Risk factors for COVID-19 associated pulmonary aspergillosis and outcomes in patients with acute respiratory failure in a respiratory sub-intensive care unit.

BACKGROUND: COVID-19-associated pulmonary aspergillosis (CAPA) is burdened by high mortality. Data are lacking about non-ICU patients. Aims of this study were to: (i) assess the incidence and prevalence of CAPA in a respiratory sub-intensive care unit, (ii) evaluate its risk factors and (iii) impact on in-hospital mortality. Secondary aims were to: (i) assess factors associated to mortality, and (ii) evaluate significant features in hematological patients. MATERIALS AND METHODS: This was a single-center, retrospective study of COVID-19 patients with acute respiratory failure. A cohort of CAPA patients was compared to a non-CAPA cohort. Among patients with CAPA, a cohort of hematological patients was further compared to another of non-hematological patients. RESULTS: Three hundred fifty patients were included in the study. Median P/F ratio at the admission to sub-intensive unit was 225 mmHg (IQR 155-314). 55 (15.7%) developed CAPA (incidence of 5.5%). Eighteen had probable CAPA (37.3%), 37 (67.3%) possible CAPA and none proven CAPA. Diagnosis of CAPA occurred at a median of 17 days (IQR 12-31) from SARS-CoV-2 infection. Independent risk factors for CAPA were hematological malignancy [OR 1.74 (95%CI 0.75-4.37), p = 0.0003], lymphocytopenia [OR 2.29 (95%CI 1.12-4.86), p = 0.02], and COPD [OR 2.74 (95%CI 1.19-5.08), p = 0.014]. Mortality rate was higher in CAPA cohort (61.8% vs 22.7%, p < 0.0001). CAPA resulted an independent risk factor for in-hospital mortality [OR 2.92 (95%CI 1.47-5.89), p = 0.0024]. Among CAPA patients, age > 65 years resulted a predictor of mortality [OR 5.09 (95% CI 1.20-26.92), p = 0.035]. No differences were observed in hematological cohort. CONCLUSION: CAPA is a life-threatening condition with high mortality rates. It should be promptly suspected, especially in case of hematological malignancy, COPD and lymphocytopenia.

The antimicrobial potential of adarotene derivatives against Staphylococcus aureus strains.

Multidrug-resistant (MDR) pathogens are severely impacting our ability to successfully treat common infections. Here we report the synthesis of a panel of adarotene-related retinoids showing potent antimicrobial activity on Staphylococcus aureus strains (including multidrug-resistant ones). Fluorescence and molecular dynamic studies confirmed that the adarotene analogues were able to induce conformational changes and disfunctions to the cell membrane, perturbing the permeability of the phospholipid bilayer. Since the major obstacle for developing retinoids is their potential cytotoxicity, a selected candidate was further investigated to evaluate its activity on a panel of human cell lines. The compound was found to be well tolerated, with IC50 5-15-fold higher than the MIC on S. aureus strains. Furthermore, the adarotene analogue had a good pharmacokinetic profile, reaching a plasma concentration of about 6 µM after 0.5 h after administration (150 mg/kg), at least twice the MIC observed against various bacterial strains. Moreover, it was demonstrated that the compound potentiated the growth-inhibitory effect of the poorly bioavailable rifaximin, when used in combination. Overall, the collected data pave the way for the development of synthetic retinoids as potential therapeutics for hard-to-treat infectious diseases caused by antibiotic-resistant Gram-positive pathogens.

Anti-Biofilm Activity of Oleacein and Oleocanthal from Extra-Virgin Olive Oil toward Pseudomonas aeruginosa.

New antimicrobial molecules effective against Pseudomonas aeruginosa, known as an antibiotic-resistant "high-priority pathogen", are urgently required because of its ability to develop biofilms related to healthcare-acquired infections. In this study, for the first time, the anti-biofilm and anti-virulence activities of a polyphenolic extract of extra-virgin olive oil as well as purified oleocanthal and oleacein, toward P. aeruginosa clinical isolates were investigated. The main result of our study was the anti-virulence activity of the mixture of oleacein and oleocanthal toward multidrug-resistant and intermediately resistant strains of P. aeruginosa isolated from patients with ventilator-associated pneumonia or surgical site infection. Specifically, the mixture of oleacein (2.5 mM)/oleocanthal (2.5 mM) significantly inhibited biofilm formation, alginate and pyocyanin production, and motility in both P. aeruginosa strains (p < 0.05); scanning electron microscopy analysis further evidenced its ability to inhibit bacterial cell adhesion as well as the production of the extracellular matrix. In conclusion, our results suggest the potential application of the oleacein/oleocanthal mixture in the management of healthcare-associated P. aeruginosa infections, particularly in the era of increasing antimicrobial resistance.

Mortality Attributable to Bloodstream Infections Caused by Different Carbapenem-Resistant Gram-Negative Bacilli: Results From a Nationwide Study in Italy (ALARICO Network).

BACKGROUND: Our aim was to analyze mortality attributable to carbapenem-resistant (CR) gram-negative bacilli (GNB) in patients with bloodstream infections (BSIs). METHODS: Prospective multicentric study including patients with GNB-BSI from 19 Italian hospitals (June 2018-January 2020). Patients were followed-up to 30 days. Primary outcomes were 30-day mortality and attributable mortality. Attributable mortality was calculated in the following groups: Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales, metallo-ß-lactamases (MBL)-producing Enterobacterales, CR-Pseudomonas aeruginosa (CRPA), CR-Acinetobacter baumannii (CRAB). A multivariable analysis with hospital fixed-effect was built to identify factors associated with 30-day mortality. Adjusted OR (aORs) were reported. Attributable mortality was calculated according to the DRIVE-AB Consortium. RESULTS: Overall, 1276 patients with monomicrobial GNB BSI were included: 723/1276 (56.7%) carbapenem-susceptible (CS)-GNB, 304/1276 (23.8%) KPC-, 77/1276 (6%) MBL-producing CRE, 61/1276 (4.8%) CRPA, and 111/1276 (8.7%) CRAB BSI. Thirty-day mortality in patients with CS-GNB BSI was 13.7% compared to 26.6%, 36.4%, 32.8% and 43.2% in patients with BSI by KPC-CRE, MBL-CRE, CRPA and CRAB, respectively (P < .001). On multivariable analysis, age, ward of hospitalization, SOFA score, and Charlson Index were factors associated with 30-day mortality, while urinary source of infection and early appropriate therapy resulted protective factors. Compared to CS-GNB, MBL-producing CRE (aOR 5.86, 95% CI 2.72-12.76), CRPA (aOR 1.99, 95% CI 1.48-5.95) and CRAB (aOR 2.65, 95% CI 1.52-4.61) were significantly associated with 30-day mortality. Attributable mortality rates were 5% for KPC-, 35% for MBL, 19% for CRPA, and 16% for CRAB. CONCLUSIONS: In patients with BSIs, carbapenem-resistance is associated with an excess of mortality, with MBL-producing CRE carrying the highest risk of death.

Genotypic Evolution of Klebsiella pneumoniae Sequence Type 512 during Ceftazidime/Avibactam, Meropenem/Vaborbactam, and Cefiderocol Treatment, Italy.

In February 2022, a critically ill patient colonized with a carbapenem-resistant K. pneumoniae producing KPC-3 and VIM-1 carbapenemases was hospitalized for SARS-CoV-2 in the intensive care unit of Policlinico Umberto I hospital in Rome, Italy. During 95 days of hospitalization, ceftazidime/avibactam, meropenem/vaborbactam, and cefiderocol were administered consecutively to treat 3 respiratory tract infections sustained by different bacterial agents. Those therapies altered the resistome of K. pneumoniae sequence type 512 colonizing or infecting the patient during the hospitalization period. In vivo evolution of the K. pneumoniae sequence type 512 resistome occurred through plasmid loss, outer membrane porin alteration, and a nonsense mutation in the cirA siderophore gene, resulting in high levels of cefiderocol resistance. Cross-selection can occur between K. pneumoniae and treatments prescribed for other infective agents. K. pneumoniae can stably colonize a patient, and antimicrobial-selective pressure can promote progressive K. pneumoniae resistome evolution, indicating a substantial public health threat.

Multiplicity of blaKPC Genes and pKpQIL Plasmid Plasticity in the Development of Ceftazidime-Avibactam and Meropenem Coresistance in Klebsiella pneumoniae Sequence Type 307.

In 2021, Klebsiella pneumoniae sequence type 307 (ST307) strains causing pulmonary and bloodstream infections identified in a hospital in Rome, Italy, reached high levels of resistance to ceftazidime-avibactam (CZA). One of these strains reached high levels of resistance to both CZA and carbapenems and carried two copies of blaKPC-3 and one copy of blaKPC-31 located on plasmid pKpQIL. The genomes and plasmids of CZA-resistant ST307 strains were analyzed to identify the molecular mechanisms leading to the evolution of resistance and compared with ST307 genomes at local and global levels. A complex pattern of multiple plasmids in rearranged configurations, coresident within the CZA-carbapenem-resistant K. pneumoniae strain, was observed. Characterization of these plasmids revealed recombination and segregation events explaining why K. pneumoniae isolates from the same patient had different antibiotic resistance profiles. This study illustrates the intense genetic plasticity occurring in ST307, one of the most worldwide-diffused K. pneumoniae high-risk clones.

Topical antibiotic prophylaxis before intravitreal injections: a pilot study.

PURPOSE: To explore whether topical antibiotic prophylaxis in patients scheduled for intravitreal injections achieves surface sterility in a greater proportion of subjects as compared to povidone-iodine alone. MATERIAL AND METHODS: A randomized, triple-blind clinical trial. POPULATION: patients scheduled for intravitreal injections for maculopathy. INCLUSION CRITERIA: any sex and race, age 18 years and above. Subjects were randomized into 4 groups: the first group applied chloramphenicol (CHLORAM), the second netilmicin (NETILM), the third a commercial ozonized antiseptic solution (OZONE), and the fourth applied no drops (CONTROL). OUTCOME VARIABLE: percentage of non-sterile conjunctival swabs. Specimens were collected before and after the application of 5% povidone-iodine moments before the injection. RESULTS: Ninety-eight subjects (33.7% females, 64.3% males), mean age: 70.2 ± 9.3 years (54-91). Before povidone-iodine, both the CHLORAM and NETILM group showed a lower percentage of non-sterile swabs (61.1% and 31.3% respectively), as compared to the OZONE (83.3%) and CONTROL (86.5%) groups (p < .04). However, this statistical difference was lost after the application of povidone-iodine for 3 min. Percentage of non-sterile swabs in each group after applying 5% povidone-iodine: CHLORAM 11.1%, NETILM 12.5%, CONTROL 15.4%, OZONE 25.0%. This was not statistically significant (p > .05). CONCLUSIONS: Topical antibiotic prophylaxis with chloramphenicol or netilmicin drops decreases the bacterial load on the conjunctiva. However, after the application of povidone-iodine, all groups showed a significant reduction in the percentage of non-sterile swabs, and this value was comparable among all groups. For this reason, authors conclude that povidone-iodine alone is sufficient and prior topical antibiotic prophylaxis is not indicated.

The effect of the electromagnetic field on metabolic-active bacterial biofilm experimentallyinduced on titanium dental implants.

Microbial biofilm is of paramount importance in the development of mucositis or peri-implantitis in patients with dental implants. This study was designed to investigate whether an electromagnetic field at high frequency waves directly applied on 33 titanium implants could remove experimentally-induced Enterococcus faecalis bacterial biofilm. A specially designed device (X-IMPLANT) was used to generate the electromagnetic field, with output power of 8 W, supply frequency (action/pause) 3/2s, and an output frequency of 625±5% kHz in plastic devices containing the biofilm-covered implants immersed in sterile saline. The bacterial biofilm on both treated and untreated control implants was quantitatively measured by phenol red-based Bio-Timer-Assay reagent. The kinetic analysis of the curves showed that the electrical treatment generated by the X-IMPLANT device completely removed the bacterial biofilm after 30 minutes of treatment (p<0.01). Elimination of the biofilm was also confirmed by chromatic observation in the macro-method. Our data seem to indicate that the procedure could be considered for clinical application in peri-implantitis to counteract bacterial biofilm on dental implants.

Interplay between Klebsiella pneumoniae producing KPC-31 and KPC-3 under treatment with high dosage meropenem: a case report.

The objective was to study ceftazidime-avibactam resistant and susceptible Klebsiella pneumoniae isolated from a patient admitted to the Policlinico Umberto I of Rome for SARS-CoV2. Data on the evolution of patient's conditions, antimicrobial therapies, and microbiological data were collected. Whole-genome sequencing performed by Illumina and Nanopore sequencing methods were used to type the strains. During the hospitalization, a SARS-CoV2-infected patient was colonized by a KPC-producing K. pneumoniae strain and empirically treated with ceftazidime-avibactam (CZA) when presenting spiking fever symptoms. Successively, ST2502 CZA-resistant strain producing the KPC-31 variant gave a pulmonary infection to the patient. The infection was treated with high doses of meropenem. The KPC-31-producing strain disappeared but the patient remained colonized by a KPC-3-producing K. pneumoniae strain. An interplay between highly conserved KPC-31- and KPC-3-producing ST2502 strains occurred in the SARS-CoV2 patient during the hospitalization, selected by CZA and carbapenem treatments, respectively.

Exposure to b-LED Light While Exerting Antimicrobial Activity on Gram-Negative and -Positive Bacteria Promotes Transient EMT-like Changes and Growth Arrest in Keratinocytes.

While blue LED (b-LED) light is increasingly being studied for its cytotoxic activity towards bacteria in therapy of skin-related infections, its effects on eukaryotic cells plasticity are less well characterized. Moreover, since different protocols are often used, comparing the effect of b-LED towards both microorganisms and epithelial surfaces may be difficult. The aim of this study was to analyze, in the same experimental setting, both the bactericidal activity and the effects on human keratinocytes. Exposure to b-LED induced an intense cytocidal activity against Gram-positive (i.e, Staphylococcus aureus) and Gram-negative (i.e., Pseudomonas aeruginosa) bacteria associated with catheter-related infections. Treatment with b-LED of a human keratinocyte cell line induced a transient cell cycle arrest. At the molecular level, exposure to b-LED induced a transient downregulation of Cyclin D1 and an upregulation of p21, but not signs of apoptosis. Interestingly, a transient induction of phosphor-histone γ-H2Ax, which is associated with genotoxic damages, was observed. At the same time, keratinocytes underwent a transient epithelial to mesenchymal transition (EMT)-like phenotype, characterized by E-cadherin downregulation and SNAIL/SLUG induction. As a functional readout of EMT induction, a scratch assay was performed. Surprisingly, b-LED treatment provoked a delay in the scratch closure. In conclusion, we demonstrated that b-LED microbicidal activity is associated with complex responses in keratinocytes that certainly deserve further analysis.

Evolutionary Trajectories toward Ceftazidime-Avibactam Resistance in Klebsiella pneumoniae Clinical Isolates.

From January 2019 to April 2020, 32 KPC-producing, ceftazidime-avibactam (CZA)-resistant Klebsiella pneumoniae strains were isolated in a university hospital in Rome, Italy. These strains belonged to the sequence type 512 (ST512), ST101, and ST307 high-risk clones. Nine different CZA-resistant KPC-3 protein variants were identified, five of them never previously reported (KPC-66 to KPC-70). Among the nine, KPC-31, KPC-39, KPC-49, KPC-66, KP-68, KPC-69, and KPC-70 showed amino acid substitutions, insertions, and deletions in the Ω loop of the protein. KPC-29 has a duplication, while the novel KPC-67 has a triplication, of the KDD triplet in the 270-loop, a secondary loop of the KPC-3 protein. Genomics performed on contemporary resistant and susceptible clones underlined that these novel mutations emerged in blaKPC-3 genes located on conserved plasmids: in ST512, all blaKPC-3 mutant genes were located in pKpQIL plasmids, while the three novel blaKPC-3 mutants identified in ST101 were on FIIk-FIA(HI1)-R plasmids. Selection also promoted multiplication of the carbapenemase gene copy number by transposition, recombination, and fusion of resident plasmids. When expressed in Escherichia coli recipient cells cloned in the high-copy-number pTOPO vector, the Ω loop mutated variants showed the CZA-resistant phenotype associated with susceptibility to carbapenems, while KPC variants with insertions in the 270-loop showed residual activity on carbapenems. The investigation of CZA resistance mechanisms offered the unique opportunity to study vertical, horizontal, and oblique evolutionary trajectories of K. pneumoniae high-risk clones.

The role of teicoplanin in the treatment of SARS-CoV-2 infection: A retrospective study in critically ill COVID-19 patients (Tei-COVID study).

Teicoplanin has a potential antiviral activity expressed against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and was suggested as a complementary option to treat coronavirus disease 2019 (COVID-19) patients. In this multicentric, retrospective, observational research the aim was to evaluate the impact of teicoplanin on the course of COVID-19 in critically ill patients. Fifty-five patients with severe COVID-19, hospitalized in the intensive care units (ICUs) and treated with best available therapy were retrospectively analysed. Among them 34 patients were also treated with teicoplanin (Tei-COVID group), while 21 without teicoplanin (control group). Crude in-hospital Day-30 mortality was lower in Tei-COVID group (35.2%) than in control group (42.8%), however not reaching statistical significance (p = .654). No statistically significant differences in length of stay in the ICU were observed between Tei-COVID group and control group (p = .248). On Day 14 from the ICU hospitalization, viral clearance was achieved in 64.7% patients of Tei-COVID group and 57.1% of control group, without statistical difference. Serum C-reactive protein level was significantly reduced in Tei-COVID group compared to control group, but not other biochemical parameters. Finally, Gram-positive were the causative pathogens for 25% of BSIs in Tei-COVID group and for 70.6% in controls. No side effects related to teicoplanin use were observed. Despite several limitations require further research, in this study the use of teicoplanin is not associated with a significant improvement in outcomes analysed. The antiviral activity of teicoplanin against SARS-CoV-2, previously documented, is probably more effective at early clinical stages.

Fast and reliable diagnosis of XDR Acinetobacter baumannii meningitis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Bacterial meningitis is a medical emergency needing quick and timely diagnosis. Even though meningitis caused by Acinetobacter baumannii is relatively rare, it is associated with high mortality rates especially in neurosurgery patients and represents a serious therapeutic problem due to the limited penetration of effective antibiotics into the cerebrospinal fluid. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) has been effectively used as a rapid method for microbial identification. In this case report we identified A. baumanni by MALDI-TOF technique directly from the CSF drawn from the external ventricular drainage of a patient with severe confusional state and signs of meningism. Simultaneously the antibiotic susceptibility test was performed by automated method from the pellet of the broth-enriched sample. The MALDI-TOF technique allowed microbial identification in less than 30 minutes, and the susceptibility test result was available in eight hours, thus allowing a fast diagnosis ready for prompt and targeted antimicrobial therapy.

Nosocomial-acquired and community-onset Clostridium difficile infection at an academic hospital in Italy: Epidemiology, recurrences and toxin genes distribution.

Toxinogenic Clostridium difficile infection (CDI) is a leading cause of infectious diarrhea. In this retrospective cohort study the molecular epidemiology of hospital-acquired and community-associated CDI was investigated in patients admitted to a tertiary care hospital. CD in stools samples was revealed by a two steps diagnostic algorithm, firstly screening for positivity to GDH antigen and thereafter RT-PCR analysis. Increased CDI incidence was observed ranging from 1.70episodes/10000patient-days in the 1st year, to 2.62 in the 2nd year, mostly hospitalized in the medicine wards, followed by outpatients (5.74 and 5.12episodes/10.000patient-days respectively). CDI positive were older than CDI negative patients and presented increased trend of diarrhea episodes as the patients' age increased. RT-PCR positive patients (n° = 314) were classified according to the CD toxin producing genes in three groups (1-3, carrying tcdB, both tcdB and cdt, and the two genes plus the deletion Δ117 of tcdC, respectively). The incidence of the group 2 and 3 increased statistically with the age of the patients showing correlation with the gender. Higher frequency of patients belonging to group 1 and group 3 was observed in the medical wards. Of note was the high incidence of group 3 in outpatients. Interestingly, patients with previous health care contacts had higher risk (RR = 1.88) of being infected by CD strains with higher toxicity than community patients. Recurrence rate was 15.9%. In conclusion the knowledge of the toxigenic profiles and of their relationships to gender, age and wards distribution may help the clinicians in the clinical management of the disease.

The Correlation Between Biofilm Production and Catheter-Related Bloodstream Infections Sustained by Candida. A Case Control Study.

Biofilm forming capacity of yeasts colonizing the intravenous devices is considered a key factor involved in the pathogenesis of Candida catheter-related bloodstream infections (CCRBSI). The biofilm production of strains of Candida spp. isolated both from the CVC and from the blood of patients with CCRBSI was compared to that of strains isolated from patients not having CCRBSI. Results, expressed in terms of Biofilm Index (BI), revealed that biofilm-producing strains were isolated in the CCRBSI group with a frequency significantly higher than in the non-CCRBSI group (χ2 = 4.25, p = 0.03). The species more frequently cultured was C. parapsilosis complex (including C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis). When this species was isolated from the CVC tip cultures of the CCRBSI group it showed BIs significantly (p = 0.05) higher than those found in the non-CCRBSI group. All the strains of C. tropicalis isolated from the CCRBSI group produced biofilm. Instead most of the isolates of C. glabrata were non-producers. The cumulative BI of non-albicans Candida strains isolated from CCRBSI patients was significantly higher than that of non-albicans strains cultured from patients non-CCRBSI (χ2 = 6.91; p = 0.008). C. albicans was a biofilm producer both in the CCRBSI and in the non-CCRBSI group. When isolated from the blood it showed enhanced biofilm production in the CCRBSI group only, while when colonizing the CVC it displayed high BIs both in the CCRBSI group and in non-CCRBSI group. Our data seem to indicate that the biofilm production capacity should be considered in the clinical management of CCRBSI.

Management and treatment of Magnusiomyces capitatus (Geotrichum capitatum) pleural infection in a non-neutropenic patient with posaconazole. A new therapeutic opportunity?

Magnusiomyces capitatus may cause uncommon yet severe infections, especially in patients with haematologic disorders. Diagnosis may be difficult and time-consuming and newer approaches are required including the MALDI-TOF technique implemented with the detection of fungal antigens in the body fluids. The recommended treatment includes amphotericin B alone or in combination with flucytosine. We describe a case of a non-neutropenic patient with M. capitatus pleural infection, as identified by MALDI-TOF, positivity for galactomannan antigen in the BAL fluid, and successfully treated with oral posaconazole in single therapy.

In vivo evolution to hypermucoviscosity and ceftazidime/avibactam resistance in a liver abscess caused by Klebsiella pneumoniae sequence type 512.

Carbapenemase-producing Klebsiella pneumoniae represents a major public health issue globally. Isolates with resistance to the newest drugs, like ceftazidime/avibactam (CZA), are increasingly reported. In this study, we analyzed the evolution of KPC-3-producing sequence type (ST) 512 K. pneumoniae strains isolated at three different times (hospitalization days 45, 56, and 78) from the same patient, two of which were observed in a pericholecystic liver abscess. The three K. pneumoniae isolates (295Kp, 304Kp, and hmv-318Kp) from the same patient were subjected to antimicrobial susceptibility testing, whole-genome sequencing, sedimentation assay, biofilm measurement, serum resistance assay, macrophage phagocytosis, and adhesion assays. KPC-producing isolate hmv-318Kp exhibited carbapenem susceptibility, hypermucoviscous (hmv) colony phenotype and CZA resistance. Virulence markers of hypervirulent Klebsiella were absent. Two non-synonymous mutations were identified in the hmv-318Kp genome comparing with isogenic strains: a single-nucleotide polymorphism (SNP) occurred in the pKpQIL plasmid, changing blaKPC-3 in the blaKPC-31 gene variant, conferring CZA resistance; and a second SNP occurred in the wzc gene of the capsular biosynthesis cluster, encoding a tyrosine kinase, resulting in the F557S Wzc protein mutation. The Klebsiella pneumoniae strain exhibiting an hmv phenotype (hmv-Kp) phenotype has been previously associated with amino acid substitutions occurring in the Wzc tyrosin kinase protein. We observed in vivo evolution of the ST512 strain to CZA resistance and acquisition of hypermucoviscosity. The pathogenetic role of the detected Wzc substitution is not fully elucidated, but other Wzc mutations were previously reported in hmv K. pneumoniae. Wzc mutants may be more frequent than expected and an underreported cause of hypermucoviscosity in K. pneumoniae clinical isolates. IMPORTANCE: Here we describe the evolution of KPC-3-producing ST512 K. pneumoniae isolated at three different times from the same patient of which the last one, from a biliary abscess, showed CZA resistance by KPC-31 production and manifested hmv colony phenotype. Hypervirulent Klebsiella pneumoniae (hv-Kp) isolates are increasingly reported worldwide. Their hypervirulent traits are associated with the presence of rmpA/A2 genes and an hmv. In this study, we identified an hmv-Kp that lacked the rmpA-D cluster but showed an amino acid substitution in the Wzc tyrosin kinase protein, involved in the capsular biosynthesis. This hmv-Kp strain emerged in vivo and evolved resistance to ceftazidime/avibactam resistance in a liver abscess of a patient. Our findings suggest that wzc mutations may be underreported, making it challenging to distinguish hv-Kp from "classic" K. pneumoniae with an hmv phenotype.

Risk factors for acute kidney injury in critically ill patients receiving high intravenous doses of colistin methanesulfonate and/or other nephrotoxic antibiotics: a retrospective cohort study.

INTRODUCTION: Use of colistin methanesulfonate (CMS) was abandoned in the 1970s because of excessive nephrotoxicity, but it has been reintroduced as a last-resort treatment for extensively drug-resistant infections caused by gram-negative bacteria (Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumonia). We conducted a retrospective cohort study to evaluate risk factors for new-onset acute kidney injury (AKI) in critically ill patients receiving high intravenous doses of colistin methanesulfonate and/or other nephrotoxic antibiotics. METHODS: The cohort consisted of 279 adults admitted to two general ICUs in teaching hospitals between 1 April 2009 and 30 June 2011 with 1) no evidence on admission of acute or chronic kidney disease; and 2) treatment for more than seven days with CMS and/or other nephrotoxic antimicrobials (NAs, that is, aminoglycosides, glycopeptides). Logistic regression analysis was used to identify risk factors associated with this outcome. RESULTS: The 279 cases that met the inclusion criteria included 147 patients treated with CMS, alone (n = 90) or with NAs (n = 57), and 132 treated with NAs alone. The 111 (40%) who developed AKI were significantly older and had significantly higher Simplified Acute Physiology Score II (SAPS II) scores than those who did not develop AKI, but rates of hypertension, diabetes mellitus and congestive heart failure were similar in the two groups. The final logistic regression model showed that in the 147 patients who received CMS alone or with NAs, onset of AKI during the ICU stay was associated with septic shock and with SAPS II scores ≥43. Similar results were obtained in the 222 patients treated with CMS alone or NAs alone. CONCLUSIONS: In severely ill ICU patients without pre-existing renal disease who receive CMS high-dose for more than seven days, CMS therapy does not appear to be a risk factor for this outcome. Instead, the development of AKI was strongly correlated with the presence of septic shock and with the severity of the patients as reflected by the SAPS II score.

Ceftazidime-avibactam resistance in Klebsiella pneumoniae sequence type 37: a decade of persistence and concealed evolution.

The first reports of carbapenem-resistant Enterobacterales in our hospital date back to 2006. In that period, few ertapenem-resistant but meropenem-susceptible Klebsiella pneumoniae isolates belonging to sequence type (ST) 37 were retrieved from clinical samples. These strains produced the CTX-M-15 extended spectrum ß-lactamase, OmpK35 was depleted due to a nonsense mutation, and a novel OmpK36 variant was identified. Yet, starting from 2010, Klebsiella pneumoniae carbapenemase (KPC)-producing ST512 isolates started prevailing and ST37 vanished from sight. Since 2018 the clinical use of the combination of ceftazidime-avibactam (CZA) has been introduced in clinical practice for the treatment of bacteria producing serine-ß-lactamases, but KPC-producing, CZA-resistant K. pneumoniae are emerging. In 2021, four CZA-resistant ST37 isolates producing KPC variants were isolated from the same number of patients. blaKPC gene cloning in Escherichia coli was used to define the role of those KPC variants on CZA resistance, and whole genome sequencing was performed on these isolates and on three ST37 historical isolates from 2011. CZA resistance was due to mutations in the blaKPC genes carried on related pKpQIL-type plasmids, and three variants of the KPC enzyme have been identified in the four ST37 strains. The four ST37 isolates were closely related to each other and to the historical isolates, suggesting that ST37 survived without notice in our hospital for 10 years, waiting to re-emerge as a CZA-resistant K. pneumoniae clone. The ancestor of these contemporary isolates derives from ST37 wild-type porin strains, with no other mutations in chromosomal genes involved in conferring antibiotic resistance (parC, gyrA, ramR, mgrB, pmrB).

Atopic dermatitis-derived Staphylococcus aureus strains: what makes them special in the interplay with the host.

Background: Atopic dermatitis (AD) is a chronic inflammatory skin condition whose pathogenesis involves genetic predisposition, epidermal barrier dysfunction, alterations in the immune responses and microbial dysbiosis. Clinical studies have shown a link between Staphylococcus aureus and the pathogenesis of AD, although the origins and genetic diversity of S. aureus colonizing patients with AD is poorly understood. The aim of the study was to investigate if specific clones might be associated with the disease. Methods: WGS analyses were performed on 38 S. aureus strains, deriving from AD patients and healthy carriers. Genotypes (i.e. MLST, spa-, agr- and SCCmec-typing), genomic content (e.g. virulome and resistome), and the pan-genome structure of strains have been investigated. Phenotypic analyses were performed to determine the antibiotic susceptibility, the biofilm production and the invasiveness within the investigated S. aureus population. Results: Strains isolated from AD patients revealed a high degree of genetic heterogeneity and a shared set of virulence factors and antimicrobial resistance genes, suggesting that no genotype and genomic content are uniquely associated with AD. The same strains were characterized by a lower variability in terms of gene content, indicating that the inflammatory conditions could exert a selective pressure leading to the optimization of the gene repertoire. Furthermore, genes related to specific mechanisms, like post-translational modification, protein turnover and chaperones as well as intracellular trafficking, secretion and vesicular transport, were significantly more enriched in AD strains. Phenotypic analysis revealed that all of our AD strains were strong or moderate biofilm producers, while less than half showed invasive capabilities. Conclusions: We conclude that in AD skin, the functional role played by S. aureus may depend on differential gene expression patterns and/or on post-translational modification mechanisms rather than being associated with peculiar genetic features.