First Detection of Carbapenem-Resistant Escherichia fergusonii Strains Harbouring Beta-Lactamase Genes from Clinical Samples

Recently discovered extraintestinal Escherichia fergusonii obtained from non-clinical samples has exhibited the potential for acquiring multiple beta-lactamase genes, just like many extraintestinal Escherichia coli strains. Albeit, they are often omitted or classified as E. coli. This study aimed to, therefore, identify carbapenem-resistant extended-spectrum beta-lactamase (ESBL) producing E. fergusonii isolates from clinical samples, determine their evolutionary relatedness using 16S rRNA sequencing analysis and screen for beta-lactamase genes. A total of 135 septic wound samples were obtained from patients on referral at a General Hospital in Lagos, Nigeria. For the phenotypic identification of isolates from culture-positive samples, morphological, and physiological tests were carried out. Identities of the isolates harbouring beta-lactamase genes were assigned to their genus strains using the 16S rRNA sequencing. The Kirby Bauer disc diffusion technique and double-disc synergy test were used to screen isolates for multidrug resistance and ESBL production. Carbapenem-resistant ESBL producing isolates were screened for beta-lactamase genes in a polymerase chain reaction. Three E. fergusonii isolates (CR11, CR35 and CR49) were obtained during this study. E. fergusonii strains were motile, non-lactose and non-sorbitol fermenting but positive for cellobiose and adonitol fermentation. The I6S rRNA assigned the phenotypically identified isolates to E. fergusonii species. All three isolates were multidrug-resistant, carbapenem-resistant and ESBL producers. Isolates CR11 and CR35 harboured cefotaximase (CTX-M) and temoniera (TEM) beta-lactamase genes while CR49 harboured sulfhydryl variable (SHV) beta-lactamase gene. We herein report the detection of multiple beta-lactamase genes in carbapenem-resistant ESBL producing E. fergusonii from clinical samples.


Results
A total of three E. fergusonii isolates designated CR11, CR35 and CR49 were classified to distinguish them from other Escherichia species associated from wound infections. Isolates were motile, non-lactose fermenters, positive for cellobiose and adonitol but negative for sorbitol fermentation (Table 1). Following 16S rRNA sequencing analysis, the phenotypically identified isolates were assigned to E. fergusonii specie group. The isolates; CR11, CR35 and CR49 were renamed as E. fergusonii ADE4 (MH040118), E. fergusonii ADE11 (MH04009393) and E. fergusonii ADE18 (MH040100), respectively. In Figure 1, the phylogenetic tree constructed using partial 16S rDNA sequences of 1349 bp (E. fergusonii ADE4), 1390 bp (E. fergusonii ADE11) and 1385 bp (E. fergusonii ADE18), showed that all three isolates obtained from this study shared mostly the same close relatives. The sequence similarity percentages of Escherichia fergusonii ADE4 when compared with other 16S rRNA gene sequences in Genbank were 99.33% to Escherichia fergusonii ATCC 35469 (NR_027549. 1    Antibiotics susceptibility patterns of CR11, CR35 and CR49 based on CLSI interpretative criteria showed multidrug resistance. All three isolates exhibited 100% resistance to all beta-lactam antibiotics, including the extended-spectrum beta-lactam (ceftriaxone, cefuroxime and ceftazidime) and carbapenem (imipenem and meropenem) antibiotics. Isolates CR11 and CR35 were also resistant to other antibiotics class such as fluoroquinolones (levofloxacin, Ofloxacin, ciprofloxacin) and nitrofurantoin. All three isolates expressed ESBL production. Polymerase chain reaction (PCR) revealed that CR11, CR35 and CR49 encoded different beta-lactamase genes ( Table 2). Isolates CR11 and CR35 harboured CTX-M type beta-lactamase ( Figure 2) and TEM beta-lactamase genes while Antibiotics susceptibility patterns of CR11, CR35 and CR49 based on CLSI interpretative criteria showed multidrug resistance. All three isolates exhibited 100% resistance to all beta-lactam antibiotics, including the extended-spectrum beta-lactam (ceftriaxone, cefuroxime and ceftazidime) and carbapenem (imipenem and meropenem) antibiotics. Isolates CR11 and CR35 were also resistant to other antibiotics class such as fluoroquinolones (levofloxacin, Ofloxacin, ciprofloxacin) and nitrofurantoin. All three isolates expressed ESBL production. Polymerase chain reaction (PCR) revealed that CR11, CR35 and CR49 encoded different beta-lactamase genes ( Table 2). Isolates CR11 and CR35 harboured CTX-M type beta-lactamase ( Figure 2) and TEM beta-lactamase genes while CR49 harboured SHV beta-lactamase gene. No carbapenemase gene was present in the three isolates from this study. CR49 harboured SHV beta-lactamase gene. No carbapenemase gene was present in the three isolates from this study.

Discussion
This study to the best of our knowledge, reports the first isolation of carbapenem-resistant ESBL producing E. fergusonii of clinical importance, contrary to the first published study on the isolation of E. fergusonii from drinking water sources, in Nigeria [28]. Some studies suggest that E. fergusonii had existed long before the first identification in 1985 but were often omitted or misidentified as E. coli, due to the low discriminatory power of phenotypic methods [7,28]. Previous studies have also reported several inconsistencies with the use of biochemical methods including API 20E and Vitek 2 (Vitek System; bioMérieux, Marcy l'Etoile, France and bioMerieux, Inc., Durham, NC), for identification of E. fergusonii [29,30]. Recently, the ability to ferment cellobiose and adonitol and inability to ferment lactose and sorbitol among the Escherichia sp. has been reported to be specific for E. fergusonii and can be a guide for differentiating E. fergusonii from other

Discussion
This study to the best of our knowledge, reports the first isolation of carbapenem-resistant ESBL producing E. fergusonii of clinical importance, contrary to the first published study on the isolation of E. fergusonii from drinking water sources, in Nigeria [28]. Some studies suggest that E. fergusonii had existed long before the first identification in 1985 but were often omitted or misidentified as E. coli, due to the low discriminatory power of phenotypic methods [7,28]. Previous studies have also reported several inconsistencies with the use of biochemical methods including API 20E and Vitek 2 (Vitek System; bioMérieux, Marcy l'Etoile, France and bioMerieux, Inc., Durham, NC), for identification of E. fergusonii [29,30]. Recently, the ability to ferment cellobiose and adonitol and inability to ferment lactose and sorbitol among the Escherichia sp. has been reported to be specific for E. fergusonii and can be a guide for differentiating E. fergusonii from other members of the genus Escherichia [7]. Escherichia fergusonii isolates obtained from our study were motile, utilised cellobiose and adonitol but did not ferment lactose and sorbitol. The first published biochemical profile of E. fergusonii revealed that they are motile and non-lactose fermenters, confirming our findings [31]. The study of Balqis et al. [32] and Maheux et al. [16] however reported varying patterns for lactose fermentation and motility. Not much information on phylogenetic studies of E. fergusonii are currently available, but phylogenetic analysis from this study revealed that E fergusonii ADE4, ADE11 and ADE18 shared the same clade with the genus Escherichia and Shigella. This is similar to previous reports on Escherichia fergusonii [31,33,34]. The genus Escherichia and Shigella are considered members of the same pathogenic lineage [35][36][37][38][39]. Naveena et al. [40] reported that Shigella and E. coli are phylogenetically the same but separated based on their clinical relevance and biochemical differences. Shigella and Escherichia are closely related genera with >99% 16S rDNA sequence similarity [40]. Whole-genome phylogeny analysis carried out by Sims and Kim [41], reported clustering of several members of the genus Shigella with different Escherichia phylogroups. Maheux et al., 2014 also reported the difficulty of using gene sequence alone in distinguishing non-E. coli Escherichia spp. strains in a group comprising E. coli, Shigella spp., E. fergusonii, and E. albertii. However, the use of a polyphasic identification method that combines 16S rDNA sequencing analysis with phenotypic methods has helped to differentiate E. fergusonii from Shigella sp., in this present study. Although both genera have very similar phenotypic traits, several differences in the biochemical characteristics of E. fergusonii have helped to differentiate them from Shigella sp. Escherichia fergusonii obtained from this study were motile, with the ability to ferment cellobiose and adonitol (Table 1), whereas members of the genus Shigella are non-motile, unable to ferment cellobiose and adonitol [42]. Also, E. fergusonii from this study were non-lactose fermenters (Table 1), unlike the slow lactose-fermenting S. sonnei [36].
The three carbapenem-resistant ESBL producing E. fergusonii stains obtained in this present study were resistant to imipenem, meropenem, ceftriaxone, ceftazidime and cefuroxime antibiotics. This finding is consistent with several studies that reported multiple resistance to broad-spectrum beta-lactam antibiotics in E. fergusonii isolated from animals and humans [17,19,31,[43][44][45]. Although Savini et al. [17] reported that resistance to broad-spectrum cephalosporin antibiotics by non-ESBL producing E. fergusonii from their study was due to non-compliance of the patient to the use of cephalosporin antibiotics administered, the E. fergusonii strains from this study were ESBL producers. Hence, resistance to broad-spectrum cephalosporins observed in this study was likely due to the presence of beta-lactamase resistance genes detected in the E. fergusonii strains. Escherichia fergusonii ADE4 and ADE11 strains harboured CTX-M genes (See Table 2). Our finding is similar to the first report of E. fergusonii from a farm animal in South Korea, harbouring CTX-M gene [45]. Our study could, however, be the first documented report of CTX-M gene in E. fergusonii from human samples. The SHV and TEM beta-lactamase genes were also detected in Escherichia fergusonii ADE18 and Escherichia fergusonii ADE4/ADE11, respectively. This corresponds with the study that reported SHV-12 as the first beta-lactamase gene detected in E. fergusonii from a human specimen [19]. Resistance to imipenem and meropenem antibiotics was observed as well in this study, supporting the first report on carbapenem-resistant E. fergusonii strains among 23 E. fergusonii isolates obtained from non-human primates [7]. The first report of carbapenem-resistant E. fergusonii from human samples is likely to be this study. None of the screened carbapenemase genes was present within the three E. fergusonii strains from this study. Resistance to carbapenem antibiotics could have been influenced by the beta-lactamase genes harboured by the E. fergusonii strains or was conferred by other carbapenemase gene types (e.g. OXA-48) not investigated in this study.
The limitation of this study was due to limited resources. Beta-lactamase genes detected during this study were not sequenced to determine their spectrum of activity and some carbapenemase genes were not screened. Despite these limitations, this study has shown that E. fergusonii can be a reservoir for the spread of antibiotics resistance and a threat to the effective treatment of bacterial infections they cause.

Culture and Phenotypic Identification of Clinical Isolates
A total of 135 samples were obtained from septic wound patients on referral to Government general hospital Odan, Lagos, Nigeria. Wound swabs were obtained using sterile swab sticks, labelled appropriately and transported to the laboratory within 2-4 h of collection for further analysis. Samples were cultured on sterile MacConkey agar plates (Rapid Lab, UK), using the streak plate method and incubated for 24 h at an optimum temperature of 37 • C. Positive cultures were subcultured on MacConkey agar to obtain pure isolates [46]. All isolates were phenotypically identified following standard routine morphological and cultural characteristics, as well as biochemical tests such as Gram stain reaction, sulphur reduction, indole, motility, methyl red, voges-Proskauer, urease, oxidase, sugar fermentation and citrate utilisation test [46]. Pure isolates phenotypically identified as E. coli. were stored in 20% Luria-Bertani (LB) broth (HIMEDIA, India) at −80 • C for long-term storage while working stocks for further analysis were held at 4 • C. Escherichia sp. were further screened for the ability to utilise cellobiose, sorbitol and adonitol. Motile isolates positive for cellobiose and adonitol fermentation but negative for lactose and sorbitol utilisation were designated CR11, CR35 and CR49.

Genotypic Identification of Isolates
A discrete colony of CR11, CR35 and CR 49 in overnight broth cultures were used for the 16S rRNA gene sequencing method [47]. Genomic DNA of isolates CR11, CR35 and CR 49 were extracted using a commercial genomic DNA extraction kit (AidLab, China), following manufacturer's instruction. Bacteria universal primer (27F: f5 -AGAGTTTGATCCTGGCTCAG-3 and 1492R: r5 -GGTTACCCTTGTTACGACTT-3 ) were used to amplify 16S rRNA gene of selected carbapenem-resistant ESBL producing isolates, in a simplex polymerase chain reaction (PCR), as described previously [48]. Briefly, initial denaturation of PCR mixture was carried out at 94 • C for 5 min, followed by 35 cycles at 94 • C for 30 s, 52 • C for 30 s, 68 • C for 60 s and a final extension at 68 • C for 5 min. 30 s. The PCR products were analysed by electrophoresis in Tris-borate EDTA buffer and viewed using UV fluorescence in a gel image documentation system. PCR pleural was purified using a PCR clean-up kit (Zymo Research, U.S.A.) [48] and eluted for sequencing at Macrogen Corp, Maryland, USA. Nucleotide sequences were determined using automated sequencer (Applied Biosystems SeqStudio Genetic Analyzer) and the dye-deoxy termination procedure. All 16S rRNA gene sequences obtained were edited using Bioedit (version 7.0.26) software and aligned by multiple sequence alignment technique using CLUSTAL W. The obtained bacterial DNA sequence was compared with other 16S rRNA genes in the GenBank, using the NCBI Basic Local alignment search tools BLAST-n program and a phylogenetic tree was constructed using Neighbour-Joining method in MEGA version 7.0.26 according to Saitou and Nei [49].

Detection of ESBL Production
Double disc synergy test (DDST) was carried out using Oxoid TM (United Kingdom) purchased augmentin (amoxicillin-clavulanate) and extended-spectrum cephalosporin antibiotics, following the recommended European Committee on Antimicrobial Susceptibility Testing breakpoint [51,52]. Ceftazidime (CAZ, 10 µg) and cefotaxime (CTX, 5 µg) disc alone and in combination with amoxicillin-clavulanate (AMX, 20/10 µg) were used to detect ESBL activity in isolates with reduced susceptibility to extended-spectrum cephalosporins. Mueller-Hinton agar (MHA) plates prepared according to manufacturer's standard were seeded with isolate (CR11, CR35 and CR49) suspensions of 0.5 McFarland turbidity standard. Antibiotic discs were placed with sterile forceps 25mm apart, centre to centre of the plates and incubated aerobically for 18 h at 37 • C. Escherichia coli ATCC 25922 strain was used as control. ESBL results were interpreted following the zone diameter interpretive clinical standard of the European Committee on Antimicrobial Susceptibility Testing. An increased inhibition zone diameter of ≤ 20mm centre to centre, augmented in the direction of amoxicillin-clavulanate antibiotics was recorded positive for ESBL production.

DNA Extraction and Genotypic Detection of β-lactamase Genes
DNA was extracted from CR11, CR35 and CR 49. DNA extraction was carried out using TENS (Tris-HCl; 10 mM (pH 8.0), EDTA; 1 mM, NaOH; 0.1 N, SDS; 0.5% (w/v)) miniprep [53,54]. Discrete colonies of overnight cultures were inoculated into 1.5 mL of Luria Bertani (LB) broth (HIMEDIA, India) in 2 mL Eppendorf tubes and incubated overnight for 18 h at 37 • C. Eppendorf tubes containing overnight cultures were spinned in a C1008-C centrifuge (Benchmark Scientific, USA) at 10,000 rpm for 1 min, to harvest cells. Supernatants were discarded, and bacterial pellets were resuspended in 1000 µL of phosphate-buffered saline (PBS), to remove cellular debris. Tubes containing resuspended cells were centrifuged at 10,000 rpm for 1 min, and supernatants were discarded. Aliquots of 300 µL of TENS buffer were added to bacterial pellets, gently inverted 3-4 times, to lyse bacterial cells. Aliquots of 150 µL 3M NaOAc (pH 5.6) were added, and the tubes were gently inverted 3-4 times to precipitate DNA. The tubes were centrifuged at 13,000 rpm for 5 min to pellet white precipitates. Clear supernatants containing DNA were carefully removed and transferred into new sterile Eppendorf tubes, using sterile 200 µL pipettes. Aliquots of 900 µL of 95% ethanol were added to clear supernatants and tubes were inverted. The tubes were centrifuged at maximum speed for 2 min to pellet DNA. Supernatants were discarded, and 500 µL of 70% ethanol were pipetted into tubes to wash the DNA pellets. The obtained DNA pellets were centrifuged for 2 min, ethanol solutions were discarded, and DNA pellets were dried in a CentriVap DNA [53,54] concentrator 79820 (Labconco, USA). DNA was dissolved in 50 µL 10mM Tris-HCl (pH 8) and chilled on ice for further experiment. DNA was screened for eight different beta-lactamase genes, including ESBLs (SHV, TEM and CTX-M genes), Oxacillinase (OXA) and carbapenemases (IMP, VIM, KPC and NDM genes). Beta-lactamase genes were screened for using specific primers (Table 3) as previously described [55][56][57][58]. For polymerase chain reaction (PCR) amplification of beta-lactamase genes in a thermocycler C100 Touch (Bio-RAD, USA), 5 µL aliquot of DNA template was added to a 20 µL master mix. Master mix contained 0.5 µL of deoxynucleotide (dNTPs) solution (New England Biolabs, UK), 0.5 µL each of primer pairs (forward and reverse), 0.125 µL Thermus aquaticus YT-1 (Taq) DNA polymerase (New England Biolabs, UK), 2.5 µL 10x standard Taq reaction buffer (New England Biolabs, UK) and 16.375 µL nuclease-free water (New England Biolabs, UK). The PCR mixture was preheated at 94 • C for 5 min, followed by 35 cycles at 94 • C for 30 s, 30 s at annealing temperature (Table 3), 60 s at 72 • C, and 72 • C for 7 min. About 4 µL of amplified PCR mixture was resolved on agarose (1%) gel electrophoresis stained with 4 µL ethidium bromide. Expected amplicon size was visualised under a UV fluorescence in a gel image documentation system 220 (Bio-Rad, UK).

Statistical Analysis
Results from this study were analysed using the SPSS version 20.0 statistical package. Results were presented in tables, figures and dendrogram.

Conclusions
The results of 16S rRNA gene sequencing analysis and phylogeny were consistent with the phenotypic identification of carbapenem-resistant ESBL-producing strains obtained from this study. However, for molecular identification and determination of evolutionary relationships, the 16S rRNA gene analysis method alone was not sufficient to adequately distinguish E. fergusonii stains from other closely related species. The use of partial 16S rDNA sequences (1349-1390 bp) of the three isolates for the construction of the phylogenetic tree might have contributed to the difficulty associated with differentiating Shigella from E. fergusonii, genotypically in this study. Further molecular analysis such as DNA-DNA hybridisation or multilocus sequence typing (MLST) is recommended to complement 16S rRNA gene analysis, for revealing molecular differences at the species level. Molecular characterisation of resistance genes detected during this study will as well throws more light on their location and clinical relevance.