Antimicrobial susceptibility profile of clinically relevant Bacteroides, Phocaeicola, Parabacteroides and Prevotella species, isolated by eight laboratories in the Netherlands.

OBJECTIVES: Recently, reports on antimicrobial-resistant Bacteroides and Prevotella isolates have increased in the Netherlands. This urged the need for a surveillance study on the antimicrobial susceptibility profile of Bacteroides, Phocaeicola, Parabacteroides and Prevotella isolates consecutively isolated from human clinical specimens at eight different Dutch laboratories. METHODS: Each laboratory collected 20-25 Bacteroides (including Phocaeicola and Parabacteroides) and 10-15 Prevotella isolates for 3 months. At the national reference laboratory, the MICs of amoxicillin, amoxicillin/clavulanic acid, piperacillin/tazobactam, meropenem, imipenem, metronidazole, clindamycin, tetracycline and moxifloxacin were determined using agar dilution. Isolates with a high MIC of metronidazole or a carbapenem, or harbouring cfiA, were subjected to WGS. RESULTS: Bacteroides thetaiotaomicron/faecis isolates had the highest MIC90 values, whereas Bacteroides fragilis had the lowest MIC90 values for amoxicillin/clavulanic acid, piperacillin/tazobactam, meropenem, imipenem and moxifloxacin. The antimicrobial profiles of the different Prevotella species were similar, except for amoxicillin, for which the MIC50 ranged from 0.125 to 16 mg/L for Prevotella bivia and Prevotella buccae, respectively. Three isolates with high metronidazole MICs were sequenced, of which one Bacteroides thetaiotaomicron isolate harboured a plasmid-located nimE gene and a Prevotella melaninogenica isolate harboured a nimA gene chromosomally.Five Bacteroides isolates harboured a cfiA gene and three had an IS element upstream, resulting in high MICs of carbapenems. The other two isolates harboured no IS element upstream of the cfiA gene and had low MICs of carbapenems. CONCLUSIONS: Variations in resistance between species were observed. To combat emerging resistance in anaerobes, monitoring resistance and conducting surveillance are essential.

Characterization of mobile genetic elements in multidrug-resistant Bacteroides fragilis isolates from different hospitals in the Netherlands.

OBJECTIVES: Five human clinical multidrug-resistant (MDR) Bacteroides fragilis isolates, including resistance to meropenem and metronidazole, were recovered at different hospitals in the Netherlands between 2014 and 2020 and sent to the anaerobic reference laboratory for full characterization. METHODS: Isolates were recovered from a variety of clinical specimens from patients with unrelated backgrounds. Long- and short-read sequencing was performed, followed by a hybrid assembly to study the presence of mobile genetic elements (MGEs) and antimicrobial resistance genes (ARGs). RESULTS: A cfxA gene was present on a transposon (Tn) similar to Tn4555 in two isolates. In two isolates a novel Tn was present with the cfxA gene. Four isolates harbored a nimE gene, located on a pBFS01_2 plasmid. One isolate contained a novel plasmid carrying a nimA gene with IS1168. The tetQ gene was present on novel conjugative transposons (CTns) belonging to the CTnDOT family. Two isolates harbored a novel plasmid with tetQ. Other ARGs in these isolates, but not on an MGE, were: cfiA, ermF, mef(EN2), and sul2. ARGs harboured differed between isolates and corresponded with the observed phenotypic resistance. CONCLUSIONS: Novel CTns, Tns, and plasmids were encountered in the five MDR B. fragilis isolates, complementing our knowledge on MDR and horizontal gene transfer in anaerobic bacteria.

Antimicrobial susceptibility profiles of anaerobic bacteria, isolated from human clinical specimens, within different European and surrounding countries. A joint ESGAI study.

OBJECTIVES: Studies on the antimicrobial susceptibility profile of anaerobic bacteria are underrepresented in the literature. Within this study we aim to give an extensive overview of the differences in antimicrobial susceptibility profiles between different European and surrounding countries. METHODS: Minimal inhibitory concentration (MIC) data of different antibiotics were collected from 10 participating laboratories, representing an equal number of countries. All MIC's were determined using Etest, according to the protocol used by the participating laboratory. Anaerobic genera represented by at least 10 clinical isolates were included in the study. RESULTS: Each country tested different antibiotics, sometimes depending on the kind of infection and/or the anaerobic species isolated. All countries tested clindamycin and metronidazole. Resistance rates differed remarkably between the different countries. Especially in Kuwait, resistance was high for all tested antibiotics. Unexpected metronidazole resistance was observed for Finegoldia magna isolates, Peptoniphilus isolates and Eggerthella lenta isolates. CONCLUSIONS: Due to the extensive differences in antimicrobial susceptibility profile of anaerobic bacteria isolated within different countries, we strongly recommend to perform this kind of study on a regular basis.

Prevalence of antimicrobial resistance genes in Bacteroides spp. and Prevotella spp. Dutch clinical isolates.

OBJECTIVES: The prevalence of resistance genes in two important anaerobic genera, Bacteroides and Prevotella, was assessed by applying PCR specifically directed to genes of interest. METHODS: A total of 101 Bacteroides spp. and 99 Prevotella spp. human clinical isolates were identified using MALDI-TOF MS. The presence of the resistance genes cfxA, cepA, cfiA, tetQ, ermF and nim, was assessed. Prevalence of resistance genes was compared with the phenotypic resistance against amoxicillin, clindamycin, meropenem and metronidazole. RESULTS: Even though the majority of the Bacteroides isolates (95.0%) showed resistance towards amoxicillin, only 52/101 of the isolates harboured one of the resistance genes, accounting for this resistance. Within the genus Prevotella the presence of cfxA (50/99) almost perfectly matched the amoxicillin resistance (48/99). No difference in prevalence of the ermF gene (16/101 and 9/99) and clindamycin resistance (16/101 and 10/99) was observed within Bacteroides and Prevotella, respectively. Two isolates of Prevotella were resistant to metronidazole. One harboured the nim gene. One metronidazole-susceptible isolate of Bacteroides harboured a nim gene. Within the Bacteroides and Prevotella genera, 6/101 strains and 5/99 isolates harboured three different resistance genes, respectively, among them tetQ. TetQ is often located on a conjugative transposon, increasing the chance of horizontal gene transfer between isolates. CONCLUSIONS: An unknown mechanism in Bacteroides non-fragilis isolates causes resistance to ß-lactam antibiotics. The fact that the prevalence of the tetQ gene among Prevotella is increasing and the existence of isolates harbouring three resistance genes are worrisome developments.

Three metronidazole-resistant Prevotella bivia strains harbour a mobile element, encoding a novel nim gene, nimK, and an efflux small MDR transporter.

Objectives: In this study we assess the antibiotic resistance genes in three metronidazole-resistant Prevotella bivia clinical isolates. Methods: Strains were whole-genome sequenced. De novo assembly was performed and genes were annotated in RAST. Manual adjustments were made, when required, to the annotation and length of the genes. Results: In all three strains a novel nim gene, nimK, was encountered located on a mobile genetic element (MGE). The nimK gene was associated with an IS1380 family transposase. On the same MGE, genes encoding an efflux small MDR (SMR) transporter were present and were associated with a crp/fnr regulator. Conclusions: This is the first description of the presence of a novel nim gene in metronidazole-resistant P. bivia clinical isolates. This gene is co-located with an efflux SMR transporter on an MGE, which has been named Tn6456 (MG827401). The identification of these resistance genes on an MGE is worrisome, since this indicates the horizontal gene transfer of antibiotic and/or biocide resistance from one strain to the other.

An overview of the data obtained during the validation of an optimized MALDI-TOF MS Biotyper database for the identification of anaerobic bacteria.

This data in brief article presents the data obtained during the validation of the optimized Biotyper Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) database. The validation was performed by the different expertise laboratories, collaborating within the European Network for the Rapid Identification of Anaerobes (ENRIA) project, using 6309 human clinical anaerobic bacterial strains. Different databases were compared with each other; the db 5989 database (V5 database); the V5 database complimented with Main Spectral Profiles (MSPs) of ENRIA strains added to the next update of the database; and the V5 database complimented with the MSPs of all anaerobic clinical isolates collected within the ENRIA project. For a comprehensive discussion of the full dataset, please see the research article that accompanies this data article (Veloo et al., 2018) [1].

Validation of MALDI-TOF MS Biotyper database optimized for anaerobic bacteria: The ENRIA project.

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.

Assessing the clinical relevance of Fenollaria massiliensis in human infections, using MALDI-TOF MS.

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.

Conserved Citrullinating Exoenzymes in Porphyromonas Species.

Porphyromonas gingivalis is one of the major oral pathogens implicated in the widespread inflammatory disorder periodontitis. Moreover, in recent years, P. gingivalis has been associated with the autoimmune disease rheumatoid arthritis. The peptidylarginine deiminase enzyme of P. gingivalis (PPAD) is a major virulence factor that catalyzes the citrullination of both bacterial and host proteins, potentially contributing to production of anticitrullinated protein antibodies. Considering that these antibodies are very specific for rheumatoid arthritis, PPAD appears to be a link between P. gingivalis, periodontitis, and the autoimmune disorder rheumatoid arthritis. PPAD was thus far considered unique among prokaryotes, with P. gingivalis being the only bacterium known to produce and secrete it. To challenge this hypothesis, we investigated the possible secretion of PPAD by 11 previously collected Porphyromonas isolates from a dog, 2 sheep, 3 cats, 4 monkeys, and a jaguar with periodontitis. Our analyses uncovered the presence of secreted PPAD homologues in 8 isolates that were identified as Porphyromonas gulae (from a dog, monkeys, and cats) and Porphyromonas loveana (from sheep). In all 3 PPAD-producing Porphyromonas species, the dominant form of the secreted PPAD was associated with outer membrane vesicles, while a minor fraction was soluble. Our results prove for the first time that the citrullinating PPAD exoenzyme is not unique to only 1 prokaryotic species. Instead, we show that PPAD is produced by at least 2 other oral pathogens.

A multi-center ring trial for the identification of anaerobic bacteria using MALDI-TOF MS.

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.

The optimization and validation of the Biotyper MALDI-TOF MS database for the identification of Gram-positive anaerobic cocci.

Gram-positive anaerobic cocci (GPAC) account for 24%-31% of the anaerobic bacteria isolated from human clinical specimens. At present, GPAC are under-represented in the Biotyper MALDI-TOF MS database. Profiles of new species have yet to be added. We present the optimization of the matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) database for the identification of GPAC. Main spectral profiles (MSPs) were created for 108 clinical GPAC isolates. Identity was confirmed using 16S rRNA gene sequencing. Species identification was considered to be reliable if the sequence similarity with its closest relative was ≥98.7%. The optimized database was validated using 140 clinical isolates. The 16S rRNA sequencing identity was compared with the MALDI-TOF MS result. MSPs were added from 17 species that were not yet represented in the MALDI-TOF MS database or were under-represented (fewer than five MSPs). This resulted in an increase from 53.6% (75/140) to 82.1% (115/140) of GPAC isolates that could be identified at the species level using MALDI-TOF MS. An improved log score was obtained for 51.4% (72/140) of the strains. For strains with a sequence similarity <98.7% with their closest relative (n = 5) or with an inconclusive sequence identity (n = 4), no identification was obtained by MALDI-TOF MS or in the latter case an identity with one of its relatives. For some species the MSP of the type strain was not part of the confined cluster of the corresponding clinical isolates. Also, not all species formed a homogeneous cluster. It emphasizes the necessity of adding sufficient MSPs of human clinical isolates.

Anaerococcus nagyae sp. nov., isolated from human clinical specimens.

We describe a new Anaerococcus species isolated from human clinical specimens. Analyses of 16S rRNA gene sequences of three strains showed <98% similarity with its closest relative Anaerococcus octavius. Phylogenetically the isolated strains form a cluster and can be differentiated from other species of the genus Anaerococcus based on its phenotypic characteristics and its MALDI-TOF MS profile. We propose the name Anaerococcus nagyae, with A. nagyae DSM101193 (accession number KU043522) as the type strain.

Anaerococcus degenerii sp. nov., isolated from human clinical specimens.

Four clinical isolates of gram-positive strict anaerobic cocci were isolated from four different human mixed anaerobic infections. The taxonomical status of the four strains could not be established using standard identification techniques. The biochemical features of the strains were established and their taxonomic position was determined using 16S rRNA sequencing. The four strains form a homogeneous phenotypical and genotypical cluster. A new Anaerococcus species is proposed for these isolates: Anaerococcus degenerii sp. nov. The type strain is UMCG-104(T) = DSM29674(T) (accession number AM176528).

Antibiotic susceptibility profiles of anaerobic pathogens in The Netherlands.

The antibiotic susceptibility profile of the Bacteroides fragilis group, Gram-positive anaerobic cocci (GPAC), Fusobacterium spp., Prevotella spp., Veillonella spp. and Bilophila wadsworthia for amoxicillin, amoxicillin-clavulanic acid, clindamycin and metronidazole was determined. Human clinical isolates were isolated between 2011 and 2013 at the Microbiological Diagnostic Laboratory of the University Medical Center Groningen, The Netherlands and subjected to MALDI-TOF MS identification and susceptibility testing using E-test for MIC determination. Differences in clindamycin susceptibility between species of the B. fragilis group and GPAC were observed, with Bacteroides ovatus and Peptoniphilus harei having the highest resistance rates. Compared to other European countries, in The Netherlands the MIC90 for clindamycin of fusobacteria is low. Metronidazole resistance was first encountered in the genus Prevotella in 2013, but not in species of GPAC as reported in Belgium and Bulgaria. The differences in clindamycin resistance between the different European countries and reports of metronidazole resistance within the genera Prevotella and GPAC warrant more extensive susceptibility studies on anaerobic pathogens.

The influence of incubation time, sample preparation and exposure to oxygen on the quality of the MALDI-TOF MS spectrum of anaerobic bacteria.

With matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), bacteria can be identified quickly and reliably. This accounts especially for anaerobic bacteria. Because growth rate and oxygen sensitivity differ among anaerobic bacteria, we aimed to study the influence of incubation time, exposure to oxygen and sample preparation on the quality of the spectrum using the Bruker system. Also, reproducibility and inter-examiner variability were determined. Twenty-six anaerobic species, representing 17 genera, were selected based on gram-stain characteristics, growth rate and colony morphology. Inter-examiner variation showed that experience in the preparation of the targets can be a significant variable. The influence of incubation time was determined between 24 and 96 h of incubation. Reliable species identification was obtained after 48 h of incubation for gram-negative anaerobes and after 72 h for gram-positive anaerobes. Exposure of the cultures to oxygen did not influence the results of the MALDI-TOF MS identifications of all tested gram-positive species. Fusobacterium necrophorum and Prevotella intermedia could not be identified after >24 h and 48 h of exposure to oxygen, respectively. Other tested gram-negative bacteria could be identified after 48 h of exposure to oxygen. Most of the tested species could be identified using the direct spotting method. Bifidobacterium longum and Finegoldia magna needed on-target extraction with 70% formic acid in order to obtain reliable species identification and Peptoniphilus ivorii a full extraction. Spectrum quality was influenced by the amount of bacteria spotted on the target, the homogeneity of the smear and the experience of the examiner.

Evaluation of three selective media for isolation of Aggregatibacter actinomycetemcomitans.

BACKGROUND AND OBJECTIVE: Aggregatibacter actinomycetemcomitans is a pathogen in oral and nonoral infections. Detection and quantification of this pathogen can be performed using selective culture techniques. The aim of this study was to establish the efficacy of two known selective media in their ability to select and support the growth of A. actinomycetemcomitans. MATERIAL AND METHODS: Trypticase soy bacitracin vancomycin (TSBV) medium and brain-heart infusion agar with vancomycin (Dentaid-1), as well as a modified Dentaid-1 medium (in which the brain-heart infusion agar was substituted with brain-heart infusion broth), were compared. Two-hundred and eighteen clinical samples were used to establish the recovery rate, the number of colony-forming units (CFUs) of A. actinomycetemcomitans as well as the total number of CFUs on the three different types of medium. In addition, the numbers of gram-negative aerobic rods and yeasts were determined. RESULTS: Both types of Dentaid-1 medium showed a higher recovery of A. actinomycetemcomitans compared with TSBV. However, these differences did not reach statistical significance. The total number of CFUs of A. actinomycetemcomitans recovered was significantly higher on Dentaid-1 compared with TSBV (p = 0.029). The mean number of gram-negative aerobic rods recovered was statistically higher on both types of Dentaid-1 medium in comparison with TSBV. Low numbers of yeasts were recovered occasionally on all test plates. CONCLUSION: Dentaid-1 is a low-cost effective alternative to TSBV for the isolation and growth of A. actinomycetemcomitans from clinical samples, such as dental plaque, which contain a complex microflora.

Antibiotic susceptibility profiles of oral pathogens.

Periodontitis is a bacterial disease that can be treated with systemic antibiotics. The aim of this study was to establish the antibiotic susceptibility profiles of five periodontal pathogens to six commonly used antibiotics in periodontics. A total of 247 periodontal bacterial isolates were tested for susceptibility to the six antibiotics using the Etest method. MIC(50) and MIC(90) values (minimum inhibitory concentrations for 50% and 90% of the organisms, respectively) were calculated. Both European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) breakpoints were used in the study to interpret results. ß-Lactamase production was tested when amoxicillin resistance was found. MIC(90) values of the anaerobic bacteria were all well below breakpoint values, except for three isolates of Prevotella intermedia and one isolate of Fusobacterium nucleatum that were resistant to amoxicillin (CLSI breakpoints); these isolates were ß-lactamase-positive. Two isolates of the capnophilic Aggregatibacter actinomycetemcomitans appeared to be amoxicillin-resistant but failed to show ß-lactamase activity. Comparison with a previous study from The Netherlands showed minor differences in susceptibility profiles, but the MIC(90) values of A. actinomycetemcomitans for amoxicillin, clindamycin, azithromycin and tetracycline were higher. Geographical differences in the susceptibility profiles of Porphyromonas gingivalis and A. actinomycetemcomitans between European countries were noted. Comparison of European susceptibility profiles with that of a South American country (Colombia) revealed a much higher resistance in the latter. Owing to these differences in susceptibility profiles, it is of concern to regularly perform surveillance studies on antibiotic resistance.

Assessment of the microbiota of a mixed infection of the tongue using phenotypic and genotypic methods simultaneously and a review of the literature.

We assessed the microbiota of a tongue abscess in which twelve different aerobic and anaerobic bacteria were identified using fluorescent in situ hybridisation (FISH), sequencing of the 16S rRNA gene and phenotypic methods. By applying the 16S rRNA based probes directly on the clinical material, a quick insight of the bacteria present was obtained and the species which were not cultured but present in the abscess were identified.

The identification of anaerobic bacteria using MALDI-TOF MS.

Matrix Assisted Laser Desorption and Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has gained more and more popularity for the identification of bacteria. Several studies show that bacterial diagnosticis is being revolutionized by the application of MALDI-TOF MS. For anaerobic bacteria, MALDI-TOF MS has been used for the identification of Prevotella spp., Fusobacterium spp., Clostridium spp., Bacteroides spp. and Gram-positive anaerobic cocci. However, to identify bacteria reliably, an extensive database is essential. For routine identification of anaerobic bacteria available databases need to be optimised.