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Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) can misidentify Cutibacterium namnetense and Cutibacterium modestum as Cutibacterium acnes. We now describe how such MALDI-TOF MS misidentification explains previous reports of C. acnes isolates that could not be characterised using a multiplex PCR phylotyping assay. • MALDI-TOF MS can misidentify C. namnetense and C. modestum as C. acnes. • Such misidentification confuses multiplex PCR phylotyping results for C. acnes. • C. modestum will lead to a negative result. • C. namnetense will only generate a 16S rRNA gene amplicon (pattern G). • Researchers should be aware of these aberrant results when using this typing method. [ABSTRACT FROM AUTHOR]

The purpose of this study was to identify microorganisms isolated from various periapical tissue diseases using Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) and classify them via an unsupervised machine learning approach. A total of 150 patients with various apical conditions and teeth in need of endodontic retreatment were divided into five groups, including Retreatment, Acute Apical Abscess, Chronic Apical Abscess, Acute Apical Periodontitis, and Chronic Apical Periodontitis. Samples were collected from root canals using paper points after agitating with a #10 K file then microorganisms were identified using MALDI-TOF-MS. Data were analyzed using a hierarchical clustering method. Quadruple clusters and dendrograms were formed according to similarities and dissimilarities. A total of 80 species were identified representative of six different phyla. The most similar microorganism species identified were: ''Enterococcus faecalis'' between 21 and 23-year-old female cases in Retreatment group; ''Lactobacillus rhamnosus'' between 20 and 18-year-old male cases in Symptomatic Apical Abscess cases; ''Lactobacillus paracasei'' between 26 and 40-year-old male cases in Asymptomatic Apical Abscess cases; ''Enterococcus faecalis'' between 48 and 50-year-old female cases in Symptomatic Apical Periodontitis cases; ''Lactobacillus rhamnosus'' between 48 and 60-year-old male cases in Asymptomatic Apical Periodontitis cases. MALDI-TOF MS can be considered a fast and high-throughput screening technique for microbial species identification in endodontics. Thus, it will provide valuable data for future research designs regarding periapical tissue diseases. As the MALDI-TOF MS database expands and comprehensive data becomes available, the relationship between microbial profiles and disease progression will become increasingly apparent. • Eighty species were identified by MALDI-TOF MS in various periapical tissue diseases quickly and practically. • An unsupervised machine learning algorithm was applied for grouping species related to periapical tissue diseases. • The hierarchical clustering approach provides better conclusive results underlying causes of periapical tissue diseases. [ABSTRACT FROM AUTHOR]

The objectives of this study were to report on the antimicrobial susceptibility of 276 clinically significant anaerobic bacteria belonging to the major genera isolated between May 2017 and November 2018 in a tertiary hospital in Granada (Spain) and to describe key clinical features of the patients. Species identification was performed by MALDI-TOF MS. Antimicrobial susceptibility tests were performed against penicillin, amoxicillin-clavulanic, imipenem, meropenem, moxifloxacin, clindamycin, metronidazole, vancomycin, and piperacillin-tazobactam using the gradient diffusion technique and EUCAST breakpoints (except for moxifloxacin). The most frequent anaerobes were Bacteroides (29.7%; n = 82), Clostridioides difficile (15.9%, n = 44), Prevotella (10.8%, n = 30), and Propionibacterium (10.7%, n = 25). Metronidazole was not universally active against all genera tested, and some isolates showed resistance to this drug. Almost all tested anaerobes were susceptible to carbapenems and amoxicillin-clavulanate except for Clostridioides difficile (resistance rate of 94%) and Bacteroides (19%), respectively. High overall resistance rates to clindamycin were observed, especially for genera Finegoldia (54%), Bacteroides (49%), and Prevotella (40%). Resistance rates to carbapenems and amoxicillin-clavulanate were very low for the majority of tested genera but were high for Clostridioides difficile and Bacteroides spp., respectively. Resistance to clindamycin was very high, especially for Bacteroides , Finegoldia magna , Prevotella and Peptoniphilus. Routine antimicrobial susceptibility testing for anaerobes contributes information on the global situation and allows empirical therapies to be selected in accordance with local data on resistant strains. • Metronidazole was not universally active against all anaerobes. • Resistance rates to carbapenems and amoxicillin-clavulanate were very low. • Resistance rates of almost all genera to clindamycin were very high. • Routine antimicrobial susceptibility testing is highly recommended. [ABSTRACT FROM AUTHOR]

We report a case of a 58-year-old male patient who underwent several surgeries following an accident. The bacterium Robinsoniella peoriensis was detected independently in multiple samples from both the right talus and tibia. The bacterium could only be identified using 16S rRNA gene sequencing. • Robinsoniella peoriensis was detected in several independent bone samples of both the patient's talus and tibia. • Main spectra of Robinsoniella peoriensis were already included in the database of the MALDI TOF MS. Our isolate however, could only be identified using sequencing of the 16S rRNA gene, but not MALDI TOF MS. • Robinsoniella peoriensis was not recognized as pathogenic microorganism. The antimicrobial therapy was therefore not adequate. • Whole genome sequencing revealed genes, which may provide an explanation for the resistance against penicillin and levofloxacin. [ABSTRACT FROM AUTHOR]

The purpose of this study was to assess drug susceptibility of clinical B. fragilis strains and to determine any correlation between drug resistance and the presence of specific genes. Antimicrobial susceptibility was assessed using E-tests. All isolates were analyzed with the PCR technique for the presence of antibiotic resistance genes (cepA , cfxA , cfiA , ermF , ermB , ermG , nim), insertion sequences elements (IS 1186 , IS 1187 , IS 1188 , IS 942), and enterotoxin-encoding genes (bft). Susceptibility tests yielded the following rates of resistance to the evaluated antibiotics: penicillin G (100%), clindamycin (22.5%), cefoxitin (6.3%), amoxicillin/clavulanic acid (1.8%). All strain were susceptible to imipenem, and metronidazole. The following antibiotic resistance genes were detected in the evaluated isolates: cepA (in 96.4% of isolates), cfxA (in 12.6%), cfiA (in 1.8%), and ermF (in 25.2%). Genes ermB , ermG , and nim were not found. The presence of the cepA gene showed no correlation with the penicillin G MIC. However, we observed a high correlation between cefoxitin MIC values and the presence of gene cfxA as well as a nearly complete correlation between clindamycin MIC values and the presence of gene ermF. The presence of a bft gene was detected in 14.4% of the analyzed B. fragilis isolates; with the bft -1 allele found in 75%, bft -2 in 25%, and bft -3 in none of the isolates. Antibiotic susceptibility profiles of enterotoxin gene-positive isolates in our study did not differ from those of enterotoxin gene-negative isolates. • Antibiotic susceptibility profile of B. fragilis in Poland. • B. fragilis resistance to certain drugs may pose therapeutic problems. • erm F genes are the most common among clindamycin resistant Bacteroides isolates. • B. fragilis maintain high susceptibility to imipenem and metronidazole. [ABSTRACT FROM AUTHOR]

Abstract Infant botulism was rarely reported in China. The second reported event of the disease including three cases occurred in 2015. In the present study, one (the third case) of the three cases was identified and investigated to trace the sources of transmission. Samples from feces and foodstuffs were used to isolate Clostridium botulinum strains. Each isolate was obtained from the baby's feces and opened powdered infant rice cereal, respectively. In this case, the C. botulinum strains were identified and characterized by combined mouse bioassay, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and high-throughput sequencing including single nucleotide polymorphisms (SNP). Results showed that the disease was caused by a type B strain of C. botulinum. Strains associated with this case as well as isolates from stored and historical samples were phylogenetically analyzed and compared. C. botulinum type B isolates from the infant feces and from an opened container of infant rice cereal were indistinguishable, suggesting that opened container of infant rice cereal is likely to be the source of transmission of spores to the infant. It is not clear that how the opened container was contaminated and the child was exposed since environmental testing was not performed. This study provides detailed information about usage of the three methods and references for dealing with other associated cases. Highlights • An Infant Botulism Case was investigated. • The disease was caused by a type B strain of C. botulinum. • Mouse lethality assay, MALDI-TOF MS and high-throughput sequencing were used. • The opened container of rice cereal is likely to be the source for transmission. [ABSTRACT FROM AUTHOR]

Abstract In 2013, we adopted MALDI-TOF MS using the Bruker Biotyper system for identification of anaerobic bacteria into our routine clinical practice. Here, we describe our experience with the use of MALDI-TOF MS for anaerobic bacterial identification, highlighting its value in replacing the more costly and time-consuming 16S ribosomal RNA gene PCR plus sequencing-based approach as the primary method of anaerobic bacterial identification. We also describe our more recent experience with the use of early/rapid MALDI-TOF MS for identification of anaerobic bacteria performed on short incubation (4–6 h) plated aerobic media from anaerobic blood culture bottles positive for Gram-negative bacilli. Highlights • For anaerobes, MALDI-TOF MS offers faster turnaround time at a lower cost than 16S rRNA gene PCR plus sequencing. • The 10% differential rule aids in identification of anaerobic bacteria via Bruker MALDI-TOF MS. • Limited MALDI-TOF MS identification of Gram-negative anaerobes by brief outgrowths on blood broth subcultures is possible. [ABSTRACT FROM AUTHOR]

Abstract 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. Highlights • Direct identification from positive blood culture bottle is reliable for anaerobic bacteria. • It is influenced by the sample preparation protocol/method. • It works equally well for BacT/ALERT-FN Plus and BACTEC-Lytic bottle type. [ABSTRACT FROM AUTHOR]

Abstract Despite the wide implementation of MALDI-TOF MS for the rapid and reliable identification of most microorganisms, some taxonomic groups such as the Porphyromonas genus remain largely untested. In this study we evaluated the performance of MALDI-TOF MS on this genus using a collection of 39 isolates sent for routine identification to our institution over a 16-year period. All of them were identified by DNA-sequencing analysis of the 16S rRNA gene plus the hsp60 gene when the previous one did not yield species-level assignment. MALDI-TOF MS provided correct identification at least at the genus level of 21/39 isolates (53.9%). Twelve isolates were correctly identified at the species level with a score value ≥ 2.0 and 9 more with score values < 2.0 and ≥ 1.7. The species most represented in the database (P. gingivalis and P. somerae) lay within this category. However, the species poorly represented in this database (P. asaccharolytica and P. uenonis) were mostly identified with lower scores (1.35–1.67) or remained unidentified by MALDI-TOF MS. The addition of two P. asaccharolytica reference spectra to our in-house library allowed 72.9% of genus-level identifications with 17/37 isolates (45.9%) identified with score values ≥ 2.0. Our results showed a high level of correlation between MALDI-TOF MS and DNA-based identification for Porphyromonas spp. strains at the species level, even with score values < 2.0. The reliability provided by MALDI-TOF MS increased when the database was fed with spectra from the species poorly represented in the commercial database. Highlights • MALDI-TOF MS provided correct identification at least at the genus level of 21/39 isolates (53.9%). • Species level identification was achieved for the species well represented in the available database. • The addition of MSPs from P. asaccharolytica allowed the identification of all isolates from this species with score > 1.95. • 72.9% correlation between MALDI-TOF with the in-house library and DNA-based identification at the genus level was shown. • Interestingly, 8 isolates could not be resolved as either P.asaccharolytica or P. uenonis by DNA sequencing or MALDI-TOF. [ABSTRACT FROM AUTHOR]

Abstract This study evaluated the MBT-ASTRA for antimicrobial susceptibility testing of Bacteroides fragilis with different classes of antibiotics. MALDI-TOF MS peak AUCs from suspensions with B. fragilis with and without an antibiotic were used to calculate the relative growth (AUC "with antibiotic" divided by "without antibiotic"). Antimicrobial susceptibility testing of B. fragilis ATCC 25285 (susceptible) and B. fragilis O18 (resistant) was demonstrated with a clear difference of the relative growth between susceptible and resistant. The MBT-ASTRA needs further development and assessment but could be a relatively easy and inexpensive method for rapid antimicrobial susceptibility testing in specific cases of infection with B. fragilis. Highlights • We evaluated the MBT-ASTRA for antimicrobial susceptibility testing of B. fragilis. • Different classes of antibiotics were tested with a susceptible and a resistant strain of B. fragilis. • The MBT-ASTRA demonstrated a clear difference of the relative growth between the susceptible and the resistant B. fragilis. • The MBT-ASTRA could be a easy and inexpensive method for rapid antimicrobial susceptibility testing of B. fragilis. [ABSTRACT FROM AUTHOR]

Abstract The genus Clostridium is of high clinical relevance, as some species may cause rapid and even lethal infections. Thus, a timely identification of these anaerobic bacteria is desirable. Conventional identification methods rely on biochemical properties of these organisms, however, establishing these is time-consuming and not always reliable. Alternatively, 16S rRNA gene sequence based diagnostic methods may be used, but they are expensive and not ubiquitously available. This study was designed to assess the possibility to identify Clostridium species employing the matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). For this purpose, 848 Clostridium strains representing 42 species were analyzed with the VITEK® MS instrument (bioMérieux, Marcy l'Etoile, France), comparing mass spectra derived from these organisms with the spectra provided in the available database. 90.3% of the strains were correctly identified at species level and another 3.6% at genus level. Since the number of Clostridium species included in the database was rather limited (21 altogether), the spectra obtained were also analyzed employing the Shimadzu Pro Series software. Thus, it became possible to create a dendrogram of the species included in this study. Highlights • 848 Clostridium strains representing 42 different species were analyzed by MALDI-TOF using the VITEK® MS instrument. • 90.3% of the strains were correctly identified at species level and another 3.6% at genus level. • The incubation time of cultures had a high impact on the spectra obtained. [ABSTRACT FROM AUTHOR]

Abstract Atopobium rimae and Parvimonas micra are both Gram-positive anaerobes involved infrequently in human infections. We report a polymicrobial anaerobic bacteremia caused by these microorganisms. A 43-year-old woman receiving coadjuvant chemotherapy due to a retroperitoneal leiomiosarcoma presented with nausea, vomiting, abdominal pain and fever (38 °C). The two blood cultures resulted in isolation of A. rimae and P. micra , being identified at species level by matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) technology with high log scores. The microorganisms were susceptible to penicilllin, amoxicillin-clavulanate, piperacillin-tazobactam, clindamycin, metronidazole, imipenem, and moxifloxacin. Treatment with levofloxacin was started and subsequently it was changed to piperacillin/tazobactam plus metronidazole and completed for 10 days, but the patient died days later due to her underlying disease. Highlights • Atopobium rimae and Parvimonas micra caused polymicrobial bloodstream infection. • Anaerobic bacteremia is especially associated with malignant processes. • Mass spectrometry method may help to diagnose rare anaerobe pathogens on species level. • A. rimae and P. micra bloodstream isolates were highly susceptible to antimicrobials. [ABSTRACT FROM AUTHOR]

Abstract Within the ENRIA project, several 'expertise laboratories' collaborated in order to optimize the identification of clinical anaerobic isolates by using a widely available platform, the Biotyper Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Main Spectral Profiles (MSPs) of well characterized anaerobic strains were added to one of the latest updates of the Biotyper database db6903; (V6 database) for common use. MSPs of anaerobic strains nominated for addition to the Biotyper database are included in this validation. In this study, we validated the optimized database (db5989 [V5 database] + ENRIA MSPs) using 6309 anaerobic isolates. Using the V5 database 71.1% of the isolates could be identified with high confidence, 16.9% with low confidence and 12.0% could not be identified. Including the MSPs added to the V6 database and all MSPs created within the ENRIA project, the amount of strains identified with high confidence increased to 74.8% and 79.2%, respectively. Strains that could not be identified using MALDI-TOF MS decreased to 10.4% and 7.3%, respectively. The observed increase in high confidence identifications differed per genus. For Bilophila wadsworthia , Prevotella spp., gram-positive anaerobic cocci and other less commonly encountered species more strains were identified with higher confidence. A subset of the non-identified strains (42.1%) were identified using 16S rDNA gene sequencing. The obtained identities demonstrated that strains could not be identified either due to the generation of spectra of insufficient quality or due to the fact that no MSP of the encountered species was present in the database. Undoubtedly, the ENRIA project has successfully increased the number of anaerobic isolates that can be identified with high confidence. We therefore recommend further expansion of the database to include less frequently isolated species as this would also allow us to gain valuable insight into the clinical relevance of these less common anaerobic bacteria. Highlights • European Network for the Rapid Identification of Anaerobes (ENRIA). • Validation of a for anaerobic bacteria optimized MALDI-TOF MS database. • The increase in high confidence identification differed per genus. • Further expansion of the database for the identification of less common species is recommended. [ABSTRACT FROM AUTHOR]

Abstract Within the European Network for the Rapid Identification of Anaerobes (ENRIA) project eight clinical isolates of Fenollaria massiliensis were encountered. In this study a more extensive description of this species is given and the MALDI-TOF MS database is optimized for its identification. F. massiliensis is an anaerobic Gram positive rod with the tendency to decolorize quickly. It is mostly encountered in clinical samples from the groin region. Less common and non-valid species are not represented in the MALDI-TOF MS database. Therefore, F. massiliensis can only be identified by laboratories performing 16S rDNA gene sequencing. The addition of less common and non-valid species to the database will give insight in their clinical relevance. Highlights • Fenollaria massiliensis seems to be a human pathogen. • An extensive description of the biochemical features of Fenollaria massiliensis. • Addition of reference spectra of Fenollaria massiliensis to the MALDI-TOF MS database enables its identification. • Adding reference spectra of uncommon anaerobic species, to the MALDI-TOF MS database, assesses their clinical relevance. [ABSTRACT FROM AUTHOR]

Clostridium innocuum is a Gram-positive anaerobic spore-forming bacillus that has been identified as part of the normal intestinal microbiota. This bacterium has been rarely associated with human infections, and only few severe infections have been reported until now. In this work, we report on four patients with bacteremia due to C. innocuum , which were well identified by MALDI-TOF MS. Moreover, a review of the previous published cases of bacteremia due to this anaerobic bacterium has been performed. • Clostridium innocuum is an uncommon cause of bacteremia. • C. innocuum is a significant cause of extra-intestinal infection. • MALDI-TOF MS is a good approach to identify C. innocuum. • Malignancies and diabetes mellitus are risk factors. [ABSTRACT FROM AUTHOR]

Bacteroides pyogenes is a Gram-negative obligate anaerobe, saccharolytic, non-motile, non-pigment-producing and non-spore-forming rod. Reports of human infections caused by B. pyogenes are scarce, with approximately 30 cases documented in scientific literature. The aim of this study was to describe the clinical characteristics of 8 different patients and to study the in vitro antibiotic susceptibility of the strains, as well as to evaluate the in vivo activity of the prescribed treatments. We conducted a descriptive retrospective study by searching all B. pyogenes isolates from January 2010 to March 2023 at the Basurto University Hospital. This included all cases, both in monomicrobial or polymicrobial cultures. Among the eight patients, three experienced severe infections such as bacteremia and osteomyelitis. All the strains were susceptible to amoxicillin/clavulanic acid, piperacillin/tazobactam, imipenem, meropenem, clindamycin, metronidazole and moxifloxacin. • B. pyogenes is a commensal of the oral cavity of cats and dogs. • In this work, half of the B. pyogenes infections were related to cat bites. • Identification of B. pyogenes isolates was succesfully performed by MALDI-TOF MS. • B. pyogenes isolates showed low MICs to all tested antibiotics. [ABSTRACT FROM AUTHOR]

The main study objective was to evaluate the correlation between matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequencing results for the identification of anaerobes. A retrospective study was conducted of all anaerobic bacteria isolated from clinically significant specimens. MALDI-TOF (Bruker Byotyper) and 16S rRNA gene sequencing were performed in all strains. Identifications were considered correct when the concordance with gene sequencing was ≥99%. The study included 364 isolates of anaerobic bacteria: 201 (55.2%) Gram-negative and 163 (44.8%) Gram-positive, mostly belonging to the genus Bacteroides. Isolates were largely obtained from blood cultures (128/35.4%) and intra-abdominal samples (116/32.1%). Overall, 87.3% of isolates were identified at species level using the version 9 database (89.5% of Gram-negative and 84.6% of Gram-positive anaerobic bacteria). All isolates belonging to the species B. fragilis sensu stricto were correctly identified by MALDI-TOF MS, but five cases of Phocaeicola (Bacteroides) dorei were misidentified as Phocaeicola (Bacteroides) vulgatus; all Prevotella isolates were correctly identified at the genus level, and most were correctly identified at the species level. Among Gram-positive anaerobes, 12 Anaerococcus species were not identified by MALDI-TOF MS, while six cases identified as Peptoniphilus indolicus were found to belong to other genera/species. MALDI-TOF is a reliable technique for identifying most anaerobic bacteria, although the database needs frequent updating to identify rare, infrequent, and newly discovered species. • Overall correct identification of anaerobes using Biotyper software was 87.3%. • Five Phocaeicola (B.) dorei were misidentified as Phocaeicola (B.) vulgatus. • All Prevotella isolates were correctly identified at genus level. • Twelve Anaerococcus species were not identified by MALDI-TOF MS. • Six Peptoniphilus indolicus were found to belong to other genera/species. [ABSTRACT FROM AUTHOR]

We report the case of a 69-year-old man admitted for septic shock secondary to necrotic pneumoniae complicated by thoracic empyema of fatal issue. Microbiological examination of pleural liquid revealed a mixed anaerobic flora involving Campylobacter rectus and Actinomyces meyeri. Campylobacter rectus is an infrequent anaerobic pathogen of oral origin To our knowledge, this is the first case report of fatal C. rectus - associated thoracic empyema, and only the second reported case in which identification was successfully performed by MALDI-TOF MS. [ABSTRACT FROM AUTHOR]

Inter-laboratory reproducibility of Matrix Assisted Laser Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF MS) of anaerobic bacteria has not been shown before. Therefore, ten anonymized anaerobic strains were sent to seven participating laboratories, an initiative of the European Network for the Rapid Identification of Anaerobes (ENRIA). On arrival the strains were cultured and identified using MALDI-TOF MS. The spectra derived were compared with two different Biotyper MALDI-TOF MS databases, the db5627 and the db6903. The results obtained using the db5627 shows a reasonable variation between the different laboratories. However, when a more optimized database is used, the variation is less pronounced. In this study we show that an optimized database not only results in a higher number of strains which can be identified using MALDI-TOF MS, but also corrects for differences in performance between laboratories. [ABSTRACT FROM AUTHOR]

Animal botulism is primarily due to botulinum neurotoxin (BoNT) types C, D or their chimeric variants C/D or D/C, produced by Clostridium botulinum group III, which appears to include the genetically indistinguishable Clostridium haemolyticum and Clostridium novyi . In the present study, we used matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI TOF MS) to identify and characterize 81 BoNT-producing Clostridia isolated in 47 episodes of animal botulism. The instrument's default database, containing no entries for Clostridium botulinum , permitted reliable identification of 26 strains at the genus level. Although supplementation of the database with reference strains enhanced the instrument's ability to identify the neurotoxic strains at the genus level, resolution was not sufficient to recognize field strains at species level. Characterization by MALDI TOF confirmed the well-documented phenotypic and genetic differences between Clostridium botulinum strains of serotypes normally implicated in human botulism (A, B, E, F) and other Clostridium species able to produce BoNTs type C and D. The chimeric and non-chimeric field strains grouped separately. In particular, very low similarity was found between two non-chimeric type C field strains isolated in the same outbreak and the other field strains. This difference was comparable with the differences among the various Clostridia species included in the study. Characterization by MALDI TOF confirmed that BoNT-producing Clostridia isolated from animals are closely related and indistinguishable at the species level from Clostridium haemolyticum and Clostridium novyi reference strains. On the contrary, there seem to be substantial differences among chimeric and some non-chimeric type C strains. [ABSTRACT FROM AUTHOR]

The genus Fusobacterium contains currently 13 species presenting as non-sporing, obligate anaerobic, Gram-negative fusiform rods. Fusobacterium ulcerans was discovered in 1988 causing tropical ulcers. We present the case of a patient with diverticulitis complicated with bacteremia. Both aerobic bottles were positive at 20 and 24 h, while one anaerobic bottle was positive at 36 h. Escherichia coli and Fusobacterium ulcerans were identified from subcultures by MALDI-TOF MS with a score of 2.02 and 2.35, respectively. The 16S rRNA gene was sequenced in order to confirm the identification of F. ulcerans with a 100% homology to the reference strain. The patient was treated with 4 g/0,5 g of IV piperacillin/tazobactam and later with 1 g/0,2 g of amoxicillin/clavulanic during 7 days with good clinical evolution. • First case of F. ulcerans causing polymicrobial bacteremia in a patient with diverticulitis. • Most cases of F. ulcerans are related to infected ulcers in tropical climates. • Identification through biochemical tests is not adequate in this species. • MALDI-TOF MS can be used to identification of Fusobacterium spp.. • 16S rRNA sequencing must be used to confirm the identification. [ABSTRACT FROM AUTHOR]

The objectives of this study were to describe differences between bloodstream infections involving Gram-positive (GP) and Gram-negative (GN) anaerobic bacteria. Patients with clinically significant anaerobic bacteremia detected between October 2016 and July 2022 in a tertiary hospital in Granada (Spain) were retrospectively included. Species identification was performed by MALDI-TOF MS and/or molecular methods. The association between variables was analyzed using contingency tables, applying the chi-square test when expected frequencies were adequate and the Fisher exact test when not. Variables were gathered at the time of the first positive blood culture. Out of 237 cases of anaerobic bloodstream infections detected, 127 (53.6%) were GN. Crude mortality was 20.3%, corresponding to 48 patients who died of causes directly attributable to bacteremia. The presence of malignant disease (p = 0.011), abdominal and/or pelvic surgery (p = 0.001), and transplantation (p = 0.008) were significantly associated with bacteremia due to GN bacteria, while the presence of diabetes mellitus was significantly associated with bacteremia due to GP bacteria (p = 0.022). The presence of both septic shock and mortality was more frequently associated with bacteremia due to GN versus GP bacteria. The association of certain variables with the presence of bloodstream infections due to GP or GN anaerobic bacteria may assist in selecting the optimal empirical therapeutic approach and improving the outcome of patients with these types of infection. • Some variables can be associated with bacteremia due to GP or GN anaerobic bacteria. • Malignancies are more frequently associated with bacteremia due to GN anaerobes. • Diabetes mellitus is more frequently associated with bacteremia due to GP anaerobes. • Acquiring septic shock is more frequently associated with bacteremia due to GN anaerobes. [ABSTRACT FROM AUTHOR]

During the past decade, the clinically relevant genus Prevotella has expanded considerably. Prevotella species can be isolated from nearly all types of oral infections but also from various non-oral infections. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been introduced in clinical microbiology laboratories as a convenient method for identifying bacterial isolates from clinical specimens. Here we tested the diagnostic accuracy of a total of 123 oral Prevotella isolates, selected based on their biochemical profile, by Bruker MALDI-TOF MS. Partial 16S rRNA sequencing was used as a reference method. The performance of MALDI-TOF MS to identify the isolates to the genus level was excellent with 100.0% accuracy, while a good identification rate of 88.6% was achieved to the species level with a log score of ≥2.0. The isolates representing P. aurantiaca and P. jejuni , which are currently missing from the MALDI BioTyper database, were identified correctly to the genus level. Of the 123 isolates, one P. pallens isolate (0.8%) was identified with a score variation of 1.7–1.999. Overall, biochemical testing produced a high proportion (70.7%) of incorrect identifications within different species. MALDI-TOF MS offers a reliable and rapid method for the identification of Prevotella species included in the database. [ABSTRACT FROM AUTHOR]

Bacteoides finegoldii is a Gram-negative, rod-shaped and obligate anaerobic bacteria isolated in human feces during studies of intestinal microbiota. We present a case report in which B. finegoldii was isolated and identified from the blood culture of a 93-year-old patient with abdominal pain. Microbiological identification was performed by MALDI-TOF MS and confirmed later by 16S rRNA sequencing. An abdomino-pelvic CT scan was conducted, showing a mass of neoplastic appearance that infiltrated the sigmoid colon and bladder, probably producing a colo-vesical fistula. Up to now, this is the first report of B. finegoldii causing human infection. • Bacteroides finegoldii is a comensal of human feces. • The first report of B. finegoldii causing human infection. • Abdominal route is a frequent source of infections caused by Bacteroides spp. • MALDI-TOF MS is a rapid and usefull tool at identification of Bacteroides spp. • 16S rRNA sequencing is needed to confirm the identification in most cases. [ABSTRACT FROM AUTHOR]

Information regarding the use of MALDI-TOF MS as an alternative to conventional laboratory methods for the rapid and reliable identification of bacterial isolates is still limited. In this study, MALDI-TOF MS was evaluated on 295 anaerobic isolates previously identified by 16S rRNA gene sequencing and with biochemical tests (Rapid ID 32A system, BioMérieux). In total, 85.8% of the isolates were identified by MALDI-TOF MS at the species level vs 49.8% using the Rapid ID 32A system (p < 0.0001). None of the isolates was discordantly identified at the genus level using MALDI-TOF MS and only 9 of them could not be identified using the method. Thus, our results show that MALDI-TOF MS is a robust and reliable tool for the identification of anaerobic isolates in the microbiology laboratory. Its implementation will reduce the turnaround time for a final identification and the number of isolates that require 16S rRNA sequencing. [ABSTRACT FROM AUTHOR]

Diagnostic laboratories are urged to take advantage of novel technological advancements to provide standardized and high-throughput information for clinicians; however, total laboratory automation (TLA) has only recently been introduced in clinical microbiology in the last 10–12 years. The introduction of total laboratory automation comes with certain advantages and drawbacks that need to be assessed before the introduction of such systems in the diagnostic workflow that includes the detection of anaerobic bacteria. For several reasons, there is yet to be a manufacturer to fully address the issue of anaerobes in the setting of laboratory automation; the aim of the present paper is to address some of the issues associated with anaerobes in lab automation. • An overview of issues regarding implementation of total laboratory automation and anaerobic bacteriology. [ABSTRACT FROM AUTHOR]

Abstract We report a case of bloodstream infection with the anaerobic bacterium Ruminococcus gnavus (R. gnavus), associated with intestinal perforation in a patient undergoing chemotherapy for multiple myeloma and cancer of the sigmoid colon. Gram staining of positive anaerobic blood cultures revealed both diplococci and short chains of gram-positive cocci. MALDI-TOF MS done directly on the blood culture bottle identified the bacterium as R. gnavus , and 16S rRNA gene sequencing confirmed the identification. Highlights • First case reported of blood stream infection by Ruminococcus gnavus identified directly from the blood culture bottle. • MALDI-TOF is useful for identifying Ruminococcus gnavus. • The patient was successfully treated with piperacillin/tazobactam. [ABSTRACT FROM AUTHOR]

Our large regional laboratory routinely provides a definitive identification (ID) for 800-1,200 anaerobic bacteria per annum that cause invasive human infections. An increasing number of isolates (i.e., 10 to 13%) recovered from clinical specimens from these cases were more unusual or rarely isolated genera and/or species (i.e., ≤5 individual cases/annum). VITEK® MS (MALDI-TOF MS) is done initially on all anaerobic bacteria, but rare isolates undergo in-house PCR/sequencing when proteomics provides a wrong ID or no results despite repeat testing. A clinical microbiologist in consultation with the Infectious Diseases service approves molecular analyses. This multi-year comparison (2014-19) of the performance of MALDI-TOF MS and 16S rRNA gene sequencing using the IDNS® SmartGene bacterial dataset shows both method's abilities to provide a genus-level and/or species-level ID for rare isolates. 489 rare anaerobes were recovered from a variety of clinical specimens: 57% blood cultures, 19% other sterile fluids, 14% sterile tissues, 8% deep wounds/abscesses, and 2% prosthetic implants. 16S rRNA gene sequencing gave an accurate genus-vs. species level ID for 487/489 (99.6%) and 401/489 (82.0%) of isolates respectively. Accurate genus-vs species-level ID were obtained by MALDI-TOF MS for 269/489 (53.4%) and 187/489 (37.3%) of isolates respectively. MALDI-TOF MS gave wrong or no results for 35.1% of Gram-negative anaerobic cocci (GNAC), 62% of Gram-negative anaerobic bacilli (GNAB), 30.8% of Gram-positive anaerobic cocci (GPAC) and 46.3% of Gram-positive anaerobic bacilli (GPAB). Neither method gave an ID for one GNAB and one GPAC isolate. MALDI-TOF MS genus-level ID of GNAC and genus/species-level ID of GPAB improved during the study but its performance remained stable for genus- or species-level ID of other organism groups. MALDI-TOF MS provides accurate ID for most common anaerobes, but molecular analyses need to be available for rare isolates. Large complex laboratories should have a workflow for sending rare isolates for 16S rRNA gene sequencing in invasive cases where a definitive ID is clinically required. • An increasingly diverse array of rare anaerobes cause invasive human infections. • Proteomics improves accurate identification of commonly encountered anaerobes. • VITEK® MS database versions from 2014-19 identified ∼50% of the rare anaerobes. • VITEK® MS database(s) should be updated with diverse spectra for rare anaerobes. • 16S rRNA gene sequencing accurately identified anaerobes when proteomics failed. [ABSTRACT FROM AUTHOR]

Eggerthella lenta is a Gram-positive anaerobic bacillus. Improved diagnostics and increased awareness of rare pathogens have revealed its potential to cause serious invasive infections. In this study, 18 clinical E. lenta isolates derived from positive blood cultures were included. Underlying problems of the patients were in the majority of cases related to the gastrointestinal tract. The performance of two MALDI-TOF MS systems, i.e. Bruker and Vitek MS, in identification of E. lenta was analyzed. In addition, the minimal inhibitory concentrations for clinically relevant antimicrobial agents were determined by routine procedures using E-test. 17 of the 18 E. lenta isolates investigated in this study were correctly identified to species level by the Bruker MS system, while the Vitek MS system identified all 18 isolates. Antimicrobial sensitivity towards the tested agents was in general good. However, high resistance rates were observed for penicillin G and piperacillin-tazobactam based on EUCAST breakpoints. [ABSTRACT FROM AUTHOR]

We retrospectively analyzed seven patients with Actinotignum schaalii bacteremia in a tertiary hospital in Japan. Pyelonephritis was the most frequent source of bacteremia, followed by Fournier's gangrene and pyometra. All patients with pyelonephritis had underlying urological conditions, ureteral stents, nephrostomy, ureteral stones, or ureterocele. • Actinotignum schaalii is being recognized as a cause of urinary tract infection. • We retrospectively analyzed seven patients with A. schaalii bacteremia. • Pyelonephritis was the most frequent source of A. schaalii bacteremia. • All patients with pyelonephritis had underlying urological conditions. [ABSTRACT FROM AUTHOR]

Eggerthia catenaformis is a Gram-positive bacilli and an anaerobic and non-spore-forming bacterium, which rarely causes infections in humans. We present a case of peritonitis caused by E. catenaformis in a peritoneal dialysis patient. The isolate was identified as E. catenaformis with the MALDI-TOF MS method as in other cases in the literature. To the best of our knowledge, this is the first case of peritonitis caused by E. catenaformis in a human host. • Eggerthia catenaformis (E. catenaformis) is a Gram-positive bacilli, which was named as Lactobacillus catenaformis before 2011. • It is an anerobic, non-spore-forming bacterium belonging to the Erysipelotrichaceae family. It was first detected in human feces in 1935. • To date, only six cases of infection caused by E. catenaformis have been reported. • MALDI-TOF MS plays an important role in a convenient identification of anaerobic bacteria such as E. catenaformis. • Microbiologist and clinicians should be aware of the possibility that the pathogen may cause a severe infection rather than being a mere contaminant. [ABSTRACT FROM AUTHOR]

Introduction of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized bacterial identification in the last decade. In 2013, MALDI-TOF MS was implemented for the identification of anaerobic bacteria at our laboratory. This study analyzed the impact of MALDI-TOF MS on the number of different anaerobic genera and species identified in diagnostics. 155 anaerobic human clinical isolates, representing the most frequently isolated anaerobic species at our laboratory, were identified by conventional biochemical methods and by a Bruker MALDI Biotyper (Bruker Daltonics, Bremen, Germany). Discrepancies were resolved by partial 16S rRNA gene sequence analysis. In addition, we compared the frequencies of anaerobic genera and species prior to the implementation of MALDI-TOF MS from 2008 to 2012 to the frequencies of anaerobes from 2013 to 2020 when MALDI-TOF MS was used for identification. The diversity of anaerobic bacteria increased from 12 genera and 20 species in 2012, before the introduction of MALDI-TOF MS, to 16 genera and 31 species in 2013 and to 20 genera and 41 species in 2020 when MALDI-TOF MS was used as primary identification method. MALDI-TOF MS allowed species assignment within closely related species such as the Bacteroides fragilis group in accordance with 16S identification, and correctly identified newly described anaerobic species. Introduction of MALDI-TOF MS identification increased genus and species diversity of the reported anaerobes at our laboratory. Updates to the MALDI-TOF MS database and new species descriptions will further increase the diversity of anaerobic bacteria isolated from infectious processes. • Anaerobic species distribution in a routine diagnostic laboratory over 13 years. • Implementation of MALDI-TOF MS increases anaerobic bacterial diversity in diagnostics. • MALDI-TOF MS identification of anaerobes is highly reliable. [ABSTRACT FROM AUTHOR]

Phocaeicola (Bacteroides) dorei is a Gram-negative anaerobic bacillus that is rarely isolated from human specimens. Its accurate identification can be hampered by its close taxonomic relationship with Bacteroides vulgatus. We report on two patients with bacteremia due to P. (B.) dorei , which was initially identified as B. vulgatus by MALDI-TOF MS. . Phocaeicola (Bacteroides) dorei is a rare cause of human infection.. Two cases of bloodstream infection due to P (B.) dorei are reported in detail. MALDI TOFF MS failed to identify P (B.) dorei. . Molecular techniques are needed to differentiate P (B.) dorei from B. vulgatus [ABSTRACT FROM AUTHOR]

Ruminococcus gnavus is frequently found among human gut microbiome. However, human bloodstream infections by R. gnavus have been reported only three times. Clinical details were lacking for one case; the other two cases with concurrent bacteremia in patients with diverticulitis. We report a case of R. gnavus bloodstream infection in a patient with a gall bladder perforation suggesting its association with damage to the gastrointestinal tract. R. gnavus was misidentified using biochemical test but 16S rRNA sequencing and Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were useful for correct identification. With the advancement of identification method in clinical laboratory, more frequent identification of R. gnavus from clinical specimens is expected. [ABSTRACT FROM AUTHOR]

Abstract: A total of 73 chicken and calves isolates were diagnosed using matrix-assisted laser desorption ionization-time-of flight mass spectrometry (Maldi-Tof MS). After a preliminary subtractive screening based on the high acid tolerance at pH 2.5 and bile resistance at 0.3% oxgall, twenty isolates belonging to the species Lactobacillus salivarius, Lactobacillus agilis, Lactobacillus reuteri, Lactobacillus murinus and Lactobacillus amylovorus were in vitro screened for the safety assessment and probiotic properties, including antibiotics susceptibility patterns, biochemical activity and potential for competitive exclusion of biofilm producing pathogens determined by crystal violet and/or quantitative Fluorescent in situ Hybridisation (FISH) assays utilizing 5′Cy 3 labelled probe Enter1432 for enteric group. Antibiotic susceptibility testing was performed according to the ISO norm 10932. The sixteen strains were susceptible to certain antimicrobial agents, except for two chicken (L. salivarius 12K, L. agilis 13K) and two calves (L. reuteri L10/1, L. murinus L9) isolates with the presence non wild-type ECOFFs (epidemiological cut-off) for gentamicin (≥256 μg ml−1), tetracycline (≥128 μg ml−1), kanamycin (≥256 μg ml−1) and streptomycin (≥96 μg ml−1). The two referenced chicken isolates gave positive aac(6′)Ie-aph(2″)Ia and tet(L) PCR results. The wild-type ECOFFs isolates were subjected to the apiZYM analysis for enzyme profile evaluation and amino acid decarboxylase activities determined by qualitative plate method and multiplex PCR for the detection of four genes involved in the production of histamine (histidine decarboxylase, hdc), tyramine (tyrosine decarboxylase, tyrdc) and putrescine (via eithers ornithine decarboxylase, odc, or agmatine deiminase, agdi). From examined strains only two chicken isolates (L. reuteri 14K; L. salivarius 15K) had no harmful β-glucuronidase, β-glucosidase activities connected with detrimental effects in the gastrointestinal tract and together no amino acid decarboxylase activities and no genes associated with biogenic amines production though only chicken L. salivarius 15K whole cells and acid supernatants shown strong suppressive potential against biofilm-forming Klebsiella and Escherichia coli. Our results highlight that above-mentioned isolate L. salivarius 15K fulfils the principle requirements of a qualified probiotic and may be seen as a reliable candidate for further validation studies in chicken. [Copyright &y& Elsevier]

Abstract: Today resistance against carbapenems is considered an emerging problem in Bacteroides fragilis. Carbapenemase activities produced by aerobic bacteria have been detected by looking at hydrolysis of carbapenems with MALDI-TOF MS, but this technique was never used for anaerobic bacteria. We have developed a protocol for detection and verification of carbapenemase production in B. fragilis within 2.5 h. Twenty-eight strains of B. fragilis were tested. Of the sixteen cfiA-positive strains all showed hydrolysis of ertapenem, whereas the twelve cfiA-negative strains showed no hydrolysis. Ertapenem hydrolysis could be inhibited with 2,6-Pyridinecarboxylic acid (DPA) in all cfiA-positive strains, verifying the presence of the metallo-beta-lactamase. Here we show a rapid way to detect carbapenemase activities of B. fragilis strains. [Copyright &y& Elsevier]

Lachnoanaerobaculum spp. is an obligate anaerobic, gram-positive, spore-forming, rod-shaped bacillus. Here, we report the first known case of bacteremia due to L. orale , which was detected in a patient with acute lymphoblastic leukemia. A 69-year-old man developed neutropenic fever with severe stomatitis during chemotherapy for leukemia. The bacteria strain isolated from blood culture was successfully identified as L. orale via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Susceptibility testing revealed that the strains showed low minimum inhibitory concentrations (MICs) of beta-lactams, clindamycin, and metronidazole, but higher MICs of fluoroquinolones. The present case study indicates that Lachnoanaerobaculum can be a cause of human infection, including bloodstream infection. • Lachnoanaerobaculum orale- induced bacteremia was detected in a patient with leukemia. • L. orale was successfully identified by MALDI-TOF-MS. • L. orale may have higher MIC values for fluoroquinolones. [ABSTRACT FROM AUTHOR]

Two commercial MALDI-TOF MS systems were used to identify 18 isolates, belonging to the Peptoniphilus genus; also the 16S rRNA sequencing identity was compared against the MALDI-TOF MS system results. Bruker Biotyper system provided higher accuracy than Vitek MS system, however, adding spectra could allow a more reliable species level identification. • Discrepancies in the identification of Peptoniphilus by MALDI-TOF MS were studied. • Incorrectly assigned reference strains in database can lead to misidentification. • Two closely species could be reported as a complex by Bruker MALDI-TOF MS. • Spectral databases should be updated by suppliers to improve identification rates. [ABSTRACT FROM AUTHOR]

Abstract: Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is widely used today for species determination of bacteria and fungi in routine microbiological laboratories, and can also be used for subtyping of bacteria, such as Bacteroides fragilis. Propionibacterium acnes is frequently referred to as an anaerobic skin commensal of relatively low pathogenicity. In addition to its accepted pathogenic role in acne, P. acnes is now emerging as an important opportunistic pathogen in many other clinical situations, including late-stage prosthetic joint infections, osteomyelitis, endocarditis, endophthalmitis, post-neurosurgical infections and possibly prostate cancer. At the population genetic level, P. acnes can be differentiated into a number of distinct phylogroups, known as types IA1, IA2, IB, IC, II and III, which may be associated with different types of infections and clinical conditions. The aim of the present study was to evaluate MS-based typing for resolution of these genetic groups after routine identification by MALDI-TOF MS (Bruker MALDI Biotyper). The software package ClinProTools 2.2 was used to analyze the protein based mass spectra of reference strains belonging to types IA, IB, IC, II and III. Phylogroup-specific peaks and peak shifts were then identified visually. In addition, peak variations between the different types of P. acnes were investigated by using FlexAnalysis 3.3 software (Bruker). A differentiating library was created, which was used to type further 48 clinical isolates of P. acnes. Typing data obtained by MALDI-TOF MS were then compared with the results from Multilocus Sequence Typing (MLST). Most of the clinical isolates (n = 19) belonged to the type IA grouping according to MALDI-TOF MS. By MLST, all isolates were identified as type IA1. Twenty-one clinical isolates belonged to the type IB cluster based on both MALDI-TOF MS and MLST typing. Eight clinical isolates were identified as type II strains by both typing methods and all the type III reference strains could be distinguished by the presence of a unique type III-specific peak (7238 Da) by the MALDI-TOF MS. Our study demonstrates that MALDI-TOF MS is a reliable and powerful tool for rapid identification and typing of P. acnes strains from the main genetic divisions of the species. [Copyright &y& Elsevier]

Abstract: In the present study, two pre-analytic processes for mass spectrometric bacterial identification were compared: the time-consuming reference method, chemical extraction, and the direct smear technique directly using cultured colonies without any further preparation. These pre-analytic processes were compared in the identification of a total of 238 strains of anaerobic bacteria representing 34 species. The results showed that 218/238 strains were identified following chemical extraction, 185 identifications (77.7%) were secured to both genus and species [log(score) > 2.0] whereas 33 identifications (14%) were secured to genus only [log(score) between 1.7 and 2.0]. Following direct smear, 207/238 anaerobic bacteria were identified, 158 identifications (66.4%) were secured to both genus and species [log(score) > 2.0] whereas 49 identifications were secured to genus only [log(score) between 1.7 and 2.0]. Twenty strains were not identified [log(score) < 1.7] by MALDI-TOF MS following chemical extraction whereas 31 strains were not identified with the direct smear technique. Although direct smear led to a significant decrease of the log(score) values for the Clostridium genus and the Gram positive anaerobic bacteria (GPAC) group (p < 0.0001, Wilcoxon test), identification to both species and genus were not changed. However these differences were not statistically significant (p = 0.1, Chi square). Therefore, MALDI-TOF MS identification following the direct smear technique appears to both non-inferior to the reference method and relevant for anaerobic bacteria identification. [Copyright &y& Elsevier]

All 50 Clostridium difficile strains were definitely identified by Vitek2 system, Rapid ID 32A system, and MALDI-TOF. For 18 non-difficile Clostridium strains, the identification results were correct in 0, 2, and 17 strains by Vitek2, Rapid ID 32A, and MALDI-TOF, respectively. MALDI-TOF could be used as the primary tool for identification of Clostridium species. [ABSTRACT FROM AUTHOR]

The aim of this study was to describe the differences in antimicrobial susceptibility to moxifloxacin between European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) in anaerobic microorganisms. Overall, resistance to moxifloxacin appears to be high in almost all groups of anaerobes, but enormous differences in susceptibility rates between these two committees could be observed. • Resistance to moxifloxacin differs according to the breakpoints used. • Higher susceptibility was observed when CLSI breakpoints were applied. • Susceptibility rates were highest in Clostridium spp. • Overall resistance rates to moxifloxacin have increased last years. [ABSTRACT FROM AUTHOR]

To perform surveillance of cfiA -positive Bacteroides fragilis using new subtyping software module, MALDI Biotyper Subtyping Module (MBT Subtyping Module), on MALDI-TOF MS system, and to evaluate the detection ability of the module. cfiA -positive strains were presumed using the module against B. fragilis isolated between 2006 and 2019. The cfiA gene was confirmed using PCR. In cfiA -positive B. fragilis , the insertion sequence (IS) elements were examined and the MBT STAR-BL assay was performed to examine meropenem hydrolysis activity. Of the 396 B. fragilis strains included, the MBT Subtyping Module detected 33 presumptive cfiA -positive strains (8.3%), of which 32 harbored the cfiA gene. The sensitivity and specificity of the MBT Subtyping Module for detecting cfiA -positive B. fragilis were 100.0% and 99.7%, respectively. Of the 32 strains harboring the cfiA gene, seven strains possessed IS elements, which were thought to induce high cfiA expression. Meropenem hydrolysis was detected in all seven strains that were positive for both cfiA and IS elements, and they exhibited resistance to meropenem and imipenem. The overall non-susceptibility rates to meropenem and imipenem were 84.8% and 36.4%, respectively, in the 33 presumptive cfiA -positive strains. The MBT Subtyping Module can detect cfiA -positive B. fragilis rapidly and accurately, supporting its use for surveillance of cfiA -positive B. fragilis in clinical settings. • The MBT Subtyping Module detected cfiA -positive B. fragilis on the MALDI-TOF MS system. • The rate of cfiA -positive B. fragilis was higher than carbapenem resistance rates. • The cfiA -positive B. fragilis exhibited decreased carbapenem susceptibility. [ABSTRACT FROM AUTHOR]

Abstract: Diverse techniques were applied to effect the identification and classification of isolated clostridial strains. Nevertheless, the correct identification of clostridial strains remains a laborious, time-consuming task which entails a not inconsiderable degree of expertise. In addition to this, traditional methods based on the metabolic properties of the bacteria require rigorously standardized media and growth conditions to assure the attainment of reproducible results. Although DNA-based methods, like the PCR of a species specific gene, are known to yield precise and reproducible results, their degree of effectivity is circumscribed by the fact that even the incidence of a toxin encoding gene is not necessarily linked to nor consequently indicative of the presence of an infectious disease. Moreover, most of these methods postulate an initial assumption concerning the expected bacterial species involved before the choice of PCR primer for use can be made. Consequently, the scope of these methods is restricted to that of targeted analyses. The 16S rDNA sequencing which is assumed to be the gold standard for bacterial classification having the unequivocal advantage of being capable of determining even uncultivable bacteria is nonetheless a time-consuming and costly technique. In the present study we describe the utilization of matrix-assisted laser desorption and ionization–time-of-flight mass spectrometry (MALDI-TOF MS) for whole cell fingerprinting in combination with a dedicated bioinformatic software tool to distinguish between various clostridial species. Total 64 clostridial strains of 31 different species each displayed a mass spectrum unique to the strain involved, to the effect that it was also possible not only to differentiate between the strains examined, but also to establish to which species the individual strains belonged to. Starting with a single colony it was possible to correctly identify a Clostridium species within minutes. It was even possible to identify species which are normally difficult to differentiate by traditional methods, such as C. chauvoei and C. septicum. With the results obtained we were able to assemble a dendrogram of the Clostridium species which showed considerable similarities to dendrograms based upon 16S rDNA sequencing data. To conclude, our findings indicate that, inasmuch as the MALDI–TOF MS technology employed is based on a high-quality reference database, it may serve as an effective tool for the swift and reliable identification and classification of Clostridia. [Copyright &y& Elsevier]

• Bowel tract pathologies appear to facilitate bloodstream infection caused by anaerobic bacteria. • Due to their difficult identification, Eubacterium spp. and Butyricimonas spp are probably under-reported human pathogens. • Expansion of the MALDI-TOF MS database is mandatory for accurate identification of uncommon bacteria. [ABSTRACT FROM AUTHOR]

The objectives of this study were to report 10 episodes of clinically significant bacteremia caused by species of the genus Anaerococcus isolated between July 2018 and February 2021 from the microbiology laboratory of a tertiary hospital in Granada (Spain). None of the isolates were identified by MALDI-TOF MS, and the definitive species identification was performed by 16 S rRNA gene sequencing. No reference spectra of the Anaerococcus species were present in the MALDI-TOF MS database. Eight isolates were finally identified as A. octavius , one isolate as A. tetradius and the other as A. urinomassiliensis. The majority of these infections were seen in patients aged >70 years. Risk factors for anaerobic infection were observed in eight patients, especially diabetes mellitus, surgery, and the presence of cancer. Fever was present in all patients. Three patients died, but only one death was attributed to the infection. Mean detection time of positive blood cultures was 47.5 h (range 24–92 h). Antimicrobial susceptibility to penicillin, amoxicillin-clavulanate, imipenem, moxifloxacin, clindamycin, metronidazole, and piperacillin-tazobactam was tested using the gradient diffusion technique and EUCAST breakpoints (except for moxifloxacin). No resistance to amoxicillin-clavulanate, metronidazole, imipenem, or piperacillin-tazobactam was detected; however, the majority of isolates were resistant to clindamycin. When MALDI-TOF MS does not provide a correct identification at genus or species level, as in some isolates of Gram-positive anaerobic cocci, microbiologists should perform an additional confirmatory technique, such as gene sequencing analysis, to obtain a definitive diagnosis. • MALDI-TOF MS could not correctly identify some Anaerococcus species. • Molecular techniques can help to achieve a definitive identification of Anaerococcus. • Bacteremia due to Anaerococcus spp is mostly seen in older patients. • Overall, antimicrobial resistance rates are low for this genus. [ABSTRACT FROM AUTHOR]

Clostridioides difficile is an important organism causing healthcare-associated infections. It has been documented that specific strains caused multiple outbreaks globally, and patients infected with those strains are more likely to develop severe C. difficile infection (CDI). With the appearance of a variant strain, BI/NAP1 ribotype 027, responsible for several outbreaks and high mortality rates worldwide, the epidemiology of the CDI changed drastically in the United States, Europe, and some Latin American countries. Although the epidemic strain 027 was not yet detected in Brazil, there are ribotypes exclusively found in the country, such as, 131, 132, 133, 135, 142 and 143, which are responsible for outbreaks in Brazilian hospitals and nursing homes. Although PCR-ribotyping is the most used method in epidemiology studies of C. difficile , it is not available in Brazil. This study aimed to develop and validate an in-house database for detecting C. difficile ribotypes, usually involved in CDI in Brazilian hospitals, by using MALDI-TOF MS. A database with 19 different ribotypes, 13 with worldwide circulation and 6 Brazilian-restricted, was created based on 27 spectra readings of each ribotype. After BioNumerics analysis, neighbor-joining trees revealed that spectra were distributed in clusters according to ribotypes, showing that MALDI-TOF MS could discriminate all 19 ribotypes. Moreover, each ribotype showed a different profile with 42 biomarkers detected in total. Based on their intensity and occurrence, 13 biomarkers were chosen to compose ribotype-specific profiles, and in silico analysis showed that most of these biomarkers were uncharacterized proteins or well-conserved peptides, such as ribosomal proteins. A double-blind assessment using the 13 biomarkers correctly assigned the ribotype in 73% of the spectra analyzed, with 94%–100% of correct hits for 027 and for Brazilian ribotypes. Although further analyses are required, our results show that MALDI-TOF MS might be a reliable, fast and feasible alternative for epidemiological surveillance of C. difficile in Brazil. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract: We present the results from 16S sequencing and phenotypic tests for differentiation of Bacteroides dorei from Bacteroides vulgatus and Bacteroides ovatus from Bacteroides xylanisolvens, which was not possible with MALDI-TOF MS. Testing with β-glucosidase could differentiate B. dorei from B. vulgatus and a negative catalase reaction could identify B. xylanisolvens. [Copyright &y& Elsevier]

Eggerthia catenaformis is a Gram-positive anaerobic rod, which has been rarely reported in human diseases. We report the second case of bacteremia due to this microorganism in an elderly patient. A 73-year-old man, without underlying diseases presented with fever, odynophagia and swelling of the cervical lymph node for several days. Culture of drained cervical fluid resulted in the isolation of Raoultella ornithinolytica and Streptococcus anginosus. Anaerobic blood cultures yielded a rare anaerobic microorganism, identified as Eggerthia catenaformis. No resistance to tested antimicrobials was documented. Treatment with drainage and several antibiotic regimens was established, and the general condition of the patient improved, at two months of follow-up. • Eggerthia catenaformis is rarely associated with human infections. • E. catenaformis causes infection in patients with dental alterations. • Mass spectrometry is a useful tool in the diagnosis of this species. • Our strain was susceptible to all antibiotics tested. [ABSTRACT FROM AUTHOR]