Céline Plainvert

Erika Matuschek

Nicolas Dmytruk

Marine Gaillard

Amandine Frigo

Yassine Ballaa

Eddy Biesaga

Gunnar Kahlmeter

Claire Poyart

Asmaa Tazi
email: asmaa.tazi@aphp.fr.

Microbiological Epidemiology of Invasive Infections Due to Non-Beta-Hemolytic Streptococci, France, 2021

Keywords: non-beta-hemolytic streptococci, viridans streptococci, streptococcal infections, antibiotic susceptibility, beta-lactam resistance, epidemiology Streptococcus salivarius, and Streptococcus sanguinis groups

Non-beta-hemolytic streptococci (NBHS), also referred to as viridans streptococci, represent an underestimated cause of human invasive diseases. Their resistance to antibiotics, including beta-lactam agents, often complicate their therapeutic management. A prospective multicenter study was conducted by the French National Reference Center for Streptococci between March and April 2021 to describe the clinical and microbiological epidemiology of invasive infections due to NBHS, excluding pneumococcus. A total of 522 NBHS invasive cases were collected. Distribution among streptococcal groups was: Streptococcus anginosus (33%), Streptococcus mitis (28%), Streptococcus sanguinis (16%), Streptococcus bovis/equinus (15%), Streptococcus salivarius (8%), and Streptococcus mutans (,1%). Median age of infection was 68 years old (range ,1 day to 100 years). Cases were more frequent in male patients (gender ratio M/F 2.1:1) and manifested mainly as bacteremia without focus (46%), intra-abdominal infections (18%) and endocarditis (11%). All isolates were susceptible to glycopeptides and displayed low-level inherent gentamicin resistance. All isolates of the S. bovis/equinus, S. anginosus, and S. mutans groups were susceptible to beta-lactams. Conversely, nonsusceptibility to beta-lactams was found in 31%, 28%, and 52% of S. mitis, S. salivarius, and S. sanguinis isolates, respectively. The screening for beta-lactam resistance using the recommended one unit benzylpenicillin disk screening failed to detect 21% of resistant isolates (21/99). Last, overall resistance rates to the alternative anti-streptococcal molecules clindamycin and moxifloxacin were 29% (149/522) and 1.6% (8/505), respectively.

IMPORTANCE NBHS are recognized as opportunistic pathogens particularly involved in infections of the elderly and immunocompromised patients. This study underlines their importance as common causes of severe and difficult-to-treat infections such as endocarditis. Although species of the S. anginosus and S. bovis/equinus groups remain constantly susceptible to beta-lams, resistance in oral streptococci exceeds 30% and screening techniques are not fully reliable. Therefore, accurate species identification and antimicrobial susceptibility testing by MICs determination appears essential for the treatment of NBHS invasive infections, together with continued epidemiological surveillance.

and can be responsible for a wide range of diseases [START_REF] Facklam | What happened to the streptococci: overview of taxonomic and nomenclature changes[END_REF][START_REF] Doern | It's not easy being green: the viridans group streptococci, with a focus on pediatric clinical manifestations[END_REF]. Streptococcus mutans and Streptococcus sobrinus are commonly associated with dental caries and odontogenic infections [START_REF] Abranches | Biology of oral streptococci[END_REF]. Besides, NBHS are a frequent cause of bacteremia in immunocompromised patients resulting from underlying malignancy or cancer therapy, ranging from the first to the fifth cause of bloodstream infections in neutropenic patients with hematological malignancies and solid tumors, and accounting for 8% to 26% of the cases [START_REF] Marin | Bloodstream infections in neutropenic patients with cancer: differences between patients with haematological malignancies and solid tumours[END_REF][START_REF] Gudiol | Changing aetiology, clinical features, antimicrobial resistance, and outcomes of bloodstream infection in neutropenic cancer patients[END_REF][START_REF] Carvalho | Bloodstream infections in neutropenic patients with haematological malignancies[END_REF]. NBHS, mainly of the S. bovis/equinus, S. mitis, S. salivarius, and S. sanguinis groups, are also reported as the first or second cause of infective endocarditis worldwide, accounting for approximately 20% of the cases [START_REF] Abranches | Biology of oral streptococci[END_REF][START_REF] Murdoch | International Collaboration on Endocarditis-Prospective Cohort Study (ICE-PCS) Investigators. 2009. Clinical presentation, etiology, and outcome of infective endocarditis in the 21st century: the International Collaboration on Endocarditis-Prospective Cohort Study[END_REF][START_REF] Ambrosioni | Hospital Clinic Infective Endocarditis Investigators. 2017. The changing epidemiology of infective endocarditis in the twenty-first century[END_REF][START_REF] Selton-Suty | Preeminence of Staphylococcus aureus in infective endocarditis: a 1-year population-based survey[END_REF]. Additionally, isolates of the S. anginosus group are causative agents of bacteremia, pyogenic infections, and abscesses in immunocompetent adults and children [START_REF] Pilarczyk-Zurek | The clinical view on Streptococcus anginosus groupopportunistic pathogens coming out of hiding[END_REF]. Similar to group A and group B beta-hemolytic streptococci (Streptococcus pyogenes and Streptococcus agalactiae, respectively), an increasing incidence of S. anginosus group bacteremia has been reported over the past decades, reaching 3.7 cases per 100,000 inhabitants in 2017 in Canada [START_REF] Laupland | Streptococcus anginosus group bloodstream infections in the western interior of British Columbia, Canada[END_REF]. Infections occur in the absence of any underlying condition or comorbidity in approximately 50% of the cases, are usually polymicrobial mainly in association with obligate anaerobes, and involve various sites, including the abdomen, lower and upper respiratory tract, and the brain [START_REF] Kobo | The pyogenic potential of the different Streptococcus anginosus group bacterial species: retrospective cohort study[END_REF][START_REF] Furuichi | Sites of infection associated with Streptococcus anginosus group among children[END_REF]. Among the three species which constitute this group (S. anginosus, Streptococcus intermedius, and Streptococcus constellatus), S. anginosus is the most commonly isolated in both bloodstream infections and abscesses (50% to 70% of the cases) [START_REF] Kobo | The pyogenic potential of the different Streptococcus anginosus group bacterial species: retrospective cohort study[END_REF][START_REF] Furuichi | Sites of infection associated with Streptococcus anginosus group among children[END_REF][START_REF] Jiang | Clinical characteristics of infections caused by Streptococcus anginosus Group[END_REF].

Beta-lactam antibiotics remain the first-line antibiotics for NBHS invasive infections [START_REF] Habib | 2015 ESC Guidelines for the management of infective endocarditis: the Task Force for the Management of Infective Endocarditis of the European Society of Cardiology (ESC)[END_REF]. Nonetheless, penicillin-resistant isolates were described as early as the 1960s [START_REF] Naiman | Penicillin-resistant bacteria in the mouths and throats of children receiving continuous prophylaxis against rheumatic fever[END_REF][START_REF] Sprunt | Penicillin resistant alpha Streptococci in pharynx of patients given oral penicillin[END_REF] and increasing rates of resistance are reported since the 1980s [START_REF] Doern | It's not easy being green: the viridans group streptococci, with a focus on pediatric clinical manifestations[END_REF][START_REF] Doern | Emergence of high rates of antimicrobial resistance among viridans group streptococci in the United States[END_REF][START_REF] Chun | Species-specific difference in antimicrobial susceptibility among viridans group streptococci[END_REF]. As in Streptococcus pneumoniae, the loss of susceptibility results from mutations in the genes encoding the penicillin-binding proteins which alter their affinity for one or more betalactam molecules. Resistance rates greatly vary between streptococcal groups with penicillin nonsusceptible isolates reaching rates as high as 35% in the S. mitis and S. sanguinis groups while remaining extremely rare in the S. anginosus group [START_REF] Chun | Species-specific difference in antimicrobial susceptibility among viridans group streptococci[END_REF][START_REF] Sader | Ceftaroline activity tested against viridans group streptococci from US hospitals[END_REF][START_REF] Chagneau | Screening for b-lactam resistance by penicillin G in the Streptococcus anginosus group challenged by rare strains with altered PBPs[END_REF]. Thus, resistance to beta-lactam antibiotics is very common in certain streptococcal species and frequently complexifies the therapeutic management of difficult-to-treat infections such as bone and joint infections or infective endocarditis.

Several antibiotics can be used as alternative or adjunctive therapy to beta-lactams in case of allergy, resistance, or in difficult-to-treat infections. These antibiotics include macrolides, lincosamides, and streptogramins (MLS), rifampicin, linezolid, and glycopeptides. Whereas NBHS resistance to MLS, together with that to beta-lactams, is an evolving problem, linezolid and glycopeptides remain usually active [START_REF] Chun | Species-specific difference in antimicrobial susceptibility among viridans group streptococci[END_REF][START_REF] Ergin | Macrolide resistance mechanisms and in vitro susceptibility patterns of viridans group streptococci isolated from blood cultures[END_REF].

Altogether, the epidemiology of NBHS invasive infections is most often studied in specific clinical settings or by considering a single species or group of species. Besides, large studies of NBHS susceptibility to antibiotics appear relatively scarce. Here, we report at the species level the epidemiology of more than 500 NBHS invasive infections, with the exclusion of pneumococcal infections. We describe for each NBHS group the clinical manifestations and the susceptibility to antibiotics, including five beta-lactam agents, MLS, and glycopeptides using disk diffusion (DD), gradient tests, and broth microdilution (BMD).

RESULTS

Microbiological epidemiology of NBHS invasive infections. Invasive isolates of NBHS were prospectively referred to the French National Reference Centre for Streptococci (Strep-NRC, https://cnr-strep.fr) by correspondents located throughout France. A total of 522 invasive NBHS was reported between March and April 2021. All isolates were identified by mass spectrometry except 27 (5.2%) which required sodA sequencing [START_REF] Poyart | Identification of streptococci to species level by sequencing the gene encoding the manganese-dependent superoxide dismutase[END_REF]. Eventually, 22 different species were identified, each of them consisting of one to 97 isolates (Table S1). The 10 most prevalent species accounted for 85% of cases and included, in the S. anginosus group, S. anginosus (97; 18.6%), S. constellatus (53; 10.2%), and S. intermedius (22; 4.2%); in the S. mitis group, Streptococcus oralis (83; 15.9%), and S. mitis (38; 7.3%); in the S. sanguinis group, S. sanguinis (36; 6.9%), and Streptococcus parasanguinis (29; 5.6%); in the S. bovis/equinus group, Streptococcus gallolyticus (30; 5.7%), and Streptococcus pasteurianus (24; 4.6%); and in the S. salivarius group, S. salivarius (30; 5.7%).

The overall gender ratio M/F was of 2.1:1 and the median age was 68 years old (Table 1). Thirty-two cases (6.1%) were pediatric. Bacteremia without focus (n = 241, 46.2%), intra-abdominal infections (n = 95; 18.2%), endocarditis (n = 58; 11.1%), pulmonary infections (n = 51; 9.2%), and bone and joint infections (n = 26; 5.0%) accounted for 90% of infections. The clinical manifestations varied significantly according to NBHS groups (Fig. 1; Table 1), isolates from the S. anginosus group being overrepresented in Susceptibility to beta-lactams. The MICs of benzylpenicillin (PEN), amoxicillin (AMX), cefotaxime (CTX), and ceftriaxone (CRO) were determined by BMD and gradient Etest for all isolates except 17 S. intermedius isolates for which MICs could only be measured by Etest because of insufficient growth in Muller-Hinton for Fastidious organisms (MH-F) broth. The agreement between BMD and Etest is shown in Fig. S1. The overall categorical agreement (average for all tested agents) was $ 94.9% (Table S2). A significant bias between the two techniques was observed for CTX and CRO for which MICs were lower with Etest in 24% and 67% of the isolates, respectively.

For the sake of clarity, only the BMD results of beta-lactam susceptibility are presented below, to which the Etest results of the 17 S. intermedius isolates with insufficient growth in MH-F broth are added. All isolates from the S. anginosus, S. bovis/equinus, and S. mutans groups were susceptible to PEN (Fig. 2) and to all the beta-lactam agents tested (Table 2). Conversely, 31.1% (46/148), 28.2% (11/39), and 51.9% (42/81) of the isolates from the S. mitis, S. salivarius, and S. sanguinis groups showed intermediate susceptibility or resistance to at least one beta-lactam, respectively (Fig. 3; Table 2). The different resistant phenotypes are shown in Table S3 and varied from resistance to a single beta-lactam agent to cross-resistance to all the beta-lactam agents tested. Overall, CTX remained the most frequently active agent, with resistance rates (MIC . 0.5 mg/L) of 18.2%, 2.5%, and 13.6% in the S. mitis, S. salivarius, and S. sanguinis group, respectively.

Screening for beta-lactam resistance using disk diffusion. The screening for beta-lactam resistance is based on DD testing of a 1-unit PEN disk where isolates showing an inhibition zone diameter $ 21 mm (European Committee on Antimicrobial Susceptibility Testing [EUCAST] Breakpoint Tables v. [START_REF] Furuichi | Sites of infection associated with Streptococcus anginosus group among children[END_REF].0) can be categorized as susceptible to beta-lactam agents. We analyzed the performances of the screening for beta-lactam resistance with BMD as the reference method using this breakpoint and the one corresponding to the study-period ($18 mm, EUCAST Breakpoint Tables v. 12.0). Using the 18 mm breakpoint (Fig. 4), 40/505 isolates (7.9%) were falsely categorized as susceptible to beta-lactams. The sensitivity, specificity, positive, and negative predictive values for the detection of beta-lactam resistance were 59.6%, 100%, 100%, and 91.0%, respectively, with the lowest performances for the S. sanguinis group (Table S4). The failure to detect beta-lactam resistance was observed for a variety of phenotypes, including cross-resistance to all tested molecules (Table S5). Changing the breakpoint to 21 mm improved the detection of beta-lactam resistance, lowering false susceptibility to 4.2% (21/505) isolates and increasing sensitivity to 78.8% (Table S4 to S6).

Susceptibility to macrolides, lincosamides, and streptogramins. Susceptibility to MLS was studied using complementary methods based on DD and BMD. Susceptibility to clindamycin was measured by DD and BMD and inducible resistance to MLS was detected by DD according to EUCAST guidelines. Moreover, in the absence of specific European guidelines, susceptibility to macrolides was studied according to the guidelines of the French Society for Microbiology [START_REF]Comité de l'Antibiogramme de la Société Française de Microbiologie[END_REF]. The results of both methods showed some discrepancies regarding clindamycin susceptibility (Table 3; Fig. S2), as 28.5% of isolates (149/522) were found resistant using DD compared to 18.6% using BMD (94/ 505). Given that all resistant phenotypes observed by DD were confirmed at the genotypic level, DD testing was considered as the reference method for MLS susceptibility testing. Hence, the detection of MLS resistance using clindamycin MIC alone by BMD showed poor sensitivity, leading to very major errors in 9.9% of cases (50/505).

The resistance rates to MLS ranged from 30.8% in the S. anginosus group (53/172) to 68.8% in the S. bovis/equinus group (53/78). Resistant phenotypes and genotypes were differentially distributed among NBHS groups, the constitutive resistance to macrolides, lincosamides, and streptogramins B (cMLS B phenotype) being dominant in the S. anginosus and S. bovis/equinus groups (79% and 57%, respectively), whereas the sole resistance to macrolides (M phenotype) was the most frequent in the S. mitis, S. salivarius, and S. sanguinis groups (62%, 71%, and 57%, respectively; Table 3). Resistance to lincosamides and combined resistance to lincosamides and streptogramins A (L/LS A phenotype) was frequent among MLS resistant isolates of the S. anginosus and S. bovis/equinus groups (17% and 42%, respectively). Susceptibility to other antibiotics. Table 4 and Fig. S2 summarize antimicrobial susceptibility testing by BMD to the other antibiotics. All the isolates displayed intrinsic lowlevel resistance to gentamicin with MICs # 32 mg/L and were susceptible to vancomycin and teicoplanin (Table 4 and data not shown). Dalbavancin MIC distribution was modal and ranged from # 0.004 to 0.125 mg/L, in contrast to that of rifampicin which was bimodal and ranged from #0.008 to $8 mg/L. Only 48.1% of the S. bovis/equinus isolates were reported as wild-type considering the EUCAST v 12.0 ECOFF at 0.125 mg/L. However, a one dilution-higher ECOFF at 0.25 mg/L (EUCAST v 13.0) categorizes all isolates but 11 (2.1%) as wild-type. Susceptibility to moxifloxacin was assessed by BMD and all but 8 isolates (1.6%) were reported as wild-type (ECOFF at 0.5 mg/L).

The frequency of tetracycline resistance, interpreted by DD according to the French guidelines in the absence of specific EUCAST guidelines [START_REF]Comité de l'Antibiogramme de la Société Française de Microbiologie[END_REF], varied according to the streptococcal group, ranging from 7.7% in the S. salivarius group (3/39) to 70.1% in the S. bovis/ equinus group (54/77) (Table 5). In the S. mitis, S. salivarius, and S. sanguinis groups, resistance was conferred almost exclusively by tet(M) whereas other determinant such as tet(L) and tet(O) were more common in the S. anginosus and S. bovis/equinus groups (Table 5).

Overall, multidrug-resistant isolates, defined as non-wild-type toward three or more classes of antibiotics, amounted to 4.8% (25/505), and included isolates that combined beta-lactam, clindamycin and tetracycline resistance.

Analysis of NBHS isolates collected in 2022. The specific study conducted in 2021 allowed us to collect and analyze more than 500 NBHS isolates. We sought to update these results through the analysis of the isolates sent on a regular basis to the Strep-NRC in 2022. A total of 79 NBHS invasive isolates were collected, which were addressed mainly for species identification and confirmation of specific resistance phenotypes. Despite the small size of this collection and the recruitment biases, we compared their characteristics to those of the 2021 collection.

The 79 isolates included 26 isolates of the S. anginosus group (33%), 21 isolates of the S. bovis/equinus (27%) and the S. mitis group (27%), seven isolates of the S. sanguinis group (8%), and four isolates of the S. salivarius group (5%). Their distribution and clinical characteristics seem similar to the 2021 collection (Table S7). All isolates of the S. anginosus and S. bovis/equinus groups remained susceptible to beta-lactam antibiotics as attested by gradient tests (Table S8). Isolates of the S. mitis group displayed a higher rate of resistance than in 2021 (14/21, 67%) but most of the isolates were sent for confirmation of their resistance phenotype, some of them displaying particularly high MICs (.32 mg/L) for all tested molecules. Last, similar rates and patterns of resistance to MLS (Table S9) and tetracycline (Table S10) were observed between 2021 and 2022 for the different streptococcal groups. Resistance rates of the S. salivarius and S. sanguinis groups were not interpretable because of the small size of the collection.

DISCUSSION

In this prospective survey, we studied more than 600 NBHS invasive isolates in France, including (i) an unbiased collection of 522 isolates responsible for invasive infections in 2021 from an ad hoc study conducted by the Strep-NRC and (ii) a collection of 79 isolates representing the total NBHS invasive isolates sent to the Strep-NRC by its correspondents in 2022 on a voluntary and routine basis, mainly for confirmation of species identification and antibiotic resistance phenotype. We gained a precise picture of NBHS infections and antimicrobial susceptibility at the species level for which the main findings are summarized below. The vast majority of cases occurred in the elderly, most often in male patients. Bacteremia without focus, intra-abdominal infections, and endocarditis accounted for 75% of the cases. Although all viridans streptococcal groups were involved, 85% of the cases were caused by not more than 10 species, with significant associations between specific clinical manifestations and NBHS groups. Susceptibility to antimicrobial agents was highly variable among NBHS isolates. Because the collection mode over the two study-periods was different, and because the isolates analyzed in the second study-period are very unlikely to be representative of the population of human infective isolates, analyzing the evolutionary trends regarding antibiotic susceptibility would not be reliable. Therefore, only the general features highlighted by the 2021 collection are discussed. Overall, whereas all isolates of the S. anginosus and S. bovis/equinus groups were susceptible to beta-lactams, intermediate susceptibility and resistance were encountered in approximately 30% isolates of the S. mitis and S. salivarius groups and in more than 50% isolates of the S. sanguinis group. Resistance to MLS was also very common among all streptococcal groups, always exceeding 30% of isolates. Comprehensive studies on NBHS invasive infections are scarce. Still, regarding the clinical characteristics, our study confirms the previously described associations between the S. anginosus group with abscesses and pleural fluid infections [START_REF] Pilarczyk-Zurek | The clinical view on Streptococcus anginosus groupopportunistic pathogens coming out of hiding[END_REF][START_REF] Kobo | The pyogenic potential of the different Streptococcus anginosus group bacterial species: retrospective cohort study[END_REF], together with the very high risk-association (.30%) between S. bovis/equinus and S. sanguinis groups bloodstream infections with endocarditis, illustrating the representativeness of our collection [START_REF] Claridge | Streptococcus intermedius, Streptococcus constellatus, and Streptococcus anginosus ("Streptococcus milleri group") are of different clinical importance and are not equally associated with abscess[END_REF][START_REF] Chamat-Hedemand | Prevalence of infective endocarditis in streptococcal bloodstream infections is dependent on streptococcal species[END_REF]. Our results also underline an increased susceptibility of male patients and of the elderly to NBHS infections, in agreement with specific reports on invasive S. anginosus group infections whose incidence increases with age and whose gender ratio usually exceeds 1.5 [START_REF] Laupland | Streptococcus anginosus group bloodstream infections in the western interior of British Columbia, Canada[END_REF][START_REF] Kobo | The pyogenic potential of the different Streptococcus anginosus group bacterial species: retrospective cohort study[END_REF][START_REF] Furuichi | Sites of infection associated with Streptococcus anginosus group among children[END_REF]. These observations can be linked to the higher prevalence of NBHS infections in adults suffering from underlying diseases which are overrepresented in the elderly [START_REF] Carvalho | Bloodstream infections in neutropenic patients with haematological malignancies[END_REF] and to the overrepresentation of male patients in endocarditis which might account for the overrepresentation of males in NBHS infective endocarditis. Nevertheless, the increased susceptibility of male patients was observed in all clinical manifestations of NBHS infections and whatever streptococcal group considered, reflecting similar gender and age susceptibility as that described for S. pneumoniae infections [START_REF] Giefing-Kröll | How sex and age affect immune responses, susceptibility to infections, and response to vaccination[END_REF].

Considering susceptibility to antibiotics, resistance rates to beta-lactams and MLS among NBHS species were similar to those reported elsewhere [START_REF] Chun | Species-specific difference in antimicrobial susceptibility among viridans group streptococci[END_REF][START_REF] Singh | Antibiotic susceptibility patterns of viridans group streptococci isolates in the United States from 2010 to 2020[END_REF][START_REF] Brenciani | Genetic determinants and elements associated with antibiotic resistance in viridans group streptococci[END_REF]. Resistance rates to beta-lactams of 30% were described in the United States among S. mitis isolates in a study including over 4,800 S. mitis, S. oralis, and nonspeciated NBHS isolates from bloodstream infections between 2010 and 2020 [START_REF] Singh | Antibiotic susceptibility patterns of viridans group streptococci isolates in the United States from 2010 to 2020[END_REF]. Resistance rates were stable over the 10-year study period. Marron et al. also found a ceftriaxone resistance rate over 30% in NBHS strains, including 80% S. mitis strains in isolates from blood cultures of neutropenic patients with cancer [START_REF] Kim | Antibiotic resistance of viridans group streptococci isolated from dental plaques[END_REF]. In agreement with our results, higher resistance rates among isolates of the S. salivarius and S. sanguinis groups were also previously described, reaching up to 70% isolates [START_REF] Kim | Antibiotic resistance of viridans group streptococci isolated from dental plaques[END_REF]. In contrast to these high rates of beta-lactam resistance, all strains remained susceptible to vancomycin as described by others and dalbavancin activity was in agreement with previous studies on nonspeciated NBHS isolates [START_REF] Singh | Antibiotic susceptibility patterns of viridans group streptococci isolates in the United States from 2010 to 2020[END_REF][START_REF] Kim | Antibiotic resistance of viridans group streptococci isolated from dental plaques[END_REF][START_REF] Marron | High rates of resistance to cephalosporins among viridans-group streptococci causing bacteraemia in neutropenic cancer patients[END_REF][START_REF] Sader | Antimicrobial activity of dalbavancin against Gram-positive bacteria isolated from patients hospitalized with bloodstream infection in United States and European medical centers (2018-2020)[END_REF]. In our study, none of the isolates displayed high-level resistance to gentamicin, also in agreement with other studies where high-level resistance rates were below 2% [START_REF] Kim | Antibiotic resistance of viridans group streptococci isolated from dental plaques[END_REF]. Concerning MLS and tetracycline, previous studies on NBHS isolates from diverse flora and streptococcal groups reported resistance as common (.10%), with the highest resistance rates among the S. bovis/equinus group [START_REF] Kobo | The pyogenic potential of the different Streptococcus anginosus group bacterial species: retrospective cohort study[END_REF][START_REF] Furuichi | Sites of infection associated with Streptococcus anginosus group among children[END_REF][START_REF] Singh | Antibiotic susceptibility patterns of viridans group streptococci isolates in the United States from 2010 to 2020[END_REF][START_REF] Brenciani | Genetic determinants and elements associated with antibiotic resistance in viridans group streptococci[END_REF][START_REF] Seppala | Antimicrobial susceptibility patterns and macrolide resistance genes of viridans group streptococci from normal flora[END_REF][START_REF] Pompilio | An overview on Streptococcus bovis/Streptococcus equinus complex isolates: identification to the species/subspecies level and antibiotic resistance[END_REF].

Our study emphasizes the critical impact of recommendations for the interpretation of antibiotic susceptibility testing and the efforts needed for their worldwide harmonization. France and most European countries, including Germany, the Netherlands, Norway, Sweden, United Kingdom, and others agreed to create common European guidelines known as the EUCAST guidelines. These are followed by many countries outside Europe such as Australia, Brazil, China, Morocco, and South Africa. On the other hand, the United States, Canada, and other countries follow the Clinical and Laboratory Standard Institute (CLSI) guidelines [START_REF] Cusack | Time to switch from CLSI to EUCAST? A Southeast Asian perspective[END_REF]. CLSI and EUCAST cooperate more and more frequently through dedicated study groups but have no common clinical breakpoint process and antibiotic susceptibility testing interpretation might be different when using one guideline or another. For instance, interpretation criteria for some antimicrobials might be missing in the EUCAST guidelines and present in the CLSI guidelines. In such cases, the French guidelines might refer to previous national guidelines or to the CLSI guidelines [START_REF]Performance standards for antimicrobial susceptibility testing[END_REF], which was the case in our study for the interpretation of NBHS susceptibility to erythromycin and tetracycline. Moreover, the clinical breakpoints and the screening methods for the detection of resistance might also differ, as exemplified by beta-lactam susceptibility testing for NBHS. In the EUCAST guidelines, screening for decreased susceptibility and resistance to beta-lactams relies on the PEN 1 unit disk, and we demonstrated that it was improved with the recent change from $18 to $21 mm in the EUCAST Breakpoint Tables. Conversely, the CLSI guidelines specify that NBHS isolated from normally sterile sites should be tested for PEN susceptibility using an MIC method. In our study, the screening for beta-lactam resistance using PEN 1 unit disk remained poorly sensitive despite the change introduced in the v 13.0 Breakpoint Table, also emphasizing the need for accurate testing by MIC measurements in invasive and difficult-to-treat infections due to NBHS.

Overall, this study provides a detailed analysis at the species level of the epidemiology of NBHS infections in France. Notable strengths are (i) the representativeness of our cohort, (ii) the exhaustive analysis of the isolates at the species level and the inclusion of a wide variety of antimicrobial agents for antibiotic susceptibility testing and (iii) the combination of multiple techniques for antibiotic susceptibility testing. First of all, the French Strep-NRC network includes more than 200 laboratories located throughout France, which brings a nationwide coverage to the study. Correspondents were asked to send all NBHS isolates isolated from normally sterile sites without any selection during a 2-month study period in 2021, in order to ensure maximum completeness of the collection. Second, all isolates were identified at the species level, assigned to a streptococcal group, and antibiotic susceptibility testing included 5 beta-lactam molecules, MLS, three glycopeptides, and four more antimicrobial agents. Last, antibiotic susceptibility testing was performed using complementary techniques, including BMD and gradient test for beta-lactams, and BMD and DD for MLS, thus reinforcing the robustness of the results. Besides, microbiological analyses were performed in a unique laboratory, discrepant results were controlled in two independent laboratories and resistance to MLS and tetracycline was confirmed by the identification of resistance genes. However, some limitations must be considered. The initial study was carried out over a short period of 2 months, which possibly introduced biases regarding the clinical epidemiology and the species collected, all the more so as it coincided with containment measures related to the COVID-19 epidemic in France. Notably, the number of isolates among the mutans group was very limited. We added to this study the isolates sent in 2022 by our correspondents on a routine basis, mainly for expertise regarding species identification and antibiotic resistance profiles, but the number of invasive isolates collected was very limited, disabling any comparison between the two periods. Considering antimicrobial susceptibility, the genetic supports of resistance to beta-lactams and moxifloxacin were not investigated, but will be the subject of future studies relying on whole-genome sequencing.

Altogether, this study highlights the wide spectrum of diseases caused by NBHS and the close relationship between specific groups, clinical manifestations, and antimicrobial resistance profiles. Comparing the resistance profiles and the genetic determinants between NBHS groups allowed us to identify both distinct and shared patterns that reflect common ecological niches and relationships with human and animal reservoirs, such as the high prevalence of resistance to beta-lactams and efflux-mediated resistance to macrolides in streptococcal species of the oral cavity (S. mitis, S. salivarius, and S. sanguinis groups) [START_REF] Brenciani | Genetic determinants and elements associated with antibiotic resistance in viridans group streptococci[END_REF][START_REF] Seppala | Antimicrobial susceptibility patterns and macrolide resistance genes of viridans group streptococci from normal flora[END_REF] and of cMLS B and L/LS A phenotypes in streptococcal species of the intestinal tract (S. anginosus and S. bovis/equinus groups) [START_REF] Pompilio | An overview on Streptococcus bovis/Streptococcus equinus complex isolates: identification to the species/subspecies level and antibiotic resistance[END_REF]. Importantly, our findings shed the light on the extremely high prevalence of resistance to beta-lactams in NBHS species of the oral cavity, which can furthermore display unusual phenotypes which might not be detected by benzylpenicillin testing alone. Therefore, accurate antibiotic susceptibility testing of beta-lactams by MICs measurements seems mandatory for the treatment of NBHS invasive infections. Besides, the possible emergency of beta-lactam resistance in NBHS of the intestinal tract and of multidrug-resistant isolates in NBHS requires a continuous surveillance of NBHS susceptibility to antimicrobials.

In conclusion, despite limitations mainly regarding the short 2-month study period, this work provides a first accurate overview of the epidemiology of NBHS invasive infections in France and will serve as a basis for future studies aiming at monitoring NBHS epidemiology. This work highlights the high level of beta-lactam nonsusceptibility in invasive streptococcal isolates belonging to the S. mitis, S. salivarius, and S. sanguinis groups, provides evidence of the diversity of antibiotic susceptibility patterns among NBHS, and emphasizes the need for a continuous surveillance of NBHS epidemiology and antibiotic susceptibility.

MATERIALS AND METHODS

Isolates and data collection. Invasive isolates of NBHS were prospectively referred to the French Strep-NRC (https://cnr-strep.fr) by correspondents located throughout France. Participants were asked to send all invasive NBHS (excluding pneumococci) isolated between March and April 2021. Besides, isolates are regularly sent on a routine basis by the correspondents for expertise, mainly for confirmation of species identification and study of antibiotic resistance. Clinical data (age, sex, clinical manifestations) were retrieved from questionnaires. Only isolates responsible for invasive infections, e.g., isolated from a normally sterile site, were considered.

Isolates were identified by MALDI-TOF mass spectrometry (Microflex; Bruker Daltonics, Bremen, Germany) using a local database enriched with NBHS spectra which includes 20, 30, and seven reference spectra for the difficult-to-identify species S. mitis, S. oralis, and S. pseudopneumoniae. Species confirmation was performed using sequencing of the sodA gene when mass spectrometry identification was ambiguous [START_REF] Poyart | Identification of streptococci to species level by sequencing the gene encoding the manganese-dependent superoxide dismutase[END_REF]. Isolates which could not be identified at the species level were excluded from the analysis.

Antibiotic susceptibility testing. MIC of PEN, AMP, AMX, CTX, CRO, gentamicin, clindamycin, rifampicin, moxifloxacin, vancomycin, and dalbavancin were determined by BMD for NBHS isolated between March and April 2021 using a custom microtiter plate (SWE1SPEC, Thermo Fisher Scientific, Basingstoke, UK) and in-house prepared broth in the form of MH-F according to EUCAST instructions [START_REF]Media preparation for EUCAST disk diffusion testing and for determination of MIC values by the broth microdilution method[END_REF]. MIC of PEN, AMX, CTX, and CRO were also determined by gradient tests (Etest, bioMérieux, Marcy-l' Etoile, France) on MH-F agar (bioMérieux, Marcy-l' Etoile, France) for all NBHS isolates. Susceptibility to PEN, AMP, gentamicin, erythromycin, clindamycin, tetracycline, vancomycin, and teicoplanin was also determined by DD (bioMérieux, Marcy-l' Etoile, France) according to EUCAST guidelines (http://www.eucast.org) for all NBHS isolates.

Quality controls using S. pneumoniae ATCC 49619 were performed weekly. Determinants for resistance to MLS and tetracyclines were detected by multiplex PCR [START_REF] Hays | Changing epidemiology of group B Streptococcus susceptibility to fluoroquinolones and aminoglycosides in France[END_REF][START_REF] Malhotra-Kumar | Multiplex PCR for simultaneous detection of macrolide and tetracycline resistance determinants in streptococci[END_REF].

Data interpretation and analysis. Antibiotic susceptibility testing results were interpreted according to EUCAST Breakpoint Tables v. [START_REF] Furuichi | Sites of infection associated with Streptococcus anginosus group among children[END_REF].0 unless otherwise specified [START_REF]European Committe on Antimicrobial Susceptibility Testing[END_REF][START_REF]European Committe on Antimicrobial Susceptibility Testing[END_REF]. When no breakpoints were available, data were interpreted according to epidemiological cut-off values (ECOFFs) or to the Committee on Antimicrobial Susceptibility Testing of the French Society for Microbiology (CA-SFM) [START_REF]Comité de l'Antibiogramme de la Société Française de Microbiologie[END_REF]. For beta-lactam susceptibility testing, results of Etests and DD were compared to reference BMD. Discrepant results and unusual phenotypes were double-checked in two independent laboratories. To allow comparison with BMD, Etest values were rounded up to the nearest 2-fold dilution. Very major errors, major errors, and minor errors were calculated as described by the Clinical and Laboratory Standards Institute [START_REF]Development of in vitro susceptibility testing criteria and quality control parameters[END_REF]. Overall categorical agreement against BMD reference MICs was evaluated for DD and Etest interpretations. Absolute and essential agreement, as well as the bias, were calculated for Etest according to ISO 20776-2 [START_REF]and in vitro diagnostic test systems -Susceptibility testing of infectious agents and evaluation of performance of antimicrobial susceptibility test devices -Part 2: evaluation of performance antimicrobial susceptibility test devices against reference broth micro-dilution[END_REF].

Statistical analysis. Categorical variables are expressed as numbers and percentages. Statistical analyses were performed using chi-square test. A P-value ,0.05 was regarded as significant.

FIG 1

 1 FIG 1 Distribution of NBHS groups according to the clinical manifestations of adult invasive infections. The proportion of the different NBHS groups according to the main clinical manifestations of invasive infections in adults are represented. Total number of cases: bacteremia without focus, n = 221; intraabdominal infections, n = 93; endocarditis, n = 56; pulmonary infections, n = 49, bone and joint infections, n = 26; skin and soft tissue infections, n = 20.

FIG 2

 2 FIG 2 Distribution of PEN MIC values within each NBHS group (n = 522). MIC values of PEN were determined by BMD for all strains except for 17 S. intermedius isolates for which the MIC to PEN was obtained by gradient Etest because of insufficient growth in MH-F broth. The black vertical dotted line indicates the breakpoint for susceptibility in the context of infective endocarditis (0.125 mg/L), and the orange and red dotted lines indicate the EUCAST v 13.0 clinical breakpoints (0.25 and 2 mg/L). CLSI, Clinical and Laboratory Standards Institute; EUCAST, European Committee on Antimicrobial Susceptibility Testing.

FIG 3

 3 FIG 3 Susceptibility to beta-lactam agents in streptococcal isolates of the mitis, salivarius, and sanguinis groups. Susceptibility to benzylpenicillin (PEN), amoxicillin (AMX), cefotaxime (CTX), and ceftriaxone (CRO) was measured by BMD. Results were interpreted according to EUCAST v13.0 clinical breakpoints. The number of isolates in each group was: S. mitis, n = 148; S. salivarius, n = 39; S. sanguinis, n = 81. EUCAST, European Committee on Antimicrobial Susceptibility Testing; S, susceptible; I, susceptible increased exposure; R, resistant.

FIG 4

 4 FIG 4 Screening for beta-lactam resistance using the PEN 1U diskpredictive value for resistance to PEN (A), AMP (B), AMX (C), CTX (D), and CRO (E) as measured by MIC values determined by BMD in 505 NBHS invasive isolates. Screening cut-off values as recommended by EUCAST and a breakpoint of 18 mm (EUCAST guidelines v 12.0, black dotted line) and 21 mm (EUCAST guidelines v 13.0, red dotted line). Critical concentrations: PEN, 0.25 to 2 mg/L; AMP/AMX, 0.5 to 2 mg/L; CTX/CRO, 0.5 mg/L.

TABLE 1

 1 Clinical characteristics of the 522 non-beta-hemolytic streptococci invasive infections Percentages are relative to the total isolates within each non-beta-hemolytic Streptococcus group.

	Streptococcus group	anginosus	bovis/equinus	mitis	mutans	salivarius	sanguinis	Total	P value
	Sex ratio M/F	1.9:1	2.2:1	1.7:1	4:1	3.3:1	2.4:1	2.1:1	ND
	Age, median	64 y	82 y	65 y	73 y	66 y	71 y	y	ND
	(min to max)	(2 m to 95 y)	(1 d h to 96 y)	(3 m to 100 y)	(26 y to 86 y)	(,1 d to 90 y)	(5 d to 94 y)	(,1 d to 100 y)
	Age, interquartile range, in years	51 to 73	69 to 85	51 to 76	26 to 76	55 to 72	57 to 81	to 79	ND
	Total, n (%)	172 (33.0%)	77 (14.8%)	148 (28.4%)	5 (1.0%)	39 (7.5%)	81 (15.5%)	(100%)	0.0023 f
	Adult cases ($18 y), n (%) a	165 (95.9%)	74 (96.1%)	135 (91.2%)	5 (100%)	34 (87.2%)	77 (95.1%)	(93.9%)	,0.0001 g
	Bacteremia without focus	57 (33.1%)	42 (54.5%)	68 (45.9%)	3 (60%)	16 (41.0%)	35 (43.2%)	(42.3%)	0.011
	Intra-abdominal infections	41 (23.8%)	11 (14.3%)	16 (10.8%)	0	10 (25.6%)	15 (18.5%)	(17.8%)	0.024
	Endocarditis	3 (1.7%)	12 (15.6%)	18 (12.2%)	2 (40%)	2 (5.1%)	19 (23.5%)	(10.7%)	,0.0001
	Pulmonary infections	26 (15.1%)	1 (1.3%)	14 (9.5%)	0	3 (7.7%)	5 (6.2%)	(9.4%)	0.010
	Bone and joint infections	13 (7.6%)	3 (3.9%)	7 (4.7%)	0	0	3 (3.7%)	(5.0%)	ND
	Skin and soft tissue infections	14 (8.1%)	2 (2.6%)	3 (2.0%)	0	1 (2.6%)	0	(3.8%)	ND
	Urinary tract infections b	4 (2.3%)	1 (1.3%)	3 (2.0%)	0	0	0	(1.5%)	ND
	Upper respiratory tract infections b	4 (2.3%)	0	4 (2.7%)	0	0	0	(1.5%)	ND
	Central nervous system infections	1 (0.6%)	1 (1.3%)	0	0	2 (5.1%)	0	(0.8%)	ND
	Others c	2 (1.2%)	1 (1.3%)	2 (1.4%)	0	0	0	(1.0%)	ND
	Neonates and infants (birth to #1 y),	1 (0.6%)	3 (3.9%)	2 (1.4%)	0	4 (10.3%)	1 (1.2%)	(2.1%)	ND
	n (%) a
	Early Onset Sepsis (0 to 3 d)	0	2 (2.6%)	0	0	2 (5.1%)	1 (1.2%)	(1.0%)	ND
	Late Onset Sepsis (4 to 89 d)	0	0	0	0	2 (5.1%)	0	(0.4%)	ND
	Others d	1 (0.6%)	1 (1.3%)	2 (1.4%)	0	0	0	(0.8%)	ND
	Pediatric cases (1 to 18 y), n (%) a	6 (3.5%)	0	11 (7.4%)	0	1 (2.6%)	3 (3.7%)	(4.0%)	ND
	Bacteremia without focus	1 (0.6%)	0	9 (6.1%)	0	1 (2.6%)	2 (2.5%)	(2.5%)	ND
	Others e	5 (2.9%)	0	2 (1.4%)	0	0	1 (1.2%)	(1.5%)	ND
	Including vascular-and catheter-related bloodstream infection (n = 3), endophthalmitis (n = 1), and upper-genital infection (n = 1).
	d Including pyelonephritis (n = 2), bacteremia without focus (n = 1), pulmonary infections (n = 1).
	e Including intra-abdominal infections (n = 2), endocarditis (n = 2), upper respiratory tract infections (n = 2), pulmonary infections (n = 1), and central nervous system infection (n = 1).

a b Bacteremia-associated infections. c f P-value for the distribution among adult, neonates and infants, and pediatric cases. g P-value for the distribution of clinical manifestations among adult cases. h d, days; m, months; y, years; ND, not determined. intra-abdominal (41/93 episodes, 44.1%) and pulmonary (26/49 episodes, 53.1%) infections, whereas isolates from the S. bovis/equinus, S. mitis, and S. sanguinis groups were overrepresented in endocarditis (12/56, 21.4%; 18/56, 32.1%; and 19/56, 33.9%, respectively).

TABLE 2

 2 Beta-lactam agents: susceptibility and MIC distributions of invasive non-beta-hemolytic Streptococcus isolates

	Streptococcus	Antimicrobial	MIC a (mg/L), n									MIC interpretation b , n (%)
	group (n; %)	agent	£0.008 0.016 0.032 0.064 0.125 0.25 0.5 1	2	‡4 S	I	R
	anginosus	Benzylpenicillin	13	23	18	92	26	0	0	0	0	0	172 (100%) 0	0
	(172; 33.0%)	Ampicillin c	4	3	17	31	51	47	2	0	0	0	155 (100%) 0	0
		Amoxicillin	4	6	19	40	56	45	2	0	0	0	172 (100%) 0	0
		Cefotaxime	0	4	10	13	41	95	9	0	0	0	172 (100%) NA d	0
		Ceftriaxone	0	2	6	11	27	54	72 0	0	0	172 (100%) NA	0
	bovis/equinus	Benzylpenicillin	0	0	2	55	19	1	0	0	0	0	77 (100%)	0	0
	(77; 14.8%)	Ampicillin	0	0	0	14	62	1	0	0	0	0	77 (100%)	0	0
		Amoxicillin	0	0	0	17	59	1	0	0	0	0	77 (100%)	0	0
		Cefotaxime	0	0	1	20	52	4	0	0	0	0	77 (100%)	NA	0
		Ceftriaxone	0	0	1	18	10	46	2	0	0	0	77 (100%)	NA	0
	mitis	Benzylpenicillin	12	17	28	14	15	20	11 9	5	17 106 (71.6%) 25 (16.9%) 17 (11.5%)
	(148; 28.4%)	Ampicillin	0	8	15	46	8	20	11 11 8	21 108 (73.0%) 19 (12.8%) 21 (14.2%)
		Amoxicillin	1	10	25	37	17	16	4	11 8	19 110 (74.3%) 19 (12.8%) 19 (12.8%)
		Cefotaxime	2	10	11	30	29	21	18 3	9	15 121 (81.8%) NA	27 (18.2%)
		Ceftriaxone	0	5	13	14	41	17	23 10 7	18 113 (76.4%) NA	35 (23.6%)
	mutans	Benzylpenicillin	0	2	3	0	0	0	0	0	0	0	5(100%)	0	0
	(5; 1.0%)	Ampicillin	0	0	0	5	0	0	0	0	0	0	5(100%)	0	0
		Amoxicillin	0	0	0	5	0	0	0	0	0	0	5(100%)	0	0
		Cefotaxime	0	0	2	3	0	0	0	0	0	0	5(100%)	NA	0
		Ceftriaxone	0	0	0	2	3	0	0	0	0	0	5(100%)	NA	0
	salivarius	Benzylpenicillin	0	0	1	8	1 0	1 0	2	6	1	1	2 9(74.4%)	9 (23.1%)	1 (2.6%)
	(39; 7.5%)	Ampicillin	0	1	0	5	1 0	9	6	5	2	1	3 1(79.5%)	7 (18.0%)	1 (2.6%)
		Amoxicillin	0	1	2	6	8	9	8	2	2	1	3 4(87.2%)	4 (10.3%)	1 (2.6%)
		Cefotaxime	1	0	6	8	9	8	6	1	0	0	3 8(97.4%)	NA	1 (2.6%)
		Ceftriaxone	0	0	1	9	9	6	9	4	1	0	3 4(87.2%)	NA	5 (12.8%)
	sanguinis	Benzylpenicillin	4	8	0	1	2 1	1 5	1 4 1 0 6	2	4 9(60.5%)	30 (37.0%) 2 (2.5%)
	(81; 15.5%)	Ampicillin	0	0	3	9	1	7	2 1 1 6 1 3 1 1 4 1(50.6%)	29 (35.8%) 11 (13.6%)
		Amoxicillin	0	0	0	10	3	9	20 20 14 5	42 (51.9%)	34 (42.0%) 5 (6.2%)
		Cefotaxime	0	1	11	20	13	16	9	9	1	1	70 (86.4%)	NA	11 (13.6%)
		Ceftriaxone	0	0	6	10	26	10	13 8	7	1	65 (80.2%)	NA	16 (19.8%)

a MICs determined by broth microdilution except in the case of 17 S. intermedius isolates (anginosus group) for which MICs could only be measured by gradient Etest because of insufficient growth in MH-F broth. b MICs interpretation according to EUCAST v 13.0. Susceptible isolates in shaded area, resistant isolates underlined. c Ampicilllin MICs were not determined for 17 S. intermedius isolates (anginosus group), neither by broth microdilution because of insufficient growth in MH-F broth nor by gradient Etest. d NA, not applicable; S, susceptible; I, susceptible increased exposure; R, resistant.

TABLE 3

 3 Macrolides, lincosamides and streptogramins (MLS): susceptibility, resistance phenotypes and genotypes of invasive non-beta-hemolytic Streptococcus isolates interpreted according to the guidelines on Antibiotic Susceptibility Testing of the French Society for Microbiology (CA-SFM).Minimum inhibitory concentrations of clindamycin were measured by BMD and interpreted according to EUCAST clinical breakpoint table v 13.0. S. intermedius isolates could not be tested by BMD but only by DD because of insufficient growth in MH-F broth. These included 5 MLS resistant isolates of which 4 cMLS B phenotypes and 1 L phenotype.Including erm (T) (5 in the bovis/equinus group), lnu (A) (2 in the anginosus group), lnu (D) (1 in the mitis group), lnu (E) (1 in the anginosus group), and lsa (B) (3 in the anginosus group and 1 in the sanguinis group).

	Streptococcus Resistant Disk diffusion phenotype a , n (%) Clinda Rb n (%) Resistance determinants, n (%) c	group isolates, n (%) L/lS A g M iMLS B cMLS B DD d BMD e mef erm(A) erm(B) lnu(C) lsa(A) Others f	anginosus (n = 172) 53 (30.8%) 9 (17.0%) 2 (3.8%) 0 (0%) 42 (79.2%) 51 (29.7%) 33 (21.3%) 3 (5.7%) 6 (11.3%) 35 (66.0%) 0 (0%) 9 (17.0%) 6 (11.3%)	bovis/equinus (n = 77) 53 (68.8%) 22 (41.5%) 0 (0%) 1 (1.9%) 30 (56.6%) 53 (68.8%) 20 (26.0%) 0 (0%) 3 (5.7%) 23 (43.4%) 11 (13.4%) 2 (3.8%) 5 (9.4%)	mitis (n = 148) 53 (35.8%) 2 (3.8%) 33 (62.3%) 2 (3.8%) 16 (30.2%) 20 (37.8%) 18 (34.0%) 38 (71.7%) 6 (11.3%) 11 (20.8%) 0 (0%) 0 (0%) 1 (1.9%)	mutans (n = 5) 0 NA NA NA NA 0 0 NA NA NA NA NA NA	salivarius (n = 39) 17 (43.6%) 0 (0%) 12 (70.6%) 0 (0%) 5 (29.4%) 5 (12.8%) 4 (10.3%) 13 (76.5%) 0 (0%) 5 (29.4%) 0 (0%) 0 (0%) 0 (0%)	sanguinis (n = 81) 46 (56.8%) 1 (2.2%) 26 (56.5%) 0 (0%) 19 (41.3%) 20 (24.7%) 19 (23.5%) 28 (60.9%) 4 (8.7%) 15 (32.6%) 0 (0%) 1 (2.2%) 1 (2.2%)	Total e (n = 522) 222 (42.5%) 34 (15.3%) 73 (32.9%) 3 (1.4%) 112 (50.7%) 149 (28.5%) 94 (18.6%) 82 (36.9%) 19 (8.6%) 89 (40.1%) 11 (5.0%) 12 (5.4%) 13 (5.9%)	A , iMLS B and cMLS B phenotypes.	g BMD, broth microdilution; Clinda R A , resistance to lincosamides and streptogramin A; M, efflux-mediated resistance to C14 and C15 macrolides; iMLSB, , clindamycin resistance; DD, disk diffusion; L, resistance to lincosamides; LS	inducible resistance to macrolides, lincosamides, and streptogramin B; cMLS

a DD phenotypes were b c Percentages are relative to total resistant isolates within each group. d Including the L/LS e A total of 17 f B , constitutive resistance to macrolides, lincosamides, and streptogramin B; NA, not applicable.

TABLE 4

 4 Non-beta-lactam agents: susceptibility and MIC range of invasive non-beta-hemolytic Streptococcus isolates

	Streptococcus	Antimicrobial	MIC (mg/L)	MIC interpretation (%)
	group (n, %)	agent	Range	S/wild-type a,b	R/non-wild-type a
	anginosus (155, 30.7%)	Dalbavancin	#0.004 to 0.064	100	0
		Moxifloxacin	#0.032 to 0.25	100	0
		Rifampicin	#0.008 to $8	96.1	3.9
		Vancomycin	0.25 to 1	100	0
	bovis/equinus (77, 15.2%)	Dalbavancin	0.008 to 0.125	NA	NA
		Moxifloxacin	0.125 to $8	97.4	2.6
		Rifampicin	0.032 to 0.25	100	0
		Vancomycin	0.25 to 0.5	100	0
	mitis (148, 29.3%)	Dalbavancin	#0.004 to 0.125	NA	NA
		Moxifloxacin	0.064 to 4	98.6	1.4
		Rifampicin	0.016 to $8	98.6	1.4
		Vancomycin	0.25 to 1	100	0
	mutans (5, 1.0%)	Dalbavancin	0.032 to 0.06	NA	NA
		Moxifloxacin	0.125	100	0
		Rifampicin	0.016 to 0.032	100	0
		Vancomycin	0.5 to 1	100	0
	salivarius (39, 7.7%)	Dalbavancin	#0.004 to 0.125	NA	NA
		Moxifloxacin	#0.032 to 0.5	100	0
		Rifampicin	0.032 to $8	89.7	10.3
		Vancomycin	0.25 to 1	100	0
	sanguinis (81, 16.0%)	Dalbavancin	#0.004 to 0.06	NA	NA
		Moxifloxacin	0.066 to $8	95.1	4.9
		Rifampicin	0.032 to $8	96.3	3.7
		Vancomycin	0.25 to 1	100	0

a Dalbavancin, and vancomycin were interpreted according to EUCAST clinical breakpoints v 13.0 (S or R). Moxifloxacin and rifampicin were interpreted as wild-type or nonwild-type in accordance with EUCAST ECOFFs (https://mic.eucast.org/; reviewed September 2022). b S, susceptible; R, resistant; NA, not applicable (no breakpoint).

TABLE 5

 5 Tetracyclines: susceptibility and resistance determinants of invasive non-beta-hemolytic Streptococcus isolates a

		Resistant	Resistance determinant, n (%) b
	Streptococcus group	isolates, n (%)	tet(M)	tet(L)	tet(O)	tet(T)	Unknown
	anginosus, n = 172	52 (30.2%)	42 (80.8%)	0 (0%)	7 (13.5%)	1 (1.9%)	2 (3.8%)
	bovis/equinus, n = 77	54 (70.1%)	37 (68.5%)	18 (33.3%)	16 (20.8%)	0 (0%)	0 (0%)
	mitis, n = 148	24 (16.2%)	23 (95.8%)	0 (0%)	1 (4.2%)	0 (0%)	0 (0%)
	mutans, n = 5	0 (0%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)
	salivarius, n = 39	3 (7.7%)	3 (100%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)
	sanguinis, n = 81	25 (30.9%)	24 (96.0%)	0 (0%)	0 (0%)	0 (0%)	1 (0%)
	Total, n = 522	158 (30.3%)	129 (81.6%)	18 (11.4%)	24 (15.2%)	1 (0.6%)	3 (1.9%)

a Interpreted according to the guidelines on Antibiotic Susceptibility Testing of the French Society for Microbiology. b Percentages are relative to total resistant isolates within each group. Non-Beta-Haemolytic Streptococcus Invasive Infections, France Microbiology Spectrum Month YYYY Volume XX Issue XX 10.1128/spectrum.00160-23

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Month YYYY Volume XX Issue XX 10.1128/spectrum.00160-23

[START_REF] Pilarczyk-Zurek | The clinical view on Streptococcus anginosus groupopportunistic pathogens coming out of hiding[END_REF].1128/spectrum.00160-23

ACKNOWLEDGMENTS

We thank all the correspondents of the French National Reference Center for Streptococci.

Part of this work has been presented at the 41st RICAI annual meeting, Paris, France and at the 32nd ECCMID annual meeting, Lisbon, Portugal.

This work was partially supported by EUCAST, Assistance Publique -Hôpitaux de Paris and Santé publique France.

We declare no conflicts of interest. Writingoriginal draft: A.T. and C.Pl.; writingreview and editing: A.T., C.Pl., G.K., and E.M.; conceptualization: A.T., C.Pl., and C.Po.; methodology: A.T., C.Pl., G.K., and E.M.; data acquisition: A.F., C.Pl., E.B., N.D., M.G., and Y.B.; data curation: A.F., C.Pl., N.D., and M.G.; supervision: A.T.; funding acquisition: A.T., C.Pl., and C.Po.


Tingqiang Yang
email: yang_tq@szu.edu.cn

Shuang Yang

Wei Jin

Yule Zhang
email: hzhang@szu.edu.cn

Nicolae Barsan

Anne Hemeryck

Wageh Swelm

Ahmed A Al-Ghamdi

Yueli Liu

Jing Zhou

Wen Chen
email: chenw@whut.edu.cn

Han Zhang

Prof Wen

Prof Han

Density Functional Investigation on Alpha-MoO3 (100): Amines Adsorption and Surface Chemistry

Keywords: DFT, α-MoO3 (100), amines adsorption, surface chemistry, gas sensing TOC Graph

The (100) surface of alpha-MoO3 (α-MoO3) should possess overwhelmingly more exposed Mo than the (010), and the exposed Mo has been extensively considered as active site for amine adsorption. However, α-MoO3 (100) has drawn little attention concerning amine sensing mechanism. In this research, adsorption of ammonia (NH3), monomethylamine (MMA), dimethylamine (DMA), and trimethylamine (TMA) is systematically investigated by density functional theory (DFT). All of these four molecules have high affinity to α-MoO3 (100) through interaction between the N and the exposed Mo, and the affinity is mainly influenced by number of methyl group and number of N-connected H. Adsorption and dissociation of water and oxygen molecule on stoichiometric or defective α-MoO3 (100) surface are then simulated to fully understand surface chemistry of α-MoO3 (100) in practical condition. At low temperature α-MoO3 (100) must be covered with lots of water molecule; the water can desorb or dissociate into hydroxyl groups at high temperature. Oxygen vacancy (VO) can be generated considering the annealing process during sensor device fabrication; VO must be filled with O2 molecule, which can further interact with adsorbed water nearby to form hydroxyl groups. According to this research, α-MoO3 (100) must be the active surface for amine sensing and its surface chemistry is well understood. In near future, further reaction and interaction will be simulated at α-MoO3 (100), and much more attention should be paid to α-MoO3 (100) not only theoretically but also experimentally.

Introduction

Alpha-MoO3 (α-MoO3) has drawn intensive attention in gas sensor mainly because of its outstanding sensing performance for amine gases, such as ammonia (NH3), trimethylamine (TMA) and triethylamine. [START_REF] Yang | High Sensitivity and Good Selectivity of Ultralong MoO3 Nanobelts for Trimethylamine Gas[END_REF][START_REF] Kwak | Molybdenum Trioxide (α-MoO3) Nanoribbons for Ultrasensitive Ammonia (NH3) Gas Detection: Integrated Experimental and Density Functional Theory Simulation Studies[END_REF][START_REF] Cho | Ultraselective and Ultrasensitive Detection of Trimethylamine Using MoO3 Nanoplates Prepared by Ultrasonic Spray Pyrolysis[END_REF][START_REF] Chu | Trimethylamine Sensing Properties of Sensors Based on MoO3 Microrods[END_REF][START_REF] Sui | An Ultraselective and Ultrasensitive Tea Sensor Based on α-MoO3 Hierarchical Nanostructures and the Sensing Mechanism[END_REF][START_REF] Sui | Construction of Three-Dimensional Flower-Like α-MoO3 with Hierarchical Structure for Highly Selective Triethylamine Sensor[END_REF][START_REF] Srinivasan | Chemi-Resistive Sensing of Methylamine Species Using Twinned α-MoO3 Nanorods: Role of Grain Features, Activation Energy and Surface Defects[END_REF] In our previous research, α-MoO3 nanobelts have been prepared and have shown extremely high response and superb selectivity to TMA. [START_REF] Yang | High Sensitivity and Good Selectivity of Ultralong MoO3 Nanobelts for Trimethylamine Gas[END_REF] For sensing reactions at α-MoO3 surface, the TMA was considered to be oxidized by lattice oxygen of α-MoO3 into N2, CO2 and H2O according to gas chromatography. [START_REF] Yang | High Sensitivity and Good Selectivity of Ultralong MoO3 Nanobelts for Trimethylamine Gas[END_REF] In term of active site for amines adsorption at α-MoO3 surface, it has been extensively believed that the N atom of amines interacts with exposed Mo atom at surface through coordination between the lone-pair electrons of N and empty 4d orbital of Mo. [START_REF] Kwak | Molybdenum Trioxide (α-MoO3) Nanoribbons for Ultrasensitive Ammonia (NH3) Gas Detection: Integrated Experimental and Density Functional Theory Simulation Studies[END_REF][START_REF] Cho | Ultraselective and Ultrasensitive Detection of Trimethylamine Using MoO3 Nanoplates Prepared by Ultrasonic Spray Pyrolysis[END_REF][START_REF] Chu | Trimethylamine Sensing Properties of Sensors Based on MoO3 Microrods[END_REF][START_REF] Sui | An Ultraselective and Ultrasensitive Tea Sensor Based on α-MoO3 Hierarchical Nanostructures and the Sensing Mechanism[END_REF] direction. [START_REF] Chithambararaj | Hydrothermally Synthesized h -MoO3 and α-MoO3 Nanocrystals: New Findings on Crystal-Structure-Dependent Charge Transport[END_REF] There are three structurally distinct oxygen atoms in each octahedron, i.e. single-coordinated terminal oxygen (Ot) forming the shortest bond with Mo, the double-coordinated asymmetric oxygen (Oas) respectively forming one long and one short bond with two Mo, and the triple-coordinated symmetric oxygen (Os) forming two equal short bonds with two Mo in the same sublayer and one longest bond with Mo in the other sublayer, as shown in Figure 1d.

The weak interaction between the layers usually results α-MoO3 nanomaterials in sheet-or belt-like morphology with the (010) plane being the most exposed surface. Similarly, our previous α-MoO3 nanobelts are dominantly exposed with the (010) plane. At the same time, it is worth noting that (100) plane also takes up a large portion for the nanobelt, and (001) can be ignored. [START_REF] Yang | High Sensitivity and Good Selectivity of Ultralong MoO3 Nanobelts for Trimethylamine Gas[END_REF] Because of highest percentage of (010) plane at α-MoO3 surface, α-MoO3 (010) has been experimentally and theoretically investigated for a long period. Figure S1a shows the atomic structure of the α-MoO3 (010) surface. It is reasonable that the geometry of the surface (010) is identical to that inside the bulk because all of Ot at (010) plane are singly coordinated with Mo and no bond needs to be broken to form α-MoO3 (010) surface. [START_REF] Papakondylis | Ab Initio Study of the Structure of theα-MoO3 Solid and Study of the Adsorption of H2O and Co Molecules on Its (100) Surface[END_REF] Although Kwak et al. have theoretically demonstrated that NH3 adsorption on the oxygen vacancy site of α-MoO3 (010) surface is much more feasible than stoichiometric one, 2 both experimental and theoretical result have demonstrated that the oxygen vacancy, especially Ot vacancy (VOt), is unstable or difficult to be generated. [START_REF] Tokarz-Sobieraj | Properties of Oxygen Sites at the MoO3(010) Surface: Density Functional Theory Cluster Studies and Photoemission Experiments[END_REF][START_REF] Agarwal | Oxygen Vacancy Formation on α-MoO3 Slabs and Ribbons[END_REF][START_REF] Mei | Density Functional Theory Study of Acetaldehyde Hydrodeoxygenation on MoO3[END_REF] Even though other theoretical research has reported that Oas can stay at its original place when Ot is removed, [START_REF] Coquet | The (010) Surface of Alpha-MoO3, a DFT + U Study[END_REF][START_REF] Chen | The Chemical Nature of Surface Point Defects on MoO3(010): Adsorption of Hydrogen and Methyl[END_REF] there is little dispute that the formation energy of VOt is so high that it is difficult to generate abundant Ot vacancies at α-MoO3 (010). Therefore, it is not advisable to consider α-MoO3 (010) as active surface for amines adsorption.

As mentioned, α-MoO3 (100) plane also takes up a large portion of the surface, especially for our nanobelts. 1 Figure S1b shows the atomic structure of α-MoO3 (100) surface, which is identically cleaved according to other theoretical research. [START_REF] Papakondylis | Ab Initio Study of the Structure of theα-MoO3 Solid and Study of the Adsorption of H2O and Co Molecules on Its (100) Surface[END_REF][START_REF] Agarwal | Oxygen Vacancy Formation on α-MoO3 Slabs and Ribbons[END_REF] It is clear that half of the α-MoO3 (100) surface is exposed with Mo atoms. According to field emission scanning electron microscope (FESEM) images of our α-MoO3 nanobelts, the width/thickness ratio of the nanobelts is even smaller than 10, as shown in Figure S2.

Additionally, the formation energy of VOas at α-MoO3 (100) is lower than that of VOt at α-MoO3 (010), which means VOas is even easier to be generated at α-MoO3 (100). [START_REF] Agarwal | Oxygen Vacancy Formation on α-MoO3 Slabs and Ribbons[END_REF] Therefore, the α-MoO3 (100) must possess overwhelmingly more exposed Mo atom at than the (010) for our nanobelts.

Actually, α-MoO3 (100) has already drawn some interest in olefin catalysis field. 17

Computational details

Spin-unrestricted DFT calculations within a periodic supercell were performed by Dmol3 package. [START_REF] Delley | An All-Electron Numerical Method for Solving the Local Density Functional for Polyatomic Molecules[END_REF][START_REF] Delley | From Molecules to Solids with the Dmol3 Approach[END_REF] General gradient approximation (GGA) in the form of a Perdew-Burke-Ernzerhof (PBE) was selected for the exchange-correlation functional, and a DFT-D correction was executed to consider van der Waals interaction among bilayers of α-MoO3 as well as the weak interaction between gas molecules and α-MoO3

Results and discussion

Amines adsorption at stoichiometric α-MoO3 (100) surface

With assurance of α-MoO3 (100) being active surface for amines adsorption, DFT calculation was performed to investigate adsorption site, adsorption energy and charge transfer for four amines, i.e. NH3, MMA, DMA, and TMA. Mo. Nevertheless, there are still some differences. As listed in Table 1, comparisons are made on the adsorption energy, the N-Mo distance and the charge transfer of these amines. For the adsorption at terrace, Eads (in absolute value) increases with the number of methyl groups increasing from 0 to 2, i.e. from NH3 to DMA, while Eads decreases when it goes to 3, i.e. TMA. The increase from NH3 to DMA can be mainly ascribed to electron-releasing effect of methyl group. The N atom is more electron-abundant in the amine molecule with more methyl group, which renders N atom more affinitive to Lewis site of Mo. Additionally, heavier molecule somehow facilitates van der Waals interaction. In term of the Eads reduction from DMA to TMA, it can be ascribed to the three methyl groups which have a repulsive effect with the surface bonds and prevent the N to coordinate with Mo. Hence, the distance between the N and Mo is 2.558 Å, longer than that of the others, as shown in Table 1. Furthermore, the low Eads also results from the lack of hydrogen bond interaction between TMA, which is has no H atom connected to N, and the surface Oas.

The repulsion effect between methyl group and Mo-Ot bond can be verified by comparing two MMA adsorption configurations. the large molecules, such as DMA and TMA, at terrace. Interestingly, Eads of TMA at channel is very approaching to that at terrace, even though the N is still distant from the Mo (3.934 Å). The reason can be that more H atoms in the methyl groups can interact with more Oas and Ot. at the surface when TMA is at channel.

The Mulliken charge is also analyzed for each configuration, as listed in Table 1. Firstly, they are all positive, suggesting electron donation from adsorbed amines to α-MoO3 (100) surface. Secondly, comparing the Mulliken charge of the four amines at terrace, we find that DMA donates electron at most, followed by MMA, NH3 and TMA, which shows the same trend as Eads. Nevertheless, for TMA at channel, the Mulliken charge reaches 0.498 |e|, much higher than others. As mentioned before, it can be attributed to the interaction between the H atoms in the methyl group with the surface Oas and Ot. Except TMA, the Mulliken charge at channel is slightly higher than that at terrace.

Based on above result, the amine molecules are highly affinitive to α-MoO3 (100) surface and can donate many electrons once adsorbed. This somehow can explain why the resistance of n-type α-MoO3 decreases when amine gas is injected. [START_REF] Yang | High Sensitivity and Good Selectivity of Ultralong MoO3 Nanobelts for Trimethylamine Gas[END_REF][START_REF] Kwak | Molybdenum Trioxide (α-MoO3) Nanoribbons for Ultrasensitive Ammonia (NH3) Gas Detection: Integrated Experimental and Density Functional Theory Simulation Studies[END_REF][START_REF] Cho | Ultraselective and Ultrasensitive Detection of Trimethylamine Using MoO3 Nanoplates Prepared by Ultrasonic Spray Pyrolysis[END_REF][START_REF] Chu | Trimethylamine Sensing Properties of Sensors Based on MoO3 Microrods[END_REF][START_REF] Sui | An Ultraselective and Ultrasensitive Tea Sensor Based on α-MoO3 Hierarchical Nanostructures and the Sensing Mechanism[END_REF][START_REF] Sui | Construction of Three-Dimensional Flower-Like α-MoO3 with Hierarchical Structure for Highly Selective Triethylamine Sensor[END_REF][START_REF] Srinivasan | Chemi-Resistive Sensing of Methylamine Species Using Twinned α-MoO3 Nanorods: Role of Grain Features, Activation Energy and Surface Defects[END_REF][START_REF] Pandeeswari | Nanostructured α-MoO3 Thin Film as a Highly Selective Tma Sensor[END_REF] However, adsorption is only the first step for gas sensing reaction, and continuous dissociation or reaction at the surface can be more significant for understanding sensing mechanism.

O2, H2O, and VO at α-MoO3 (100) surface

Instead of continuing to simulate decomposition or reaction of these adsorbed amines, we would like to investigate the surface chemistry of at α-MoO3 (100) with taking O2, H2O and VO into consideration. It must be kept in mind that there is never a perfect surface. It is necessary to consider whether and how can Mo sites be exposed under gas sensing measurement condition, in which a large number of O2 and H2O molecules must interact with the surface. O2 concentration in air is about 21 % (210,000 ppm) and H2O concentration at 50 % relative humidity at 25 °C is about 10, 000 ppm, 23 which are both much higher than amine concentration in sensing measurement.

The effect of the oxygen molecule in air cannot be excluded for understanding gas sensing mechanism.

For some metal oxides, reducing gas is believed to be oxidized by adsorbed oxygen, but for some others, it is lattice oxygen. [START_REF] Yang | Investigation on the Transformation of Absorbed Oxygen at ZnO {10-10} Surface Based on a Novel Thermal Pulse Method and Density Functional Theory Simulation[END_REF][START_REF] Yang | Surface Reactions of CH3OH, NH3 and CO on ZnO Nanorod Arrays Film: DFT Investigation for Gas Sensing Selectivity Mechanism[END_REF][START_REF] Kim | Catalyst-Free Highly Sensitive SnO2 Nanosheet Gas Sensors for Parts Per Billion-Level Detection of Acetone[END_REF][START_REF] Hübner | Influence of Oxygen Backgrounds on Hydrogen Sensing with SnO2 Nanomaterials[END_REF][START_REF] Boepple | Impact of Heterostructures on Hydrogen Sulfide Sensing: Example of Core-Shell Cuo/CuFe2O4 Nanostructures[END_REF][START_REF] Wang | Synthesis and Enhanced H2S Gas Sensing Properties of α-MoO3/CuO P-N Junction Nanocomposite[END_REF] Lattice oxygen of α-MoO3 is abundant and active, and some have believed it is lattice oxygen that oxidize reducing gas molecules, [START_REF] Yang | High Sensitivity and Good Selectivity of Ultralong MoO3 Nanobelts for Trimethylamine Gas[END_REF][START_REF] Sui | Construction of Three-Dimensional Flower-Like α-MoO3 with Hierarchical Structure for Highly Selective Triethylamine Sensor[END_REF] while others adsorbed oxygen. [START_REF] Sui | An Ultraselective and Ultrasensitive Tea Sensor Based on α-MoO3 Hierarchical Nanostructures and the Sensing Mechanism[END_REF][START_REF] Srinivasan | Chemi-Resistive Sensing of Methylamine Species Using Twinned α-MoO3 Nanorods: Role of Grain Features, Activation Energy and Surface Defects[END_REF] Hence, the O2 adsorption at stoichiometric α-MoO3 (100) is simulated, as shown in Figure S6. It is found that Eads is 0.57 eV and 0.47 eV for adsorption at terrace and channel site of α-MoO3 (100) and O2 molecule is still of ~3 Å distance from the exposed Mo site. Therefore, O2 adsorption on stoichiometric α-MoO3 (100) surface is not favorable.

As mentioned before, humidity has also to be considered. It has been discovered that H2O molecule has huge effects on gas sensing properties of metal oxides, and certainly for α-MoO3. [START_REF] Yang | High Sensitivity and Good Selectivity of Ultralong MoO3 Nanobelts for Trimethylamine Gas[END_REF][START_REF] Hübner | Influence of Humidity on CO Sensing with p-Type CuO Thick Film Gas Sensors[END_REF][START_REF] Degler | Investigations on the Temperature-Dependent Interaction of Water Vapor with Tin Dioxide and Its Implications on Gas Sensing[END_REF][START_REF] Wicker | Ambient Humidity Influence on CO Detection with SnO2 Gas Sensing Materials. A Combined DRIFTS/DFT Investigation[END_REF] Water adsorption at α-MoO3 (100) has already been theoretically investigated, and it has found that Eads is about -1 eV for full coverage of H2O (each exposed Mo with one H2O molecule). [START_REF] Papakondylis | Ab Initio Study of the Structure of theα-MoO3 Solid and Study of the Adsorption of H2O and Co Molecules on Its (100) Surface[END_REF] Herein, H2O adsorption at α-MoO3 (100) in different coverages (1/8, 1/4, 1/2 and full) as well as its dissociation are simulated, as

shown in Figure 3. The adsorption energy and Mulliken charge of these configurations are listed in Table 2.

The result demonstrates that water molecule tends to be adsorbed at channel site at low coverage (1/8 and 1/4) with its O atom bonding to the exposed Mo and its two H atoms having hydrogen bond interaction with surface Oas, as shown in Figure 3a-d. When the coverage increases to 1/2, the water molecule cannot be entirely adsorbed at channel, and half of them terrace, as shown in Figure 3e. Figure 3f shows the relaxed configuration of four water molecules at channel site, and it can be found that some of the water molecules rotate a little to a lower-energy state in which one H can interact with Oas at channel and the other H can interact with Ot at terrace. This suggests that the hydrogen bond interaction between H atom and the Oas at channel is reduced if there are eight H atoms from four water but only four Oas. Moving half of water molecules to terrace can render stronger hydrogen bond interaction. for n-type α-MoO3, which has already been verified by our previous research. [START_REF] Yang | High Sensitivity and Good Selectivity of Ultralong MoO3 Nanobelts for Trimethylamine Gas[END_REF] From Table 2, it can also be seen that less electronic effect is induced as the number of adsorbed water molecules increases. dissociate with Ea and Ereact of 0.78 eV and 0.57 eV, compared with 1.05 eV and 0.76 eV at terrace. From the atomic structures on the right, it can be seen that the reason why the dissociated water is more stable at the channel than at the terrace is that the formed HOas group of the former can have hydrogen bond interaction with nearby Ot in terrace with a distance of 2.044 Å. Based on the calculation of water adsorption, it can be deduced that there must be numerous water molecules previously adsorbed at α-MoO3 (100) in measurement condition, especially at room or relatively low temperature. The adsorption site for water is same as that for amines, thus the existence of water should prevent the adsorption of amines. However, if working temperature is elevated, some of water molecules may be desorbed, which can provide site for amine adsorption. The water at the terrace is more likely to desorb because the Eads at terrace is somewhat lower than that at channel. In contrast, the molecules at channel the may either be desorbed or dissociate.

Besides O2 and H2O, VO must also be taken into consideration, because the annealing process during material synthesis and device fabrication probably leads to VO formation. Co-adsorption of O2 and H2O around VOas(t) is also of high possibility, because there are three exposed Mo atoms very close to each other. Figure 6 shows co-adsorption and dissociation of both O2 and H2O at α-MoO3 (100) with one VOas(t). The energy for the co-adsorption is -1.59 eV, suggesting the high feasibility, even though it is a little lower than the sum of Eads of single O2 (-0.63 eV) and single H2O (-

Conclusion

It has been rationally analyzed that α-MoO3 (100) should possess overwhelmingly more exposed Mo than α-MoO3 (010) based on previous experimental and theoretical investigation. Accordingly, in this research, amines adsorption as well as interaction between O2, H2O and VO at α-MoO3 (100) surface are systematically investigated by density functional theory calculation. All four amine molecules have high affinity to the α-MoO3 (100) surface through interaction between the N and the exposed Mo, while the affinity is mainly influenced by number of methyl group, number of N-connected H. Regarding the surface chemistry, α-MoO3 (100) under practical conditions must be covered with many water molecules at low temperature; besides, O2 molecule filling VOas and hydroxyl group can also be present at α-MoO3 (100) under practical gas sensing measurement if considering interaction between O2, H2O and VO.

Figure 1 d

 1 Figure 1 (a) Primitive cell of α-MoO3; primitive cell in (b) ball-stick and (c) polyhedron display style

- 18

 18 Papakondylis et al. have theoretically explored the adsorption of H2O and CO on α-MoO3 (100). 9 However, little attention has been paid to amine adsorption at α-MoO3 (100). the theoretical research by Papakondylis et al. in early 1996 is still far from understanding the surface chemistry of α-MoO3 (100) under gas sensing measurement condition. In this work, for the first time, amines adsorption at α-MoO3 (100) surface is theoretically investigated by density functional calculation. The adsorption energy and charge transfer of NH3, monomethylamine (MMA), dimethylamine (DMA), and TMA are calculated and compared. Additionally, to fully understand surface chemistry of α-MoO3 (100) under amine sensing measurement condition, the adsorption and dissociation of water and oxygen molecule on stoichiometric or defective α-MoO3 (100) surface are simulated.

  Figure S1b and Figure S2. The supercell is in size 13.86 × 14.95 × 37.36 Å 3 with 240 O atoms and 80 Mo atoms in total. A 15 Å vacuum layer is introduced to avoid interaction among the slabs in [100] direction

  Figure 2a and b shows adsorption configurations of NH3 at terrace of α-MoO3 (100). It is similar to what has been believed that N atom bonds to the exposed Mo atom by offering its lone-pair electron to the empty 4d orbital of Mo. Additionally, two H atoms of NH3 may have hydrogen bond interaction with adjacent Oas. The adsorption energy is -1.47 eV, suggesting high ability of α-MoO3 (100) to adsorb NH3. Figure 2c-h shows the adsorption configurations of MMA, DMA, and TMA at terrace of α-MoO3 (100). All of these adsorptions mainly originate from the interaction between the N and exposed

Figure 2

 2 Figure 2 Adsorption configuration at terrace of α-MoO3 (100): (a, b) NH3, (c, d) MMA, (e, f) DMA, and (g,

Figure

  Figure 3gwith three water rotating is somewhat more stable than that in Figure3hwith only one rotating molecule. Notably, when the three rotate, the remaining molecule does not need to rotate because it can

Figure 3

 3 Figure 3 Water adsorption at α-MoO3 (100) in (a, b) 1/8, (c, d) 1/4, (e, f) 1/2 and (g, h) full coverage: (a) At channel and (b) at terrace site; (c) two both at channel and (d) one at channel and the other terrace; (e) all at

  The adsorbed water may also dissociate when temperature is elevated. Herein, dissociation of one water molecule at terrace or channel of α-MoO3 (100) is simulated. As clearly shown in Figure4, the molecularly adsorbed water is more stable than dissociated water either at terrace or at channel according to the relative energy and energy barrier. Nonetheless, the water molecule at channel takes more possibility to

Figure 4

 4 Figure 4 Water dissociation process at terrace and channel of α-MoO3 (100). The left shows relative energy

  that α-MoO3 (100) with one VOt(tf) is relaxed to the same configuration as that with one VOas(t), as can be seen in Figure5b and d. Movie S1 shows the process more clearly. This process is similar to what has happened at α-MoO3 (010) in other theoretical research. 11-14 Therefore, VOas(t) is most likely to generate. The relaxed configuration with VOt(te) is also very interesting, and Figure 5e and Movie S2 show that the exposed Mo is dragged inside by two Oas underneath.

Figure 5

 5 Figure 5 (a) α-MoO3 (100) surface showing five kinds of oxygen, and (b-f) the relaxed configuration of α-MoO3 (100) with five different vacancies.

Figure 6

 6 Figure 6 Adsorption and dissociation of both O2 and H2O at α-MoO3 (100) with one VOas(t). The left shows

FigureFigure S2 Figure S5 Figure S6 Figure S7 Figure S8

 S2S5S6S7S8 Figure S1 (a) (4 × 4) supercell of α-MoO3 (010) surface, (b) (1 × 4) supercell of α-MoO3 (100) surface

Figure S9

 S9 Figure S9 Water adsorption at α-MoO3 (100) with one VOas(t): (a, b) at vacancy and (c-f) beside vacancy but

Table 1

 1 Adsorption energy, distance between the N and the Mo, and Mulliken charge for amines adsorbed at terrace or channel site of α-MoO3 (100).

	Amines		NH3	MMA	DMA	TMA
	Number of methyl group	0	1	2	3
	Eads (eV)	Terrace Channel	-1.47 -1.66	-1.70 -1.80	-1.77 -1.69	-1.37 -1.36
	N-Mo (Å)	Terrace Channel	2.343 2.338	2.345 2.346	2.401 2.442	2.558 3.934
	Mulliken charge (|e|)	Terrace Channel	0.283 0.285	0.308 0.301	0.319 0.316	0.271 0.498
	Besides terrace, amine molecules can also be adsorbed at channel, as shown in Figure S5. The

adsorption energy is also listed in Table

1

. By comparing the Eads at terrace and channel site, it can be concluded that small amine molecules, such as NH3 and MMA, tend to be adsorbed at the channel, while

Table 2

 2 Average adsorption energy and Mulliken charge for one water molecule at α-MoO3 (100) in different coverage (Superscripts of a-h represents the configuration in

Figure 3a-h.)

	Coverage	1/8	1/4	1/2	Full
	Eads (eV)	-1.20 a -1.13 b	-1.19 c -1.17 d	-1.16 e -1.15 f	-1.14 g -1.11 h
	Mulliken charge (|e|)	0.192 a 0.182 b	0.187 c 0.187 d	0.184 e 0.169 f	0.174 g 0.175 h

1

  Figure5ashows five kinds of oxygen at α-MoO3 (100) surface. Oas(t) and Oas(c) represent Oas at terrace and channel, and Os(t) represents Os at terrace. Notably, there are two kinds of Ot even only at the terrace, one of them is connected with fully coordinated Mo, which is labeled Ot(tf), and the other is connected with exposed Mo, which is labeled Ot(te). Figure5b-f shows the relaxed configuration of α-MoO3 (100) with five different vacancies, i.e.

	VOas(t), VOas(c), VOt(tf), VOt(te), and VOs(t), whose vacancy formation energy is respectively 1.23 eV, 1.30 eV,
	1.23 eV, 1.35 eV, and 3.75 eV. Interestingly, E[VOas(t)] is same as the E[VOt(tf)], and the underlying reason is

  The dissociation of O2 in turn facilitates dissociation of H2O. The H-O bond closest to dissociated O2 elongates to 0.995 Å, and the other 0.990 Å. The former is easier to rupture by transferring the H to the Oas, which is dissociated from O2, with Ea and Ereact of 0.83 eV and 0.34 eV. The Ea and Ereact are 0.22 eV and 0.42 eV lower than those of water dissociation at terrace of stoichiometric α-MoO3 (100). The HOas is not stable, and the H can be easily (Ea = 0.23 eV) captured by the other O atom dissociated from O2, If the temperature is high enough, adsorbed water may dissociate to form a terminal HO and rooted HOas group. Because of the annealing process, VOas(t) is likely to be generated, but can be easily filled with O2 molecule. The adsorbed O2 molecule at VOas(t) may dissociate with the nearby H2O, and this finally leads to two terminal hydroxyl groups at surface.

	1.06 eV) adsorption at or around VOas(t). Additionally, the presence of H2O facilitates the dissociation of O2 with Ea and Ereact of 1.57 eV and 1.03 eV, which is respectively 0.07 eV and 0.15 eV lower than those with absence formed. Therefore, in practive, α-MoO3 (100) must already be covered with lots of water molecule, especially of H2O. reaching a more stable configuration. Finally, α-MoO3 (100) surface with two terminal hydroxyl groups is at low temperature.

Acknowledgements

This research is supported by the Hubei Provincial Natural Science Foundation of China (No. 2020CFB188), Sanya Science and Education Innovation Park of Wuhan University of Technology (No. 2020KF0030), National Natural Science Foundation of China (No. 62171331), the Key Project of Department of Education of Guangdong Province (No. 2018KCXTD026) and the Deanship of Scientific Research (DSR) at King Abdulaziz University of Jeddah (No. KEP-MSc-70-130-42). Thanks to the computational support by Research Centre for Materials Genome Engineering in Wuhan University of Technology.

Supporting Information

Surface chemistry of α-MoO3 (100) and its active site for amines adsorption through density functional calculation