Yann Dumont
email: y-dumont@chu-montpellier.fr

Lucas Bonzon

Anne-Laure Michon

Christian Carriere

Marie-Noëlle Didelot

Chrislène Laurens

Beatrice Renard

Alida C M Veloo

Sylvain Godreuil

Hélène Jean-Pierre

Epidemiology and microbiological features of anaerobic bacteremia in two French University hospitals

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Anaerobic bacteria are implicated in 2 to 6% of bacteremia episodes [START_REF] Tan | Clinical characteristics and antimicrobial susceptibilities of anaerobic bacteremia in an acute care hospital[END_REF][START_REF] De Keukeleire | Anaerobic bacteraemia: a 10-year retrospective epidemiological survey[END_REF][START_REF] Kim | Anaerobic bacteremia: impact of inappropriate therapy on mortality[END_REF][START_REF] Ng | Anaerobic bacteraemia revisited: species and susceptibilities[END_REF]. Conflicting data have been published on their incidence (increase, decrease or stability) during the last decades [START_REF] De Keukeleire | Anaerobic bacteraemia: a 10-year retrospective epidemiological survey[END_REF][START_REF] Fenner | Is the incidence of anaerobic bacteremia decreasing? Analysis of 114,000 blood cultures over a ten-year period[END_REF][START_REF] Lassmann | Reemergence of anaerobic bacteremia[END_REF][START_REF] Lazarovitch | Decrease in anaerobe-related bacteraemias and increase in Bacteroides species isolation rate from 1998 to 2007: A retrospective study[END_REF]. Mortality caused by bacteremia implicating anaerobic bacteria (BIAB) is high, from 13 to 25% [START_REF] Tan | Clinical characteristics and antimicrobial susceptibilities of anaerobic bacteremia in an acute care hospital[END_REF][START_REF] De Keukeleire | Anaerobic bacteraemia: a 10-year retrospective epidemiological survey[END_REF][START_REF] Urbán | Five-year retrospective epidemiological survey of anaerobic bacteraemia in a university hospital and rewiew of the literature[END_REF][START_REF] Vena | Are incidence and epidemiology of anaerobic bacteremia really changing?[END_REF][START_REF] Ngo | Population-based assessment of the incidence, risk factors, and outcomes of anaerobic bloodstream infections[END_REF], and even higher in patients who are inappropriately treated [START_REF] Kim | Anaerobic bacteremia: impact of inappropriate therapy on mortality[END_REF]. They should especially be taken into account in centers specialized in cancer treatment [START_REF] Tan | Clinical characteristics and antimicrobial susceptibilities of anaerobic bacteremia in an acute care hospital[END_REF][START_REF] De Keukeleire | Anaerobic bacteraemia: a 10-year retrospective epidemiological survey[END_REF][START_REF] Kim | Anaerobic bacteremia: impact of inappropriate therapy on mortality[END_REF][START_REF] Ng | Anaerobic bacteraemia revisited: species and susceptibilities[END_REF][START_REF] Vena | Are incidence and epidemiology of anaerobic bacteremia really changing?[END_REF]. Antimicrobial resistance in BIAB remains rare: most of anaerobic bacteria are susceptible to metronidazole, to the association of amoxicillin and clavulanic acid, and to penems [START_REF] Tan | Clinical characteristics and antimicrobial susceptibilities of anaerobic bacteremia in an acute care hospital[END_REF][START_REF] Ng | Anaerobic bacteraemia revisited: species and susceptibilities[END_REF][START_REF] Ngo | Population-based assessment of the incidence, risk factors, and outcomes of anaerobic bloodstream infections[END_REF][START_REF] Lee | Multicenter study of antimicrobial susceptibility of anaerobic bacteria in Korea in 2012[END_REF][START_REF] Wang | Trends in the susceptibility of commonly encountered clinically significant anaerobes and susceptibilities of blood isolates of anaerobes to 16 antimicrobial agents, including fidaxomicin and rifaximin, 2008-2012, northern Taiwan[END_REF]. Resistance to clindamycin and fluoroquinolones has been more frequently reported, although about half of the isolates are still susceptible [START_REF] Tan | Clinical characteristics and antimicrobial susceptibilities of anaerobic bacteremia in an acute care hospital[END_REF][START_REF] Ng | Anaerobic bacteraemia revisited: species and susceptibilities[END_REF][START_REF] Lee | Multicenter study of antimicrobial susceptibility of anaerobic bacteria in Korea in 2012[END_REF][START_REF] Wang | Trends in the susceptibility of commonly encountered clinically significant anaerobes and susceptibilities of blood isolates of anaerobes to 16 antimicrobial agents, including fidaxomicin and rifaximin, 2008-2012, northern Taiwan[END_REF].

Importantly, a clone of multi-resistant Bacteroides fragilis has been recently described in Europe [START_REF] Sóki | Emergence and evolution of an international cluster of MDR Bacteroides fragilis isolates[END_REF]. The most frequent sources of BIAB are intra-abdominal and female genital tract infections [START_REF] Brook | The role of anaerobic bacteria in bacteremia[END_REF]. Multiple conditions have been associated with higher risk of BIAB, such as solid and hematological malignancies and surgery of the gastro-intestinal tract or the urogenital system [START_REF] Brook | The role of anaerobic bacteria in bacteremia[END_REF].

BIAB incidence in different centers has not been compared in recent studies. This could be a limit in epidemiology studies because the combination and frequency of local risk factors could influence their incidence and spread. Therefore, the aim of our study was to compare BIAB incidence, their microbial epidemiology, and the resistance to antibiotics of the involved anaerobic bacteria in two different French hospitals.

Material and methods

Blood cultures were received from two different hospital centers: Montpellier University Hospital (MUH), a training hospital of 2000 beds, and the Institute of Cancer in Montpellier (ICM), a center dedicated to cancer treatment with 210 beds.

For every positive blood culture identified in our bacteriology laboratory between 1 June 2015 and 31 December 2016, the following data were retrieved: patients' demographic data (anonymized at the first step of data analysis except birth date), hospital service where the patient was hospitalized at sampling time, and results of the identification and antibacterial susceptibility testing (AST) assays of the isolated bacteria. AST data for anaerobic isolates (categorization in term of susceptibility and minimum inhibitory concentrations (MIC)) were retrieved from the SIRWEB sofware database.

From these data, all putative bacteremia episodes were determined for each patient and each bacterial species, using a bioinformatics pipeline. Briefly, a 5-day interval between two positive blood cultures was chosen to define two separate bacteremia episodes. For each episode, only the species with i) at least one positive blood culture for aerobic and anaerobic species that are known to be systematic pathogens in blood cultures (e. g., Staphylococcus aureus, Enterobacterales, Bacteroides spp., etc.), ii) at least two positives blood cultures for an aerobic species known as possible contaminant and with comparable AST results (i.e., coagulase-negative Staphylococcus or Corynebacterium spp.), or iii) the presence of more than one positive blood culture for an anaerobic species known as probable contaminant (e.g., Cutibacterium acnes) were retained (see supplementary data to see which species are associated to which part of the algorithm). Fungi were excluded. Each bacteremia episode was associated with the service where the earliest positive blood culture was sampled.

Episodes occurring in patients younger than 16 years old were automatically excluded.

During this period, blood cultures were always concomitantly performed in BacT/Alert FA (aerobic) and FN Plus (anaerobic) bottles, containing antimicrobial-absorbing beads. Bottles were incubated and screened using the BacT/ALERT 3D system. Aerobic bottles were incubated for 5 days (standard conditions), or for 10 and 21 days when fungemia and endocarditis, respectively, were suspected. Anaerobic bottles were incubated in standard conditions for 7 days, or for 21 days when endocarditis was suspected. For each positive blood culture, the bacteria retrieved after subculture were identified Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) using a Bruker Microflex system (Bruker Daltonics, Bremen, Germany) and the Bruker database. The European Network for the Rapid Identification of Anaerobes (ENRIA) database was also interrogated when needed [START_REF] Veloo | ENRIA workgroup, Validation of a for anaerobic bacteria optimized MALDI-TOF MS biotyper database: The ENRIA project[END_REF]. AST was performed using the disk diffusion method according to the recommendations by the "Comité de l'Antibiogramme de la Société Française de Microbiologie" (Antibiogram Committee of the French Microbiology Society; CA-SFM) for aerobic bacteria (version 2015) and for anaerobic bacteria (version 2013). For anaerobes, AST was carried out only for species with known possible resistance (mostly Bacteroides) and in specific contexts. The MIC values were determined using strips for amoxicillin-clavulanic acid, cefoxitin, metronidazole and tigecyclin. Resistance for other antibiotics was tested using discs: piperacillin 75 µg, piperacillin 75 µg + tazobactam 10 µg, imipenem 10 µg, moxifloxacin 5 µg, rifampicin 30 µg and clindamycin 2 UI. For each genus and species, and for each antibiotic, the number of AST, the percentage of susceptible isolates, and when possible their MIC50, MIC90 and MIC range were calculated. Multiple resistances (i.e., association of acquired resistance against at least three categories of antibiotics, adapted from the definitions for Enterobacterales (former Enterobacteriaceae) and Pseudomonas aeruginosa [START_REF] Magiorakos | Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance[END_REF]) in the same isolate were verified manually. Categories of molecules used were penicillins + β-lactams inhibitors (amoxicillin-clavulanic acid, piperacillin-tazobactam), cephamycins (cefoxitin), and carbapenems (imipenem), fluoroquinolones (moxifloxacin), ansamycins (rifampicin), lincosamides (clindamycin), nitroimidazole derivatives (metronidazole), and cyclins (tigecyclin). Statistical analyses were performed with the scipy package v1.3.0, in python 3.6.

The scripts used are published on GitHub (DOI 10.5281/zenodo.3601471). The different dataset can be requested to the corresponding, upon motivated request.

Results

Among the 82 463 records of blood cultures retrieved, 2 465 episodes of bacteremia were identified, including 144 BIAB episodes (5.8%). Bacteremia was poly-microbial in 301 episodes (12.2%), including 46 episodes involving at least one anaerobe (31.5% of all BIAB), and 13 episodes involving only anaerobes (9.0% of all BIAB episodes). Poly-microbial bacteremia was significantly associated with the presence of anaerobes (p<0.001, chi² test).

Overall, the mean number of bacteria species isolated in each episode was 2.29, and increased to 2.61 for BIAB episodes (range: 1 to 6 and 1 to 5, respectively).

The number of bacteremia episodes was 2040 at MUH and 425 at ICM among which 4.9% (n=100) and 10.4% (n=44) were BIAB, respectively (p<0.001; chi² test). In hospital services with more than 10 episodes of bacteremia, BIAB frequency ranged from 0 to 27.3% (mean: 4.1%) at MUH and from 4.2 to 19.2% (mean: 10.8%) at ICM. At MUH, the highest BIAB frequency was detected in services specialized in abdominal surgery (including an intensive care unit) and at the clinical hematology department. At ICM, the highest frequency was observed in the intensive care unit and in the two surgical departments.

Among the blood cultures, 26 different genera and 56 different species of anaerobic bacteria were identified (table 1). The most frequently detected genera were Bacteroides (n=64), Clostridium (n=18), Lactobacillus (n=13), and Eggerthella (n=13). The most frequently observed anaerobic species were Bacteroides fragilis (37 episodes), Bacteroides thetaiotaomicron (n=15), and Eggerthella lenta (n=13). The B. fragilis group (BFG) was detected in 68 episodes (46.6% of all BIAB episodes). All the identified Bacteroides species belonged to the BFG.

AST was performed in 106 isolates. Metronidazole was the most frequently tested antibiotic, and the only tested antibiotic in 20 isolates (18.9%). Only one isolate (B. fragilis) was resistant to metronidazole. The AST results for metronidazole are summarized in table 2. Sixty-seven isolates with AST belonged to the BFG (AST was not performed on 3 isolates).

The results of these isolates are summarized in table 3. AST was not realized in 3 cases.

Globally, isolates from the BFG were highly susceptible to piperacillin-tazobactam, imipenem, rifampicin and metronidazole (susceptibility rates of 97.0%, 98.5%, 97.0%, and 98.5%, respectively). Results for amoxicillin-clavulanic acid, cefoxitin and tigecyclin were slightly lower (92.5%, 90.8%, and 91.0%, respectively). Five of the six isolates non-susceptible to cefoxitin were identified as B. thetaiotaomicron. Isolates were inconstantly susceptible to piperacillin, moxifloxacin and clindamycin (50.7%, 58.2% and 50.8%, respectively).

Multi-drug resistance was detected in seven Bacteroides isolates (6.6% of all anaerobic bacteria isolates with AST, and 10.4% of all Bacteroides with AST; table 4). Multi-drug resistance was mostly due to acquired resistance to moxifloxacin (n=7/7), clindamycin (n=6/7), and tigecyclin (n=4/7). Some isolates had paradoxical results: susceptible to amoxicillin-clavulanic acid and piperacillin-tazobactam and intermediate to imipenem, or susceptible to amoxicillin-clavulanic acid and resistant to piperacillin-tazobactam.

Discussion

This study found an important difference in BIAB frequency in two French hospital centers, and in the different services of each hospital (from 0% to 27.3%). Anaerobe presence in bacterial cultures related to bacteremia episodes was significantly associated with polymicrobial bacteremia. AST data analysis showed that resistance to the most frequently used anti-anaerobe antibiotics was rare, although a small fraction of multi-drug resistant isolates was detected.

The difference between hospital centers could easily be explained by the different patient populations treated. MUH is a multi-disciplinary teaching hospital in which some wards are specialized in cancer treatment, but others (such as rheumatology, neurology, and cardiology) display very few risk factors for BIAB. Conversely, ICM is a center specialized in cancer treatment. Therefore, the higher BIAB frequency at ICM could be explained by the fact that malignancies are one of the prominent risk factors for BIAB [START_REF] Urbán | Five-year retrospective epidemiological survey of anaerobic bacteraemia in a university hospital and rewiew of the literature[END_REF][START_REF] Brook | The role of anaerobic bacteria in bacteremia[END_REF]. Comparison of service-specific data shows higher frequency heterogeneity at MUH than ICM. This may be again explained by the difference in the incidence of risk factors among MUH services. This is also supported by the high BIAB frequency in some MUH services, sometimes higher than those observed at ICM. In this study, BIAB occurrence was reported as the percentage of bacteremia episodes with anaerobic bacteria among all bacteremia episodes. Other approaches have been used in other studies, such as the number of BIAB cases per year, per patient-days, per inhabitant or per admission; or the number of positive anaerobic cultures per sampled blood cultures; or the percentage of anaerobic bacteria isolated from blood cultures per bacteria [START_REF] De Keukeleire | Anaerobic bacteraemia: a 10-year retrospective epidemiological survey[END_REF][START_REF] Kim | Anaerobic bacteremia: impact of inappropriate therapy on mortality[END_REF][START_REF] Ng | Anaerobic bacteraemia revisited: species and susceptibilities[END_REF][START_REF] Fenner | Is the incidence of anaerobic bacteremia decreasing? Analysis of 114,000 blood cultures over a ten-year period[END_REF][START_REF] Lassmann | Reemergence of anaerobic bacteremia[END_REF][START_REF] Urbán | Five-year retrospective epidemiological survey of anaerobic bacteraemia in a university hospital and rewiew of the literature[END_REF][START_REF] Vena | Are incidence and epidemiology of anaerobic bacteremia really changing?[END_REF][START_REF] Ngo | Population-based assessment of the incidence, risk factors, and outcomes of anaerobic bloodstream infections[END_REF].

Our approach should be independent of the total number of patients, the length of hospital stay, and the frequency or indications of blood cultures because the number of BIAB episodes was normalized to the total number of bacteremia cases. The overall BIAB frequency at MUH was comparable to that reported by De Keukeleire et al. [START_REF] De Keukeleire | Anaerobic bacteraemia: a 10-year retrospective epidemiological survey[END_REF] in a Belgian university hospital. On the other hand, the overall frequency at ICM was much higher than that at those in other recent studies that were not performed in cancer-specific centers.

However, the main bias of our approach is the algorithm used to differentiate true bacteremia episodes from contaminations, and one episode from another. The number of bottles with the same bacteria and isolation of the same bacteria with the same antibiotic susceptibility pattern in multiple bottles are frequently used to detect false positives in blood cultures [START_REF] Lamy | How to optimize the use of blood cultures for the diagnosis of bloodstream infections? A state-of-the art[END_REF], but misinterpretation is always possible. In our study, antibiotic susceptibility patterns were used for aerobic bacteria because in our laboratory, AST of coagulase-negative staphylococci is systematically performed on two different isolates and the results are compared to conclude whether this is a true bacteremia or a contamination. However, episodes with different antibiotic susceptibility patterns are sometimes considered as real episodes by the medical team in charge, since no other explanation for the sepsis can be found. Contamination of multiple blood culture samples by the same strain is also possible, and may be falsely interpreted as a real bacteremia. Hopefully, these two error types are rare, and may cancel each other out on a large scale. An alternative way to determine whether a positive blood culture is due to a true bacteremia or a contamination would have been to use the time-to-positivity of the blood culture, which relates to the biomass present in the blood culture. However, the time-to-positivity depends on several factors e.g. time of transportation, volume of blood in each blood culture, antimicrobial therapy administered before sampling. These factors are not standardly recorded in the participating hospitals and are therefore unknown. Another bias in the algorithm is the interval between two positive blood cultures in order to consider it as two separate bacteremia episodes. The 5-day interval was chosen because all bacteremia episodes must be evaluated and treated rapidly. When treatment is not appropriate, other blood cultures may be performed within this interval. A longer period, on the other hand, could falsely create multi-bacterial episodes in the case of episodes occurring close together.

The frequency of poly-microbial bacteremia in the BIAB group observed in our study (31.9%) is comparable to that of other studies (from 12.9% to 38%) [START_REF] Tan | Clinical characteristics and antimicrobial susceptibilities of anaerobic bacteremia in an acute care hospital[END_REF][START_REF] De Keukeleire | Anaerobic bacteraemia: a 10-year retrospective epidemiological survey[END_REF][START_REF] Kim | Anaerobic bacteremia: impact of inappropriate therapy on mortality[END_REF][START_REF] Vena | Are incidence and epidemiology of anaerobic bacteremia really changing?[END_REF][START_REF] Ngo | Population-based assessment of the incidence, risk factors, and outcomes of anaerobic bloodstream infections[END_REF].

In our study, BIAB microbial epidemiology was similar to that of previous works. Bacteroides spp. was the most frequently implicated genus, followed by Clostridium spp. [START_REF] Tan | Clinical characteristics and antimicrobial susceptibilities of anaerobic bacteremia in an acute care hospital[END_REF][START_REF] De Keukeleire | Anaerobic bacteraemia: a 10-year retrospective epidemiological survey[END_REF][START_REF] Kim | Anaerobic bacteremia: impact of inappropriate therapy on mortality[END_REF][START_REF] Ng | Anaerobic bacteraemia revisited: species and susceptibilities[END_REF][START_REF] Vena | Are incidence and epidemiology of anaerobic bacteremia really changing?[END_REF]. The distribution of the different species included in the BFG was identical to what published previously: B. fragilis was the most frequent, followed by B. thetaiotaomicron, P. distasonis, and then B. ovatus and B. vulgatus [START_REF] Tan | Clinical characteristics and antimicrobial susceptibilities of anaerobic bacteremia in an acute care hospital[END_REF][START_REF] De Keukeleire | Anaerobic bacteraemia: a 10-year retrospective epidemiological survey[END_REF][START_REF] Kim | Anaerobic bacteremia: impact of inappropriate therapy on mortality[END_REF][START_REF] Umemura | Clinical characteristics associated with mortality of patients with anaerobic bacteremia[END_REF]. This distribution is similar to that of the BFG species in infections described by Nagy et al., except for P. distasonis, but this was attributed by the authors to a taxonomic change [START_REF] Nagy | Antimicrobial susceptibility of Bacteroides fragilis group isolates in Europe: 20 years of experience[END_REF]. Cutibacterium frequency was as low as in the study by Vena et al., where this genus was only considered when more than two samples were positive, because it is considered to be a frequent blood culture contaminant [START_REF] Vena | Are incidence and epidemiology of anaerobic bacteremia really changing?[END_REF]. On the other hand, Lactobacillus spp. was more frequently isolated compared to other studies.

The ENRIA MALDI-TOF database allowed us to identify other genera unfrequently found in BIAB, such as Tissierella praeacuta. It should be noticed that the recovery of anaerobic bacteria differs between the different blood culture systems. With each system, some species are more easily retrieved, resulting in distribution differences [START_REF] Almuhayawi | The performance of the four anaerobic blood culture bottles BacT/ALERT-FN, -FN Plus, BACTEC-Plus and -Lytic in detection of anaerobic bacteria and identification by direct MALDI-TOF MS[END_REF][START_REF] Mueller-Premru | Performance of two blood culture systems to detect anaerobic bacteria. Is there any difference?[END_REF]. This is particularly true for Finegoldia magna, rarely described in the literature, and C. acnes [START_REF] Mueller-Premru | Performance of two blood culture systems to detect anaerobic bacteria. Is there any difference?[END_REF]. E. lenta detection has been reported by Bact/ALERT users [START_REF] Kim | Anaerobic bacteremia: impact of inappropriate therapy on mortality[END_REF][START_REF] Ngo | Population-based assessment of the incidence, risk factors, and outcomes of anaerobic bloodstream infections[END_REF][START_REF] Simmon | Genotypic diversity of anaerobic isolates from bloodstream infections[END_REF] as in our study, but not by users of the BACTEC system.

AST was mostly performed on isolates identified as belonging to the BFG (63% of AST assays), due to its frequency of isolation in BIAB (46.6%). As this group is known for its high antibiotic resistance rates [START_REF] Schuetz | Antimicrobial resistance and susceptibility testing of anaerobic bacteria[END_REF], this could have led to a distortion of the results toward higher resistance rates. Yet, these rates remained low for the most prevalent anti-anaerobic bacteria antibiotics (piperacillin-tazobactam, metronidazole, imipenem), and are comparable with those of other studies, even though they were performed in Asia [START_REF] Tan | Clinical characteristics and antimicrobial susceptibilities of anaerobic bacteremia in an acute care hospital[END_REF][START_REF] Ng | Anaerobic bacteraemia revisited: species and susceptibilities[END_REF][START_REF] Lee | Multicenter study of antimicrobial susceptibility of anaerobic bacteria in Korea in 2012[END_REF][START_REF] Wang | Trends in the susceptibility of commonly encountered clinically significant anaerobes and susceptibilities of blood isolates of anaerobes to 16 antimicrobial agents, including fidaxomicin and rifaximin, 2008-2012, northern Taiwan[END_REF]. The breakpoints used in our study are those of the CA-SFM version 2013 that have not been updated since the adoption of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) methods. EUCAST (v8.1) only proposes breakpoints for anaerobic bacteria (the same as those used by CA-SFM 2013), but has none for cefoxitin, tigecyclin, moxifloxacin and rifampicin. Therefore, our AST results should be interpreted with caution while waiting for new guidelines for anaerobic bacteria AST.

In the BFG, the frequency of susceptibility to clindamycin (50.8%) was equivalent to that of previous studies on anaerobic bacteria [START_REF] Tan | Clinical characteristics and antimicrobial susceptibilities of anaerobic bacteremia in an acute care hospital[END_REF][START_REF] Ng | Anaerobic bacteraemia revisited: species and susceptibilities[END_REF][START_REF] Lee | Multicenter study of antimicrobial susceptibility of anaerobic bacteria in Korea in 2012[END_REF][START_REF] Wang | Trends in the susceptibility of commonly encountered clinically significant anaerobes and susceptibilities of blood isolates of anaerobes to 16 antimicrobial agents, including fidaxomicin and rifaximin, 2008-2012, northern Taiwan[END_REF], but lower than that published for European collections in other contexts [START_REF] Nagy | Antimicrobial susceptibility of Bacteroides fragilis group isolates in Europe: 20 years of experience[END_REF][START_REF] Mayne | In vitro activity of tigecycline and comparators against organisms associated with intra-abdominal infections collected as part of TEST (2004-2009)[END_REF][START_REF] Rodloff | In vitro activity of tigecycline and comparators against a European collection of anaerobes collected as part of the Tigecycline Evaluation and Surveillance Trial (T.E.S.T.)[END_REF]. This could be due to the increasing prevalence of clindamycin resistance in anaerobic bacteria in Europe [START_REF] Rodloff | In vitro activity of tigecycline and comparators against a European collection of anaerobes collected as part of the Tigecycline Evaluation and Surveillance Trial (T.E.S.T.)[END_REF]. The BFG susceptibility to moxifloxacin was low (58.2%), as described in the literature [START_REF] Genzel | Activity of the investigational fluoroquinolone finafloxacin and seven other antimicrobial agents against 114 obligately anaerobic bacteria[END_REF]. A small percentage of isolates were non-susceptible to tigecyclin, as previously reported [START_REF] Rodloff | In vitro activity of tigecycline and comparators against a European collection of anaerobes collected as part of the Tigecycline Evaluation and Surveillance Trial (T.E.S.T.)[END_REF].

Seven multi-drug resistant BFG isolates were found in this study. None had a resistance profile compatible with the clone described by Sóki et al. (most were susceptible to piperacillin-tazobactam or imipenem, and all to metronidazole), thus cfiA and nim genes may not be present, or not expressed [START_REF] Sóki | Emergence and evolution of an international cluster of MDR Bacteroides fragilis isolates[END_REF]. In our study, multi-drug resistance was mostly due to resistance to moxifloxacin and clindamycin, associated with resistance to at least another antibiotic. Major anti-anaerobic bacteria antibiotics were not affected in these isolates. The definition of multi-drug resistance for anaerobic bacteria used in our study was interpolated from that for aerobic bacteria proposed by Magiorakos et al.: "acquired non-susceptibility to at least one agent in three or more antimicrobial categories" [START_REF] Magiorakos | Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance[END_REF]. This definition has a major pitfall, as resistance to moxifloxacin and clindamycin in the BFG is frequent (58.2% and 50.8%, respectively), the cumulative probability of having at least one resistance within any of the six other categories of antibiotics is high (28.0% in our dataset, independently of the results for moxifloxacin and clindamycin), inflating the percentage of multi-drug resistant isolates, although these isolates remains susceptible to numerous antibiotics. Thus, criteria for multi-drug resistance in Bacteroides should be clearly established with differences between major (for example metronidazole, imipenem) and minor (moxifloxacin, clindamycin) molecules. Moreover, a new definition for extensively drug-resistant Bacteroides should be created.

To conclude, BIAB frequency was nearly two-fold higher at ICM, although some MUH services had higher frequencies. This underlines the need for clinicians to take into account the factors leading to an higher risk of BIAB, which can be a deadly condition [START_REF] Tan | Clinical characteristics and antimicrobial susceptibilities of anaerobic bacteremia in an acute care hospital[END_REF][START_REF] De Keukeleire | Anaerobic bacteraemia: a 10-year retrospective epidemiological survey[END_REF][START_REF] Urbán | Five-year retrospective epidemiological survey of anaerobic bacteraemia in a university hospital and rewiew of the literature[END_REF][START_REF] Vena | Are incidence and epidemiology of anaerobic bacteremia really changing?[END_REF][START_REF] Ngo | Population-based assessment of the incidence, risk factors, and outcomes of anaerobic bloodstream infections[END_REF]. In vitro susceptibility of the implicated anaerobic bacteria remains high, therefore empirical antimicrobial therapy against anaerobic bacteria should be effective. The development of multi-drug resistance is a concern because these molecules are mainly broad-spectrum antibiotics. Therefore, AST must be performed as frequently as possible, particularly for isolates belonging to the BFG, and new strategies should be developed to reduce the interval between blood culture positivity and AST results. (0.7) AB anaerobic bacteria, BIAB bacteriemia involving anaerobic bacteria. a Genera are in bold. When only one species was isolated in a genus, lines were merged. Genus and species in a genus are ordered from the most to the least frequent. "sp" species were either not specified in the ENRIA database, or results were not sufficient to discriminated among species. b As multiple species of the genus could be found in a BIAB episode, the sum of the occurences of all species from a genus can be higher than the occurence of that genus. .032-8 NA: not applicable: the percentage of susceptible results was not calculated for n=1, and MIC50 and MIC90 were not calculated for n<5. AMC, amoxicillin-clavulanic acid; AST, antibiotic susceptibility testing; CLI, clindamycin; CXI, cefoxitin; IMI imipenem; MIC, minimum inhibitory concentration; MOX, moxifloxacin; MET, metronidazole; PIP, piperacillin; PIT, piperacillin-tazobactam; RIF, rifampicin; TIG, tigecycline.

Four

  isolates showed intermediate susceptibility to amoxicillin-clavulanic acid (n=1 B. fragilis, n=1 Dysgonomonas capnocytophagoides, and n=2 B. thetaiotaomicron), and two Parabacteroides distasonis isolates were resistant. One B. thetaiotaomicron was resistant to piperacillin-tazobactam and two isolates (n=1 Bacteroides ovatus and n=1 Veillonella parvula) showed intermediate susceptibility. Susceptibility to imipenem was intermediate in three isolates (n=1 B. thetaiotaomicron and n=2 Lactobacillus rhamnosus).

Table 1 : Anaerobic bacteria found in bacteriemia episodes involving anaerobic bacteria

 1

	(n=144)

Table 2 : Results of antibiotic susceptibility testing of metronidazole

 2

		Metronidazole AST results
		(breakpoint > 4 mg/L)
	Genus and species of anaerobic bacteria isolated in blood cultures	n susceptible/n tested (%)	MIC50 MIC90 MIC range or value
	Bacteroides	61/62 (98.4)	.5	1.5	.064-6
	-fragilis	34/35 (97.1)	.38	1	.094-6
	-thetaiotaomicron	13/13 (100)	.75	1.25	.25-2
	-vulgatus	5/5 (100)	.38	1.5	.064-2
	-ovatus	4/4 (100)	NA	1.5	.5-2
	-uniformis	3/3 (100)	NA	NA	.19-.5
	-finegoldii	1/1 (NA)	NA	NA	1.5
	-nordii	1/1 (NA)	NA	NA	.19
	Clostridium	11/11 (100)	.25	1.5	<.016-2
	-ramosum	2/2 (100)	NA	NA	.125-.75
	-tertium	2/2 (100)	NA	NA	.5-.75
	-cadaveris	2/2 (100)	NA	NA	.047-.047
	-clostridioforme	1/1 (NA)	NA	NA	<.016
	-perfringens	2/2 (100)	NA	NA	1.5-2
	-glycolicum	1/1 (NA)	NA	NA	.032
	-innocuum	1/1 (NA)	NA	NA	1.5
	Fusobacteriumnucleatum	1/1 (NA)	NA	NA	.016
	Prevotella	3/3 (100)	NA	NA	.019-.75
	-buccae	2/2 (100)	NA	NA	.19-.75
	-melaninogenica	2/2 (100)	NA	NA	.5-.5
	Parabacteroides distasonis	5/5 (100)	1.5	3	.75-4
	Veillonella	2/2 (100)	NA	NA	.75-1
	-parvula	1/1 (NA)	NA	NA	.75
	-sp	1/1 (NA)	NA	NA	1
	Dialister pneumosintes	1/1 (NA)	NA	NA	2
	Porphyromonas asaccharolytica	1/1 (NA)	NA	NA	.032
	Ruminococcus gnavus	1/1 (NA)	NA	NA	.064
	Tissierella praeacuta	1/1 (NA)	NA	NA	.064
	TOTAL	87/88 (98.9)	.5	1.5	<.016-6
	NA: not applicable: the percentage of susceptible results was not calculated for n=1, and MIC50 and MIC90 were not
	calculated for n<5.
	AST antibiotic susceptibility typing.

Table 3 : Results of antibiotic susceptibility testing for Bacteroides fragilis group's isolates Results of AST per antibiotic

 3

		Number of susceptible results/total number of results (percentage of susceptible results) ± MIC50, MIC90 and range for
							MIC/value
	Genus and species of
	anaerobic bacteria isolated in	AMC (MIC)		PIP	PIT		CXI (MIC)		IMI
	blood cultures
	Breakpoints (CASFM 2013)	S ≤ 4 mg/L ; R > 8 mg/L	R > 18 mm	S ≥ 21 mm R < 19 mm	R > 32 mg/L		S ≥ 24 mm R < 17 mm
	Bacteroides	59/62 (95.2) .75 4 .094-8 32/62 (51.6) 60/62 (96.8) 54/60 (90.0)	8 32 1.5->256 61/62 (98.4)
	-fragilis	34/35 (97.1) .5 3 .094-6 22/35 (62.9) 35/35 (100)	33/34 (97.1)	6 24 1.5->256 35/35 (100)
	-thetaiotaomicron	11/13 (84.6) 2	4	.25-8	4/13 (30.8) 12/13 (92.3)	8/13 (61.5)	32 64	12-64	12/13 (92.3)
	-vulgatus	5/5 (100)	1	3 .094-4	2/5 (40.0)	5/5 (100)	5/5 (100)	4 16	2-16	5/5 (100)
	-ovatus	4/4 (100) NA NA .19-2	3/4 (75.0)	3/4 (75.0)	4/4 (100)	NA NA	12-24	4/4 (100)
	-uniformis	3/3 (100) NA NA .25-3	1/3 (33.3)	3/3 (100)	3/3 (100)	NA NA	2-3	3/3 (100)
	-finegoldii	1/1 (NA)	NA NA	2	0/1 (NA)	1/1 (NA)	1/1 (NA)	NA NA	6	1/1 (NA)
	-nordii	1/1 (NA)	NA NA	1.5	0/1 (NA)	1/1 (NA)	1/1 (NA)	NA NA	32	1/1 (NA)
	Parabacteroides distasonis	3/5 (60.0) 2 16	1-24	2/5 (40.0)	5/5 (100)	5/5 (100)	24 24	16-24	5/5 (100)
	TOTAL	62/67 (92.5) 1	4 .094-24 34/67 (50.7) 65/67 (97.0) 59/65 (90.8)	8 32 1.5->256 66/67 (98.5)

Table 3 : Results of antibiotic susceptibility testing for Bacteroides fragilis group's isolates (continued)

 3 (61.5) 12/13 (92.3) 4/13 (30.8) 13/13 (100) .75 1 .25-2 11/13 (84.6) .

	Results of AST per antibiotic

Table 4 : Antibiotic susceptibility testing of multi-drug resistant Bacteroides fragilis group's isolates Species AMC PIT CXI IMI MOX RIF CLI MET TIG N. of families with resistance

 4 -clavulanic acid; PIT, piperacillin-tazobactam; CXI, cefoxitin; IMI, imipenem; MOX, moxifloxacin; RIF, rifampicin; CLI clindamycin; MET, metronidazole; TIG, tigecycline.

	B. thetaiotaomicron	S	S	R	I	R	R R	S	S	5
	B. fragilis	I	S	S	S	R	S R	S	I	4
	B. thetaiotaomicron	I	S	R	S	R	S	S	S	I	4
	B. thetaiotaomicron	I	S	R	S	R	S R	S	S	4
	B. thetaiotaomicron	S	R	S	S	I	S R	S	S	3
	B. fragilis	S	S	S	S	R	S R	S	I	3
	B. thetaiotaomicron	S	S	S	S	R	S R	S	I	3
	AMC, amoxicillin