Contemporary Isolates Research Articles

Rickettsia rickettsii subsp. californica subsp. nov., the etiologic agent of Pacific Coast tick fever.

The etiologic agent of Pacific Coast tick fever (PCTF), a moderately severe tickborne illness that resembles Rocky Mountain spotted fever (RMSF), was first isolated in 1966 from specimens of Dermacentor occidentalis (the Pacific Coast tick) obtained in California. For several decades, this bacterium was identified ambiguously as the unclassified spotted fever group Rickettsia species 364-D, Rickettsia 364, or Rickettsia philipii. However, none of these epithets satisfied criteria of formal bacterial nomenclature. Data developed from mouse serotyping studies performed 45 years ago, and multi-locus sequence typing several decades later, indicated that this bacterium was similar to, but distinct from isolates of Rickettsia rickettsii, the etiological agent of RMSF. We applied an integrative taxonomic approach, combining phenotypic, ecological, and clinical data with whole genome sequencing of 11 contemporary isolates of this pathogen to identify it as a distinct subspecies of R. rickettsii, and propose the name Rickettsia rickettsii subsp. californica subsp. nov.

Revealing hidden viruses inducing similar yellowing symptoms or remaining asymptomatic in cucurbit crops

AbstractMixed viral infections occur frequently in plants, leading to diseases that can be difficult to diagnose, especially when an unknown virus is hidden behind expression of symptoms common to other viruses. We monitored aphid‐ and whitefly‐transmitted viruses in two cucurbit crops (melon and watermelon) for three consecutive seasons (2021–2023) across three production regions in Spain, focusing on the common yellowing and mosaic symptoms. A total of 984 symptomatic leaf samples from 246 field plots were tested for 10 frequently found cucurbit viruses. Cucurbit aphid‐borne yellows virus (CABYV) was the most prevalent virus infecting both cucurbit crops over the three seasons. Additionally, occurrence of watermelon mosaic virus (WMV) in samples with mosaic symptoms was significant. However, an important proportion of yellowing symptomatic samples tested negative for known viruses. Using a sequence‐independent approach, we identified a novel polerovirus, Pepo aphid‐borne yellows virus (PABYV), which was widespread, infecting both crops. Analysis of our long‐term cucurbit frozen‐sample collection revealed that PABYV had emerged in Spain in 2018, possibly unnoticed as its yellowing symptoms were similar to CABYV, with which it was frequently associated. Additionally, we found the cryptic Cucumis melo endornavirus (CmEV) in all tested melon samples from 2011 and, for the first time, in pumpkin. Genetic characterization of CABYV, PABYV and CmEV populations revealed a replacement of ancient CABYV isolates by contemporary ones, while PABYV and CmEV isolates were genetically homogenous among their populations. This study underlines the need for continuous surveillance and further investigation into common symptoms of mixed viral infections.

Investigating Anthrax-Associated Virulence Genes among Archival and Contemporary Bacillus cereus Group Genomes.

Bacillus anthracis causes anthrax through virulence factors encoded on two plasmids. However, non-B. anthracis organisms within the closely related, environmentally ubiquitous Bacillus cereus group (BCG) may cause an anthrax-like disease in humans through the partial adoption of anthrax-associated virulence genes, challenging the definition of anthrax disease. To elucidate these phenomena and their evolutionary past, we performed whole-genome sequencing on non-anthracis BCG isolates, including 93 archival (1967-2003) and 5 contemporary isolates (2019-2023). We produced annotated genomic assemblies and performed a pan-genome analysis to identify evidence of virulence gene homology and virulence gene acquisition by linear inheritance or horizontal gene transfer. At least one anthrax-associated virulence gene was annotated in ten isolates. Most homologous sequences in archival isolates showed evidence of pseudogenization and subsequent gene loss. The presence or absence of accessory genes, including anthrax-associated virulence genes, aligned with the phylogenetic structure of the BCG core genome. These findings support the hypothesis that anthrax-associated virulence genes were inherited from a common ancestor in the BCG and were retained or lost across different lineages, and contribute to a growing body of work informing public health strategies related to anthrax surveillance and identification.

Current trend of biapenem susceptibility and disc diffusion breakpoints in Enterobacterales and Pseudomonas aeruginosa

IntroductionBiapenem has been recently approved by the Drug Controller General of India for the treatment of complicated urinary tract infections (cUTI). However, there are no assessment studies that evaluate the in-vitro activity of biapenem against contemporary ESBL-producing Indian Enterobacterales isolates. To determine the activity of biapenem against contemporary ESBLs and/or OXA-1/ampC producing Enterobacterales and Pseudomonas aeruginosa isolates. MethodologyIsolates were tested for susceptibility to biapenem and its comparators using the broth microdilution method. Presence of ESBLs (SHV, TEM, CTX-M) genes, OXA-1, and ampC genes (ACC, ACT, DHA, CIT/CMY, FOX) using multiplex PCR. ResultsAgainst ESBL with OXA-1 and/or ampC-producing E. coli, ESBL-K. pneumoniae, and cephalosporin-resistant P. aeruginosa, biapenem showed in-vitro activity similar to that of meropenem. Overall, a biapenem disc concentration of 10 μg provided no error rates for testing E. coli, K. pneumoniae, and P. aeruginosa isolates. ConclusionIt is more accurate to test biapenem at a 10 μg disc concentration and apply more stringent disc diffusion breakpoints for interpretation.

Efficacies of omadacycline + amikacin + imipenem and an all-oral regimen omadacycline + clofazimine + linezolid in a mouse model of M. abscessus lung disease.

Treatment outcomes for Mycobacteroides abscessus (Mab, also known as Mycobacterium abscessus) disease are still unsatisfactory, mainly due to issues with drug toxicity, tolerability, and efficacy. Treating Mab disease is challenging due to its high baseline antibiotic resistance, initial requirement for intravenous therapy, and poor medication tolerance. Omadacycline, a new tetracycline, is active against Mab. Since any new antibiotic effective against Mab is expected to be used in combination with other antibiotics, we evaluated the efficacy of two triple-drug combinations comprising omadacycline, omadacycline + amikacin + imipenem, and omadacycline + clofazimine + linezolid against two contemporary Mab clinical isolates in a mouse model of Mab lung disease. Antibiotic administration was initiated 1-week post-infection and was given daily, with Mab burden in the lungs at treatment completion serving as the endpoint. Omadacycline alone moderately reduced Mab levels and maintained better health in mice compared to untreated ones, which typically suffered from the infection. The omadacycline + clofazimine + linezolid combination showed immediate bactericidal activity and enhanced efficacy over 6 weeks, particularly against the more resistant strain (M9507). However, the clofazimine + linezolid combination lacked early bactericidal activity. When combined with amikacin and imipenem, omadacycline did not improve the regimen's effectiveness over 4 weeks of treatment. Our study showed that omadacycline + clofazimine + linezolid exhibited significant bactericidal activity over an extended treatment duration. However, adding omadacycline to amikacin and imipenem did not improve regimen effectiveness against the evaluated clinical isolates within 4 weeks. Further research in Mab disease patients is needed to determine the most effective omadacycline-containing regimen.IMPORTANCEMycobacteroides abscessus is a common environmental bacterium that causes infections in people with compromised lung function, including those with bronchiectasis, cystic fibrosis, chronic obstructive pulmonary disease, and weakened immune systems, especially among older individuals. Treating M. abscessus disease is challenging due to the limited effectiveness and toxicity of current antibiotics, which often require prolonged use. Omadacycline, a new antibiotic, shows promise against M. abscessus. Using a mouse model that mimics M. abscessus disease in humans, we studied the effectiveness of including omadacycline with recommended antibiotics. Adding omadacycline to clofazimine and linezolid significantly improved treatment outcomes, rapidly clearing the bacteria from the lungs and maintaining effectiveness throughout. This oral combination is convenient for patients. However, adding omadacycline to amikacin and imipenem did not improve treatment effectiveness within 4 weeks. Further study with M. abscessus patients is necessary to optimize omadacycline-based treatment strategies for this disease.

Resistance to aztreonam-avibactam among clinical isolates of Escherichia coli is primarily mediated by altered penicillin-binding protein 3 and impermeability

This study was conducted to investigate decreased susceptibility (minimum inhibitory concentrations [MICs] 0.25–4 mg/L) and resistance (MICs > 4 mg/L) to aztreonam-avibactam (ATM-AVI). Contemporary non-replicate clinical isolates of carbapenemase-producing Escherichia coli (CP-EC) (n=90) and ESBL-producing E. coli (EP-EC) (n=12) were used. CP-EC belonged to 25 distinct sequence types (STs) and all EP-EC belonged to ST405. All strains were isolated from 2019 to 2022 at the Karolinska University Laboratory, Stockholm, Sweden. ATM-AVI MICs were determined using broth microdilution. The EUCAST epidemiological cut-off value of 0.125 mg/L was used to define the wild type (WT). Whole-genome sequences (Illumina) were analysed for detecting resistance determinants among WT vs. non-WT isolates. Among 102 isolates, 40 (39%) and 62 (61%) were WT and non-WT, respectively. Among non-WT isolates, resistance was noted for 20 and decreased susceptibility for 42. Resistance was observed among 14/47 New Delhi metallo-β-lactamase (NDM)-producers, 5/43 OXA-48 group producers, and 1/12 EP-EC. Decreased susceptibility was observed among 29/47 NDM, 13/43 OXA-48 group, and 3/12 EP-EC. Resistant isolates predominantly belonged to ST405, followed by STs 410, 361, 167, 617, and 1284. Penicillin-binding protein 3 (PBP3) inserts (YRIK/YRIN) were observed in 20/20 and CMY-42 in 5/20 resistant isolates. Several mutations in the ftsI (encoding PBP3) and regulatory genes of outer membrane proteins (OmpC and OmpF) and efflux pumps (AcrAB-TolC) were detected. A ≥ 2-fold reduction in MICs was observed among 20/20 vs. 7/20 isolates tested in the presence of the membrane permeabiliser, polymyxin B nanopeptide (PMBN) and efflux inhibitor, phenylalanine arginine β-naphthylamide (PAβN), respectively. In conclusion, resistance to ATM-AVI is a result of interplay of various determinants, including target alterations, deactivating enzymes, and decreased permeability.

Multistate Sensitivity Monitoring of Fusarium virguliforme to the SDHI Fungicides Fluopyram and Pydiflumetofen in the United States.

Sudden death syndrome (SDS), caused by Fusarium virguliforme, is an important yield-limiting disease of soybean (Glycine max). From 1996 to 2022, cumulative yield losses attributed to SDS in North America totaled over 25 million metric tons, which was valued at over US $7.8 billion. Seed treatments are widely used to manage SDS by reducing early season soybean root infection by F. virguliforme. Fluopyram (succinate dehydrogenase inhibitor [SDHI] - FRAC 7), a fungicide seed treatment for SDS management, has been registered for use on soybean in the United States since 2014. A baseline sensitivity study conducted in 2014 evaluated 130 F. virguliforme isolates collected from five states to fluopyram in a mycelial growth inhibition assay and reported a mean EC50 of 3.35 mg/liter. This baseline study provided the foundation for the objectives of this research: to detect any statistically significant change in fluopyram sensitivity over time and geographical regions within the United States and to investigate sensitivity to the fungicide pydiflumetofen. We repeated fluopyram sensitivity testing on a panel of 80 historical F. virguliforme isolates collected from 2006 to 2013 (76 of which were used in the baseline study) and conducted testing on 123 contemporary isolates collected from 2016 to 2022 from 11 states. This study estimated a mean absolute EC50 of 3.95 mg/liter in isolates collected from 2006 to 2013 and a mean absolute EC50 of 4.19 mg/liter in those collected in 2016 to 2022. There was no significant change in fluopyram sensitivity (P = 0.1) identified between the historical and contemporary isolates. A subset of 23 isolates, tested against pydiflumetofen under the same conditions, estimated an absolute mean EC50 of 0.11 mg/liter. Moderate correlation was detected between fluopyram and pydiflumetofen sensitivity estimates (R = 0.53; P < 0.001). These findings enable future fluopyram and pydiflumetofen resistance monitoring and inform current soybean SDS management strategies in a regional and national context.

Cefepime/sulbactam combination as a treatment alternative for extended spectrum beta-lacatmase producing Enterobacterales: A multicentric study from India

BackgroundPiperacillin/tazobactam has limited activity against Enterobacterales, which concurrently express ESBL/ampC/OXA-1 beta-lactamases, which are widely prevalent in India. Thus, there is a severe dearth for carbapenem sparing options that aggravate more dependent carbapenem therapy. In the Indian market, various cefepime/BLI combinations in diiferent fixed ratios were introduced to clinical practice. We investigated the activity of cefepime with increased sulbactam ratios against contemporary isolates of ESBL/OXA-1 expressing Enterobacterales. MethodsNon-duplicate contemporary ESBL and/or OXA-1 expressing E. coli (n = 117) and K. pneumoniae (n = 71) isolates were included in this study. The MIC of cefepime/sulbactam and its comparators were determined using a broth microdilution method. The presence of ESBL and OXA-1 genes were identified using multiplex PCR. ResultsAgainst ESBL and OXA-1 co-producing E. coli, cefepime combined with sulbactam at 8 mg/L, inhibited 82% of the isolates. Cefoperazone susceptibility against ESBL-E. coli increased from 52% at 4 mg/L to 77% at 8 mg/L with sulbactam. When tested against K. pneumoniae co-expressing ESBL and OXA-1, cefepime susceptibility was 77% at 4 mg/L sulbactam and 88% at 8 mg/L sulbactam. PTZ consistently showed limited activity against ESBL co-harbouring OXA-1 (E. coli, 43% and K. pneumoniae, 61%). ConclusionThe present study demonstrates that adding an increased sulbactam concentration enhances cefepime's activity against contemporary ESBL/OXA-1 expressing Enterobacterales. A reformulated cefepime/sulbactam (2 g/2 g) combination may be the best carbapenem sparing option until cefepime/tazobactam and cefepime/enmetazobactam become available in the future.

Pandemic Risk Assessment for Swine Influenza A Virus in Comparative In Vitro and In Vivo Models.

Swine influenza A viruses pose a public health concern as novel and circulating strains occasionally spill over into human hosts, with the potential to cause disease. Crucial to preempting these events is the use of a threat assessment framework for human populations. However, established guidelines do not specify which animal models or in vitro substrates should be used. We completed an assessment of a contemporary swine influenza isolate, A/swine/GA/A27480/2019 (H1N2), using animal models and human cell substrates. Infection studies in vivo revealed high replicative ability and a pathogenic phenotype in the swine host, with replication corresponding to a complementary study performed in swine primary respiratory epithelial cells. However, replication was limited in human primary cell substrates. This contrasted with our findings in the Calu-3 cell line, which demonstrated a replication profile on par with the 2009 pandemic H1N1 virus. These data suggest that the selection of models is important for meaningful risk assessment.

The complete genome sequence of a new fowl adenovirus (Fadv-4) strain from a recent outbreak in a chicken farm in Iran.

The whole genomic sequence of fowl adenovirus C (FAdV-4) strain FAdV-4/Pasouk, isolated from chickens with hepatitis-hydropericardium syndrome (HHS) from an outbreak in Iran, has been deposited in GenBank under accession number ON652872. Notably, this FAdV-4 isolate exhibited significant genetic similarities to contemporary isolates originating from China, indicating a shared ancestry.

Sulbactam-durlobactam: a β-lactam/β-lactamase inhibitor combination targeting Acinetobacter baumannii.

Sulbactam-durlobactam is a pathogen-targeted β-lactam/β-lactamase inhibitor combination that has been approved by the US FDA for the treatment of hospital-acquired and ventilator-associated bacterial pneumonia caused by susceptible isolates of Acinetobacter baumannii-calcoaceticus complex (ABC) in patients 18years of age and older. Sulbactam is a penicillin derivative with antibacterial activity against Acinetobacter but is prone to hydrolysis by β-lactamases encoded by contemporary isolates. Durlobactam is a diazabicyclooctane β-lactamase inhibitor with activity against Ambler classes A, C and D serine β-lactamases that restores sulbactam activity both in vitro and in vivo against multidrug-resistant ABC. Sulbactam-durlobactam is a promising alternative therapy for the treatment of serious Acinetobacter infections, which can have high rates of mortality.

Whole-Genome sequencing in routine Mycobacterium bovis epidemiology - scoping the potential.

Mycobacterium bovis the main agent of bovine tuberculosis (bTB), presents as a series of spatially-localised micro-epidemics across landscapes. Classical molecular typing methods applied to these micro-epidemics, based on genotyping a few variable loci, have significantly improved our understanding of potential epidemiological links between outbreaks. However, they have limited utility owing to low resolution. Conversely, whole-genome sequencing (WGS) provides the highest resolution data available for molecular epidemiology, producing richer outbreak tracing, insights into phylogeography and epidemic evolutionary history. We illustrate these advantages by focusing on a common single lineage of M. bovis (1.140) from Northern Ireland. Specifically, we investigate the spatial sub-structure of 20 years of herd-level multi locus VNTR analysis (MLVA) surveillance data and WGS data from a down sampled subset of isolates of this MLVA type over the same time frame. We mapped 2108 isolate locations of MLVA type 1.140 over the years 2000-2022. We also mapped the locations of 148 contemporary WGS isolates from this lineage, over a similar geographic range, stratifying by single nucleotide polymorphism (SNP) relatedness cut-offs of 15 SNPs. We determined a putative core range for the 1.140 MLVA type and SNP-defined sequence clusters using a 50 % kernel density estimate, using cattle movement data to inform on likely sources of WGS isolates found outside of core ranges. Finally, we applied Bayesian phylogenetic methods to investigate past population history and reproductive number of the 1.140 M. bovis lineage. We demonstrate that WGS SNP-defined clusters exhibit smaller core ranges than the established MLVA type - facilitating superior disease tracing. We also demonstrate the superior functionality of WGS data in determining how this lineage was disseminated across the landscape, likely via cattle movement and to infer how its effective population size and reproductive number has been in flux since its emergence. These initial findings highlight the potential of WGS data for routine monitoring of bTB outbreaks.

Historic methicillin-resistant Staphylococcus aureus: expanding current knowledge using molecular epidemiological characterization of a Swiss legacy collection.

Few methicillin-resistant Staphylococcus aureus (MRSA) from the early years of its global emergence have been sequenced. Knowledge about evolutionary factors promoting the success of specific MRSA multi-locus sequence types (MLSTs) remains scarce. We aimed to characterize a legacy MRSA collection isolated from 1965 to 1987 and compare it against publicly available international and local genomes. We accessed 451 historic (1965-1987) MRSA isolates stored in the Culture Collection of Switzerland, mostly collected from the Zurich region. We determined phenotypic antimicrobial resistance (AMR) and performed whole genome sequencing (WGS) using Illumina short-read sequencing on all isolates and long-read sequencing on a selection with Oxford Nanopore Technology. For context, we included 103 publicly available international assemblies from 1960 to 1992 and sequenced 1207 modern Swiss MRSA isolates from 2007 to 2022. We analyzed the core genome (cg)MLST and predicted SCCmec cassette types, AMR, and virulence genes. Among the 451 historic Swiss MRSA isolates, we found 17 sequence types (STs) of which 11 have been previously described. Two STs were novel combinations of known loci and six isolates carried previously unsubmitted MLST alleles, representing five new STs (ST7843, ST7844, ST7837, ST7839, and ST7842). Most isolates (83% 376/451) represented ST247-MRSA-I isolated in the 1960s, followed by ST7844 (6% 25/451), a novel single locus variant (SLV) of ST239. Analysis by cgMLST indicated that isolates belonging to ST7844-MRSA-III cluster within the diversity of ST239-MRSA-III. Early MRSA were predominantly from clonal complex (CC)8. From 1980 to the end of the twentieth century, we observed that CC22 and CC5 as well as CC8 were present, both locally and internationally. The combined analysis of 1761 historic and contemporary MRSA isolates across more than 50 years uncovered novel STs and allowed us a glimpse into the lineage flux between Swiss-German and international MRSA across time.

Descriptive analysis of targeted carbapenemase genes and antibiotic susceptibility profiles among carbapenem-resistant Acinetobacter baumannii tested in the Antimicrobial Resistance Laboratory Network-United States, 2017-2020.

Acinetobacter baumannii is a Gram-negative bacillus that can cause severe and difficult-to-treat healthcare-associated infections. A. baumannii can harbor mobile genetic elements carrying genes that produce carbapenemase enzymes, further limiting therapeutic options for infections. In the United States, the Antimicrobial Resistance Laboratory Network (AR Lab Network) conducts sentinel surveillance of carbapenem-resistant Acinetobacter baumannii (CRAB). Participating clinical laboratories sent CRAB isolates to the AR Lab Network for characterization, including antimicrobial susceptibility testing and molecular detection of class A (Klebsiella pneumoniae carbapenemase), class B (Active-on-Imipenem, New Delhi metallo-β-lactamase, and Verona integron-encoded metallo-β-lactamase), and class D (Oxacillinase, blaOXA-23-like, blaOXA-24/40-like, blaOXA-48-like, and blaOXA-58-like) carbapenemase genes. During 2017‒2020, 6,026 CRAB isolates from 45 states were tested for targeted carbapenemase genes; 1% (64 of 5,481) of CRAB tested for targeted class A and class B genes were positive, but 83% (3,351 of 4,041) of CRAB tested for targeted class D genes were positive. The number of CRAB isolates carrying a class A or B gene increased from 2 of 312 (<1%) tested in 2017 to 26 of 1,708 (2%) tested in 2020. Eighty-three percent (2,355 of 2,846) of CRAB with at least one of the targeted carbapenemase genes and 54% (271 of 500) of CRAB without were categorized as extensively drug resistant; 95% (42 of 44) of isolates carrying more than one targeted gene had difficult-to-treat susceptibility profiles. CRAB isolates carrying targeted carbapenemase genes present an emerging public health threat in the United States, and their rapid detection is crucial to improving patient safety.IMPORTANCEThe Centers for Disease Control and Prevention has classified CRAB as an urgent public health threat. In this paper, we used a collection of >6,000 contemporary clinical isolates to evaluate the phenotypic and genotypic properties of CRAB detected in the United States. We describe the frequency of specific carbapenemase genes detected, antimicrobial susceptibility profiles, and the distribution of CRAB isolates categorized as multidrug resistant, extensively drug-resistant, or difficult to treat. We further discuss the proportion of isolates showing susceptibility to Food and Drug Administration-approved agents. Of note, 84% of CRAB tested harbored at least one class A, B, or D carbapenemase genes targeted for detection and 83% of these carbapenemase gene-positive CRAB were categorized as extensively drug resistant. Fifty-four percent of CRAB isolates without any of these carbapenemase genes detected were still extensively drug-resistant, indicating that infections caused by CRAB are highly resistant and pose a significant risk to patient safety regardless of the presence of one of these carbapenemase genes.

A novel antibiotic class targeting the lipopolysaccharide transporter

Carbapenem-resistant Acinetobacter baumannii (CRAB) has emerged as a major global pathogen with limited treatment options1. No new antibiotic chemical class with activity against A. baumannii has reached patients in over 50 years1. Here we report the identification and optimization of tethered macrocyclic peptide (MCP) antibiotics with potent antibacterial activity against CRAB. The mechanism of action of this molecule class involves blocking the transport of bacterial lipopolysaccharide from the inner membrane to its destination on the outer membrane, through inhibition of the LptB2FGC complex. A clinical candidate derived from the MCP class, zosurabalpin (RG6006), effectively treats highly drug-resistant contemporary isolates of CRAB both in vitro and in mouse models of infection, overcoming existing antibiotic resistance mechanisms. This chemical class represents a promising treatment paradigm for patients with invasive infections due to CRAB, for whom current treatment options are inadequate, and additionally identifies LptB2FGC as a tractable target for antimicrobial drug development.

Interhospital Spread of blaVIM-1- and blaCTX-M-15-Producing K. pneumoniae ST15 on an IncR Plasmid in Southern Spain.

In 2014-2015, the main CTX-M-15- and OXA-48-producing clone in our region was ST15. Recently, K. pneumoniae ST15 isolates co-producing VIM-1 and CTX-M-15 were detected in several hospitals. The aim was to study the emergence and acquisition of this carbapenemase. Between 2017 and 2019, four hospitals submitted twenty-nine VIM-1- and CTX-M-15-producing K. pneumoniae ST15 isolates to our laboratory. Seven representatives of each XbaI PFGE pulsotype were sequenced using short- and long-read technologies. RAST, CGE databases, and Pathogenwatch were used for resistance determinants and capsule-type analysis. Plasmid comparison was performed with Easyfig2.1. Phylogenetic analysis included other contemporary ST15 isolates from Spain. The 29 isolates were clustered into seven different pulsotypes. The selected genomes, from three hospitals in two different provinces, were clustered together (fewer than 35 alleles) and differed by more than 100 alleles from other ST15 isolates obtained in the region. These seven isolates harbored one IncR plasmid (200-220 kb) with a common backbone and four regions flanked by IS26: one contained blaVIM-1, another contained blaCTX-M-15, the third contained blaOXA-1, and the fourth harbored heavy-metal-tolerance genes. The two initial plasmids, from two different centers, were identical, and rearrangement of four regions was observed in the five subsequent plasmids. Our findings showed the first intercenter dissemination of IncR plasmids carrying blaVIM-1, blaCTX-M-15, and metal-tolerance genes mediated by a new lineage of K. pneumoniae ST15. Two different capture events of the blaVIM-1 gene or different IS26-mediated plasmid rearrangements from a common ancestor may explain plasmid variations.

Antifungal susceptibility testing following the CLSI M27 document, along with the measurement of MFC/MIC ratio, could be the optimal approach to detect amphotericin B resistance in Clavispora (Candida) lusitaniae. Susceptibility patterns of contemporary isolates of this species.

Antifungal susceptibility testing (AST) is crucial in clinical settings to guide appropriate therapy. Nevertheless, discrepancies between treatment response and some results still persist, particularly in detecting resistance to amphotericin B (AMB) in Clavispora (Candida) lusitaniae. This study aimed to assess the susceptibility patterns of 48 recent isolates of C. lusitaniae to 9 antifungal agents and explore the feasibility of using a CLSI reference-based method to identify AMB resistance. Microdilution techniques revealed a wide range of minimal inhibitory concentration (MIC) values for azole antifungals, while echinocandins and AMB exhibited a narrow range of MIC values, with all strains considered wild-type for the tested polyene and echinocandins. However, when agar diffusion (ellipsometry) was employed for AST, certain strains displayed colonies within the inhibition ellipse, indicating potential resistance. Interestingly, these strains did not respond to AMB treatment and were isolated during AMB treatment (breakthrough). Moreover, the evaluation of AMB minimum fungicidal concentrations (MFCs) indicated that only the strains with colonies inside the ellipse had MFC/MIC ratios ≥ 4, suggesting reduced fungicidal activity. In conclusion, this study confirms the effectiveness of ellipsometry with RPMI-1640 2% glucose agar for detecting AMB resistance in C. lusitaniae. Additionally, the proposed approach of culturing "clear" wells in the microdilution method can aid in uncovering resistant strains. The findings highlight the importance of appropriate AST methods to guide effective treatment strategies for deep-seated candidiasis caused by C. lusitaniae. Further collaborative studies are warranted to validate these findings and improve the detection of AMB clinical resistance.

2790. Activity of Cefiderocol and Comparator Agents Against Pediatric Isolates of Enterobacterales, Pseudomonas aeruginosa, Acinetobacter baumannii-calcoaceticus species complex, and Stenotrophomonas maltophilia from the SENTRY Surveillance Program (2020-2022)

Abstract Background Cefiderocol (CFDC) is a siderophore cephalosporin with broad activity against Gram-negative bacteria including multi-drug resistant isolates. The in vitro activity of CFDC and comparator agents was evaluated against pediatric (0-17 years old) isolates collected in 2020–2022 as part of the SENTRY Antimicrobial Surveillance Program. In vitro Activity of Cefiderocol and Comparator Agents Against Pediatric Isolates Methods 2,249 Enterobacterales (ENT), 707 P. aeruginosa, 194 Acinetobacter baumannii-calcoaceticus complex (ABC) and 220 S. maltophilia from the USA and Europe were tested for susceptibility (%S) by broth microdilution with cation-adjusted Mueller-Hinton broth (CAMHB). Iron-depleted CAMHB was applied for CFDC. Comparators included newer β-lactam/β-lactamase inhibitor (BL/BLI) combinations ceftazidime-avibactam (CZA), ceftolozane-tazobactam (C/T), imipenem-relebactam (I-R) and meropenem-vaborbactam (MVB) as well as ampicillin/sulbactam (SAM) meropenem (MEM) and colistin (CST). %S was interpreted according to 2023 CLSI &amp; EUCAST breakpoints. Carbapenem non-susceptible (CarbNS) was defined as non-susceptibility to imipenem and MEM. Results All agents displayed &amp;gt;96 %S for ENT while CFDC (MIC50/90, 2/4 mg/L; 91.7 %S) was the most active agent against CarbNS ENT. P. aeruginosa susceptibilities to CFDC and BL/BLI combinations were &amp;gt;97.0%. CFDC was the most potent agent against CarbNS P. aeruginosa with MIC50/90 values of 0.12/0.25 mg/L and 100 %S &amp; 98.5 %S per CLSI &amp; EUCAST breakpoints, respectively. ABC susceptibility to CFDC was &amp;gt;97% per CLSI &amp; EUCAST while the susceptibility for MEM was 87.1% (CLSI &amp; EUCAST) and SAM was 80.9% (CLSI). CFDC (MIC50/90, 0.25/1 mg/L; 95.7 %S) displayed better in vitro potency in CarbNS ABC as compared to SAM (MIC50/90, 32/ &amp;gt;64 mg/L; 17.4 %S CLSI) and CST (MIC50/90, 0.5/ &amp;gt;8 mg/L; 78.3%S EUCAST). Among pediatric S. maltophilia, CFDC was the most active agent with MIC50/90, 0.06/0.25 and 100 %S per CLSI &amp; EUCAST breakpoints. Conclusion CFDC was a highly active β-lactam against contemporary pediatric isolates of Enterobacterales, P. aeruginosa, ABC, and S. maltophilia, including CarbNS subsets for which treatment options are limited. These data suggest CFDC may be a valuable treatment for serious Gram-negative infections in pediatric patients. Disclosures Sean T. Nguyen, PharmD, Shionogi: Employee|Shionogi, Inc: Employee Boudewijn L. DeJonge, PhD, Shionogi Inc.: Employee Jason J. Bryowsky, PharmD, MS, Shionogi Inc.: Employee Anne Henriksen, PhD, Shionogi: Employee Christopher M. Longshaw, PhD, Shionogi BV: Employee Joshua Maher, PhD, AbbVie: Grant/Research Support|Affinity Biosensors: Grant/Research Support|AimMax Therapeutics, Inc: Grant/Research Support|Alterity Therapeutics: Grant/Research Support|Amicrobe, Inc: Grant/Research Support|Arietis Pharma: Grant/Research Support|Armata Pharmaceuticals, Inc: Grant/Research Support|Astrellas Pharma, Inc.: Grant/Research Support|Basilea Pharmaceutica AG: Grant/Research Support|Becton Dickinson And Company: Grant/Research Support|bioMerieux, Inc: Grant/Research Support|Boost Biomes: Grant/Research Support|Diamond V: Grant/Research Support|Fedora Pharmaceuticals, Inc: Grant/Research Support|Iterum Therapeutics plc: Grant/Research Support|Johnson &amp; Johnson: Grant/Research Support|Kaleido Biosciences, Inc.: Grant/Research Support|Meiji Seika Pharma Co. Ltd.: Grant/Research Support|National Institutes of Health: Grant/Research Support|Pfizer Inc.: Grant/Research Support|Roche Holding AG: Grant/Research Support|Shionogi Inc.: Grant/Research Support|Summmit Therapeutics, Inc.: Grant/Research Support|Zoetis Inc: Grant/Research Support Rodrigo E. Mendes, PhD, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|Cipla: Grant/Research Support|Entasis: Grant/Research Support|GSK: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support Miki Takemura, n/a, Shionogi &amp; Co., Ltd.: Stocks/Bonds Yoshinori Yamano, PhD, Shionogi HQ: Employee

Bacitracin enhances ceftriaxone susceptibility of the high-level ceftriaxone-resistant gonococcal FC428 clone

ABSTRACT Neisseria gonorrhoeae is a multidrug-resistant bacterial pathogen for which ceftriaxone mono-antimicrobial therapy and ceftriaxone plus azithromycin dual-antimicrobial therapy are the only remaining effective therapies. However, ceftriaxone efficacy is threatened by the global dissemination of the high-level ceftriaxone-resistant FC428 clone, while azithromycin has in recent years been removed from the recommended dual therapy in some countries as a result of increasing (high-level) azithromycin resistance. Inclusion of an alternative second antimicrobial could be an effective strategy to protect ceftriaxone from (further) resistance development. In this study, we investigated gonococcal susceptibility to bacitracin and determined its synergistic anti-gonococcal activity with ceftriaxone. Gonococcal susceptibility to bacitracin was investigated for 449 contemporary clinical isolates using the agar dilution method. Bacitracin displayed consistent activity against the N. gonorrhoeae strain collection, with a minimum inhibitory concentration (MIC) range of 2–32 mg/L and a MIC 90 of 32 mg/L. Furthermore, bacitracin was bactericidal in time-kill assays, particularly against the tested FC428-associated isolate, which showed 10 5 -fold inactivation after 8 hours of exposure to a 4× MIC dose. Importantly, bacitracin and ceftriaxone displayed synergistic activity, with a fractional inhibitory concentration index ≤0.5 for the majority of tested strains. Finally, spot assays showed strong synergistic activity against the FC428-associated isolates, with up to 200-fold reduced plating efficacy for bacitracin combined with ceftriaxone compared with ceftriaxone only. In conclusion, bacitracin shows synergistic anti-gonococcal activity with ceftriaxone and might therefore be an interesting candidate for inclusion in a dual-antimicrobial therapy with ceftriaxone. IMPORTANCE Ceftriaxone-based antimicrobial therapies for gonorrhea are threatened by waning ceftriaxone susceptibility levels and the global dissemination of the high-level ceftriaxone-resistant gonococcal FC428 clone. Combination therapy can be an effective strategy to restrain the development of ceftriaxone resistance, and for that purpose, it is important to find an alternative antimicrobial to replace azithromycin, which has recently been removed in some countries from the recommended ceftriaxone plus azithromycin dual-antimicrobial therapy. Ideally, the second antimicrobial should display synergistic activity with ceftriaxone. We hypothesized that bacitracin might display synergistic activity with ceftriaxone because of their distinct mechanisms targeting bacterial cell wall synthesis. In this study, we showed that bacitracin indeed displays synergistic activity with ceftriaxone against Neisseria gonorrhoeae . Importantly, strains associated with the FC428 clone appeared to be particularly susceptible to the bacitracin plus ceftriaxone combination, which might therefore be an interesting dual therapy for further in vivo testing.

Impact of piperaquine resistance in Plasmodium falciparum on malaria treatment effectiveness in The Guianas: a descriptive epidemiological study

Plasmodium falciparum is an apicomplexan parasite responsible for lethal cases of malaria. According to WHO recommendations, P falciparum cases are treated with artemisinin-based combination therapy including dihydroartemisinin-piperaquine. However, the emergence of resistant parasites against dihydroartemisinin-piperaquine was reported in southeast Asia in 2008 and, a few years later, suspected in South America. To characterise resistance emergence, a treatment efficacy study was performed on the reported patients infected with P falciparum and treated with dihydroartemisinin-piperaquine in French Guiana (n=6, 2016-18). Contemporary isolates collected in French Guiana were genotyped for P falciparum chloroquine resistance transporter (pfCRT; n=845) and pfpm2 and pfpm3 copy number (n=231), phenotyped using the in vitro piperaquine survival assay (n=86), and analysed through genomic studies (n=50). Additional samples from five Amazonian countries and one outside the region were genotyped (n=1440). In field isolates, 40 (47%) of 86 (95% CI 35·9-57·1) were resistant to piperaquine in vitro; these phenotypes were more associated with pfCRTC350R (ie, Cys350Arg) and pfpm2 and pfpm3 amplifications (Dunn test, p<0·001). Those markers were also associated with dihydroartemisinin-piperaquine treatment failure (n=3 [50%] of 6). A high prevalence of piperaquine resistance markers was observed in Suriname in 19 (83%) of 35 isolates and in Guyana in 579 (73%) of 791 isolates. The pfCRTC350R mutation emerged before pfpm2 and pfpm3 amplification in a temporal sequence different from southeast Asia, and in the absence of artemisinin partial resistance, suggesting a geographically distinctive epistatic relationship between these genetic markers. The high prevalence of piperaquine resistance markers in parasite populations of the Guianas, and the risk of associated therapeutic failures calls for caution on dihydroartemisinin-piperaquine use in the region. Furthermore, greater attention should be given to potential differences in genotype to phenotype mapping across genetically distinct parasite populations from different continents. Pan American Health Organization and WHO, French Ministry for Research, European Commission, Santé publique France, Agence Nationale de la Recherche, Fundação de Amparo à Pesquisa do Estado do Amazonas, Ministry of Health of Brazil, Oswaldo Cruz Foundation, and National Institutes of Health. For the French and Portuguese translations of the abstract see Supplementary Materials section.