-kill assays, respectively. RESULTS: Eighteen patients with CRAB pneumonia or bacteraemia were included. Treatment regimens included high-dose ampicillin-sulbactam, meropenem, plus polymyxin B (SUL/MEM/PMB; 72%), SUL/PMB plus minocycline (MIN; 17%) or other combinations (12%). Clinical resolution was achieved in 50% of patients and 30-day mortality was 22% (4/18). Seven patients had recurrent infections, during which further antimicrobial resistance to SUL or PMB was not evident. PMB/SUL was the most active two-drug combination by checkerboard. Paired isolates collected before and after treatment with SUL/MEM/PMB did not demonstrate new gene mutations or differences in the activity of two- or three-drug combinations. CONCLUSIONS: Use of three-drug regimens for severe CRAB infections among COVID-19 resulted in high rates of clinical response and low mortality relative to previous studies. The emergence of further antibiotic resistance was not detected phenotypically or through WGS analysis. Additional studies are needed to elucidate preferred antibiotic combinations linked to the molecular characteristics of infecting strains.

During chronic cystic fibrosis (CF) infections, evolved Pseudomonas aeruginosa antibiotic resistance is linked to increased pulmonary exacerbations, decreased lung function, and hospitalizations. However, the virulence mechanisms underlying worse outcomes caused by antibiotic resistant infections are poorly understood. Here, we investigated evolved aztreonam resistant P. aeruginosa virulence mechanisms. Using a macrophage infection model combined with genomic and transcriptomic analyses, we show that a compensatory mutation in the rne gene, encoding RNase E, increased pyoverdine and pyochelin siderophore gene expression, causing macrophage ferroptosis and lysis. We show that iron-bound pyochelin was sufficient to cause macrophage ferroptosis and lysis, however, apo-pyochelin, iron-bound pyoverdine, or apo-pyoverdine were insufficient to kill macrophages. Macrophage killing could be eliminated by treatment with the iron mimetic gallium. RNase E variants were abundant in clinical isolates, and CF sputum gene expression data show that clinical isolates phenocopied RNase E variant functions during macrophage infection. Together these data show how P. aeruginosa RNase E variants can cause host damage via increased siderophore production and host cell ferroptosis but may also be targets for gallium precision therapy.

BACKGROUND: There is a growing interest in Klebsiella variicola as a causative pathogen in humans, though its clinical features and the impact of co-infection or secondary infection with COVID-19 remain unknown. CASE PRESENTATION: A 71-year-old man presented with fever, altered mental status and generalized weakness and was admitted to ICU due to severe COVID-19 pneumonia. He was newly diagnosed with type II diabetes mellitus upon admission. On hospital day 3, his respiratory status deteriorated, requiring invasive mechanical ventilation. On hospital day 10, superimposed bacterial pneumonia was suspected and subsequently, broad-spectrum antibiotics were administered for the associated bloodstream infection. On hospital day 13, despite administration of active antibiotics and appropriate source control, he decompensated and died. The causative organism isolated from blood cultures was initially reported as K. pneumoniae, but it was identified as K. variicola by a genetic analysis. A representative isolate (FUJ01370) had a novel multilocus sequence typing allelic profile (gapA-infB-mdh-pgi-phoE-rpoB-tonB: 16-24-21-27-52-17-152), to which sequence type 5794 was assigned (GenBank assembly accession: GCA_019042755.1). CONCLUSIONS: We report a fatal case of respiratory and bloodstream infection due to K. variicola complicating severe COVID-19. Co-infection or secondary infection of K. variicola in COVID-19 is likely under-recognized and can be fulminant as in this case.

BACKGROUND: Limited treatment options exist for patients with mild-to-moderate coronavirus disease 2019 (COVID-19), irrespective of vaccination history or risk status. Ensitrelvir is a novel oral severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3C-like (3CL) protease inhibitor. While phase 2 studies of ensitrelvir have demonstrated promising results in treating mild-to-moderate COVID-19, evaluation of its clinical efficacy due to shifting vaccination status and emergence of the Omicron variant represents significant challenges. Here, we describe the protocol for a phase 3 study designed to evaluate the efficacy and safety of ensitrelvir in patients with mild-to-moderate COVID-19, regardless of risk status or vaccination history. METHODS: This is a multicenter, randomized, double-blind, placebo-controlled, phase 3 study. Patients with mild-to-moderate COVID-19 within 120 hours from onset will be randomized in a 1:1:1 ratio into 3 treatment arms-ensitrelvir 125 mg (375 mg loading dose on Day 1), ensitrelvir 250 mg (750 mg loading dose on Day 1), and placebo. The study interventions will be administered orally, once-daily, for 5 days. The primary endpoint will be the time to resolution of 5 symptoms of COVID-19 (stuffy or runny nose, sore throat, cough, feeling hot or feverish, and low energy or tiredness), and the key secondary endpoints will include the change from baseline on Day 4 in the amount of SARS-CoV-2 viral ribonucleic acid (RNA) and the time to first negative SARS-CoV-2 viral titer. The primary population for the primary and key secondary endpoints will be patients with <72 hours from COVID-19 onset to randomization and, subsequently, patients in entire patient population (<120 hours) in the ensitrelvir 125 mg group. Closed testing procedure will be used for the primary and key secondary endpoints in both the primary and entire patient populations. All safety assessments and adverse events (AE) will be reported. DISCUSSION: In a post hoc analysis of the phase 2b study, compared with placebo, ensitrelvir demonstrated a reduced time to resolution of 5 symptoms in patients with mild-to-moderate COVID-19. Through this study, we intend to validate and establish the efficacy and safety of ensitrelvir in patients with mild-to-moderate COVID-19.

BACKGROUND: Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is a global threat, but the distribution and clinical significance of carbapenemases are unclear. The aim of this study was to define characteristics and outcomes of CRPA infections and the global frequency and clinical impact of carbapenemases harboured by CRPA. METHODS: We conducted an observational, prospective cohort study of CRPA isolated from bloodstream, respiratory, urine, or wound cultures of patients at 44 hospitals (10 countries) between Dec 1, 2018, and Nov 30, 2019. Clinical data were abstracted from health records and CRPA isolates were whole-genome sequenced. The primary outcome was 30-day mortality from the day the index culture was collected. We compared outcomes of patients with CRPA infections by infection type and across geographic regions and performed an inverse probability weighted analysis to assess the association between carbapenemase production and 30-day mortality. FINDINGS: We enrolled 972 patients (USA n=527, China n=171, south and central America n=127, Middle East n=91, Australia and Singapore n=56), of whom 581 (60%) had CRPA infections. 30-day mortality differed by infection type (bloodstream 21 [30%] of 69, respiratory 69 [19%] of 358, wound nine [14%] of 66, urine six [7%] of 88; p=0·0012) and geographical region (Middle East 15 [29%] of 52, south and central America 20 [27%] of 73, USA 60 [19%] of 308, Australia and Singapore three [11%] of 28, China seven [6%] of 120; p=0·0002). Prevalence of carbapenemase genes among CRPA isolates also varied by region (south and central America 88 [69%] of 127, Australia and Singapore 32 [57%] of 56, China 54 [32%] of 171, Middle East 27 [30%] of 91, USA ten [2%] of 527; p<0·0001). KPC-2 (n=103 [49%]) and VIM-2 (n=75 [36%]) were the most common carbapenemases in 211 carbapenemase-producing isolates. After excluding USA patients, because few US isolates had carbapenemases, patients with carbapenemase-producing CRPA infections had higher 30-day mortality than those with non-carbapenemase-producing CRPA infections in both unadjusted (26 [22%] of 120 vs 19 [12%] of 153; difference 9%, 95% CI 3-16) and adjusted (difference 7%, 95% CI 1-14) analyses. INTERPRETATION: The emergence of different carbapenemases among CRPA isolates in different geographical regions and the increased mortality associated with carbapenemase-producing CRPA infections highlight the therapeutic challenges posed by these organisms. FUNDING: National Institutes of Health.

Bartonella species are hemotropic, facultative intracellular bacteria, some of which cause zoonoses, that are widely disseminated among many mammals, including humans. During infection in humans, vascular endothelial cells play a crucial role as a replicative niche for Bartonella, and some are capable of promoting vascular proliferation. Along with well-studied pathogenic factors such as a trimeric autotransporter adhesin BadA or VirB/D4 type IV secretion system, bacteria-secreted protein BafA is also involved in Bartonella-induced vasoproliferation. Genes encoding BafA orthologs have been found in the genomes of most Bartonella species, but their functionality remains unclear. In this study, we focused on three cat-derived zoonotic species (B. henselae, B. koehlerae, and B. clarridgeiae) and two rodent-derived species (B. grahamii and B. doshiae) and compared the activity of BafA derived from each species. Recombinant BafA proteins of B. henselae, B. koehlerae, B. clarridgeiae, and B. grahamii, species that also cause human disease, induced cell proliferation and tube formation in cultured endothelial cells, while BafA derived from B. doshiae, a species that is rarely found in humans, showed neither activity. Additionally, treatment of cells with these BafA proteins increased phosphorylation of both vascular endothelial growth factor receptor 2 and extracellular signal-regulated kinase 1/2, with the exception of B. doshiae BafA. Differential bafA mRNA expression and BafA secretion among the species likely contributed to the differences in the cell proliferation phenotype of the bacteria-infected cells. These findings suggest that the biological activity of BafA may be involved in the infectivity or pathogenicity of Bartonella species in humans.

We evaluated the impact of carbapenem shortage on antimicrobial practice and patient outcome at a tertiary care center. During the shortage, hospital antimicrobial practice could be safely managed through additional antimicrobial stewardship measures including treatment guidance and mandatory preauthorization. Antimicrobial shortage may present a unique opportunity for promoting antimicrobial stewardship.

Background: Antimicrobial resistance surveillance is essential for empiric antibiotic prescribing, infection prevention and control policies and to drive novel antibiotic discovery. However, most existing surveillance systems are isolate-based without supporting patient-based clinical data, and not widely implemented especially in low- and middle-income countries (LMICs). Methods: A Clinically-Oriented Antimicrobial Resistance Surveillance Network (ACORN) II is a large-scale multicentre protocol which builds on the WHO Global Antimicrobial Resistance and Use Surveillance System to estimate syndromic and pathogen outcomes along with associated health economic costs. ACORN-healthcare associated infection (ACORN-HAI) is an extension study which focuses on healthcare-associated bloodstream infections and ventilator-associated pneumonia. Our main aim is to implement an efficient clinically-oriented antimicrobial resistance surveillance system, which can be incorporated as part of routine workflow in hospitals in LMICs. These surveillance systems include hospitalised patients of any age with clinically compatible acute community-acquired or healthcare-associated bacterial infection syndromes, and who were prescribed parenteral antibiotics. Diagnostic stewardship activities will be implemented to optimise microbiology culture specimen collection practices. Basic patient characteristics, clinician diagnosis, empiric treatment, infection severity and risk factors for HAI are recorded on enrolment and during 28-day follow-up. An R Shiny application can be used offline and online for merging clinical and microbiology data, and generating collated reports to inform local antibiotic stewardship and infection control policies. Discussion: ACORN II is a comprehensive antimicrobial resistance surveillance activity which advocates pragmatic implementation and prioritises improving local diagnostic and antibiotic prescribing practices through patient-centred data collection. These data can be rapidly communicated to local physicians and infection prevention and control teams. Relative ease of data collection promotes sustainability and maximises participation and scalability. With ACORN-HAI as an example, ACORN II has the capacity to accommodate extensions to investigate further specific questions of interest.

The incidence of seasonal infections due to respiratory viruses other than severe acute respiratory coronavirus virus 2 (SARS-CoV-2) has declined due to heightened public infection prevention measures against coronavirus disease 2019 (COVID-19). We describe an outbreak of human coronavirus OC43 infection that occurred at a long-term care facility and whose clinical features were indistinguishable from COVID-19.