Detection of Multidrug-resistant Extended-spectrum Beta-lactamase-producing Enterobacteria from Community Infections in the city of Reynosa, Tamaulipas Mexico

Production of extended spectrum beta-lactamases (ESBL) is one of the main problems related to antimicrobial resistance worldwide, with the CTX-M, TEM and SHV types standing out as the most prevalent. These enzymes are usually related to plasmids which facilitates their horizontal genetic transmission. In the northeast region of Tamaulipas their clinical prevalence is unknown. Therefore, the aim of this work was to dene the molecular epidemiology of ESBL-producing Enterobacteriaceae in clinical strains collected in Reynosa Tamaulipas, Mexico. A selection of 123 Enterobacteriaceae strains from different clinical patients were collected from August 2018 to December 2019. These strains were phenotypically identied by double disk synergy tests (DDST) and subsequently subjected to polymerase chain reaction for the detection and amplication of the bla TEM , bla SHV , bla CTX−M−1 , bla CTX−M−2 , bla CTX−M−9 and bla CTX−M−8/25 genes. Lastly, antimicrobial resistance proles were determined by plate diffusion method and their capacity to transfer this sort of resistance by conjugation was assessed. Our results showed a prevalence of 48.78% (60/123) of ESBL-producing enterobacteria, with the bla TEM and bla CTX−M−1 genes most commonly detected in 76.67% (46/60) and 58.33% (35/60), respectively. Additionally, a 68.33% (41/60) of these ESBL-producing Enterobacteriaceae were multidrug-resistant, while 51.67% (31/60) were able to transfer some genes related to ESBL production, being bla CTX−M−1 the most common. This is the rst study in the region that evaluates ESBL production in clinical Enterobacteriaceae strains, as well as the content of genes related to this phenotype and the ability to transfer this type of antimicrobial resistance.


Introduction
Antimicrobial resistance is currently a great challenge in the clinical environment, since it complicates the treatment, control, and prevention of a growing number of infections [1,2,3]. Infections related to multidrug-resistant bacteria (MDR) have led to the use of broad-spectrum antibiotics such as third and fourth generation cephalosporins and carbapenems, causing an increase on selective pressure that has favored the proliferation and dissemination of resistant strains [4,5]. Extended-spectrum beta-lactamases (ESBL) producing Enterobacteriaceae are considered by the World Health Organization (WHO) as priority microorganisms for the development of new antibiotics, as well as for microbial resistance monitoring [6]. ESBL production confers resistance to most penicillins, to rst, second, and third generation cephalosporins and to monobactams [7,8,9]. ESBLs belong to classes A and D of Ambler's beta-lactamases classi cation and are characterized by containing a serine residue in its active site, in addition to being strongly inhibited by classical inhibitors such as clavulanic acid [10]. ESBLs groups includes nine structural families: TEM, SHV, CTX-M, PER, VEB, GES, TLA, BES and OXA [11]. The TEM, SHV and CTX-M families are the most prevalent worldwide reported in hospital and community infections [6,12,13]. ESBLs contained in conjugative plasmids facilitate their mobility through horizontal gene transfer [11,14,15] and in turn, these plasmids may contain resistance related genes to other antibiotics, such as quinolones, aminoglycosides, tetracyclines and even colistin, which increases the chances of failure in treatment schemes [16,17,18].
ESBL-producing Enterobacteriaceae were originally associated with infections related to the hospital environment, however, cases of community-associated infections have increased [5], and ESBL-producing Enterobacteriaceae have been reported in sources, such as water, soil, livestock, pets and in healthy human carriers [15,19]. In the northeast region of Tamaulipas, the presence of ESBL-producing Enterobacteriaceae has been reported in animal origin food and super cial waters [20]. However, there is little information regarding the prevalence of such microorganisms of clinical origin. The aim of the present work was to de ne the molecular epidemiology of ESBLproducing Enterobacteriaceae in clinical strains isolated in the city of Reynosa, Tamaulipas in the northeast region of Mexico, in addition to identify the genotype related to ESBL production, determine their microbial resistance pro le and lastly, to evaluate their capacity to transfer this resistance.
Antimicrobial resistance pro les of the ESBL-producing strains are shown in Table 2. We observed a high resistance to the beta-lactams included in this study, with resistance to CTX being the most common found in 96.67% (58/60) of ESBL-producing Enterobacteriaceae. We can highlight high ranges of resistance to STX, quinolones and TE; while AN turned out to be the most effective antibiotic against the ESBLproducing strains studied in this work. On the other hand, it should be noted that 66.67% (40/60) of the ESBL-producing strains presented the MDR phenotype since it showed resistance to 3 or more classes of antibiotics used in this work.
ESBL Horizontal Gene Transfer.
The conjugation assay was successful (   Discussion ESBL-mediated resistance in Enterobacteriaceae is one of the greatest threats to public health at present, increasing morbidity and mortality of many infectious diseases, in addition to increasing health costs. There are different reports in Mexico regarding the presence and mobility of ESBL-producing strains and the genes associated with their production [12,19,21,22,2,25]. However, in the northeast region of Mexico only the environmental distribution of these organisms has been elucidated [20]. Our results showed that 48.78% of analyzed Enterobacteriaceae presented the characteristic ESBL-production phenotype, being E. coli and K. pneumoniae the most common species which presented this phenotype; similar results have been reported in different countries [26,27]. Although K. pneumoniae has stood out as one of the most common producers of ESBLs, it should be noted that in recent years, ESBLproducing E. coli has gained importance worldwide [28,29]. Most of the ESBL-producing isolates were isolated from diabetic foot wounds and urinary tract infections (UTIs). As for UTIs, Enterobacteriaceae, speci cally E. coli stands out as one of the main etiological agents of these sort of infections [30], while in diabetic foot infections E. coli and K. pneumoniae stands out together with Pseudomonas aeruginosa, Enterococcus spp. Staphylococcus aureus as common infectious agents related to these wounds. In addition to this, cephalosporins are part of the empirical antimicrobial treatment which could compromise said therapy by prolonging the morbidity of these conditions [30,31].
Regarding ESBL-related genes, most of the analyzed strains in the present work were found to be related to the TEM type and group 1 of the CTX-M type, while those of the SHV type were found to be less prevalent; although it should be mentioned that the prevalence of ESBL genes varies greatly depending on the geographical region [32]. An interesting fact is that ESBLs of TEM and SHV types had been the main described genetic antimicrobial resistance determinants during much of the 2000s, however, we reported a very low prevalence of the SHV type and an increase in the CTX-M type, which is considered an emerging beta-lactamase. This may be due to the fact that CTX-M type ESBLs come from an environmental Enterobacteriaceae, which increases the possibilities of acquiring a CTX-M-producing Enterobacteriaceae in the community [28]. An 8.33% (5/60) of analyzed strains presented the ESBL-related phenotype but lacked the searched ESBLs in this work, however, the presence of some different ESBL that was not included in the present work is not discarded.
Lastly, 51. 67% (31/60) of analyzed Enterobacteriaceae in this work had the ability to transfer resistance to 3rd. generation cephalosporins, being the CTX-M-1 type beta-lactamase the predominant transferred type in 23/31 trans-conjugating strains, in comparison with the TEM type (13/31). This is relevant due to the CTX-M type beta-lactamase can be mobilized by environmental Enterobacteriaceae, and it has been reported that sub-therapeutic concentrations of cephalosporins can increase their ability to transfer genes related to CTX-M beta-lactamases [33,34]. In addition, 66.67% (40/60) of the ESBL-producing strains showed resistance to more than 3 antibiotic families, which indicates the spread of MDR phenotype in ESBL-producing Enterobacteriaceae which could drastically reduce treatment options [4]. Amikacin stood out as the most effective antibiotic, which has already been reported in previous works and this can constitute a treatment option of infections caused by these antimicrobial-resistant microorganisms [35,36].
To our knowledge, this is the rst work that focuses on studying production of ESBLs, genomic type and evaluation of horizontal gene transfer potential of ESBL genes in clinical Enterobacteriaceae strains in the northeast region of Tamaulipas. Described ndings can give us an insight of the current outlook regarding the state of resistance to 3rd generation cephalosporins and other families of antibiotics in the region. Collected data will allow us to develop possible treatment schemes and to evaluate key points related to the current antimicrobial resistance in the region.

Methods
Strain Selection.
Strains were obtained from the Environment-Microorganism Interaction Laboratory's biobank of the Centro de Biotecnología Genómica of the Instituto Politécnico Nacional and private laboratories of the city of Reynosa, Tamaulipas. A total of 123 Enterobacteriaceae strains were selected from bronchial aspiration samples (6), vaginal exudates (5), expectoration samples (5), stool samples (14), wounds (6), lithotomy (1), urine samples (37)  Conjugation tests were performed using the sodium azide-resistant E. coli strain J53 as a recipient. Both, the donor strain and the recipient strain were grown separately at 37 ºC overnight in Luria Bertani broth (BD Becton Dickinson & Co.). Conjugation tests were carried out mixing equal volumes in a 1:1 ratio of the donor and the recipient strains for their subsequent incubation at 37 °C for 4 hours. After incubation, they were cultured on tryptic soy agar medium plates supplemented with 2 µg./mL. of CTX and 100 µg./mL. of sodium azide to eliminate donor strains. Positive strains were assayed for the detection of ESBL production-related genes.