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Molecular Characterization of Carbapenemase-Producing Enterobacterales in Children with Diarrhea in Rural Burkina Faso

René Dembélé^1,2*, Issiaka Soulama³, Wendpoulomdé Aimé Désiré Kaboré¹, Ali Konat鹈, Assèta Kagambèga^1,4, David Coulibaly N'Golo⁵, Oumar Traoré^1,2, Abdoulaye Seck^6,7, Alfred Sababenedyo Traoré¹, Nathalie Guessennd^8,9, Amy Gassama-Sow¹⁰ˆ and Nicolas Barro¹

¹ Laboratory of Molecular Biology, Epidemiology and Surveillance of Bacteria and Viruses Transmitted by Food/Center for Research in Biological, Food and Nutritional Sciences/Graduate School of Science and Technology/University Joseph KI-ZERBO, 03 BP 7021 Ouagadougou 03, Burkina Faso.

² Training and Research Unit in Applied Sciences and Technologies/University of Dedougou, BP 176 Dedougou, Burkina Faso.

³ National Centre for Research and Training on Malaria, 01 BP 2208 Ouagadougou 01, Burkina Faso.

⁴ Institute of Sciences, 01 BP 1757 Ouagadougou 01, Burkina Faso.

⁵ Molecular Biology Platform, Pasteur Institute of Abidjan, Ivory Coast, 01 BP 490 Abidjan 01.

⁶ Laboratory of Biological and Medical Analyzes, Pasteur Institute of Dakar, Dakar, Senegal.

⁷ Faculty of Medicine, Pharmacy and Odontology, University of Cheikh Anta DIOP of Dakar, Senegal.

⁸ Laboratory of Bacteriology-Virology, Unit of Antibiotics, Natural Substances and Surveillance of Resistance of Microorganisms to Antimicrobials/ Pasteur Institute of Abidjan, Ivory Coast, 01 BP 490 Abidjan 01.

⁹ Laboratory of Bacteriology-Virology/Unit of Training and Research of Medical Sciences/University Felix Houphouet BOIGNY, 01 BP V34 Abidjan 01, Ivory Coast.

¹⁰ Unit of Experimental Bacteriology, Pasteur Institute of Dakar, 36 avenue Pasteur, BP 220, Dakar, Senegal.

ˆAli Konaté and Amy Gassama-Sow are deceased

Conflict of Interest: The authors stipulate that they have no conflict of interest in regard to this study.

INTRODUCTION

New antimicrobial resistance mechanisms are emerging and spreading globally, hampering our ability to effectively treat common infectious diseases. This has extended illness, disability and increased death rates ¹. As a result, antimicrobial resistance represents a major challenge for public health worldwide. One of the most worrying threat is the emergence and rapid dissemination of carbapenem resistant Gram-negative bacteria, following the spread of carbapenemase-producing Enterobacterales (CPE) ^{2, 3}. In bacteria of animal and human origins, beta-lactam resistance, which includes resistance to extended-spectrum beta-lactams, has now been increasingly observed ⁴. In Enterobacteriaceae, the carbapenemases have been previously classified into the three following classes : class A [ie K. pneumoniae carbapenemase (KPC) enzymes], Class B [ie metallo-beta-lactamases (MBL)] including New Delhi metallo-β-lactamase (NDM), Verona integrin-encoded metallo-β-lactamase (VIM), Imipenemase (IMP), and Class D [ie oxacillinase (OXA)-48 and related variants] ^5-7. Enterobacteriaceae can be resistant to carbapenems through intrinsic or acquired mechanisms. The chromosomally-encoded mechanisms can occur by (a) production of chromosomal carbapenemases from the group of class A serine carbapenemases ⁸ or (b) efflux pumps or (c) reduction in outer membrane permeability through porin loss ⁹. The acquired resistance is a plasmid-mediated mechanism through which the mobile carbapenemases are easily transmitted between bacteria ¹⁰.

The recent spread of CPE is one of the major public health threats worldwide ¹¹ because carbapenems are among the main stay of therapy for treating severe infections directly related to multidrug-resistant bacteria producing extended-spectrum β-lactamases (ESBLs) ¹². Carbapenemases are defined as β-lactamases that hydrolyze almost all beta-lactam antibiotics. According to some recent studies, the most prevalent carbapenemases in Enterobacteriaceae are bla_KPC (Ambler class A), bla_VIM, bla_IMP, bla_NDM (class B) and bla_OXA-48 like (class D) ^{11, 13}. Although several studies reported the occurrence of carbapenemases producing bacteria in Africa ¹², to our best knowledge, no study focusing on the molecular characterization of Carbapenemase-producing E. coli and Salmonella has been undertaken in Burkina Faso. Therefore, the objective of the present study was to carry out a molecular characterization of carbapenemase-Producing Escherichia coli and Salmonella isolates recovered from children in two rural hospitals in Burkina Faso.

MATERIAL AND METHODS

Study design and population

A prospective cross sectional study was conducted to determine the serotypes and antimicrobial susceptibility of diarrheagenic E. coli and Salmonella among children visiting hospitals in rural settings of Burkina Faso.

Settings

This study was conducted in north (Gourcy, distance 140 km) and western (Boromo, distance 185 km) of the capital Ouagadougou, Burkina Faso (Fig. 1). The main sources of income in these rural settings are subsistence farming, animal husbandry and small scale trade.

Specimen collection

Two hundred and seventy five (275) faecal samples were taken in 2009-2010 by trained health staff personnel using a swab transport system (M40 transystemAmies agar gel without charcoal; Copan Italia Spa, Brescia, Italy) and transported to laboratory within 24h of their collection for analysis. A questionnaire was used to collect demographic information (e.g., age and sex) of each patient).

Salmonella isolation and serotyping

Selenite broth (Emapol, Pologne) was used for the enrichment of specimens followed by an incubation at 37°C for 18h. Subsequently, samples were cultured on Hecktoen Enteric agar (Liofilchem, Italy) and incubated at 37°C for 24h. The identity of typical-looking Salmonella colonies on Hektoen was examined by using orthonitrophenyl-ß-D-galactopyranoside (ONPG), citrate, mannitol, lysine decarboxylase tests and the Kliger Hajna medium (Liofilchem, Italy). Finally the isolates were confirmed by API 20E (BioMérieux, Marcy l’Etoile, France). All Salmonella isolates were serotyped by the Salmonella Reference Laboratory. Isolates were serotyped with the somatic O and flagellar H anti-sera according to the Kauffman White scheme ¹⁴.

1. coli isolation and identification

Stool samples were plated on eosin methylene blue agar (Liofilchem, Italy), and the plates were incubated at +37°C for 18–24 h. After incubation, the suspected colonies were selected and streaked onto Mueller Hinton agar plate (Liofilchem, Italy). Confirmation was carried out by a biochemical microbiology method based on negative urease (Bio-Rad, France), negative citrate (Liofilchem, Italy), positive indole (Bio-Rad, France), positive lactose (Liofi lchem, Italy), and positive orthonitrophenyl-β-D-galactopyranoside (ONPG) (bioMerieux, France). E. coli strains isolated were confirmed by API 20E (bioMérieux, France).

The 16-plex PCR was used to detect simultaneously 16 genes from the five main pathogroups of E. coli (enterohemoragic E. coli: EHEC, enteropathogenic E. coli: EPEC, enteroaggregative E. coli: EAEC, enteroinvasive E. coli: EIEC and enterotoxigenic E. coli: ETEC) as described by Antikainen et al. ¹⁵. The genes investigated and primers used are listed in Table 1 ^15-17.

Antimicrobial susceptibility testing

Antibiotic susceptibility was determined on Mueller–Hinton agar (Liofilchem, Italy) using the standard disc diffusion procedure as described by the European Committee of Antimicrobial Susceptibility Testing (EUCAST) ¹⁸. Nineteen antibiotics belonging to 7 different families (Table 2) were tested : amoxicillin (25 µg), amoxicillin-clavulanic acid (20/10 µg), ceftriaxone (30 µg), cefotaxime (30 µg), cefepime (30 µg), cefixime (10 µg), piperacillin (75 µg), piperacillin-tazobactam (100 +10 µg), imipenem (10 µg), tetracycline (30 µg), chloramphenicol (30 µg), trimethoprim-sulfametoxazole (1.25 ± 23.75 µg), aztreonam (30 µg), colistin sulfate (50 µg), ciprofloxacin (5 µg), nalidixic acid (30 µg), gentamicin (15 µg), netilmicin (10 µg) and tobramycin (10 µg) (Bio-Rad, France). The diameters of the antibiotic susceptibility halos were recorded according to the recommendations of EUCAST. Intermediate (I) susceptibility of pathovars was classified as resistant (R).

Detection of antibiotic resistance genes

The strains that were resistant to imipenem and amoxicillin/clavulanate were PCR screened. DNA for PCR analysis was extracted from the isolates using the heat lysis method ¹⁹. A loopful of bacterial growth from Mueller Hinton agar (Liofilchem, Italy) plate was suspended in 1 ml of sterile water, and the mixture was boiled for 10 min at +100 °C and centrifuged for 10 min at 12000 rpm at +4°C. The obtained supernatant was collected and used for PCR reactions. Multiplex PCR assays were carried out using oligonucleotides (Table 3) to detect the presence of genes of the bla_KPC, bla_VIM, bla_IMP, bla_TEM, bla_SHV, bla_OXA and bla_CTX-M types in all E. coli and Salmonella strains of the study.

About 2.5 μl of supernatant were added to 22.5 μl reaction mixture containing 5U of Taq DNA polymerase (Accu Power, Korea), deoxyribonucleic triphosphate (10 mM), buffer GC (10X), MgCl2 (25 mM), and PCR primers (10 μM). We performed PCR conditions as followed: 5min at +94°C, followed by 35 amplification cycles of +94°C for 30s, 59±4 °C for 60s and +72°C for 60s with a final extension of +72°C for 10min on a thermal cycler (Gene Amp 9700, Applied Biosystems). Our reaction products were separated by electrophoresis in (1.5% weight/volume) agarose gel, stained with a Redsafe solution (Prolabo, France) and visualized under ultraviolet (UV) light (Gel Logic 200).

Ethics approval and consent to participate

Permission to conduct the study was obtained from the hospital authorities of Burkina Faso, and informed verbal consent was obtained from the parents/guardians of every child before sample collection. The National Ethical Committee (s) of Burkina Faso (N ° 2009-39) approved the study protocol.

RESULTS

Bacterial isolates

Of the 275 stool samples, five isolates were confirmed as E. coli strains: 3 EAEC, and 2 atypical EPEC. Nine Salmonella isolates were detected belonging to the following serotypes: Salmonella Poona, S. Typhimurium, S. Ouakam, S. Virchow, S. Duisburg and S. Hvittingfoss.

Antimicrobial resistance

The five E. coli strains were resistant to amoxicillin-clavulanic acid, amoxicillin and tetracycline (5/5); four strains were resistant to trimetoprim-sulfametoxazol, colistin-sulfate and piperacillin (4/5); three strains showed resistance to cefotaxim, ceftriaxone, aztreonam, cefixime and cefepime (3/5). Whereas, resistance to chloramphenicol and also to imipenem was shown in two of isolates (2/5).

All the nine Salmonella strains were resistant to amoxicillin (9/9), eight were resistant to amoxicillin-clavulanic acid (8/9), six were resistant to tetracycline, cefixime and cefepime (6/9); five were resistant to ceftriaxone, cefotaxim and colistin-sulfate (5/9). Less than five strains were resistant to aztreonam, trimetoprim-sulfametoxazol and piperacillin while no resistance was shown to imipenem (Fig. 2).

Carbapenemase genes

The results of PCR amplification of bla_KPC, bla_VIM, bla_IMP, bla_TEM, bla_SHV, bla_OXA and bla_CTX-M genes in E. coli and Salmonella isolates are given in table 4. bla_KPC gene (Fig. 4), bla_VIM gene and bla_IMP-2 gene were carried by two E. coli isolates which were resistant to imipenem. bla_OXA gene (Fig. 3) was found in all the five E. coli strains (5/5) and in three of the Salmonella strains (3/9). bla_CTX-M gene (Fig. 4) was shown in one E. coli strain and in none of the Salmonella. bla_TEM and bla_SHV were reported in none of the E. coli and Salmonella strains.

All E. coli strains were isolated in children of one year old while the sex distribution was 4/5 for male and 1/5 for female. The Salmonella were reported in children of one year old (4/9), two years old (2/9) and three years old (3/9) with a sex distribution of 5/9 for male and 4/9 for female (table 3).

Figure 1: Map of Burkina Faso. In red = Gourcy and Boromo where the study was conducted.

https://commons.wikimedia.org/wiki/File:Burkina_Faso,_administrative_divisions_-_de_-_monochrome.svg

Figure 2: Resistance to individual antimicrobial among E. coli and Salmonella strains.

Figure legend: AMC = Amoxicillin- clavulanic acid, AMX = Amoxicillin, CTX = Cefotaxime, ATM = Aztreoname, IPM = Imipenem, CRO = Ceftriaxone, FEP = Cefepime, CFM = Cefixime, TET = Tetracycline, CHL = Chloramphenicol, SXT = Trimethoprim-sulfametoxazole CIP = Ciprofloxacine, NAL = nalidixic acid, CST = Colistin sulfate, GMI = Gentamicin, TZP = Piperacillin-tazobactam, PIP = Piperacillin, NTM = Netilmicin, TMN = Tobramycin, I = Intermediate, R = Resistant.

Figure 3: blaOXA gene detected in E. coli.

Figure legend : Lane M : hyperlader (100 bp), Lane 1 : blaOXA positive control (813 pb), Lane 2-8 : positive samples for blaOXA gene (813 pb), Lane T : negative sample.

Figure 4. blaCTX-M and  blaKPC genes detected in E. coli.

Figure legend: Lane M = hyperlader 100 bp; Lane A = blaCTX-M positive control (544 pb), Lane B, D et E = positive samples for blaCTX-M  gene (544 pb), Lane B, C, D et E = positive samples for blaKPC  gene (300 pb), T: negative sample.

Table 1 : Oligonucleotides primers used for multiplex PCR reaction

Table 2 : Zones of inhibition of the tested antibiotics

Table 3: Oligonucleotides used to amplify carbapenemases genes

Table 4: Carbapenemase-producing Enterobacteriaceae

DISCUSSION

Increasing numbers of antibiotic-resistant Enterobacteriaceae are responsible of serious problems in infection control. This phenomenon also contributes to the global spread of Carbapenemase- producing bacteria becoming therefore especially worrisome ²⁰. It has been shown that Enterobacterales spp., such as Escherichia coli and Klebsiella pneumoniae, are common human pathogens and asymptomatic colonizers of the human gastrointestinal tract and environmental niches ²¹. Our study reported for the first time the occurrence of Carbapenemase-producing E. coli and Salmonella in children with diarrhea in rural settings of Burkina Faso.

The isolated strains were mainly resistant to amoxicillin-clavulanic acid, amoxicillin, tetracycline, trimetoprim-sulfametoxazol, colistin-sulfate, piperacillin, cefotaxime, ceftriaxone, aztreonam, cefixime and cefepime (between 60% and 100%). Particularly, two E. coli harbored resistance patterns to imipenem. In contrast, no resistance to imipenem was observed in Salmonella strains. Similar results concerning E. coli resistance to imipenem were reported in India ¹². All isolated E. coli and Salmonella were 100% sensitive to netilmicin in agreement with data reported in Bangladesh ²².

In the present study, the most detected carbapenemase was bla_OXA which was encoded by eight of the isolates (57.14%). In agreement to our result, a recent review showed that OXA-48-like enzymes associated with Enterobacteriaceae are one of the most concerning developments in carbapenem resistance in the last decade and are still globally ascending ²³. bla_CTX-M gene was shown in one E. coli strain while bla_TEM and bla_SHV were reported in none of the strains. Unlike our results, studies conducted in India reported a high prevalence of CTX‑M‑type ESBL among ESBL‑E. coli isolated from clinical specimens ^{24, 25}. Interestingly, the strain encoding bla_CTX-M gene in our study also harbored bla_OXA gene. Indeed, enteric Gram-negative bacteria with the blaOXA-48-like genes could co-harbor genes encoding ESBL (bla_CTX-M, bla_SHV, bla_TEM) ²⁶. bla_KPC2, bla_VIM and bla_IMP-2 were carried by two E. coli isolates while no Salmonella strains harboured these genes. Similarly, a recent study reported no blaKPC, blaVIM and blaIMP genes on carbapenemase producing Salmonella enterica isolates in the United Kingdom but reported 2 blaOXA48 strains and one blaNDM on the same isolates ²⁷. This is expected because carbapenemase-producing Salmonella strains are rarely isolated. In contrast, resistance to carbapenems was observed in CIP-R Salmonella KentuckyX1-ST198-SGI1 isolates in which carbapenemases blaVIM−2 and blaOXA−48 have been detected ²⁸.

Our results show that carbapenemase-producing Enterobacterales (CPE) remain one of the most urgent healthcare threats. Indeed, carbapenemase-encoding genes are already widespread in many parts of the world ²³. A recent study on wastewater used for urban agriculture in Ouagadougou (Burkina Faso) concluded that raw sewage used as fertilizer could be contributing to the spread of resistance bacteria among humans and animals ²⁹. Furthermore, a study has shown that bacteria producing carbapenemase are currently spreading among pets ³ and because of the proximity between humans and animals, these bacteria can contaminate humans. For example, it has been shown that poultry flocks contribute to the global dissemination of Salmonella Kentucky ST198-X1-SGI1CIP-R strain in developing countries ²⁸. Since subsistence, farming and animal husbandry are the primary economic activities for the local populations in Boromo and Gourcy, the spread of these bacteria poses serious health concerns.

Three specific genes were detected in two E. coli strains: Klebsiella pneumoniae carbapenemase (KPC), Verona integrin-encoded metallo-β-lactamase (VIM) and Imipenemase (IMP-2). To our best of knowledge, this is the first report of bla_KPC gene in E. coli in Burkina Faso. However, KPC producers have been described, mostly from nosocomial K. pneumoniae isolates, and E. coli strains in Israel but also from other enterobacterial species ³⁰. Indeed, since their identification the first time in the USA in 1996, Klebsiella pneumoniae carbapenemases (KPCs) have spread internationally among Gram-negative bacteria, especially K. pneumoniae, although their precise epidemiology is diverse across countries and regions worldwide ³¹. Furthermore, because of its extensively identification worldwide, K. pneumoniae may have contributed to the spread of the blaKPC genes ³².

As far as the class B metallo-β-lactamases (MBLs) is concerned, our results corroborate the existing reports. Endemicity of VIM- and IMP-type enzymes has been reported in Greece, Taiwan and Japan ^{33, 8}, although outbreaks and single reports of VIM and IMP producers have been shown in many other countries ³³.

It has been shown that carbapenemases can hydrolyze almost all β-lactams, and are easily transferable among enterobacterial species ³⁴. These genes are found in multidrug-resistant isolates consistent with the result found in the present study ³⁴. Therefore, its spread in Enterobacteriaceae is a public health issue. For example, invasive infections by carbapenem-resistant strains have been found to be associated with high morbidity and mortality rates ³⁵.

Otherwise, several risk factors of colonization and infection with carbapenemase-producing Enterobacterales (CPE) including severe underlying illness, prolonged hospital stay, the presence of invasive medical devices, and antibiotic use have been shown ^36-39. According to previous studies, CPE have been associated with adverse clinical and economic outcomes such as increased mortality, increased length of stay, setting up an effective therapy scheme, decreased functional status on discharge, and increased cost of health care ^40-43. Young children (those under one year old) were severely infected with carbapenem-resistant E. coli. This is a matter of public health issues because the emergence of MBL-producing bacteria greatly limits treatment options ⁴⁴. The most frequent MBLs reported to date belong to the VIM and IMP types and have been described extensively worldwide ⁴⁵.

The main limitation of the present study consists of the low number of isolates which makes generalizability difficult. Morever, the use of phenotypic approach limited to imipenem MIC is not adapted for the detection of all carbapenemase type. For instance, enterobacteria strains carrying blaOXA48 carbapenemase could present low imipenem MIC (0.5 mg/L) suggesting the use of temocillin for phenotypic detection of blaOXA48 ⁴⁶.

CONCLUSIONS

Infections by carbapenem-resistant bacteria are difficult to treat successfully. This study highlights the need for rapid identification of MBL-producing Gram-negative species both for appropriate treatment and for timely implementation of infection control measures. In developing countries like Burkina Faso, phenotypic methods may be useful for routine detection of Carbapenemase production, particularly when PCR is not available.

ACKNOWLEDGMENT

We thank the "Réseau de Recherche sur les Maladies Entériques à Potentiel Épidémique en Afrique de l’Ouest (REMENTA)" for assistance with PCR reagents, the "Centre National de Recherche et de Formation sur le Paludisme (CNRFP)/Ouagadougou, Burkina Faso" and the "Centre National de Référence pour les Antibiotiques (CNR-Antibiotiques)/Institut Pasteur de Côte d'Ivoire" for technical support.

CONFLICT OF INTEREST

The authors stipulate that they have no conflict of interest in regard to this study.

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