Antibiotic Resistance Pattern of Bacteroides Fragilis Isolated From Clinical and Gastrointestinal Specimens

Background: Bacteroides fragilis is a part of the normal gastrointestinal ora and the most prevalent anaerobic bacteria causes’ infection. It is highly resistant to antibiotics and contains abundant antibiotic resistance mechanisms. Methods: The antibiotic resistance pattern of 78 isolates of B. fragilis (56 strains from the gastrointestinal [GI] tract and 22 strains from clinical samples) was investigated using agar dilution method. The gene encoding Bacteroides fargilis toxin bft, and antibiotic resistance genes were targeted by PCR assay. Results: The highest rate of resistance was observed for penicillin G (100%) followed by tetracycline (74.4%), clindamycin (41%) and cefoxitin (38.5%). Only a single isolate showed resistance to imipenem which contained cA and IS1186 genes. All isolates were susceptible to metronidazole. Accordingly, tetQ (87.2%), cepA (73.1%) and ermF (64.1%) were the most abundant antibiotic-resistant genes identied in this study. MIC values for penicillin, cefoxitin and clindamycin were signicantly different among isolates with the cepA, cfxA and ermF in compare with those lacking such genes. In addition, 22.7% and 17.8% of clinical and GI tract isolates had the bft gene, respectively. Conclusions: Therefore, it is of utmost importance to determine the antibiotic resistance patterns of B. fragilis periodically in different geographical areas to provide a suitable treatment prole for patients and to prevent improper antibiotic prescriptions.


Background
Bacteroides fragilis is an anaerobic, Gram-negative bacteria and a part of the human gastrointestinal microbiota but can cause severe infections in human opportunistically. Genus Bacteroides accounted for about 25% of gastrointestinal (GI) tract ora [(1, 2)]. Among various species of this genus, Bacteroides fragilis (B. fragilis) has been also introduced as the most abundant opportunistic anaerobic bacterium isolated from clinical specimens (3). The bacteria form 1-2% of the normal ora of the gastrointestinal tract and, if dislocated into other anatomical sites, develop various infections such as abdominal infections, abscesses, and bacteremia with a mortality rate of about 19% (1,4).
Relevant studies have further revealed that B. fragilis exhibits the highest antibiotic resistance and the most abundant antibiotic resistance mechanisms compared with other anaerobic bacteria in the GI tract (5). This not only makes it di cult to treat infections caused by B. fragilis, but also has the potential to act as a reservoir of antibiotic-resistant genes (6), leading to the transfer of resistance genes to other normal bacterial ora through integrated transposons, integrated genetic elements, as well as conjugative plasmids (7). In this respect, different resistance patterns of this bacterium have been so far reported from different parts of the world. There have been reports of increased resistance to carbapenems and beta-lactams among B. fragilis isolates worldwide (8)(9)(10)(11)(12). Of note, the rate of resistance to metronidazole, as an effective antibiotic against anaerobic bacteria, is about 1%, but some reference laboratories have reported a resistance rate of up to 7.5% (13)(14)(15). Also, the number of multidrugresistant B. fragilis isolates has augmented over the last decade (16-18).
Bacterial virulence factors have important roles in the pathogenicity of B. fragilis. Enterotoxigenic B. fragilis (ETBF) also produces a 20 kDa metalloprotease toxin, mainly known as B. fragilis toxin (BFT) (19,20). Studies in this line have further established that the ETBF strains are more pathogenic than nontoxigenic ones and they are associated with various diseases such as septicaemia, diarrhoea, irritable bowel syndrome (IBS), and colorectal cancer (CRC) (10,21).
However, due to the costly and time-consuming process of isolation and identi cation of B. fragilis, antibiotic susceptibility testing is not routinely performed in laboratories (22,23). Therefore, in this study antibiotic resistance pro les of B. fragilis isolated from the GI tract and clinical samples were evaluated using phenotypic and genotypic methods.

Study population
The current cross-sectional study examined two populations, the patients, and the healthy controls. This study was approved by the Ethics Committee of National Institute for Medical Research development in Iran (NO. 971329). Informed consent was obtained from all individual participants.
The patient population included people suspected of having anaerobic infection hospitalized in different wards of Imam Khomeini Hospital of Tehran, and the healthy population included people with no history of GI disease or antibiotic consumption for the past three months.
In the sampling process from the patients, 130 different clinical samples were collected from hospitalized patients in different wards of Imam Khomeini Hospital during 1 year from August 2018 to August 2019.
Sampling, culture and isolation of anaerobic bacteria were performed (24).
In the sampling process from healthy individuals, 40 biopsies of the rectum were collected by a physician during colonoscopy. To isolate B. fragilis, the biopsy sample was homogenized by mortar and pestle, and then 2-3 drops were inoculated on a plate containing Bacteroides Bile Esculin Agar (BBE) and Brucella Blood Agar (BBA) containing 5% sheep blood, vitamin K1 (0.5 mg/L) and hemin (5 mg/L) and cultured by isolation method. The cultivated plates were incubated for 48-72 hours at 37ºC under anaerobic conditions. The black-colored colonies on the BBE medium and the grown ones on the BBA medium (5-10 colonies) were subcultured on the BBA medium. Ultimately, after observing the obligate anaerobic, gramnegative, bile esculin-positive and catalase-positive coccobacilli, the isolated strains were preserved at 80ºC using 5% glycerol (25).

Identi cation of anaerobic bacteria
The anaerobic bacteria were phenotypically identi ed based on colony morphology, gram staining, and differential tests such as catalase, indole, bile disc, and nally Vitek 2 system (Biomerieux, France). Two polymerase chain reactions (PCR) were also performed to amplify the 16S rRNA gene fragment; the rst reaction to con rm the B.fragilis group and the second reaction to approve the B. fragilis species (26,27). The 16S rRNA gene was sequenced for B. fragilis strains and then submitted to the GenBank sequence database.
Antibiotic susceptibility of B. fragilis isolates The antibiotic susceptibility testing of B. fragilis isolates was conducted using agar dilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (28). The tested antibiotics included ampicillin/sulbactam, piperacillin/tazobactam, penicillin G, tetracycline, imipenem, meropenem, clindamycin, cefoxitin, and metronidazole. Different concentrations of the antibiotics were also prepared on the BBA medium containing vitamin K1 (0.5 mg/l) and hemin (5 mg/l).
Moreover, 10 µl of microbial suspension with a density of 107 colony-forming unit (CFU) ml-1 was added to the plates containing antibiotics and a negative control plate to achieve a nal dilution of 105 CFU per spot. The plates were also incubated for 48 hours at 36ºC under anaerobic conditions. After the incubation period, the Minimum Inhibitory Concentration (MIC) were calculated according to the CLSI guideline.

Identi cation of resistance genes
The presence of IS1186 and c A genes (associated with resistance to carbapenems), the cepA and cfxA genes (associated with resistance to beta-lactams), the ermF, ermB, and mefA genes (associated with resistance to clindamycin), the tetQ gene (associated with resistance to tetracycline) and the nim gene (associated with resistance to metronidazole) were determined by the PCR in B. fragilis isolates (29). In order to detect the bft gene using PCR, parts of this gene were ampli ed (30).

Statistical analysis
Data were analysed using the SPSS ver. 18.0 (SPSS Inc., Chicago, IL). The Chi-square test was performed to calculate signi cant differences between presences of antibiotic resistance genes among resistant strains in comparison to non-resistant strains. Also, Mann-Whitney test was employed to examine signi cant differences of MIC value for each antibiotic class among isolates with resistance genes in compare with isolates lacking these genes. A p-value less than 0.05 was considered as statistically signi cant.
From 40 colorectal tissue biopsies in healthy individuals, 56 B. fragilis isolates were identi ed in 24 specimens (60%). The antibiotic resistance of 78 B. fragilis isolates (22 isolates from clinical samples and 56 isolates from the GI tract of healthy individuals) was determined using the agar dilution method. Table 2 shows the antibiotic resistance pattern of B. fragilis with the MIC 50 and MIC 90 values (µg/mL). The B. fragilis isolates also had the highest resistance to penicillin (100%), tetracycline (74.4 %), clindamycin (41%) and cefoxitin (38.5%).
The tetQ, ermF, ermB, c A, cepA, cfxA, mefA, nim genes and the insertion sequence IS1186 were further searched to evaluate antibiotic resistance by the PCR. Absolute and relative frequencies of resistance and insertion sequences genes are presented in Table 3.
In this study, the tetQ (87.2%), cepA (73.1%) and ermF (64.1%) were the most abundant antibioticresistant genes. The nim and ermB genes were not detected in any of the isolates. The IS1186 sequence in the upstream region of the c A gene was detected in one isolate (1.3%); this isolate was also resistant to imipenem.
The presence of the cfxA and ermF genes were signi cantly higher in cefoxitin and clindamycin resistant isolates in compare with cefoxitin and clindamycin susceptible isolates (p=0.001, 0.000).
In addition, MIC value of penicillin, cefoxitin and clindamycin were signi cantly difference among isolate with the cepA, cfxA and ermF genes in compare with isolates lacking these genes (p=0.002, 0.000, 0.001) (Fig. 1).
The bft gene was observed in 22.7% and 17.8% of the clinical and GI isolates, respectively (Table 3).

Discussion
Bacteroidetes as a large community of gut microbiota can be isolated from human clinical specimens and lead to mixed anaerobic bacterial infections (3). Antibiotic-resistant genes also play important roles in the antibiotic resistance of B. fragilis and cause unsuccessful antibacterial therapy. In addition, the transmission of resistance genes through horizontal gene transfer, as the most common mean of acquiring resistance genes among bacteria, is another major problem. In this study, we have evaluated the prevalence of resistance genes and antibiotic resistance pro le of B. fragilis using phenotypic approaches and ampli cation of genes of interest.
In this study, B. fragilis accounted for 57.4% of anaerobic bacteria isolated from clinical samples.
The MIC 50 and MIC 90 values for ampicillin/sulbactam, piperacillin/tazobactam, metronidazole and clindamycin in clinical isolates were at least twice higher than GI isolates. One possible reason for this might be the use of antibiotics in these patients.
Although carbapenems have been considered as highly effective antibiotics in the prevention of anaerobic infections, bacterial resistance to these antibiotics has increased (6, 11,15,31). In this study, 1.3% of isolates (n = 1) were resistant to imipenem and 1.3% of isolates (n = 1) were resistant to meropenem, these isolates were collected from the GI tract of healthy individuals which could be considered as a serious risk. The emergence of carbapenem resistance has also been reported in different studies. For instance, meropenem resistance was found to be 0.5% in the United States and 2% in Europe (6, 12, 32). In a study conducted by Kohsari et al. in Iran, the resistance of B. fragilis to meropenem was 13.9% (33). Discrepancies observed in different studies regarding antibiotic resistance pro le of B. fragilis may be due to different reasons including geographical features, population study, and differences in laboratory techniques.
Resistance to carbapenems in B. fragilis is usually caused by the expression of the class B metallo-betalactamase encoded by the c A gene, located on the chromosome. Accordingly, if an insertion sequence is located in its upstream region, it will be expressed and will cause carbapenem resistance (4,34). In a study conducted by Soki et al., B. fragilis isolates (n = 10) contained the c A gene, of which seven isolates were resistant to imipenem (35). In the present study, 18.1% and 12.5% of the clinical and GI samples had the c A gene respectively. Moreover, the imipenem-resistant isolates had the c A gene and the IS1186 insertion sequence in the upstream region of the gene whereas the meropenem-resistant strain had this gene but lacked the IS1186 insertion sequence. The resistance was possibly due to expression of the silent carbapenemase gene (36), the presence of other insertion sequences in the upstream region of this gene (IS1187, IS1188, IS942) (32), or other resistance mechanisms such as membrane permeability or penicillin-binding protein (PBP) a nity (37). In addition, some isolates had the c A gene but were phenotypically sensitive to carbapenem which demonstrate the antibiotic resistance gene may not be expressed. In a study performed by Rashidian et al. in Iran, 31.5% and 20% in B. fragilis group isolate from the patients and control groups harbored c A gene, respectively (38).
Penicillins and second-generation cephalosporin resistance have also been observed in B. fragilis.
The most important mechanisms contributing to this resistance is the expression of beta-lactamases which are encoded by the cepA gene (resistance to penicillin and cephalosporins other than cefoxitin) and cfxA gene (resistance to cefoxitin) (39,40).
In this study, all the isolates (100%) were resistant to penicillin, of which 73.1% had the cepA gene. There was also meaningful difference in penicillin MIC value of isolates with cepA gene compared to isolates without cepA gene indicating the importance of this gene in resistance to penicillin. In addition, 45.5% and 35.7% of the clinical and GI isolates were respectively resistant to cefoxitin, and 22.7% and 26.8% of these isolates had the cfxA gene, respectively. The presence of the cfxA gene was signi cantly higher in cefoxitin-resistant isolates compare to cefoxitin-susceptible isolates, which was also statistically signi cant.
The rate of B. fragilis resistance to cefoxitin in recent years has been 6.8-33.3% in Europe, 12.6% in Canada, and 23% in Brazil (6, 41,42). In a study conducted by Kangaba et al. in Turkey, 28% of B. fragilis isolates and 32% of isolates from the GI tract had been found to be resistant to cefoxitin. In this study, resistance to ampicillin/sulbactam and piperacillin/tazobactam were 6.4% and 2.6%, respectively (10). In another investigation, 5.4% of B. fragilis isolates were resistant to piperacillin/tazobactam which was relatively consistent with the ndings reported by Maraki et al. (5.4%) and Yunoki et al. studies (2.8%) (15,43).
The ermB and mefA genes were also involved in the development of macrolide resistance in B. fragilis (44). The prevalence of clindamycin resistance had been further reported by 54.5% in clinical isolates and 42.9% in the GI isolates which were mainly associated with the presence of the ermF gene (40). Clindamycin resistance among B. fragilis have been reported in several countries (8, [45][46][47].
In the present study, all clindamycin-resistant isolates had the ermF genes. In addition, ve isolates had the mefA gene and three of which were clindamycin-resistant strains. The presence of the ermF gene also was higher in clindamycin-resistant isolates than clindamycin susceptible-isolates respectively, which was statistically signi cant. None of the isolates in this study had ermB gene.
The presence of tetQ gene associated with tetracycline resistance has been further reported in clinical isolates (43,48). In the present study, 81.8% and 71.4% of the clinical and GI isolates had tetracycline resistance, and 90.9% and 85.7% of these isolates had the tetQ genes, respectively.
In a study conducted by Narimani et al., 86% of the GI isolates were resistant to tetracycline, and the tetQ gene was found in 85% of the isolates (48). In the investigation by Kangaba et al. study, 72% of clinical isolates and 92% of GI isolates were resistant to tetracycline, 64% and 92% of them had the tetQ gene, respectively (10).
The metronidazole resistance rate was found to be 0-3% in different parts of the world (6, 10, 38, 49). There were no isolates resistant to metronidazole in this study and the nim gene was not detected in any isolates.
Based on previous studies, the prevalence of the bft gene was reported to be 6.2-20% in the GI isolates (37,(50)(51)(52)(53) and 18.5-38.2% in clinical isolates (53-55) which was consistent with the ndings in the present study.
Although phenotypic ndings indicated resistance to some antibiotics in this study, the PCR ndings did not con rm the presence of corresponding resistance genes in the isolates. This fact may suggest the role of other resistance mechanisms such as e ux pumps, changes in the cell wall structure, and catalytic enzymes in B. fragilis isolates (40, 56).

Conclusion
In conclusion, metronidazole, imipenem and meropenem were the most active agents against B. fragilis isolates. It was concluded that continuous monitoring of antibiotic resistance patterns of B. fragilis in different geographical areas was vital to provide a suitable treatment pro le and to prevent infection more accurately. In other words, with regard to the presence of antibiotic-resistant genes and the high risk of antibiotic-resistant strains in the GI tract of healthy people, proper prescription of antibiotics and avoidance of its arbitrary use can help prevent infection and transmission of resistant isolates.