Titolo della tesi: ANALYSIS OF DIFFERENT COMBINATIONS AGAINST MULTI-DRUG RESISTANCE GRAM-NEGATIVE BACTERIA & INTESTINAL PERMEABILITY AND MICROBIAL TRANSLOCATION DURING CORONAVIRUS DISEASE-19
PART 1. ANALYSIS OF DIFFERENT COMBINATIONS AGAINST MULTI-DRUG RESISTANCE GRAM-NEGATIVE BACTERIA
ABSTRACT
The increasing rates of antimicrobial resistance have become one of the biggest challenges putting at risk the global public health at the beginning of the third millennium, since there is a paucity of effective antimicrobials for the treatment of infections due to multi-drug resistant microorganisms. Multi-drug resistant (MDR) bacteria represent is a challenging problem of contemporary medicine, and the optimal antimicrobial therapy for MDR infection is still needed.
Aims. The study's main purpose was to investigate the activity of antibiotic and non-antibiotic compounds against a collection of clinically relevant MDR Gram-negative microorganisms. To this end, several experiments were performed: i) to analyze the in-vitro activity of Fosfomycin (FOS), alone and in combination with different antibiotics, against different MDR Gram-negative isolates; ii) to test the emergence of MEM resistance among CZA resistant MEM susceptible KPC-producing K. pneumoniae; iii) to analyze the in-vitro activity of NAC, alone and in combination with MEM, against MEM resistant NDM- producing Escherichia coli, iv) to analyze the clinical characteristics of hospitalized patients with colonization or infection due to CZA-resistant MEM-susceptible KPC-Kp and combined them with the results of in vitro analyses.
Methods. The antibacterial activity of each compound was tested by agar dilution, broth microdilution, antimicrobial gradient strip method, and agar disk-diffusion. The synergism between different antimicrobials was evaluated by double E-test, E-test - agar diffusion and checkerboard methods. The following strains were included in the in vitro tests: CZA-resistant MEM-susceptible KPC-Kp; CZA-susceptible CR-Kp; NDM-producing E. coli; CR-Ab. The antimicrobials were included in this study: fosfomycin (FOS), meropenem (MEM), aztreonam (AZT), ceftazidime/avibactam (CZA), meropenem/vaborbactam (MVB), N-acetylcysteine (NAC), ampicillin/sulbactam (AMP/SUL), amoxicillin/clavulanate (AMOX/CLAV).
Results. Overall, 24 strains were included (9 CZA-resistant MEM-susceptible KPC-Kp, 5 CZA- susceptible CR-Kp, 5 NDM-producing E. coli, 5 CR-Ab). FOS MICs50/90 were 128/128, 64/256, 32/64 and 0.25/0.25 µg/mL for CR-Kp, CZA-susceptible CR-Kp, CZA-resistant MEM-susceptible KPC-Kp and NDM-producing E. coli, respectively. In all the tested strains, FOS-containing combinations showed absence of bacterial growth at concentration of the drug easily achievable in the serum. Furthermore, the combination MEM+FOS showed full synergism in 44% of the CZA-resistant MEM-susceptible KPC-Kp strains. In these strains, MEM resistance development occurred after 7 days of antibiotic challenge (MEM MIC 32 µg/mL), with maintenance of CZA resistance. When considering non-antibiotics compounds, NAC showed high activity against NDM-producing E.coli, with MIC50/90 2.5/2.5 mg/mL Of note the combinations of MEM+NAC at sub-inhibitory concentrations showed a full synergism.
Conclusions. In this study, we performed a comprehensive in vitro study with the objective of evaluating the possible role of fosfomycin-combination therapies in the fight against MDR infections. Furthermore, when dealing with non-antibiotics compound, we showed that NAC, alone and in combination with MEM at sub-inhibitory concentrations, was highly in vitro effective. The encouraging results of these in vitro analyses should be confirmed in clinical studies.
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PART 2. INTESTINAL PERMEABILITY AND MICROBIAL TRANSLOCATION DURING CORONAVIRUS DISEASE-19
ABSTRACT
Background. Intestinal involvement has been described during Coronavirus Disease-19 (COVID-19), with up to one-third of COVID-19 patients also experiencing gastrointestinal symptoms such as diarrhea and vomiting along with the most common respiratory symptoms. However, biomarkers of microbial translocation (MT) and intestinal damage (ID) are still poorly explored in COVID-19. The main purposes of this project were to assess i) whether an alteration of gut permeability and cell integrity is present during the course of COVID-19 and, ii) if present, whether the degree of this alteration is more pronounced in severe infection.
Methods. Over a period of 6 months (March 2020–July 2020), patients with COVID-19 hospitalized at Sapienza University (Rome) were enrolled in the study. For each subject, blood samples were collected at COVID-19 diagnosis (T0) and after 7 days (T7), respectively. Patients were further divided into those requiring Intensive Care (ICU) admission or not. Markers of MT [LPB (Lipopolysacharide Binding Protein) and EndoCab IgM] and ID [I-FABP (Intestinal Fatty Acid Binding Protein)] were evaluated using enzyme-linked immunosorbent assays (ELISA) in plasma. As control group, non-hospitalized healthy donors (HDs, n=16) matched for age and sex with negative SARS-CoV-2 RNA detection were enrolled.
Results. A total of 45 patients with COVID-19 were included in the study, median age 66 (56-71) years, 27 males and 18 females. Median duration of symptoms before COVID-19 diagnosis was 3.7 (2.7-9.7) days. Among symptoms, gastrointestinal involvement was observed in 4 subjects (8.8%). Twenty-one patients (46.6%) were admitted to ICU and overall mortality was 22% (10/45). Compared to HD, a high degree of MT and ID was observed during COVID-19, which was maintained, if not increased, from T0 to T7. Similarly, patients who were admitted to ICU had higher levels of MT but not of ID than patients not admitted to ICU. At univariate analysis, high levels of LBP were associated with mortality (p=0.04), whereas at multivariable analysis only the occurrence of thrombotic event was independently associated with death (p=0.018).
Conclusions. Patients with Coronavirus Disease-19 exhibited high level of MT, especially in subjects with more severe infection. COVID-19 is associated with gut permeability and alteration of intestinal mucosa.