In vivo evolution to echinocandin resistance and increasing clonal heterogeneity in Candida auris during a difficult-to-control hospital outbreak, Italy, 2019 to 2022.

A difficult-to-control outbreak of Candida auris is ongoing in a large tertiary care hospital in Liguria, Italy, where it first emerged in 2019. In a retrospective analysis, 503 cases of C. auris carriage or infection were observed between July 2019 and December 2022. Genomic surveillance identified putative cases that no longer occurred as part of one defined outbreak and the emergence of echinocandin (pandrug) resistance following independent selection of FKS1S639F and FKS1F635Y mutants upon prolonged exposure to caspofungin and/or anidulafungin.

Recent advances in the microbiological diagnosis of bloodstream infections.

Rapid identification (ID) and antimicrobial susceptibility testing (AST) of the causative agent(s) of bloodstream infections (BSIs) are essential for the prompt administration of an effective antimicrobial therapy, which can result in clinical and financial benefits. Immediately after blood sampling, empirical antimicrobial therapy, chosen on clinical and epidemiological data, is administered. When ID and AST results are available, the clinician decides whether to continue or streamline the antimicrobial therapy, based on the results of the in vitro antimicrobial susceptibility profile of the pathogen. The aim of the present study is to review and discuss the experimental data, advantages, and drawbacks of recently developed technological advances of culture-based and molecular methods for the diagnosis of BSI (including mass spectrometry, magnetic resonance, PCR-based methods, direct inoculation methods, and peptide nucleic acid fluorescence in situ hybridization), the understanding of which could provide new perspectives to improve and fasten the diagnosis and treatment of septic patients. Although blood culture remains the gold standard to diagnose BSIs, newly developed methods can significantly shorten the turnaround time of reliable microbial ID and AST, thus substantially improving the diagnostic yield.

A new rapid method for direct antimicrobial susceptibility testing of bacteria from positive blood cultures.

BACKGROUND: Rapid identification and antimicrobial susceptibility testing (AST) of the causative agent(s) of bloodstream infections can lead to prompt appropriate antimicrobial therapy. To shorten species identification, in this study bacteria were recovered from monomicrobial blood cultures by serum separator tubes and spotted onto the target plate for direct MALDI-TOF MS identification. Proper antibiotics were selected for direct AST based on species identification. In order to obtain rapid AST results, bacteria were recovered from positive blood cultures by two different protocols: by serum separator tubes (further referred to as PR1), or after a short-term subculture in liquid medium (further referred to as PR2). The results were compared with those obtained by the method currently used in our laboratory consisting in identification by MALDI-TOF and AST by Vitek 2 or Sensititre on isolated colonies. RESULTS: The direct MALDI-TOF method concordantly identified with the current method 97.5 % of the Gram-negative bacteria and 96.1 % of the Gram-positive cocci contained in monomicrobial blood cultures. The direct AST by PR1 and PR2 for all isolate/antimicrobial agent combinations was concordant/correct with the current method for 87.8 and 90.5 % of Gram-negative bacteria and for 93.1 and 93.8 % of Gram-positive cocci, respectively. In particular, 100 % categorical agreement was found with levofloxacin for Enterobacteriaceae by both PR1 and PR2, and 99.0 and 100 % categorical agreement was observed with linezolid for Gram-positive cocci by PR1 and PR2, respectively. There was no significant difference in accuracy between PR1 and PR2 for Gram-negative bacteria and Gram-positive cocci. CONCLUSIONS: This newly described method seems promising for providing accurate AST results. Most importantly, these results would be available in a few hours from blood culture positivity, which would help clinicians to promptly confirm or streamline an effective antibiotic therapy in patients with bloodstream infections.

Rapid and reliable identification of Gram-negative bacteria and Gram-positive cocci by deposition of bacteria harvested from blood cultures onto the MALDI-TOF plate.

BACKGROUND: Rapid identification of the causative agent(s) of bloodstream infections using the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) methodology can lead to increased empirical antimicrobial therapy appropriateness. Herein, we aimed at establishing an easier and simpler method, further referred to as the direct method, using bacteria harvested by serum separator tubes from positive blood cultures and placed onto the polished steel target plate for rapid identification by MALDI-TOF. The results by the direct method were compared with those obtained by MALDI-TOF on bacteria isolated on solid media. RESULTS: Identification of Gram-negative bacilli was 100 % concordant using the direct method or MALDI-TOF on isolated bacteria (96 % with score > 2.0). These two methods were 90 % concordant on Gram-positive cocci (32 % with score > 2.0). Identification by the SepsiTyper method of Gram-positive cocci gave concordant results with MALDI-TOF on isolated bacteria in 87 % of cases (37 % with score > 2.0). CONCLUSIONS: The direct method herein developed allows rapid identification (within 30 min) of Gram-negative bacteria and Gram-positive cocci from positive blood cultures and can be used to rapidly report reliable and accurate results, without requiring skilled personnel or the use of expensive kits.

Sensitization of KPC and NDM Klebsiella pneumoniae To Rifampicin by the Human Lactoferrin-Derived Peptide hLF1-11.

A synergistic effect of non-bactericidal concentrations of the human lactoferrin (hLF)-derived peptide hLF1-11 and rifampicin against multidrug-resistant KPC (Klebsiella pneumoniae carbapenemase)-producing K. pneumoniae has been previously shown. The present study focuses on the mechanism(s) underlying this synergistic effect. The contribution of hLF1-11 and rifampicin to the synergistic effect was evaluated by killing assays with KPC K. pneumoniae cells incubated with hLF1-11 and, after washing, with rifampicin, or vice versa. Cell membrane permeability and polarization upon exposure to hLF1-11 and/or rifampicin were evaluated by ethidium bromide (EtBr) and DiBAC4(3) (bis-1,3-dibutylbarbituric acid trimethine oxonol) permeability, respectively. The effect of carbonyl cyanide m-chlorophenyl hydrazone (CCCP), an uncoupler of oxidative phosphorylation, was also evaluated. KPC K. pneumoniae cells were effectively killed after prior exposure to rifampicin for 30 to 60 min followed by treatment with hLF1-11, while no antibacterial activity was observed when cells were incubated with hLF1-11 first and then with rifampicin. EtBr accumulation increased upon exposure to hLF1-11 or the combination of hLF1-11 and rifampicin, but not upon exposure to rifampicin alone. Moreover, hLF1-11 induced a dose-dependent membrane depolarization. As expected, the antibacterial activity of hLF1-11 alone or combined with rifampicin was significantly reduced in the presence of CCCP. Furthermore, hLF1-11 and rifampicin were synergistic also against a colistin-resistant NDM (New Delhi metallo-ß-lactamase)-producing K. pneumoniae strain. The results suggest that rifampicin was accumulated by KPC cells during the 30-to-60-min incubation and that the addition of hLF1-11 sensitized bacterial cells to rifampicin by inducing a transient loss of membrane potential and increased cell membrane permeability, thus facilitating the entrance and retention of rifampicin into the cytoplasm. IMPORTANCE The present study describes a synergistic effect between rifampicin, an impermeable hydrophobic antibiotic with an intracellular target, and an hLF1-11, an antimicrobial peptide derived from human lactoferrin, against multidrug-resistant Klebsiella pneumoniae. Carbapenem-resistant K. pneumoniae has recently caused an outbreak in Tuscany, Italy, thus pressing the need for the development of new treatment options. The mechanisms underlying such a synergistic effect have been studied. The results suggest that the synergistic effect was due to the transient loss of membrane potential induced by hLF1-11 and the subsequent increase in cell membrane permeability which allowed rifampicin to enter the bacterial cell. Therefore, it is likely that a sub-inhibitory concentration of hLF1-11 can efficiently permeabilize K. pneumoniae cells to rifampicin, allowing the antibiotic to reach its intracellular target. These results encourage further exploration of possible applications of this synergistic combination in the treatment of K. pneumoniae infections.

Sensitivity of Serum Beta-D-Glucan in Candidemia According to Candida Species Epidemiology in Critically Ill Patients Admitted to the Intensive Care Unit.

Serum beta-D-glucan (BDG) determination plays an important role in the diagnosis of candidemia among critically ill patients admitted to the intensive care unit (ICU). However, BDG levels measured may be lower in the case of infections caused by some non-albicans species, such as C. parapsilosis and C. auris. The aim of this single-center study was to investigate the sensitivity of serum BDG for the diagnosis of candidemia stratified according to causative Candida species in ICU patients. This was a single-center, retrospective study, including all adult patients admitted to ICU during the period 2018-2021. All episodes of candidemia with a determination of BDG available within 3 days before or after positive blood culture were recorded. The preplanned primary objective was to investigate the sensitivity of serum BDG to detect candidemia early and the effect of different Candida species. The secondary objective was to measure serum BDG in patients with candidemia from different Candida species. In total, 146 candidemia episodes in 118 patients were analyzed. Median BDG value for C. albicans candidemia (182 pg/mL) was higher than that observed for C. parapsilosis (78 pg/mL, p = 0.015) and C. auris (48 pg/mL, p = 0.022). The overall sensitivity of BDG for the diagnosis of candidemia was low (47%, 95% CI 39-55%). In conclusion, in critically ill patients admitted to ICU, serum BDG levels for candidemia were different among species, with lower levels confirmed for C. parapsilosis and C. auris. Serum BDG sensitivity for early detection of candidemia was lower than previously reported in other ICU populations.

T2Bacteria and T2Resistance Assays in Critically Ill Patients with Sepsis or Septic Shock: A Descriptive Experience.

The use of rapid molecular tests may anticipate the identification of causative agents and resistance determinants in the blood of critically ill patients with sepsis. From April to December 2021, all intensive care unit patients with sepsis or septic shock who were tested with the T2Bacteria and T2Resistance assays were included in a retrospective, single center study. The primary descriptive endpoints were results of rapid molecular tests and concomitant blood cultures. Overall, 38 combinations of T2Bacteria and T2Resistance tests were performed. One or more causative agent(s) were identified by the T2Bacteria assay in 26% of episodes (10/38), whereas negative and invalid results were obtained in 66% (25/38) and 8% (3/38) of episodes, respectively. The same pathogen detected by the T2Bacteria test grew from blood cultures in 30% of cases (3/10). One or more determinant(s) of resistance were identified by the T2Resistance assay in 11% of episodes (4/38). Changes in therapy based on T2Bacteria and/or T2Resistance results occurred in 21% of episodes (8/38). In conclusion, T2Bacteria/T2Resistance results can influence early treatment decisions in critically ill patients with sepsis or septic shock in real-life practice. Large, controlled studies remain necessary to confirm a favorable impact on patients' outcomes and antimicrobial stewardship interventions.

Fluorescence Lifetime Imaging Microscopy of Porphyrins in Helicobacter pylori Biofilms.

Bacterial biofilm constitutes a strong barrier against the penetration of drugs and against the action of the host immune system causing persistent infections hardly treatable by antibiotic therapy. Helicobacter pylori (Hp), the main causative agent for gastritis, peptic ulcer and gastric adenocarcinoma, can form a biofilm composed by an exopolysaccharide matrix layer covering the gastric surface where the bacterial cells become resistant and tolerant to the commonly used antibiotics clarithromycin, amoxicillin and metronidazole. Antimicrobial PhotoDynamic Therapy (aPDT) was proposed as an alternative treatment strategy for eradicating bacterial infections, particularly effective for Hp since this microorganism produces and stores up photosensitizing porphyrins. The knowledge of the photophysical characteristics of Hp porphyrins in their physiological biofilm microenvironment is crucial to implement and optimize the photodynamic treatment. Fluorescence lifetime imaging microscopy (FLIM) of intrinsic bacterial porphyrins was performed and data were analyzed by the 'fit-free' phasor approach in order to map the distribution of the different fluorescent species within Hp biofilm. Porphyrins inside bacteria were easily distinguished from those dispersed in the matrix suggesting FLIM-phasor technique as a sensitive and rapid tool to monitor the photosensitizer distribution inside bacterial biofilms and to better orientate the phototherapeutic strategy.

The in vitro Photoinactivation of Helicobacter pylori by a Novel LED-Based Device.

The rise of antibiotic resistance is the main cause for the failure of conventional antibiotic therapy of Helicobacter pylori infection, which is often associated with severe gastric diseases, including gastric cancer. In the last years, alternative non-pharmacological approaches have been considered in the treatment of H. pylori infection. Among these, antimicrobial PhotoDynamic Therapy (aPDT), a light-based treatment able to photoinactivate a wide range of bacteria, viruses, fungal and protozoan parasites, could represent a promising therapeutic strategy. In the case of H. pylori, aPDT can exploit photoactive endogenous porphyrins, such as protoporphyrin IX and coproporphyrin I and III, to induce photokilling, without any other exogenous photosensitizers. With the aim of developing an ingestible LED-based robotic pill for minimally invasive intragastric treatment of H. pylori infection, it is crucial to determine the best illumination parameters to activate the endogenous photosensitizers. In this study the photokilling effect on H. pylori has been evaluated by using a novel LED-based device, designed for testing the appropriate LEDs for the pill and suitable to perform in vitro irradiation experiments. Exposure to visible light induced bacterial photokilling most effectively at 405 nm and 460 nm. Sub-lethal light dose at 405 nm caused morphological changes on bacterial surface indicating the cell wall as one of the main targets of photodamage. For the first time endogenous photosensitizing molecules other than porphyrins, such as flavins, have been suggested to be involved in the 460 nm H. pylori photoinactivation.

Spectroscopic characterization and fluorescence imaging of Helicobacter pylori endogenous porphyrins.

Conventional antimicrobial strategies have become increasingly ineffective due to the rapid emergence of antibiotic resistance among pathogenic bacteria. In order to overcome this problem, antimicrobial PhotoDynamic Therapy (PDT) is considered a promising alternative therapy. PDT has a broad spectrum of action and low mutagenic potential. It is particularly effective when microorganisms present endogenous photosensitizing pigments. Helicobacter pylori (Hp), a pathogen notoriously responsible of severe gastric infections (chronic gastritis, peptic ulcer, MALT lymphoma and gastric adenocarcinoma), produces and accumulates the photosensitizers protoporphyrin IX and coproporphyrin, thus it might be a suitable target of antimicrobial PDT. With the aim to design and develop an ingestible LED-based robotic pill for intragastric phototherapy, so that irradiation can be performed in situ without the use of invasive endoscopic light, photophysical studies on the Hp endogenous photosensitizers were carried out. These studies represent an important prerequisite in order to select the most effective irradiation conditions for Hp eradication. The photophysical characterization of Hp porphyrins, including their spectroscopic features in terms of absorption, steady-state and time-resolved fluorescence, was performed on bacterial extracts as well as within planktonic and biofilm growing Hp cells.

The N-Terminus of Human Lactoferrin Displays Anti-biofilm Activity on Candida parapsilosis in Lumen Catheters.

Candida parapsilosis is a major cause of hospital-acquired infection, often related to parenteral nutrition administered via catheters and hand colonization of health care workers, and its peculiar biofilm formation ability on plastic surfaces. The mortality rate of 30% points to the pressing need for new antifungal drugs. The present study aimed at analyzing the inhibitory activity of the N-terminal lactoferrin-derived peptide, further referred to as hLF 1-11, against biofilms produced by clinical isolates of C. parapsilosis characterized for their biofilm forming ability and fluconazole susceptibility. hLF 1-11 anti-biofilm activity was assessed in terms of reduction of biofilm biomass, metabolic activity, and observation of sessile cell morphology on polystyrene microtiter plates and using an in vitro model of catheter-associated C. parapsilosis biofilm production. Moreover, fluctuation in transcription levels of genes related to cell adhesion, hyphal development and extracellular matrix production upon peptide exposure were evaluated by quantitative real time RT-PCR. The results revealed that hLF 1-11 exhibits an inhibitory effect on biofilm formation by all the C. parapsilosis isolates tested, in a dose-dependent manner, regardless of their fluconazole susceptibility. In addition, hLF 1-11 induced a statistically significant dose-dependent reduction of preformed-biofilm cellular density and metabolic activity at high peptide concentrations only. Interestingly, when assessed in a catheter lumen, hLF 1-11 was able to induce a 2-log reduction of sessile cell viability at both the peptide concentrations used in RPMI diluted in NaPB. A more pronounced anti-biofilm effect was observed (3.5-log reduction) when a 10% glucose solution was used as experimental condition on both early and preformed C. parapsilosis biofilm. Quantitative real time RT-PCR experiments confirmed that hLF 1-11 down-regulates key biofilm related genes. The overall findings suggest hLF 1-11 as a promising candidate for the prevention of C. parapsilosis biofilm formation and to treatment of mature catheter-related C. parapsilosis biofilm formation.

Inhibition of Candida albicans Biofilm Formation by the Synthetic Lactoferricin Derived Peptide hLF1-11.

The aim of this study was to evaluate the in vitro activity of the synthetic peptide hLF1-11 against biofilm produced by clinical isolates of Candida albicans with different fluconazole susceptibility. The antibiofilm activity of the peptide hLF1-11 was assessed in terms of reduction of biofilm cellular density, metabolic activity and sessile cell viability. The extent of morphogenesis in hLF1-11 treated and untreated biofilms was also investigated microscopically. Transcription levels of genes related to cell adhesion, hyphal development and extracellular matrix production were analysed by qRT-PCR in hLF1-11 treated and untreated biofilms. Exogenous dibutyryl-cAMP (db-cAMP) was used to rescue morphogenesis in cells exposed to the peptide. The results revealed that hLF1-11 exhibited an inhibitory effect on biofilm formation by all C. albicans isolates tested in a dose-dependent manner, regardless of their fluconazole susceptibility. Visual inspection of treated or untreated biofilm cells with an inverted microscope revealed a significant reduction in hyphal formation by hLF1-11 treated cells, as early as 3 hours of incubation. Moreover, hLF1-11 showed a reduced activity on preadherent cells. hLF1-11 induced the down-regulation of biofilm and hyphal-associated genes, which were predominantly regulated via the Ras1-cAMP-Efg1 pathway. Indeed, exogenous db-cAMP restored morphogenesis in hLF1-11 treated cells. The hLF1-11 peptide significantly inhibited biofilm formation by C. albicans mainly at early stages, interfering with biofilm cellular density and metabolic activity, and affected morphogenesis through the Ras1-cAMP-Efg1 pathway. Our findings provide the first evidence that hLF1-11 could represent a potential candidate for the prevention of biofilm formation by C. albicans.