Bacterial Adaptive Memory in Methicillin-Resistant Staphylococcus aureus from Endotracheal Tubes.

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.

A long-lasting porcine model of ARDS caused by pneumonia and ventilator-induced lung injury.

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 < 150 mmHg. Four animals (pneumonia-without-VILI group) were protectively ventilated 3 h before inoculum and thereafter. Gas exchange, respiratory mechanics, hemodynamics, microbiological studies and inflammatory markers were analyzed during the 96-h experiment. During necropsy, lobar samples were also analyzed. RESULTS: All animals from pneumonia-with-VILI group reached Berlin criteria for ARDS diagnosis until the end of experiment. The mean duration under ARDS diagnosis was 46.8 ± 7.7 h; the lowest PaO2/FiO2 was 83 ± 5.45 mmHg. The group of pigs that were not subjected to VILI did not meet ARDS criteria, even when presenting with bilateral pneumonia. Animals developing ARDS presented hemodynamic instability as well as severe hypercapnia despite high-minute ventilation. Unlike the pneumonia-without-VILI group, the ARDS animals presented lower static compliance (p = 0.011) and increased pulmonary permeability (p = 0.013). The highest burden of P. aeruginosa was found at pneumonia diagnosis in all animals, as well as a high inflammatory response shown by a release of interleukin (IL)-6 and IL-8. At histological examination, only animals comprising the pneumonia-with-VILI group presented signs consistent with diffuse alveolar damage. CONCLUSIONS: In conclusion, we established an accurate pulmonary sepsis-induced ARDS model.

Determining the most accurate 16S rRNA hypervariable region for taxonomic identification from respiratory samples.

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.

Resistance mechanisms and molecular epidemiology of Pseudomonas aeruginosa strains from patients with bronchiectasis.

BACKGROUND: Non-cystic fibrosis bronchiectasis (BE) is a chronic structural lung condition that facilitates chronic colonization by different microorganisms and courses with recurrent respiratory infections and frequent exacerbations. One of the main pathogens involved in BE is Pseudomonas aeruginosa. OBJECTIVES: To determine the molecular mechanisms of resistance and the molecular epidemiology of P. aeruginosa strains isolated from patients with BE. METHODS: A total of 43 strains of P. aeruginosa were isolated from the sputum of BE patients. Susceptibility to the following antimicrobials was analysed: ciprofloxacin, meropenem, imipenem, amikacin, tobramycin, aztreonam, piperacillin/tazobactam, ceftazidime, ceftazidime/avibactam, ceftolozane/tazobactam, cefepime and colistin. The resistance mechanisms present in each strain were assessed by PCR, sequencing and quantitative RT-PCR. Molecular epidemiology was determined by MLST. Phylogenetic analysis was carried out using the eBURST algorithm. RESULTS: High levels of resistance to ciprofloxacin (44.19%) were found. Mutations in the gyrA, gyrB, parC and parE genes were detected in ciprofloxacin-resistant P. aeruginosa strains. The number of mutated QRDR genes was related to increased MIC. Different ß-lactamases were detected: blaOXA50, blaGES-2, blaIMI-2 and blaGIM-1. The aac(3)-Ia, aac(3)-Ic, aac(6″)-Ib and ant(2″)-Ia genes were associated with aminoglycoside-resistant strains. The gene expression analysis showed overproduction of the MexAB-OprM efflux system (46.5%) over the other efflux system. The most frequently detected clones were ST619, ST676, ST532 and ST109. CONCLUSIONS: Resistance to first-line antimicrobials recommended in BE guidelines could threaten the treatment of BE and the eradication of P. aeruginosa, contributing to chronic infection.

Molecular characterization of methicillin-resistant Staphylococcus aureus clinical strains from the endotracheal tubes of patients with nosocomial pneumonia.

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.

Mucoid Pseudomonas aeruginosa alters sputum viscoelasticity in patients with non-cystic fibrosis bronchiectasis.

INTRODUCTION AND AIM: Pseudomonas aeruginosa could acquire a mucoid phenotype due to mutations in mucA (mucoid Pseudomonas aeruginosa - mPA) that is a hallmark of poor prognosis in patients with bronchiectasis. Despite the higher prevalence of Pseudomonas aeruginosa in bronchiectasis, how mPA and non-mucoid Pseudomonas aeruginosa (non-mPA) phenotypes could affect viscoelastic properties of sputum is unknown. Our aim was to determine the relationship between Pseudomonas aeruginosa phenotypes isolation, the viscoelastic properties of sputum and the clinical outcomes in patients with bronchiectasis. METHODS: A cross-sectional study was conducted of sputum samples obtained by spontaneous expectoration and sent for microbiology and rheology analysis. Elasticity and viscosity were measured at two oscillatory frequencies (1 and 100 rad/s). Socio-demographic and clinical data were recorded. RESULTS: We analyzed 17 patients with mPA, 14 with non-mPA and 17 with no organism reported (NOR). Compared with the NOR group, the mPA group showed higher elasticity (median 10.30 vs. 5.70, p = 0.023), viscosity (2.40 vs. 1.50, p = 0.039), and stiffness (10.70 vs. 6.00, p = 0.024). Values in the mPA group tended to be higher compared with non-mPA. Clinically, the mPA group showed greater hospitalizations during the previous year and greater affected lobes than the non-mPA and NOR groups. CONCLUSIONS: The mPA phenotype is associated with increased elasticity, viscosity and stiffness of bronchiectatic sputum. Viscoelastic properties could be used as a marker of poor mucociliary clearance in mPA, with potentially important clinical implications.