Bloodstream Infections in Pediatric Oncology Patients: Bacterial Pathogen Distribution and Antimicrobial Susceptibility at the University Hospital Centre Zagreb, Croatia-A 5-Year Analysis.

The epidemiology of bacterial pathogens causing bloodstream infections (BSIs) in pediatric hematology/oncology patients is changing and resistance to antimicrobial agents is globally spread. We retrospectively assessed demographic, clinical, and microbiologic data of BSIs during a 5-year period at a pediatric hematology/oncology unit from January 1, 2017, to December 31, 2021, at the University Hospital Centre Zagreb, Zagreb, Croatia. In 66 pediatric patients with malignancies, 93 BSI episodes were registered and 97 bacterial isolates were cultured. The Gram-positive versus Gram-negative ratio was 67 (69.1%) versus 30 (30.9%). Coagulase-negative staphylococci (48; 49.6%) were the most frequent isolates, followed by Enterobacterales (17; 17.5%) and Staphylococcus aureus (6; 6.2%). Multidrug resistance isolates included extended spectrum ß-lactamase producers (n=3). Resistance rates to piperacillin/tazobactam, cefepime, and meropenem in Gram-negative isolates were 15.4%, 14.3%, and 0.0%, respectively. Gram-positive bacteria are the most common cause of BSI in our patients. Resistance rates to piperacillin/tazobactam and cefepime in Gram-negative isolates make meropenem a better choice for empirical antimicrobial treatment. As national and hospital data may differ, the surveillance of pathogen distribution and antimicrobial susceptibility in pediatric hematology/oncology wards is necessary to adjust empirical treatment accordingly.

CAN IgG ANTIBODIES DIRECTED AGAINST DIFFERENT TARGETS SOLVE THE PROBLEM OF FALSE-POSITIVE RESULTS IN SARS-CoV-2 SEROLOGY?

In addition to RT-PCR assays, serology testing that has been recognized as a useful tool to assess the spread of infection in the population is considered successful and important strategy in the control of the global pandemic of SARS-CoV-2 infection. Now, a great number of manufacturers offer their serologic tests on the market. When interpretating the results, the rate of seroprevalence should be taken in consideration because it may influence the positive predictive value, as well as cross-reactivity with other coronaviruses in case of assays with poorly designed antigens. We present results of 11 patients with different clinical background and tested with two different serologic tests, DIAPRO (ELISA; Sesto San Giovanni, Italy) and VIDAS (ELFA; BioMerieux, Marcy I'Etoile, France). The results obtained by the former test showed ten of these patients to be IgG positive and one patient was IgG weakly positive with different confidence index. The latter test discriminated positive results with medium confidence index on the former test as negative. The results obtained with two serology tests were concordant with the observation that the results with medium confidence index may indicate cross-reactivity.

European confederation of medical mycology expert consult-An ECMM excellence center initiative.

OBJECTIVES: Difficult-to-treat invasive fungal infections require infectious diseases expert consultation to improve treatment outcome and increase survival rates. METHODS: The European Confederation of Medical Mycology (ECMM) intends to provide expert help free of charge by a newly founded ECMM Expert Consultation Service for medical centres around the globe seeking advice when there is no fungal infection consultant available. The expert consult will provide recommendations and broad expertise on difficult-to-treat invasive fungal infections (eg azole-resistant Aspergillus species, Candida auris, mucormycosis) to improve diagnostic and therapeutic management and outcome. RESULTS: The initiative plans global outreach through video conferencing between ECMM Excellence Centers and treating physicians. FungiScope® registries will be used to structure case information and to evaluate the impact of the collegial advice system at regular intervals. Advice will follow recent guidelines, and EQUAL Scores will be used to measure guideline adherence. CONCLUSIONS: Infectious diseases expert consultation should be an integral component of care for patients with difficult-to-treat invasive fungal infections. The ECMM Expert Consult will attend to this matter on a global scale.

Activity of fosfomycin against nosocomial multiresistant bacterial pathogens from Croatia: a multicentric study.

AIM: To determine in vitro susceptibility of multiresistant bacterial isolates to fosfomycin. METHODS: In this prospective in vitro study (local non-random sample, level of evidence 3), 288 consecutively collected multiresistant bacterial isolates from seven medical centers in Croatia were tested from February 2014 until October 2016 for susceptibility to fosfomycin and other antibiotics according to Clinical and Laboratory Standards Institute methodology. Susceptibility to fosfomycin was determined by agar dilution method, while disc diffusion was performed for in vitro testing of other antibiotics. Polymerase chain reaction and sequencing were performed for the majority of extended spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae (K. pneumoniae) and carbapenem-resistant isolates. RESULTS: The majority of 288 multiresistant bacterial isolates (82.6%) were susceptible to fosfomycin. The 236 multiresistant Gram-negative isolates showed excellent susceptibility to fosfomycin. Susceptibility rates were as follows: Escherichia coli ESBL 97%, K. pneumoniae ESBL 80%, Enterobacter species 85.7%, Citrobacter freundii 100%, Proteus mirabilis 93%, and Pseudomonas aeruginosa 60%. Of the 52 multiresistant Gram-positive isolates, methicillin-resistant Staphylococcus aureus showed excellent susceptibility to fosfomycin (94.4%) and vancomycin-resistant enterococcus showed low susceptibility to fosfomycin (31%). Polymerase chain reaction analysis of 36/50 ESBL-producing K. pneumoniae isolates showed that majority of isolates had CTX-M-15 beta lactamase (27/36) preceded by ISEcp insertion sequence. All carbapenem-resistant Enterobacter and Citrobacter isolates had blaVIM-1 metallo-beta-lactamase gene. CONCLUSION: With the best in vitro activity among the tested antibiotics, fosfomycin could be an effective treatment option for infections caused by multiresistant Gram-negative and Gram-positive bacterial strains in the hospital setting.

Evaluation of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry in Identification of Nontuberculous Mycobacteria.

BACKGROUND/AIMS: Species-level identification of nontuberculous mycobacteria (NTM) is important in making decisions about the necessity and choice of antimicrobial treatment. The reason is predictable clinical significance and the susceptibility profile of specific NTM species. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is recognized as a diagnostic tool for routine identification of bacteria and yeasts in the clinical laboratory based on protein fingerprint analysis. The aim of the study was to evaluate MALDI-TOF MS in the identification of NTM. METHODS: A total of 25 NTM isolates from liquid cultures were identified with both polymerase chain reaction (PCR)-based hybridization assay and MALDI-TOF MS at the University Hospital Center Zagreb. RESULTS: PCR-based hybridization assay identified 96% (24/25) and MALDI-TOF MS 80% (20/25) of tested NTM isolates. Five isolates with no reliable MALDI-TOF MS identification belonged to the Mycobacterium avium-intracellulare complex. Seventy percent (14/20) of NTM isolates successfully identified with MALDI-TOF MS had a score higher than 2.0, indicating reliable species identification. CONCLUSION: MALDI-TOF MS is a promising tool for the identification of NTM. With a further improvement of the protein extraction protocol, especially regarding the M. avium-intracellulare complex, MALDI-TOF MS could be an additional standard method for identification of NTM.

[EVOLUTION OF BETA-LACTAM ANTIBIOTIC RESISTANCE IN ENTEROBACTER SPP. IN CROATIA].

Enterobacter spp. develops resistance to expanded-spectrum cephalosporins by induction or derepression of chromosomal AmpC ß-lactamase, or production of extended-spectrum ß-lactamases (ESBLs) or carbapenemases. The aim of the study was to analyze the mechanisms of resistance to expanded-spectrum cephalosporins and the evolution of resistance mechanism during the study period (2008­2011) on a collection of 58 randomly collected Enterobacter spp. strains from three hospital centers in Croatia and Bosnia and Herzegovina during 2008-2010. The antibiotic susceptibility was determined by broth microdilution method according to CLSI. Resistance genes were determined by PCR. Plasmids were characterized by PCR-based replicon typing (PBRT). The hypothesis of the study was that there will be multiple mechanisms of ceftazidime resistance involved, from inducible and derepressed AmpC ß-lactamases to extended-spectrum ß-lactamases and carbapenemases at the end of the study. The isolates from different centers were expected to express different phenotypes and mechanisms of resistance. The study showed the predominance of derepressed AmpC ß-lactamases combined with ESBLs belonging to CTX-M family as a mechanism of resistance to expanded-spectrum cephalosporins. The emergency of MBLs was reported in the last year of the study in University Hospital Center Zagreb. The plasmids encoding ESBLs belonged to different incompatibility groups. This points out to the evolution of ß-lactam resistance in Enterobacter spp. from derepressed AmpC ß-lactamases and ESBL to carbapenemases.

Nationwide Survey of Klebsiella Pneumoniae Strains Producing CTX-M Extended-spectrum ß-lactamases in Croatia.

Extended-spectrum ß-lactamases (ESBL) producing bacteria have been increasingly reported in both hospital and community patients. Production of ESBLs is the major mechanism of resistance to oxymino-cephalosporins and aztreonam in Gram-negative bacteria. Recently a new family of ESBLs with predominant activity against cefotaxime (CTX-M ß-lactamases) has been reported. Over 80 CTX-M enzymes have been described so far, which can be grouped into five main subgroups according to amino acid sequence identity (CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9 and CTX-M-25). In some countries, CTX-M ß-lactamases are the most prevalent types of ESBLs, for instance in Russia, Greece, Spain, Switzerland, Japan, Taiwan, China and Argentina. These enzymes have been identified in countries near Croatia such is Italy, Hungary and Austria. The aim of this study was to determine the prevalence and the types of CTX-M ß lactamases produced by Klebsiella pneumoniae clinical isolates collected from October 2006 to January 2007 from both community- and hospital-based isolates were included (Figure 1.). 128 ESBL isolates were subjected to further analysis: screening with double disc diffusion test and confirmed by ESBL E test.

First Report of NDM-1-Producing Acinetobacter guillouiae.

BACKGROUND: Acinetobacter spp. is an opportunistic pathogen that has demonstrated increasing relevance in nosocomial infections. Carbapenem-resistant strains have been reported worldwide. METHODS: Since 2014, screening for metallo-ß-lactamases (MBLs) in all Acinetobacter spp. isolates using phenotypic methods and PCR has been implemented at the University Hospital Center Zagreb. RESULTS: The bacterial strain was isolated from the drain of a child hospitalized in a paediatric intensive care unit and identified as Acinetobacter guillouiae using a MALDI TOF automated system. The strain was resistant to meropenem, ceftazidime, cefotaxime, ceftriaxone, cefepime, sulbactam/ampicillin, gentamicin and ciprofloxacin, intermediately susceptible to piperacillin/tazobactam and imipenem, and susceptible to amikacin and colistin. The Hodge test and combined disk test with EDTA were positive. The MICs of meropenem and imipenem were not reduced by cloxacillin, but a small reduction of two dilutions was observed following the addition of sodium chloride, which indicated that OXA-58 was produced. PCR and sequencing of chromosomal DNA from boiled colonies revealed blaOXA-58 and blaNDM-1 genes. CONCLUSION: This is the first report of NDM-1 in Acinetobacter spp. in Croatia. The early detection of these genes will aid in the prevention and in the achievement of adequate infection control by limiting the spread of these organisms.

Impact of Pericoronary Microbiota Composition on Course of Recovery after Third Molar Alveotomy.

Although the role of microbiota has been investigated in relation to different oral diseases, it is unknown if its composition has any effect on the course of recovery after third molar alveotomy. Our aim was to determine the influence of patient clinical characteristics as well as pericoronary microbiota composition on the course of recovery after a semi-impacted third molar alveotomy. Thirty-six patients were included and samples obtained with paper points, swabs, and tissue samples were analyzed using DNA hybridization and culture methods. Among the 295 organisms detected, the most frequent were Streptococcus spp. (22.4%; 66/295) followed by Fusobacterium spp. (11.9%; 35/295), and T. forsythia (9.1%; 27/295). A comparison of microbiota composition in patients with better and worse recovery did not show significant differences. Worse recovery outcomes were more frequent in patients with a grade 2 self-assessment of oral health (p = 0.040) and better recovery courses were observed in patients with a grade 4 self-assessment (p = 0.0200). A worse recovery course was statistically significant more frequently in patients with previous oral surgical procedures (p = 0.019). Although we demonstrate that worse recovery outcomes were more frequent when certain bacteria were detected, there was no statistically significant difference. Further research is needed to identify microbial profiles specific to the development of worse outcomes after a third molar alveotomy.

Bacteriuria in Paediatric Oncology Patients: Clinical Features, Distribution and Antimicrobial Susceptibility of Bacterial Pathogens at University Hospital Centre Zagreb, Croatia over a 4-Year Period.

Bacteriuria in paediatric oncology patients have not been well studied. This retrospective study analysed clinical features, distribution and antimicrobial susceptibility of bacterial pathogens cultured from urine in paediatric oncology patients over a 4-year period (2019-2022). A total of 143 episodes of bacteriuria were documented in 74 patients. Neutropenia was present in 17.5% (25/143), symptoms in 25.9% (37/143) and urinary catheter in 7.0% (10/143) episodes. Symptomatic bacteriuria episodes were statistically significantly more frequent in patients with neutropenia (p = 0.0232). The most common bacterial pathogens were Escherichia coli (n = 49; 32.2%), Klebsiella spp. (n = 34; 22.4%), Pseudomonas aeruginosa (n = 22; 14.5%) and Enterococcus spp. (n = 21; 13.8%). Extended-spectrum ß-lactamases-producing (ESBL) Enterobacterales were found in 11 episodes (11/143; 7.7%) with the highest proportion among Klebsiella pneumoniae isolates (n = 7/34; 20.6%). No carbapenem-resistant Enterobacterales, multidrug-resistant P. aeruginosa or vancomycin-resistant Enterococcus spp. were found. The most important novelties are demonstrating P. aeruginosa as one of the prominent bacteriuria pathogens in this patient population, presence of ESBL isolates and carbapenem-resistant P. aeruginosa later during hospitalization highlights the need for appropriate antimicrobial treatment. However, because of the small number of symptomatic patients, further studies are needed to clarify the importance of including urine culture in the diagnostic process in patients with febrile neutropenia.

What's New in Prevention of Invasive Fungal Diseases during Hospital Construction and Renovation Work: An Overview.

The goal of the overview was to give insight into the recent data of invasive fungal diseases (IFDs) associated with construction and renovation in healthcare settings as well as the recent evidence about available prevention and infection control measures. The number of studies describing IFD outbreaks associated with construction or renovation is on the rise again. Applying adequate prevention measures is still a challenge not just for healthcare workers but also for architects and construction workers as well. The role of multidisciplinary teams in the planning and monitoring of prevention measures cannot be overemphasized. Dust control is an inevitable part of every prevention plan. HEPA filters are helpful in the prevention of fungal outbreaks in hematologic patients, but further studies are needed to clarify the extent in which they contribute as specific control measures. The cut-off value for a "threating" level of fungal spore contamination still remains to be defined. The value of antifungal prophylaxis is difficult to assess because other preventive measures are simultaneously applied. Recommendations are still based on few meta-analyses, a large number of descriptive reports, and the opinion of respective authorities. Outbreak reports in the literature are a valuable resource and should be used for education as well as for preparing outbreak investigations.

Evaluation and clinical impact of MALDI Biotyper Mycobacteria Library v6.0 for identification of nontuberculous mycobacteria by MALDI-TOF mass spectrometry.

Major challenges in the identification of non-tuberculous mycobacteria (NTM) by MALDI-TOF MS include protein extraction protocol and updating of the NTM database. The aim of this study was to evaluate MALDI Biotyper Mycobacteria Library v6.0 (Bruker Daltonics GmbH, Bremen, Germany) for identification of clinical NTM isolates and its impact on clinical management. NTM isolates cultivated from clinical samples in 101 patients were identified simultaneously by PCR-reverse hybridization (Hain Lifescience GmbH, Nehren, Germany) as a routinely used reference molecular method and using MALDI Biotyper Microflex LT/SH after protein extraction. Each isolate was applied to eight spots, and mean scores were used in analysis. MALDI-TOF MS obtained correct identification to the species level for 95 (94.06%) NTM isolates. The majority of correctly identified isolates (92/95; 96.84%) were identified with high-confidence score of ≥1.80 and only 3.16% (3/95) with a score of <1.80. Mean value ± SD of RGM NTM isolates (2.127 ± 0.172) was statistically significant higher in comparison to SGM NTM isolates (2.027 ± 0.142) with a p value of 0.007. In comparison to PCR-reverse hybridization, discordant identification results by MALDI-TOF MS were found in six (6/101; 5.94%) NTM isolates for which clinical data were analyzed. We demonstrated a high confidence NTM identifications using Mycobacterium Library v 6.0 on routine clinical isolates. This is the first study that analyzed MALDI-TOF MS identification results of NTM isolates in the context of clinical data, and it showed that MALDI-TOF MS with its updated databases could help clarify the epidemiology, clinical characteristics, and course of infections caused by less frequent NTM species.

Increasing trend in enterococcal bacteraemia and vancomycin resistance in a tertiary care hospital in Croatia, 2017-2021.

BACKGROUND: The epidemiology of enterococcal bacteraemia has changed worldwide and vancomycin-resistant enterococci increasingly cause healthcare-associated infections) with limited treatment options. Studies show heterogeneity among countries, regions and individual hospitals. METHODS: We retrospectively analysed enterococcal bacteraemia with Enterococcus faecalis and E. faecium at the largest hospital in Croatia, University Hospital Centre Zagreb from January 2017 to December 2021. RESULTS: A total of 432 cases of enterococcal bacteraemia were identified with 256 (59.3%) due to E. faecalis and 176 (40.7%) to E. faecium. Enterococcal bacteraemia occurred more frequently in men (n = 270; 62.5%) and the median age of all patients was 62 years (IQR: 0-92). We found statistically significant increase in the incidence trend of bacteraemic episodes with an annual percent change of 20.9% (95% confidence interval 14.3 to 27.8; p = .002) predominantly due to an increase of E. faecalis bacteraemia. The majority of patients (362/432; 83.8%) had healthcare-associated infections and 38.0% (165/432) of patients were in the intensive care unit. The proportion of vancomycin-resistant enterococcal bacteraemia increased from 12.7% (n = 8/63) in 2017 to 25.7% (n = 29/113) in 2021, statistically significant increasing trend (p = .0455), mainly due to an increased proportion of vancomycin-resistant E. faecium (p = .0169). CONCLUSIONS: This is the first study describing the trends in enterococcal bacteraemia and vancomycin-resistance in enterococci in Croatia. We found a rising trend in enterococcal bacteraemia and in the proportion of vancomycin resistance and identified the most vulnerable patient groups, notably intensive care unit patients.

Description of Mycobacterium pinniadriaticum sp. nov., isolated from a noble pen shell (Pinna nobilis) population in Croatia.

Introduction: Shortly before the mass mortality event of the noble pen shell (Pinna nobilis) population in the south-eastern Adriatic coast, two rapidly growing Mycobacterium strains CVI_P3T (DSM 114013 T, ATCC TSD-295 T) and CVI_P4 were obtained from the organs of individual mollusks during the regular health status monitoring. Methods: The strains were identified as members of the genus Mycobacterium using basic phenotypic characteristics, genus-specific PCR assays targeting the hsp65 and 16S rRNA genes and the commercial hybridization kit GenoType Mycobacterium CM (Hain Lifescience, Germany). MALDI-TOF mass spectrometry did not provide reliable identification using the Bruker Biotyper Database. Results and discussion: Genome-wide phylogeny and average nucleotide identity (ANI) values confirmed that the studied strains are clearly differentiated from their closest phylogenetic relative Mycobacterium aromaticivorans and other validly published Mycobacterium species (ANI ≤ 85.0%). The type strain CVI_P3T was further characterized by a polyphasic approach using both phenotypic and genotypic methods. Based on the phenotypic, chemotaxonomic and phylogenetic results, we conclude that strains CVI_P3T and CVI_P4 represent a novel species, for which the name Mycobacterium pinniadriaticum sp. nov. is proposed.

Quality of MALDI-TOF mass spectra in routine diagnostics: results from an international external quality assessment including 36 laboratories from 12 countries using 47 challenging bacterial strains.

OBJECTIVES: Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is a widely used method for bacterial species identification. Incomplete databases and mass spectral quality (MSQ) still represent major challenges. Important proxies for MSQ are the number of detected marker masses, reproducibility, and measurement precision. We aimed to assess MSQs across diagnostic laboratories and the potential of simple workflow adaptations to improve it. METHODS: For baseline MSQ assessment, 47 diverse bacterial strains, which are challenging to identify by MALDI-TOF MS, were routinely measured in 36 laboratories from 12 countries, and well-defined MSQ features were used. After an intervention consisting of detailed reported feedback and instructions on how to acquire MALDI-TOF mass spectra, measurements were repeated and MSQs were compared. RESULTS: At baseline, we observed heterogeneous MSQ between the devices, considering the median number of marker masses detected (range = [2-25]), reproducibility between technical replicates (range = [55%-86%]), and measurement error (range = [147 parts per million (ppm)-588 ppm]). As a general trend, the spectral quality was improved after the intervention for devices, which yielded low MSQs in the baseline assessment as follows: for four out of five devices with a high measurement error, the measurement precision was improved (p-values <0.001, paired Wilcoxon test); for six out of ten devices, which detected a low number of marker masses, the number of detected marker masses increased (p-values <0.001, paired Wilcoxon test). DISCUSSION: We have identified simple workflow adaptations, which, to some extent, improve MSQ of poorly performing devices and should be considered by laboratories yielding a low MSQ. Improving MALDI-TOF MSQ in routine diagnostics is essential for increasing the resolution of bacterial identification by MALDI-TOF MS, which is dependent on the reproducible detection of marker masses. The heterogeneity identified in this external quality assessment (EQA) requires further study.

Whole genomes from bacteria collected at diagnostic units around the world 2020.

The Two Weeks in the World research project has resulted in a dataset of 3087 clinically relevant bacterial genomes with pertaining metadata, collected from 59 diagnostic units in 35 countries around the world during 2020. A relational database is available with metadata and summary data from selected bioinformatic analysis, such as species prediction and identification of acquired resistance genes.

Value of rapid aetiological diagnosis in optimization of antimicrobial treatment in bacterial community acquired pneumonia.

In 80 adult patients with community acquired pneumonia (CAP) conventional microbiological methods, polymerase chain reaction (PCR) and serum C-reactive protein (CRP) levels were performed and the appropriateness of the empirical antimicrobial treatment was evaluated according to bacterial pathogen detected. The aetiology was determined in 42 (52.5%) patients, with Streptococcus pneumoniae as the most common pathogen. PCR applied to bronchoalveolar lavage (BAL) provided 2 and PCR on sputum samples 1 additional aetiological diagnosis of CAP The mean CRP values in the S. pneumoniae group were not significantly higher than in the group with other aetiological diagnoses (166.89 mg/L vs. 160.11 mg/L, p = 0.457). In 23.8% (10/42) of patients with determined aetiology, the empirical antimicrobial treatment was inappropriate. PCR tests need further investigation, particularly those for the atypical pathogens, as they are predominant in inappropriately treated patients. Our results do not support the use of CRP as a rapid test to guide the antimicrobial treatment in patients with CAP.

In vitro susceptibility of carbapenem-resistant Enterobacterales to eravacycline - the first report from Croatia.

The main obstacle in treatment of infections caused by carbapenem-resistant Enterobacterales (CRE) are limited treatment options. The novel antimicrobial agents other than ß-lactams with activity not being dependent on ß-lactamase class are especially important. Eravacycline (ERV) is the first fully synthetic fluorocycline indicated for the treatment of complicated intra-abdominal infections in adults. Eighty CRE isolates at the University Hospital Centre Zagreb, Croatia were examined for susceptibility to ERV by disc diffusion method and minimal inhibitory concentration (MIC). Total of 54 (54/80; 67.5%) isolates were susceptible to ERV with MIC50 of ≤0.5 µg/mL and MIC90 of 4 µg/mL. Susceptibility of OXA-48 positive isolates was not significantly higher in comparison with NDM positive (P = 0.539) and VIM positive (P = 0.7805) isolates. ERV is possible alternative to novel ß-lactamase inhibitor combinations for treatment of CRE infections with antimicrobial susceptibility testing of CRE isolate to ERV in particular patient as condicio sine qua non before administration.

Sepsityper® Kit versus In-House Method in Rapid Identification of Bacteria from Positive Blood Cultures by MALDI-TOF Mass Spectrometry.

In order to further accelerate pathogen identification from positive blood cultures (BC), various sample preparation protocols to identify bacteria with MALDI-TOF MS directly from positive BCs have been developed. We evaluated an in-house method in comparison to the Sepsityper® Kit (Bruker Daltonics, Bremen, Germany) as well as the benefit of an on-plate formic acid extraction step following positive signal by the BACTECTM FX system. Confirmation of identification was achieved using subcultured growing biomass used for MALDI-TOF MS analysis. A total of 113 monomicrobial positive BCs were analyzed. The rates of Gram-positive bacteria correctly identified to the genus level using in-house method and Sepsityper® Kit were 63.3% (38/60) and 81.7% (49/60), respectively (p = 0.025). Identification rates at species level for Gram-positive bacteria with in-house method and Sepsityper® kit were 30.0% (18/60) and 66.7% (40/60), respectively (p < 0.001). Identification rates of Gram-negative bacteria were similar with the in-house method and Sepsityper® Kit. Additional on-plate formic acid extraction demonstrated significant improvement in the identification rate of Gram-positive bacteria at both genus and species level for both in-house (p = 0.001, p < 0.001) and Sepsityper® Kit methods (p = 0.007, p < 0.001). Our in-house method is a candidate for laboratory routines with Sepsityper® Kit as a back-up solution when identification of Gram-positive bacteria is unsuccessful.