Molecular detection of extended spectrum β-lactamase genes in Escherichia coli clinical isolates from diarrhoeic children in Kano, Nigeria.

The increase in antimicrobial resistance in developed and developing countries is a global public health challenge. In this context β-lactamase production is a major contributing factor to resistance globally. The aim of this study was to determine the prevalence of phenotypic and genotypic extended spectrum β-lactamases (ESBLs) in 296 E. coli isolates recovered from diarrhoeic children younger than five years in Kano whose susceptibility profile against 7 antimicrobials had been determined. The E. coli isolates were subjected to double disc synergy test for phenotypic ESBLs detection and ESBL associated genes (blaCTX-M, blaTEM and blaSHV) were detected using conventional PCR. Phenotypically, 12.8% (38/296) E. coli isolates presented a ESBLs phenotype, with a significantly higher proportion in isolates from females compared with males (P-value = 0.024). blaCTX-M 73.3% and blaTEM 73.3% were the predominant resistance genes in the ESBLs positive E. coli (each detected in 22/30 isolates, of which 14 harboured both). In addition, 1/30 harboured blaCTX-M + blaTEM + blaSHV genes simultaneously. This study demonstrates the presence of ESBLs E. coli isolates in clinically affected children in Kano, and demonstrates the circulation of blaCTX-M and blaTEM associated with those phenotypes. Enactment of laws on prudent antibiotic use is urgently needed in Kano.

Introduction

Hospital-based surveillance systems have reported an increase in the distribution of antibiotic resistant microorganisms in both developed and developing countries worldwide [1]. The ability to control infectious diseases, which includes diarrhoea caused by Escherichia coli, is currently endangered by this phenomenon, which has been declared as a global threat to public health [2]. Infections caused by resistant microorganisms often fail to respond to conventional antibiotics resulting in prolonged morbidity and higher mortality [2]. Even though antibiotics are not recommended for treatment of diarrhoea caused by E. coli, diarrhoeagenic E. coli (DEC) [3, 4] can carry antimicrobial resistance genes that may be acquired through horizontal transfer from other resistant isolates within the same or other genus [5]. β-lactamase production in Gram negative bacteria is the most important contributing factor to β-lactam resistance [6], and has undergone evolution over time since its first appearance [7]. Newer β-lactamase-producing enterobacteriaceae have been isolated from clinical settings in different parts of the world [5] carrying factors such as plasmid-mediated cephamycinases, extended spectrum β-lactamases (ESBLs), and carbapenemases [8], and thus demonstrating the variability in the potential mechanisms behind this phenotype. ESBLs have the ability to hydrolyse penicillins, cephalosporins and monobactams, but not cephamycins and carbapenems [9]. They are inhibited by classical and newly developed β- lactamase inhibitors such as clavulanic acid, sulbactam, tazobactam, avibactam, relebactam or nacubactam, among others [5, 7, 10].

ESBLs producing E. coli are a frequent cause of community and hospital acquired infections, thus creating an increasing public health challenge [5], and are one of the leading causes of infections worldwide [11]. Acquired ESBLs emerged in the 1980s as derivatives of blaTEM (named after the patient Temoneira) and blaSHV (sulfhydryl reagent variable) enzyme types [5, 12]. The genes encoding these enzymes are plasmid mediated and therefore easily transmissible between bacteria of the same or different species. This phenomenon may be favoured by the extensive use of β-lactam antibiotics in human medicine [7]. The ESBLs genotype has been associated with hospital and community outbreaks in a pandemic manner [12].

Diarrhoeal disease is a major cause of morbidity and the second most important cause of mortality in children less than 5 years of age [13]. Diagnosis of the causative agents is very important for proper management and surveillance purposes. There is however limited epidemiological information on the presence and distribution of ESBLs producing E. coli from diarrheic children in Nigeria. Detection of ESBLs phenotypes and genotypes in a paediatric population within a geographic area is very important as emergence of ESBLs producing E. coli leads to carbapenems reliance as the alternative treatment option. Since there are no previous reports on molecular detection of ESBL associated genes among diarrhoeic children in Kano, here we aimed at investigating the prevalence of ESBLs phenotypes and the associated resistance genes in E. coli isolates from diarrhoeic children in this state in Nigeria.

Materials and methods

Study population

The study was conducted in Kano state, Nigeria. Specimens were collected from outpatient diarrhoeic children under five years attending three major hospital: Bichi General Hospital (BGH), Murtala Muhammad Specialist Hospital (MMSH) and Wudil General Hospital (WGH). Additional information of the sampled population is provided elsewhere [14]. Diarrhoea is defined as the passage of three or more loose or watery stool in 24 hours.

Ethical consideration

Rectal swab specimens were obtained after obtaining informed consent from the parent and legal guardian of the children. Earlier on, ethical approval was granted by the Kano State Ministry of Health with Ref: MOH/Off/797/T.I/186.

Sample collection, bacterial isolation and antimicrobial susceptibility

Two hundred and ninety six E. coli isolates recovered from swabs from children with diarrhoea were obtained as described elsewhere [14]. Results from the antimicrobial susceptibility tests against Cefuroxime (CXM) 30 μg, Ceftazidime (CAZ) 30 μg, Cefotaxime (CTX) 30 μg, Amoxicillin-clavullanic acid (AMC) 30 μg, Gentamicin (CN) 10 μg, trimethoprim-sulfamethoxazole (SXT) 25 μg, and ciprofloxacin (CIP) 5 μg in all isolates has been previously reported [14]. Phenotypic ESBLs screening and confirmation was carried out on all 296 E. coli as recommended by CLSI [15]. The screening was carried out using Ceftazidime 30 μg disc (Oxoid, UK): a zone of inhibition ≤ 22mm was considered suggestive of ESBLs production and positive isolates were further investigated using the double disc synergy test with a combination of three antibiotic discs (ceftriaxone, amoxicillin-clavulanic acid and ceftazidime). A ≥ 5 mm increase in the inhibition zone for either antibiotic towards the amoxicillin-clavulanic acid with a dumbbell shape was considered indicative of an ESBLs phenotype. In addition, Tetracycline (TET) 30 μg and Imipenem 10 μg antibiotics (Oxoid UK) were tested on the ESBLs positive E. coli. Results were recorded as susceptible, intermediate and resistant according to the reference zone of inhibition of each antibiotic according to CLSI [15]. Isolates expressing ESBLs phenotype were preserved in Tryptic soy broth (TSB) (Biomerieux France) supplemented with 20% glycerol at -80°C until further testing.

DNA extraction

All E. coli isolates that were previously preserved in TSB+20% glycerol were cultured onto Eosin Methylene Blue (EMB) agar at 37°C for 18–24 hours. One loop full of E. coli from the EMB plates was suspended in about 2 ml of sterile distilled water in an eppendorf tube; the bacterial suspension was boiled at 100°C in a water bath for 10 minutes and centrifuged at 13,000 rpm for 1minute as previously described [16]. The supernatant was used as DNA template for PCR.

Detection of ESBLs associated genes by PCR

The molecular detection of ESBLs associated genes (blaSHV, blaTEM and blaCTX-M) was carried out using conventional multiplex PCRs in the majority of the ESBL presumptive E. coli isolates. Primers used were previously described by Monstein et al. [17]. The primer mix for the detection of ESBLs associated genes was prepared in an eppendorf tube by adding 210 μl of ultrapure water, 5μl uidA- Forward primer + 5μl uidA–Reverse primer, 5μl SHV—Forward primer + 5μl SHV–Reverse primer, 5μl CTX-M–Forward primer + 5μl CTX-M–Reverse primer, 5μl TEM–Forward primer + 5μl TEM–Reverse primer. Eppendorf tubes containing 22 μl of the reaction mixture were used for the PCRs (8 μl of water (Biorad) + 10 μl Mastermix (Bio-Rad) + 2μl of the mix primer + 2 μl of DNA). The ultrapure PCR cycling conditions were as follows: initial denaturation for 15 seconds at 95°C, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 1 minute 30 seconds, elongation at 72°C for 2 minutes and final elongation at 72°C for 10 minutes. Biorad MyCycler PCR thermal cycler was used to run the PCR cycles. The post amplification products were analysed using 2% agarose gel electrophoresis. Gel Doc XR+ Imaging system (Bio-Rad) was used in viewing the gel after exposure to UV light.

Isolates positive in the PCR for detection of blaTEM genes were subjected to another PCR for amplification and sequencing of the complete gene in order to identify the subtypes present. In this case the primer mix consisted of 5μl TEMSEQ forward primer + 5μl TEMSEQ reverse primer [18] + 240μl ultrapure water, and the remaining steps as described above. PCR cycling conditions consisted of initial denaturation for 15 seconds at 95°C, 40 cycles of denaturation at 95°C for 30 seconds, annealing at 50°C for 1 minute 30 seconds, elongation at 72°C for 1 minute and final elongation at 72°C for 10 minutes. Successfully amplified PCR products were purified using Illustra ExoProStar 1-Step, and sequenced. Sequences were analyzed using Bioedit [19] and MEGA X [20] and identified by comparing them with the NCBI database using BLAST [21].

Results

An ESBL phenotype was detected in 38 (12.84%) of the collection of 296 E. coli retrieved from the rectal swab specimens. ESBLs phenotype was significantly (chi-square test, P-value = 0.024) more common among E. coli recovered from female patients (17.83%, 23/129) compared with samples from males (8.98%, 15/167). Thirty out of the 38 phenotypic ESBLs positive E. coli (due to inability to recover eight isolates) were screened for ESBLs associated genes (blaSHV, blaTEM and blaCTX-M): all 30 isolates tested positive for at least one of the three resistance associated gene targets; blaCTX-M and blaTEM were detected in 73.3% (22/30), and blaSHV was detected in 6.66% (2/30) of the E. coli screened. The blaTEM sequences of 16/22 isolates carrying this gene were obtained, revealing two variants: 13 sequences matched perfectly with a previously published blaTEM-1 sequence (genbank NG050145.1), and the other three had just one single nucleotide polymorphism in position 396).

The susceptibility patterns of the ESBLs producing E. coli and the resistance pattern of E. coli based on the presence of the ESBLs associated genes are presented in Tables 1 and 2. All the ESBLs positive E. coli were resistant to tetracycline (except for only 2 isolates in the phenotypic group with intermediate resistance) and susceptible to imipenem, while intermediate levels of resistance were found for the rest of the antimicrobials. Proportion of resistance to all antimicrobials in isolates harbouring ESBL genes was very similar or identical to that found in the phenotypic-positive ESBLs isolates, although the percentage of isolates harbouring a MDR resistance profile (simultaneous resistance to three or more antimicrobial families) was higher among genotypically confirmed isolates (90.0%) compared with total ESBL presumptive isolates (78.9%) (Table 1). When comparing isolates with either blaCTX-M or blaTEM (n = 7 in both cases), a higher proportion of resistant isolates for all antimicrobials except AMC and SXT were found for those harbouring the former (Table 1). Interestingly, the simultaneous presence of both blaCTX-M and blaTEM led to higher proportion of resistance compared with the presence of either of the genes in several cases (Table 2).

Table 1

Antibiotic susceptibility pattern of ESBLs producing E. coli.

	ESBLs Phenotypic (N = 38)				ESBLs Genotypic (N = 30)
Antibiotics	S	I	R	MDR	S	I	R	MDR
	n (%)	n (%)	n (%)	n (%)	n (%)	n (%)	n (%)	n (%)
Cefuroxime	2 (5.3)	9 (23.7)	27 (71.1)		1 (3.3)	7 (23.3)	22 (73.3)
Cefotaxime	3 (7.9)	7 (18.4)	28 (73.7)		3 (10.0)	5 (16.7)	22 (73.3)
Amox-Clav	3 (7.9)	7 (18.4)	28 (73.7)		1 (3.3)	5 (16.7)	24 (80.0)
Ceftazidime	4 (10.5)	8 (21.1)	26 (68.4)		3 (10.0)	5 (16.7)	22 (73.3)
Ciprofloxacin	20(52.6)	5 (13.2)	13 (34.2)	30(78.9)	14(46.7)	5 (16.7)	11 (36.7)	27(90.0)
Gentamycin	29(76.3)	0 (0.0)	9 (23.7)		22(73.3)	0 (0.0)	8 (26.7)
Cotrimoxazole	3 (7.9)	0 (0.0)	35 (92.1)		3 (10.0)	0 (0.0)	27 (90.0)
Tetracycline	0 (0.0)	2 (6.7)	36 (94.7)		0 (0.0)	0 (0.0)	30(100.0)
Imipenem	38 (100)	0 (0.0)	0 (0.0)		30 (100)	0 (0.0)	0 (0.0)

S-Susceptible, I-Intermediate, R-Resistant, MDR- Multi-Drug Resistant, Amox-Clav- Amoxicillin-clavulanic acid.

Table 2

Antibiotics resistance pattern according to detected associated ESBLs genes.

Antibiotics		CXM	CTX	CAZ	AMC	CIP	CN	SXT	MDR
Associated Gene	N(%)	n(%)	n(%)	n(%)	n(%)	n(%)	n(%)	n(%)	n(%)
blaCTX-M Only	7(23.33)	5(71.4)	5(71.4)	5(71.4)	5(71.4)	4(57.1)	2(28.6)	6(85.7)	5(71.4)
blaTEM Only	7(23.33)	2(28.6)	3(42.9)	3(42.9)	6(85.7)	0(0.0)	2(28.6)	6(85.7)	6(85.7)
blaSHV Only	1(3.33)	1(100.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	1(100.0)	1(100.0)
blaCTX-M+blaTEM	14(46.67)	13(92.9)	13(92.9)	13(92.9)	12(85.7)	7(50.0)	4(28.6)	13(92.9)	13(92.9)
blaCTX-M+blaTEM+blaSHV	1(3.33)	1(100.0)	1(100.0)	1(100.0)	1(100.0)	0(0.0)	0(0.0)	1(100.0)	1(100)

Key: CXM-Cefuroxime, CAZ-Ceftazidime, AMC- Amoxicillin-clavulanic acid, CIP-Ciprofloxacin, CN- Gentamycin, SXT-Trimethoprim-sulfamethoxazole, N-Total sample screened, n-Number obtained.

Discussion

The spread of plasmid-encoded extended-spectrum β-lactamase (ESBLs) genes [5], conferring resistance to third-generation cephalosporins including aztreonams [9] is considered a major contributor to the ongoing emergence of antimicrobial resistance. The proportion of E. coli isolates displaying an ESBL-phenotype among those recovered from diarrhoeic children in Kano found here was 12.8%, which is in the range of values found in diarrhoeic children in other African countries like Kenya 12% [22] and Libya 13.4% [23]. Interestingly, very different values were found in isolates also retrieved from diarrhoeic children in Iran [24] (25.9%), China [5] (5.6%) or USA [25] (7%). In developing countries, patients often receive antibiotics treatment without antibiotic susceptibility testing or prescription which will exert a selective pressure on the existing E. coli, whereas in developed countries strategies for reducing antimicrobial use have been put in place [24]. E. coli recovered from female subjects had a significantly higher probability of ESBLs production, although the reason for this difference could not be established in this study. However, this result is in agreement with previous reports by Vatopoulos et al. [26] that found that E. coli bearing transferable Resistance plasmids was more often associated with antibiotic resistance among female than male. ESBLs positive strains from our study were highly resistant to trimethoprim/sulphamethoxazole, which is similar to the reports of Miao et al. [27] and Valenza et al. [28]. All phenotypic ESBLs producing strains were susceptible to imipenem in agreement with reports from Tanzania [29], Libya [23], China [5, 27], and in contrast with the study of Hamprecht et al. [30] in Germany. The full susceptibility of isolates to imipenem may be attributable to the relatively low or non-usage of carbapenem drugs among the population. All the ESBLs positive isolates (genotypic) were resistant to tetracycline, similar to the report from Egypt [31] and Libya [23] (88.9% resistance), what could be due to an extensive use of this drug among human and animals.

blaCTX-M was very prevalent among the ESBL-positive isolates, in agreement with previous studies suggesting this gene is widespread worldwide [5, 7, 23, 27, 32–35], although blaTEM-1 was also found in approximately three quarter of all ESBL isolates tested. No data on the presence of ESBL associated genes was available for clinical isolates in Nigeria; however blaCTX-M and blaTEM were also the predominant ESBLs associated genes in E. coli recovered from 200 cattle and 150 pigs in Nigeria [36]. Our detection rate is in the range of values reported from Egypt [31] (73.7% blaCTX-M), Turkey [37] (73.43% blaTEM) and USA [25] (74% blaCTX-M). This study also shows that the carriage of multiple bla genes complicates the phenotypic interpretation of the resistance phenotypes, which is related to complex antimicrobial resistance [38]. Isolates with multiple combinations of bla genes and especially those carrying blaCTX-M+blaTEM and blaCTX-M+blaTEM+blaSHV were resistant to a larger number of β-lactams (>92% resistance). blaCTX-M in combination with bla gene was found in 46.7% of the isolates, similar to the findings of Harada et al. [39], which reported 48.8% of E. coli carried blaCTX-M+blaTEM in clinical isolates from Japanese tertiary hospital, but higher than the result of Tawfick et al. [31] who found only 21.7% of E. coli isolates from diarrhoeic stool in Egypt carrying both blaCTX-M and blaTEM.

In summary, our study demonstrates the presence of both phenotypically and genotypically ESBLs E. coli positive isolates in diarrheic children in Kano. This is a matter of concern since their presence was associated with high levels of phenotypic resistance. Performing antibiotic susceptibility testing on clinical isolates before antibiotic prescription could help to mitigate the emergence of antimicrobial resistance, and therefore regulations helping to control the sale of antibiotics by patent medicine stores and prudent antibiotic usage by clinicians is urgently needed in Nigeria.

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14 Feb 2020

PONE-D-19-35058

Molecular Detection of Extended Spectrum β-lactamase Genes in Escherichia coli clinical isolates from Diarrhoeic Children in Kano, Nigeria

PLOS ONE

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Reviewer #1: The study presents important data for local epidemiology. However, the absence of sequencing allows interpretation bias and reduces the impact of the results.

1.- Not all TEM variants are ESBL. Therefore, sequencing the complete orf is essential to identify the variant before relating its presence to the ESBL phenotype, especially in strains where only this gene was identified by PCR.

2. From the epidemiological point of view it is important the establishment of the variant of each investigated gene and the ST of at least all ESBL isolates.

Additionally, I strongly recommend discussing the significant difference between the presence of E. coli ESBL in men and women.

Reviewer #2: Dear author,

Please review next little details

38 E. coli, diarrhoeagenic E. coli (DEC) --> could you defined it? As far as I know DEC does not exist, maybe EAEC, EHEC or EPEC.

42 Newer β-lactamase-producing enterobacteriaceae have been

43 isolated from clinical settings in different parts of the world (Bai et al., 2017) carrying factors such

44 as plasmid-mediated cephamycinases, extended spectrum β-lactamases (ESBLs), and

45 carbapenemases (Jacoby & Munoz-Price, 2005).

What do you mean?

They are inhibited by classical β- lactamase inhibitors such as

48 clavulanic acid, sulbactam and tazobactam (Pitout, 2013; Reuland et al., 2013).

Please upgrade with new inhibitors and combinations

99 20% glycerol were cultured onto EMB

100 agar at 37°C for 18-24 hours

Could you define the medium? ingredients or composition?

Table 1: Antibiotic Susceptibility 143 Pattern of ESBLs Producing E. coli

curiosity.....

what do you think or consider that can be the cause of this dis-match between phe- and geno-type

did you have some false negative??

Please correct some errors among both tables (1 and 2), CTX, CIP, STX and MDR not match the n (%)

f.ex CTX = 22(73.3) and table 2 sum 23

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Reviewer #1: Yes: David Ortega-Paredes

Reviewer #2: Yes: Torres-Sangiao, E

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12 Jun 2020

Dear Editor,

We would like to thank the reviewers for their careful consideration of our manuscript. We have modified the paper according to their suggestions, and detail responses follow:

Reviewer#1

1.- Not all TEM variants are ESBL. Therefore, sequencing the complete orf is essential to identify the variant before relating its presence to the ESBL phenotype, especially in strains where only this gene was identified by PCR.

We agree with the point made by the reviewer, and in order to follow the suggestion we have now sequenced the complete blaTEM gene in the isolates that were positive in the PCR. This is now indicated in material and methods (lines 119-127) and results (lines 136-139).

2. From the epidemiological point of view it is important the establishment of the variant of each investigated gene and the ST of at least all ESBL isolates.

We have identified the subtypes of the blaTEM genes identified in this study, but unfortunately the determination of the sequence type of the studied isolates is beyond the scope of the study and we have not been able to do so due to lack of resources.

Additionally, I strongly recommend discussing the significant difference between the presence of E. coli ESBL in men and women.

We thank the reviewer for the comment, we have added a comment on gender differences following the reviewer’s suggestions in lines 169-173

Reviewer#2

Line 38: E. coli, diarrhoeagenic E. coli (DEC) --> could you defined it? As far as I know DEC does not exist, maybe EAEC, EHEC or EPEC.

We appreciate the reviewers comment on this, DEC was designated as a group by Kaper and Nataro, 1998 (Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev. (1998)

11:142–201. doi: 10.1128/CMR.11.1.142) and since their pronouncement of DEC as a group, several articles and published books had recognized DEC as bacterial group.

Lines 42-45: Newer β-lactamase-producing enterobacteriaceae have been isolated from clinical settings in different parts of the world (Bai et al., 2017) carrying factors such as plasmid-mediated cephamycinases, extended spectrum β-lactamases (ESBLs), and carbapenemases (Jacoby & Munoz-Price, 2005). What do you mean?

We appreciate the comment of the reviewer. We are reviewing the antibiotic resistance trend in the β–lactam agents. We highlight how resistance move from penicillinase to β-lactamases before the newer β-lactamases. We have completed the sequence to clarify our point in lines 136-139.

Lines 47-48: They are inhibited by classical β- lactamase inhibitors such as clavulanic acid, sulbactam and tazobactam (Pitout, 2013; Reuland et al., 2013). Please upgrade with new inhibitors and combinations

We appreciate the comment of the reviewer, newer inhibitors now included along with a more updated reference in Line 48

Line 99: 20% glycerol were cultured onto EMB agar at 37°C for 18-24 hours. Could you define the medium? ingredients or composition?

Thank you for the comment. EMB medium is a selective/differential enteric media containing eosin and methylene-blue. The full name has been added to the manuscript line 94-95.

Table 1: Antibiotic Susceptibility 143 Pattern of ESBLs Producing E. coli. curiosity..... what do you think or consider that can be the cause of this dis-match between phe- and geno-type. did you have some false negative??

The phenotypic identification of the ESBLs producing isolates was conducted in Nigeria, due to some challenges beyond our control, we could not recover 8 of the phenotypic ESBLs positive when the isolates were due to be transported to Madrid. Therefore, even though there were 38 ESBL-producing isolates (according to their phenotype) only 30 could be subjected to the PCRs for detection of the genes, and all of them were positive for at least one of them.

Please correct some errors among both tables (1 and 2), CTX, CIP, STX and MDR not match the n (%). f.ex CTX = 22(73.3) and table 2 sum 23

We appreciate the reviewer for the comment, we have double checked the tables all errors corrected

Submitted filename: Response to reviewers.docx

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9 Jul 2020

PONE-D-19-35058R1

Molecular Detection of Extended Spectrum β-lactamase Genes in Escherichia coli clinical isolates from Diarrhoeic Children in Kano, Nigeria

PLOS ONE

Dear Dr. Kayode Saka,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Aug 23 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Monica Cartelle Gestal, PhD

Academic Editor

PLOS ONE

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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 #2: (No Response)

**********

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 #2: Yes

**********

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

Reviewer #2: 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 #2: 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 #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 #2: Dear authors,

I have no comments to add, though my point of view the I have recommend to publish this manuscript as letter, not as original report.

I hope you understand

My best

**********

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 #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.]

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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

6 Aug 2020

Dear Editor

Thank you for the kind consideration of our manuscript.

Submitted filename: Response to Reviewer.docx

Click here for additional data file.

13 Oct 2020

PONE-D-19-35058R2

Molecular Detection of Extended Spectrum β-lactamase Genes in Escherichia coli clinical isolates from Diarrhoeic Children in Kano, Nigeria

PLOS ONE

Dear Dr. SAKA,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Nov 27 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Monica Cartelle Gestal, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Additional Editor Comments (if provided):

[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 #3: 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 #3: Partly

**********

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

Reviewer #3: Yes

**********

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 #3: 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 #3: 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 #3: Interesting document, however, some recommendations are submitted

Comments:

1. Line- 87-88: The double disc synergy is not recommended by CLSI to confirm the presence o ESBL enzymes in Enterobacterales.

2.In table number 1. The Augmentin is a commercial name of amoxicillin-clavulanic acid. It should be changed.

3.- The characterization of CTX-M Beta-lactamases in humans samples must be complemented with DNA sequencing.

**********

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 #3: 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.]

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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

15 Oct 2020

Dear Editor.

We would like to thank the reviewer for the careful consideration of our manuscript. We have modified the manuscript according to the suggestions and response follow:

Reviewer #3

1. Line- 87-88: The double disc synergy is not recommended by CLSI to confirm the presence o ESBL enzymes in Enterobacterales.

We appreciate the comment of the reviewer, the information now corrected to reflect our choice of using the double disc synergy test for more specificity in the confirmation of ESBLs phenotype in E. coli.

2. In table number 1. The Augmentin is a commercial name of amoxicillin-clavulanic acid. It

should be changed.

Thank you for the comment, we have made the correction.

3. The characterization of CTX-M Beta-lactamases in humans samples must be complemented

with DNA sequencing.

We agree with the submission of the reviewer, we could not go beyond typing blaTEM genes due to financial constraints.

Submitted filename: Response to Reviewer.docx

Click here for additional data file.

17 Nov 2020

Molecular Detection of Extended Spectrum β-lactamase Genes in Escherichia coli clinical isolates from Diarrhoeic Children in Kano, Nigeria

PONE-D-19-35058R3

Dear Dr. SAKA,

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

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Monica Cartelle Gestal, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

24 Nov 2020

PONE-D-19-35058R3

Molecular Detection of Extended Spectrum β-lactamase Genes in Escherichia coli Clinical Isolates from Diarrhoeic Children in Kano, Nigeria.

Dear Dr. Saka:

I'm 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.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Monica Cartelle Gestal

Academic Editor

PLOS ONE