Benefits of Aerosolized Phages for the Treatment of Pneumonia Due to Methicillin-Resistant Staphylococcus aureus: An Experimental Study in Rats.

BACKGROUND: The optimal method for delivering phages in the context of ventilator-associated pneumonia (VAP) is unknown. In the current study, we assessed the utility of aerosolized phages (aerophages) for experimental methicillin-resistant Staphylococcus aureus (MRSA) pneumonia. METHODS: Rats were ventilated for 4 hours before induction of pneumonia. Animals received one of the following: (1) aerophages; (2) intravenous (IV) phages; (3) a combination of IV and aerophages; (4) IV linezolid; or (5) a combination of IV linezolid and aerophages. Phages were administered at 2, 12, 24, 48, and 72 hours, and linezolid was administered at 2, 12, 24, 36, 48, 60, and 72 hours. The primary outcome was survival at 96 hours. Secondary outcomes were bacterial and phage counts in tissues and histopathological scoring of the lungs. RESULTS: Aerophages and IV phages each rescued 50% of animals from severe MRSA pneumonia (P < .01 compared with placebo controls). The combination of aerophages and IV phages rescued 91% of animals, which was higher than either monotherapy (P < .05). Standard-of-care antibiotic linezolid rescued 38% of animals. However, linezolid and aerophages did not synergize in this setting (55% survival). CONCLUSIONS: Aerosolized phage therapy showed potential for the treatment of MRSA pneumonia in an experimental animal model and warrants further investigation for application in humans.

Dynamics of bacterial pathogens at the driveline exit site in patients with ventricular assist devices: A prospective, observational, single-center cohort study.

BACKGROUND: Driveline infections (DLIs) at the exit site are frequent in patients with left ventricular assist devices (LVADs). The dynamics from colonization to infection are yet to be investigated. We combined systematic swabbing at the driveline exit site and genomic analyses to study the dynamics of bacterial pathogens and get insights into DLIs pathogenesis. METHODS: A prospective, observational, single-center cohort study at the University Hospital of Bern, Switzerland was performed. Patients with LVAD were systematically swabbed at the driveline exit site between June 2019 and December 2021, irrespective of signs and symptoms of DLI. Bacterial isolates were identified and a subset was whole-genome sequenced. RESULTS: Fifty-three patients were screened, of which 45 (84.9%) were included in the final population. Bacterial colonization at the driveline exit site without manifestation of DLI was frequent and observed in 17 patients (37.8%). Twenty-two patients (48.9%) developed at least one DLI episode over the study period. Incidence of DLIs reached 2.3 cases per 1000 LVAD days. The majority of the organisms cultivated from exit sites were Staphylococcus species. Genome analysis revealed that bacteria persisted at the driveline exit site over time. In four patients, transition from colonization to clinical DLI was observed. CONCLUSIONS: Our study is the first to address bacterial colonization in the LVAD-DLI setting. We observed that bacterial colonization at the driveline exit site was a frequent phenomenon, and in a few cases, it preceded clinically relevant infections. We also provided acquisition of hospital-acquired multidrug-resistant bacteria and the transmission of pathogens between patients.

Tick-borne encephalitis affects sleep-wake behavior and locomotion in infant rats.

BACKGROUND/AIMS: Tick-borne encephalitis (TBE) is a disease affecting the central nervous system. Over the last decade, the incidence of TBE has steadily increased in Europe and Asia despite the availably of effective vaccines. Up to 50% of patients after TBE suffer from post-encephalitic syndrome that may develop into long-lasting morbidity. Altered sleep-wake functions have been reported by patients after TBE. The mechanisms causing these disorders in TBE are largely unknown to date. As a first step toward a better understanding of the pathology of TBEV-inducing sleep dysfunctions, we assessed parameters of sleep structure in an established infant rat model of TBE. METHODS: 13-day old Wistar rats were infected with 1 × 106 FFU Langat virus (LGTV). On day 4, 9, and 21 post infection, Rotarod (balance and motor coordination) and open field tests (general locomotor activity) were performed and brains from representative animals were collected in each subgroup. On day 28 the animals were implanted with a telemetric EEG/EMG system. Sleep recording was continuously performed for 24 consecutive hours starting at day 38 post infection and visually scored for Wake, NREM, and REM in 4 s epochs. RESULTS: As a novelty of this study, infected animals showed a significant larger percentage of time spend awake during the dark phase and less NREM and REM compared to the control animals (p < 0.01 for all comparisons). Furthermore, it was seen, that during the dark phase the wake bout length in infected animals was prolonged (p = 0.043) and the fragmentation index decreased (p = 0.0085) in comparison to the control animals. LGTV-infected animals additionally showed a reduced rotarod performance ability at day 4 (p = 0.0011) and day 9 (p = 0.0055) and day 21 (p = 0.0037). A lower locomotor activity was also seen at day 4 (p = 0.0196) and day 9 (p = 0.0473). CONCLUSION: Our data show that experimental TBE in infant rats affects sleep-wake behavior, leads to decreased spontaneous locomotor activity, and impaired moto-coordinative function.

Efficacy assessment of a novel endolysin PlyAZ3aT for the treatment of ceftriaxone-resistant pneumococcal meningitis in an infant rat model.

BACKGROUND: Treatment failure in pneumococcal meningitis due to antibiotic resistance is an increasing clinical challenge and alternatives to antibiotics warrant investigation. Phage-derived endolysins efficiently kill gram-positive bacteria including multi-drug resistant strains, making them attractive therapeutic candidates. The current study assessed the therapeutic potential of the novel endolysin PlyAZ3aT in an infant rat model of ceftriaxone-resistant pneumococcal meningitis. METHODS: Efficacy of PlyAZ3aT was assessed in a randomized, blinded and controlled experimental study in infant Wistar rats. Meningitis was induced by intracisternal infection with 5 x 107 CFU/ml of a ceftriaxone-resistant clinical strain of S. pneumoniae, serotype 19A. Seventeen hours post infection (hpi), animals were randomized into 3 treatment groups and received either (i) placebo (phosphate buffered saline [PBS], n = 8), (ii) 50 mg/kg vancomycin (n = 10) or (iii) 400 mg/kg PlyAZ3aT (n = 8) via intraperitoneal injection. Treatments were repeated after 12 h. Survival at 42 hpi was the primary outcome; bacterial loads in cerebrospinal fluid (CSF) and blood were secondary outcomes. Additionally, pharmacokinetics of PlyAZ3aT in serum and CSF was assessed. RESULTS: PlyAZ3aT did not improve survival compared to PBS, while survival for vancomycin treated animals was 70% which is a significant improvement when compared to PBS or PlyAZ3aT (p<0.05 each). PlyAZ3aT was not able to control the infection, reflected by the inability to reduce bacterial loads in the CSF, whereas Vancomycin sterilized the CSF and within 25 h. Pharmacokinetic studies indicated that PlyAZ3aT did not cross the blood brain barrier (BBB). In support, PlyAZ3aT showed a peak concentration of 785 µg/ml in serum 2 h after intraperitoneal injection but could not be detected in CSF. CONCLUSION: In experimental pneumococcal meningitis, PlyAZ3aT failed to cure the infection due to an inability to reach the CSF. Optimization of the galenic formulation e.g. using liposomes might enable crossing of the BBB and improve treatment efficacy.

Efficacy of phage therapy in preclinical models of bacterial infection: a systematic review and meta-analysis.

BACKGROUND: Antimicrobial resistance of bacterial pathogens is an increasing clinical problem and alternative approaches to antibiotic chemotherapy are needed. One of these approaches is the use of lytic bacterial viruses known as phage therapy. We aimed to assess the efficacy of phage therapy in preclinical animal models of bacterial infection. METHODS: In this systematic review and meta-analysis, MEDLINE/Ovid, Embase/Ovid, CINAHL/EbscoHOST, Web of Science/Wiley, Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Reviews, and Google Scholar were searched from inception to Sept 30, 2021. Studies assessing phage efficacy in animal models were included. Only studies that assessed the efficacy of phage therapy in treating established bacterial infections in terms of survival and bacterial abundance or density were included. Studies reporting only in-vitro or ex-vivo results and those with incomplete information were excluded. Risk-of-bias assessment was performed using the Systematic Review Centre for Laboratory Animal Experimentation tool. The main endpoints were animal survival and tissue bacterial burden, which were reported using pooled odds ratios (ORs) and mean differences with random-effects models. The I2 measure and its 95% CI were also calculated. This study is registered with PROSPERO, CRD42022311309. FINDINGS: Of the 5084 references screened, 124 studies fulfilled the selection criteria. Risk of bias was high for 70 (56%) of the 124 included studies; therefore, only studies classified as having a low-to-moderate risk of bias were considered for quantitative data synthesis (n=32). Phage therapy was associated with significantly improved survival at 24 h in systemic infection models (OR 0·08 [95% CI 0·03 to 0·20]; I2=55% [95% CI 8 to 77]), skin infection (OR 0·08 [0·04 to 0·19]; I2 = 0% [0 to 79]), and pneumonia models (OR 0·13 [0·06 to 0·31]; I2=0% [0 to 68]) when compared with placebo. Animals with skin infections (mean difference -2·66 [95% CI -3·17 to -2·16]; I2 = 95% [90 to 96]) and those with pneumonia (mean difference -3·35 [-6·00 to -0·69]; I2 = 99% [98 to 99]) treated with phage therapy had significantly lower tissue bacterial loads at 5 ±â€ˆ2 days of follow-up compared with placebo. INTERPRETATION: Phage therapy significantly improved animal survival and reduced organ bacterial loads compared with placebo in preclinical animal models. However, high heterogeneity was observed in some comparisons. More evidence is needed to identify the factors influencing phage therapy performance to improve future clinical application. FUNDING: Swiss National Foundation and Swiss Heart Foundation.

Searching for synergy: combining systemic daptomycin treatment with localised phage therapy for the treatment of experimental pneumonia due to MRSA.

OBJECTIVE: Bacteriophages (or phages) are viruses which infect and lyse bacteria. The therapeutic use of phages (phage therapy) has regained attention in the last decades as an alternative strategy to treat infections caused by antimicrobial-resistant bacteria. In clinical settings it is most likely that phages are administered adjunct to antibiotics. For successful phage therapy it is therefore crucial to investigate different phage-antibiotic combinations in vivo. This study aimed to elucidate the combinatorial effects of systemic daptomycin and nebulised bacteriophages for the treatment of experimental pneumonia due to methicillin-resistant Staphylococcus aureus (MRSA). RESULTS: Using a rat model of ventilator-associated pneumonia caused by MRSA, the simultaneous application of intravenous daptomycin and nebulised phages was not superior to aerophage therapy alone at improving animal survival (55% vs. 50%), or reducing bacterial burdens in the lungs, or spleen. Thus, this combination does not seem to be of benefit for use in patients with MRSA pneumonia.

Isolation and characterization of bacteriophages from the human skin microbiome that infect Staphylococcus epidermidis.

Phage therapy might be a useful approach for the treatment of nosocomial infections; however, only few lytic phages suitable for this application are available for the opportunistic pathogen, Staphylococcus epidermidis. In the current study, we developed an efficient method to isolate bacteriophages present within the human skin microbiome, by using niche-specific S. epidermidis as the host for phage propagation. Staphylococcus epidermidis was identified on the forehead of 92% of human subjects tested. These isolates were then used to propagate phages present in the same skin sample. Plaques were observable on bacterial lawns in 46% of the cases where S. epidermidis was isolated. A total of eight phage genomes were genetically characterized, including the previously described phage 456. A total of six phage sequences were unique, and spanned each of the major staphylococcal phage families; Siphoviridae (n = 3), Podoviridae (n = 1) and Myoviridae (n = 2). One of the myoviruses (vB_SepM_BE06) was identified on the skin of three different humans. Comparative analysis identified novel genes including a putative N-acetylmuramoyl-L-alanine amidase gene. The host-range of each unique phage was characterized using a panel of diverse staphylococcal strains (n = 78). None of the newly isolated phages infected more than 52% of the S. epidermidis strains tested (n = 44), and non-S. epidermidis strains where rarely infected, highlighting the narrow host-range of the phages. One of the phages (vB_SepM_BE04) was capable of killing staphylococcal cells within biofilms formed on polyurethane catheters. Uncovering a richer diversity of available phages will likely improve our understanding of S. epidermidis-phage interactions, which will be important for future therapy.

Clinical presentation of 54 patients with endogenous hyperinsulinaemic hypoglycaemia: a neurological chameleon (observational study).

BACKGROUND Important causes of endogenous hyperinsulinaemic hypoglycaemia (EHH) in adult patients are insulinoma and adult nesidioblastosis. Data on main symptoms in EHH are scarce and controversial. We analysed main symptoms of patients with EHH in the framework of two prospective studies investigating glucagon-like peptide-1 receptor imaging. METHODS Patients were referred from secondary European endocrine centres and endocrinologists. Inclusion criteria were biochemically proven EHH (glucose <2.5 mmol/l in the presence of inadequate insulin and C-peptide levels) with neurological hypoglycaemic symptoms. Demographic characteristics and aetiologies of the patients with EHH were retrieved. Main symptoms were categorised into neurological, sympathicoadrenal (sweating, tremor, palpitation, hunger, shivering and pallor) and nonspecific other symptoms (nonspecific asthenia, weight gain, gastrointestinal symptoms and headaches). Neurological symptoms were subdivided into moderately impaired consciousness (confusion, dizziness, somnolence and delirium), visual, speech and sensorimotor impairment, severely impaired consciousness (loss of consciousness and apathy), attention deficit, seizures and personality changes. Biochemical assessment and duration of EHH at the end of a fasting test were recorded. RESULTS Fifty-four patients with full documentation were included in the analysis (74% female; mean age 54 years, range 22­84). Median duration from onset of symptoms to diagnosis of EHH was 12 months (range 0­120). Fifty (92.6%) patients had neurological symptoms, including moderately impaired consciousness (46.3%), visual, speech and sensorimotor function impairment (44.4%), severely impaired consciousness (37%), attention deficit (31.5%), seizures (16.7%) and personality change (13%). Sympathicoadrenal symptoms were present in 33 (61.1%) patients. Nonspecific other symptoms occurred in 36 (66.7%) patients. 43 patients (79.6%) suffered from symptoms of at least two different categories. CONCLUSIONS Clinical symptoms of EHH are characterised by a wide variety of mainly different neurological symptoms ("neurological chameleon"). EHH should be considered as a differential diagnosis in many neurological disorders. Trial registration numbers NCT00937079 & NCT02127541

Streptococcus pneumoniae capsule determines disease severity in experimental pneumococcal meningitis.

Streptococcus pneumoniaebacteria can be characterized into over 90 serotypes according to the composition of their polysaccharide capsules. Some serotypes are common in nasopharyngeal carriage whereas others are associated with invasive disease, but when carriage serotypes do invade disease is often particularly severe. It is unknown whether disease severity is due directly to the capsule type or to other virulence factors. Here, we used a clinical pneumococcal isolate and its capsule-switch mutants to determine the effect of capsule, in isolation from the genetic background, on severity of meningitis in an infant rat model. We found that possession of a capsule was essential for causing meningitis. Serotype 6B caused significantly more mortality than 7F and this correlated with increased capsule thickness in the cerebrospinal fluid (CSF), a stronger inflammatory cytokine response in the CSF and ultimately more cortical brain damage. We conclude that capsule type has a direct effect on meningitis severity. This is an important consideration in the current era of vaccination targeting a subset of capsule types that causes serotype replacement.