Recent Trends in Antimicrobial Resistance among Anaerobic Clinical Isolates

Anaerobic bacteria are normal inhabitants of the human commensal microbiota and play an important role in various human infections. Tedious and time-consuming, antibiotic susceptibility testing is not routinely performed in all clinical microbiology laboratories, despite the increase in antibiotic resistance among clinically relevant anaerobes since the 1990s. β-lactam and metronidazole are the key molecules in the management of anaerobic infections, to the detriment of clindamycin. β-lactam resistance is usually mediated by the production of β-lactamases. Metronidazole resistance remains uncommon, complex, and not fully elucidated, while metronidazole inactivation appears to be a key mechanism. The use of clindamycin, a broad-spectrum anti-anaerobic agent, is becoming problematic due to the increase in resistance rate in all anaerobic bacteria, mainly mediated by Erm-type rRNA methylases. Second-line anti-anaerobes are fluoroquinolones, tetracyclines, chloramphenicol, and linezolid. This review aims to describe the up-to-date evolution of antibiotic resistance, give an overview, and understand the main mechanisms of resistance in a wide range of anaerobes.


Introduction
Anaerobes are well known to be an important part of the normal human intestinal, vaginal, oral, and skin microbiota [1]. Anaerobic bacteria are also opportunistic pathogens that could be involved in various types of human infections in association with aerobic bacteria, such as brain abscesses, intraabdominal, skin, or pelvic infections. They can also cause monomicrobial infections such as bacteremia, deep tissue infections, and bone and joint infections. These infections are associated with severe morbidity and a high rate of mortality [2,3]. In addition to the well-known anaerobes such as Bacteroides spp. or Clostridium perfringens, new genera and species are regularly described through improvements in culture and identification techniques and implicated in severe human infections. Clinically relevant Gram-negative anaerobic bacteria include Bacteroides fragilis group, Prevotella spp., Fusobacterium spp., and Veillonella spp. [4]. The main Gram-positive bacilli isolated from clinical samples are Cutibacterium spp., especially C. acnes (formerly known as Propionibacterium acnes), which is involved in chronic bone and joint infections. Additionally, Clostridium spp. (apart from Clostridioides difficile) are responsible for many types of severe infections, such as gas gangrene. Actinomyces spp., isolated in deep tissue infections, is also noteworthy [5][6][7]. Finegoldia magna and Parvimonas micra are the two most isolated Gram-positive anaerobic cocci (GPAC).
Due to technical and financial constraints associated with the identification and culture of anaerobic bacteria, microbiology and antibiotic susceptibility testing (AST) of anaerobes isolates are rarely routinely performed in clinical microbiology laboratories [1]. Therefore, the treatment of anaerobic infections has long been empirical, which has led to therapeutic failures and the emergence of resistance [8]. Over the last two decades, a growing number of studies around the world have focused on describing the epidemiology of resistance in anaerobic bacteria, with a worldwide increase despite differences between countries [9]. However, AST has been performed differently in laboratories according to countries, most of the time without following CLSI and EUCAST methods [4]. For example, Asian laboratories mostly used micro-dilution-based techniques, while the majority of laboratories in Europe and the US employed gradient diffusion strips or agar dilution, which is the reference method. Breakpoints for AST interpretation also sometimes differ between EUCAST and CLSI guidelines. This diversity of practices could lead to great variability in results and make studies difficult to compare. Moreover, there is a scarcity of recent comprehensive epidemiological data with a significant number of strains.
The aim of our study was to describe the evolution of resistance among anaerobic clinical isolates and summarize the mechanisms of resistance to the main antibiotics used in the treatment of anaerobic infections. To analyze the evolution of resistance epidemiology, articles published on PubMed between 2013 and 2023 were selected. Specifically, we included only those articles in which the authors used MALDI-TOF mass spectrometry or sequencing to identify anaerobes. This review only focused on clinical isolates, whereas data about anaerobes isolated from healthy people wAS not included. We also excluded results concerning C. difficile as the therapeutic management of infections caused by this pathogen is species-specific.
Carbapenem resistance in B. fragilis is primarily promoted by the class-B metallocarbapenemase CfiA, encoded by a chromosomal gene recovered in some strains. In terms of intra-species diversity, B. fragilis can be classified into two subgroups based on the presence or absence of the cfiA and cepA genes. These subgroups are referred to as division I (cfiA-) and division II (cfiA+). Subgroup division is achieved by DNA-DNA hybridization and ribotyping, while detection is now available by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy [39][40][41]. In a retrospective study, Ferløv-Schwensen et al. observed an increase in division II bacteroides among clinical isolates (2.8% vs. 7.8%) between 1973-1991 and 2002-2015, potentially due to the overuse of carbapenems [42]. Recent studies have reported a similar prevalence of cfiA in Europe, ranging between 8 and 16% [24,34,43]. In Asia, close rates have been reported, with a prevalence of 15% and 22% in Japan and China, respectively [33,44]. Curiously, a higher prevalence of division II (cfiA+) has been reported in bloodstream infections compared to other clinical isolates [43]. The gene cfiA is not always correlated with phoneticphenotypic resistance related to low-level expression. Insertion sequences upstream of the gene, mainly belonging to the IS1380 family, are the cornerstone to induce their overexpression, which leads to phenotypic resistance to β-lactams [38,45,46]. It should be noted that the use of meropenem (not imipenem) +/− EDTA allows the detection of cfiA+ strains with low-level expression [47]. In the non-fragilis Bacteroides group, only cfxA is generally recovered, and an unknown mechanism provides resistance to β-lactams in non-cfxA isolates [34].
β-lactamases in other anaerobes are less studied, but penicillinases are outlined, mainly by phenotypic approaches in Fusobacterium spp., Porphyromonas spp., and Clostridium spp. [52][53][54][55]. Target modification by alteration of penicillin-binding proteins (PBPs) promotes cefoxitin resistance in the Bacteroides fragilis group. In fact, the modification of PBPA or PBP3 appears to play a greater role than hydrolysis by CfxA [37,56,57]. A decrease in imipenem susceptibility was also associated with PBP2Bfr modifications [58]. In C. perfringens, PBP alterations following β-lactams exposure result in decreased affinity of B-lactams for PBP1 but an overproduction of PBP6 related to phenotypic resistance to penicillin G and ceftriaxone [59,60].
In Veillonella spp., PBP modification leads to high-level resistance to TZP (MIC >128 mg/L) in β-lactamase-negative isolates, whereas ampicillin remains active (MIC = 0.5-4 mg/L) due to a retained affinity for PBP [61]. In B. fragilis, derepression of bmeABC coding for a RND efflux pump can trigger the extrusion of ampicillin, cefoperazone, and cefoxitin [62]. Resistance induced by porin loss remains poorly studied, while in B. thetaiotaomicron, it has been suggested that resistance to AMC may be related to a defect in the expression or absence of a porin [63]. Moreover, loss of porin associated with PBP alteration may be co-induced following cefoxitin exposure leading to ampicillin and cephalosporin resistance in B. thetaiotaomicron [57].

Metronidazole
Metronidazole, a drug of the 5-nitroimmidazole family, is an old molecule introduced in the 1960s but is still one of the most important antibiotics for the management of anaerobic infections. Mechanistically, metronidazole is a prodrug that remains inactive until absorbed by passive diffusion and activated intracellularly. The reduction in the nitro group leads to the formation of a toxic metabolite that interacts with several macromolecules in the cell, mainly DNA, leading to strand breaks and cell death. Enzymatic reduction is principally driven by pyruvate ferredoxin/flavodoxin oxydoreductase (PFOR) and occurs only at low oxygen concentrations [64,65]. In 1978, the first clinical isolate of B. fragilis selected by long-term treatment with metronidazole was described [66]. Currently, the prevalence of resistance to metronidazole is relatively low in Gram-negative bacilli, with higher proportions in Bacteroides spp., Parabacteroides spp. (<3%), and Prevotella spp. (<5%) [13,15,[17][18][19][20][21][22][23][24]27,28]. Some countries reported a higher prevalence of resistance in Prevotella spp., such as Germany and Spain, where 12% (3/25) and 7% (2/30) of isolates were resistant, respectively [26,30]. In other species, resistance is globally lower, but higher resistance rates have been sporadically reported in small studies in Veillonella spp. (23-27%, 3/14 and 3/11), Peptoniphilus spp. (13%, 3/21), Anaerococcus spp. (13%, 4/31), P. micra (9-12%, 1/11 and 2/17), and Clostridium spp. (8%, 3/37) [19,21,30,31]. Some resistant isolates of Phorphyromonas spp. and Alistipes spp. have also been reported [26]. Grampositive bacteria such as Actinomyces spp., Cutibacterium spp., and Propionibacterium spp. are intrinsically resistant to metronidazole. This is probably due to the absence of the PFOR system or an alternative pathway of pyruvate catabolism [67]. However, susceptible isolates have rarely been reported in this species (<5%), while higher susceptible rates were observed in the US for Actinomyces spp. (16.2%) [13,18,22,28]. Over the past years, resistance to metronidazole appears to be stable and rare, which allows this old antibiotic to maintain a key role in the management of infections caused by anaerobes [13,25]. However, it remains difficult to evaluate resistance evolution worldwide considering the different clinical breakpoints of CLSI (≥32 mg/L) and EUCAST (>4 mg/L). The ranges of MIC, MIC 50 , and MIC 90 are synthetized in Tables 3 and 4.    [31] Resistance to metronidazole occurs through a complex mechanism mediated by decreased drug activation, limited absorption, or increased DNA repair. One of the most important and well-studied mechanisms is the pro-drug inactivation via nitro-imidazole reductase coding by nim genes that transform metronidazole into a non-toxic compound. Up to now, 12 nim genes (nimA to nimL) sharing > 50% nucleotide similarity have been detected worldwide, mainly in Bacteroides spp. and Prevotella spp. [67,69]. Few studies focused on the prevalence of nim genes, and 2-3% of Bacteroides spp. carry them with a wide diversity (nimA-H, nim J, and nimL) [13,70]. Higher rates have been reported recently in India, where 61% of isolates were positive for nim genes (34/56) [71]. In Prevotella spp., the prevalence ranges from 3.7 to 5.3% (nimA-C, nimI, and nim K) [67,72,73]. In other Gram-negative anaerobes, nim genes have been reported in Fusobacterium spp. (nimD), Porphyromonas spp. (nimC), and Veillonella spp. (nimE) [67,73,74]. In Gram-positive bacteria, nimB carriage was reported in Peptostreptococcus spp., F. magna, Anaerococcus prevotti, and P. micra [75]. These resistance genes may be chromosomally located or harbored by plasmids, which may raise concerns about the spread of these resistance genes among anaerobic bacteria [4,67]. The nim genes are not always correlated with phenotypic antibiotic resistance (MIC = 1-6 mg/L), which is related to the low-level expression [76]. However, it is well recognized that high levels of resistance can be conferred by these "silent" genes, especially after metronidazole exposure, and insertion sequence (IS) is the cornerstone of their over-expression [76,77]. Other resistance mechanisms have been described, but their relevance in clinical isolates remains difficult to estimate. Metronidazole resistance in Bacteroides spp. can be related to complex metabolic changes in metabolism, such as a decrease in PFOR activity associated with an overproduction of LDH activity, an increase in rhamnose catabolism, or a modification of iron transport [78][79][80][81]. Another way to escape the antibiotic effect is the metronidazole extrusion by BmeABC, a RND family efflux transporter of Bacteroides spp. [62]. Overexpression of the DNA repair system RecA in B. fragilis can overcome DNA damage caused by metronidazole [82].

Clindamycin
Clindamycin is a bacteriostatic lincosamide active against Gram-positive cocci and anaerobes. This antibiotic is widely used in many countries due to its low cost, good tolerability, and the existence of susceptibility-testing recommendations. Because of its bacteriostatic activity, clindamycin is not recommended in the treatment of severe infections, for which bactericidal antibiotics such as β-lactams or metronidazole are preferred. Clindamycin, due to its broad spectrum of activity against anaerobes, has traditionally been used as a standard treatment for non-severe anaerobic infections. However, over the past 20 years, numerous cases of resistance have emerged, leading to clindamycin being one of the antibiotics with the highest rates of resistance among all anaerobic bacterial species [4].
In the 1990s, the prevalence of clindamycin resistance in Bacteroides/Parabacteroides spp. was less than 10% in European countries, the US, and Canada. However, over a span of 20 years, this resistance has more than tripled, reaching a prevalence of 35% in the US, Canada, and Europe [11][12][13]83,84]. Studies with more recent data confirmed that the rates of clindamycin resistance remained high and variable from one region to another. This variability is even more important as not all authors used the same AST techniques and breakpoint values. Indeed, CLSI's recommendations differ from those of EUCAST.. In recent studies that considered all Bacteroides and Parabacteroides species, the resistance rate remained a concern and reached 33.5% in Italy, 49% in Spain and France, and 80% in Korea [21,30,85,86]. Curiously, clindamycin resistance was much lower in Scandinavian countries and Ireland; authors described a prevalence of around 17% [27,87,88]. For B. fragilis, the resistance rate was between 20 and 40% in European and North American countries and 50% in Asian countries, but there were many differences between countries [17][18][19]22,28,89]. In Japan, Ueda et al. described a significant increase in clindamycin resistance between 2010 (40.7%) and 2018-2019 (61.1%) [16]. In contrast, Kierkowska et al. showed that clindamycin resistance in Poland slightly decreased, from 22.5% to 18.3% between the 2007-2012 and 2003-2017 periods [15]. For other Bacteroides species belonging to the fragilis group, all studies described a higher rate of resistance (44% in the US, more than 50% in Canada, European, and Asian countries) [16][17][18][19][21][22][23]28,33]. The prevalence of clindamycin resistance was also important in Prevotella spp. strains, regardless of the country considered. It was around 30-40% in European countries and in the US, except in Ireland, where Ali et al. reported a higher rate (66.9%), and in Germany, where Wolf et al. reported a lower rate (24%) [17,19,[26][27][28]30,89]. In Asia, the resistance rate was also significant (45% in Korea and 62.1% in Japan) [21,23]. Fusobacterium spp., Veillonella spp., and Porphyromonas spp. are also involved in anaerobic infections and are typically susceptible to clindamycin. Clindamycin resistance rates varied according to the regions, ranging from 6.7 to 25% and from 0 to 17% for Fusobacterium spp. and Veillonella spp., respectively [17,19,[21][22][23][26][27][28]30,68]. In other Gram-negative anaerobes, such as Porphyromonas spp. or Alistipes spp., clindamycin resistance was rarely described [23,26,27].
Studies about antibiotic resistance in Gram-positive anaerobes are fewer in number. In European and North American studies on Cutibacterium spp. involved in infectious diseases, the authors reported a low prevalence of clindamycin resistance. Indeed, the resistance rate was about 8% or less in Spain, Italy, Ireland, and Austria, and around 10% in the US and Canada [18,19,22,27,28,30]. In Japan, Koizumi et al. described variable resistance rates according to species. Cutibacterium avidum was the most resistant species, with a resistance rate of 25%, which is consistent with other studies on Cutibacterium species [90,91]. As Actinomyces spp. is difficult to cultivate, AST is rarely performed in routine microbiology laboratories, and few studies on antibiotic resistance are available. Nevertheless, clindamycin resistance now appears to be substantial in most of the studies published. Except for Ireland, where Ali et al. described a resistance rate of 7.5%, resistance rates ranged from 18 to 44% in Europe and North America [18,22,27,28,30,31,92]. For Clostridium species other than C. difficile, as for other bacteria, the susceptibility to clindamycin was variable according to the country and AST techniques. Globally, most of the studies described a moderate activity of clindamycin against Clostridium spp., with resistance rates ranging from 16 to 30% in the US, Europe, and Korea [19,21,22,28,31,68]. Two European studies found lower resistance rates. In Ireland, Ali et al. observed 8.4% of resistant strains, similar to Sarvari et al. in Hungary [7,27]. Of all the non-difficile Clostridium species, C. perfringens is the most frequently isolated. Clindamycin resistance appeared to be lower than in other Clostridium species. In the US and Europe, authors described 10% or less of resistant strains, except in Greece, where Maraki et al. found a resistance rate of 40% [14,19,28,31,68]. Forbes et al. published a resistance rate of 14.6% in Canada. Limited studies have specifically investigated GPAC isolates, with the majority of them presenting data on GPAC as a whole without differentiating between specific species. However, it is worth noting that susceptibility to clindamycin appears to exhibit heterogeneity among GPAC species [29]. Except in Korea, clindamycin has limited activity against F. magna. Resistance rates were around 25% in the US, Spain, France, and Italy; around 35% in Canada; and above 50% in Greece [19,21,22,28,29,31,68]. On the other hand, a similar trend was described by the same authors for Peptoniphilus spp., P. micra, and P. anaerobius, which appeared to be more susceptible to clindamycin with resistance rates of 10% or less [22,[28][29][30][31]68]. The ranges of MIC, MIC 50, and MIC 90 are synthetized in Tables 5 and 6.   Resistance to macrolides, lincosamides, and streptogramins (MLS) in Gram-negative anaerobes is mainly mediated by rRNA methylases encoded by erm genes (Table 7). In Bacteroides group and Prevotella spp., the majority of clindamycin-resistant strains harbored erm(F) gene [33,88]. Both erm(G) and erm(B) were also identified in some resistant isolates [70]. It is important to note that the presence of erm genes is not systematically correlated with phenotypic resistance to clindamycin. Indeed, Eitel et al. and Hashimoto et al. found that the prevalence of erm(F) was not significantly different in clindamycinsusceptible or clindamycin-resistant Bacteroides isolates. This implies that the expression of several resistance genes is necessary to observe phenotypic resistance [24,33]. Some other genes, lin(A), mef (A), and msrSA, may also be responsible for MLS resistance in Bacteroides, but they are rarely searched compared to erm genes. The lin(A) gene is located on a transposon (NBU2) and encodes for an O-nucleotidyltransferase, responsible for clindamycin and lincomycin resistance [24,93]. The msrSA and mef (A) genes are not specific to Bacteroides and mediate efflux-pump mechanisms [24]. Limited data are available on resistance mechanisms in Gram-positive anaerobes. As in Gram-negative anaerobes, clindamycin resistance seems to be mainly mediated by erm genes but data differed from one study to another. Koieumi et al. have shown that erm(X) is more prevalent than 23S rRNA mutations in Cutibacterium clinical isolates, especially in C. avidum [90]. In contrast, erm(X) was not detected in any resistant strain in the Oprica et al. study, suggesting that several mechanisms are involved [94]. Little is known about the genetic basis of resistance to MLS in GPAC. Only three studies have been published on clindamycin resistance mechanisms, and all three linked the resistance to erm gene expression, specifically erm(A), subclass erm(TR), and erm(B) [29,95,96]. Other mechanisms are probably involved, and more studies are needed to understand MLS resistance in Gram-positive anaerobes.  Clostridium spp.,

Fluoroquinolones
The class of fluoroquinolones includes many molecules, but due to their difficulties in reaching target sites, they are usually considered ineffective against anaerobes [4]. Moxifloxacin is currently the only antibiotic that demonstrates activity against anaerobic bacteria. However, since its approval by the FDA, numerous species have developed resistance mechanisms against this drug. In their review, Boyanova et al. observed a worldwide significant increase in resistance to moxifloxacin between 2000 and 2010, in Bacteroides/Parabacteroides spp. and Clostridium spp. [11,13,25,84,97]. In 2017, Gajdacs et al. reported similar results [4]. This upward trend has since been confirmed. In recent studies about Bacteroides/Parabacteroides spp., the prevalence of moxifloxacin resistance was between 30 and 42% [21,30,85]. In Asian and European countries, resistant strains represented about 20% and 30% of B. fragilis and non-fragilis Bacteroides isolates, respectively [16,17,21,23,24]. In the US, Hastey et al. observed higher resistance rates (26% for B. fragilis and 30% for the B. fragilis group without B. fragilis) [14]. Resistance was also significant in other Gram-negative anaerobes such as Fusobacterium spp. and Prevotella spp. However, the results of different studies varied considerably, with resistance rates ranging from 7 to 50% for Fusobacterium spp. and from 9 to 88% for Prevotella spp. It is important to note that, as for other antibiotics, AST was performed differently according to laboratories. Additionally, breakpoint values for AST interpretation differed between EUCAST and CLSI guidelines, making interpretation and comparison of studies difficult [98]. Indeed, a recent Italian study using the EUCAST criteria showed that 81% of Bacteroides spp. strains were resistant to moxifloxacin, whereas using the CLSI criteria, only 40% of these same strains were categorized as resistant [99]. Moreover, the authors used the agar dilution method to perform AST, which is recommended by EUCAST and CLSI guidelines and observed the highest resistance rates. This suggests that resistance rates determined by E-test or microdilution may be underestimated. Nevertheless, the prevalence of moxifloxacin resistance in anaerobes appears to be increasing worldwide.
Resistance to moxifloxacin in the B. fragilis group mainly resulted from chromosomal mutations (gyrA, parC) leading to target modification or active efflux [4,8,100]. Some studies have suggested that the multidrug efflux pump BexA could also be involved in moxifloxacin resistance in B. fragilis, but it remains unclear. Indeed, in Eitel et al. and Rong et al. studies, bexA was detected in some moxifloxacin-susceptible strains but not constantly resistant strains [24,38]. In B. thetaiotaomicron, bexA is overexpressed and confers constitutive low-level resistance to fluoroquinolones [101]. Resistance mechanisms in other anaerobic bacteria have been little explored until now.

Chloramphenicol
Chloramphenicol is a bacteriostatic antibiotic with good in vitro activity against most anaerobic bacteria. It has long been used in severe anaerobic and central nervous system (CNS) infections because of its lipid solubility and excellent diffusion in the CNS [102]. Currently, chloramphenicol is rarely used because of its toxicity. Side effects associated with chloramphenicol, particularly hematological complications but also cardiac and neurological issues, are infrequent but can be severe. Hematological side effects include bone marrow suppression and hemolytic anemia, which can have serious consequences. Additionally, rare cases of fatal aplastic anemia have been reported, and these effects are often irreversible [103]. Due to its limited use, chloramphenicol susceptibility is rarely tested routinely and few data are published. In vitro chloramphenicol resistance seemed to be exceptional and mainly described in Bacteroides spp. strains, even with breakpoint values that differed between EUCAST and CLSI guidelines [17,19,21,23,31,86]. Some therapeutic failures have been reported in patients with anaerobic sepsis treated with chloramphenicol, suggesting that in vitro susceptibility may not be correlated with clinical efficacy [104]. In Bacteroides spp., resistance to chloramphenicol is plasmid-mediated by the gene cat, which codes for an acetyltransferase that inactivates the drug [4,103,104].

Tetracyclines
Tetracycline is a bacteriostatic broad-spectrum antibiotic. Although it was initially active on anaerobes, this molecule now has limited usefulness due to the development of resistance in most anaerobic bacteria. These resistances were described a long time ago [105]. Some recent studies also observed high resistance rates in most anaerobes, although tetracycline susceptibility is rarely tested routinely [4,21,23]. Minocycline and doxycycline are second-generation tetracyclines that appeared to be more active than tetracycline, but as AST was not always performed in laboratories, few data are available and some resistance is described [90,106,107]. Since many resistance mechanisms affecting tetracycline can also affect minocycline and doxycycline, it is advisable to perform AST before using them. However, CLSI and EUCAST do not give clinical breakpoints for these drugs since there is no correlation between MIC and clinical outcome. This makes the use of these molecules even more difficult. Tigecycline is a third-generation tetracycline active against Gram-positive and Gram-negative anaerobes and approved to treat complicated infections, including those due to anaerobes [108][109][110]. As for minocycline and doxycycline, there are no breakpoint values available for MIC interpretation. Several studies described low tigecycline MICs for Gram-positive and Gram-negative anaerobes, suggesting an absence or a low level of resistance [17,26,31,89]. However, some resistant B. fragilis strains have been observed [24,83,85].
Tetracycline resistance involves three types of mechanisms in anaerobes: the efflux pump, ribosomal protection or modification, and enzymatic degradation, most of which are mediated by tet genes. More than 20 tetracycline-resistant efflux genes are identified, but only tet(A) to tet(E) and tet(K)-(L), which confer resistance to tetracycline and doxycycline, have been so far described in anaerobes, mainly in the B. fragilis group [4]. In addition, tet(M), tet(O), tet(Q), tet(W), and tet(32) are coding for ribosomal protection proteins associated with tetracycline resistance in anaerobic bacteria and confer resistance to tetracycline, doxycycline, and minocycline [4,24,[111][112][113]. Finally, tet(X) is associated with enzymatic degradation [114]. Tigecycline escapes most of the mechanisms of resistance to first-and second-generation tetracyclines due to its slightly different conformation. Few strains with elevated MICs have been identified among anaerobes, and resistant mechanisms still remain unclear [4,115,116].

Other Agents
Other antibiotics appeared to be active in vitro against anaerobic bacteria, although they are rarely tested routinely and more data on their in vitro activity and clinical effectiveness are needed. Linezolid and tedizolid are oxazolidinone antibiotics, mainly active on Gram-positive bacteria. Studies showed that linezolid had good activity against some Gram-negative anaerobes, including Bacteroides spp., Prevotella spp., Fusobacterium nucleatum, and Gram-positive anaerobes [29,92,[117][118][119]. A clinical report confirmed linezolid's clinical effectiveness in treating anaerobic infections, but there is a lack of published clinical data [120]. Recently, linezolid-resistant isolates of B. fragilis (MIC = 8 mg/L) and C. perfringens (MIC = 16 mg/L) were identified from zoonotic samples. Genomic analysis reveals the presence of cfr(C), previously described in C. difficile encoding Cfr(C), a 23S ribosomal methylase linked to cross-resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramins A (the so-called PhLOPS A resistance phenotype) [121]. In B. fragilis, cfr(C) was located on Tn6994 in combination with other resistance genes (nimL, ant(9), ant(6)-1a, erm(G), and tet(Q)), which may lead to the diffusion of multi-drug resistance across anaerobes [122,123].
Studies testing vancomycin showed that vancomycin had good activity against Grampositive anaerobes. No resistance was described among Gram-positive cocci with low MIC [18,29,31,68]. Gram-positive bacilli also appeared susceptible to vancomycin. Koizumi et al. and Parisio et al. described no resistant Cutibacterium spp. strains, while recent Italian studies reported a low resistance rate (nearly 5%) in Clostridium spp. However, as isolates were not identified at the species level, this resistance rate could be influenced by species intrinsically resistant to vancomycin, such as C. ramosum or C. innocuum. Additionally, Steininger et al. showed that vancomycin is also effective in vitro against Actinomyces spp. [90,92,124].

Discussion
As anaerobic infections are often polymicrobial and mixed with aerobic bacteria, detection, identification, and AST of anaerobic bacteria are cumbersome and not systematically performed in routine microbiological laboratories. However, these bacteria are fully accepted as major pathogens associated with significant morbidity and mortality, especially as their resistance to antibiotics is increasing worldwide. Interpreting and comparing studies becomes challenging due to the difficulties associated with implementing the AST methods recommended by international guidelines. Additionally, the heterogeneity in breakpoint values used for AST interpretation between European and American recommendations further complicates the process. Despite this, global antibiotic resistance surveillance studies have been increasingly published over the past 20 years [11,83]. In all studies, authors observed an alarming increase in antibiotic resistance, and antibiotics that were once considered first-line treatments, such as clindamycin or metronidazole, appear less and less effective. Newer antibiotics, such as tigecycline and linezolid, seemed to be effective against anaerobes with no or low resistance levels. However, more published data are needed to complete our knowledge about them. Our current understanding of the resistance mechanisms that mediate antibiotic resistance in anaerobes is limited, with the majority of studies focusing primarily on Bacteroides spp. As a result, there is a need to gather comprehensive data on resistance mechanisms in other anaerobic bacteria to enhance our understanding of this phenomenon. In addition, several studies have shown that the presence of resistance genes is not systematically correlated with phenotypic resistance, and conversely, some susceptible strains harbored resistance genes. This suggests that resistance in anaerobes is complex and that further studies are needed. Worryingly, multidrug-resistant anaerobes have been described in many bacterial species, especially the B. fragilis group, with strains that simultaneously harbored a large number of resistance genes, some of which encode multidrug efflux pumps [125][126][127]. In the future, the therapeutic management of these infections may become challenging.

Conclusions
In conclusion, antibiotic resistance in anaerobes is increasing worldwide, and AST should be performed as far as possible to avoid therapeutic failure. An increasing volume of epidemiological and mechanistic data is being published, highlighting the importance of ongoing laboratory studies on anaerobes. Such research is crucial to fully understanding and preventing resistance in these bacteria.
Funding: This research received no external funding.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest:
The authors declare no conflict of interest.