Advances in drug resistance and resistance mechanisms of four colorectal cancer-associated gut microbiota

Mechanisms of β-lactamase-mediated antibiotic resistance

It was shown that the FUS-1 enzyme found in F. nucleatum is the first of its class D β-lactamase-producing enzymes (Dupin et al., 2015). Class D β-lactamase genes, often identified as intrinsic resistance determinants in environmental bacteria, occur in mobile genetic elements carried by clinically important pathogenic bacteria (Yoon & Jeong, 2021), Reported FUS-1 genotype of F. nucleatum from a clinical isolate of human pathogenic F. nucleatum (Voha et al., 2006).

Other resistance mechanisms

Studies have shown that exposure to a particular antibiotic interferes with the susceptibility of F. nucleatum to several antibiotics and may reduce susceptibility to antibiotics with similar mechanisms of action or the same resistance mechanism (de Souza Filho et al., 2012). de Souza Filho et al. (2012) showed that selected β-lactam strains were also much less susceptible to chloramphenicol and metronidazole. However, it has been shown that while resistance to β-lactam antibiotics in most Gram-negative bacteria is mediated by β-lactamase production, other mechanisms of antibiotic resistance include changes in penicillin-binding proteins, decreased permeability, or increased efflux pump activity (Huemer et al., 2020), Thus, in the study by de Souza Filho et al. (2012), it was again noted that no significant differences were observed in the antimicrobial drug susceptibility patterns of ampicillin and ampicillin-sulbactam, which suggests that cross-resistance between β-lactams, chloramphenicol, and metronidazole may indicate the induction of common mechanisms of resistance, such as changes in cell wall permeability. This suggests that there is a mechanistic association between antibiotics and that perhaps a combination of antibiotics may be effective in treating resistant strains. Studies have shown that antibiotic combinations can be more effective than the use of a single antibiotic because our bodies harbor a variety of microbiota, including aerobic, anaerobic, and facultative bacteria. Baumgartner & Xia (2003) demonstrated that combining metronidazole with penicillin V increased the activity of metronidazole (Baumgartner & Xia, 2003; Jacinto et al., 2008). A study from the United States showed that F. nucleatum utilizes type IV pili to facilitate the natural ability to import DNA and transfer genes horizontally, and found that F. nucleatum ATCC 23726 was able to ingest genomic DNA containing a chloramphenicol resistance gene and subsequently induced horizontal transfer of the gene to the chromosome of a wild-type strain (Sanders et al., 2023).

Antibiotic resistance mechanisms in E. coli

E. coli is one of the most common bacterial species in our intestinal system. A total of 60% of E. coli isolates isolated from biopsy samples of CRC patients were resistant to multiple antibiotics by Tariq et al. (2022). A study from Mexico showed that E. coli bacteria isolated from colorectal disease tissues were highly resistant to more than 10 antibiotics, including ampicillin, tetracycline, and ciprofloxacin (Canizalez-Roman et al., 2021). This suggests that there may be a greater correlation between CRC development and drug-resistant strains of E. coli.

Drug resistance mediated by mutations in drug resistance genes

First, it has been shown that mutations in drug-resistant genes are one of the main mechanisms for the development of drug resistance in E. coli, including spontaneous mutations, hypermutations, and adaptive mutations (Pulingam et al., 2022). Among them, Rodríguez-Verdugo, Gaut & Tenaillon (2013) showed that spontaneous mutations can be driven by, for example, interfering with DNA replication, and that resistance to rifampicin in E. coli is achieved by mutations in the rpoB gene encoding an RNA polymerase; hypermutation confers an evolutionary advantage to the bacterial species during adaptation to new environments or stressful conditions (Oliver & Mena, 2010); adaptive mutations refer to mutations at the transcriptional level that occur in the bacterial genome to adapt to changes in the survival environment, and when the environmental stress is lifted, the bacterial genome returns to its original condition (Fernández & Hancock, 2012; Pulingam et al., 2022).

According to the study, AmpR and AmpC are encoded by the ampR and ampC genes, respectively, suggesting that the ampR and ampC genes are related, and that AmpR serves as a transcriptional activator that binds to the cis-trans region upstream of the ampC gene promoter, thus acting to regulate AmpC (Philippon et al., 2022). Since the transcriptional regulator (AmpR) expressing AmpC is reportedly not present in E. coli, by what mechanism is the AmpC gene regulated? Haenni, Châtre & Madec (2014) showed that there are five highly conserved mutations in the promoter of AmpC, while the mutations in the attenuator are much more frequent, which are mainly attributed to spontaneous mutations in the promoter and attenuator (Haenni, Châtre & Madec, 2014; Kakoullis et al., 2021). This suggests to us that using promoter or attenuator mutation sites as drug targets may be an effective strategy to deal with AmpC-type E. coli drug resistance.

Hypermutagenic bacteria are microorganisms that have a stronger affinity for spontaneous mutations due to DNA repair defects or avoidance system errors (Oliver & Mena, 2010), resulting in greater adaptability to antibiotics. Hypermutagenic phenotypes of E. coli have been reported earlier (Denamur et al., 2002). According to studies, the mismatch repair system (MMR) is particularly important in the phenomenon of hypermutation, as it is not only one of the main causes of bacterial hypermutation, but also one of the main factors in the progression of CRC (Jin & Sinicrope, 2022). It has been shown that the most commonly mutated gene in strains with hypermutated phenotypes of E. coli is the mutHLS gene of the DNA methyl-orientation MMR pathway (Ellington et al., 2006).

Adaptive mutation denotes a temporary increase in the viability of a bacterium when it is attacked by an antibiotic, mainly due to changes in the bacterial genome or protein expression as a result of other environmental factors to which the bacterium is subjected, such as the nutrient conditions to which it is subjected or the sub-inhibitory concentration of the antibiotic itself (Fernández & Hancock, 2012). Simply put, adaptive mutations may be the induced mechanism by which bacteria produce genetic variability in a stressful state (McKenzie et al., 2000). It was shown that E. coli exposed to sublethal concentrations of streptomycin induced the expression of recA- and umuDC-independent mutant phenotypes on transfected M13 single-stranded DNA (Pulingam et al., 2022).

β-lactamase-mediated drug resistance

Another major mechanism by which E. coli develops resistance to β-lactam antibiotics is mediated by β-lactamase activity (Bush, 2018). The Ambler system based on sequence information indicates that β-lactamases are classified into four distinct classes called A, B, C, and D (Tooke et al., 2019). Epidemiologic investigations have shown that the prevalent strains have different rates and mechanisms of resistance at different times, in different regions, and in different populations (Xing, Bin & Lei, 2020). Strains with class A extended-spectrum β-lactamase genotypes (AmpA-type β-lactamases) are the most common, and class C β-lactamase (AmpC-type β-lactamases) genotypes are highly resistant, which has attracted extensive interest from researchers (Poirel et al., 2018).

Class A β-lactamases include penicillinase type 1 (PC1) (Tooke et al., 2019), TEM (named after Temoneira, the patient from whom the isolate originated) (Datta & Kontomichalou, 1965), sulfhydryl variant (SHV) (Chaves et al., 2001), Cefotaximase (CTX-M) (Bauernfeind, Schweighart & Grimm, 1990) and Klebsiella pneumoniae carbapenemases (KPC) (Rapp & Urban, 2012), it has been shown that the key to the ability of these class A β-lactamases to render antibiotics less effective is their ability to propagate on plasmids and other mobile genetic elements in a range of Gram-negative bacteria, as well as the fact that they broaden their spectrum of activity as new substrates are discovered in the clinic, which is also referred to in clinical terms as “extended-spectrum” phenotypic β-lactamases (ESBLs) (Tooke et al., 2019). The production of extended-spectrum β-lactamases is the main reason for the resistance of E. coli to β-lactam antibiotics. At present, the most reported extended spectrum β-lactamases are CTX-M type enzymes, which can be divided into five categories: CTX-M-1, CTX-M-2, CTX-M-8-8, CTX-M-9, and CTX-M-25 (Seo & Lee, 2021).

Finally, with the emergence of multidrug-resistant strains, some highly effective antibiotics have become “antibiotics of last resort”, with polymyxin E considered to be the last line of defense against multidrug-resistant and carbapenem-resistant Gram-negative bacteria, but in recent years there have been an increasing number of reports of colistin (polymyxin E) resistant bacteria (Hussein et al., 2021). Liu et al. (2016) revealed by whole plasmid sequencing that polymyxin E resistance may be caused by the plasmid-mediated mcr-1 gene. Dadashi et al. (2022) showed that the prevalence of colistin-resistant E. coli containing the mcr-1 resistance gene was reported to be 66.72%, 25.49%, 5.19%, 2.27%, and 0.32% in Asia, Europe, the Americas, Africa, and Oceania. Tigecycline and colistin are the last antibiotics against carbapenem-resistant bacteria, and it was found that a plasmid encoding the colistin resistance gene, mcr-1, and the tigecycline-resistance enzyme, tet (X6), existed in the same strain of E. coli, and that the presence of the two plasmids made E. coli co-resistant to these two classes of antibiotics (Xu et al., 2021b). Researchers predicted that the emergence of plasmids co-integrating mcr-1 and tet (X4) would pose a significant threat to humans, Lu et al. (2021) obtained seven evolutionary plasmids carrying mcr-1 and tet (X4) in vitro and further demonstrated that the plasmids could be inherited. Shafiq et al. (2022) detected a broadly resistant E. coli isolate co-carrying plasmid-mediated blaNDM-5 and tet (X4) genes.

Horizontal gene transfer-mediated drug resistance

Tigecycline is used as a broad-spectrum glycylcycline antibiotic for the treatment of E. coli infections, but tigecycline-resistant strains have emerged clinically. The mechanism of resistance involves flavin-dependent monooxygenase (tetX), and studies have shown that the emergence of tetX can increase resistance to tigecycline (Li et al., 2016). The main resistance mechanism is that tet (X3/X4) can directly inactivate tigecycline potency through hydroxylation of carbon 11a (Cui et al., 2020). As tet (X3/X4) is present on mobile plasmids, this leads to horizontal transfer of resistance across strains and species. Studies suggest that high levels of plasmid-mediated tigecycline resistance genes tet (X3) and tet (X4) emerged in 2019, which poses a significant threat to global public health (Li et al., 2023).

It was shown that conjugative plasmid-mediated horizontal gene transfer is the main mechanism mediating the spread of antibiotic resistance genes in E. coli, conjugation plasmids are the main vectors for spreading antibiotic resistance and are elements that can mediate their own transmission through conjugation (Mota-Bravo et al., 2023; Palomino et al., 2023). Minja, Shirima & Mshana (2021) showed that out of 51 blaCTX-M-15 positive donor isolates, 45 transferred the plasmid via conjugation. It has been shown that E. coli performs horizontal gene transfer mainly through DNA released by cell lysis, and that it can transfer DNA to different bacteria by secreting vesicles loaded with plasmid DNA into the environment (Cooper, Tsimring & Hasty, 2017; Maeusli et al., 2020). According to Kulkarni, Nagaraj & Jagannadham (2015) the presence of antibiotics and stress response caused by changes in the host environment lead to the formation of vesicles in E. coli strains, and vesicles isolated from E. coli protect the bacteria from membrane-active antibiotics (colistin and polymyxin B), suggesting that vesicles protect the bacteria from the growth inhibitory effects of certain antibiotics.

Drug resistance in E. coli mediated by efflux pumps

As early as the 1990s, the drug-resistant nodular differentiation family (RND) was identified in E. coli and is represented by the AcrAB-TolC pump in E. coli (Fig. 5), which mediates bacterial multidrug resistance. RND is located in the inner membrane, and as a transporter protein, it must interact with periplasmic bridging proteins and outer membrane channels in order to excrete drugs directly across the inner, periplasmic, and outer membranes to the outside of the cell membrane (Li, Plésiat & Nikaido, 2015).

In the presence of E. coli AcrAB-TolC, drug efflux through the cell membrane forms an effective permeability barrier due to the presence of low-permeability pore proteins (i.e., “slow pore proteins”), which are capable of generating multidrug resistance (Li, Plésiat & Nikaido, 2015). It was shown that E. coli AcrAB-TolC is regulated by the multiple antibiotic resistance manipulator Mar, which is expressed as two separate transcriptional units, one of which is MarRAB, controlled by MarO, which specifies a Mar repressor (MarR), an activator (MarA) and a small protein (MarB), who are respectively encoded by marR, marA and marB, with MarB located downstream of MarA (Alekshun & Levy, 1999; Weston et al., 2018). Under normal conditions, MarR represses the MarRAB operon by binding to the two palindromic sequences of marO, but when antibiotics are encountered, repression of MarR is disrupted and transcription of marRAB occurs. It has been shown that de-repression of the marRAB operon results in the expression of MarA: each regulator promoter has a binding site called a “marbox” binding site, MarA undergoes positive feedback when it binds to DNA sequences upstream of the marbox, the repressor site of MarR, so this represses marR and allows marA to be activated. MarA expression promotes the activation of several genes in its regulator, including the AcrAB and TolC genes, which increases drug efflux and lead to multidrug resistance (Martin et al., 1999; Weston et al., 2018). It was shown that drug resistance aspects were affected in strains lacking the gene encoding the AcrAB-TolC multidrug efflux pump (ΔtolC or ΔacrB) (Kobylka et al., 2020).

Drug resistance mediated by mutations in drug resistance genes

Resistance to β-lactam antibiotics in E. faecalis occurs by two mechanisms: production of β-lactamases and alteration of the affinity of penicillin-binding proteins for β-lactam antibiotics (Ono, Muratani & Matsumoto, 2005). Studies have shown that resistance to β-lactam antibiotics in E. faecalis can be mediated by the production of a non-inducible β-lactamase (Herrera-Hidalgo et al., 2023). The presence of penicillin-binding protein 4 (PBP4) in E. faecalis results in a low affinity for β-lactam antibiotics, which leads to a certain degree of resistance to β-lactam antibiotics in E. faecalis (Urban-Chmiel et al., 2022), and PBP4 is considered to be the key molecular basis for the resistance of E. faecalis to β-lactam antibiotics (Lazzaro et al., 2021). Epidemiologic data suggest that the progressive increase in resistance to β-lactam antibiotics in E. faecalis is attributable to overexpression of PBP4 (Lazzaro et al., 2021). PBP4 belongs to the class of transpeptidases involved in the formation of the peptidoglycan layer; whereas β-lactam antibiotics block peptidoglycan biosynthesis via PBP4 acylation (Moon et al., 2018; Timmler et al., 2022). Further studies revealed that PBP4-mediated resistance to β-lactam antibiotics in E. faecalis was associated with the CroRS two-component signaling system (TCS) (Kellogg et al., 2017). Timmler et al. (2022) showed a correlation between PBP4 and the CroR system, the exact mechanism of which requires further experimental confirmation.

Vancomycin is commonly used for severe drug-resistant Gram-positive bacterial infections (National Institute of Diabetes and Digestive and Kidney Diseases, 2012a), and plays a twofold role in the adjuvant treatment of CRC: on the one hand, vancomycin depletes butyrate-producing bacteria in the gut, thereby enhancing the efficacy of radiotherapy; on the other hand, it inhibits the bacteria that convert primary bile acids into secondary bile acids, thereby enhancing the efficacy of anticancer therapy (Singh et al., 2020; Yang et al., 2023). The ability of E. faecalis to cause the development of CRC has been certified, but recent studies have found that vancomycin-resistant E. faecalis plays a more pronounced role in the proliferation, migration, and angiogenesis of CRC cells compared to E. faecalis (Zhang et al., 2024a). Since the first discovery of vancomycin-resistant E. faecalis clone sequence type 796 (ST796) in Australia in 2011, drug-resistant strains are now widely reported worldwide (Li, Walker & De Oliveira, 2022). A total of nine vancomycin resistance cluster genes of the Van family have been identified, with VanA and VanB being the most common among clinical isolates (Raza et al., 2018; Zalipour, Esfahani & Havaei, 2019). Taji et al. (2019) showed that vanA gene was detected in 37.7% of E. faecalis isolates. Strateva et al. (2019) tested and characterized an isolated strain of E. faecalis and found that the vanA gene cluster was on a segregating overlapping cluster with two repetitive IS1216E sequences around its flanks, followed by transfer experiments by filter mating assay using E. faecalis JH2-2 as a receptor strain, which showed unsuccessful results in terms of transferring vancomycin resistance, suggesting that the possible location of the vanA gene cluster at the chromosomal position. According to the study, the Tn1549 transposon carries the vanB operon on it (Simar et al., 2023). Although there are fewer reports on VanB, it has attracted much attention from scholars because of its high detection rate (Sadowy, 2021).

The main mechanism of resistance to linezolid in enterococci involves the G2576T mutation in the 23S rRNA gene (Rodríguez-Noriega et al., 2020), and other mechanisms are mutations in the L3 and L4 ribosomal proteins as well as in two plasmid vector genes (cfr and optrA) (Arias & Murray, 2012). According to research, optrA is located in a new gene cluster containing the chloramphenicol output gene fexA. The protein product of optrA belongs to the ATP binding cassette (ABC)—F protein superfamily, and its resistance is mediated by ribosome protection. Compared with other gene determinants such as cfr or 23S rRNA and ribosomal protein mutations, mutations in optrA are a common cause of oxazolidinone resistance in E. faecalis (Roy et al., 2020). The research results of Deshpande et al. (2018) showed that isolates with chromosome localization of optrA exhibited different array structures. The flank regions of the optrA arrays of E. faecalis from Thailand and isolates from France were different. From the results of gene array analysis, it can be seen that with the continuous spread of optrA, a large degree of gene rearrangement is occurring, and the core genetic elements remain similar. However, in isolates from different geographical locations, their positions in the array are not the same (Deshpande et al., 2018). Therefore, the monitoring of the flanking regions of the optrA array is of great clinical importance.

Horizontal gene transfer-mediated drug resistance

The genome of E. faecalis was found to be highly plastic, and resistance to other antibiotics, such as high levels of aminoglycoside resistance, high levels of ampicillin resistance, and vancomycin resistance, is readily acquired through mutations in resistance genes or through horizontal transfer of genetic elements conferring resistance determinants (García-Solache & Rice, 2019). It was confirmed that transposons constitute the majority of the mobile genetic elements present in the genome of E. faecalis, and that Tn916, as the first confirmed conjugative transposon, carries tetracycline resistance and is able to be transferred to the chromosome of the recipient cell or to a conjugative plasmid by transposition, and that transposon incorporation into the conjugative plasmid increases the frequency of transfer (García-Solache & Rice, 2019).

Data from the China Antimicrobial Drug Surveillance Network (CDSN) showed that the high-level gentamicin resistance rate in E. faecalis ranged from 28.8% to 61.4% from 2005 to 2017, and was mainly mediated by the bifunctional enzyme encoded by the fused aac(6′)-aph(2″) gene in E. faecalis, 6′-acetyltransferase-2″-phosphotransferase (Ferretti, Gilmore & Courvalin, 1986). The aac(6′)-aph(2″) gene is plasmid-borne in most cases and is located on the E. faecalis Tn5281-like transposon (Zhang et al., 2018). The non-truncated form of Tn5281 consists of a central region containing the aac(6′)-aph(2″) gene flanked by inserted inverted repeats of sequence IS256, whereas the truncated form is the aac(6′)-aph(2″) 3′- or 5′-end, or both lacking IS256 (Zhang et al., 2018). Daikos et al. (2003) showed that 24 out of 30 isolates containing the truncated form transferred gentamicin resistance, while only 3 out of 34 isolates containing the nontruncated form transferred gentamicin resistance, suggesting that the truncated variant is mobile and more effective in transferring gentamicin resistance.

It has been shown that the bacterial type IV secretion systems (T4SSs) are a functionally diverse translocation superfamily, and that one of its major functional subfamilies is the conjugation system that mediates DNA transfer between bacteria, and that the conjugation system can propagate mobile genetic elements that typically encode bacterial resistance to antibiotics (Costa et al., 2021). The transferable plasmids of multidrug-resistant E. faecalis are T4SS with a functional plasmid-encoded (PE-T4SS) and a chromosome-encoded T4SS (CE-T4SS); compared with PE-T4SS, CE-T4SS exhibits different characteristics in protein structure and can mediate large genome-wide gene transfer (Hua et al., 2022). The study by Hua et al. (2022) identified a widely distributed CE-T4SS in E. faecalis, and to better understand the process of gene transfer, the researchers analyzed the oriT element (Hua et al., 2022). At the initiation site of horizontal gene transfer, the researchers identified four putative oriT with reverse complementary structural domains, oriT1-oriT4, and hypothesized experimentally that oriT4 is the required initiation site for horizontal gene transfer mediated by CE-T4SS in D5165. The investigators selected CE-T4SS+ reference strain ATCC 19433 as a donor and a popular erythromycin-resistant ST179 strain, S6008, as a recipient, suggesting that CE-T4SS induces gene transfer in the host (Hua et al., 2022).

While the previous discussion was about horizontal transfer of drug resistance genes in Gram-negative bacteria through membrane vesicles, we learned some results about whether membrane vesicles can work in Gram-positive strains of E. faecalis, which is a Gram-positive bacterium. Initial studies on whether membrane vesicles produced by E. faecalis carry out the transfer of drug-resistant genes showed that there was no detection that E. faecalis could carry out horizontal gene transfer via membrane vesicles (Afonina et al., 2021). However, a recent study by Zhao et al. (2024) showed that a strain of E. faecalis carrying a linezolid resistance gene could successfully transfer the resistance gene to another strain of E. faecalis via a membrane vesicle, and that the recipient strain still had the potential to transmit the resistance gene.

Multidrug efflux system-mediated drug resistance

EfrAB, a heterodimeric multidrug ATP-binding cassette (ABC efflux system family) transporter protein, causes endogenous resistance to antibiotics including fluoroquinolones in Enterococcus spp. (Shiadeh et al., 2020). Shiadeh et al. (2020) showed that ciprofloxacin-resistant E. faecalis isolates showed varying degrees of overexpression of efrA and efrB genes. A study by Esfahani et al. (2020) found no significant relationship between the upregulation of the expression of the efflux pump and the level of minimal inhibitory concentration, and the researchers found isolates without any mutations in the expression of efflux genes but with drug resistance, and furthermore, 23 homologs of the ABC family of transporter proteins were detected in E. faecalis isolates. The above evidence suggests that the development of fluoroquinolone resistance may be the result of ABC family transporter proteins but not necessarily EfrA or EfrB.

Tetracycline is one of the most commonly used broad-spectrum antibiotics, and many bacteria have developed resistance to this antibiotic, the most common mechanism involves membrane-associated proteins (TetA), which exclude the antibiotic from the bacterial cell before inhibiting peptide elongation (Ramos et al., 2005). According to research reports, tetracycline repressor protein (TetR) family proteins affect the tolerance of E. faecalis to tetracycline by controlling the expression of tetracycline efflux pump genes, such as the regulation of efflux pumps by the tetA gene (Gu et al., 2020). TetR proteins control the expression of the tet gene, the product of which confers bacterial resistance to tetracycline (Ramos et al., 2005). Research has shown that tetR and tetA are adjacent and oriented differently, and their gene products tightly control the expression of tetA and tetR (Ramos et al., 2005), TetR homodimers block the promoter of efflux pump genes by binding to repeat palindromic sequences in the upstream gene intergenic region using helix α1 to α3. In the presence of tetracycline, TetR homodimers interact with tetracycline and magnesium to form protein ligand complexes, which cause conformational changes in the TetR ligand complex, leading to the release of TetR homodimers from the promoter of efflux pump genes. This separation activates the expression of tetracycline related efflux pumps and squeezes tetracycline out of bacterial cells (Gu et al., 2020).