Annals of Clinical Microbiology and Antimicrobials最新文献

Background: Antimicrobial resistance (AMR) poses a significant threat to pediatric health; therefore, precise identification of pathogens as well as AMR is imperative. This study aimed at comprehending antibiotic resistance patterns among critically ill children with infectious diseases admitted to pediatric intensive care unit (PICU) and to clarify the impact of drug-resistant bacteria on the prognosis of children.

Methods: This study retrospectively collected clinical data, identified pathogens and AMR from 113 children's who performed metagenomic next-generation sequencing for pathogen and antibiotic resistance genes identification, and compared the clinical characteristic difference and prognostic effects between children with and without AMR detected.

Results: Based on the presence or absence of AMR test results, the 113 patients were divided into Antimicrobial resistance test positive group (AMRT+, n = 44) and Antimicrobial resistance test negative group (AMRT-, n = 69). Immunocompromised patients (50% vs. 28.99%, P = 0.0242) and patients with underlying diseases (70.45% vs. 40.58%, P = 0.0019) were more likely to develop resistance to antibiotics. Children in the AMRT + group showed significantly increased C-reaction protein, score of pediatric sequential organ failure assessment and pediatric risk of mortality of children and longer hospital stay and ICU stay in the AMRT + group compared to the AMRT+- group (P < 0.05). Detection rate of Gram-negative bacteria was significantly higher in the AMRT + group rather than Gram-positive bacteria (n = 45 vs. 31), in contrast to the AMRT- group (n = 10 vs. 36). Cephalosporins, β-lactams/β-Lactamase inhibitors, carbapenems and sulfonamides emerged as the most common types of drug resistance in children. Resistance rates to these antibiotics exhibited considerable variation across common pathogens, including Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii.

Conclusions: The development of drug resistance in bacteria will significantly affect the prognosis of patients. The significant differences in drug resistance of common pathogenic bacteria indicate that identification of drug resistance is important for the rational use of antibiotics and patient prognosis.

Introduction: Colonisation and infection with Carbapenem-resistant Enterobacterales (CRE) in healthcare settings poses significant risks, especially for vulnerable patients. Genomic analysis can be used to trace transmission routes, supporting antimicrobial stewardship and informing infection control strategies. Here we used genomic analysis to track the movement and transmission of CREs within clinical and environmental samples.

Methods: 25 isolates were cultured from clinical patient samples or swabs, that tested positive for OXA-48-like variants using the NG-Test® CARBA-5 test and whole genome sequenced (WGS) using Oxford Nanopore Technologies (ONT). 158 swabs and 52 wastewater samples were collected from the ward environment. 60 isolates (matching clinical isolate genera; Klebsiella, Enterobacter, Citrobacter and Escherichia) were isolated from the environmental samples using selective agar. Metagenomic sequencing was undertaken on 36 environmental wastewater and swab samples.

Results: 21/25 (84%) clinical isolates had > 1 bla_OXA gene and 19/25 (76%) harboured > 1 bla_NDM gene. Enterobacterales were most commonly isolated from environmental wastewater samples 27/52 (51.9%), then stick swabs 5/43 (11.6%) and sponge swabs 5/115 (4.3%). 11/60 (18%) environmental isolates harboured > 1 bla_OXA gene and 1.9% (1/60) harboured bla_NDM-1. bla_OXA genes were found in 2/36 (5.5%) metagenomic environmental samples.

Conclusions: Potential for putative patient-patient and patient-ward transmission was shown. Metagenomic sampling needs optimization to improve sensitivity.

Non-O1/non-O139 Vibrio cholerae (NOVC) strains are a distinct group of Vibrio cholerae that do not cause epidemic cholera. NOVC infections usually cause mild forms of gastroenteritis, and rarely severe (extra)intestinal infections, mostly affecting immunocompromised patients. Here, we describe the clinical course of a patient with NOVC bacteremia causing multiple liver abscesses, after drinking from a freshwater well in a non-coastal area. This case highlights the potential of a V. cholerae strain, that is phylogenetically distinct from the current pandemic cholera strain, to cause severe extra-intestinal infections, including liver abscesses.

Background: Drug susceptibility testing (DST) for Nocardia spp. is essential to initiate effective antibiotic therapy. Currently, the only recommended technique is the determination of minimum inhibitory concentrations (MICs) by microdilution. This method can be tedious to perform, despite the availability of ready-to-use plates. Herein, the aim was to determine the critical inhibition diameters specific to Nocardia spp.

Methods: MICs of 134 Nocardia isolates were determined by microdilution. Interpretative categories (Susceptible/Intermediate/Resistant) were determined using Clinical and Laboratory Standards Institute breakpoints. In parallel, disk diffusion DST was performed. Receiver-operating-characteristic (ROC) curves were constructed to determine the inhibition diameter value that best discriminated between susceptible and non-susceptible strains (intermediate/resistant). The category agreement (CA), the rate of major (maj) and very major (vmj) discrepancies between microdilution and disk diffusion method was calculated.

Results: For tobramycin, the critical diameter of 19 mm (diameter ≤ 19 mm = resistant strain; diameter > 19 mm = susceptible strain) provided a CA of 98.5%, 0.0% vmj, and 2.9% maj discrepancies, reaching strictly the acceptable performance criteria defined by the U.S. Food and Drug Administration (FDA). For amikacin, the critical diameter of 25 mm (diameter ≤ 25 mm = resistant strain; diameter > 25 mm = susceptible strain) provided a CA of 98.5%, 0.0% vmj, and 1.5% maj discrepancies. For imipenem, excluding N. farcinica and N. cyriacigeorgica, the critical diameter of 29 mm (diameter ≤ 29 mm = resistant strain; diameter > 29 mm = susceptible strain), provided a CA of 98.6%, 0.0% vmj, and 0.0% maj discrepancies. Despite an estimated vmj rate 0.0%, the 95%-confident-interval exceeded the FDA criteria due to an insufficient number of amikacin/imipenem-resistant strains. For other tested antibiotics (ciprofloxacin, moxifloxacin, amoxicillin-clavulanate, ceftriaxone, cotrimoxazole, linezolid), the FDA criteria were not reached.

Conclusions: Although the FDA criteria were mostly unmet, disk diffusion DST was suitable to accurately categorize Nocardia isolates into interpretative categories for the aminoglycosides and imipenem only, excluding species N. farcinica and N. cyriacigeorgica.

In June 2022, a 73-year-old man with a history of laryngeal and esophageal carcinoma was admitted to the emergency unit with sudden fever, confusion, and general condition deterioration. Initial assessments showed a fever of 38.5 °C, elevated C-reactive protein (CRP) at 209 mg/L, and a neutrophil count of 10.4 G/L, with negative results for urine analysis, blood cultures, and multiple infectious pathogens, including Legionella pneumophila, pneumococcal antigen, and SARS-CoV-2. Computed tomography (CT) scans revealed no significant infectious focus.Empirical treatment with Ceftriaxone and Ciprofloxacin was initiated. Despite treatment, the patient's condition remained unchanged, and a lumbar puncture revealed turbid cerebrospinal fluid (CSF) with 14,300 white blood cells (WBC)/mm³, predominantly neutrophils, elevated proteins, and decreased glucose. Gram staining suggested Neisseria meningitidis, but further testing was necessary. Antibiotic therapy was switched to Cefotaxime and Dexamethasone, and the patient was transferred to the Tropical and Infectious Disease Unit.Multiplex PCR assays and additional CSF tests were negative for common pathogens. Sequencing of 16S ribosomal RNA identified Gemella sp. The patient's condition improved with continued Cefotaxime treatment, and he recovered without neurological sequelae. Subsequent dental CT revealed poor dental hygiene but no signs of osteo-meningeal breach or bone lysis.A literature review identified 22 reported cases of central nervous system (CNS) infections caused by various Gemella species from 1980 to 2022. Of these, 59% presented with meningitis, and 41% had additional encephalitis or brain abscesses. Complete recovery occurred in 77% of cases, with 9% resulting in neurological damage and another 9% in fatal outcomes. Relapses occurred in 14% of the cases. The review highlighted that CNS infections by Gemella spp. primarily affect immunocompromised adults with ENT (ear nose throat) or neurological breaches, although some cases involved healthy individuals.This case underscores the diagnostic challenges posed by uncommon pathogens like Gemella and highlights the utility of molecular microbiology in identifying causative agents, thus guiding appropriate treatment. The patient's history of ENT and esophageal cancers, along with recent radiotherapy and chemotherapy, likely contributed to the infection's development. The case emphasizes the importance of thorough investigation in febrile confusion cases and the potential role of Gemella spp. in CNS infections.

Background: During prolonged FDC therapy, the emergence of FDC non-susceptibility in CRAB has been reported. Here, we report a transmission cluster of FDC-non-susceptible CRAB in four patients, all naïve to FDC treatment, characterized by a premature stop codon and amino acid deletion in the PirA protein.

Methods: CRAB strains obtained from patients admitted in a single medicine ward of the IRCCS Fondazione Ospedale Maggiore Policlinico between March and July 2024 were analyzed by WGS and antimicrobial susceptibility testing. Phylogenetic analysis was used to assess their genetic relatedness.

Results: Between March and July 2024, an outbreak of 33 CRAB was observed among hospitalized patients in a single ward at IRCCS. Genomic analysis, available in 29 cases, revealed that 24 isolates belonged to ST208/1806, 4 to ST369, and one to ST195/1816 (according to the Oxford scheme). FDC susceptibility was affected only in the four ST369 isolates (Kirby-Bauer disk diffusion diameter: 13 mm; UMIC^® method MIC: 4 mg/L), all characterized by a premature stop codon followed by a 52 amino acid deletion located between the amino acids 377 and 428 of the siderophore-drug receptor PirA. No other relevant mutations were detected in the iron-uptake genes. Core-genome ML tree including ST369 reference strains revealed that the four ST369 isolates were highly related and formed a distinct cluster (SNP distance: 3 [IQR: 1-6]). Of note, the four isolates were collected from four FDC-naïve individuals, two experiencing a CRAB-mediated infection.

Conclusions: Our findings alert about the circulation of clones carrying modified siderophore-drug receptors without evidence of previous FDC treatment and support the importance of testing FDC susceptibility appropriately before its administration.

Background: The permeability of the outer membrane barrier modulates the susceptibility of microorganisms to antimicrobial agents. Loss or structural alterations of porins contribute to decreased antibiotic concentration of multiple antimicrobial agents. Precise definition of porin profiles is of critical importance to understand the role of porins in antimicrobial resistance. The objectives of this study are to compare the expression patterns of major outer membrane proteins (OMP) of clinical isolates of Klebsiella pneumoniae obtained with Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight mass spectrometry (MALDI-TOF/MS), with those obtained with sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and to correlate porin expression patterns with the sequences of porins genes defined with whole genome sequencing (WGS).

Methods: The OMP profiles of 26 clinical isolates of K. pneumoniae and of strain ATCC 13883 (wild-type) and ATCC 700603 (producing SHV-18) have been determined using both SDS-PAGE and MALDI-TOF/MS. SDS-PAGE was performed using both homemade and commercial gels, and protein bands were identified by liquid chromatography coupled to mass spectrometry. A rapid extraction method was used to analyse OMPs by MALDI-TOF/MS. The sequences of porin genes were obtained by WGS and mutations were defined by BLAST.

Results: Same results were obtained for all strains either using SDS-PAGE or MALDI-TOF/MS. SDS-PAGE showed protein bands of ~ 35, ~36, and ~ 37 kDa, identified as OmpA, OmpK36 and OmpK35, respectively. By MALDI-TOF/MS, peaks at ~ 35,700 (OmpA), ~ 37,000 (OmpK35), and ~ 38,000 (OmpK36) m/z were detected. ompK35 was intact in nine wild-type isolates and was truncated in 13 isolates, but OmpK35 was not observed in 3 isolates without mutations in ompK35. One point mutation was detected in another isolate and multiple mutations were detected in the remaining isolate. ompK36 was truncated in two isolates lacking this protein and presented one point mutation (n = 1) or multiple mutations in the remaining isolates.

Conclusion: MALDI-TOF/MS was reliable for porin detection, but because of the complex regulation of porin genes, WGS cannot always anticipate protein expression, as observed with SDS-PAGE and MALDI-TOF/MS.

This study elucidates the in vivo genetic mechanisms contributing to the emerging resistance to carbapenem in Shewanella algae through a lens of adaptive microbial evolution. Leveraging PacBio amplification-free sequencing, we tracked the evolution of β-lactam resistance in clinical isolates from a persistent S. algae bacteremia case amidst antimicrobial therapy. Our investigation spotlighted a recurrent G547W mutation in the sensor histidine kinase (pdsS), which was associated with the overexpression of an OmpA-like protein (pdsO) within a proteobacteria-specific sortase system. Intriguingly, we observed a recurrent switch between wild-type and G547W alleles, revealing an adaptive expansion and contraction of underlying cell subpopulations in response to β-lactam exposure. Comparative transcriptome analyses further demonstrated the overexpression of genes pivotal for membrane integrity, biofilm formation, immune evasion, and β-lactamase activation in resistant samples. This underscores the pre-existence of resistant cells at minuscule frequencies even without antibiotic pressure, potentially explaining the within-host emergence of resistance during antibiotic treatments. Our findings provide pivotal insights into the dynamic genetic adaptations of S. algae under therapeutic pressures, unmasking intricate resistance mechanisms and highlighting the critical role of subpopulation dynamics in treatment outcomes.

Introduction: Diabetes mellitus is a chronic metabolic disorder characterized by persistent hyperglycemia due to impaired insulin production or utilization, leading to severe health complications. Diabetic foot ulcers (DFUs) represent a major complication, often exacerbated by polymicrobial infections involving Staphylococcus aureus and Acinetobacter baumannii. These pathogens, notorious for their resistance to antibiotics, complicate treatment efforts, especially due to biofilm formation, which enhances bacterial survival and resistance. This study explores the synergistic effects of combining gentamicin, imipenem, and fucoidan, a sulfated polysaccharide with antimicrobial properties, against both planktonic and biofilm forms of S. aureus and A. baumannii.

Methods: Isolates of S. aureus and A. baumannii were collected from DFUs and genetically confirmed. Methicillin resistance in S. aureus was identified through disk diffusion and PCR. Biofilm formation, including dual-species biofilms, was analyzed using the microtiter plate method. The antimicrobial efficacy of gentamicin, imipenem, and fucoidan was assessed by determining the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), minimum biofilm inhibitory concentration (MBIC), and minimum biofilm eradication concentration (MBEC). Synergistic interactions were evaluated using the fractional inhibitory concentration index (FICi) and fractional bactericidal concentration index (FBCi). The expression of biofilm-associated genes (icaA in S. aureus and bap in A. baumannii) was analyzed, and the cytotoxicity of fucoidan was assessed.

Results: The study revealed that 77.4% of S. aureus and all A. baumannii isolates showed multidrug resistance. Among 837 tested conditions for dual-species biofilm formation, 72 resulted in strong biofilm formation and 67 in moderate biofilm formation. The geometric mean MIC values for gentamicin were 12.2 µg/mL for S. aureus, 22.62 µg/mL for A. baumannii, and 5.87 µg/mL for their co-culture; for imipenem, they were 19.84, 9.18, and 3.70 µg/mL, respectively, and for fucoidan, 48.50, 31.20, and 19.65 µg/mL, respectively. The MBC values for gentamicin were 119.42, 128, and 11.75 µg/mL; for imipenem, they were 48.50, 14.92, and 8 µg/mL; and for fucoidan, they were 88.37, 62.62, and 42.48 µg/mL. The MBIC values were 55.71, 119.42, and 18.66 µg/mL for gentamicin; 68.59, 48.50, and 25.39 µg/mL for imipenem; and 153.89, 101.49, and 53.53 µg/mL for fucoidan. The MBEC values were 315.17, 362.03, and 59.25 µg/mL for gentamicin; 207.93, 157.58, and 74.65 µg/mL for imipenem; and 353.55, 189.46, and 99.19 µg/mL for fucoidan. When cultured in planktonic form, the geometric mean FICi and FBCi values indicated additive effects, while co-culture showed FICi values of ≤ 0.5, suggesting a synergistic interaction. Treatment with gentamicin and fucoidan led to significant downregulation of the icaA

Background: Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a significant threat to immunocompromised populations, including lung transplant recipients. This study investigates mixed CRKP strains carrying either bla_KPC-2 or bla_KPC-33 following ceftazidime-avibactam (CAZ/AVI) exposure, particularly in the context of lung transplantation. Mixed CRKP strains with shifting resistance phenotypes were frequently identified in patients exposed to CAZ/AVI. We aimed to elucidate the transitional state of bla_KPC variants by selecting CAZ/AVI-sensitive and -resistant CRKP strains from a lung transplantation patient.

Methods: The bla_KPC-variant-carrying CRKP strains were collected from lung transplant recipients exposed to CAZ/AVI in less than two years. Antibiotic susceptibility testing (AST) was conducted using microbroth dilution, and whole-genome sequencing (WGS) was used to identify genotypes and resistance mechanisms. Limiting dilution, drop-plate, and in vitro induction experiments determined bla_KPC-variant changes during CAZ/AVI administration. qPCR primers/probes were designed to identify bla_KPC-2 mutations.

Results: Among 104 lung transplant recipients infected by bla_KPC-harboring CRKP strains and receiving CAZ/AVI, 10 (9.6%) experienced changing resistance phenotypes. The limiting dilution method found that Patient 10's CRKP strains carried either bla_KPC-2 or bla_KPC-33. The drop-plate experiment showed differing growth patterns on CAZ/AVI mediums. The in vitro induction experiment demonstrated shifting from bla_KPC-2 to bla_KPC-33.

Conclusions: The study identified a "transitional state" of the mixed CRKP strains carrying either bla_KPC-2 or bla_KPC-33 in CAZ/AVI-exposed patients. Molecular diagnostics are crucial for identifying mixed strains and the transitional state of bla_KPC variants, guiding treatment decisions in this complex landscape.