Antimicrobial resistance profiles and molecular characterization of Escherichia coli strains isolated from healthy adults in Ho Chi Minh City, Vietnam

In this study, we attempted to isolate Escherichia coli from healthy adults in Ho Chi Minh City, Vietnam, and characterized its antimicrobial resistance profile, extended-spectrum β-lactamase (ESBL) genotype, phylogenetic grouping and virulence gene profile. A total of 103 E. coli isolates were obtained, and most of them were antimicrobial resistant such to streptomycin (80.6%), tetracycline (67.0%), ampicillin (65.0%), sulfamethoxsazole/trimethoprim (48.5%), nalidixic acid (43.7%), chloramphenicol (34.0%), cefotaxime (15.5%), ciprofloxacin (15.5%), kanamycin (12.6%), ceftazidime (10.7%), fosfomycin (4.9%) and gentamicin (2.9%). However, all these E. coli strains were susceptible to imipenem. Surprisingly, of 103 strains, 74 (71.8%) and 43 (41.7%) strains showed resistance to more than 3 and 5 classes of antimicrobials, respectively. Furthermore, 10 E. coli strains were ESBL-producers and positive for blaCTX-M genes (7 for blaCTX-M-9 and 3 for blaCTX-M-1), while five were additionally positive for blaTEM genes. S1-nuclease pulsed-field gel electrophoresis analysis revealed that 7 and 3 strains of E. coli carry blaCTX-M genes on their large plasmid and chromosome, respectively. Phylogenetic analysis exhibited that majority of the E. coli strains was grouped into A (44.7%), followed by B1 (23.3%), B2 (18.4%) and D (13.6%). Virulence genes associated with diarrheagenic E. coli, such as astA, EAF, eaeA, elt and eagg were also detected in ESBL-producing E. coli as well as antimicrobial resistant strains. These data suggest that commensal E. coli of healthy human could be a reservoir for antimicrobial resistance determinants and some of them might be harmful to human.

doi: 10.1292/jvms.  investigated in only children [14], raw food samples [49], household and small chicken farms [31]. However, comprehensive studies regarding analysis of antimicrobial resistance, ESBL type and virulence gene profile of commensal E. coli in adults were not conducted.
The study of the commensal E. coli population from healthy individuals is of great significance to understand and monitor the prevalence, distribution and carriage of pathogenic traits and antimicrobial resistance. The potentially pathogenic microbes with antimicrobial determinants may spread to other individuals not only from human but also animal and animal originated foods. On the other hand, the antimicrobial determinants may be transferred to more pathogenic bacteria in this niche. Keeping these factors in mind, this study was designed to characterize the commensal E. coli strains isolated from healthy adults of Ho Chi Minh City, Vietnam, for the presence of virulence gene, antimicrobial resistance profile and ESBL detection.

Bacterial isolates
A total of 103 stool specimens were collected from healthy adults whose ages ranged from 20 to 70 years in 3 urban districts (73 adults in 3, 6 and Tan Phu) and 1 suburban district (30 adults in Cu Chi) in Ho Chi Minh City, Vietnam, during March to November 2013. It was confirmed that none of the study participants had history of taking antimicrobials at least 3 months before the sample collection. All people whose samples were collected gave their informed consent for the participation in this study. Stool specimens were inoculated onto CHROMagar Orientation (CHROMagar Paris France, Paris, France). After incubation at 37°C for 22 ± 2 hr, a dark pink to reddish colony was selected and then identified to be E. coli by the IMViC tests [19]. One E. coli strain was collected from each sample and used for further analysis.

Determination of antimicrobial susceptibility
Antimicrobial susceptibility of the E. coli strains against ampicillin (AMP), cefotaxime (CTX), ceftazidime (CAZ), imipenem (IPM), nalidixic acid (NAL), ciprofloxacin (CIP), gentamicin (GEN), kanamycin (KAN), streptomycin (STR), fosfomycin (FOS), tetracycline (TET), trimethoprim/sulfamethoxazole (SXT) and chloramphenicol (CHL) was examined by disk diffusion method [11] on Mueller-Hinton agar (Becton Dickinson and Co., Sparks, NV, U.S.A.) using commercially available disks (Becton Dickinson and Co.). Charaterization of E. coli strains as susceptible, intermediate resistant or resistant was based on the size of the inhibition zones according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [10]. E. coli strain ATCC 29522 was used as a control strain in the susceptibility test. MDR was defined as resistance to at least one antimicrobial agent in three or more classes of antimicrobial agents [6].

ESBL phenotype and genotype confirmation
ESBL phenotype was analyzed by a double-disk diffusion test using CTX, CAZ, CTX/clavulanic acid and CAZ/clavulanic acid [10]. ESBL (bla CTX-M , bla TEM and bla SHV ) genes were determined by a multiplex PCR as described previously [27]. Genotype of bla CTX-M genes was further analyzed by PCR using four primer sets targeting the CTX-M-1, CTX-M-2, CTX-M-8 and CTX-M-9 groups [40].

Plasmid profile and location of bla CTX-M gene
Plasmid profiles of ESBL-producing E. coli strains were examined by S1-nuclease-PFGE with some modifications [3]. Briefly, a single genomic DNA block was incubated at 37°C for 40 min with 4 units of S1 nuclease (Thermo Fisher Scientific, Waltham, MA, U.S.A.) in 200 µl of 30 mM sodium acetate buffer (pH 4.6) including 50 mM NaCl, 1.0 mM zinc acetate and 5% (v/v) glycerol. The digested DNA was electrophoresed on a 1.0% pulsed-field certified agarose (Bio-Rad Laboratories Inc., Hercules, CA, U.S.A.) in 0.5X TBE (45 mM Tris, 45 mM boric acid and 1 mM EDTA) buffer at 6 V/cm 2 , with a switch time of 2-60 sec for 21 hr using the CHEF Mapper machine (Bio-Rad Laboratories Inc.). XbaI-digested Salmonella serotype Braenderup H98121 was used as a molecular size marker.
Location of bla CTX-M genes was analyzed by Southern hybridization. The resolved plasmids and genomic DNA by S1-PFGE gels were transferred onto nylon membranes (Perkin Elmer, Waltham, MA, U.S.A.). The membrane was hybridized with a specific 32 P-labeled DNA probe as described below. The bla CTX-M-1 and bla CTX-M-9 gene-probes were prepared by PCR using primers and conditions as described elsewhere [40] and labeled by random priming method using MultiPrime DNA Labeling System (GE Healthcare UK, Buckinghamshire, U.K.) with [α-32 P]-dCTP (111 TBq/mmol) (Perkin Elmer). Radioactivity was visualized by the BAS FLA-3000 system (GE Healthcare, Tokyo, Japan).

Phylogenetic characterization
Phylogenetic group of E. coli strains was determined by using a multiplex PCR as reported previously [9].

Antimicrobial susceptibility
A total of 103 E. coli strains including 73 from urban districts and 30 from suburban district were successfully isolated from 103 healthy adults in Ho Chi Minh City, Vietnam, and further analyzed for their antimicrobial susceptibility to 13 different drugs including AMP, CTX, CAZ, IPM, NAL, CIP, GEN, KAN, STR, FOS, TET, SXT and CHL. In this study, E. coli strains showing intermediate antimicrobial resistance were interpreted as resistant. As shown in Table 1, most of the E. coli strains were resistant to STR (80.6%), TET (67.0%), AMP (65.0%), SXT (48.5%) and NAL (43.7%), although all E. coli strains were susceptible to IPM. Moreover, 96.1% of the E. coli strains (99/103) were resistant to one or more antimicrobial agents. Notably, MDR was detected in 71.8% (74/103) of the tested strains. In addition, extensive MDR, which was defined to be resistant against at least five different classes of antimicrobial agents, was detected in 41.7% (43/103) strains. There was no significant difference in rate of MDR of E. coli strains isolated from healthy adults in urban (54/73; 74.0%) and suburban districts (20/30; 66.7%).

ESBL characterization and its genotype
Since 51, 5 and 11 E. coli strains were resistant to AMP alone, AMP and CTX, AMP, CTX and CAZ, respectively, these strains might produce ESBL. Indeed, 10 out of the 67 strains were confirmed to be ESBL-producing E. coli. Among 10 ESBLproducing E. coli strains, 9 were isolated from healthy adults in urban districts, while 1 was from healthy adult in suburban district. Subsequently, ESBL genes were characterized for these 10 ESBL-producing E. coli strains, and the result is summarized in Table  2. The bla CTX-M genes were detected in all the ESBL-producing E. coli. Moreover, five CTX-M-positive E. coli strains additionally possessed bla TEM genes, but none of the strains were positive for the bla SHV genes. Analysis of genotype for the bla CTX-M genes Table 1. Antimicrobial resistant E. coli strains isolated from healthy adults fecal samples (n=103)

Plasmid profiles and location of bla CTX-M genes
In general, the ESBL genes are predominantly located on plasmids and are easily disseminated. To see if ESBL genes are located on plasmid or chromosome, plasmid profile and location of bla CTX-M genes were analyzed for the 10 ESBL-producing E. coli strains by PFGE with S1-nuclease digestion and Southern hybridization. As shown in Fig. 1, there was at least one large plasmid ranging from 50 to 244 kb in size and a variation of plasmid profiles in ESBL-producing E. coli.
The Southern hybridization of S1-PFGE gel demonstrated that most of the ESBL-producing E. coli (7/10 isolates) harbored the bla CTX-M genes on a large plasmid (ranging from 50 to 200 kb in size) as shown in Fig. 1. Other 3 strains, however, carried the bla CTX-M genes on chromosome. Moreover, among the 5 strains harboring both bla CTX-M and bla TEM genes, 3 strains carried these genes on the same plasmid (data not shown).

Virulence gene profile
Distribution of virulence genes associated with DEC was examined, and the result is shown in Table 2. Of 103 E. coli strains, none of the strains were positive for bfpA, est, stx1, stx2, invE and daaD genes. However, astA was most prevalent and detected in 15 strains, followed by EAF (n=5), elt (n=3), eagg (n=2) and eae (n=1), indicating that some of the E. coli strains isolated from healthy adults are potentially virulent to human. In addition, 7 astA genes were detected in combination with EAF (n=2), elt (n=3), eagg (n=1) and eaeA (n=1). Fifteen out of 18 E. coli strains harboring virulence genes were MDR. Thus, the strains carrying both virulence and antimicrobial resistance genes may pose a serious threat to public health. Besides, 8, 7 and 3 E. coli strains harboring virulence genes belonged to the phylogenetic groups A, B1 and D, respectively (Table 2). Furthermore, 2 out of these 18 E. coli strains harboring virulence genes were isolated from healthy adults in suburban districts.

DISCUSSION
Emergence of antimicrobial resistant bacteria has been increasingly recognized as a serious problem in the world. Recently, a number of studies regarding prevalence of antimicrobial resistant E. coli including ESBL producer in not only hospitalized patients but also healthy people have been reported in many parts of the world [1, 4, 15, 21-23, 32, 42, 47]. However, there was less report regarding the comprehensive analysis of antimicrobial resistant E. coli strains isolated from healthy children in Vietnam [14].
In the present study, therefore, we attempted to isolate E. coli from healthy adults in Ho Chi Minh City in Vietnam for further characterization of their antimicrobial resistance, ESBL genotype and its location, phylogenetic groups and virulence gene profiles. A total of 103 E. coli were successfully isolated, and most of the strains showed resistance to one or more antimicrobials. The results of antimicrobial susceptibility in this study are in accordance with a previous study of healthy children in FilaBavi, near Hanoi, Vietnam, except for CIP [14] in which only 6 antimicrobials, such as AMP, CIP, CHL, NAL, TET, and SXT were examined and the isolated E. coli strains were resistant to AMP (65%), TET (74%), CHL (40%), NAL (27%) and SXT (68%). However, <1% of the E. coli strains isolated in FilaBavi were resistant to CIP [14], although 15.5% strains were resistant to CIP in this study. Surprisingly, 72% of the E. coli strains were MDR, and furthermore, 42% of the E. coli strains were extensive MDR. This high rate of MDR was observed in the E. coli strains isolated from healthy adults in both urban and suburban districts in Ho Chi Minh City. These percentages are quite high in comparison to those observed in a French study [32], but consistent with those reported from Korea and Bolivia [2,8]. These results revealed a high risk of fecal carriage of antimicrobial resistant E. coli strains in healthy adults in Ho Chi Minh City, Vietnam. One reason could be due to improper use of antimicrobials for human in Vietnam where antimicrobials can be purchased over the counter without a prescription and there is no sanction for non-compliance of drug law regulations [28]. Alternatively, it could be due to improper use of antimicrobials for food-animals [7,48] and veterinary medicine. The phenomenon of over usage of antimicrobials and thus emergence of MDR is an emerging problem worldwide, especially in developing countries. It could be also possible that antimicrobial resistant E. coli might be transmitted from food-animals and raw foods to human. The previous studies from southern Vietnam suggested that food-animals and raw foods could be a potential reservoir of antimicrobial resistance bacteria that disseminated to human [31,49]. It was found that commensal E. coli isolates on household and small-scale chicken farms in the Mekong Delta of Vietnam have high prevalence of resistance to TET, AMP, SXT, and CHL ( >60%) and MDR (81.3%), indicating the high risk of transmission of antimicrobial-resistant E. coli to community [31]. It was also reported that 61.6% of E. coli isolates from raw foods in the marketplace in Ho Chi Minh City were MDR and some of the tested strains contained a pool of mobile genetic elements for dissemination of antimicrobial resistant genes to human [49]. The occurrence rate of ESBL-producing E. coli in this study was lower than that of community from Hanoi, Vietnam (46.7%) [46], Thailand (65.7%) [23] and China (50.5%) [21], but consistent with that of community in Libya (13.4%) [1], Tunisia (7.3%) [4], Japan (6.4%) [22], Germany (6.3%) [47], France (6.0%) [32], Netherlands (7.8%) [42] and Switzerland (5.8%) [15]. Higher doi: 10.1292/jvms.  prevalence of ESBL-producing E. coli in Hanoi, Vietnam, and other countries could be due to differences in the isolation protocols. In those studies, β-lactam supplemented agar media were used for the isolation of the E. coli. ESBLs can be broadly divided into three groups, such as TEM, SHV and CTX-M, and CTX-M β-lactamases are the most widespread and frequently detected in E. coli [39]. In addition, genotype of CTX-Ms was found to be different and specific between regions in the world [50]. In this study, bla CTX-M-9 was found to be the most predominant group in healthy adults in Ho Chi Minh City, which is similar in northern Vietnam [5], Thailand [23] and China [21]. Whereas bla CTX-M-1 group was the most predominant β-lactamase gene in ESBLproducing E. coli in healthy carriage in European countries, such as Germany, France, Netherland and Switzerland [15,32,42,47]. In Ho Chi Minh City, it has been reported that bla CTX-M-9 and bla CTX-M-1 groups were the two most predominant types among ESBL-producing E. coli strains from foods [29] and patients with nosocomial infections in hospitals [45]. These data suggest that bla CTX-M-9 and bla CTX-M-1 groups might be circulating among foods, healthy adults and clinical settings in Ho Chi Minh City, Vietnam.
It is well known that plasmid plays an important role in the horizontal transfer of antimicrobial resistant genes in microbes including ESBL [12,25]. In this study, bla CTX-M genes were detected on plasmids, whose sizes are ranged from about 50 to 200 kb, in 7 out of 10 ESBL-producing E. coli strains. Our data indicate that ESBL-producing E. coli in healthy carriage could be a potential reservoir and source of dissemination of plasmid-mediated ESBL genes.
Phylogenetic group of E. coli strains isolated from healthy adults was investigated. It has been reported that the intestinal pathogenic E. coli are mostly classified into phylogenetic groups A, B1 and D [41], whereas strains belonging to phylogenetic group B2 are highly virulent and associated with extra-intestinal infections [38]. Most of the E. coli strains isolated in this study were identified to be phylogenetic group A followed by groups B1, B2 and D. This result is consistent with many of the previous reports that E. coli from community mainly belonged to phylogenetic group A [9,47]. Notably, the majority of MDR E. coli strains in this study belonged to phylogenetic group A (34/74 strains). Furthermore, 13 of the MDR E. coli strains belonged to phylogenetic group B2, suggesting that these strains might have a potential virulence capacity and could pose a risk to public health.
Furthermore, virulence gene profile analysis indicated that indeed 18 E. coli strains carried one or two virulence genes associated with DEC (Table 2). Among 11 virulence genes tested, astA was most prevalent, and some of the strains carried eae, EAF, elt and eagg in addition to astA. astA encoding EAST1 was initially detected in enteroaggregative E. coli (EAEC) [26] and thought to be a specific virulence factor of EAEC. However, astA has been detected in E. coli strains belonging to various pathotypes [41], and significance of EAST1 in diarrhea is not still clear. On the other hand, there are several reports regarding association of E. coli containing only astA among various virulence genes for DEC with a few outbreaks all over the world [16,33,34,51], indicating  MDR E. coli having DEC-associated virulence genes, in particular astA-positive MDR E. coli strains, might be harmful to human.
In conclusion, most of the E. coli strains isolated from healthy adults in Ho Chi Minh City, Vietnam, are MDR including extensive MDR. Some of them have virulence genes associated with DEC. Together with phylogenetic analysis, these data suggest that E. coli strains in healthy adults in southern Vietnam could be reservoir of antimicrobial resistance genes including ESBL and might be harmful to human.