High-Risk Clone of Klebsiella pneumoniae Co-Harbouring Class A and D Carbapenemases in Italy.

Background: Carbapenem-resistant Klebsiella pneumoniae (CR-Kp) is endemic globally, causing severe infections in hospitalized patients. Surveillance programs help monitor and promptly identify the emergence of new clones. We reported the rapid spread of a novel clone of K. pneumoniae co-harbouring class A and D carbapenemases in colonized patients, and the potential risk factors involved in the development of infections. Methods: Rectal swabs were used for microbiological analyses and detection of the most common carbapenemase encoding genes by real-time PCR (i.e., blaKPC, blaOXA-48, blaNDM, blaVIM, and blaIMP). All strains co-harbouring KPC and OXA-48 genes were evaluated. For each patient, the following variables were collected: age, sex, length and ward of stay, device use, and outcome. Clonality of CR-Kp was assessed by preliminary pulsed field gel electrophoresis (PFGE), followed by multi-locus sequence typing (MLST) analyses. Results: A total of 127 isolates of K. pneumoniae co-harbouring KPC and OXA-48 were collected between September 2019 and December 2020. The median age (IQR) of patients was 70 (61-77). More than 40% of patients were admitted to intensive care unit (ICU). Around 25% of patients developed an invasive infection, the majority of which were respiratory tract infections (17/31; 54.8%). ICU stay and invasive infection increased the risk of mortality (OR: 5.39, 95% CI: 2.42-12.00; OR 6.12, 95% CI: 2.55-14.69, respectively; p-value ≤ 0.001). The antibiotic susceptibility test showed a resistance profile for almost all antibiotics considered. Monoclonal origin was confirmed by PFGE and MLST showing a similar restriction pattern and belonging to ST-512. Conclusions: We report the spread and the marked antibiotic resistance profiles of K. pneumoniae strains co-producing KPC and OXA-48. Further study could clarify the roles of clinical and microbiological variables in the development of invasive infection and increasing risk of mortality, in colonized patients.

Antibiotic Susceptibility of Environmental Legionella pneumophila Strains Isolated in Northern Italy.

Legionella pneumophila is ubiquitous in aquatic environments and responsible for severe pneumonia in humans through inhalation of aerosol containing Legionella spp. Macrolides and fluoroquinolones are frequently used antimicrobials, but treatment failures are increasingly being reported. As susceptibility testing is not routinely performed, this study aimed to determine the minimum inhibitory concentrations (MICs) on 58 environmental Legionella pneumophila strains (24 of serogroup 1 and 34 of non-serogroup 1) isolated in Northern Italy. MICs of azithromycin, erythromycin, ciprofloxacin, levofloxacin, and rifampicin were determined by the microdilution method using buffered yeast extract broth supplemented with α-ketoglutarate (BYEα). Seventy-five percent of Legionella pneumophila isolates showed MIC values below the tentative highest MICs indicated by the European Committee on Antimicrobial Susceptibility Testing (EUCAST); rifampicin was the most active agent with MIC90 values below 0.008 mg/L. Interestingly, one isolate was tested and found to be PCR-positive for the azithromycin LpeAB active efflux system, further confirmed by the reserpine/resazurin microtiter assay. In conclusion, this study has provided additional susceptibility data for environmental Legionella pneumophila isolates from Northern Italy demonstrating, in general, low MICs values for the tested antimicrobials, although one strain tested was shown to possess the LpeAB resistance determinant, indicating that future surveillance studies are warranted.

Evaluation of an Environmental Transport Medium for Legionella pneumophila Recovery.

The collection and storage of water-related matrices such as biofilm from collection to processing are critical for the detection of Legionella pneumophila by cultural and molecular tests. SRK™ is a liquid medium that acts both as an antimicrobial neutralizing agent and a transport medium for bacterial culture enumeration and is useful to maintain the stability of the sample from collection to analysis. The aims of this study were to evaluate Legionella pneumophila viability and bacterial nucleic acids' stability in SRK™ medium over time at different storage conditions. Artificial bacterial inoculates with an approximate concentration of 104, 103 and 102 CFU/mL were made using Legionella pneumophila certified reference material suspended in SRK™ medium. Bacteria recovery was analyzed by cultural and molecular methods at time 0, 24 and 48 h at room temperature and at 0, 24, 48 and 72 h at 2-8 °C, respectively. SRK™ medium supported Legionella pneumophila culture viability with CFU counts within the expected range. The recovery after 72 h at 2-8 °C was 83-100% and 75-95% after 48 h at room temperature. Real-time PCR appropriately detected Legionella pneumophila DNA at each temperature condition, dilution and time point. Results demonstrated a good performance of SRK™ medium for the reliable recovery of environmental Legionella.

In vitro bactericidal activity of 0.6% povidone-iodine eye drops formulation.

PURPOSE: To evaluate the bactericidal activity of a diluted povidone-iodine formulation (0.6%) in comparison with the most used 5% povidone-iodine solution ophthalmic preparation. METHODS: In vitro bactericidal activity comparison between 0.6% povidone-iodine versus 5% povidone-iodine formulations, against these bacteria: Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 43300, Staphylococcus epidermidis ATCC 12228, linezolid-resistant Staphylococcus epidermidis α99 strain, a clinical isolate, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922. RESULTS: About 0.6% povidone-iodine formulation was demonstrated to be faster than 5% povidone-iodine preparation in killing Gram-positive as well as Gram-negative bacteria. Against a linezolid-resistant methicillin-resistant Staphylococcus epidermidis strain, 0.6% povidone-iodine formulation showed the best antiseptic efficacy requirement of 3-log10 reduction in bacterial load, if compared with the 5% povidone-iodine formulation. CONCLUSION: Our investigation has demonstrated that the more diluted 0.6% preparation was more rapidly bactericidal than the 5% povidone-iodine formulation, most probably due to the fact that dilution from 5% to 0.6% increases the amount of free iodine. While our finding must be confirmed by in vivo clinical studies, this fact constitutes an intriguing news for what concerns the use of povidone-iodine eye drops in the ocular surface treatment before intravitreal injections as well as ophthalmic surgery.

4-Alkyliden-azetidinones modified with plant derived polyphenols: Antibacterial and antioxidant properties.

Antimicrobial resistance is one of the major and growing concerns in hospital- and community acquired infections, and new antimicrobial agents are therefore urgently required. It was reported that oxidative stress could contribute to the selection of resistant bacterial strains, since reactive oxygen species (ROS) revealed to be an essential driving force. In the present work 4-alkylidene-azetidinones, a new class of antibacterial agents, were functionalized with phytochemical polyphenolic acids such as protocatechuic, piperonyl, caffeic, ferulic, or sinapic acids and investigated as dual target antibacterial-antioxidant compounds. The best candidates showed good activities against multidrug resistant clinical isolates of MRSA (MICs 2-8 µg/mL). Among the new compounds, two revealed the best antioxidant capacity with TEAC-DPPH and TEAC-ABTS being significantly more active than Trolox®.

Design and Synthesis of 4-Alkylidene-ß-lactams: Benzyl- and Phenethyl-carbamates as Key Fragments to Switch on Antibacterial Activity.

The emergence of multidrug-resistant bacterial strains is particularly important in chronic pathologies such as cystic fibrosis (CF), in which persistent colonization and selection of resistant strains is favored by the frequent and repeated use of antibacterial agents. Staphylococcus aureus is a common pathogen in CF patients that has an associated increased multidrug resistance. In previous studies we demonstrated that the presence of a 4-alkylidene side chain directly linked to a ß-lactam appeared to strengthen the potency against S. aureus, especially against methicillin-resistant S. aureus (MRSA) strains. In the present study, 21 new 4-alkylidene-ß-lactams were synthesized and evaluated for antibacterial activity. We designed the new compounds to have aryl, benzyl, or phenethyl-carbamate groups on the C3 hydroxyethyl side chain. We found a correlation between biological activity and the nitrogen substituent of the carbamate group, and two phenethyl-carbamate ß-lactams were shown to be valuable antibacterial agents against selected linezolid-resistant strains, with a minimum inhibitory concentrations of 2-4 mg L-1 .

Resistance to linezolid in Staphylococcus spp. clinical isolates associated with ribosomal binding site modifications: novel mutation in domain V of 23S rRNA.

Linezolid is the main representative of the oxazolidinones, introduced in 2000 in clinical practice to treat severe Gram-positive infections. This compound inhibits protein synthesis by binding to the peptidyl transferase centre of the 50S bacterial ribosomal subunit. The aim of this study was to characterize 12 clinical strains of linezolid-resistant Staphylococcus spp. isolated in Northern Italy. All isolates of Staphylococcus spp. studied showed a multi-antibiotic resistance phenotype. In particular, all isolates showed the presence of the mecA gene associated with SSCmec types IVa, V or I. Mutations in domain V of 23S rRNA were shown to be the most prevalent mechanism of linezolid resistance: among these a new C2551T mutation was found in S. aureus, whilst the G2576T mutation was shown to be the most prevalent overall. Moreover, three S. epidermidis isolates were shown to have linezolid resistance associated only with alterations in both L3 and L4 ribosomal proteins. No strain was shown to harbor the previously described cfr gene. These results have shown how the clinical use of linezolid in Northern Italy has resulted in the selection of multiple antibiotic-resistant clinical isolates of Staphylococcus spp., with linezolid resistance in these strains being associated with mutations in 23S rRNA or ribosomal proteins L3 and L4.

Molecular typing of XDR Acinetobacter baumannii strains in an Italian ICU.

OBJECTIVE: To investigate the antimicrobial susceptibility and clonal relationship of Acinetobacter baumannii strains isolated in an Italian ICU. DESIGN: Epidemiological, observational, retrospective, longitudinal study. SETTING AND PARTICIPANTS: The ICU of the University Hospital of Sassari, Italy. MAIN OUTCOME MEASURES: Pulsed Field Gel Electrophoresis (PFGE) and Multi Locus Sequence Typing (MLST) were used to evaluate the genomic features of the isolated strains. RESULTS: Drug susceptibility testing for all isolated strains showed the same resistance pattern, characterized by resistance to the most important antibiotics, with the only exception of colistin. PFGE showed a very poor between-strain variability; three distinct clusters, 11, 4, and 1 isolates in size, were identified (Dice's coefficient: 92.11%). MLST showed that all isolated strains belonged to sequence type 2 (ST2). All isolates collected from the environment and the human samples were positive for the following genes: blaOXA-23, blaOXA-51-like, blaVIM-like, blaIMP-like, andISAba1; however, blaOXA-24-like, blaOXA-58-like, and blaNDM-like were not detected. CONCLUSIONS: The survey identified XDR strains belonging to the same cell clone, confirming the wide circulation and environmental persistence of this microorganism.