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A total of 94 E. coli strains were identified in 490 fecal samples from patients with diarrhea and 53 fecal samples from healthy individuals between 2019 and 2021. Among them, 70 strains were from the diarrheal group, and 24 were from healthy donors.
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The drug resistance rates of the 94 E. coli strains were 29.79% for macrolides, 40.43% for tetracyclines, 71.28% for β-lactams, 65.96% for quinolones, 32.98% for aminoglycosides, 35.11% for sulfonamides, 8.51% for chloramphenicols, 3.19% for polypeptides, and 26.60% for compounds. There were 64 (68.09%) MDR strains, including 41 strains (43.62%) resistant to ≥ 5 antibiotics and 9 strains (E21, E38, E43, E44, E45, E48, E68, E73, and E9) resistant to ≥ 9 antibiotics. The sensitivity of the strains from the healthy group to AMS (P = 0.003, P < 0.05), ETP (P = 0.000, P < 0.05), CZA (P = 0.000, P < 0.05), CTX (P = 0.011, P < 0.05), and AZI (P = 0.026, P < 0.05) was significantly higher than that of the diarrheal group. No significant difference in MDR was observed between the diarrheal and healthy groups. Drug resistance rates are listed in Table 1.
Table 1. Drug resistance of 94 E. coli strains
Antibiotic Toltel antibiotic resistance n (%) Diarrheagenic E. coli (n = 70) Common E.coli (n = 24) P Antibiotic resistance n (%) No antibiotic resistance n (%) Antibiotic resistance n (%) No antibiotic resistance n (%) Ampicillin (AMP) 63 (67.02) 49 (70.00) 21 (30.00) 14 (58.33) 10 (41.67) 0.294 Ampicillin/sulbactam (AMS) 25 (26.60) 24 (34.29) 46 (65.71) 1 (4.17) 23 (95.83) 0.004 Tetracycline (TET) 36 (38.30) 25 (35.71) 45 (64.29) 11 (45.83) 13 (54.17) 0.379 Meropenem (MEM) 2 (2.13) 2 (2.86) 68 (97.14) 0 24 (100) 0.986 Polymyxin E (CT) 2 (2.13) 1 (1.43) 69 (98.57) 1 (4.17) 23 (95.83) 1.000 Ertapenem (ETP) 30 (31.91) 30 (42.86) 40 (57.14) 0 24 (100) < 0.001 Ceftazidime/avibactam (CZA) 26 (27.66) 26 (37.14) 44 (62.86) 0 24 (100) < 0.001 Tigecycline (TIG) 7 (7.45) 7 (1.00) 63 (90.00) 0 24 (100) 0.279 Cefotaxime (CTX) 26 (27.66) 24 (34.29) 46 (65.71) 2 (8.33) 22 (91.67) 0.014 Ceftazidime (CAZ) 7 (7.45) 7 (1.00) 63 (90.00) 0 24 (100) 0.246 Ciprofloxacin (CIP) 39 (41.49) 30 (42.86) 40 (57.14) 9 (37.50) 15 (62.50) 0.646 Azithromycin (AZI) 28 (29.79) 25 (35.71) 45 (64.29) 3 (12.50) 21 (87.50) 0.032 Chloramphenicol (CHL) 8 (8.51) 8 (11.43) 62 (88.57) 0 24 (100) 0.191 Nalidixic acid (NAL) 53 (56.38) 38 (54.29) 32 (45.71) 15 (62.50) 9 (37.50) 0.484 Streptomycin (STR) 31 (32.98) 23 (32.86) 47 (67.14) 8 (33.33) 16 (66.67) 0.966 Trimethoprim/sulfamethoxazole (SXT) 33 (35.11) 25 (35.71) 45 (64.29) 8 (33.33) 16 (66.67) 0.833 Amikacin (AMK) 0 0 70 (100) 0 24 (100) / Multidrug resistance (MDR) 64 (68.09) 49 (70.00) 21 (30.00) 15 (62.50) 9 (37.50) 0.496 -
Ninety kinds of ARGs were identified in the 94 strains. All kinds of ARGs were constructed and divided into five drug resistance categories, from the most to least: efflux pump conferring antibiotic resistance (41), antibiotic inactivation or hydrolase (36), target-site changes (6), protein-altering cell wall charge conferring antibiotic resistance (5), and others (2). We then analyzed the relationship between MDR and ARGs. A comparison between the number of kinds of ARGs and MDR showed that there was a positive correlation, i.e., the higher the number of ARGs, the higher the MDR rate. However, an exception was found for E57, a sensitive strain that surprisingly contained more than 50 kinds of ARGs. Detailed information is shown in Figure 1. In total, among the strains containing 30–40 kinds of ARGs, 5 of the 9 strains were multidrug resistant. Among the strains containing 40–50 kinds of drug ARGs, 39 of the 63 strains were multidrug resistant. Among the strains containing 50–60 kinds of ARGs, 17 of the 19 strains were multidrug resistant.
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This study analyzed the correlation between each of the 90 ARGs and MDR, revealing that only the following 8 ARGs are significantly associated with MDR. These eight ARGs are: gadW, dfrA17, mphA, aadA5, qnrS1, AAC(3)-IId, sul1, and TEM.1 (Table 2). The remaining 82 ARGs were not significantly associated with MDR.
Table 2. Eight drug resistance genes with significant correlation with MDR
ARGs χ2 P gadW 4.990 0.025* dfrA17 9.728 0.002** mphA 11.262 0.001** aadA5 9.038 0.003** qnrS1 5 .845 0.016* AAC(3)-IId 10.650 0.001** sul1 5.591 0.018* TEM.1 8.249 0.004** Note. *P < 0.05; **P < 0.01. ARGs, antibiotic resistance genes; MDR, multidrug resistance. -
To better predict the gene combinations associated with MDR, the RF classifier approach was utilized to identify MDR risk markers. The optimal gene combination pattern of TEM.1 + baeR + mphA + mphB + QnrS1 + AAC.3-IId was associated with MDR by the RF method. An RF classifier model with 64 MDR and 30 non MDR controls was constructed to assess the potential of ARGs as predictors of multidrug resistance in E. coli. Six ARGs (TEM.1 + baeR + mphA + mphB + QnrS1 + AAC.3-IId) were found to comprise an influential group characteristics (top six features by relative influence), predicting MDR by a ten-fold cross-validation of the RF model (Figure 2). These six predictors achieved a classification accuracy of 85.19%. The MDR index reached an area under the receiver operating characteristic curve (AUC) of 0.917 with a 95% confidence interval (CI) of 0.6627–0.9581 between the MDR and non-MDR controls (P = 0.02754; Figure 3). The data indicated that the classifier model based on the six predictors achieved the predicted potential for the investigation of the risk of MDR in E. coli.
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Based on the seven allelic differences, 94 E. coli strains were divided into 50 STs, of which two strains were named new STs, STn1 and STn2, because they failed to match any known ST. STs of E. coli isolates showed diverse distributions, with ST10 (10.64%) as the main epidemic type, followed by ST1491 (6.38%), ST48 (6.38%), and ST69 (5.32%), with MDR rates of 50.00% (5/10), 50.00% (3/6), 83.33% (5/6), and 100% (5/5). There were 28 STs (41 strains) with a 100% MDR, and 7 STs (20 strains) with a 50.00%–100% MDR. In addition, more than 50% of strains in four STs were resistant to > 4 types of antibiotics, including ST10 (50.00%), ST48 (50.00%), ST4 (66.70%), and ST73 (66.70%). Through a comparison between healthy and diarrheal patients, we found that ST48, ST137, ST4, ST73, ST69, ST38, and ST131 had more strains with MDR in the diarrheal group. ST38, ST69, ST131, and ST10 showed more strains with MDR in the healthy group. We also found that 70.00% (49/70) of the strains from the diarrheal group and 62.50% (15/24) of the strains from the healthy group had MDR. No difference in MDR was found between the two populations, indicating that the proportion of strains in the healthy group was relatively high. Therefore, the analysis was conducted according to the following conditions: i.e. diarrhea MDR, health MDR, diarrhea non MDR, and health non MDR. The results indicated that the MDR ratios of ST69 and ST131 were higher in the healthy group than in the diarrheal group. In ST69, MDR strains accounted for 60.00% of the participants in the healthy group and 40.00% in the diarrheal group. The healthy and diarrheal groups MDR rates for ST131 were 66.70% and 33.30%, respectively. In addition, more than 50% of the strains in four STs were resistant to > 4 types of antibiotics, including ST10 (50.00%), ST48 (50.00%), ST4 (66.70%), and ST73 (66.70%). Two new STs, namely STn1 and STn2, were identified in this study, with the representative strains E71, a strain with MDR from the diarrheal group, and H95, a strain with MDR from the healthy group (Figure 4).
Figure 4. Distribution of STs and MDR in the healthy and diarrheal groups. ST, sequence types; MDR, multidrug resistance
According to Clermont’s method, 94 E. coli strains could be divided into groups A (44 strains, 46.81%), B1 (11 strains, 11.70%), B2 (14 strains, 14.89%), C (1 strain, 1.06%), D (20 strains, 21.28%), E (2 strains, 2.13%), F (1 strain, 1.06%), and U (1 strain, 1.06%), with MDR rates of 59.09% (26/44), 63.64% (7/11), 78.57% (11/14), 0% (0/1), 80% (16/20), 100% (2/2), 100% (1/1), and 100% (1/1), respectively. Figure 5 shows the distribution of the groups and MDR. The Figure highlights that MDR groups are generally surrounded by MDR groups, and non MDR groups are also surrounded by non MDR groups. Because group B was more pathogenic than the other groups[21, 22], we analyzed group B in detail. The B2 group, including ST569, ST73, ST131, ST589, and ST1092, was an MDR group (100%). ST1193 in B2 had MDR (50%). ST582 in B2 did not have MDR (100%). ST29, ST3285, ST1196, ST155, ST316, ST641 in B1 had MDR (100%). ST327, ST20, ST517, ST40 in B1 were non MDR (100%).
We established an SNP tree for E. coli based on the number of SNP genes. We found that the MDR strains were distributed across all clades. No significant differences in MDR were observed among the clades. Moreover, Figure 6 shows that group A and D are the main groups. This represents the relationship between MDR and the six-gene combination. We can clearly see that the strains with TEM.1 + baeR + mphA + mphB + QnrS1 + AAC.3-IId genes positive combination all had MDR.
doi: 10.3967/bes2023.050
Genotyping Characteristics of Human Fecal Escherichia coli and Their Association with Multidrug Resistance in Miyun District, Beijing
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Abstract:
Objective To explore the genotyping characteristics of human fecal Escherichia coli (E. coli) and the relationships between antibiotic resistance genes (ARGs) and multidrug resistance (MDR) of E. coli in Miyun District, Beijing, an area with high incidence of infectious diarrheal cases but no related data. Methods Over a period of 3 years, 94 E. coli strains were isolated from fecal samples collected from Miyun District Hospital, a surveillance hospital of the National Pathogen Identification Network. The antibiotic susceptibility of the isolates was determined by the broth microdilution method. ARGs, multilocus sequence typing (MLST), and polymorphism trees were analyzed using whole-genome sequencing data (WGS). Results This study revealed that 68.09% of the isolates had MDR, prevalent and distributed in different clades, with a relatively high rate and low pathogenicity. There was no difference in MDR between the diarrheal (49/70) and healthy groups (15/24). Conclusion We developed a random forest (RF) prediction model of TEM.1 + baeR + mphA + mphB + QnrS1 + AAC.3-IId to identify MDR status, highlighting its potential for early resistance identification. The causes of MDR are likely mobile units transmitting the ARGs. In the future, we will continue to strengthen the monitoring of ARGs and MDR, and increase the number of strains to further verify the accuracy of the MDR markers. -
Key words:
- E. coli /
- Multidrug resistance /
- Whole-genome sequencing /
- Antibiotic resistance genes /
- Random forest
The authors have no conflicts of interest to declare.
&These authors contributed equally to this work.
注释:1) AUTHOR CONTRIBUTIONS: 2) CONFLICTS OF INTEREST: -
Table 1. Drug resistance of 94 E. coli strains
Antibiotic Toltel antibiotic resistance n (%) Diarrheagenic E. coli (n = 70) Common E.coli (n = 24) P Antibiotic resistance n (%) No antibiotic resistance n (%) Antibiotic resistance n (%) No antibiotic resistance n (%) Ampicillin (AMP) 63 (67.02) 49 (70.00) 21 (30.00) 14 (58.33) 10 (41.67) 0.294 Ampicillin/sulbactam (AMS) 25 (26.60) 24 (34.29) 46 (65.71) 1 (4.17) 23 (95.83) 0.004 Tetracycline (TET) 36 (38.30) 25 (35.71) 45 (64.29) 11 (45.83) 13 (54.17) 0.379 Meropenem (MEM) 2 (2.13) 2 (2.86) 68 (97.14) 0 24 (100) 0.986 Polymyxin E (CT) 2 (2.13) 1 (1.43) 69 (98.57) 1 (4.17) 23 (95.83) 1.000 Ertapenem (ETP) 30 (31.91) 30 (42.86) 40 (57.14) 0 24 (100) < 0.001 Ceftazidime/avibactam (CZA) 26 (27.66) 26 (37.14) 44 (62.86) 0 24 (100) < 0.001 Tigecycline (TIG) 7 (7.45) 7 (1.00) 63 (90.00) 0 24 (100) 0.279 Cefotaxime (CTX) 26 (27.66) 24 (34.29) 46 (65.71) 2 (8.33) 22 (91.67) 0.014 Ceftazidime (CAZ) 7 (7.45) 7 (1.00) 63 (90.00) 0 24 (100) 0.246 Ciprofloxacin (CIP) 39 (41.49) 30 (42.86) 40 (57.14) 9 (37.50) 15 (62.50) 0.646 Azithromycin (AZI) 28 (29.79) 25 (35.71) 45 (64.29) 3 (12.50) 21 (87.50) 0.032 Chloramphenicol (CHL) 8 (8.51) 8 (11.43) 62 (88.57) 0 24 (100) 0.191 Nalidixic acid (NAL) 53 (56.38) 38 (54.29) 32 (45.71) 15 (62.50) 9 (37.50) 0.484 Streptomycin (STR) 31 (32.98) 23 (32.86) 47 (67.14) 8 (33.33) 16 (66.67) 0.966 Trimethoprim/sulfamethoxazole (SXT) 33 (35.11) 25 (35.71) 45 (64.29) 8 (33.33) 16 (66.67) 0.833 Amikacin (AMK) 0 0 70 (100) 0 24 (100) / Multidrug resistance (MDR) 64 (68.09) 49 (70.00) 21 (30.00) 15 (62.50) 9 (37.50) 0.496 Table 2. Eight drug resistance genes with significant correlation with MDR
ARGs χ2 P gadW 4.990 0.025* dfrA17 9.728 0.002** mphA 11.262 0.001** aadA5 9.038 0.003** qnrS1 5 .845 0.016* AAC(3)-IId 10.650 0.001** sul1 5.591 0.018* TEM.1 8.249 0.004** Note. *P < 0.05; **P < 0.01. ARGs, antibiotic resistance genes; MDR, multidrug resistance. -
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