Molecular Characterization of Carbapenemase-Producing Escherichia coli and Salmonella in children with diarrhea in rural Burkina Faso.


 Background In recent years, Carbapenemase-producing Enterobacteriaceae (CPE) resistance to antibiotics has dramatically increased leading to limitations of their treatment options. In the present study, we investigated the occurrence of carbapenemase-producing Escherichia coli and Salmonella in rural Burkina Faso, using bacterial strains obtained from previous studies. Results Diarrheagenic Escherichia coli (DEC) strains was identified using 16-plex Polymerase Chain Reaction (PCR), whereas antibiotic susceptibility was realized using the disk diffusion method. Furthermore, multiplex PCR assays were used to characterize bla KPC, bla VIM and bla IMP genes in carbapenemase-producing E . coli and Salmonella . The study highlighted high resistance rates of the identified bacteria to common antibiotics. Likewise, two strains of E . coli were imipenem resistant with Carbapenemase-encoding genes. The genes detected were Klebsiella pneumoniae carbapenemase (KPC), Verona integrin-encoded metallo-β-lactamase (VIM) and Imipenemase (IMP-2) reaching a rate of 40% each. However, no Carbapenemase-encoding genes were detected in Salmonella isolates. Conclusions This study showed that for a real-time infection control and prompt application of antimicrobial chemotherapy, characterization of carbapenemase-producing Enterobacteriaceae in patients is crucial.


Background
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 Gramnegative bacteria, following the spread of carbapenemase-producing Enterobacteriaceae (CPE) [2,3].In bacteria of animal and human origins, beta-lactam resistance, which includes resistance to extendedspectrum beta-lactams, has now been increasingly observed [4].In Enterobacteriaceae, the carbapenemases have been previously classi ed 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][6][7].Enterobacteriaceae can be resistant to carbapenems through intrinsic or acquired mechanisms.The intrinsic mechanisms can occur by (a) production of chromosomal carbapenemases from the group of class A serine carbapenemases [8] or (b) e ux 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 (Table 1; [10]).

Table 1
The plasmid-mediated mechanisms of resistance [10] Type of mechanism of resistance Examples 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 de ned as βlactamases 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.

Antimicrobial susceptibility testing and ESBL production
Antibiotic susceptibility was determined on Mueller-Hinton agar using the standard disc diffusion procedure as described by the European Committee of Antimicrobial Susceptibility Testing (EUCAST) [19].
Nineteen antibiotics belonging to 7 different families were tested : amoxicillin (

Detection of antibiotic resistance genes
DNA for PCR analysis was extracted from the isolates using the heat lysis method [20].A loopful of bacterial growth from Mueller Hinton agar (Lio lchem, 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 and bla IMP types in carbapenemase-producing E. coli and Salmonella strains [21,22].

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 [23].Indeed, it is a major public health concern, mainly within communities.Our study reported for the rst time the occurrence of carbapenemase-producing E. coli 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, ce xime 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].Susceptibility to netilmicin and cipro oxacin appears to follow the general trend observed elsewhere in the world.
Our nding on overall prevalence of E. coli harboring carbapenemase genes in the 275 children was 1.81%.This result is similar to 2.5% reported in pets in Africa [3] suggesting that bacteria producing carbapenemase are currently spreading among these pets [3] and because of the proximity between humans and animals, these bacteria can contaminate humans.Indeed, animals could be reservoirs of gene transmission to humans.For example, it has been shown that poultry ocks contribute to the global dissemination of Salmonella Kentucky ST198-X1-SGI1CIP-R strain in developing countries [24].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.
Oatherwises, we detected no carbapenemase genes in Salmonella strains.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 [24].
Three main genes were detected in E. coli strains: Klebsiella pneumoniae carbapenemase (KPC), Verona integrin-encoded metallo-β-lactamase (VIM) and Imipenemase (IMP-2) with a rate of 40% each.To our best of knowledge, this is the rst 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 [25].As K. pneumoniae was identi ed extensively worldwide, a study suggested that it may have contributed to the spread of the blaKPC genes [26].
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 [27,8], although outbreaks and single reports of VIM and IMP producers have been shown in many other countries [27].
It has been shown that carbapenemases can hydrolyze almost all β-lactams, and are easily transferable among enterobacterial species [28].These genes are found in multidrug-resistant isolates consistent with the result found in the present study [28].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 [29].

Figures
Figures

Figure 1 Map
Figure 1

Figure 2 Resistance
Figure 2 The genes investigated and primers used are listed in.A questionnaire was used to collect demographic information (e.g., age and sex) of each patient).