Prevalence of O25b-ST131 Escherichia coli Clone: Fecal Carriage of Extended-Spectrum β -Lactamase and Carbapenemase—Producing Isolates in Healthy Adults in Tehran,Iran

Abstract

Fecal carriage of multidrug-resistant Escherichia coli, particularly sequence type 131 (ST131), is becoming a global concern. This study aimed at determining the prevalence rate and molecular epidemiology of extended-spectrum β-lactamase-producing E. coli (ESBL-Ec), carbapenemase-producing E. coli (CPEc), ceftazidime/avibactam (CAZ/AVI)-resistant E. coli, and ST131 isolates in healthy fecal carriers in Tehran, Iran. Among 540 samples studied, 233 (43.1%) carried ESBL-Ec, with the majority (93.9%) harboring the bla_CTX-M. The carriage rate of CPEc was 2.5% (n = 14/540), and bla_NDM gene was the predominant carbapenemase gene. Most CPEc isolates (n = 11/14) was shown to be resistant to CAZ/AVI. Among ESBL-Ec/CPEc, 7.3% (n = 17/233) belonged to E. coli ST131 clone, which was identified by polymerase chain reaction and confirmed by multilocus sequence typing. The ST131 isolates genetically typed by pulsed-field gel electrophoresis were heterogeneous and four different plasmids were detected by plasmid typing, with the IncFIA/FIB being the major type. Our findings disclose that the presence of carbapenem-resistant ST131 isolates, which are also resistant to CAZ/AVI, contributes to the spread of resistant strains in the community. Therefore, screening and monitoring of such resistant clone in healthy people is necessary.

Introduction

Multidrug-resistant (MDR) Gram-negative bacilli have the ability to colonize the intestinal tract of humans and can give rise to fatal infections.¹ However, the prevalence rate of these drug-resistant organisms, particularly MDR Escherichia coli, a leading cause of diseases, mortality, and social costs, has escalated over the past few years.² Antimicrobial resistance of the gut flora is troublesome due to the transfer of the resistance genes to other pathogens.³

The widespread use of carbapenems, as the last drug option for treating infections caused by MDR isolates, has resulted in the expansion of carbapenem-resistant E. coli (CREc) and carbapenemase-producing E. coli (CPEc) isolates.⁴ To fight CREc, limited treatment options, such as colistin and ceftazidime/avibactam (CAZ/AVI), have been adopted.¹ CAZ/AVI is active against strains bearing Ambler class A (bla_KPC), class C (bla_AmpC), and some class D β-lactamases (bla_OXA-48), but it is inactive against metallo-β-lactamases, namely bla_NDM, bla_IMP, and bla_VIM.⁵ The emergence of E. coli isolates resistance to colistin mainly driven by the horizontal transfer of mcr-1 bearing plasmids leads to a clinical threat.¹

Spread of extended-spectrum β-lactamase (ESBL)-producing E. coli (ESBL-Ec), such as sequence type 131 (ST131), in the community has raised a major global health concern owing to the increased frequency of virulence factors and varied antimicrobial resistances.⁶ ST131 E. coli isolates belong mostly to the O25b serogroup and are known as MDR. These isolates are often recognized as broad-spectrum cephalosporin-resistant E. coli_.⁷ In addition, resistance to carbapenems and CAZ/AVI in ST131 E. coli can lead to a crisis in the treatment of related infections. In this regard, our study was undertaken to investigate the prevalence of ESBL-Ec/CPEc isolates, the patterns of colistin and CAZ/AVI-resistance in CPEc isolates, and characterization and plasmid analysis of ST131 clone E. coli in healthy fecal carriers in Tehran, Iran.

Materials and Methods

Sample collection and microbiological analysis

Sampling was conducted in Tehran, the capital city of Iran, from February to July 2018. The stool samples were collected from 540 subjects, in the age range between 15 and 35 years old (working age), who attended community health centers. The cases had neither diarrhea nor history of antibiotic use or hospitalization 3 months before the initiation of the survey. The present work was approved by the Research and the Ethics Committee of the Pasteur Institute of Iran, Tehran (approval code: B9321).

By using a sterile swab, the stool samples were inoculated in Cary-Blair transport media. The identification of ESBL-Ec and CPEc isolates was carried out by culturing fecal swabs on CHROMagar ESBL and CHROMagar Carba media (CHROMagar, Paris, France), respectively. Each red colony grown on the plates was selected and identified by using standard microbiologic techniques, including the morphologic appearance of the colony, Gram's staining, catalase test, and oxidase test, along with other biochemical tests (carbohydrate utilization, indole production, urea hydrolysis, citrate utilization, and amino acid decarboxylation).⁸ Phenotypic confirmation of ESBL-Ec isolates was conducted by a combination disk test (CDT) recommended by the Clinical and Laboratory Standards Institute (CLSI). Klebsiella pneumoniae ATCC 700603 (ESBL positive) and E. coli ATCC 25922 (ESBL negative) were used as the control strains.

Antimicrobial susceptibility testing

The antimicrobial susceptibility of the isolates was tested with 15 various antibiotics by using Mueller Hinton agar and Kirby–Bauer disk diffusion technique following the recommendations of CLSI.⁹ The antibiotic disks employed in this study included ampicillin (10 μg), aztreonam (30 μg), trimethoprim-sulfamethoxazole (SXT; 25 μg), ciprofloxacin (5 μg), nalidixic acid (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), gentamicin (10 μg), imipenem (10 μg), meropenem (10 μg), ertapenem (10 μg), piperacillin-tazobactam (100/10 μg), ampicillin-sulbactam (10/10 μg), amoxicillin-clavulanic acid (20/10 μg), and amikacin (30 μg), all of which were procured from Mast Group Ltd. (Merseyside, United Kingdom). MDR was defined as acquired resistance to at least one agent in three or more antimicrobial categories.¹⁰ The minimum inhibitory concentrations (MICs) of meropenem, cefotaxime, ciprofloxacin, and CAZ/AVI in CREc were determined by Etest method (AB bioMérieux, France) as per the protocol provided by the manufacturer. The CREc isolates were resistant to ertapenem, imipenem, and meropenem with an MIC ≥4 μg/mL against meropenem. Colistin MICs were obtained by the broth microdilution method in CREc. E. coli ATCC 25922 and Proteus mirabilis ATCC 12453 were applied as control strains.⁹

Antibiotic-resistant genes

For genomic DNA extraction, bacterial isolates were harvested in Luria-Bertani broth (Merck, Germany), and the DNA was extracted by the boiling method.¹¹ The quality of the DNA was confirmed by using a NanoDrop (Thermo Scientific, Roskilde, Denmark), and its purity was determined by calculating the ratio of absorbance at 260 nm divided by the reading at 280 nm. The phenotypically characterized ESBL-Ec and CREc isolates were analyzed for detecting the β-lactamase and carbapenemase genes. The molecular detection of the isolates harboring ESBL genes (bla_CTX-M, bla_CTX-M-15, and bla_CTX-M-14) was conducted by using the polymerase chain reaction (PCR) and sequencing.¹² All the ertapenem-, imipenem-, and meropenem-resistant isolates were screened by PCR for detecting bla_KPC, bla_NDM, bla_OXA-₄₈, bla_IMP, bla_VIM genes, and carbapenemase-harboring isolates selected as CPEc were screened for mcr-1 as colistin-resistant genes.¹³ The PCR products were sequenced, and the obtained sequences of the amplified target sites were aligned and compared with those in the National Centre for Biotechnology Information database (www.ncbi.nlm.nih.gov), using the BLAST program.

Molecular characterization of ST131 clone

The ST131 clone was identified by PCR of ST131-specific single-nucleotide polymorphisms in mdh and gyrB genes among ESBL-Ec/CPEc isolates.¹⁴ The detection of O25b and O16 serogroups was performed by using specific primers for the rfb region (O-antigen gene cluster).¹⁵ The isolates identified as ST131 by PCR and CPEc isolates were further analyzed by applying multilocus sequence typing (MLST) according to the Pasteur scheme using eight housekeeping genes (PutP, trpA, PabB, trpB, uidA, polB, icdA, and dinB).¹⁶ To assess the genetic relatedness of ST131 isolates, their chromosomal DNA was digested by the restriction enzyme XbaI and then subjected to pulsed-field gel electrophoresis (PFGE) analysis according to the standard protocol.¹⁷ The DNA of Salmonella enterica serotype Braenderup strain H9812 was also treated with XbaI and used as a molecular weight standard. The dendrogram panel was constructed by the aid of Gelcompare II software. Isolates with a dice similarity index ≥80% were considered to be in the same PFGE cluster.

Plasmid analysis

The plasmids of CPEc and ST131 isolates were extracted by using a plasmid kit (GeneAll Biotechnology, Seoul, Korea). Plasmid replicon typing in CPEc and ST131 isolates was accomplished by multiplex PCR, to detect 18 plasmid replicon types (IncW, T, A/C, K, B/O, X, Y, F, FIA, FIB, FIC, HI1, HI2, I1-I gamma, L/M, N, P, and FIIA).¹⁸ The ability of the isolates to transfer the antibiotic resistance genes was analyzed by a filter mating experiment between the ST131 E. coli isolates as donor and azide-resistant E. coli J53 (J53 AZr) sensitive to 21 antibiotics as recipient.¹⁹ MacConkey agar plates supplemented with sodium azide (150 mg/L) and cefotaxime (2 mg/L) were utilized to select the transconjugants. Antimicrobial susceptibilities of the transconjugants were evaluated by using the disk diffusion method, and the transfer of the resistance genes was confirmed by PCR. Plasmid incompatibility in transconjugant was determined by a PCR-based replicon typing method, as described earlier.¹⁸

Statistical analysis

Data analysis was carried out by the R software version 3.3.3 and interpreted according to the frequency distribution and percentage. The comparison between different groups was made by using chi-square test and independent t-test. Data with the p-value ≤0.05 (95% confidence interval) were regarded as statistically significant.

Results

Sample collection and antimicrobial susceptibility analysis

Among 540 fecal samples from healthy individuals, 44.4% (n = 240) third-generation cephalosporin-resistant E. coli were obtained. Finally, 43.1% (n = 233) third-generation cephalosporin-resistant isolates were confirmed as ESBL-Ec by CDT. In addition, 2.5% (n = 14) CREc isolates were detected. The antibiotic susceptibility patterns of ESBL-Ec/CREc isolates are represented in Table 1. The ESBL-Ec/CREc isolates showed resistance to cephalosporins, sulfonamides, and fluoroquinolones so that 40.3% (n = 94/233) of these isolates were classified as MDR isolates. In addition, four CREc isolates were found to be resistant to colistin (MIC = 4 μg/mL), whereas the mcr-1 gene was not detected. A total of 78.5% (n = 11/14) of CREc isolates were resistant to CAZ/AVI (MIC ≥128 μg/mL).

Table 1.

Antibiotic Resistance and Frequency of bla_CTX-M-_15/_CTX-M-₁₄ Genes Among Non-ST131 and ST131 Extended-Spectrum β-Lactamase-Producing Escherichia coli Isolates

Antibiotic	Total, n = 233	Non-ST131, n = 216	ST131, n = 17	p
Antibiotic	n (%)	n (%)	n (%)	p
Ampicillin	231 (99.1)	214 (99.1)	17 (100)	0.82
Cefotaxime	228 (97.8)	211 (97.6)	17 (100)	0.371
Ceftazidime	210 (90.1)	195 (90.2)	15 (88.2)	0.734
Aztreonam	170 (72.9)	156 (72.2)	14 (82.3)	0.365
Trimethoprim-sulfamethoxazole	169 (72.5)	154 (71.2)	15 (88.2)	0.071
Nalidixic acid	121 (51.9)	106 (49.1)	15 (88.2)	0.002^*
Ciprofloxacin	75 (32.2)	67 (31)	8 (48.7)	0.183
Amoxicillin-clavulanic acid	48 (20.6)	40 (18.5)	8 (48.7)	0.005^*
Ampicillin-sulbactam	44 (18.9)	39 (18.5)	5 (29.4)	0.249
Amikacin	23 (9.9)	18 (8.3)	5 (29.4)	0.005^*
Gentamicin	36 (15.5)	33 (15.3)	3 (17.6)	0.795
Piperacilin-tazobactam	20 (8.5)	18 (7.7)	2 (11.8)	0.155
Ertapenem	28 (12.1)	26 (12.03)	2 (11.8)	0.973
Imipenem	12 (5.2)	11 (5.09)	1 (5.9)	0.955
Meropenem	12 (5.2)	11 (5.09)	1 (5.9)	0.955
ESBL genes
bla_CTX-M	219/233 (93.9)	202/216 (93.5)	17/17 (100)	0.752
bla_CTX-M-15	198/219 (90.4)	183/202 (90.6)	15/17 (88.2)	0.696
bla_CTX-M-14	15/219 (6.8)	14/202 (6.9)	1/17 (5.9)	0.904
bla_CTX-M-15/bla_CTX_-M-14	6/219 (2.7)	5/202 (2.4)	1/17 (5.9)	0.371

^* The statistically significant values.

ESBL, extended-spectrum β-lactamase.

Antibiotic-resistant genes

Among the 219 harboring bla_CTX-M gene E. coli isolates surveyed, 198 (90.4%) harbored bla_CTX-M-15 gene and 15 (6.8%) harbored bla_CTX-M-14 gene. Moreover, 6 (2.7%) ESBL-Ec isolates carried both the bla_CTX-M14 and bla_CTX-M-15 genes. Among 14 CREc isolates, 11 (78.5%) harbored bla_NDM gene, whereas 50% (n = 7/14) carried bla_NDM-5 and 28.5% (n = 4/14) harbored bla_NDM-1 gene. Besides, bla_OXA-48 gene was detected in 28.5% (n = 4/14) of the CREc isolates; one of the CREc isolates harbored both bla_NDM-5 and bla_OXA-48 genes. None of the CREc isolates carried bla_KPC, bla_IMP, and bla_VIM genes. All the harboring carbapenemase genes in CREc isolates (n = 14, 2.5%) were considered as CPEc.

Molecular characterization of ST131 clone

A total of 17 (7.3%) out of 233 ESBL-Ec/CPEc isolates were identified as ST131 by using the PCR technique. All the isolates were detected as O25b-ST131 clone by specific PCR for O25b antigen, and none of them belonged to O16 serogroup. Moreover, the entire ST131 clone isolates were confirmed by the Pasteur MLST scheme (Fig. 1). The antimicrobial susceptibility profile of the ST131 isolates is depicted in Table 1. Among the 17 ST131 isolates, 13 (76.5%) were MDR, whose resistance to at least 3 classes of antibiotics, that is, cephalosporin, sulfonamide, and fluoroquinolone, was confirmed. Significant differences were explored in the distribution of resistance to amikacin (p = 0.005), amoxicillin-clavulanic acid (p = 0.005), and nalidixic acid (p = 0.002) between ST131 and non-ST131 isolates (Table 1). Two isolates were carbapenemase producing: One of them co-harbored bla_OXA-48 and bla_NDM-5 genes, and the other one carried bla_NDM-5 gene; however, both isolates were resistant to CAZ/AVI. Eight distinct Pasteur sequence types (PSTs) distributed among 14 CPEc isolates were found by MLST analysis; PST indicates the ST under the Pasteur scheme typing. PST490 (21.4%, n = 3/14) and PST2 (21.4%, n = 3/14) were the main types, followed by PST43 (14.2%, n = 2/14), PST629 (14.2%, n = 2/14), PST3 (7.1%, n = 1/14), PST19 (7.1%, n = 1/14), PST500 (7.1%, n = 1/14), and PST267 (7.1%, n = 1/14). Pasteur MLST of ST131 isolates also indicated that the 88.2% (n = 15/17) of the ST131 isolates belonged to PST43, and only 11.8% (n = 2/17) were PST568. By applying 80% similarity of cut-off point, PFGE analyzing 17 ST131 isolates demonstrated 17 pulsotypes, which were classified into 17 singletons with unique patterns numbered from P1 to P17 (Fig. 1).

FIG. 1.

PFGE patterns showing the genetic relatedness of the fecal Escherichia coli ST131 isolates. The dendrogram was constructed by using Dice similarity coefficient and UPGMA. The scale bar at the top indicates the similarity coefficient (%). CAZ/AVI, ceftazidime/avibactam; CIP, Ciprofloxacin; CTX, Cefotaxime; MICs, minimum inhibitory concentrations; PFGE, pulsed-field gel electrophoresis; PST, pasteur sequence type; UPGMA, unweighted pair group method using arithmetic averages.

Plasmid analysis

A variety of plasmid replicon types were detected in 14 CPEc isolates, in which there were 4 types of incompatible groups, comprising 5 (35.7%) IncFIIs, 3 (21.4%) IncA/C, 2 (14.2%) IncL/M, and 1 (7.1%) IncFIA replicon types, as well as 3 untypeable isolates. Four different incompatible groups found in 17 ST131 isolates included 6 (35.2%) IncFIA/IncFIB, 5 (29.4%) IncFIA, 3 (17.6%) IncFIB, 2 (11.7%) IncFIIs, and 1 (5.8%) IncL/M. Three transconjugants (17.6%, n = 3/17) were obtained after performing gene transferring for ST131 clone isolates in the filter-mating test by using the E. coli J53 AZr as a recipient. Two transconjugants displayed resistance to ampicillin, amoxicillin-clavulanic acid, cefotaxime, and ceftazidime, but not to gentamicin, aztreonam, and ciprofloxacin; however, the donor isolates were resistant to all the mentioned antibiotics. Further, these two transconjugants displayed greater MIC of cefotaxime (MIC ≥32 μg/mL) relative to the recipient (MIC = 0.1 μg/mL). The bla_CTX-M and IncFIIs genes were detected in the aforementioned transconjugants by PCR. The third transconjugant showed resistance to ampicillin, cefotaxime, and ceftazidime, but this resistance was not observed in imipenem, meropenem, and ertapenem, compared with the donor strain, which was resistant to the antibiotics of the panel. This transconjugant also displayed greater MIC values for meropenem (MIC = 1 μg/mL), as compared with the recipient (MIC = 0.1 μg/ml). Successful transferring of the bla_OXA-48 gene and IncL/M plasmid replicon type to the transconjugants was confirmed by PCR.

Discussion

E. coli is the predominant facultative anaerobe species in Enterobacterales of the gut flora.⁴ Resistance to antibiotics in commensal flora is a critical health concern, as gut-colonizing resistant E. coli can be the origin of extraintestinal infections at later stages.^20,21 Our results revealed that the prevalence rate of ESBL-Ec differed from the rates reported previously in Iran. In our study, the fecal carriage rate of healthy subjects (43.1%) was lower than that of ESBL-Ec in hospitalized patients (60.3%) and higher than that of healthy children (36.2%).^22,23 The different prevalence rates of ESBL-Ec fecal carriage in healthy people have been addressed worldwide and ranged from about 50% in Thailand and 23% in Russia to 21.5% in South Korea.^24–26 This variation might be due to the dissimilarities between the socioeconomic state, poor sanitation, weak infection and prevention systems, age groups, method used, and low quality of antibiotics.

In the current work, the bla_CTX-M genes were highly prevalent in the identified ESBL-Ec isolates, with bla_CTX-M-15 being the predominant subtype; this finding is in congruence with those reported by former studies.^27,28 In addition, in our study, bla_CTX-M-14 was found to be infrequent, but it is a predominant subtype in East Asia.¹⁸

The rate of colonization with CPEc in the present study was 2.5%, which was lower than the fecal carriage rate reported from Iranian hospitalized patients (16.8%).²⁹ A major resistance mechanism to carbapenems in CREc isolates is the production of carbapenemases.³⁰ We detected bla_NDM-1, bla_NDM-5, and bla_OXA-48 carbapenemase genes in carbapenem-resistant isolates from healthy people. With the emergence of the resistance to carbapenems, the treatment of the infections caused by such bacteria will be challenging. There are some new functional antibiotics, such as CAZ/AVI, against CREc isolates.⁵ However, our results revealed that the CAZ/AVI MICs for 78.5% of CPEc isolates were up to 128 μg/mL. All the CAZ/AVI-resistant isolates were bla_NDM producing, whereas the sensitive isolates (21.4%) were non-NDM producers. Although CAZ/AVI is now unavailable in Iran, it can be applied for the treatment of non-NDM CPEc isolates, and identification of the dominant carbapenemase gene circulating in CPEc isolates in any region would help enter a new drug into the treatment panel.

At present, in Iran, MDR isolates resistant to carbapenems are treated with colistin. Plasmid-transmitted resistance to colistin has been reported earlier; nonetheless, we could not detect the carriage of mcr-1 gene in our isolates.¹ This observation signifies the need for subsequent studies to identify additional variants of mcr gene, or even other possible resistance factors.³¹ Accordingly, the identification of MDR E. coli in the feces of healthy individuals is a matter of serious concern due to the high risk of their dissemination to other individuals, particularly the distribution of ST131 clone.³²

International ST131 E. coli has been reported from varied countries, and the prevalence rate of asymptomatic carriage has been shown to be different.^33,34 The present study displayed the low prevalence rate (7.3%) of the intestinal colonization by the ST131 E. coli in healthy adults in the community. A study conducted on clinical specimens in Iran reflected a high prevalence rate of ST131 E. coli isolates in patients with urinary tract infection (26.9%).³⁵ The discrepancy between the prevalence rates of ST131 E. coli isolates likely arises from the specimen types and the higher exposure of the patients to the antibiotics in the hospital. Our results exhibited that all the ST131 E. coli isolates belonged to O25b serogroup, which supports the finding reported by a former study.⁴ As mentioned earlier, gut colonization by ST131 E. coli is associated with subsequent infections²⁰; therefore, molecular screening of ST131 E. coli, as an indicator to predict the risk of infection, might be helpful.³⁶

Besides eight MLST loci defined by the Pasteur scheme among O25b-ST131 E. coli isolates, different PSTs, such as PST43, PST527, and PST568, have reported elsewhere.⁴ PST43, based on the Achtman scheme, was the dominant PST in our ST131 isolates, which agrees with a study conducted earlier.³⁷ In addition to PST2, PST490 and PST43 were the most prevalent PST isolates identified in our carbapenemase-producing isolates. Moreover, two PSTs detected in our study, PST43 and PST2, have been recognized as carbapenemase-producing PSTs in Spain and Ecuador, indicating that not only the ST131 but also some other STs with high resistance to antibiotics in asymptomatic carriers need to be taken into account.^16,37 Amoxicillin/clavulanate or piperacillin/tazobactam are appropriate alternatives to carbapenems for treating infections caused by ESBL-Ec isolates.³⁸ However, our ST131 isolates displayed a high nonsusceptibility rate to amoxicillin/clavulanate; therefore, their efficacy for the treatment of E. coli infection remains to be determined. Our study revealed that bla_CTX-M-15 in ST131 isolates is predominant (84.7%) without a significance correlation. Hence, it seems that different STs have a role in expanding this gene by horizontal gene transfer. The high genetic variability within the ST131 E. coli clonal group has been demonstrated by using molecular typing approaches.³⁹ The genetic diversity of PFGE profiles, varied PST, and dissimilar antimicrobial susceptibilities can evidence the rapid release of this high-risk ST from various resources in community, a finding that is comparable with that of another study.⁴

Different pulsotypes and plasmid replicon types in ST131 E. coli in our survey might be the indication of the plasticity of the ST131 E. coli genome, as implied by other researchers.³² The ESBLs and/or carbapenemase genes were detected in the transconjugants, denoting that resistance genes are transferable between bacteria and could enhance the antibiotic resistance of other pathogens or microbiota of hosts. The data provided by our study uncovered that IncL/M and IncFIIs plasmid replicon types were responsible for the bla_OXA-48 and bla_CTX-M gene dissemination among ST131 E. coli isolates. Thus, tracking particular plasmids such as IncL/M and IncFIIs group is essential to prevent the spread of antibiotic resistance.

Our study also observed the concurrent transfer of bla_OXA-48 gene and IncL/M plasmid replicon type in one of the ST131 isolates harboring bla_NDM and bla_OXA-48 genes; however, the transfer of bla_NDM and its carrier plasmid were not found in this isolate. Therefore, it seems that the bla_OXA-48 gene is carried by IncL/M plasmid, and according to studies, the bla_NDM gene could be carried on IncA/C, IncX, IncFII, IncColE, and IncQ, or nontypeable plasmids.^40–43 Our future studies, with more precise molecular methods such as plasmid mapping, could investigate the presence of varied plasmids, the location of different genes on plasmids, and the genes associated with this resistance gene.

Conclusion

The intestinal carriage of ESBL-Ec, CPEc, and ST131 E. coli is a significant challenge to public health. Persistent carriage of ESBL/carbapenemase-producing ST131 in healthy individuals might intensify the risk of sustained ESBL carriage in the community. Thus, there is seemingly a need to control the spread of ST131 clone by fecal carriage.

Footnotes

Disclosure Statement

No competing financial interests exist.

Funding Information

Pasteur Institute of Iran, Tehran, funded this research.

References

Dandachi

, Chaddad

, Hanna

, Matta

, and Daoud

. 2019. Understanding the epidemiology of multi-drug resistant gram-negative bacilli in the Middle East using a one health approach. Front. Microbiol. 10:1941.

Alizade

2018. Escherichia coli in Iran: an overview of antibiotic resistance: a review article. Iran. J. Public Health, 47:1.

Baron

S.A.

, Diene

S.M

, and Rolain

J.M.

. 2018. Human microbiomes and antibiotic resistance. Humic, 10:43–52.

Nicolas-Chanoine

M.H.

, Bertrand

, and Madec

J.Y.

. 2014. Escherichia coli ST131, an intriguing clonal group. Clin. Microbial. Rev. 27:543–574.

Temkin

, Torre-Cisneros

, Beovic

, et al. 2017. Ceftazidime-avibactam as salvage therapy for infections caused by carbapenem-resistant organisms. AAC, 61:e01964-16.

Kim

Y.A.

, Kim

J J

, Kim

, and Lee

. 2017. Community-onset extended-spectrum-β-lactamase-producing Escherichia coli sequence type 131 at two Korean community hospitals: the spread of multidrug-resistant E. coli to the community via healthcare facilities. Int. J. Infect. Dis. 54:39–42.

Kondratyeva

, Salmon-Divon

, and Navon-Venezia

. 2020. Meta-analysis of pandemic Escherichia coli ST131 plasmidome proves restricted plasmid-clade associations. Sci. Rep. 10:1–11.

Pasteran

, Mendez

, Guerriero

, Rapoport

, and Corso

. 2009. Sensitive screening tests for suspected class A carbapenemase production in species of Enterobacteriaceae. JCM, 47:1631–1639.

Wayne, P.A. C Performance Standards for Antimicrobial Susceptibility Testing

LSI

. 2016. Clinical Lab Standards Institute. M100-S27.

10.

Magiorakos

A.P.

, Srinivasan

, Carey

, et al. 2012. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin. Microbiol. Infect. 18:268–281.

11.

Hammond

D.S.

, Schooneveldt

J.M

, Nimmo

, Huygens

, and Giffard

P.M.

. 2005. blaSHV genes in Klebsiella pneumoniae: different allele distributions are associated with different promoters within individual isolates. AAC, 49:256–263.

12.

Song

, Lee

, et al. 2009. CTX-M-14 and CTX-M-15 enzymes are the dominant type of extended-spectrum β-lactamase in clinical isolates of Escherichia coli from Korea. JMM, 58:261.

13.

Han

, Shi

, Wu

, et al. 2020. Dissemination of carbapenemases (KPC, NDM, OXA-48, IMP, and VIM) among carbapenem-resistant Enterobacteriaceae isolated from adult and children patients in China. Front. Cell Infect. Microbiol. 10:314.

14.

Johnson

J.R.

, Menard

, Johnston

, et al. 2009. Epidemic clonal groups of Escherichia coli as a cause of antimicrobial-resistant urinary tract infections in Canada, 2002 to 2004. AAC 53:2733–2739.

15.

Johnson

J.R.

, Clermont

, Johnston

, et al. 2014. Rapid and specific detection, molecular epidemiology, and experimental virulence of the O16 subgroup within Escherichia coli sequence type 131. JCM, 52:1358–1365.

16.

Ortega

, Sáez

, Bautista

, et al. 2016. Carbapenemase-producing Escherichia coli is becoming more prevalent in Spain mainly because of the polyclonal dissemination of OXA-48. JAC, 71:2131–2138.

17.

Ribot

E.M.

, Fair

, Gautom

, et al. 2006. Standardization of pulsed-field gel electrophoresis protocols for the subtyping of Escherichia coli O157: H7, Salmonella, and Shigella for PulseNet. FPD, 3:59–67.

18.

Carattoli

, Bertini

, Villa

, Falbo

, Hopkins

, and Threlfall

E.J

. 2005. Identification of plasmids by PCR-based replicon typing. J. Microbiol. Methods, 63:219–228.

19.

Tark

D.S.

, Moon

D.C

, Kang

H.Y

, et al. 2017. Antimicrobial susceptibility and characterization of extended-spectrum β-lactamases in Escherichia coli isolated from bovine mastitic milk in South Korea from 2012 to 2015. JDS, 100:3463–3469.

20.

Ríos

, López

M.C

, Rodríguez-Avial

, Culebras

, and Picazo

J.J.

. 2017. Detection of Escherichia coli ST131 clonal complex (ST705) and Klebsiella pneumoniae ST15 among faecal carriage of extended-spectrum β-lactamase-and carbapenemase-producing Enterobacteriaceae. JMM, 66:169–174.

21.

Kessler

, Nisa

, Hazen

T.H

, et al. 2015. Diarrhea, bacteremia and multiorgan dysfunction due to an extraintestinal pathogenic Escherichia coli strain with enteropathogenic E. coli genes. Pathog. Dis. 73:ftv076.

22.

Aghamohammad

, Badmasti

, Shirazi

A.S

, et al. 2019. Considerable rate of putative virulent phylo-groups in fecal carriage of extended-spectrum β-lactamase producing Escherichia coli. MEEGID, 73:184–189.

23.

Mahmoodi

, Rezatofighi

S.E

, and Akhoond

M.R.

. 2020. Antimicrobial resistance and metallo-beta-lactamase producing among commensal Escherichia coli isolates from healthy children of Khuzestan and Fars provinces; Iran. BMC, 20:1–11.

24.

Niumsup

, Tansawai

, Na-Udom

, et al. 2018. Prevalence and risk factors for intestinal carriage of CTX-M-type ESBLs in Enterobacteriaceae from a Thai community. EJCMID, 37:69–75.

25.

Hong

J.S.

, Song

, Park

H.M

, et al. 2020. Molecular characterization of fecal extended-spectrum β-lactamase-and AmpC β-lactamase-producing Escherichia coli from healthy companion animals and cohabiting humans in South Korea. Front. Microbiol. 11:674.

26.

, Kozlov

, Dumpis

, et al. 2018. Large variation in ESBL-producing Escherichia coli carriers in six European countries including Russia. EJCMD, 37:2347–2354.

27.

Farra

, T. Frank, L. Tondeur, et al. 2016. High rate of faecal carriage of extended-spectrum β-lactamase-producing Enterobacteriaceae in healthy children in Bangui, Central African Republic. Clin. Microbiol. Infect. 22:891. e1–e891. e4.

28.

Quiñones

, Aung

M.S

, Carmona

, et al. 2020. High prevalence of CTX-M Type Extended-Spectrum Beta-Lactamase genes and detection of NDM-1 carbapenemase gene in extraintestinal pathogenic Escherichia coli in Cuba. Pathogens, 9:65.

29.

Solgi

, Badmasti

, Aminzadeh

, et al. 2017. Molecular characterization of intestinal carriage of carbapenem-resistant Enterobacteriaceae among inpatients at two Iranian university hospitals: first report of co-production of blaNDM-7 and blaOXA-48. EJCMID, 36:2127–2135.

30.

Nordmann

2014. Carbapenemase-producing Enterobacteriaceae: overview of a major public health challenge. MEDMAL, 44:51–56.

31.

Yin

, Li

, Shen

, et al. 2017. Novel plasmid-mediated colistin resistance gene mcr-3 in Escherichia coli. MBio. 8:e00543-17.

32.

Mathers

A.J.

, Peirano

, and Pitout

J.D.

. 2015. The role of epidemic resistance plasmids and international high-risk clones in the spread of multidrug-resistant Enterobacteriaceae. Clin. Microbiol. Rev. 28:565–591.

33.

Ouédraogo

A.S.

, Sanou

, Kissou

, et al. 2017. Fecal carriage of Enterobacteriaceae producing extended-Spectrum Beta-lactamases in hospitalized patients and healthy community volunteers in Burkina Faso. MDR, 23:63–70.

34.

Leflon-Guibout

, Blanco

, Amaqdouf

, Mora

, Guize

, and Nicolas-Chanoine

M.H

. 2008. Absence of CTX-M enzymes but high prevalence of clones, including clone ST131, among fecal Escherichia coli isolates from healthy subjects living in the area of Paris, France. JCM 46:3900–3905.

35.

Rasoulinasab

, Shahcheraghi

, Feizabadi

M.M

, et al. 2020. Distribution of pathogenicity island markers and H-antigen types of Escherichia coli O25b/ST131 isolates from patients with urinary tract infection in Iran. Microbiol. Drug Resist. 27:3.

36.

Sasaki

, Hirai

, Niki

, et al. 2010. High prevalence of CTX-M β-lactamase-producing Enterobacteriaceae in stool specimens obtained from healthy individuals in Thailand. JAC, 65:666–668.

37.

Chiluisa-Guacho

, Escobar-Perez

, and Dutra-Asensi

. 2018. First detection of the CTXM-15 producing Escherichia coli O25-ST131 pandemic clone in Ecuador. Pathogens, 7:42.

38.

Hawkey

P.M.

, Warren

R.E

, Livermore

D.M

, et al. 2018. Treatment of infections caused by multidrug-resistant gram-negative bacteria: report of the British society for antimicrobial chemotherapy/healthcare infection society/British infection association joint working party. JAC, 73:iii2-iii78.

39.

Mora

, Dahbi

, López

, Mamani

, et al. 2014. Virulence patterns in a murine sepsis model of ST131 Escherichia coli clinical isolates belonging to serotypes O25b: H4 and O16: H5 are associated to specific virotypes. PONE, 9:e87025.

40.

Paskova

, Medvecky

, Skalova

, et al. 2018. Characterization of NDM-encoding plasmids from Enterobacteriaceae recovered from Czech hospitals. Front. Microbiol. 9:1549.

41.

Solgi

, Badmasti

, Giske

C.G

, Aghamohammad

, and Shahcheraghi

. 2018. Molecular epidemiology of NDM-1 and OXA-48-producing Klebsiella pneumoniae in an Iranian hospital: clonal dissemination of ST11 and ST893. JAC, 73:1517–1524.

42.

Voulgari

, Gartzonika

, Vrioni

, et al. 2014. The Balkan region: NDM-1-producing Klebsiella pneumoniae ST11 clonal strain causing outbreaks in Greece. JAC, 69:2091–2097.

43.

Sonnevend

, Al Baloushi

, Ghazawi

, et al. 2013. Emergence and spread of NDM-1 producer Enterobacteriaceae with contribution of IncX3 plasmids in the United Arab Emirates. JMM, 62:1044–1050.