Genome Sequences of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli Recovered from Mid-Stream Urine Samples in Accra, Ghana

Escherichia coli, a member of the commensal intestinal microbiota, is a significant aetiology of urinary tract infections (UTIs) and has a propensity for acquiring multidrug resistance characteristics, such as extended-spectrum beta-lactamases (ESBLs). Despite the increase in the incidence of ESBL-producing E. coli infections in sub-Saharan Africa, routine ESBL detection in Ghana is often absent, and molecular data on ESBL genotypes is scarce. Eleven ESBL-producing E. coli recovered from mid-stream urine samples were subjected to antimicrobial susceptibility testing and whole-genome sequence analyses. All isolates exhibited multidrug resistance, demonstrating phenotypic resistance to third-generation cephalosporins, such as cefotaxime, ceftazidime, and cefpodoxime. Three isolates demonstrated resistance to norfloxacin (a fluoroquinolone), and one isolate demonstrated intermediate resistance to ertapenem (a carbapenem). Analysis of the draft genomes identified multiple antimicrobial resistance genes including ESBL genotypes blaTEM-1B/TEM-190 (6/11 and 1/11, respectively), blaCTX-M-15/CTX-M-3 (7/11 and 1/11) and blaOXA-1/OXA-181 (3/11 and 1/11). The strains belong to 10 different serotypes and 10 different multilocus sequence types. This study provides information on phenotypic resistance in 11 ESBL E. coli from Ghana and AMR genotypes within their genomes.


Introduction
Escherichia coli is a natural member of the human microbiota and is a significant aetiology of urinary tract infections (UTIs) [1].E. coli is able to exchange genetic material, including antibiotic resistance determinants, which may be plasmids or other mobile genetic elements, with other intestinal commensals [2].One important group of antibiotic resistance determinants is beta-lactamases (encoded by bla genes), which hydrolyse the beta-lactam ring in penicillin and first-and second-generation cephalosporins (1GC and 2GC, respectively) [3][4][5][6][7].
There is an increase in the incidence of infections caused by ESBL-producing E. coli globally and in sub-Saharan Africa [12,13].Previous studies in Ghana have indicated high positivity rates (49.3-61.1%)for ESBLs among E. coli isolates recovered from clinical sources [14].This is of public health concern, particularly because ESBL detection among Gram-negative bacterial species is not routinely performed in the majority of the clinical microbiology laboratories in Ghana.In addition, very little is known about the antimicrobial resistance and virulence gene content of ESBLs in the country, leaving noticeable gaps in antimicrobial resistance surveillance data [15].This study, therefore, characterised 11 ESBL-producing E. coli using whole-genome sequencing and antimicrobial susceptibility testing to augment existing knowledge.

Phenotypic Investigations
Eleven (11) archived E. coli isolates that were recovered from mid-stream urine samples (from two hospitals in Accra, Ghana, during laboratory-based surveillance conducted between April 2017 and March 2018) and were confirmed by colonial morphology, Gram staining and Matrix-assisted Laser Desorption/Ionisation Time of Flight (MALDI-TOF) Mass Spectrometry (Bruker Daltonics, Bremen, Germany) using Biotyper™ system 2.0 software at the genus [log(score) 1.7-2.0)]and species [log(score) ≥ 2.0] levels.Antimicrobial susceptibility testing was performed using the Kirby-Bauer disc diffusion method and interpreted following the Clinical and Laboratory Standards Institute (CLSI) guidelines [16].E. coli ATCC 25922 and K. pneumoniae ATCC 700603 were used as control strains.ESBL was confirmed by the double-disc method with cefotaxime, ceftazidime, and cefepime, alone and in combination with clavulanic acid [17].Inhibition zone differences of ≥5 mm between the single and the combination discs for cefotaxime, ceftazidime, or cefepime were regarded as positive for ESBL production.

Overview of the Identified Genomes
Genome analysis for genes linked to antibiotic resistance against two curated databases, ResFinder and CARD, identified 28 and 70 genes, respectively (Figure 1A and 1B, respectively).β-lactamase genes from Class A (bla TEM , and bla CTX-M ) and Class D (bla OXA ) were identified; bla CTX-M-15 was identified in 7/11 isolates, bla CTX-M-3 was identified in an eighth isolate (0232).Additionally, bla TEM was present in 7/11 isolates-while CARD labeled all these as bla TEM-181 (Figure 1B), ResFinder distinguished the bla TEM of Isolate 0232 as bla TEM-35 , but those of the other six isolates as bla TEM-1B (Figure 1A).Protein sequence analysis identified bla TEM-35 in Isolate 0232 and bla TEM-1B in Isolates 0190, 0350, 0456, 0320, 0549, and 0492 (Figure 1A); bla TEM-1B differs from bla TEM-181 because of an A182V protein substitution.The bla OXA-1 gene was present in three isolates (0232, 0135, and 0198); however, Isolate 0232 had, in addition, bla OXA-181 (Figure 1); bla OXA-181 belongs to the OXA-48-like family carbapenem-hydrolysing Class D β-lactamases.PlasmidFinder identified putative plasmids through the identification of replicon sequences; however, it was not possible to link the AMR genes with these plasmids.
Analysis of genotypic and phenotypic data revealed a wide diversity within the limited isolate collection with the exception of Isolates 0513 and 0533 (Table 1, Figure 1).Isolate 0513 and 0533 were both predicted to be ST131 and had the same AST profiles (Table 1).Additionally, the AMR genotypes identified were identical (Figure 1).
Isolate 0232 was an outlier from the other E. coli isolates when clustering by AMR genes and was predicted to contain six or seven CDSs (CARD and ResFinder, respectively, Supplementary Tables S2 and S3) that were not found in the other ten isolates (Figure 1A).Isolate 0232 was also the only strain to harbour two bla OXA genes, and was also demonstrated to be intermediately resistant to ertapenem, but sensitive to ceftazidime (Table 1, Figure 1).Additionally, it was the only strain predicted to have bla CTX-M-3 , which is only one amino acid different from bla CTX-M-15 (Asp240Gly); however, the difference in the omega loop region of bla CTX-M-15 results in increased ceftazidime hydrolysis and antibiotic resistance compared with bla CTX-M-3 .Isolate 0320 was also identified to carry bla CTM-X-15 but was ceftazidime-sensitive.
strated to be intermediately resistant to ertapenem, but sensitive to ceftazidime (Table 1, Figure 1).Additionally, it was the only strain predicted to have blaCTX-M-3, which is only one amino acid different from blaCTX-M-15 (Asp240Gly); however, the difference in the omega loop region of blaCTX-M-15 results in increased ceftazidime hydrolysis and antibiotic resistance compared with blaCTX-M-3.Isolate 0320 was also identified to carry blaCTM-X-15 but was ceftazidime-sensitive.
(A) (B)  Isolate 0350 was the only strain susceptible to ampicillin, cefuroxime, and ceftriaxone despite possessing bla TEM-1B , the most common plasmid-mediated β-lactamase of ampicillin resistance.The bla TEM-1B protein sequence was identical in all six strains, with no mutations or frameshifts present in Isolate 0350.Genome analysis suggested that Isolate 0350 had only a few differences in AMR genes from its nearest and resistant neighbor (Figure 1); ResFinder only identified a lack of drfA14 compared with Isolate 0456, and CARD suggested Isolate 0350 was lacking acrS, catI, dfrA14, mdtM, and ugd, but contained a cpxA compared with Isolate 0456.These genes are not involved or targets for β-lactam resistance.
Three strains demonstrated resistance to norfloxacin (a fluroquinolone), which is primarily mediated through non-synonymous mutations in the DNA gyrase (gyrA/B) or topoisomerase (parC) genes.Analysis of the draft genomes demonstrated that compared with norfloxacin-sensitive isolates 0190, 0320, and 0513, resistant isolates 0198 and 0232 have a GyrA A83L and D87N mutation.Isolate 0198 also has an additional GyrA D78E mutation.No mutations were seen in the GyrB of resistant isolates compared to the sensitive consensus sequence.In ParC, Isolates 0198 and 0232 contained an S80I compared with the sensitive consensus sequence.Isolate 0465, which had phenotypic resistance to norfloxacin, did not have non-synonymous mutations within these genes compared to the sensitive strains.

Discussion
In this study, we expanded on the limited knowledge of ESBLs from Ghana.Resistance of isolates to ampicillin, cefuroxime, ceftriaxone, cefotaxime, cefpodoxime, and ceftazidime is consistent with the characteristics of ESBL-producing organisms.Moreover, high levels of resistance (88.9-100%) against ampicillin, cefuroxime, and cefotaxime have been reported among E. coli isolates in previous studies conducted in the country [23].It is interesting that the proportion of ESBL-producing E. coli resistant to norfloxacin in the current study (3/11, 27%) is comparable to the pooled proportion (6/15, 40%) reported for ESBL-producing and non-producing E. coli isolated from urine samples in a previous study in Ghana [24].No isolate was resistant to ertapenem despite the presence of carbapenem resistance determinants bla OXA-1 in three strains.A single isolate (0232) carried two bla OXA penemases did show intermediate resistance to ertapenem, and the low level of resistance may be due to this antimicrobial being rarely used in Ghana.However, it is concerning that one strain demonstrated intermediate resistance to ertapenem despite its limited use clinically.
The bla CTX-M and bla TEM genes were common among the ESBL-producing E. coli isolates detected in the study by Donkor et al. [24].Similarly, in the study by Deku et al. [25] involving a collection of E. coli isolates recovered from a variety of clinical specimens from Ho in Ghana-blood, high vaginal swab, pleural aspirates, sputum, ear swab, wound, and urine-bla TEM-1 and bla CTX-M (bla CTX-M-1 , bla CTX-M-825 , and bla CTX-M-914 ) were the predominant genes detected among ESBL isolates.Likewise, in the study by Mahazu et al. [26], which was conducted at two major hospitals in Ghana and involved E. coli from a similar set of specimens, as well as urethral discharge, pus, ascitic fluid, semen, endocervical swabs, stool, and others, bla CTX-M genes (bla CTX-M-14 , bla CTX-M-15 , and bla CTX-M-27 ) dominated.These may explain the high proportion of cefotaxime and ampicillin resistance in the current study.The bla CTX-M-15 gene and, to a relatively lesser extent, other bla CTX-M variants, have also been reported to be prevalent in E. coli isolated from locally-bred and imported chicken in the country [27,28].Hence, it seems that the bla CTX-M genes may be widespread in the country, as confirmed in other previous reports [29,30], and could be harboured by non-typical urine E. coli pathotypes, such as the enteroinvasive, enteroaggregative, enteropathogenic, enteropathogenic, enterotoxigenic, and enterohaemorrhagic E. coli.Dissemination of AMR genes is often associated with plasmid transmission.
In the study by Mahazu et al. [26] involving E. coli isolated from multiple clinical specimens, the predominant sequence type was ST131 (25/102, 24.5%).Similarly, in a recent study in the country, ESBL-producing E. coli isolated from poultry predominantly belonged to ST10 (31/45, 69%) and, in humans, to ST131 (4/34, 12%) [12].Other studies conducted in the African setting (Nigeria and Tanzania) have also commonly detected ST10 in poultry [31,32], while others conducted in European and Asian countries have additionally identified it in humans at high frequencies (9-11%) [33][34][35].The detection of CC10 among ESBL-producing E. coli in the current study suggests a link to the livestock production chain.This is especially important as they could cyclically serve as sources of multidrug-resistant E. coli infections and reservoirs for the accumulation of multidrugresistant E. coli and other Enterobacteriaceae shed by humans.CC131 is known to occur globally, is multidrug-resistant, commonly harbours the bla CTX-M-15 gene, and is associated with pandemics [36].The two E. coli isolates that belonged to CC131 each harboured the bla CTX-M-15 gene and several other resistant genes, and could be of the same strain.Similarly, STs belonging to CC ST10 were also observed to possess several multidrug-resistant genes that may possibly explain the phenotypic resistances observed.
This study underscores the importance of enhancing antimicrobial stewardship programmes and utilising robust tools like whole-genome sequencing to generate detailed data for informing AMR surveillance efforts in the country.The high level of antimicrobial resistance could be a reflection of the prevalent overuse and misuse of antimicrobials in Ghana.
A limitation of this study was the few isolates from a single time point, which reduces the generalisability of the findings.Subsequent studies that evaluate a higher number of isolates could be helpful in validating the observations in this study.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/microorganisms12061139/s1,Table S1: Prediction of serotypes; Table S2: Identification of antibacterial genes using Abricate and the CARD database; Table S3: Identification of antibacterial genes using Abricate and the ResFinder database.Table S4: Information on plasmids harboured by the E. coli isolates.

Informed Consent Statement:
The archived isolates examined were previously collected and deidentified, so informed consent was not required for this study.

Author Contributions:
Conceptualisation, N.T.K.D.D., B.E. and R.A.S.; methodology, N.T.K.D.D., R.A.S., B.E. and B.A.; software, R.A.S. and B.A.; validation, N.T.K.D.D., B.E. and R.A.S.; formal analysis, N.T.K.D.D., B.E. and R.A.S.; investigation, N.T.K.D.D., B.E. and R.A.S.; resources, The Fleming Fellowship Fund; data curation, N.T.K.D.D., F.A.-O., C.O.-N.and F.C.N.K.; writing-original draft preparation, N.T.K.D.D., B.E. and R.A.S.; writing-review and editing, N.T.K.D.D., B.E., F.A.-O., F.A.-O., C.O.-N., B.A., F.C.N.K., E.S.D. and R.A.S.; visualisation, N.T.K.D.D., E.S.D. and F.C.N.K.; supervision, R.A.S.; project administration, N.T.K.D.D. and R.A.S.; funding acquisition, R.A.S.All authors have read and agreed to the published version of the manuscript.Funding: This research was funded by the Fleming Fund Fellowship Scheme through the London School of Hygiene and Tropical Medicine and the University of Ghana according to Grant Number RZ07.Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board of Noguchi Memorial Institute for Medical Research, University of Ghana (FW00001824).

Table 1 .
Antibiotic susceptibility and clonal information of the ESBL E. coli isolates.

Table 2 .
Overview of draft genomes.