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Abstract Background Animal models of acute respiratory distress syndrome (ARDS) do not completely resemble human ARDS, struggling translational research. We aimed to characterize a porcine model of ARDS induced by pneumonia—the most common risk factor in humans—and analyze the additional effect of ventilator-induced lung injury (VILI). Methods Bronchoscopy-guided instillation of a multidrug-resistant Pseudomonas aeruginosa strain was performed in ten healthy pigs. In six animals (pneumonia-with-VILI group), pulmonary damage was further increased by VILI applied 3 h before instillation and until ARDS was diagnosed by PaO2/FiO2

Abstract 16S rRNA gene profiling, which contains nine hypervariable regions (V1–V9), is the gold standard for identifying taxonomic units by high-throughput sequencing. Microbiome studies combine two or more region sequences (usually V3–V4) to increase the resolving power for identifying bacterial taxa. We compare the resolving powers of V1–V2, V3–V4, V5–V7, and V7–V9 to improve microbiome analyses in sputum samples from patients with chronic respiratory diseases. DNA were isolated from 33 human sputum samples, and libraries were created using a QIASeq screening panel intended for Illumina platforms (16S/ITS; Qiagen Hilden, Germany). The analysis included a mock community as a microbial standard control (ZymoBIOMICS). We used the Deblur algorithm to identify bacterial amplicon sequence variants (ASVs) at the genus level. Alpha diversity was significantly higher for V1–V2, V3–V4, and V5–V7 compared with V7–V9, and significant compositional dissimilarities in the V1–V2 and V7–V9 analyses versus the V3–V4 and V5–V7 analyses. A cladogram confirmed these compositional differences, with the latter two being very similar in composition. The combined hypervariable regions showed significant differences when discriminating between the relative abundances of bacterial genera. The area under the curve revealed that V1–V2 had the highest resolving power for accurately identifying respiratory bacterial taxa from sputum samples. Our study confirms that 16S rRNA hypervariable regions provide significant differences for taxonomic identification in sputum. Comparing the taxa of microbial community standard control with the taxa samples, V1–V2 combination exhibits the most sensitivity and specificity. Thus, while third generation full-length 16S rRNA sequencing platforms become more available, the V1–V2 hypervariable regions can be used for taxonomic identification in sputum.

Objectives: To evaluate the expression dynamics of biofilm genes in methicillin-resistant Staphylococcus aureus (MRSA) retrieved from endotracheal tubes (ETT) and to determine how gene regulation is attenuated in vitro where host–environmental factors are no longer present. Methods: Biofilm was grown (24 h) in tryptic broth soy plus 0.25% glucose for a clinical MRSA isolate in planktonic state and after sessile growth named ETT-MRSA (S2, S3, S4, S5, S6, S7). Gene expression of five biofilm-related genes (icaC, clfB, ebps, fnbB, and RNA III) was assessed consecutively from day 1 to day 4 after ETT growth through real-time PCR. 16S rRNA was used as a control. Results: The MRSA isolates retrieved from ETT were capable of producing biofilms dependent on ica. The gene expression dynamics of ETT-MRSA changed progressively compared to planktonic MRSA gene expression under both ambient air (p < 0.001) and ambient air with 5% CO2 (p < 0.001). Dynamic assessment of icaC expression in both atmospheric conditions showed progressive downregulation in vitro compared to in vivo ETT biofilms. The expression patterns of clfB and ebps genes were similar to icaC. In contrast, the expression of the RNA III gene showed progressive upregulation from day 1 to day 4 (p < 0.001). Conclusions: MRSA loses its biofilm gene expression in vitro, by adaptive features across multiple generations, as evidenced by the progressive downregulation of icaC and upregulation of RNA III. These findings underscore the significance of host–environment dependence in regulating bacterial biofilm genes, highlighting its importance in diagnostics. Bacterial strains lose their host-specific characteristics as they are cultured in vitro.

IntroductionBiofilm production is an important yet currently overlooked aspect of diagnostic microbiology that has implications for antimicrobial stewardship. In this study, we aimed to validate and identify additional applications of the BioFilm Ring Test® (BRT) for Pseudomonas aeruginosa (PA) isolates from patients with bronchiectasis (BE).Materials and methodsSputa were collected from BE patients who had at least one PA positive culture in the previous year. We processed the sputa to isolate both mucoid and non-mucoid PA, and determined their susceptibility pattern, mucA gene status, and presence of ciprofloxacin mutations in QRDR genes. The Biofilm production index (BPI) was obtained at 5 and 24 hours. Biofilms were imaged using Gram staining.ResultsWe collected 69 PA isolates, including 33 mucoid and 36 non-mucoid. A BPI value below 14.75 at 5 hours predicted the mucoid PA phenotype with 64% sensitivity and 72% specificity.ConclusionOverall, our findings suggest that the fitness-cost associated with the mucoid phenotype or ciprofloxacin resistance is shown through a time-dependent BPI profile. The BRT has the potential to reveal biofilm features with clinical implications.

Abstract Background Among all cases of nosocomial pneumonia, Staphylococcus aureus is the second most prevalent pathogen (17.8%). In Europe, 29.9% of the isolates are oxacillin-resistant. The changing epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) nosocomial infections and the decreasing susceptibility to first-line antibiotics leave clinicians with few therapeutic options. The objective of our study was to determine the antimicrobial susceptibility, the associated molecular mechanisms of resistance and the epidemiological relatedness of MRSA strains isolated from the endotracheal tubes (ETT) of intubated critically ill patients in the intensive care unit (ICU) with nosocomial pneumonia caused by Staphylococcus aureus. Methods The antimicrobial susceptibility to vancomycin, linezolid, ciprofloxacin, clindamycin, erythromycin, chloramphenicol, fusidic acid, gentamicin, quinupristin-dalfopristin, rifampicin, sulfamethoxazole/trimethoprim, and tetracycline were measured. Resistance mechanisms were then analyzed by polymerase chain reaction and sequencing. Molecular epidemiology was carried out by multi-locus sequence typing. Results S. aureus isolates were resistant to ciprofloxacin, erythromycin, gentamicin, tetracycline, clindamycin, and fusidic acid. The most frequent mutations in quinolone-resistant S. aureus strains were S84L in the gyrA gene, V511A in the gyrB gene, S144P in the grlA gene, and K401R/E in the grlB gene. Strains resistant to erythromycin carried the ermC, ermA, and msrA genes; the same ermC and ermA genes were detected in strains resistant to clindamycin. The aac(6′)-aph(2″) gene was related to gentamicin resistance, while resistance to tetracycline was related to tetK (efflux pump). The fusB gene was detected in the strain resistant to fusidic acid. The most frequent sequence types were ST22, ST8, and ST217, which were distributed in four clonal complexes (CC5, CC22, CC45, and CC59). Conclusions High levels of resistance to second-line antimicrobials threatens the treatment of nosocomial respiratory infections due to methicillin-resistant S. aureus with decreased susceptibility to linezolid and vancomycin. The wide genotypic diversity found reinforces the central role of ICU infection control in preventing nosocomial transmission.