Phenotypic and genotypic characterization of multi-
drug-resistant Escherichia coli isolates harboring
bla_CTX-M group extended-spectrum β-lactamases recovered
from pediatric patients in Shenzhen, southern China

Introduction

The swift emergence of antibiotic-resistant Enterobacteriaceae family is the major cause of hospital admission and associated morbidity and mortality in children.¹Enterobacteriaceae, predominantly Escherichia coli is a significant opportunistic pathogen causing infections in hospitals and serves as a key cause of urinary tract infections, gastrointestinal tract infections, bloodstream infection, and meningitis in humans.^2,3 E. coli is a major reservoir of the Extended-spectrum β-lactamases (ESBLs) encoding genes.⁴ ESBLs are able to hydrolyze the modern β-lactam antibiotics including third- generation Cephalosporin. A total of 350 dissimilar ESBLs variant has been identified till date. which are divided into nine separate families based on the amino acid sequences such as TEM, SHV, CTX-M, PER, VEB, GES, BES, TLA, and OXA.^5,6 Among them, TEM, SHV, CTX-M, and OXA are the major variants reported globally. Particularly bla_CTX-M has been increased rapidly and is now widely found in clinical isolates of E. coli across the world.^7,8 Some studies have demonstrated that ESBLs-producing E. coli has become an epidemic in China.^9–11 However, all these studies focused on food, environment, and adult clinical cases. So far, little is known about the epidemiology of ESBLs-producing E. coli in pediatric patients from southern China. Moreover, it is critical to provide up-to-date resistance pattern which guides the treatment decision in southern China. Therefore, this study was aimed to investigate the phenotypic and genotypic characterization of ESBLs-producing E. coli recovered from pediatric patients in Shenzhen Children’s Hospital, China.

Materials and methods

Bacterial isolation and identification

A total of 2,670 unique clinical E. coli isolates were collected (one isolate recovered from one child) between January 2014 and December 2015 from Shenzhen Children’s Hospital, China. This hospital is a major children hospital in the southern area of China. Among the 2,670 E. coli isolates, 950 (35%) were confirmed as ESBLs-producing E. coli by VITEK2 compact system (Ref. No. 27530/275660) of which 50 were randomly selected for molecular analysis. Among the 50 ESBLs- producing E. coli. isolates 32 (64%) were from male and 18 (36%) were from female, patients age ranges from 1 month to 12 years. The clinical isolation site for specimens was as follows, urine n=16, sputum n=16, pus n=12, catheter-associated n=3, blood n=2 and cerebral spinal fluid n=1(S-1).

Phenotypic detection of ESBLs production

The combination disc test was done for phenotypic detection of ESBLs production. The test was performed by using the disc of both cefotaxime and ceftazidime, alone and in combination with clavulanic acid. Control strain, which was selected from the characterized strain collection of our laboratory while ATCC25922 used as a negative control strain. The ESBLs production result was analyzed according to the Clinical and Laboratory Standards Institute (CLSI) guideline.¹²

Antimicrobial susceptibility test

Antimicrobial susceptibility was performed by VITEK@2 compact system (Biomerieux-Ref. No. 27530/275660) method for 18 antimicrobial agents, namely, Ampicillin/Sulbactam, Piperacillin, Ertapenem, Amikacin, Levofloxacin, Nitrofurantoin, Ampicillin, Cefazolin, Ceftazidime, Ceftriaxone, Cefepime, Imipenem, Cefotetan, Tobramycin, Gentamicin, and Ciprofloxacin. The results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guideline.¹²

Detection of β-lactamase and associated genes

The standard PCR was performed to detect the presence of ESBLs encoding genes: bla_TEM, bla_SHV, bla_CTX-M(variant), bla_GES, bla_CARB, bla_PER, bla_VEB, and bla_OXA using specific primers previously described.¹³ In addition, carbapenemase genes (bla_KPC and bla_NDM-1) and colistin resistance mcr-1 were determined in ESBLs-producing E. coli by PCR assay and sequencing. The specific primers were used as described in our previous study.¹⁴ The purified PCR products were sequenced commercially (Sangon Biotech-Shanghai, China). DNA Sequences were analyzed by NCBI-BLAST program.

Multi-locus sequence typing (MLST)

The sequences types (STs) were determined for ESBLs-producing E. coli isolates by MLST. PCR assay was performed to amplify internal portions of seven housekeeping genes of E. coli (adk, fumC, gyrB, icd, mdh, purA, and recA) with specific primers.¹⁵ Amplified products were sequenced commercially (Sangon Biotech-Shanghai, China). The allelic type and sequences type for all ESBLs-producing E. coli were determined with achtman scheme available at http://mlst.warwick.ac.uk/mlst/dbs/Ecoli.

Phylogenetic group detection

Major phylogenetic group of all ESBLs-producing E. coli isolates was determined by multiplex PCR assays, using the combination of three DNA markers genes (chuA, yjaA, and TspE4.C2) as described by Clermont et al¹⁶.

Plasmid transferability

Conjugation experiments were performed to analyze the horizontal gene transfer of bla_CTX-M for ESBLs-producing E. coli isolates by using streptomycin-resistant E. coli C₆₀₀ as the recipient strain. We used liquid mating assay as described earlier.¹⁷ Transconjugants were selected on Luria Bertani agar containing streptomycin 2,000 (µg/mL) and cefotaxime (32 µg/mL). Transconjugants were further tested for ESBLs production and the existence of bla_CTX-M genes by PCR.

PCR-based replicon typing

PCR-based replicon typing was performed for both plasmids from parental and transconjugant isolates. The Inc (Incompatibility) groups were determined by using specific primer introduced by Carattoli et al, in 2005.¹⁸

Results

ESBLs production and antimicrobial susceptibility

A total of n=950 shown ESBLs production by VITEK2 compact system (Ref. No. 27530/275660) of which randomly selected 50 E. coli further confirmed as ESBLs-producing E. coli by combination disc test. The result was analyzed according to the CLSI guideline. Antimicrobial susceptibility tests reflected that all of the randomly selected 50 ESBLs -producing E. coli isolates 100% were resistant to Ampicillin/sulbactam, Ceftriaxone, Cefazolin, and Ampicillin while Aztreonam (50%), Trimethoprim (60%), Gentamycin (38%), Ciprofloxacin (32%), Cefepime (30%), Cefotaxime (28%), Tobramycin (14%), and Nitrofurantoin (3,8%). However, all of the isolates were susceptible to Piperacillin, Ertapenem, Amikacin, Imipenem, and Cefotetan. The antimicrobial phylogram was analyzed by Bio-numeric software (Figure 1). The antimicrobial results indicate that all of 50 ESBLs-producing E. coli are resistant to two more than two class of antibiotics so-called as multi-drug resistant E. coli. According to the results obtained, resistance to Aztreonam and Cefepime showed a significant difference for the time period during January 2014–July 2014 and July 2014–December 2015.

Figure 1 Antimicrobial susceptibility pattern and phylogram of ESBLs producing E.coli, Red color indicates resistant; yellow color indicates intermediate resistant; Green color indicates sensitive.Abbreviations: E.coli, Escherichia coli; ESBL, extended-spectrum β-lactamases.

Molecular analysis of drug resistance genes

All of the 50 ESBLs-producing E. coli isolates were carrying bla_CTX-M genes, with the most common being bla_CTX-M-15 (18/50, 36%), followed by bla_CTX-M-14 (16/50, 32%) and bla_CTX-M-90 (9/50, 18%), bla_CTX-M-55 (3/50, 6%) bla_{CTX-M-101, 211, 27, 109} (1/50, 2%). Additionally, co-existence of other β lactamase genes was detected, bla_TEM (10/50,20%) followed by bla_SHV (8/50,16%), bla_GES (5/50,10%), bla_CARB (1/50,2%) (Table 1). The bla_PER, bla_VAB, and bla_OXA group genes were not detected in this study. It was noteworthy that all bla_CTX-M-14 gene carrying ESBLs-producing E. coli isolates were resistant to Ciprofloxacin. There was no significant difference in the prevalence of bla_CTX-M genes among the ESBLs-producing E. coli isolated from the different isolation sites or even samples. Moreover, carbapenemase-producing genes were detected, including bla_NDM-1 (14/50, 28%), bla_KPC (5/50, 10%), and most recently discovered colistin resistance mcr-1 (2/50,10%) (Table 1).

Table 1 β-lactamase encoding gene analysis and STs of ESBLs-producing E. coli

Multi-locus sequences typing and phylogenetic grouping

The extensive diversity of MLST was recorded from ESBLs-producing E. coli isolates, with a total of 30 different STs of which, ST131 (12%) was highly prevalent in Shenzhen (Table 1). E. coli ST95 clonal complex (CC) was the major complex observed among all studied isolates. The ST95CC has been usually observed from urine, sputum, and pus samples. All ST131CC isolates were recovered from general surgery wards, these results indicate that ST131CC E. coli was protuberant in the general surgery wards and key transporter for the bla_CTM-M-14 gene (Table 1). Our particular concern is that bla_CTM-M-15 gene was reported in different 15 STs in Shenzhen Children’s Hospital. This observation suggested that ESBLs-producing E. coli isolates carrying bla_CTM-M-15 gene spread in the Shenzhen region and are now widespread in Southern China. All ESBLs-producing E. coli isolates were classified into four phylogenetic groups, namely, A, B1, D, and B2. The results revealed that majority of isolates belonged to group A (54%), along with a substantial proportion for groups B1 (22%), D (8%), and B2 (6%).

Plasmid profiling

The successful transconjugants were selected from Luria Bertani agar containing streptomycin 2,000 (µg/mL) and cefotaxime (32 µg/mL). We observed that IncFIA(n=14), IncHI2 (n=10), IncFIB (n=10), IncFIIS(n=3), IncFIC(n=2), and IncFH1(n=2) “Inc” group plasmids were responsible for the horizontal gene transformation of blaCTX-M genes (S 2). Nine transconjugants isolates were not shown any “Inc” group.

Discussion

The incidence of CTX-M-type ESBLs among clinical isolates especially E. coli has noticeably increased in the earlier several years.¹⁹ To the best of our knowledge, this is the first study from southern China to precisely demonstrate the prevalence of CTXM-type ESBLs and antimicrobial susceptibility pattern of E. coli which were isolated from pediatric infectious cases. The prevalence of ESBLs-producing E. coli in our study was 35% which was lower than the across China-northwest (71.7%), southwest (61.1%), north (48.2%), and east (46.86%) reported in bloodstream infection.²⁰ The high prevalence of ESBLs-producing E. coli about 82.6% was reported from Taian, a large city in Shandong province, China. But, the prevalence of ESBLs-producing E. coli in Shenzhen, China higher than the other nations, namely, Brazil (12.8%), Chile (23.8%) and Argentina (18.1%).²¹ We have a lower prevalence of ESBLs-producing E. coli than rest of the country may due to sturdy prevention measures, however, we should pay continual attention to tackle this problem by following informed treatment decisions from past experience.

The antimicrobial susceptibility test data clearly indicate that high resistance rate of ESBLs-producing E. coli to Ceftriaxone, Cefazolin and Ampicillin (100%), Aztreonam (50%), Trimethoprim (60%), Gentamycin (38%), and Ciprofloxacin (32%) has raised serious concern and became a challenge for clinicians. Therefore, we suggest avoiding indiscriminate use of antibiotics in medical practice which will certainly lower the opportunities for the emergence of resistance. Our antimicrobial susceptibility results were comparable with another part of China, Taiwan, and Thailand.^20,22,23

We reported, bla_CTX-M-15 as the most prevalent genotype of ESBLs-producing E. coli in Shenzhen followed by bla_CTX-M-14, bla_CTX-M-90, bla_CTX-M-55. bla_CTX-M-101, bla_CTX-M-211, bla_CTX-M-27. This result indicates the diversity of CTX-M genotype of ESBLs-producing E. coli in Shenzhen, China. Similar results were reported from across China.^24,25 In our study bla_CTX-M-55 is not detected normally in pediatric patients, which means children may not be in contact with an animal since this genotype is mostly circulated via animal origin E. coli isolates.²⁶ Co-existence of CTX-M group gene and β-lactamase genes includes bla_TEM (20%) followed by bla_SHV (16%), bla_GES (10%), bla_CARB (2%) were detected in ESBLs-producing E. coli. The β-lactamase genes detection results indicate that the β-lactamase genes excluding CTX-M group decreased over the period of time comparable study reported by Jiranun Bubpamala.²³ Jiranun Bubpamala reported that CTX-M group genes continually increasing from 2007 to 2018 but other β-lactamase genes were declined. In addition, co-existence of ESBLs with either carbapenem-resistant genes bla_NDM-1 (28%), bla_KPC (10%), or most recently discovered colistin-resistant mcr-1 (10%) raises a concern about the spread of such superbugs in the Shenzhen area. Several reports showed the co-existence of carbapenem resistance genes and mcr-1 in E. coli in China.²⁷ The 41 ESBLs-producing E. coli isolates shown six different Inc plasmid groups which include IncFIA, IncHI2, IncFIB, InFIIS, IncFIC, and InFH1 similar types of plasmid group with associated with bla_CTX-M was reported in China.²⁸ Though our study was limited by the isolate numbers and geographic area however the results sufficiently implicate the need of close monitoring of such superbugs in a clinical setting for this region.

MLST results reflect that ST131 (12%) was the most common ST among the 50 ESBLs-producing E. coli isolates. Similarly, some current countrywide studies from tertiary and county hospitals have also shown that ST131 was found in 9.6%, 12.7%, and 13.4% of ESBL-producing E. coli, respectively.^8,29,30 In contrast, the percentage of ST131 ESBLs-producing E. coli is notably lower in China as compared to European and American regions, according to a community infection study in the US (53%), UK (64%), and Belgium (64%).^31,32

Conclusion

The study demonstrated that bla_CTX-M gene was dominant in ESBLs-producing E. coli at Shenzhen Children’s Hospital and was composed of a variety of subtypes. We described the ESBLs-producing E. coli has developed an increasing level of resistance to antibiotics. Our study stresses on the necessity of long-term monitoring on ESBLs-producing E. coli in hospital environments, especially in Shenzhen Children’s Hospital. National programs devoted to the health of children in China need to consider the emerging threat of ESBLs-producing bacteria, and research efforts should be devoted to focus on the molecular characterization of ESBL types as well as additional controlled studies assessing risk factors and possible outcomes for children.