1. Introduction
The emerging antimicrobial resistance (AMR) is a critical problem faced by the medical and scientific community. While extensive research has addressed AMR in aerobic and facultative anaerobic pathogens, the study on strict anaerobes has received comparatively less attention. Anaerobic Gram-negative bacteria (AGNB) comprise a significant proportion of the human microbiota and often act as secondary pathogens [
1]. AGNB are the most common anaerobes associated with infections and include some of the most antimicrobial-resistant species [
2]. These bacteria, particularly the
Bacteroides fragilis group, have exhibited notable resistance rates [
3], with some strains demonstrating multiple-drug resistance (MDR) [
4]. MDR is now being recognized among other clinical AGNB as well, which were earlier believed to be susceptible [
5]. Most of the available literature on AMR belongs to the genus
Bacteroides, and the remaining AGNB continue to take a back seat. These bacteria can serve as channels for horizontal gene transfer (HGT) and the ignored conduct may lead to the possibility of selection and transfer of resistance determinants [
6].
Understanding the extent of AMR in AGNB is essential for several reasons. AGNB are clinically significant, often associated with polymicrobial infections, and can cause severe infections such as abscesses, bacteremia, and intra-abdominal infections [
2]. The appropriate management of these infections requires accurate antimicrobial susceptibility testing (AST) and an understanding of local resistance patterns. However, the current knowledge of AMR patterns in AGNB is limited, and the existing data often deviate from the standard guidelines [
7,
8]. The changing antibiograms and the emergence of resistance determinants in AGNB mandate periodic phenotypic and genotypic AST of these bacteria. Moreover, AGNB exhibit intrinsic resistance mechanisms that differ from those observed in aerobic bacteria. They possess distinct sets of resistance genes, including those encoding resistance to critical antibiotics [
9], making effective treatment challenging. In AGNB, there has been a focus on studying AMR, especially genotypic resistance in limited species and against a restricted spectrum of antibiotics, many of which hold less therapeutic significance today.
In this study, we aimed to address these knowledge gaps and explore the prevalence of AMR among clinically significant AGNB. We assessed the phenotypic and genotypic resistance among clinically significant AGNB to at least one drug from each class of antimicrobial agents recommended by CLSI for the primary testing of anaerobes, viz., metronidazole, clindamycin, piperacillin–tazobactam, and imipenem; and to two supplemental drugs for the selective testing, viz., chloramphenicol and cefoxitin. Due to the comparable clinical efficacy and interpretive results, only one antimicrobial agent from each antimicrobial class was included [
10]. We intentionally excluded all ß-lactams since most of the
B. fragilis group members are reported as uniformly resistant to them [
11]. In this study, the resistance to each antimicrobial agent was considered, including (a) phenotypic resistance, an isolate with a “resistant” phenotype, when the MIC was greater than the breakpoint, and the data are interpreted by including the intermediate and resistant phenotypes in the resistant category; and (b) genotypic resistance, an isolate with a “resistant” genotype, when the isolate harbored the gene encoding for AMR to the given antimicrobial agent. We also studied the prevalence of the AMR determinants for the aforementioned antimicrobials, viz.,
nimE (metronidazole),
cfiA (imipenem),
cepA (piperacillin–tazobactam),
cfxA (cefoxitin),
ermF (clindamycin),
cat (chloramphenicol) and MGEs such as
cfiAIS, the insertion sequence present upstream of
cfiA gene known to upregulate the expression of the
cfiA gene and resultant imipenem resistance, and
IS1186, associated with the
cfiA expression and in many cases known to induce
nim gene-mediated metronidazole resistance.
Overall, the study findings may have important implications, with the potential to enhance clinical decision-making, inform antibiotic stewardship efforts, and shape infection control strategies tailored to AGNB-associated infections. It underscores the importance of addressing AMR challenges and improving patient outcomes.
3. Discussion
This is the first Indian study that describes the genotypic and phenotypic resistance in AGNB against six major anti-anaerobic drugs. In this study, we saw that AMR was most commonly found in
Bacteroides spp. and was reported highest against clindamycin. Over the past 40 years, resistance against clindamycin has increased drastically worldwide, especially in the Asian countries. Data from the early 1980 and 1990s shows less than 5% resistance to clindamycin [
12,
13], which has increased to 32.4% after a decade, as reported in a Europe-wide study of 13 countries [
14]. However, in the present scenario, resistance to clindamycin seems steady in the European countries [
3,
15,
16,
17], while it has increased to 50–68% in the US [
18,
19,
20] and even higher in the Asian countries (80–90%) [
15,
21,
22,
23,
24]. In our study, we detected a 33.5% resistance against clindamycin, and the results confirm a 100% association between resistant genotypes and phenotypes. The
ermF gene was found as the clindamycin resistance determinant in our isolates, different from the prevalent variants known to confer clindamycin resistance in aerobes and Gram-positive anaerobes [
8]. This may suggest that our clinical isolates have the potential to act as a reservoir of clindamycin resistance, certainly for AGNB. Our study reported a high resistance rate against clindamycin in the genus
Prevotella (36.7%) and
Fusobacterium (38.7%). The literature survey showed an even higher resistance rate in
Prevotella, i.e., up to 40% in Europe, 50% in both US and Asia, and highest in Kuwait, around 89% [
15]. In
Fusobacterium spp., the resistance rates of Asian countries such as Taiwan (31%) [
24] and Singapore (44%) [
25] are similar to ours; however, a ≤15% resistance has been reported from the rest of the world. Clindamycin, which was earlier believed to be a drug of choice for infections above the diaphragm, now calls for attention, since
Prevotella and
Fusobacterium are usual isolates of orodental and other above-diaphragm infections.
Metronidazole can be used as a drug of choice to treat anaerobic infections caused by
Fusobacterium and
Prevotella. Unfortunately, resistance to metronidazole is also becoming a topic of concern [
26]. Our study showed only one and three metronidazole-resistant isolates from these genera, respectively. In our isolates, a very low resistance was detected in
Fusobacterium spp., which was similar to various studies worldwide with a few exceptions [
27,
28]. However, the literature shows an emerging AMR to metronidazole in the genus
Prevotella [
3] and
Veillonella [
29]. Reduced susceptibility of
Veillonella isolates to metronidazole has been seen in our isolates (48.6%), which is in tune with the literature from the East Asian countries [
28,
30]. Metronidazole resistance among
B. fragilis group isolates is emerging worldwide due to the non-judicious use of the drug to treat anaerobic infections. The European data from the early 1990s showed no resistance, but in the succeeding years, there was an increase to 0.5% [
31,
32,
33]. On the contrary, a significantly higher resistance of up to 15% has been seen in many western countries [
34,
35,
36] and up to 30% from a few Asian regions [
22,
35,
36,
37,
38]. In our study, a 41.1% resistance was seen in
Bacteroides spp., whereas resistance varying from 7% to 31% has been reported in other Indian studies [
26,
39]. The main mode of metronidazole resistance is nitroimidazole reductase activity encoded by
nim genes [
40]. The studies evaluating the presence of
nim genes are limited; nevertheless, they depict a low prevalence ranging from 0.5 to 2.8% in
Bacteroides spp. [
27,
29,
40]; 0 to 5.3% in
Prevotella spp. [
27], and 0 to 5.9% in
Fusobacterium spp. [
27,
40]. Yet again, most of the literature belongs to the European countries [
40], and the geographic distribution of
nim genes in the Indian subcontinent is relatively under-explored. In our study, the
nim genes were detected in 24% (48/200) of isolates and were more prevalent in
Bacteroides spp. at 49.3% (36/73). The findings were in accordance with an Indian study, where
nim gene positivity was seen in 53% (20/38) of
Bacteroides spp. [
26]. Out of the 24% (48/200)
nim gene-positive isolates, 27% (13/48) of isolates carried the
nim genes, yet they were phenotypically susceptible, which is possibly due to the absence of IS elements that regulate the expression of the
nim genes. Of all the IS elements reported so far, the isolates were tested for the presence of IS
1186, which has been frequently studied in anaerobes [
40]. Unexpectedly
, all 200 isolates tested negative for IS
1186, which shows that possibly other IS elements are prevalent in our geographic region, or alternative non-
nim-induced mechanisms of metronidazole are more active in our isolates. The non-
nim-gene-based mechanisms of metronidazole resistance such as overexpression of efflux pump,
RecA proteins, rhamnose catabolism regulatory protein, activation of antioxidant defense systems, and deficiency of ferrous iron transporter feoAB have been described; however, the literature is scant [
40]. In our study, the ideal possibility of elucidating the non-
nim-gene-mediated resistance can be attributed to the
Veillonella genus. The genus displayed the highest resistance to metronidazole yet showed the minimum number of resistant genotypes, as only 1 of 17
Veillonella-resistant isolates harbored the
nim gene.
The emergence of metronidazole resistance has led to the use of carbapenems, particularly imipenem, for the treatment of anaerobes. Resistance to imipenem is due to a metallo-ß-lactamase encoded by the
cfiA gene, which is expressed by the upstream IS element that carries a promoter to drive the gene expression [
31]. Data obtained in the West from various studies reports an overall carbapenem resistance varying from 1 to 9.6% and
cfiA positivity of 5 to 27% in
B. fragilis [
41]. The East Asian literature shows a 7% imipenem resistance in
B. fragilis, 4% in
Fusobacterium spp. over 16 years [
24].
B. fragilis from China demonstrates a remarkably high resistance, reaching as high as 38.5% over a period of one year [
38]. In other Asian regions, resistance has increased from 0 to 24.1% in 5 years [
38,
42], whereas in our isolates, it was only 0.5%. None of our isolates were detected with the presence of IS elements; however, 10% represented “silent
cfiA carriers” as these isolates were phenotypically susceptible despite carrying the
cfiA gene.
In our study, the presence of the
cfiA gene was only restricted to
B. fragilis strains; based on that, we have categorized our
B. fragilis strains into Division I (
cfiA-negative) and Division II (
cfiA-positive). This categorization holds a potential significance as MALDI-TOF MS can successfully differentiate these strains based on the well-defined mass spectra produced by
cfiA-negative and -positive strains. The limitation is that MALDI-TOF MS can only identify the resistant genotype. As shown in a study, the IS acquisition and resultant carbapenem resistance were not differentiated via MALDI-TOF MS [
43]. In our study, 42.5% of the
B. fragilis isolates harbored silent
cfiA genes and belonged to Division II; however, the unaccompanied presence appeared to be a handicap without insertion sequences. The results show that presently, imipenem resistance is not a challenge to current treatment options. However, these isolates may act as a reservoir of imipenem resistance since
B. fragilis harbored a significant repertoire of the
cfiA gene. With time, this may pose a threat, as susceptible strains may become resistant via the one-step acquisition of mobile IS elements from non-adjacent
cfiA sites [
12].
Another reason to study the distribution of
cfiA genes is their association with a decrease in susceptibility to BL-BLIs such as piperacillin–tazobactam [
44]. Though in our isolates, we determined that out of 27.5% (55/200) piperacillin–tazobactam-resistant isolates, 12.7% (7/55) carried the
cfiA gene. We cannot comment on whether the presence of
cfiA was responsible for seven piperacillin–tazobactam-resistant phenotypes, as IS elements required for the
cfiA expression were absent, and such expression of the
cfiA gene without IS elements is not reported in the literature. However, two studies have demonstrated that the strains showing heterogeneous resistance to carbapenems may exploit a weak self-promotor of the
cfiA genes, which we think could be likely as our
cfiA-positive strains showed reduced susceptibility towards imipenem as well [
45,
46]. However, piperacillin–tazobactam resistance could be attributed to the presence of other ß-lactam genes (
cfxA) as six out of seven piperacillin–tazobactam-resistant phenotypes containing the
cfiA gene contained the
cfxA gene as well, but lacked
cepA.
In our study, piperacillin–tazobactam resistance showed a strong correlation with the presence of the
cepA gene.
cepA is ß-lactamase that can hydrolyze both penicillins and cephalosporins; however, it is frequently inhibited by ß-lactamase inhibitors [
47]. Only 19 out of 200 clinical isolates showed the presence of
cepA, which was limited to
B. fragilis only. The
cepA genes are known to provide both low- and high-level resistance to penicillins. The high-level resistance is attributable to the overexpression of the
cepA gene due to the transcriptional activation via putative IS family IS
21 [
48]. We did not study the
IS21 elements in our isolates, but all isolates harboring the
cepA gene exhibited piperacillin–tazobactam resistance; thus, we propose the presence of these elements in our isolates.
The
cfxA gene was found in 29% (16/55) of piperacillin–tazobactam-resistant phenotypes and 18.6% (27/145) of susceptible phenotypes. These results are in line with those of previous studies which have reported the presence of
cfxA in amoxicillin–clavulanate-resistant strains, thereby suggesting the differential expression of the
cfxA gene [
49]. Our results confirm a significant association between the
cfxA gene and cefoxitin resistance; however, 11/53 isolates showed resistance but did not harbor the gene. This may suggest for other resistance mechanisms, e.g
., reduced penetration of the drug through the outer membranes, upregulation of drug efflux, presence of other ß-lactamases, accumulation of mutations in the outer membrane porin molecules, and penicillin-binding proteins [
31,
49].
The
cfxA gene was seen widely distributed among non-
Bacteroides species, explaining their role in conferring resistance to ß-lactams in other anaerobic isolates, as
cepA and
cfiA were somehow restricted to
B. fragilis only. Furthermore, our results demonstrated a constrained co-existence of
cepA and
cfiA gene in
B. fragilis isolates, as all the isolates positive with
cepA gene lacked the
cfiA gene and vice versa. Similar associations have been seen in other studies, confirming the acquisition of these two genes in unique and separate events. The origin of these genes has also been discriminated on the basis of different G+C mol% content and their localization, as
cepA is chromosomal, whereas
cfiA is located on both chromosomes and plasmids [
45,
50,
51]. It is believed that the two divisions have remained continuously isolated to circumvent HGT, and gene transfer between these two divisions is unlikely [
52]. Therefore, we confirmed whether any AMR gene was found to have an affinity to any of these divisions. No significant association of any other AMR gene was markedly noted with the members of Divisions I and II.
In our study, all organisms showed 100% susceptibility to chloramphenicol. The absence of chloramphenicol resistance may reflect the occasional use of this drug in our clinical setup. For more than half of these isolates, the clustering of MICs was observed around the breakpoints (12% at 8 mg/L; 52% at 4 mg/L). Similar clustering was observed in another study as well [
53], which may pose a threat in case of MIC creep over time. None of the tested isolates harbored chloramphenicol resistance genes, thereby confirming that they do not act as a reservoir of chloramphenicol resistance.