Cutibacterium acnes clonal complexes display various growth rates in blood culture vials used for diagnosing orthopedic device-related infections.

OBJECTIVES: Blood culture bottles (BCBs) are commonly used for the diagnosis of infections associated with orthopedic devices. Although Cutibacterium acnes is an important pathogen in orthopedics, relatively little is known about its growth characteristics in BCBs. This prompted us to analyze the influence of bacterial genotype and clinical significance on time-to-detection (TTD) in BCBs. METHODS: We reviewed 59 cases of orthopedic device-related infections in which at least one intraoperative specimen yielded a pure C. acnes culture from anaerobic BCBs (BD Bactec Lytic/10 Anaerobic/F; Lytic-Ana) and/or solid media. A strain was considered infectant if the same genotype was present in two or more intraoperative samples. From these cases, we isolated a total of 72 unique C. acnes strains belonging to four multilocus sequence type clonal complexes (CCs): CC18, CC28, CC36 and CC53. Growth rate and TTD in Lytic-Ana BCB were studied under experimental conditions (inoculation of standard inoculum) and in clinical samples (inoculation of periprosthetic tissue samples). RESULTS: Median TTD values were shorter for CC53 compared to other CCs under experimental conditions (69 vs. 103 h; p < 0.001) and from clinical specimens (70 vs. 200 h; p = 0.02). Infectant strains had a shorter median TTD compared to contaminant strains in a clinical situation, while the difference was not observed under experimental conditions. CONCLUSIONS: The detection dynamics of C. acnes in Lytic-Ana BCBs were associated with genotype. Thus, TTD not only reflects the bacterial load in clinical samples, but may also reflect the intrinsic properties of the clonal complex of C. acnes.

Growth detection of Cutibacterium acnes from orthopaedic implant-associated infections in anaerobic bottles from BACTEC and BacT/ALERT blood culture systems and comparison with conventional culture media.

Cutibacterium acnes is a major etiologic agent of orthopaedic implant-associated infections (IAIs) and requires up to 14 days of incubation in an anaerobic atmosphere for growth detection. As blood culture (BC) systems are increasingly being used to monitor the growth of IAI specimens, we compared different BC media for growth detection of C. acnes. Non-duplicate C. acnes isolates (n = 99) obtained from sonicate-fluid cultures of orthopaedic IAIs from Slovenia (n = 54), conventional tissue samples of monomicrobial orthopaedic IAIs from France (n = 43) and two reference strains were inoculated to anaerobic BC bottles of two major BC systems and 3 conventional culture media types (thioglycolate broth, Schaedler and chocolate agar). Growth and time-to-detection (TTD) were recorded. Only Lytic (BACTEC) and SN (BacT/ALERT) bottles consistently detected growth of C. acnes within 14 days with 94% (n = 93) and 92% (n = 91) detection rates, respectively (p = 0.79). Lytic was superior to Plus BACTEC medium (p < 0.001), while SN was superior to all other BacT/ALERT media (p < 0.001). Mean TTD was 128 ± 43 h (61-336 h) for Lytic and 158 ± 65 h (77-336 h) for SN medium. Among the conventional media, 99% (n = 98) of the isolates grew on Schaedler agar, 96% (n = 95) in thioglycolate broth and 74% (n = 73) on chocolate agar. Inconsistent growth of C. acnes in different BC media can critically influence the detection of this major IAI pathogen. Only Lytic (BACTEC) and SN (BacT/ALERT) BC media types were consistently able to detect C. acnes within 14 days of incubation. However, visible growth was observed faster in thioglycolate broth and Schaedler agar media.

High prevalence of division II (cfiA positive) isolates among blood stream Bacteroides fragilis in Slovenia as determined by MALDI-TOF MS.

Bacteroides fragilis can be classified into division I (cfiA negative) and division II (cfiA positive) isolates. Division II isolates have a silent chromosomal carbapenemase gene (cfiA) that can become overexpressed by an insertion of a mobile genetic element and thus develop a phenotypic resistance to carbapenems. Aims of our study were (i) to determine the prevalence of B. fragilis division II (cfiA positive) isolates among blood stream and non-blood stream isolates from two major Slovenian tertiary-care hospitals and (ii) to assess its influence on phenotypic resistance to imipenem. Consecutive non-duplicate B. fragilis isolates from blood stream and non-blood stream specimens were included in the analysis from 2015 to 2017 period. Data from laboratory information system were matched with mass spectra obtained with Microflex LT instrument and MALDI Biotyper 3.1 software (Bruker Daltonik, Bremen, Germany). All mass spectra were reanalyzed using Bruker taxonomy library. Spectra with a log(score) > 2.0 were further analyzed with cfiA library that separates B. fragilis division I and II isolates based on a log(score) value difference of >0.3. Minimal inhibitory concentrations (MICs) for imipenem were determined with Etest (bioMérieux, Marcy l'Étoile, France), using supplemented Brucella agar and EUCAST breakpoints (S ≤ 2 mg/L, R > 8 mg/L). Altogether 623 consecutive B. fragilis isolates were included in the analysis; 47 (7.5%) were isolated from blood stream and 576 (92.5%) from non-blood stream specimens. Among all study isolates, 51 (8.2%) proved to belong to division II (cfiA positive). The proportions of division II isolates among blood stream and non-blood stream isolates were 14.9% and 7.6%, respectively (p = 0.081, ns). In total, 1.3% (n = 8) were non-susceptible to imipenem (MIC >2 mg/L); 4.3% (n = 2) among blood stream and 1% (n = 6) among non-blood stream isolates. All imipenem resistant isolates belonged to division II. Modal MICs (MIC range) were 0.064 mg/L (0.016 mg/L-2 mg/L) and 0.125 mg/L (0.064 mg/L-≥32 mg/L) for division I and II isolates, respectively.

Sample preparation method influences direct identification of anaerobic bacteria from positive blood culture bottles using MALDI-TOF MS.

Rapid detection and identification of anaerobic bacteria from blood is important to adjust antimicrobial therapy by including antibiotics with activity against anaerobic bacteria. Limited data is available about direct identification of anaerobes from positive blood culture bottles using MALDI-TOF mass spectrometry (MS). In this study, we evaluated the performance of two sample preparation protocols for direct identification of anaerobes from positive blood culture bottles, the MALDI Sepsityper kit (Sepsityper) and the in-house saponin (saponin) method. Additionally, we compared two blood culture bottle types designed to support the growth of anaerobic bacteria, the BacT/ALERT-FN Plus (FN Plus) and the BACTEC-Lytic (Lytic), and their influence on direct identification. A selection of 30 anaerobe strains belonging to 22 different anaerobic species (11 reference strains and 19 clinical isolates) were inoculated to 2 blood culture bottle types in duplicate. In total, 120 bottles were inoculated and 99.2% (n = 119) signalled growth within 5 days of incubation. The Sepsityper method correctly identified 56.3% (n = 67) of anaerobes, while the saponin method correctly identified 84.9% (n = 101) of anaerobes with at least log(score) ≥1.6 (low confidence correct identification), (p < 0.001). Gram negative anaerobes were better identified with the saponin method (100% vs. 46.5%; p < 0.001), while Gram positive anaerobes were better identified with the Sepsityper method (70.8% vs. 62.5%; p = 0.454). Average log(score) values among only those isolates that were correctly identified simultaneously by both sample preparation methods were 2.119 and 2.029 in favour of the Sepsityper method, (p = 0.019). The inoculated bottle type didn't influence the performance of the two sample preparation methods. We confirmed that direct identification from positive blood culture bottles with MALDI-TOF MS is reliable for anaerobic bacteria. However, the results are influenced by the sample preparation method used.

Evaluation of the routine antimicrobial susceptibility testing results of clinically significant anaerobic bacteria in a Slovenian tertiary-care hospital in 2015.

The aim of our study was to determined antimicrobial susceptibility profiles of 2673 clinically significant anaerobic bacteria belonging to the major genera, isolated in 2015 in a large tertiary-care hospital in Slovenia. The species identification was performed by MALDI-TOF mass spectrometry. Antimicrobial susceptibility was determined immediately at the isolation of the strains against: penicillin, co-amoxiclav, imipenem, clindamycin and metronidazole, using gradient diffusion methodology and EUCAST breakpoints. The most frequent anaerobes were Bacteroides fragilis group with 31% (n = 817), Gram positive anaerobic cocci (GPACs) with 22% (n = 589), Prevotella with 14% (n = 313) and Propionibacterium with 8% (n = 225). Metronidazole has retained full activity (100%) against all groups of anaerobic bacteria intrinsically susceptible to it. Co-amoxiclav and imipenem were active against most tested anaerobes with zero or low resistance rates. However, observed resistance to co-amoxiclav (8%) and imipenem (1%) is worrying especially among B. fragilis group isolates. High overall resistance (23%) to clindamycin was detected in our study and was highest among the genera Prevotella, Bacteroides, Parabacteroides, GPACs and Clostridium. Routine testing of antimicrobial susceptibility of clinically relevant anaerobic bacteria is feasible and provides good surveillance data.

Performance of two blood culture systems to detect anaerobic bacteria. Is there any difference?

We studied the performance characteristics of two blood culture (BC) bottles/systems, (i) BacT/ALERT-FN Plus/3D (bioMérieux, Marcy l'Étoile, France) and (ii) BACTEC-Lytic/9000 (Becton Dickinson, Sparks, USA) for detection of growth and time-to-positivity (TTP) against a balanced and diverse collection of anaerobic bacterial strains (n = 48) that included reference strains (n = 19) and clinical isolates (n = 29) of 32 species (15 Gram-negative and 17 Gram-positive). Standard suspension of bacteria was inoculated to each bottle in duplicates and incubated in the corresponding system. Overall, 62.5% (n = 30) of strains were detected by both BC bottle types. Comparing the two, 70.8% (n = 34) and 79.2% (n = 38) of strains were detected by BacT/ALERT-FN Plus and BACTEC-Lytic bottles, respectively (p = 0.38). Among Gram-negative anaerobes (n = 25) the detection rate was 76.0% (n = 19) vs. 92.0% (n = 23) (p = 0.22), respectively. Among Gram-positive anaerobes (n = 23) the detection rate was 65.2% (n = 15) in both bottles (p = 1). The average TTP per bottle was calculated only for the strains detected by both systems (n = 30) and was 40.85 h and 28.08 h for BacT/ALERT-FN Plus and BACTEC-Lytic, respectively (p < 0.001). The mean difference was 12.76 h (95% CI: 6.21-19-31 h). Six anaerobic strains were not detected by any system, including Gram-negative Porphyromonas gingivalis, and five Gram-positive strains: Finegoldia magna, Peptostreptococcus anaerobius, Propionibacterium acnes, Clostridium novyi and Clostridium clostridioforme. Furthermore, Eggerthella lenta and Prevotella bivia were detected only by BacT/ALERT-FN Plus, while Prevotella disiens and Prevotella intermedia were detected only by BACTEC-Lytic bottles. There were no major differences in detection rate among clinical and reference strains. Anaerobic bacteria represent a minority of BC isolates, however, far from ideal detection rate was observed in this study for both tested bottle/system combinations. Nevertheless, in those cases where both gave positive signal, BACTEC-Lytic was superior to BacT/ALERT FN Plus with 12.76 h shorter mean TTP. Improvements of media in blood culture bottles available for detection of anaerobes are warranted.

Improvement of pneumococcal pneumonia diagnostics by the use of rt-PCR on plasma and respiratory samples.

BACKGROUND: The aim of the study was to assess the performance of a real-time polymerase chain reaction (rt-PCR) assay on plasma and respiratory samples for the diagnosis of pneumococcal pneumonia. METHODS: Three hundred and forty patients (160 children and 180 adults) with community-acquired pneumonia were included prospectively from January 2011 to May 2012. Blood samples were obtained simultaneously for culture and rt-PCR targeting the lytA gene. Respiratory samples were also obtained: nasopharyngeal swab in nearly all patients and sputum or tracheal aspirate when available. RESULTS: Streptococcus pneumoniae was detected in 222 (65%) of 340 patients: 143 (89%) children and 79 (44%) adults. Pneumonia was assigned as definite pneumococcal in 96 (28.2%) of 340 patients, according to S. pneumoniae detected in blood: in 54 (33.8%) children - by rt-PCR in 51 (31.9%) and by culture in 5 (3.1%); and in 42 (23.3%) adults - by rt-PCR in 41 (22.8%) and by culture in 12 (6.7%). Pneumonia was considered as probably pneumococcal in 19 (10.6%) adults according to S. pneumoniae detected in sputum/tracheal aspirate, by rt-PCR in 19 and by culture in 5. In 18 adults and 89 children with S. pneumoniae detected only in the nasopharynx, pneumonia was considered as possibly pneumococcal; however it should be noted that nasopharyngeal colonization with S. pneumoniae is also common in children with other aetiologies of pneumonia. CONCLUSIONS: rt-PCR on plasma and other samples performed significantly better than culture for the detection of pneumococcal pneumonia (p < 0.0005) in children and adults.

Systemic Antimicrobial Treatment of Chronic Osteomyelitis in Adults: A Narrative Review.

Chronic osteomyelitis in adults is a complex condition that requires prolonged and intensive antimicrobial therapy, but evidence on optimal selection and duration of antibiotics is limited. A review of PubMed and Ovid Embase databases was conducted to identify systematic reviews, meta-analyses, retrospective and randomised controlled trials (RCTs) on antibiotic treatment outcomes in adults with chronic osteomyelitis. Three main areas of interest were investigated: short-term versus long-term antibiotic therapy, oral versus parenteral antibiotic therapy, and combination antibiotic therapy with rifampicin versus without rifampicin. A total of 36 articles were identified and findings were synthesised using a narrative review approach. The available literature suffers from limitations, including a lack of high-quality studies, inconsistent definitions, and varying inclusion/exclusion criteria among studies. Most studies are open-labelled and lack blinding. Limited high-quality evidence exists that oral therapy is non-inferior to parenteral therapy and that shorter antibiotic duration might be appropriate in low-risk patients. Studies on the impact of rifampicin are inconclusive. Further well-designed studies are needed to provide more robust evidence in these areas.

Atypical Skull-Base Osteomyelitis: Comprehensive Review and Multidisciplinary Management Viewpoints.

Atypical skull-base osteomyelitis is a rare but fatal disease that usually involves infection of the ethmoid, sphenoid, occipital, or temporal bones that form the skull base. Unlike typical (so-called otogenic), atypical skull-base osteomyelitis has no otogenic cause. Instead, some authors call atypical skull-base osteomyelitis sinonasal, since the infection most often originates from the nose and paranasal sinuses. Diagnosing and treating this disease is challenging. To assist in managing atypical skull-base osteomyelitis, a review of the most recent literature, with patient cases and multidisciplinary perspectives from otolaryngologists, neurosurgeons, radiologists, infectious disease specialists, pathologists, and clinical microbiologists, is provided in this paper.

Antimicrobial Use in Hospitalised Patients with COVID-19: An International Multicentre Point-Prevalence Study.

Studies suggest that the incidence of coinfections in patients with the coronavirus disease 2019 (COVID-19) is low, but a large number of patients receive antimicrobials during hospitalisation. This may fuel a rise in antimicrobial resistance (AMR). We conducted a multicentre point-prevalence survey in seven tertiary university hospitals (in medical wards and intensive care units) in Croatia, Italy, Serbia and Slovenia. Of 988 COVID-19 patients, 521 were receiving antibiotics and/or antifungals (52.7%; range across hospitals: 32.9-85.6%) on the day of the study. Differences between hospitals were statistically significant (χ2 (6, N = 988) = 192.57, p < 0.001). The majority of patients received antibiotics and/or antifungals within 48 h of admission (323/521, 62%; range across hospitals: 17.4-100%), their most common use was empirical (79.4% of prescriptions), and pneumonia was the main indication for starting the treatment (three-quarters of prescriptions). The majority of antibiotics prescribed (69.9%) belonged to the "Watch" group of the World Health Organization AWaRe classification. The pattern of antimicrobial use differed across hospitals. The data show that early empiric use of broad-spectrum antibiotics is common in COVID-19 patients, and that the pattern of antimicrobial use varies across hospitals. Judicious use of antimicrobials is warranted to prevent an increase in AMR.

Two-year prospective evaluation of colonization with extended-spectrum beta-lactamase-producing Enterobacteriaceae: time course and risk factors.

BACKGROUND: We wanted to determine the time course of colonization with extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae (EPE), sites of colonization and risk factors for prolonged colonization with EPE to obtain information for successful infection control measures. METHODS: Rectal swab, urine, throat swab and other clinically relevant samples (wound swab, tracheal aspirate and sputum) were obtained from each participant. Sets of follow-up samples and data about potential risk factors for prolonged colonization with EPE were collected every 3 months for 2 years. Multivariate analysis using a logistic regression model was performed to identify risk factors for prolonged colonization. RESULTS: A total of 114 patients were included in the study, 49 completed the 2-year follow-up. In all, 611 sample sets were collected, 309 (50.6%) of which were positive for ESBL. Of the positive sample sets, 90% had a rectal swab positive for ESBL, the throat swab was positive for ESBL in 17.2% of cases and urine in 36.2% of cases; 10% of positive sample sets had negative rectal swabs with EPE isolated from other sites, most often from urine. Immobility was found to be associated with prolonged carriage (≥ 12 months) of EPE. After 2 years, 15/49 (30.6%) patients were colonized with EPE. In 12/49 (24.5%) patients, transient negativity was observed. CONCLUSIONS: We found that prolonged colonization with EPE was common, especially in bedridden patients. Transient negative samples were often observed during the course of colonization. In some patients, urine can be the only positive site from which EPE are isolated.

Lactococcus garvieae septicaemia in a patient with artificial heart valves.

Lactococcus garvieae is usually an animal pathogen. Only a few cases of infections in humans have been described. We describe a case of an elderly patient with prosthetic heart valves with a septicaemia without infective endocarditis, and with a favourable clinical course.

Antibiotic prescribing in long-term care facilities for the elderly: EVIPNet evidence brief for policy, number 3

The Ministry of Health of Slovenia commissioned this evidence brief for policy, to be published under the aegis of the WHO European Evidence-informed Policy Network, to develop evidence-informed options for the country to consider in tackling the problem of prescribing broad-spectrum antibiotics to elderly people in long-term care facilities. Such treatment is taking place without proper diagnosis of infection and microbiological testing, and without indication, choice, dose or duration of therapy, thus representing the key driving factor for antimicrobial resistance. The work was carried out within the framework of the Biennial Collaborative Agreement between the Ministry of Health and WHO, involving high-level national policy institutions and national experts, and supported by the technical experts of WHO Regional Office for Europe. The Slovene National Institute for Public Health convened a working group comprising representatives from the clinical field, pharmacology, public health and health care management. The group identified, selected, appraised and synthesized relevant research evidence on the problem, three options for tackling it and considerations in implementing them. The three options are: surveillance, monitoring and audit/feedback on antibiotic consumption and antimicrobial resistance in long-term care facilities; development and implementation of guidelines and clinical pathways for diagnosing and treating infections; and continuous medical education for health care professionals and provision of health information to residents of long-term care facilities and their relatives/visitors.