Clinical Isolates Research Articles

Integrating the Transcriptome and Proteome to Postulate That TpiA and Pyk Are Key Enzymes Regulating the Growth of Mycoplasma Bovis.

Mycoplasma bovis is a global problem for the cattle industry due to its high infection rates and associated morbidity, although its pathophysiology is poorly understood. In this study, the M. bovis transcriptome and proteome were analyzed to further investigate the biology of clinical isolates of M. bovis. A differential analysis of M. bovis, a clinical isolate (NX114), and an international type strain (PG45) at the logarithmic stage of growth, was carried out using prokaryotic transcriptome and 4D-label-free quantitative non-labeled proteomics. Transcriptomics and proteomics identified 193 DEGs and 158 DEPs, respectively, with significant differences in 49 proteins/34 transcriptomic CDS post-translational protein sequences (15 jointly up-regulated and 21 jointly down-regulated). GO comments indicate membrane, cytoplasmic and ribosome proteins were important components of the total proteins of M. bovis NX114 clinical isolate. KEGG enrichment revealed that M. bovis NX114 is mainly associated with energy metabolism, the biosynthesis of secondary metabolites, and the ABC transporters system. In addition, we annotated a novel adhesion protein that may be closely related to M. bovis infection. Triosephosphate isomerase (TpiA) and Pyruvate kinase (Pyk) genes may be the key enzymes that regulate the growth and maintenance of M. bovis and are involved in the pathogenic process as virulence factors. The results of the study revealed the biology of different isolates of M. bovis and may provide research ideas for the pathogenic mechanism of M. bovis.

3,5-disubstituted pyridines with potent activity against drug-resistant Mycobacterium tuberculosis clinical isolates.

Aim: We designed and synthesized a series of compounds with a 3,5-disubstituted pyridine moiety and evaluated them against Mycobacterium tuberculosis (Mtb) and drug-resistant Mtb clinical isolates.Methodology: A library of 3,5-disubstituted pyridine was synthesized. The compounds were screened for activity against M. tuberculosis H37Rv. The optimal substitutions needed for the activity were identified through structure-activity relationship (SAR) studies.Results: From the screening studies, compounds 24 and 26 were identified as potent members of this series with Minimum Inhibitory Concentration (MIC) of 1.56μg/ml against M. tuberculosis H37Rv. These compounds did not show any inhibition against a panel of ESKAPE pathogens at >50μg/ml indicating their selective killing of M. tuberculosis H37Rv. Importantly, compound 24 showed a selectivity index of 54.64 against CHO-K1 and 78.26 against VERO cell lines, while compound 26 showed a selectivity index of 108.5 against CHO-K1 and 63.2 against VERO cell lines, respectively. Compound 24 formed a stable complex with the target protein DprE1 with predicted binding energy -8.73kcal/mol and inhibited multidrug-resistant clinical isolate of M. tuberculosis at 6.25μg/ml.Conclusion: This study identified the 3,5-disubstituted pyridine derivative 24 with potent antituberculosis activity and can be taken forward to generate new preclinical candidate.

Burkholderia multivorans Infections Associated with Use of Ice and Water from Ice Machines for Patient Care Activities - Four Hospitals, California and Colorado, 2020-2024.

Ice machines can harbor water-related organisms, and the use of ice or tap water for clinical care activities has been associated with infections in health care settings. During 2021-2022, a total of 23 cases of infection by Burkholderia multivorans (sequence type ST659) were reported at two southern California hospitals and linked to contaminated ice and water from ice machines. In addition to these 23 cases, this report also includes 23 previously unreported cases of B. multivorans ST659 infections that occurred during 2020-2024: 13 at a northern California hospital, eight at a hospital in Colorado, and two additional cases at one of the southern California hospitals. The same brand of ice machine and brands of filters, descaling, and sanitizing products were used by all four hospitals; B. multivorans was isolated from samples collected from ice machines in two of the hospitals. Whole genome sequencing indicated that all clinical and ice machine isolates were highly genetically similar (0-14 single nucleotide variant differences across 81% of the selected reference genome). Recommendations from public health officials to halt the outbreak included avoiding ice and tap water during clinical care activities. An investigation is ongoing to determine possible sources of ice machine contamination. During outbreaks of water-related organisms in health care facilities, health care personnel should consider avoiding the use of tap water, including ice and water from ice machines, for patient care.

Discovery of benzo[c]phenanthridine derivatives with potent activity against multidrug-resistant Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb), the pathogen responsible for tuberculosis (TB), is the leading cause of bacterial disease-related death worldwide. Current antibiotic regimens for the treatment of TB remain dated and suffer from long treatment times as well as the development of drug resistance. As such, the search for novel chemical modalities that have selective or potent anti-Mtb properties remains an urgent priority, particularly against multidrug-resistant (MDR) Mtb strains. Herein, we design and synthesize 35 novel benzo[c]phenanthridine derivatives (BPDs). The two most potent compounds, BPD-6 and BPD-9, accumulated within the bacterial cell and exhibited strong inhibitory activity (MIC90 ~2 to 10 µM) against multiple Mycobacterium strains while remaining inactive against a range of other Gram-negative and Gram-positive bacteria. BPD-6 and BPD-9 were also effective in reducing Mtb survival within infected macrophages, and BPD-9 reduced the burden of Mycobacterium bovis BCG in the lungs of infected mice. The two BPD compounds displayed comparable efficacy to rifampicin (RIF) against non-replicating Mtb (NR-Mtb). Importantly, BPD-6 and BPD-9 inhibited the growth of multiple MDR Mtb clinical isolates. Generation of BPD-9-resistant mutants identified the involvement of the Mmr efflux pump as an indirect resistance mechanism. The unique specificity of BPDs to Mycobacterium spp. and their efficacy against MDR Mtb isolates suggest a potential novel mechanism of action. The discovery of BPDs provides novel chemical scaffolds for anti-TB drug discovery.IMPORTANCEThe emergence of drug-resistant tuberculosis (TB) is a serious global health threat. There remains an urgent need to discover new antibiotics with unique mechanisms of action that are effective against drug-resistant Mycobacterium tuberculosis (Mtb). This study shows that novel semi-synthetic compounds can be derived from natural compounds to produce potent activity against Mtb. Importantly, the identified compounds have narrow spectrum activity against Mycobacterium species, including clinical multidrug-resistant (MDR) strains, are effective in infected macrophages and against non-replicating Mtb (NR-Mtb), and show anti-mycobacterial activity in mice. These new compounds provide promising chemical scaffolds to develop potent anti-Mtb drugs of the future.

Biology and genetic diversity of Candida krusei isolates from fermented vegetables and clinical samples in China

ABSTRACT Candida krusei, also known as Pichia kudriavzevii, is an emerging non-albicans Candida (NAC) species causing both superficial and deep-seated infections in humans. This fungal pathogen is inherently resistant to the first-line antifungal drug, fluconazole, and is widely distributed in natural environments such as soil, foods, vegetables, and fruits. In this study, we collected 86 C. krusei strains from clinical settings and traditional fermented vegetables from different areas of China. Compared to C. krusei strains from fermented vegetables, clinical isolates exhibited a higher ability to undergo filamentation and biofilm development, which could facilitate its host colonization and infections. Isolates from fermented vegetables showed higher resistance to several antifungal drugs including fluconazole, voriconazole, itraconazole, amphotericin B, and caspofungin, than clinical strains, while they were more susceptible to posaconazole than clinical strains. Although C. krusei has been thought to be a diploid organism, we found that one-fourth of clinical strains and the majority of isolates from fermented vegetables (87.5%) are triploid. Whole-genome sequencing and population genetic analyses demonstrated that isolates from clinical settings and fermented food are genetically associated, and distributed across a wide range of genetic clusters. Additionally, we found that six nucleotide substitutions at the promoter region of the ABC11 gene, encoding a multidrug efflux pump, could play a critical role in antifungal resistance in this species. Given the ubiquitous distribution of C. krusei strains in fermented vegetables and their genetic association with clinical strains, a One Health approach will be necessary to control the prevalence of this pathogen.

A-298 A Novel DNA Array Technology to Detect and Discriminate Complex Panels of Microbial Pathogens

Abstract Background UTI (Urinary Tract Infection) is a prototype for the field of polymicrobial testing. Current testing standards suggest that UTI testing may require the Detection and Discrimination of a panel of at least (@24) bacteria and fungi (@2) along with a panel of antibiotic resistance gene (ARG) markers (>12). Today, such analysis of UTI’s at the DNA level can be obtained with predicates such as large-panel qPCR reactions, e.g. the Thermo OpenArray qPCR assembly, or by NGS based microbiomics. The necessary complexity of such polymicrobial testing pushes the content limits of qPCR and, although NGS has substantial capacity to resolve complex mixtures, NGS remains relatively low throughput and requires a level of data processing that exceeds what is practical in some laboratories. Methods We have developed a new technology for UTI testing, “D3 ArrayTM-UTI”, which obviates most of the present limitations of DNA based polymicrobial testing. The process begins with urine being isolated and purified using standard automated magnetic bead DNA sample prep, already in place in most molecular laboratories, at up to 96 samples in parallel. This is followed by a single multiplex, endpoint PCR reaction on each sample, also at up to 96 samples in parallel, then automated room temperature hybridization of the PCR product in a standard 96 well format, onto a single 21x21 D3 Array at the bottom of each well. Dedicated autonomous software analyzes the hybridization data yielding the complete list of microbial pathogens and ARG’s detected in a urine specimen. The first deployment of the D3 Array-UTI test is now available to support analysis of 96 urine samples in 6hrs. The results of such preliminary testing are presented here. Results aLOD analysis has been performed with purified DNA and found to be in the 0.1-10cp/RXN range among the full set of 24 bacterial species and 12 ARG targets. Concordance analysis for bacterial Species Detection and Discrimination has been performed on 235 clinical isolates obtained from collaborators at Mako Medical laboratories (Thermo Openarray as the comparator) which averaged = 97 +/- 5% for all but two species: A urinae (88%) and M morganii (94%). Concordance analysis for Antibiotic Resistance Gene Detection and Discrimination has been performed on 384 clinical isolates also obtained from Mako Medical laboratories (Thermo Openarray as the comparator) which averaged = 98 +/-5% and was found to be >98% for all but one ARG target: gyrA point mutation (88%). Conclusions The data provided here demonstrates that the D3 Array-UTI platform can perform complex polymicrobial/ARG testing as a high throughput 96 well plate based assay, with Sensitivity and Specificity that matches or exceeds that of qPCR, using standard automation that can be deployed in any molecular laboratory. The resulting performance is unique in that Species and ARG Discrimination are readily scalable to 3-4 dozen Species per test, performed in triplicate in a single well with completely autonomous, cloud enabled data analysis, at a sample-to-answer throughput >96 samples per shift. Beta testing has been initiated among laboratory partners.

Pan-azole resistance in clinical Aspergillus fumigatus isolates carrying TR34/L98H from birds and mammals in Belgium

Aspergillosis causes significant health risks to both birds and mammals. The outcome of these infections is often poor due to delayed diagnosis and treatment failure. We investigated 152 cases of aspergillosis from birds and mammals in Belgium. Most samples originated from the taxonomic orders Artiodactyla (40.1 %) and Columbiformes (19.7 %). Five isolates (3.3 %) showed phenotypical resistance against at least one medical azole. Three of these isolates were pan-azole resistant bearing the TR34/L98H mutation. The predominance of this resistance mutation supports an environmental route for exposure and resistance selection, highlighting the importance of the One Health concept.

Tolfenpyrad Derivatives Exhibit Potent Francisella-Specific Antibacterial Activity without Toxicity to Mammalian Cells In Vitro.

Tularemia is a deadly disease caused by Francisella tularensis, an emerging intracellular bacterial pathogen that can be disseminated rapidly through aerosols and vector-borne transmission. Recent surveillance data demonstrate an increasing incidence in several countries. Although clinical isolates of Francisella strains are sensitive to currently used antibiotics, engineered or horizontal acquisition of antibiotic resistance is a constant threat to public health. Therefore, the identification of antibiotics that target previously undrugged pathways is required to safeguard human health. An environmental pesticide that is registered for use in multiple countries, tolfenpyrad, shows promising activity to block Francisella growth; however, it is not a suitable antimicrobial candidate for use in vivo due to potential toxicity in humans and other animals. In this study, we applied a structure-activity relationship approach to tolfenpyrad to generate compounds with improved antibacterial activity and reduced toxicity. Through screening of a library of derivatives, we identified analogs with improved therapeutic windows compared with tolfenpyrad. Although structural diversity exists among these analogs, they inhibit the growth of Francisella species but not other Gram-negative or Gram-positive species. These compounds block intramacrophage growth of F. novicida and pathogenesis in an in vivo arthropod model of infection. Although the biochemical activity of these drugs is unknown, they appear to target the same pathway as the parent molecule because F. novicida mutants that are resistant to tolfenpyrad are also resistant to its analogs. Taken together, these findings suggest that these tolfenpyrad-derived compounds comprise a new class of Francisella-targeted antimicrobials and merit further evaluation and development.

B-205 Performance of a Novel Fluorogenic Assay for Detection of Carbapenemase-producing Enterbacteriaceae

Abstract Background In recent years, under the selective challenge of antibiotics, the variety and number of drugresistant pathogenic microorganisms have increased significantly, which brings great challenges to clinical diagnosis and treatment, especially the infection caused by carbapenem resistant Enterobacteriaceae (CRE). Carbapenemases production is the main mechanism of drug resistance of Enterobacteriaceae to carbapenems. Drug resistance of Carbapenems can be caused by three mechanisms, resulting in the production of five major Carbapenemases. These are Klebsiella pneumoniae enzyme (KPC), New Delhi metal β-lactamase (NDM), carbapenem hydrolyzed oxalase (OXA-48 like), integrin-encoded metal β- lactamase (VIM) and IMP (Imipenemase). The real-time fluorescence quantitative PCR (qPCR) method based on molecular beacons was combined with the melting curve analysis to identify five drug resistance genes simultaneously by a single PCR reaction, with rapid detection, high sensitivity and strong specificity. Based on this principle, we developed the novel fluorogenic assay for rapid detection of Carbapenemases in multidrug-resistant Enterobacteriaceae (Dynamiker Biotechnology (Tianjin) Co., Ltd.). Methods We evaluated the performance of the novel fluorogenic assay for rapid detection of Carbapenemases in multidrug-resistant Enterobacteriaceae, including the limit of detection (LoD) and cross-reactivity, and compared it with the lateral flow immunochromatography assay (LFA). Results The LoD ranged from 75-450 CFU/mL for the five carbapenemase genes. The analytical specificity for target genes was 100%, as assessed with a panel of 15 pathogens, which indicated no cross-reactions. Comparison of qPCR and LFA results from twenty-three CRE clinical isolates with characterized carbapenemase content demonstrated a complete agreement (Table 1). Conclusions The novel fluorogenic assay for rapid detection of Carbapenemases in multidrug-resistant Enterobacteriaceae is an accurate and rapid method to identify KPC, NDM, VIM, IMP and OXA-48-like carbapenemases in the clinical microbiology laboratory, which can guide infection control programs to limit the spread of these organisms.

A-292 Validation of Aztreonam-Avibactam MIC Antimicrobial Susceptibility Test for MicroScan Dried Gram-Negative MIC Panels from a Multicenter Assessment of Enterobacterales, Pseudomonas species, and Stenotrophomonas maltophilia

Abstract Background Aztreonam-avibactam (AZA) is a novel antibiotic combination for treating Gram-negative bacterial infections, including metallo-β-lactamase-producing multidrug-resistant pathogens for which there are limited or no treatment options.1 Given the need for accurate antimicrobial susceptibility testing results, a study was conducted with clinical isolates of Enterobacterales, Pseudomonas species, and Stenotrophomonas maltophilia on an investigational MicroScan Dried Gram-negative MIC (MSDGN) panel with AZA.2 Methods Three clinical sites evaluated MSDGN MIC values to MICs from CLSI reference panels. The study included 602/671 Enterobacterales, 53/59 Pseudomonas species and 69/70 S. maltophilia isolates using Prompt or turbidity inoculation. MSDGN panels were incubated at 35 ± 1°C and read on the WalkAway, autoSCAN-4, and visually at 16-20 hours. Reference panels were prepared according to CLSI methodology, incubated 16-20 hours for Enterobacterales and Pseudomonas species, and 20-24 hours for S. maltophilia and read visually. Results Essential agreement was calculated comparing MSDGN panel MIC results against reference panel MIC results for all isolates (Table 1). Conclusions AZA MIC results for Enterobacterales, Pseudomonas species, and S. maltophilia from MSDGN panels correlate well with MICs obtained using reference panels in this study. 1Pending approval by US Food and Drug Administration (FDA); not available for in vitro diagnostic use in the US. Not for Distribution in the US. 2Pending submission and clearance by the US FDA; not yet available for in vitro diagnostic use in the US. For Investigational Use Only. The performance characteristics of this product have not been established. In development, pending achievement of CE compliance; not yet available for in vitro diagnostic use. © 2024 Beckman Coulter. All rights reserved. All other trademarks are the property of their respective owners. BEC, the stylized logo and the BEC product and service marks mentioned herein are trademarks or registered trademarks of BEC, Inc. in the US and other countries. 2023-12350

The Diverse Activities and Mechanisms of the Acylphloroglucinol Antibiotic Rhodomyrtone: Antibacterial Activity and Beyond.

Background/Objectives: The rose myrtle Rhodomyrtus tomentosa is a medicinal plant used in traditional Asian medicine. The active compound in R. tomentosa leaf extracts is rhodomyrtone, a chiral acylphloroglucinol. Rhodomyrtone exhibits an impressive breadth of activities, including antibacterial, antiviral, antiplasmodial, immunomodulatory, and anticancer properties. Its antibacterial properties have been extensively studied. Methods: We performed a comprehensive literature review on rhodomyrtone and summarized the current knowledge about this promising acylphloroglucinol antibiotic and its diverse functions in this review. Results: Rhodomyrtone shows nano to micromolar activities against a broad range of Gram-positive pathogens, including multidrug-resistant clinical isolates, and possesses a unique mechanism of action. It increases membrane fluidity and creates hyperfluid domains that attract membrane proteins prior to forming large membrane vesicles, effectively acting as a membrane protein trap. This mechanism affects a multitude of cellular processes, including cell division and cell wall synthesis. Additionally, rhodomyrtone reduces the expression of inflammatory cytokines, such as TNF-α, IL-17A, IL1β, and IL8. Generally showing low toxicity against mammalian cells, rhodomyrtone does inhibit the proliferation of cancer cell lines, such as epidermal carcinoma cells. The primary mechanism behind this activity appears to be the downregulation of adhesion kinases and growth factors. Furthermore, rhodomyrtone has shown antioxidant activity and displays cognitive effects, such as decreasing depressive symptoms in mice. Conclusions: Rhodomyrtone shows great promise as therapeutic agent, mostly for antibacterial but also for diverse other applications. Yet, bottlenecks such as resistance development and a better understanding of mammalian cell toxictiy demand careful assessment.

A-254 Increase of Stenotrophomonas maltophilia Isolates Non-Susceptible to Trimethoprim-Sulfamethoxazole and/or Levofloxacin at Brazilian Hospitals

Abstract Background Stenotrophomonas maltophilia is an emerging multidrug-resistant pathogen that exhibits intrinsic and acquired resistance to a wide variety of antimicrobial agents such as β-lactams, aminoglycosides, quinolone, tetracyclines and disinfectants. In addition to that, the increased resistance to sulfamethoxazole/trimethoprim (SMX/TMP) and Levofloxacin has been reported in the past few years, leading to few therapeutic options. This study aimed to describe the increase of Stenotrophomonas maltophilia clinical isolates recovered from Brazilian hospitals over a 6-year analysis (January 2018 to December 2023). Methods This study design was a retrospective, observational analysis based on microbiological data of emergent BGN-NF isolates detected in several Brazilian tertiary hospitals. The bacterial identification was performed by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF/MS) and the antimicrobial susceptibility profile to trimethoprim-sulfamethoxazole and levofloxacin was determined using disk-diffusion method and epsilometer method, respectively. Only one sample per patient was considered in this analysis. Results During the 6 year period, a total of 1,706 (2018; 221), (2019; 203), (2020; 294), (2021; 276), (2022; 294) and (2023; 418) Stenotrophomonas maltophilia strains were isolated from blood (n=626), respiratory tract (n=365), fluid and secretion (n=235), skin and soft tissue (n=162), urine (n=132), catheter tip (n=112), swab (n=36), bone fragment (n=29) and cerebrospinal fluid (n=9). The major frequency of S. maltophilia isolation occurred among man (52.6%) and the main age range was over 60 years (42.7%), while 0-10 comprised 16.6% of cases. Compared the 6-years period, the resistance rate to trimethoprim-sulfamethoxazole was 2.7%; 5.4%; 5.8%; 2.2%; 5.8%; 7.9% and levofloxacin was 3.6%; 3.4%; 3.1%; 3.3%; 1.7%; 2.9%, respectively. The resistance rate to trimethoprim-sulfamethoxazole and levofloxacin, simultaneously, was 0.23%. Conclusions The incidence rate of S. maltophilia infections has increased significantly, especially in the last year, followed by an increase in the pattern of resistance to trimethoprim-sulfamethoxazole. Regarding trimethoprim-sulfamethoxazole and levofloxacin co-resistance, very low rates were detected in our analysis. Our data point to a scenario wherein the presence of S. maltophilia may have been intensified by the covid-19 pandemic. Regional surveillance reports are extremely important and should be out to understand some aspects of the dynamics of bacterial resistance in both hospital and community settings.

Berberine Inhibits the Inflammatory Response Induced by Staphylococcus aureus Isolated from Atopic Eczema Patients via the TNF-α/Inflammation/RAGE Pathways.

Atopic eczema patients exhibit high levels of Staphylococcus aureus (S. aureus) skin colonization. S. aureus can stimulate macrophages and the expression of proinflammatory cytokines. Berberine (BBR), an alkaloid, attenuates S. aureus toxin production. This study investigated if BBR suppressed bacterial growth and inflammatory response induced by eczema-patient-derived S. aureus using murine macrophage (RAW 264.7) and human monocyte cell lines (U937). RAW 264.7 and U937 were treated with BBR at different concentrations and stimulated with heat-killed S. aureus (ATCC #33591) or S. aureus derived from severe eczema patients (EC01-EC10), who were undergoing topical steroid withdrawal, for 24 h. TNF-α protein levels were determined by ELISA, gene expression by qRT-PCR, cell cytotoxicity by trypan blue excursion, and reactive oxygen species (ROS) levels by fluorometric assay. BBR showed a bacteriostatic effect in S. aureus (ATCC strain #33591 and clinical isolates (EC01-EC10) and suppressed TNF-α production in RAW 264.7 and U937 cells exposed to heat-killed S. aureus (ATCC and clinical isolates) dose-dependently without any cell cytotoxicity. BBR (20 µg/mL) suppressed >90% of TNF-α production (p < 0.001), downregulated genes involved in inflammatory pathways, and inhibited S. aureus ROS production in U937 and RAW 264.7 cells (p < 0.01). BBR suppresses S. aureus-induced inflammation via inhibition of TNF-α release, ROS production, and expression of key genes involved in the inflammatory pathway.

Evaluation of a customised Sensititre YeastOne plate containing isavuconazole for antifungal susceptibility testing in Singapore

This study evaluated the performance of a customised Sensititre YeastOne (SYO) plate including isavuconazole (YIT) against existing practice (comprising SYO YO10 plate and isavuconazole gradient strip) in order to streamline the workflow for antifungal susceptibility testing in a tertiary hospital in Singapore.A total of 101 (51 yeast and 50 mould) clinical isolates were included for analysis. Isolates included in the study were recovered from a variety of body sites and reflected the case mix encountered in daily practice. Antifungal susceptibility testing was performed using three methods: YO10, YIT and gradient diffusion strip (for isavuconazole only). Reproducibility, essential agreement and categorical agreement were calculated.When YO10 and YIT plates were compared, the reproducibility was 100% for 8 common antifungals. The categorical agreement (CA) was >97% for all antifungals except for amphotericin B (89.4%), but this was attributed to seven isolates with MICs bordering the wild-type (WT) cut-off. The essential agreement (EA) obtained when testing isavuconazole using YIT versus gradient diffusion was 77.2% overall, 90.2% for yeasts and 64% for moulds.In conclusion, the YIT plate is suitable for antifungal susceptibility testing of yeasts in our laboratory. Its use for mould isolates needs to be monitored further.

Analysis of antibiotic response in Clinical Wound Pseudomonas aeruginosa isolates: Unveiling Proteome Dynamics of tobramycin tolerant phenotype

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic human pathogen, causing serious chronic infections. P. aeruginosa can adapt efficiently to antibiotic stressors via different genotypic or phenotypic strategies such as resistance and tolerance. The adaptation regulatory system is not always very well understood. In this study, we use shotgun proteomics to investigate the system-level response to tobramycin in two clinical wound P. aeruginosa isolates and PAO1. We profiled each strain for its antibiotic drug-tolerant phenotype using supra-minimum inhibitory concentrations (supra-MIC) of tobramycin and applied proteomics to investigate the protein expression profiles. The MIC revealed that all isolates were susceptible to tobramycin but at supra-MIC concentrations at stationary growth, a degree of tolerance was observed for the isolates. We identified around 40 % of the total proteins encoded by the P. aeruginosa genome and highlighted shared and unique protein signatures for all isolates. Comparative proteome profiling in the absence of antibiotic treatment showed divergent fingerprints, despite similarities in the growth behavior of the isolates. In the presence of tobramycin, the isolates shared a common response in the downregulation of proteins involved in the two-component system, whereas stress response proteins were present at higher levels. Our findings provide insight into the use of proteomic tools to dissect the system-level response in clinical isolates in the absence and presence of antibiotic stress.

Genotypic and phenotypic characterisation of respiratory syncytial virus after nirsevimab breakthrough infections: a large, multicentre, observational, real-world study

Nirsevimab, a long-acting monoclonal antibody, has been approved for the prevention of respiratory syncytial virus (RSV) infection in infants. In France, more than 210 000 single doses were administered in infants younger than 1 year during the 2023-24 season. In this context, the selection and spread of escape variants might be a concern. Here, we aimed to characterise RSV associated with breakthrough infection. We did a multicentre, national, observational study in France during the 2023-24 RSV season in RSV-infected infants (aged <1 year) who either received or did not receive a dose of nirsevimab before their first RSV season. We excluded infants with insufficient information about nirsevimab treatment or without parental consent. We used respiratory samples collected in each laboratory for full-length RSV RNA sequencing to analyse changes in the nirsevimab binding site Ø. We tested clinical RSV isolates for neutralisation by nirsevimab. We analysed F candidate substitutions by fusion-inhibition assay. Of the 695 RSV infected infants, we analysed 545 (78%) full-length RSV genome sequences: 260 (48%) from nirsevimab-treated breakthrough infections (236 [91%] RSV-A and 24 [9%] RSV-B) and 285 (52%) from untreated RSV-infected infants (236 [83%] RSV-A and 49 [17%] RSV-B). Analysis of RSV-A did not reveal any substitution in site Ø known to be associated with resistance to nirsevimab. Two (8%) of 24 RSV-B breakthrough infections had resistance-associated substitutions: F:N208D (dominant resistance-associated substitution) and a newly described F:I64M plus F:K65R combination (minority resistance-associated substitution), both of which induced high levels of resistance in the fusion-inhibition assay. This study is, to the best of our knowledge, the largest genotypic and phenotypic surveillance study of nirsevimab breakthrough infections to date. Nirsevimab breakthrough variants remain very rare despite the drug's widespread use. The detection of resistance-associated substitutions in the RSV-B F protein highlights the importance of active molecular surveillance. ANRS Maladies Infectieuses Emergentes and the French Ministry of Health and Prevention.

Genomic insights into qnrVC1 gene located on an IncP6 plasmid carried by multidrug resistant Pseudomonas aeruginosa from clinical asinine isolates

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen, causing significant global health threat due to its antimicrobial resistance. Among equines, P. aeruginosa can cause infections, particularly in the reproductive tract, leading to reproductive failure. Multidrug-resistant (MDR) P. aeruginosa has been a major concern in animal husbandry, including the donkey industry. The study aims to elucidate the phylogenetic relationship of P. aeruginosa strains isolated from donkeys with endometritis farmed in a large intensive unit in Hebei Province, China. Genes coding for multiple antimicrobial resistances were predicted by whole genomic sequencing. Multilocus sequence typing (MLST) revealed that all strains belonged to the same sequence type (ST1058). An IncP6 plasmid encoding the qnrVC1 gene, associated with quinolone resistance, was identified. Comparative genomic analysis illustrated the characteristics of the strains and genetic context of qnrVC1. This study is the first to report that these MDR P. aeruginosa asinine strains exhibited high levels of antimicrobial and metal resistance conferred by a qnrVC1-carrying plasmid. Additionally, P. aeruginosa strains with integrated mega-plasmids were identified. From a One Health perspective, the study underlined the significance of monitoring antimicrobial resistance genes in food animals, including donkeys.

Nosocomial cluster of patients infected with imipenemase-1-producing Enterobacter ludwigii.

Introduction. Imipenemase (IMI) enzymes are an uncommon class A carbapenemases that have been isolated from aquatic environments and, occasionally, from clinical isolates of Enterobacterales.Aim. We describe a cluster of three patients infected by IMI-1 carbapenemase-producing Enterobacter ludwigii (IMI-1-Elud) in a tertiary university hospital in Gran Canaria, Spain.Methodology. Antimicrobial susceptibility was determined using the Vitek2 AST-N355 card and antibiotic gradient strips. The modified carbapenem inactivation method (CIM) test was performed in cases where the ertapenem MIC value was higher than 0.125 mg l-1. The carbapenemase was identified by PCR and DNA microarray and later characterized by whole-genome next-generation sequencing (NGS) with Illumina.Results. Three patients presented thoracic or abdominal infections caused by IMI-1-Elud ST1677 from 14 June 2022 to 14 July 2022. All patients underwent at least one gastroscopy during their admission, and two of them were located in adjoining rooms. Isolates were resistant to carbapenems, colistin and fosfomycin but susceptible to ciprofloxacin. IMI/NMC-A carbapenemase was detected by PCR and hybridization test and confirmed by NGS as IMI-1. All patients underwent at least one gastroscopy, and two of them were in nearby rooms. Patients showed microbiological and clinical improvement following focus drainage and targeted antibiotic treatment with a fluoroquinolone.Conclusions. This study reports the first documented global outbreak of patients infected with IMI-1-Elud. The source appeared to be related to endoscopes. Contact transmission may also have played a role. A screening method such as the modified CIM test is crucial for detecting less common carbapenemases that might not be identified by rapid molecular or immunochromatographic tests, as these often do not include bla IMI genes, which could lead to the undetected dissemination of carbapenemase-producing Enterobacterales. Effective infection source control and targeted treatment are essential for achieving a favourable clinical outcome.

Whole-Genome Sequencing of Clinical Isolates of Klebsiella michiganensi in China carrying blaIPM-4 and blaNDM-1

AimThis study aimed to analyze the prevalence, phenotypes, and carriage of resistance genes of carbapenem-resistant Klebsiella michiganensis strains isolated in our hospital. Methodology: Four K. michiganensis strains were collected from January 2015 to December 2023. Antimicrobial susceptibility was tested using 21 antibiotics with the BD Phoenix™ M50 System. Whole-genome sequencing of the four strains was performed on an Illumina NovaSeq 6000 platform. Species identification was performed using Kleborate software, sequence type (ST) was performed using MLST, and prediction of antibiotic resistance genes and virulence genes were performed using ABRicate. PlasmidFinder was used to search for plasmids. Whole genome data from 211 strains of carbapenem-resistant K. michiganensis were downloaded from the NCBI database; together with the strains in this study, these data were used to construct an phylogenetic tree using genome single nucleotide polymorphisms (SNP). ResultsAntimicrobial susceptibility showed that K. michiganensis was highly resistant to β-lactams. For the three carbapenems, strain WF0046 was only resistant to ertapenem, while WF0047, WF0052, and WF0053 were resistant to all three carbapenems tested. The four strains of K. michiganensis only showed complete sensitivity to polymyxin E and tigecycline. The four K. michiganensis strains were found to have 43 resistance genes, and all carried carbapenem resistance genes; WF0046 carried the carbapenem resistance gene blaIMP-4, while the other three strains carried blaNDM-1. Among the other resistance genes, β-lactam resistance genes were predicted most (11 types), followed by eight types of aminoglycoside resistance genes. Of the strains characterized in this study, WF0046 belonged to ST158, WF0047 belonged to ST40, WF0052 belonged to ST533, and WF0053 belonged to ST13. These four strains, along with 211 carbapenem-resistant K. michiganensis strains downloaded from NCBI, were divided into five clades; WF0052 belonged to clade A, WF0046 belonged to clade C, and WF0047 and WF0053 both belonged to clade D. The four strains had relatively distant genetic relationships; WF0046, WF0047, and WF0053 were closely related to strains of human origin from other regions of China, while WF0052 was genetically close with a strain of K. michiganensis isolated in the United States. ConclusionThe strains of K. michiganensis from our hospital were resistant to multiple antibiotics, and all strains carried carbapenem resistance genes along with multiple other antibiotic resistance genes. The phylogenetic results showed that these four strains had distant genetic relationships and different ST types, indicating that they came from different sources and the possibility of polyclonal transmission.

Complete genome identified of clinical isolate Prototheca.

Introduction. Prototheca is an opportunistic pathogen that can infect both humans and animals, of which Prototheca wickerhamii (P. wickerhamii) being the most significant pathogenic green algae.Gap statement. The incidence of human diseases caused by Prototheca has been on the rise, yet there is a significant gap in genetic research pertaining to the pathophysiological aspects of these infections.Aim. The aim of this study is to present the whole genome data from the clinical isolate InPu-22_FZ strain and to understand its genomic characteristics through comparative genomic analysis and phylogenetic tree analysis. Functional annotation of protein-coding genes and analysis of their pathogenicity are also conducted.Methodology. We described the high-quality de novo genome assembly of the clinical isolate InPu-22_FZ strain, achieved by combining Nanopore ONT and Illumina NovaSeq sequencing technologies. Phylogenetic tree was constructed to study the evolutionary relationship between the InPu-22_FZ strain and other species. The average nucleotide identity (ANI) analysis was used to assess the similarity between different species. Additionally, the size, distribution and composition of synteny blocks were also analysed to infer the evolutionary relationships of the genomes.Results. The size of the assembled nuclear genome was 18.47 Mb with 48 contigs. Key features of the genome include high overall GC content (63.31%), high number (5478) and proportion (62.24%) of protein-coding genes and more than 96.71% of genes annotated for gene function. Phylogenetic analyses showed that the InPu-22_FZ strain and other P. wickerhamii clustered into one clade with a bootstrap value of 99% and collinearity analysis revealed high levels of collinearity with ATCC 16529. The ANI analysis revealed only a relatively high similarity (89-93%) to available P. wickerhamii genomes, suggesting the overall genomic novelty of InPu-22_FZ strain. Interestingly, the analysis of the pathogen-host interaction database unveiled and demonstrated reduced virulence of this strain, albeit it was isolated from a chronic upper-limb cutaneous infection.Conclusion. The study provides an in-depth insight into the genomic structure and biological function of the InPu-22_FZ strain, revealing the genetic basis of its pathogenicity and virulence.