antibiotics Article Profile of Enterobacteria Resistant to Beta-Lactams Andressa Liberal Santos 1, Adailton Pereira dos Santos 1,Célia Regina Malveste Ito 1 , Pedro Henrique Pereira de Queiroz 1, Juliana Afonso de Almeida 1 , Marcos Antonio Batista de Carvalho Júnior 1 , Camila Zanatta de Oliveira 2, Melissa Ameloti G. Avelino 3, Isabela Jubé Wastowski 4, Giselle Pinheiro Lima Aires Gomes 5, Adenícia Custódia Silva e Souza 6, Lara Stefânia Netto de Oliveira Leão Vasconcelos 1, Mônica de Oliveira Santos 3, Carla Afonso da Silva 1 and Lilian Carla Carneiro 1,* 1 Institute of Tropical Pathology and Public Health, Federal University of Goiás, 235 Street, Goiânia 74605-050, Brazil; [email protected] (A.L.S.); [email protected] (A.P.d.S.); [email protected] (C.R.M.I.); [email protected] (P.H.P.d.Q.); [email protected] (J.A.d.A.); [email protected] (M.A.B.d.C.J.); [email protected] (L.S.N.d.O.L.V.); [email protected] (C.A.d.S.) 2 Agency of Agricultural Defense of Paraná—ADAPAR, Goioerê 80035-050, Brazil; [email protected] 3 Medicine College, Federal University of Goiás, 235 Street, Goiânia 74690-900, Brazil; [email protected] (M.A.G.A.); [email protected] (M.d.O.S.) 4 Laranjeiras Unit, State University of Goiás, Goiânia 74705-010, Brazil; [email protected] 5 Department of Biology, Federal University of Tocantins, Square 109 North, NS15 Avenue, ALCNO-14-Plano Director North, Palmas 77001-090, Brazil; [email protected] 6 Department of Biology, Pontifical Catholic University of Goiás, 1st Avenue, Goiânia 74175-120, Brazil; [email protected] * Correspondence: [email protected]; Tel.: +55-62-32026528 Received: 28 February 2020; Accepted: 10 April 2020; Published: 15 July 2020 Abstract: A serious emerging problem worldwide is increased antimicrobial resistance. Acquisition of coding genes for evasion methods of antimicrobial drug mechanisms characterizes acquired resistance. This phenomenon has been observed in Enterobacteriaceae family. Treatment for bacterial infections is performed with antibiotics, of which the most used are beta-lactams. The aim of this study was to correlate antimicrobial resistance profiles in Enterobacteriaceae by phenotypic methods and molecular identification of 14 beta-lactamase coding genes. In this study, 70 exclusive isolates from Brazil were used, half of which were collected in veterinary clinics or hospitals Phenotypic methodologies were used and real-time PCR was the molecular methodology used, through the Sybr Green system. Regargding the results found in the tests it was observed that 74.28% were resistant to ampicillin, 62.85% were resistant to amoxicillin associated with clavalunate. The mechanism of resistance that presented the highest expression was ESBL (17.14%). The genes studied that were detected in a greater number of species were blaGIM and blaSIM (66.66% of the samples) and the one that was amplified in a smaller number of samples was blaVIM (16.66%). Therefore, high and worrying levels of antimicrobial resistance have been found in enterobacteria, and a way to minimize the accelerated emergence of their resistance includes developing or improving techniques that generate diagnoses with high efficiency and speed. Keywords: antibiogram; antimicrobial resistance; β-lactams; enterobacterias; molecular diagnosis 1. Introduction Regarding taxonomy, in relation to taxonomy, the Enterobacteriacea family has 53 genera of which more than 170 species have already been named. Among these, 26 bacterial genera have already been Antibiotics 2020, 9, 410; doi:10.3390/antibiotics9070410 www.mdpi.com/journal/antibiotics Antibiotics 2020, 9, 410 2 of 18 associated with bacterial infections in humans. Members of this family are Gram-negative, facultative anaerobic rods and most species are able to grow at 37 ◦C, although some grow more properly at 25 to 30 ◦C[1]. These microorganisms are widely distributed in nature and are found in soil, water, vegetables, in humans and vertebrates gastrointestinal tract [2]. Enterobacteriaceae represent the main group of bacteria isolated in clinical samples and are associated with a wide variety of community and hospital infections [3]. Gram-negative bacteria, specifically Enterobacteriaceae, are common causes of both community-acquired and hospital-acquired infections, including urinary tract, bloodstream, and lower respiratory tract infections [4]. Resistance among clinically important organisms to antimicrobial agents is severely threatening the repertoire of treatment options for common infections. The challenge is intensified by the fact that several of these organisms are resistant to multiple antimicrobials [5]. Infections caused by Gram-negative bacteria resistant to multiple drugs are a serious public health problem due to the scarcity of treatment options for these infections [6]. Currently, antimicrobial resistance is one of the most important factors that threaten public health [7]. Transmission between species of resistant bacteria or genetic elements of resistance from animals or the environment to humans has been reported [8,9]. Monitoring hospital environments and those related to animal husbandry and treatment has permanently entered the timeline of the most important studies and annual reports that assess the scope and level of this phenomenon [10]. Antibiotics play a key role in the success of some medical practices. Unfortunately, they tend to lose their efficacy over time due to the emergence and spread of resistance among bacterial pathogens [11]. According to Magiorakos [12], bacteria can be multi drug-resistant (MDR). MDR was classified as having acquired non-susceptibility to at least one agent in three or more antimicrobial categories. Drug resistance genes can be spread from one bacterium to another through various mechanisms such as plasmids, bacteriophages, naked DNA or transposons. Some transposons contain integrons—more complex transposons that contain a site for integrating different antibiotic resistance genes and other gene cassettes in tandem for expression from a single promoter [13]. Bacterial conjugation is the most sophisticated form of horizontal gene transfer (HGT) in bacteria and provides a platform for the spread and persistence of antibiotic resistance and virulence genes [14]. Beta-lactams are preferred because of their clinical efficacy and safety by virtue of their highly selective toxicity [15]. Resistance to beta-lactams in Enterobacteriaceae and other Gram-negative organisms is primarily mediated by beta-lactamases [16]. Beta-lactamases are enzymes that catalyze the beta-lactam hydrolysis ring leading to antimicrobial inactivation and preventing it from being active against the enzymes responsible for bacterial cell wall synthesis [17]. Antimicrobial susceptibility testing methods are divided into types based on the principle applied in each system [18]. The antibiogram provides qualitative results by categorizing bacteria as susceptible, intermediate susceptibility or resistant. Therefore, it is a tool based on the resistance phenotype of the tested microbial strain. However, inhibition of bacterial growth does not mean bacterial killing, the phenotypic method fails to distinguish between bactericidal and/or bacteriostatic effects [19]. Molecular diagnosis is another method of identifying bacterial resistance that can be applied. The molecular technique performed through nucleic acids, while requiring advancements, may allow the patients to obtain a fast examination result, within a four-hour period; thus, initiating the most appropriate antibiotic therapy. This can improve treatment outcomes for the patient and reduce empirical antimicrobial prescriptions, decreasing the duration and cost of antimicrobial treatment. Thus, technologies with the diagnosis of nucleic acids have the potential to reduce the selection of new resistances as well as to reduce the potential of existing resistances [20]. The objectives of this study are to correlate the resistance profiles of Enterobacteria using phenotypic and genotypic methodologies. The genes encoding resistance to beta-lactams are: blaSPM, blaSIM, blaVIM, blaKPC, blaSHV, blaCTX-M, blaGIM, blaOXA, blaIMP, blaNDM, blaSME, blaDHA, blaCMY and blaTEM. This study is justified because it is assumed that molecular methods improve accuracy and Antibiotics 2020, 9, 410 3 of 18 efficiency compared to the classical phenotyping method. In addition, it can be released in a short time; helping to improve the effectiveness of antibiotic therapy. 2. Results The enterobacteria from this study were isolated from samples collected in four different types of origin. Among the total bacterial isolates, 40% are from the human clinic, 20% from an animal clinic, 10% from a human hospital environment and 30% from the veterinary hospital environment. This antimicrobial resistance study in enterobacteria characterized a phenotypic profile of resistance to beta-lactam antibiotics, in which among the 70 bacterial samples studied, 52 (74.28%) were resistant to ampicillin, 44 (62.85%) were resistant to amoxicillin associated with the beta-lactamase inhibitor clavalunate, 38 (54.28%) were resistant to cefazolin, and 6 (8.57%) were resistant to cefuroxime. Table1 shows the percentage of antimicrobial resistance by sample source of Enterobacteriaceae. Table 1. Percentage of antimicrobial resistance by sample source (%). Percentage of Antimicrobial Resistance (%) Antibiotics Manual Human Human Veterinary Animal Animal Resuscitators