Multidrug-Resistant Escherichia albertii: Co-occurrence of β-Lactamase and MCR-1 Encoding Genes

Escherichia albertii is an emerging member of the Enterobacteriaceae causing human and animal enteric infections. Antimicrobial resistance among enteropathogens has been reported to be increasing in the past years. The purpose of this study was to investigate antibiotic resistance and resistance genes in E. albertii isolated from Zigong city, Sichuan province, China. The susceptibility to 21 antimicrobial agents was determined by Kirby–Bauer disk diffusion method. The highest prevalence was tetracycline resistance with a rate of 62.7%, followed by resistance to nalidixic acid and streptomycin with a rate of 56.9 and 51.0%, respectively. All isolates were sensitive or intermediate susceptible to imipenem, meropenem, amoxicillin–clavulanic acid, and levofloxacin. Among 51 E. albertii isolates, 15 were extended-spectrum β-lactamase-producing as confirmed by the double disk test. The main β-lactamase gene groups, i.e., blaTEM, blaSHV, and blaCTX-M, were detected in17, 20, and 22 isolates, respectively. Furthermore, four colistin-resistant isolates with minimum inhibitory concentrations of 8 mg/L were identified. The colistin-resistant isolates all harbored mcr-1 and blaCTX-M-55. Genome sequencing showed that E. albertii strain SP140150 carried mcr-1 and blaCTX-M-55 in two different plasmids. This study provided significant information regarding antibiotic resistance profiles and identified the co-occurrence of β-lactamase and MCR-1 encoding genes in E. albertii isolates.


INTRODUCTION
Escherichia albertii is a gram-negative facultative rod bacterium belonging to a member of the Enterobacteriaceae. It was previously recovered from stool specimens of sick Bangladeshi children and was preliminarily identified as atypical eae-positive Hafnia alvei (Albert et al., 1991(Albert et al., , 1992. In 2003, it was proposed as a new species, named E. albertii based on further genotypic and biochemical studies (Huys et al., 2003). E. albertii was reported to be the probable cause of death for redpoll finches (Carduelis flammea) in Alaska in 2004 (Oaks et al., 2010). In recent years, E. albertii was reported to be an emerging human enteropathogen associated with many sporadic infections and outbreaks in humans (Konno et al., 2012;Ooka et al., 2012Ooka et al., , 2013Asoshima et al., 2014;Murakami et al., 2014;Brandal et al., 2015;Inglis et al., 2015). Besides, it has been detected in water and raw meats of animal origin (Felfoldi et al., 2010;Maheux et al., 2014;Asoshima et al., 2015;Lindsey et al., 2015;Maeda et al., 2015;Wang et al., 2016), thus posing a high risk to public health.
Antimicrobials are one of the most successful forms of chemotherapy used in the treatment of infectious diseases by killing or inhabiting the growth of microorganisms. The most important and widely used antimicrobials are β-lactam drugs. In recent years, antimicrobial resistance among Gram-negative bacteria has been reported to be increasing (MacVane, 2017). In Enterobacteriaceae, resistance to the β-lactam is mediated by production of β-lactamase enzymes which inactivate the drugs by hydrolyzing the β-lactam ring (Bush and Bradford, 2016). Some clinically most important enzymes are as follows: (1) extended-spectrum β-lactamases (ESBLs), including SHV, TEM and CTX-M types; (2) carbapenemases, including class A (KPC types), class B metallo-β-lactamases (MBLs), and class D oxacillinases; and (3) the AmpC cephalosporinases (Bonomo, 2017).
Colistin is an antibiotic of last resort for the treatment of extensively drug-resistant Gram-negative bacteria (Kaye et al., 2016). In a recent study, the emergence of plasmid-mediated colistin resistance has been reported in E. coli, raising a great concern around the world (Liu et al., 2016). Thereafter, plasmid-mediated colistin resistance gene (mcr-1) has also been identified in other members of the Enterobacteriaceae from South America, Asia, Europe and Africa, suggesting that mcr-1 might be widespread (Olaitan et al., 2016;Al-Tawfiq et al., 2017).
To date, little is known about the antibiotic resistance of E. albertii. This study investigated the antibiotic resistance, identified the ESBL-producing and colistin-resistant isolates, and determined the distribution of β-lactamase genes and mcr-1 gene in E. albertii isolated from Zigong city, Sichuan province, China.
The minimum inhibitory concentration (MIC) of colistin was determined by broth microdilution method recommended by the joint CLSI-EUCAST Polymyxin Breakpoints Working Group 1 .

Identification of the ESBL-Producing Isolates
The double disk test was performed to confirm the ESBL phenotype. It was carried out on MHA with two pairs of disks (ceftazidime + ceftazidime/clavulanic acid and cefotaxime + cefotaxime/clavulanic acid) (BD Diagnostics, United States). The results were interpreted as recommended by the CLSI (CLSI, 2016). E. coli ATCC 25922 and Klebsiella pneumoniae ATCC 700603 were used as the quality control strains.

Detection of the β-Lactamase Gene Groups
Escherichia albertii isolates were inoculated on LB agar and incubated overnight at 37 • C. A colony was suspended in 50 µl of sterilized distilled water and boiled for 10 min. The cell suspension was centrifuged at 10,000 × g for 5 min, and the supernatant was used as template DNA. PCR was performed to screen the main β-lactamase gene groups, i.e., bla TEM , bla SHV , bla CTX−M , bla KPC , and bla NDM genes. All primers and PCR conditions used in this study were presented in Supplementary  Table S1. Each reaction tube contained 10 µl of master Mix (Qiagen, Germany), 0.5 µM of forward and reverse primers, and 1 µl of template DNA, and was made up to a total volume of 20 µl with sterile distilled water.
Detection and Sequencing of the mcr-1 Gene All 51 isolates were subjected to PCR for the presence of mcr-1 gene using primers described previously (Supplementary  Table S1). The expected PCR products were sequenced using the ABI 3730 Automated DNA Analyzer (Applied Biosystems, United States).
The mcr-1 sequences obtained in this study have been deposited in GenBank under accession numbers: KX765477-KX765480.

Plasmid Profiling
Plasmid DNA profiles of all 51 E. albertii isolates were analyzed using the S1-nuclease pulsed-field gel electrophoresis (PFGE) method. Briefly, the bacterial cells embedded in agarose were lysed by SDS/proteinase K, and then were digested with 8 U S1-nuclease at 37 • C for 30 min. Finally, each sample was resolved by PFGE in a Chef-Mapper (Bio-Rad, United States) at 14 • C, with a switch time 2.16 to 54.17 s at 6 V/cm for 18 h. Each DNA band visualized was considered as a unit length of linear plasmid. The approximate size of each plasmid was determined by comparing profiles with XbaI-digested DNA from Salmonella serotype Braenderup strain H9812 (Bai et al., 2017).

Mating Experiments
Four of the mcr-1-positive isolates were selected for conjugation experiments. Filter conjugation was carried out using E. coli J53 (sodium azide-resistant) as the recipient. The donor and recipient were grown on LB medium to an optical density at 600 nm of 0.5, mixed equally, and then incubated on sterilized filter paper for 4 h. The filter was then resuspended in LB medium, and dilutions were plated on M-H agar containing sodium azide (150 µg/mL and colistin (4 µg/mL) to select for transconjugants. Mobilization efficiency was calculated as the number of transconjugant colonies divided by the number of donor colonies (Wang et al., 2011).

Whole-Genome Sequencing
Genomic DNA was isolated from an overnight culture using the Wizard Genomic DNA purification kit (Promega, United States) according to the manufacturer's instructions. Total DNA obtained was subjected to quality control by agarose gel electrophoresis and quantified by Qubit (Life Technologies, United States). The complete genome was sequenced by single molecule real-time (SMRT) technology using the Pacific Biosciences (PacBio) sequencing platform performed at the Beijing Novogene Bioinformatics Technology, Co., Ltd. (McCarthy, 2010). The filtered reads were assembled to generate one contig without gaps using SMRT Analysis 2.3.0 (Berlin et al., 2015). The protein-coding sequences (CDSs) were predicted using GeneMarkS (Besemer et al., 2001). ARDB (Antibiotic Resistance Genes Database) 2 was used to search for antimicrobial resistance genes (Liu and Pop, 2009
Ten isolates (19.6%) were resistant to one antimicrobial agent, while the majority exhibited resistance to two or more antimicrobials tested. All isolates from diarrheal patients and healthy carriers were susceptible to imipenem, meropenem, piperacillin, cefepime, aztreonam, and levofloxacin (Supplementary Table S2).

ESBL-Producing E. albertii Isolates
Among 51 E. albertii isolates, 15 isolates from four sources, i.e., six isolates from duck intestine, six from chicken intestine, two from chicken meat and one from raw mutton samples, were ESBL-producing as confirmed by the double disk test. All isolates recovered from diarrheal patients and healthy carriers were non-ESBL-producing (Supplementary Table S2).
All ESBL-producing E. albertii isolates were resistant to piperacillin, cefuroxime, cephalothin, ceftriaxone, aztreonam, tetracycline and chloramphenicol, whereas resistance to streptomycin and nalidixic acid were observed in 14 of the 15 ESBL-producing isolates. ESBL-producing E. albertii isolates have higher resistance rate than non-ESBL-producing isolates, as expected ( Table 1).

mcr-1-Positive and Colistin-Resistant E. albertii Isolates
Four out of 51 isolates (one from raw mutton, one from raw chicken meat, and two from chicken intestine) were positive for MCR-1 encoding gene by PCR. Sequencing analysis showed that the four nucleotide sequences are identical to the first reported mcr-1 sequence in plasmid pHNSHP45 (GenBank accession number KP347127). The MICs of colistin of all 51 E. albertii isolates were determined by broth microdilution method. The four mcr-1positive isolates were colistin-resistant with MIC of 8 mg/L. The MICs of colistin of all mcr-1-negative isolates were less than 4 mg/L.

Co-occurrence of bla CTX−M and mcr-1 Genes in E. albertii Isolates
Except one isolate T150248 from healthy carrier, plasmids ranging in size from 36 to 283 kbp were identified by S1-nucleasebased PFGE in 50 E. albertii isolates. Among which, 23 isolates harbored one plasmid; 13 harbored two plasmids; 10 harbored three plasmids; three harbored four plasmids; and one isolate harbored five plasmids (Supplementary Table S2). Two different size plasmids (56 and 113 kbp) were identified in all four colistinresistant isolates, and an additional 45 kbp plasmid was present in three out of four colistin-resistant isolates ( Table 2).
All four isolates harboring mcr-1 were positive for bla CTX−M group gene (bla CTX−M−55 type) and were ESBL-producing. The four colistin-resistant isolates showed indistinguishable PFGE patterns and they were all typed as sequence type (ST) 2 | Antimicrobial resistance profiles, β-lactamase genes, and plasmid content of colistin-resistant E. albertii isolates.

Genome Features of E. albertii Strain SP140150 Harboring mcr-1
The completed genome sequence of SP140150 consists of a circular chromosome of 4,881,553 bp with an average GC content of 49.8% and three circular plasmids. Antimicrobial resistance genes were searched against these three plasmids. bla CTX−M−55 was identified in pEA-1, a plasmid of 129,356 bp in size with an average GC content of 51.9%. In addition, pEA-1 also carried chloramphenicol resistance gene cml, tetracycline resistance determinant tetA, streptomycin resistance genes aph6id and aph33ib, sulfonamide-resistant dihydropteroate synthase gene sul2, and multidrug efflux RND transporter OqxA and OqxB. None of any antimicrobial resistance genes was identified in pEA-2, the second plasmid of 57,110 bp in size with an average GC content of 46.1%. A 1626 bp CDS encoding MCR-1 was found located downstream of an insertion sequence ISApl1 in the third plasmid pEA-3. pEA-3 is 68,747 bp in size with an average GC content of 42.5%. pEA-3 possesses an IncFII-type backbone and contains 90 predicted CDSs encoding plasmid replication, maintenance and stability functions, and a type IV protein secretion system (Figure 2).

DISCUSSION
Antimicrobial resistance is an increasingly encountered phenomenon among the Enterobacteriaceae and an alarming threat to global health (Laxminarayan et al., 2013). Resistance to β-lactams is primarily because of bacterially produced β-lactamases that are able to hydrolyze the β-lactam ring (Bush and Bradford, 2016). There are several reports of porin-mediated resistance in clinical isolates of enterobacteria, mainly affecting susceptibility to β-lactams (Martinez-Martinez et al., 1996;Poirel et al., 2004;Pavez et al., 2008;Perez et al., 2013). Some E. albertii isolates in this study demonstrated resistance to four antimicrobials identified by the World Health Organization (WHO) as being of critical importance in the treatment of human infectious diseases, including piperacillin, ampicillin, cefotaxime, and cefepime (WHO, 2012). Furthermore, 29.4% of isolates demonstrated resistance to cephalothin that was identified by the WHO as highly important in human disease treatment (WHO, 2012).
Extended-spectrum β-lactamases are defined as enzymes produced by certain bacteria that are able to hydrolyze extended spectrum cephalosporin. TEMgroup (exception of TEM-1 and TEM-2), SHVgroup , and CTX-Mgroup β-lactamases are important types of ESBLs (Ghafourian et al., 2015). Among the three types, the prevalence of CTX-M is increasing in Enterobacteriaceae and predominates as a cause of extended spectrum cephalosporin resistance (Falagas and Karageorgopoulos, 2009). In the current study, the prevalence of bla CTX−M gene in E. albertii was in agreement with those reported in the other studies (Bora et al., 2014). Besides, bla CTX−M gene was mostly detected in isolates resistant to cefotaxime. Nineteen (37.3%) of E. albertii isolates carried two or three different β-lactamase gene groups, demonstrating the co-occurrence of these genes in various combinations (Shahid et al., 2011).
Carbapenem-resistant Enterobacteriaceae have been increasingly reported worldwide. The carbapenemases include NDMs, KPCs, OXA-48, and others (Tzouvelekis et al., 2012). KPCs are currently the most common cause of carbapenem resistance worldwide. The emergence of New Delhi metalloβ-lactamase (NDM-1) and its variants had raised a major public health concern. NDM-1 can hydrolyze a wide range of β-lactam antibiotics, including carbapenems (Khan et al., 2017). In China, Wang et al. (2015) has reported that Enterobacteriaceae remained susceptible to carbapenems. In the present study, none of the isolate was positive for bla KPC or bla NDM and all isolates demonstrated susceptibility to meropenem and imipenem.
Polymyxins are active against most members of the Enterobacteriaceae family, however, some are naturally resistant to polymyxins, like Proteus, Brucella, Legionella, Campylobacter, and Vibrio (Poirel et al., 2017). In additional to intrinsic resistance, mechanisms responsible for acquired resistance to polymyxins in Enterobacteriaceae have been identified, including genes encoding LPS-modifying enzymes; regulators of the PmrAB and PhoPQ two-component systems; the intrinsic regulator RamA (Poirel et al., 2017). Recently, Liu et al. (2016) had reported plasmid-mediated colistin resistance in E. coli. Thereafter, plasmid-mediated colistin resistance gene (mcr-1) FIGURE 2 | Structure of plasmid pEA-3 carrying mcr-1 from E. albertii strain SP140150. From outer circle to inner circle, each represents CDS, GC content and GC skew, respectively. The functions of corresponding CDSs are colored as indicated.
has been proved to be widespread in other members of the Enterobacteriaceae (Olaitan et al., 2016). In this study, four genetically related E. albertii isolates were positive for mcr-1 and were colistin-resistant. When the mcr-1 harboring plasmid pEA-3 sequence was compared using BLASTn to the nucleotide database at NCBI (accessed 26.01.2018), several highly similar (99% identities with query coverage over 90%) mcr-1 harboring plasmids from E. coli, Cronobacter sakazakii or Salmonella enterica were identified, suggesting that mcr-1 has also spread to E. albertii.

CONCLUSION
The present study provides significant information regarding antibiotic resistance of E. albertii from human, animal, and raw retail meats for the first time. Co-occurrence of β-lactamase and MCR-1 encoding genes in E. albertii isolates were identified. Further epidemiological assessments on the drug resistance patterns of E. albertii and determination of the molecular resistance mechanisms are needed in the treatment and prevention of both human and animal infections.

AUTHOR CONTRIBUTIONS
QL, HW, YM, JX, and YX designed the project; QL, HW, ZZ, XLiu, LZ, GY, XC, and JZ carried out the sampling work; YXu, XB, JW, XLi, NZ, SF, RF, and JL carried out the experiments and generated the data; XLi, JL, and YX analyzed the data and drafted the manuscript.
TABLE S2 | Antibiotic resistance patterns and prevalence of β-lactamase and MCR-1 encoding genes in E. albertii isolates.