Whole genome sequencing of extended-spectrum β-lactamase genes in Enterobacteriaceae isolates from Nigeria.

Extended Spectrum β-lactamase (ESBL)-producing Enterobacteriaceae are of major concern as they are implicated in multidrug resistant nosocomial infections. They are listed on a recently published global priority list of antibiotic-resistant bacteria by the World Health Organization which raises concern in both healthcare and community settings. This study aimed at determining the frequency of ESBL genes in multidrug resistant human clinical Enterobacteriaceae isolates from Edo state Nigeria and to characterize the resistance mechanisms using whole genome sequencing. A total of 217 consecutive clinical isolates of Enterobacteriaceae, selection based on inclusion criteria, were collected from March-May 2015 from three medical microbiology laboratories of hospitals in Edo state Nigeria. All isolates were analyzed using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Antibiotic susceptibility testing was performed by Kirby-Bauer method and minimum inhibitory concentration (MIC) determination by E-test method. Double disc synergy test was used to screen for the production of ESBL. Whole genome sequencing (WGS) was performed for isolate characterization and identification of resistance determinants. Out of 217 consecutive clinical Enterobacteriaceae isolates, 148 (68.2%) were multi-drug resistant. Of these multi-drug resistant isolates, 60 (40.5%) were positive for the ESBL phenotypic test and carried ESBL genes. CTX-M-15 was the predominant ESBL found, among 93.3% (n = 56/60). Thirty-two plasmid incompatibility groups and 28 known and two new sequence types were identified among the ESBL isolates. The high occurrence of CTX-M-15 with associated resistant determinants in multidrug resistant Enterobacteriaceae harboring different plasmid incompatibility groups and sequence types calls for the need of continuous monitoring of this resistance threat to reduce its public health impact. To our knowledge, this study presents the first genomic characterization of ESBL production mediated by blaCTX-M-15 in human clinical isolates of Enterobacter hormaechei, Citrobacter werkmanii and Atlantibacter hermannii from Nigeria.

Introduction

Enterobacteriaceae can cause various infections and can also be found as commensals in the gastrointestinal tract. Beta lactam antibiotics are widely used for treatment of infections caused by Enterobacteriaceae. However, increased use of these antibiotics, particularly of third generation cephalosporins, is associated with the emergence of bacterial resistance caused by extended-spectrum beta-lactamases. Extended spectrum β-lactamases are enzymes capable of hydrolyzing a wide range of extended-spectrum β-lactams, including oxyiminocephalosporins and aztreonam, but are less active against cephamycins and carbapenems [1]. ESBLs are inhibited in-vitro by β-lactamase inhibitors such as clavulanic acid and tazobactam. Gram-negative bacteria producing ESBLs often acquire associated resistance to fluoroquinolones, aminoglycosides, tetracycline and chloramphenicol [2]. ESBL genes and quinolone resistance (PMQR) genes can be co-located either on the same plasmid or on different plasmids within the same isolate [3, 4].

There are two classification systems for β-lactamases that are currently in use. They include the Ambler molecular classification and the Bush, Jacoby, and Medeiros classification. In the Ambler classification, enzymes are further categorized into four classes A, B, C, and D enzymes based on conserved motifs and protein sequence. The Bush, Jacoby, and Medeiros classification groups β-lactamases according to their substrate and inhibitor profiles. The β-lactamase mediated resistance is either mediated by plasmid or expressed chromosomally. Nevertheless, the spread of β-lactamases has been reported to be linked frequently with plasmid mediated ESBLs, especially the CTX-M family [5, 6]. The CTX-M enzymes are under the class A group of enzymes in the Ambler classification of β-lactamases. ESBL-producing Enterobacteriaceae were first described in 1983 [7] in relation with hospital-acquired infections, with resistance arising from point mutations in enzymes that are plasmid mediated like TEM-1, TEM-2 and SHV-1 [1]. CTX-M enzymes are now predominant and have rapidly spread globally. The CTX-M enzymes have been divided into six groups (CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, CTX-M-25, and KLUC, named after the first member of the group that was described). CTX-M-15 and CTX-M-14, a group 1 and 9 enzyme respectively are frequently identified globally in important microbes [8]. Klebsiella pneumoniae and Escherichia coli are the most common ESBL producing species. Transmission of ESBL-producing Enterobacteriaceae challenges healthcare facilities worldwide regarding the implementation of effective infection control measures to limit nosocomial spread. Recently, a global priority list of antibiotic resistant bacteria was published by the World Health Organization to increase and encourage research into new treatments for such pathogens. The list included extended-spectrum β-lactamase producing Enterobacteriaceae which calls for concern both in healthcare and community settings [6]. Extended-spectrum β-lactamase producing Enterobacteriaceae cause high morbidity and mortality rate and increased healthcare costs. An increasing prevalence of ESBL-Enterobacteriaceae correlates with a rise in the consumption of carbapenems [9, 10]; and this appears to have resulted in the emergence and spread of carbapenem resistance, especially in Enterobacteriaceae [11]. Carbapenemase-producing Enterobacteriaceae have been reported globally which includes a recent report of blaOXA-48, blaOXA-181, blaNDM1 in Enterobacteriaceae from Edo state, Nigeria in which some of the isolates had ESBL genes specifically CTX-M-15 [12, 13, 14].

Many reports have described and characterized ESBLs in Enterobacteriaceae [2, 7], but only a few reports originate from African countries [15, 16, 17, 18, 19, 20]. The aim of this study was to determine the frequency of ESBL genes in multidrug resistant human clinical Enterobacteriaceae isolates from Edo state Nigeria and to characterize the resistance mechanisms using whole genome sequencing.

Materials and methods

Bacterial isolates

A total of 217 consecutive clinical isolates of Enterobacteriaceae were collected from March-May 2015 from three medical microbiology laboratories of hospitals all in Edo state Nigeria (Fig 1). Isolates obtained were from samples of both in-patients and out-patients of the three different hospitals. The three hospitals are tertiary healthcare institutions with University of Benin teaching hospital having the largest number of bed capacity of over 910. Identities of the isolates were confirmed by MALDI-TOF mass spectrometry (Bruker Daltonik GmbH, Bremen, Germany) analysis.

Fig 1

Figure showing sample size of Enterobacteriaceae isolates provided by three Nigerian hospitals.

Ethical considerations

Research ethics approval was not required in the three hospitals where isolates were obtained from for the study. Only the pre-identified isolates used in the study were obtained from the microbiology laboratories. There was no contact with patients and the samples where the isolates were obtained from. Informed consent was not required by the institutions where the isolates were collected from as the data collected regarding the isolates which include patients’ age, gender and sample source of isolates was obtained from clinical records and analyzed anonymously. Data obtained was for analysis purpose and this was carried out anonymously.

Antimicrobial susceptibility testing and ESBL screening

The Kirby-Bauer susceptibility testing technique [21] was carried out and results were interpreted using European Committee on Antimicrobial Susceptibility Testing, (EUCAST) criteria [22]. The isolates were tested with 14 antibiotics: meropenem, ertapenem, ceftazidime, cefotaxime, amoxicillin/clavulanic acid, cefoxitin, cefepime, aztreonam, trimethoprim, ciprofloxacin, amikacin, piperacillin/tazobactam, chloramphenicol, levofloxacin (Oxoid, Basingstoke Hampshire, UK). The double disc synergy test was used to screen for the production of ESBLs [7]. ESBL production was screened phenotypically using cefotaxime (30 μg). The isolates were confirmed by combination disk tests with cefotaxime and ceftazidime (30 μg), with and without clavulanic acid (10 μg), as described by the CLSI guidelines [23]. A ≥ 5 mm increase in the zone diameter for cefotaxime or ceftazidime in combination with clavulanic acid versus the zone diameter of the corresponding antimicrobial agent alone defined an ESBL producer [23]. Isolates that were multidrug resistant and phenotypically positive for ESBL production were subjected to whole genome sequencing. Multidrug resistance was defined as non-susceptibility of an Enterobacteriaceae isolate to ≥1 agent of ≥3 antimicrobial categories [24]. Intrinsic resistances were not taken into account for categorization. The MICs of ampicillin, ciprofloxacin, trimethoprim were tested using E-test strips from bioMérieux (Marcy L’Etoile, France), those for amoxicillin-clavulanic acid, cefotaxime, cefepime, ceftazidime, aztreonam, amikacin using E-test strips from Oxoid (Basingstoke Hampshire, UK) for all the ESBL producing strains as previously described [25] and results were interpreted using EUCAST criteria [22].

Whole genome sequencing

Whole genome sequencing (WGS) was performed for all 60 multidrug resistant isolates, which were also positive for the ESBL phenotypic test as described above. Extraction of genomic DNA (gDNA) was carried out using the MagAttract HMW DNA extraction kit (Qiagen, Hilden, Germany). Genomic DNA was quantified on a Qubit® 2.0 Fluorometer using the dsDNA BR Assay kit (Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA) and diluted to 0.2ng/μl as recommended (Illumina sample preparation guide). The Illumina Nextera XT DNA library preparation kit (Illumina Inc, San Diego, CA, USA) was used for preparation of fragment libraries of the bacterial genomes. 1ng of gDNA was used for DNA fragment library preparation (Illumina sample preparation guide). Miseq reagent kit v3 containing the reagent cartridge and flow cell was used for a paired end sequencing using a read length of 2x300bp on an Illumina Miseq (Miseq v3.0, Illumina Inc). Pooled libraries were loaded on the reagent cartridge. Samples were sequenced for minimum targeted coverage of 100-fold using Illumina’s recommended standard protocols.

For sequence analysis the raw reads (FASTQ files) were trimmed at their 5’ and 3’ ends until an average base quality of 30 was reached in a window of 20 bases, and assembly was performed with Velvet version 1.1.04 [26] using optimized k-mer size and coverage cutoff values based on the average length of contigs with >1,000 bp. Assembled genomes were uploaded to the ResFinder 2.1 web server (http://www.genomicepidemiology.org) to identify resistance genes using a threshold of 98% identity (ID) [27]. All ESBL isolates were further typed by multilocus sequence typing (MLST; MLST 1.8, Centre for Genomic Epidemiology (CGE), Lyngby DK). The identity of ESBL Enterobacteriaceae isolates were further confirmed by performing a tetra correlation search (TCS) between draft genomes obtained in this study and JSpeciesWS reference database based on their resulting Tetra-nucleotide signature correlation index [28]. PlasmidFinder 1.3 webtool (http://cge.cbs.dtu.dk/services/PlasmidFinder) was used to analyze and classify plasmids on the draft genomes of the ESBL Enterobacteriaceae isolates using a threshold of 95% ID [29]. Plasmid replicons were detected and plasmids were assigned to Inc groups.

Nucleotide sequence accession numbers

This Whole Genome Shotgun project PRJNA600702 has been deposited at DDBJ/ENA/GenBank under the accession numbers JAAARR000000000-JAAATV000000000.

Results and discussion

Out of 217 consecutive clinical Enterobacteriaceae isolates, 148 (68.2%) were multi-drug resistant and were further investigated by whole genome sequence (WGS) analysis (Table 1).

Table 1

Antibiotic susceptibility test results showing % of resistant ESBL isolates.

Antibiotics	K. pneumoniae [n = 26]	E. coli [n = 17]	E. asburiae [n = 3]	E. cloacae [n = 5]	P.mirabilis [n = 1]	A.hermanii [n = 1]	C.werkmanii [n = 1]	E.hormaechei[n = 6]
Ceftazidime	100%[n = 26]	88%[n = 15]	100%[n = 3]	100%[n = 5]	-	100%[n = 1]	100%[n = 1]	100%[n = 6]
Cefotaxime	100%[n = 26]	100%[n = 17]	100%[n = 3]	100%[n = 5]	100%[n = 1]	100%[n = 1]	100%[n = 1]	100%[n = 5]
Amoxicillin-clavulanic acid	92%[n = 24]	47%[n = 8]	100%[n = 3]	-	-	-	100%[n = 1]	-
Cefoxitin	8%[n = 2]	35%[n = 6]	100%[n = 3]	100%[n = 5]	-	-	100%[n = 1]	100%[n = 6]
Cefepime	96%[n = 25]	82%[n = 14]	100%[n = 3]	60%[n = 3]	-	100%[n = 1]	100%[n = 1]	100%[n = 6]
Aztreonam	100%[n = 26]	94%[16]	100%[n = 3]	100%[n = 5]	-	100%[n = 1]	100%[n = 1]	100%[n = 6]
Meropenem	4%[n = 1]	-	-	-	-	-	-	-
Ciprofloxacin	65%[n = 17]	100%[n = 17]	100%[n = 3]	20%[n = 1]	100%[n = 1]	-	100%[n = 1]	83%[n = 5]
Amikacin	12%[n = 3]	-	-	20%[n = 1]	-	-	-	-
Chloramphenicol	39%[n = 10]	59%[n = 10]	100%[n = 3]	100%[n = 5]	100%[n = 1]	100%[n = 1]	100%[n = 1]	67%[n = 4]
Piperacillin-tazobactam	35%[n = 9]	18[n = 3]	-	40%[n = 2]	-	-	-	-
Ertapenem	15%[n = 4]	-	-	-	-	-	-	17%[n = 1]
Trimethoprim	92%[n = 24]	100%[n = 17]	100%[n = 3]	100%[n = 5]	100%[n = 1]	100%[n = 1]	100%[n = 1]	100%[n = 6]
Levofloxacin	54%[n = 14]	100%[n = 17]	67%[n = 2]	20%[n = 1]	100%[n = 1]	-	100%[n = 1]	83%[n = 5]

Of the multi-drug resistant isolates, sixty (40.5%) were positive for the ESBL phenotypic test and had ESBL genes detected. In total, the ESBL strains mostly were resistant to ceftazidime (88.9%), cefotaxime (93.7%), cefepime (82.5%) and aztreonam (90.5%). In addition, isolates showed resistance to amoxicillin-clavulanic acid (73%) and piperacillin-tazobactam (20.6%). MIC results confirmed data from the disc diffusion tests revealing high resistance rates among the ESBL producing isolates (S1 Table).

Whole genome sequencing revealed that 46 ESBL producing isolates harbored one single ESBL resistance gene while 14 isolates harbored double ESBL resistance genes. Ten Klebsiella pneumoniae isolates harbored the blaCTX-M-15 gene; one K. pneumoniae isolate harbored the blaSHV-28 gene, and one K.pneumoniae isolate had the blaCTX-M-11 gene. Thirteen Klebsiella pneumoniae isolates harbored both blaCTX-M-15 and blaSHV-28 gene and one Klebsiella pneumoniae isolate harbored both blaCTX-M-15 and blaSHV-33 gene. The blaCTX-M-15 gene was also detected in 15 E. coli, three Enterobacter asburiae, five Enterobacter cloacae, six Enterobacter hormaechei, one Atlantibacter hermannii, one Citrobacter werkmanii and one Proteus mirabilis respectively. One E. coli harbored the ESBL gene blaCTX-M-65 and one E. coli isolate had the blaCTX-M-14. S1 Table shows the characteristics of the ESBL producing isolates. Multilocus sequence typing (MLST) grouped the 26 ESBL producing K. pneumoniae isolates into 14 known sequence types (ST) with ST 15 being predominant (n = 6), ST 14 (n = 4), ST 340 and ST 1552 (n = 2) respectively. The 17 ESBL producing E. coli isolates were grouped into 8 known STs with ST 405 being predominant (n = 5), ST 410 (n = 2), ST 359 (n = 1), ST 5869 (n = 1), ST 156, ST 617, ST 648 and ST 131 (n = 2) respectively. MLST grouped the five E. cloacae isolates into the 3 known sequence types, ST 187, ST 156 and ST 836. Two ESBL producing E. cloacae were assigned to new sequence types. The six Enterobacter hormaechei were grouped into three different sequence types, ST 78, ST114 and ST 459 (n = 2) respectively. Eleven plasmid incompatibility groups were identified among the ESBL K. pneumoniae isolates which included the FIB, FII, FIA, P, Q1, R, HI1B, X3, ColKP3, ColpVC, Col (BS512) plasmid replicon types. The ESBL producing E. coli had 14 plasmid incompatibility groups which included the FIB, FII, FIA, FIB(AP001918), FIB(pB171), Y, I1, FII(pRSB107), X4, Col156, Col(MG828), FII(pCoo), Q1, Col (BS512) plasmid replicon types. All ESBL producing E. asburiae isolates had the FII(Yp), FIB(K) and P plasmid replicon types. Seven plasmid incompatibility groups were identified among the ESBL producing E. cloacae and E. hormachei isolates which included FII(Yp), R, FIB(K), FII(pECLA), HI2, HI2A, and A/C2 plasmid replicon types. The ESBL A. hermannii isolate had the HI2, HI2A plasmid replicon types while the C. werkmanii isolate had the FIB (pHCM2) and R plasmid replicon types. The ESBL producing P. mirabilis isolate yielded a Q1 plasmid replicon type.

ESBL producing organisms are an increasingly important cause of multi-drug resistant infections [30]. ESBL genes are often located within mobile genetic elements such as plasmids, transposons and integrons which also can contain other resistance genes, thereby conferring resistance to antimicrobials that are extensively used in animals and humans (e.g. trimethoprim, aminoglycosides, fluoroquinolones, and sulfonamides). This could play an important role in co-selection [31].

The number of multidrug resistant ESBL producing Enterobacteriaceae isolates in our study was 60 (40.5%). The blaCTX-M-15 gene was identified in 93.3% (n = 56/60) of the ESBL producing isolates. This is consistent with previous findings demonstrating the predominance of CTX-M enzymes and in particular CTX-M-15 [32, 33]. CTX-M-15 enzymes have been identified worldwide [34] including Nigeria in Klebsiella spp., Escherichia coli, Proteus spp., Pseudomonas aeruginosa and Enterobacter spp. [19, 35, 36]. There is limited data on the prevalence of ESBL-producing Enterobacteriaceae in Africa. A previous report showed a high prevalence of ESBL carriage with multiple clones in children presenting at a pediatric department in Guinea-Bissau [17]. Another report from South Africa revealed the dissemination of ESBL-producing K.penumoniae within and between wards of hospitals [15]. In Angola, a high occurrence of CTX-M-15 in diverse Enterobacteriaceae sp and non-clinical niches was reported which included Enterobacter hormaechei (n = 2) and Citrobacter werkmanii (n = 1) [20]. Recently, a high prevalence of ESBL-producing isolates was reported among K. pneumoniae strains detected in clinical specimens from a teaching hospital in Cōte d’Ivoire with paediatric patients being most frequently affected [18]. Previous reports also from Nigeria revealed ESBL production mediated by blaVEB in Providencia spp. isolated from chicken faecal samples [37] and ESBL production mediated by blaVEB in human clinical Providencia spp. and Citrobacter freundii isolates [36, 38]. To our knowledge this study presents the first genomic characterization of ESBL production mediated by blaCTX-M-15 in human clinical isolates of Enterobacter hormaechei, Citrobacter werkmanii and Atlantibacter hermannii from Nigeria. Our results show a wide dissemination of the blaCTX-M-15 gene, which could also be attributed to widespread and uncritical use of beta-lactam antibiotics. The results from this study are similar to previous studies especially from African countries where the increasing prevalence of ESBL Enterobacteriaceae especially blaCTX-M-15 has been reported [15, 16, 17, 18, 19, 20]. Reports from developing countries especially in Africa are alarming considering the diagnostic and treatment possibilities available. Multiple resistance determinants were found on the draft genome sequences of the multidrug resistant ESBL producing isolates. In this study, 87% (n = 52/60) of ESBL producers harbored PMQR determinants thereby supporting the strong association between ESBL production and quinolone resistance previously reported in Enterobacteriaceae [39]. The aac(6’)-lb-cr gene has spread rapidly among Enterobacteriaceae, and although conferring only low level resistance, it may create an environment facilitating the selection of highly resistant determinants especially in organisms harboring topoisomerase mutations. Resistance genes are also disseminated through horizontal gene transfer which is supported by the co-selection through various antimicrobials [39, 40]. The acquisition and accumulation of resistance determinants led to the emergence of multi-drug resistant ESBL producers further limiting therapeutic options.

ESBL genes of the TEM, SHV and CTX-M families can reside on large conjugative plasmids [7]. Additional resistance genes present on the same plasmid carrying bla explain the multiresistant phenotype of CTX-M 15-producing bacteria [41]. Conjugative plasmids are regarded as one of the main factors in the successful spread of CTX-M type ESBLs among Enterobacteriaceae [42]. The presence of different plasmid replicon types in ESBL producing isolates reveal their importance in the dissemination of these resistance genes. The IncF family is the most commonly reported among the ESBL isolates. IncF plasmids are low-copy number plasmids, often carrying more than one replicon [43]. Detection of this plasmid type known to evolve quickly by replicon diversification and acquisition on antibiotic resistance traits [44] increases the potential role of Enterobacteriaceae as a reservoir for ESBL genes and other resistance determinants.

Application of whole genome sequencing offers a level of discrimination and information on relatedness and resistance mechanisms that surpass previous typing methods [45, 46]. ST 15, predominant among the ESBL producing K. pneumoniae isolates, corresponds to an internationally occurring clone and has been associated with different ESBL genes and MBL genes coding for NDM and VIM [47, 48]. Recently, Izdebski et al., [49] found that ST66, ST78, ST108 and ST114 strains spread as high-risk international clones of extended spectrum cephalosporin-resistant E. cloacae. The results from the MLST analysis of this study showed two E. hormaechei isolates belonged to the international clone ST114 recognized as a high-risk lineage responsible for the dissemination of carbapenemases and ESBLs in hospital setting [49]. For the E. coli isolates, MLST analysis revealed eight different known STs, including ST131, ST405 and ST410. ST131 is a dominant international clinical clone. ST405 has been associated internationally with the carriage of ESBLs and ST410 has been reported worldwide in extra intestinal pathogens associated with resistance to fluoroquinolones, third-generation cephalosporins and carbapenems [50, 51]. The distribution of the different sequence types and the plasmid replicon types of the ESBL isolates especially the predominant ones across the three hospitals suggests a probable clonal spread of the ESBL strains both intra and inter-hospital. A limitation of this study however, is to conclude on clonal bacterial transmission between ESBL Enterobacteriaceae isolates that included seven groups of different organisms. Another limitation of this study is the lack of adequate clinical data [e.g. differentiating samples of in-patients from out-patients] that might help to identify if the frequency of ESBL carriage was community based or nosocomial.

Previously, only a few reports from Nigeria provided draft genome sequence analyses, mainly concerning multidrug-resistant E. coli strains isolated from humans and chicken [52, 53]. Whilst three ESBL E. coli poultry isolates belonged to ST6359 [52], the poultry E. coli isolates reported by Sharma et al., were assigned to ST131 and ST162 respectively. The human E. coli isolates were assigned to six different STs which included ST131, ST617, ST542, ST398, ST4143 and ST398 [53].

Conclusions

Many factors can contribute to high rates of antibiotic resistance in developing countries. These include: poor drug quality or inadequate posology, long-term treatments, misuse of antibiotics by health professionals, unskilled practitioners, self-medication (antibiotics can be purchased without prescription), unhygienic conditions accounting for the spread of resistant bacteria and inadequate surveillance programs [54, 55]. In Nigeria the collapse of the primary healthcare system coupled with the unavailability of drugs in hospitals had the effect that people often resort to purchase drugs over the counter and in some cases from roadside sellers, which exposes them to the danger of acquiring and selecting ESBL-producing organisms [56]. Unlike the situation in developed countries, the financial resources to provide alternative agents such as carbapenems are lacking in developing countries and the option to tailor therapy based on antimicrobial resistance testing is unavailable, except for a few hospitals. To control the emergence and spread of ESBL-producing Enterobactericeae, it is essential for the public to practice good hygiene habits and comply with recommendations on the proper use of antibiotics. Proper infection-control practices and barriers are essential to prevent spreading and outbreaks of ESBL-producing bacteria. At an institutional level, practices that can minimize the spread of such organisms include clinical and bacteriological surveillance of patients admitted to intensive care units and antibiotic cycling, as well as policies of restriction, especially on the empirical use of broad-spectrum antimicrobial agents such as the third- and fourth-generation cephalosporins and quinolones [57].

Results from this study could serve a step towards developing targeted strategies to control the spread of ESBL producing bacteria in Benin city and Nigeria at large. Methods should be improved to efficiently detect and track bacterial clones and plasmids that constitute the major vehicles for the spread of ESBL-mediated resistance. Ideally, such methods of detection should be accessible to medium-level diagnostic microbiology laboratories, to assure the possibility of performing interventions in real time.

Characteristics of the ESBL isolates.

(XLSX)

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Antibiotic susceptibility test result of the ESBL isolates.

(DOCX)

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Source data of the ESBL isolates.

(DOCX)

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Sequence and Plasmid replicon types of the ESBL isolates.

(XLSX)

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11 Nov 2019

PONE-D-19-26950

Molecular characterization of extended-spectrum β-lactamase genes in Enterobacteriaceae isolates from Nigeria

Dr Christiana Jesumirhewe, Department of Pharmaceutical microbiology, Igbinedion University Okada, Edo state, Nigeria

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Reviewer #1: Jesumirhewe and collaborators present an interesting manuscript with the general objective of determine the frequency of ESBL genes in multidrug resistant human clinical Enterobacteriaceae isolates from Edo state Nigeria and to characterize the resistance mechanisms. Thank you for being able to contribute to it before your presentation to the scientific community. Therefore, I suggest that the following questions be modified, deleted, or explained.

Line 1. I think authors should change in the title: Whole genome sequence of extended-spectrum β-lactamase genes in Enterobacteriaceae isolates from Nigeria.”

Improve the Abstract and Introduction. ESBL concern in both healthcare and community settings

Line 16, 64. “This study aimed at determining the frequency of ESBL genes in multidrug resistant human clinical Enterobacteriaceae isolates from Edo state Nigeria” using whole genome sequencing

Line 33. I suggest to describe Ambler classification of beta-lactamases and groups of CTX-M on the introduction.

Line 51. I would rather see table 1 summarized in a Figure instead of a table. I suppose it would be better demonstrated. The three hospitals are at a different level of care?

Why did you decide to include the strains of unidentified source; Teaching Hospital, Benin n=12, and Okada n=4

Line 74. “Flurometer” Fluorometer

Line 94. The authors reported that (41%) were positive for the ESBL phenotypic test and had ESBL genes detected. Please explain the results of Pret3577 and EC11, where no resistance pattern to B lactams was observed. In strains with ESBL and carbapenemase, the ESBL screening and confirmation performs well?

Line 115. Table 4. Please highlight in results the presence of carbapenemases NDM and Oxa-48 genes in strains Kp_852 ST15, Kp_852K ST15, Kp_872 ST340 (First cases described?)

I recommend to improve tables, columns of ST set in the beginning

Have all the sequences been deposited at GenBank?

Is it possible probable clonal spread of the ESBL strains Intra or inter-hospital

Limitations must be included

I suggest to include virulence factors and mobile genetic elements associated with ESBL and phylogeny based on core genome MLST.

I recommend to highlight the WGS findings (figures), highlight the emerging global high risk clones, and to present this manuscript in a better fashion.

Reviewer #2: The manuscript evaluates the frequency of ESBL genes in multidrug resistant human clinical Enterobacteriaceae isolates from Edo state Nigeria and characterize the resistance mechanisms. The research is correctly conducted, and the conclusions are supported by the data. However, results are not well exposed and are difficult to understand.

Materials and method

Line 66. The phenotypic method used, is not useful for microorganisms with Amp C , so some ESBL producers could not be included, and this could be considered as a limitation

Table 1. Use italics to Enterobacteriaceae. This table does not present summarized data and contains partial results. Include n values

Results: Include summary tables or figures

Table 2. Present information by isolated microorganism or at least the predominant ones. cite the % and include the n, also include the denominator of isolates tested for each antimicrobial listed

Line 93. Include n value

Table 3. summarize with MIC 50 and MIC 90 values. This information must be included as supplementary material.

Line 99 -114 summarize using tables or figures

Line 139. How many isolates were studied by MLST? Include n values

What was the resistance mechanism involved in meropenem resistance?

None S. marcescens isolate had ESBL genes?

**********

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Reviewer #1: Yes: José E Villacís

Reviewer #2: No

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7 Jan 2020

Additional Editor’s comments

This manuscript is of great interest for us, please address the questions and suggestions offered by the reviewers.

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The supporting information files have been worked on and in-text citations updated to match accordingly

Review Comments to the Author

Reviewer #1: Jesumirhewe and collaborators present an interesting manuscript with the general objective of determine the frequency of ESBL genes in multidrug resistant human clinical Enterobacteriaceae isolates from Edo state Nigeria and to characterize the resistance mechanisms. Thank you for being able to contribute to it before your presentation to the scientific community. Therefore, I suggest that the following questions be modified, deleted, or explained.

Line 1. I think authors should change in the title: Whole genome sequence of extended-spectrum β-lactamase genes in Enterobacteriaceae isolates from Nigeria.

The title of the article has been changed to Whole genome sequencing of extended-spectrum β-lactamase genes in Enterobacteriaceae isolates from Nigeria

Improve the Abstract and Introduction. ESBL concern in both healthcare and community settings

The abstract and introduction has been improved on. Concern about ESBL in both healthcare and community settings have been highlighted

Line 16, 64. “This study aimed at determining the frequency of ESBL genes in multidrug resistant human clinical Enterobacteriaceae isolates from Edo state Nigeria” using whole genome sequencing

The sentence has been edited in the revised manuscript

Line 33. I suggest to describe Ambler classification of beta-lactamases and groups of CTX-M on the introduction.

Ambler classification of beta-lactamases have been described and groups of CTX-M mentioned on the introduction

Line 51. I would rather see table 1 summarized in a Figure instead of a table. I suppose it would be better demonstrated. The three hospitals are at a different level of care?

Table 1 is now presented as a figure. The 3 hospitals are tertiary healthcare institutions.

Why did you decide to include the strains of unidentified source; Teaching Hospital, Benin n=12, and Okada n=4

I included strains of unidentified source [Data not available} because some of these strains are important i.e had the esbl resistant gene and associated resistant determinants. Removing them from the number of isolates would not give a true picture of the frequency of resistant determinants present in the sample size

Line 74. “Flurometer” Fluorometer

Fluorometer spelling corrected

Line 94. The authors reported that (41%) were positive for the ESBL phenotypic test and had ESBL genes detected. Please explain the results of Pret3577 and EC11, where no resistance pattern to B lactams was observed. In strains with ESBL and carbapenemase, the ESBL screening and confirmation performs well?

There was a mix-up in our earlier report. Isolates that were positive for the ESBL phenotypic test and had ESBL genes detected are sixty [40%]. Pret3577 was sensitive to the ESBL phenotypic test but had the ESBL gene detected by WGS. EC 11 was resistant to both ceftazidime and cefotaxime. The ESBL screening and confirmation performed well in strains with ESBL and carbapenemase

Line 115. Table 4. Please highlight in results the presence of carbapenemases NDM and Oxa-48 genes in strains Kp_852 ST15, Kp_852K ST15, Kp_872 ST340 (First cases described?)

I recommend to improve tables, columns of ST set in the beginning

Have all the sequences been deposited at GenBank?

Is it possible probable clonal spread of the ESBL strains Intra or inter-hospital

Limitations must be included

I suggest to include virulence factors and mobile genetic elements associated with ESBL and phylogeny based on core genome MLST.

I recommend to highlight the WGS findings (figures), highlight the emerging global high risk clones, and to present this manuscript in a better fashion.

The carbapenemases have been earlier reported along with a few other isolates that are not ESBL producers as indicated in the manuscript with tracked changes. The aim of this study was to determine the frequency of ESBL genes in multidrug resistant human clinical Enterobacteriaceae isolates from Edo state Nigeria. Tables have been improved on. Table S1 gives the characteristics of the ESBL isolates. Sequences have been deposited at the GenBank and accession numbers included on Table S1.Yes there is a possibility of probable clonal spread of the ESBL strains especially intra-hospital. The WGS findings have been re-presented. A limitation in this study is to conclude on clonal bacterial transmission between the ESBL Enterobacteriaceae isolates that included seven groups of different organisms. However, the presence of different plasmid replicon types in ESBL producing isolates reveals their importance in the dissemination of these resistance genes.

Reviewer #2: The manuscript evaluates the frequency of ESBL genes in multidrug resistant human clinical Enterobacteriaceae isolates from Edo state Nigeria and characterize the resistance mechanisms. The research is correctly conducted, and the conclusions are supported by the data. However, results are not well exposed and are difficult to understand.

The results have been represented and are now easy to understand

Materials and method

Line 66. The phenotypic method used, is not useful for microorganisms with Amp C , so some ESBL producers could not be included, and this could be considered as a limitation

The phenotypic method used was based on CLSI guidelines. All ESBL producers detected phenotypically were confirmed in the WGS.Only one isolate had ESBL detected by WGS but not detected phenotypically

Table 1. Use italics to Enterobacteriaceae. This table does not present summarized data and contains partial results. Include n values

Table 1 has been re-presented as a figure

Results: Include summary tables or figures

Results have been re-presented

Table 2. Present information by isolated microorganism or at least the predominant ones. cite the % and include the n, also include the denominator of isolates tested for each antimicrobial listed

Information has been re-presented showing the % of ESBL isolates resistant to the various antibiotics with n included and the denominator of isolates tested for each antimicrobial listed

Line 93. Include n value

n value has been included

Table 3. summarize with MIC 50 and MIC 90 values. This information must be included as supplementary material.

The MIC values are included as supporting information now.The MIC values are presented only for the 60 ESBL isolates that are multidrug resistant. MIC values were not determined for the entire 218 enterobacteriaceae isolates. The E-test has been reported to be considered a reliable method to determine antimicrobial susceptibility testing and it gives results which are at least as accurate as those obtained by the broth dilution method. As our ESBL isolates are not up to 100 and the Enterobacteriaceae isolates are a collection of 7 different group of isolates calculating MIC50 and MIC90 may not be applicable here

Line 99 -114 summarize using tables or figures

Line 99-114 has been summarized and tables represented. The genetic characteristics of the ESBL isolates are presented in the supporting information with other details.

Line 139. How many isolates were studied by MLST? Include n values

All the multidrug resistant isolates that are ESBL producers were studied by MLST, n=60, The sensitive isolate that genotypically had ESBL detected was also studied by MLST, n=1.Some of the isolates did not have available sequence types.

What was the resistance mechanism involved in meropenem resistance?

None S. marcescens isolate had ESBL genes?

No S. marcescens isolate had ESBL genes. The carbapenemases have been earlier reported along with a few other isolates that are not ESBL producers as indicated in the manuscript with tracked changes. The aim of this study was to determine the frequency of ESBL genes in multidrug resistant human clinical Enterobacteriaceae isolates from Edo state Nigeria.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

4 Feb 2020

PONE-D-19-26950R1

Whole genome sequencing of extended-spectrum β-lactamase genes in Enterobacteriaceae isolates from Nigeria

PLOS ONE

Dear Dr jesumirhewe,

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[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

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Reviewer #1: N/A

Reviewer #2: Yes

**********

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Line 36. Enterobacteriaceae

Line 38. Specie of Providencia spp

Line 88. Repeated “Fig 1. Figure showing sample size of Enterobacteriaceae isolates provided by three Nigerian hospitals”

Line 90. Incomplete sentence

Line 94. patients’

According to the answer The ESBL screening and confirmation performed well in strains with ESBL and carbapenemase. Please consider that strains with ampC and carbapenemases can affect the performance of ESBL screening test

Line 87. Please include that the 3 hospitals are tertiary healthcare institutions, number of beds

Line 116- 128 Consider to rewrite the paragraph. There are words repeated “manufacturer’s instructions”

Line 195. Specie of Providencia spp

Line 233. The study concludes a probable clonal spread of the ESBL strains especially intra-hospital. Does WGS gave you some evidence? If not please consider the limitations.

The findings in the study can support a better conclusion in the abstract and the manuscript

Reviewer #2: The manuscript have been improved in quality, but there are some comments

Line 77. Please use additional bibliography to support this statement

Line 79. Please check the bibliography number 21

Line 87. Include that are tertiary hospitals. As a comment, Figure 1 does not improve the manuscript

I have some questions about isolates:

All isolates were obtained from hospitalized patients? Please specify if not

Line 134: specify which isolates were further studied with MLST. The results are just for K.pneumoniae, E.coli and E. cloacae?

Results

Piperacillin-tazobactam for K. pneumonia n = 9

results about imcompatibility groups and ST shoould be presented in a resume table. The current version is hard to read

Discussion

The manuscript does not highlight well their results. Include some data about ESBL in other countries from Africa and compare them.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: José E Villacís

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

25 Feb 2020

Reviewer No1

Line 36. Enterobacteriaceae

Correction has been effected on the revised manuscript

Line 38. Specie of Providencia spp

The revised manuscript has been updated. We decided to strike out Providencia spp as part of the reported ESBL isolates as it was sensitive to the ESBL phenotypic screening but we detected CTX-M-15 by sequencing. However during our submission of the sequences to NCBI also the sequence had issues that were challenging to resolve so we decided not to include it in the study anymore. The genome submission to NCBI gave a better insight with regards to the isolates.

Line 88. Repeated “Fig 1. Figure showing sample size of Enterobacteriaceae isolates provided by three Nigerian hospitals”

Correction has been effected on the revised manuscript.

Line 90. Incomplete sentence

Sentence has been updated in the revised manuscript.

Line 94. patients

Correction has been effected on the revised manuscript

According to the answer The ESBL screening and confirmation performed well in strains with ESBL and carbapenemase. Please consider that strains with ampC and carbapenemases can affect the performance of ESBL screening test

Noted. The 60 ESBL isolates presented including the ones with carbapenemases were positive to both the phenotypic screening for ESBL and the sequencing. Alongside other resistance genes only carbapenemases were detected in the ESBL isolates. No ampC was detected in any of the ESBL isolates.

Line 87. Please include that the 3 hospitals are tertiary healthcare institutions, number of beds

Revised manuscript has been updated

Line 116- 128 Consider to rewrite the paragraph. There are words repeated “manufacturer’s instructions”

Paragraph has been revised.

Line 195. Specie of Providencia spp

Manuscript has been revised. Providencia isolate is no longer included in the study. Reasons have been stated earlier in the rebuttal letter.

Line 233. The study concludes a probable clonal spread of the ESBL strains especially intra-hospital. Does WGS gave you some evidence? If not please consider the limitations.

Using the data obtained from the MLST assignment and plasmid incompatibility grouping, the distribution of the sequence types and the plasmid replicon types especially the predominant ones of the ESBL isolates across the three hospitals suggests a probable clonal spread of the ESBL strains both intra and inter-hospital. A limitation of this study however is to conclude on clonal bacterial transmission between ESBL Enterobacteriaceae isolates that included seven groups of different organisms.

The findings in the study can support a better conclusion in the abstract and the manuscript

Conclusion in the abstract and manuscript have been revised

Reviewer No 2

Line 77. Please use additional bibliography to support this statement

Additional bibliography has been added to the revised manuscript.

Line 79. Please check the bibliography number 21

Bibliography no 21 has been revised

Line 87. Include that are tertiary hospitals. As a comment, Figure 1 does not improve the manuscript

The manuscript has been revised indicating the hospitals are tertiary healthcare institutions. The figure was included in response to another reviewer’s comment from the previous revision.

All isolates were obtained from hospitalized patients? Please specify if not

Line 134: specify which isolates were further studied with MLST. The results are just for K.pneumoniae, E.coli and E. cloacae?

Isolates were obtained from samples of both in-patients and out-patients of the three different hospitals. All ESBL isolates were further studied with MLST. Sequence type numbers were not available for other isolates not presented. Some isolates had unknown sequence types which are submitted for curation of sequence type number.

Results

Piperacillin-tazobactam for K. pneumonia n = 9

Correction has been updated in the revised manuscript

results about imcompatibility groups and ST shoould be presented in a resume table. The current version is hard to read

Table has been revised. Result about incompatibility groups and ST has been represented in a separate table.

Discussion

The manuscript does not highlight well their results. Include some data about ESBL in other countries from Africa and compare them.

Discussion has been revised highlighting the results properly. Data about ESBL in other countries from Africa have been included and compared with the study

Submitted filename: rebuttal letter 1.docx

Click here for additional data file.

18 Mar 2020

Whole genome sequencing of extended-spectrum β-lactamase genes in Enterobacteriaceae isolates from Nigeria

PONE-D-19-26950R2

Dear Dr. jesumirhewe,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

Monica Cartelle Gestal, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Line 36-37. The report of blaCTX-M-15 in clinical isolates of Providencia spp., Citrobacter freundii and Atlantibacter hermannii is important, however the results of WGS, plasmid profiles an ST related to the dissemintation of CTX-M are more relevant.

Line 79. Please include what carbapenemases were recently reported. KPC, NDM, OXA-48 ???

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: José E Villacís

31 Mar 2020

PONE-D-19-26950R2

Whole genome sequencing of extended-spectrum β-lactamase genes in Enterobacteriaceae isolates from Nigeria

Dear Dr. Jesumirhewe:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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