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Abstract Mycoplasma hominis can be a part of human urogenital tract microbiome, and it is a frequent cause of urogenital infections. In rare cases, it can also cause extragenital infections, especially in immunocompromised patients. In this case series, we report two cases and provide a literature review of extragenital infections caused by M. hominis in patients with hypogammaglobulinemia. Patient 1 was a 61-year-old woman with diffuse large B-cell lymphoma who, after rituximab-containing chemotherapy and CAR-T therapy, developed M. hominis spondylodiscitis. Patient 2 was a 50-year-old woman with congenital hypogammaglobulinemia who developed disseminated M. hominis infection involving pleura, muscles, and right ankle. Antibiotic therapy with levofloxacin and doxycycline for 10 weeks in patient 1 and with levofloxacin alone for 6 weeks in patient 2 led to infection resolution. The literature review identified 14 additional cases reporting M. hominis extragenital infection in patients with hypogammaglobulinemia. M. hominis should also be suspected as an etiological agent of extragenital infection in patients with B-cell immunodeficiency with a clinical picture of persistent, standard-culture negative infection, particularly with arthritis or abscess formation. Even if M. hominis can grow on standard bacterial medium, in suspected cases molecular methods should be promptly used for correct diagnostic work-up and successful therapy.

ABSTRACT 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-β-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.

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.

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.

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 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.

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.

Introduction: In order to improve the outcome of patients, reduce length of stay, costs and resources engaged in diagnostics, more rapid reports are requested to the clinical microbiologists.The purpose of this study is to assess the impact on workflow of MALDI-TOF technology, recently made available for use in routine diagnostics. Methods:The work list by the management information system is sent to the instrument MALDI-TOF, where are held at least three successive analytic sessions: the first includes bacteria isolated from CSF, blood cultures, and cases already reported as serious/urgent, the second includes all other germs isolated, the third, microorganisms that require extraction with trifluoroacetic acid (TFA) or formic acid (FA) for identification.The results of each session direct to the execution of different types of susceptibility testing. Results:The times of microbial identifications are reduced by 24 or 48 hours and made available to the clinician for the rational empirical therapy.The reagent costs are reduced by 40%.The subcultures were reduced by 80%, and microscopic examinations by 50%.The antibiotic susceptibility tests were immediately performed with the most appropriate method, based on the knowledge of local epidemiology and microbial species. Conclusion:The bacteriology is the less automated discipline among the clinical laboratory activities and results of diagnostic tests are poorly well-timed. The new interpretative algorithms of MALDI-TOF spectra, now available, allow the correct identification of bacteria in near real time, completely eliminating the wait is necessary for biochemical identification and guiding the operator in selecting the most appropriate antibiotic susceptibility tests. This technology makes work more rapid, economic and efficient, eliminating errors and, together with effective computerization of data, transforms the information content of the microbiological report, making it much more effective for the therapy and the patient care.