The Beta-Lactam Resistome Expressed by Aerobic and Anaerobic Bacteria Isolated from Human Feces of Healthy Donors

Antibiotic resistance is a major health problem worldwide, causing more deaths than diabetes and cancer. The dissemination of vertical and horizontal antibiotic resistance genes has been conducted for a selection of pan-resistant bacteria. Here, we test if the aerobic and anaerobic bacteria from human feces samples in health conditions are carriers of beta-lactamases genes. The samples were cultured in a brain–heart infusion medium and subcultured in blood agar in aerobic and anaerobic conditions for 24 h at 37 °C. The grown colonies were identified by their biochemical profiles. The DNA was extracted and purified by bacterial lysis using thermal shock and were used in the endpoint PCR and next generation sequencing to identify beta-lactamase genes expression (OXA, VIM, SHV, TEM, IMP, ROB, KPC, CMY, DHA, P, CFX, LAP, and BIL). The aerobic bacterias Aeromonas hydrophila, Citrobacter freundii, Proteus mirabilis, Providencia rettgeri, Serratia fonticola, Serratia liquefaciens, Enterobacter aerogenes, Escherichia coli, Klebsiella pneumoniae, Pantoea agglomerans, Enterococcus faecalis, and Enterobacter cloacae, the anaerobic bacteria: Capnocytophaga species, Bacteroides distasonis, Bifidobacterium adolescentis, Bacteroides ovatus, Bacteroides fragilis, Eubacterium species, Eubacterium aerofaciens, Peptostreptococcus anaerobius, Fusobacterium species, Bacteroides species, and Bacteroides vulgatus were isolated and identified. The results showed 49 strains resistant to beta-lactam with the expression of blaSHV (10.2%), blaTEM (100%), blaKPC (10.2%), blaCYM (14.3%), blaP (2%), blaCFX (8.2%), and blaBIL (6.1%). These data support the idea that the human enteric microbiota constitutes an important reservoir of genes for resistance to beta-lactamases and that such genes could be transferred to pathogenic bacteria.


Introduction
The indiscriminate and unconscious use of antibiotics in the clinic, agriculture, and livestock have been increased the selection pressure in the worldwide microbiome, inducing resistant, multiresistant, and pan-resistant pathogen bacteria selection [1][2][3]. This problem Pharmaceuticals 2021, 14, 533 2 of 15 reached a maximal level during the last two years where the antibiotic use has increased drastically due to the pandemic spread of SARS-CoV-2, since more than 70% of COVID-19 patients have received complimentary treatment with antibiotics drugs [4,5].
Bacterial resistance to antibiotics is a situation that has been increasing during the last decades, converting into a wide world public health problem [6,7], that complicates the treatment of the infection while increases the mortality rate in both nosocomial or community-acquired infectious diseases [8,9]. This resistance is reaching a critical point, as the increase in these resistant, multi-resistant, and pan-resistant pathogenic strains causes serious medical complications [10].
The lactam groups of molecules are integrated into four families, i.e., beta-lactam [11], gamma-lactam [12], delta-lactam [13], and epsilon-lactam [14], with a specific action and not necessarily like antibiotics. Specifically, the beta-lactams drugs show antibiotic effects in a wide bacterial variety. They are characterized by a beta-lactam ring presence, which confers the antimicrobial effect by the transpeptidases and carboxypeptidases inactivation (the proteins responsible for the biosynthesis of the cell wall) through their binding to Penicillin Binding Protein (PBPs) receptors [15]. The beta-lactam family is made up of various members, such as penicillins B and G, amoxicillin, ampicillin, cephalosporins (cephalothin, cephalexin, ceftriaxone, and cefepime), carbapenems (imipenem, meropenem, ertapenem), aztreonbactam, monobactam, and some others. The indiscriminate and unconscious use of these antibiotics in daily clinical practice has caused an increase in multiresistant Enterobacteriaceae strains, especially Escherichia coli and Klebsiella pneumoniae, turning this situation into a serious global health problem [16]. During the last decade that there has been a significant increase in the presence of multi-resistant strains worldwide, mainly in the European Union. The European Read for Antimicrobial Resistance Surveillance (EARSNet) has been identified pan-resistant Escherichia coli and Klebsiella pneumoniae strains [17,18]. The bacteria have various resistance mechanisms, mainly associated with plasmid assimilation, which allow cross-resistance against a wide range of antibiotics [19]. The most widely described mechanism of antibiotic resistance that is mediated by plasmid transfer is the enzymatic degradation of the beta-lactam ring through the expression of betalactamases [20]. The genes encoding for the beta-lactamases enzymes have been widely spread among the bacterial population, through mobile genetic elements, which confer a plasmid-mediated multi-resistance [21,22]. These multi-resistance bacteria complicate the patient treatments and condition the type of antibiotics that can be administrated for the treatment of common infections [23]. The first-line antibiotic treatment regimens for gastrointestinal and airway infections usually include beta-lactams drugs, such as ampicillin, amoxicillin, or third generation cephalosporins (Ceftriaxone), but the resistance determines the treatment that can be given to patients and decreases the effectiveness significantly of these treatments. The Carbapenems (ertapenem, meropenem, imipenem) have been widely used as an alternative for the treatment of these resistant bacteria, however, in recent years, resistant bacteria strains to these antibiotics have been identified [24]. The main causes associated with this increase in bacterial resistance are the error in the medical prescription, self-medication, incomplete antibiotics treatment, and the use of these drugs by the agricultural and livestock industry for the treatment of crops for human consumption [25].
For these reasons, we test the hypothesis that the Enterobacteriaceae integrating the human gastrointestinal microbiome are carriers of beta-lactamases genes in healthy individuals who have not received antibiotic treatment during their last year of life.

Beta-Lactamases Gene Family Identification
The endpoint PCR results showed that 100 % of the obtained bacterial strain carries at least a gene of Beta-Lactamases, identified the gene families transcribed and the phenotype responsible for Beta-Lactam resistance: 49 strains were positive to blaTEM (100%), five positive strains for the blaSHV (10.2%), five positive strains for blaKPC (10.2%), seven positive strains for blaCYM (14.3%), one positive strain for blaP (2%), four positive strain for blaCFX (8.2%), and three positive strains for blaBIL (6.1%) gene families (Table 4). No members of gene families blaOXA, blaVIM, blaIMP, blaROB, blaCTX, blaDHA, and blaLAP were found in all obtained strains from feces of human and healthy conditions. These data were also confirmed by whole-genome sequencing of isolated strains (Tables 3 and 4). The isolated strains from human feces in healthy conditions showed a restricted pattern of resistance to beta-lactam drugs. The individual analysis showed that of the blaTEM was present in all (100% n = 49) of isolated strains, from these the 77.5% (n = 38) was carrier only for this gene family, the other 25.5% of isolated strains showed a mixed genotype for multiple gene families encoding beta-lactamase and specific for each strain, conformed from two (blaTEM + blaBIL, blaTEM + blaCYM, or blaTEM + blaP) to five (blaTEM + blaSHV + blaKPC + blaCYMX + blaDHA + blaBIL) gene families of beta-lactamases (Table 5).

Discussion
The wide use of antibiotics in the clinic, agriculture, and livestock induce a high selection pressure in the worldwide microbiome allowing the resistant, multi-resistant, and pan-resistant bacteria selection and colonization of the gut of human and some other animals including insects and seafoodn [1][2][3]. In the same way, contamination affects vegetables for human consumption [26][27][28][29][30][31][32][33][34][35][36][37]. Even though many of these bacteria do not produce pathology, they are capable of transferring these antibiotic resistances mechanisms to human gut microbiota members and pathogenic bacteria, making it a reservoir for antibiotic resistance genes [37]. Following this idea, our research group characterized the beta-Lactam resistome carried by the aerobic and anaerobic Enterobacteriaceae from healthy medical students of Mexico City.

Enterobacteriaceae Characterization
The data analysis demonstrates the presence of multiple members of aerobic and anaerobic beta-lactam resistant Enterobacteriaceae, the identified bacteria distribution was as follows. Aerobic: Aeromonas hydrophila, Citrobacter freundii, Proteus mirabilis, Providencia rettgeri, Serratia fonticola, and Serratia liquefaciens. Each one represents 3.7% of all beta-lactam resistant aerobic Enterobacteriaceae, the Enterobacter aerogenes represent 7.4%, Enterococcus faecalis was present in 14.8%, Enterobacter cloacae were in the 22.2% of all isolated strands, the more abundant Enterobacteriaceae were Escherichia coli, Klebsiella pneumoniae in and Pantoea agglomerans were found in the 11.1% each one (Figure 1, Tables 1 and 2). The founded species from human feces are in concordance with previous results were reported the repertoire and variations of human gut microbiota, they characterized 113 different bacteria, including Gram-positive bacteria (Bifidobacterium, Eubacterium, Peptostreptococcus, Ruminococcus, Lactobacillus, and Clostridium genera) and Gram-negative bacteria (Bacteroides, Fusobacteria genera) [38]. All our bacterial culture data were corroborated by NGS sequencing show 98% of concordance in the final bacterial identification. These data are supported by previously published works. Our NGS data are in concordance with other NGS studies that found 71% of all bacterial community was conformed by Firmicutes, 9% was Actinobacteria, while Bacteroidetes, Proteobacteria, and Cyanobacteria represent a relatively low abundance ranging from 3% to 7%, the bacterial families found were: Lactobacillacea, Ruminococcacea, Lancnospiraceacea, Clostridiaceacea. Streptococceacea, Streptophyta, Staphylococceacea, Verrucomicrobiaceace, and Enterobacteriaceae among others [39]. However, the composition of the intestinal microbiota varies depending on the type of diet [40]. The obtained results from feces samples reveal the potentially pathogenic enterobacteria presence in the human digestive tract in healthy conditions, their presence as the gastrointestinal resident microbiome, and not necessarily associated with an active gut infection.
To determine the potential of these batteries to cause infection, it would be necessary to study other factors, such as virulence and pathogenesis genes expressed by each bacterium, bacterial load, and subjects' immunological status.

Beta-Lactam Gene Families
Due to the isolated Enterobacteriaceae being isolated in presence of the beta-Lactam drug in the culture media (Table 2), all strains were expected to be carriers at least one member of the different gene families that code for beta-Lactamases. The endpoint PCR identified the gene families transcribed and the phenotype responsible for Beta-Lactam resistance: 100% of the isolated strain were positive to blaTEM, 10.2% positive for the blaSHV, 10.2% positive to blaKPC, 10.2% positive for blaCYM (14.3%), 2% positive to blaP, 8.2% positive to blaCFX and 6.1 positive blaBIL gene family (Tables 3 and 4), no strand positives to members of blaOXA, blaVIM, blaIMP, blaROB, blaCTX, blaDHA, and blaLAP gene families were found (Tables 3 and 4). These results accord with previous works, finding that Enterobacteriaceae expressed a member of blaCTX, blaSHV, and blaTEM genes families [41], while around the world the main reported beta-lactamase families in the multidrug-resistant Enterobacteriaceae are: blaKPC, blaTEM, blaOKP, blaOXA, blaSHV, blaVIM and blaNDM [42], in agreement with our results.
The data analysis showed multiple members of aerobic and anaerobic Enterobacteriaceae that show multi-resistance molecular mechanisms to beta-lactams drugs, mediated by unique resistance genes or conforming a beta-lactam resistome, in asymptomatic individuals. This can be widely explained by mechanisms such as antibiotics self-medication, abuse, and error in their medical prescription, as well as lack of adherence to antibiotic treatment, including via the consumption of contaminated water [45][46][47] or foods, mainly meats [48,49], fruits [50], and vegetables [26,51]. Moreover, the agriculture [52] and livestock industries [53,54] indiscriminately use the beta-lactam drugs to maximize production [55,56].

Bioinformatic Analysis and Primer Design
The bioinformatic analysis and primer design were previously described and reported by our research group [26]. Briefly, we obtained the beta-Lactamases integrons and DNA sequences from the GenBank of NCBI, all reported sequences of beta-lactamases we found were used to design primer for the PCR. The DNA sequence alignments were made with ClustalW v.2 software (http://www.clustal.org/clustal2/ accessed on 10 April 2021) [27][28][29], to obtain the phylogenetic trees by using FigTree V1.4.0 software (http://tree.bio.ed.ac.uk/software/figtree/ accessed 10 April 2021) [30]. The conserved sequences found in the alignment were used to design a set of specific and degenerate primers (PerPrimer v1.

Sampling
Twenty volunteers (10 men and 10 women) were selected for this study. All of them were medicine students from Mexico City enrolled at the Anahuac University, aged between 20 and 22 years, with no history of beta-lactam antibiotics treatment in the last year or have been suffering diarrhea during that period. Donors were provided with the sample collection material, which consisted of plastic containers, gloves, a cardboard paper sheet, and wooden spatulas; all the material was sterile. The donors were asked to deposit a portion of their stool on the cardboard sheet and from this, they collect with the wooden spatula, an approximate volume of 10 cubic cm, and place it in the sterile container. The time elapsed from when the sample was taken to its beginning of processing in the laboratory was not more than 2 h, this in order not to alter, as far as possible, the bacterial viability. Then, five g of each human feces samples were weighed and cultured in BHI (brain and heart infusion broth) (Becton Dickinson, Franklin Lakes, NJ) incubated at 37 • C for 24 h, in both aerobic and anaerobic conditions.

Bacterial Isolation
For the anaerobic conditions, the BHI tubes containing samples were immediately placed in the BD BBL ™ GasPak ™ anaerobic jar, one envelope of BD BBL ™ CO2 gas generators and another of BD BBL ™ GasPak ™ anaerobic indicator were placed on them. Later they were incubated at 37 • C during 24 h. For the aerobic conditions, the tubes with BHI containing the samples were directly incubated at 37 • C for 24 h. After is incubation, 10 µL from the BHI were taken, subcultured in MacConkey agar (Becton Dickinson, Franklin Lakes, NJ, USA), and incubated at 37 • C for 24 h. The obtained colonies were subcultured in blood agar and incubated at 37 • C for 24 h, in both aerobic or anaerobic conditions.

Biochemical Bacterial Identification
The automatized bacterial identification was made according to previously reported by our research group. Briefly, bacterial suspensions were made, by depositing two or three medium-sized colonies (2 to 3 mm) in BBL™ Crystal™ Inoculum Broth (Becton Dickinson, Franklin Lakes, NJ, USA). Said inoculum was adjusted to a Mac Farland 1.0 scale (Expected CFU/mL 3.0 × 10 8 ). The inoculum was deposited in BBL™ Crystal™ Enteric/Nonfermenter or Anaerobe ID Kit plates, incubated at 37 • C for 18 h, without CO 2 and 40-60% humidity. Finally, the plates were read by the BBL™ Crystal™ AutoReader (Becton Dickinson, Franklin Lakes, NJ, USA) and the results were analyzed with the BBL™ Crystal™ MIND v.5.05 Software (Becton Dickinson, Franklin Lakes, NJ, USA) [26].

Antimicrobial Susceptibility Testing
The antimicrobial susceptibility testing was done following a previously reported method [26]. Briefly, the pure colonies obtained from blood agar were resuspended in bacterial suspensions, and were made depositing two or three medium-sized colonies  Five mL of Luria-Bertani broth (LB) were inoculated with an isolated bacterial colony and incubated overnight at 37 • C with continuous shaking (200 rpm), the bacterial culture was centrifuged at 4000 rpm for five minutes at room temperature, bacterial the pellet was resuspended on 1 mL of sterile free-RNAse and free-DNAse deionized water, the bacterial suspension was heated at 94 • C for 10 min followed by ice shock. Finally, the sample of lysed bacterial was kept at -80 • C until use [26].

Genomic DNA Extraction
The DNA extraction was done following a previously reported method [26]. Briefly, 500 µL of bacterial culture was obtained from a tested single bacterial colony for genomic DNA isolation by RTP pathogen kit (Invitek, Germany) following the manufacturer's instruction. The eluted DNA solution was quantified by absorbance and its integrity was verify by 2% agarose gel electrophoresis. The sample was kept at -20 • C until use.

Plasmid DNA Extraction
The plasmid DNA extraction was done following a previously reported method [26], by using plasmid DNA isolation by using PureLink HiPure Plasmid DNA Purification Kit (Invitrogen, USA) following the manufacturer's instructions. Plasmid DNA solution was quantified by digital spectrophotometry by using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) and its integrity was verified by electrophoresis in 2 % agarose gel, the sample was kept at -20 • C until use.

Endpoint PCR
The endpoint PCR to identify the β-Lactamases gene families was made by using previously reported specific and degenerated primes (Table 7)  The PCR products were analyzed by electrophoresis in a 2% Agarose gel prestained with ethidium bromide and the image was digitized in a GelLogistic 3000 photodocumenter.

Whole and Plasmid DNA Sequencing
The DNA sequencing was performed following a previously reported method by our research group [26], the indexed libraries that were prepared using a standard Illumina Nextera XT DNA Sample Preparation Kit (FC-131-1096) for small genomes and were sequenced on the MiSeq platform (Illumina; San Diego, CA, USA). Adapters and barcodes were trimmed by the default setting in the Illumina experiment manager, generating 300-bp paired-end reads. The quality of the unprocessed reads was assessed using FastQC High Throughput Sequence QC Report v:0.11.5 (Babraham Bioinformatics, Babraham Institute; Cambridge, UK) [32]. A minimum Q score of more than 30 for at least 85% of all reads was attained. All reads were mapped using BWA-MEM aligner version 0.7.7-r441 (Wellome trust, Sanger Institute, Hinxton, UK) [33] and SAMtools version 1.3.1 (Wellome trust, Sanger Institute, Hinxton, UK). The NOVO genome assembly was done using the SPAdes Genome Assembler software version 3.11 (CAB, St. Petersburg State University, St. Petersburg, Russia) [34]. The metagenomic analysis for the taxonomic classification of bacteria was done by using the software Kraken taxonomic sequence classification system Version 0.10.5-beta (CCB, Johns Hopkins University, Baltimore, MD, USA) [35]. The betalactamase genes were identified by the comparative analysis while using the Basic Local Alignment Search Tool (BLAST, NCBI-NIH, Bethesda, MD, USA) [36]. Table 7. Primers sequences and amplicon size for beta-lactamase gene family [26].

Conclusions
The human gut bacteriome is an important reservoir and mediator for the environmental beta-lactam resistome. The cumulative beta-lactam resistome in the Enterobacteriaceae is indicative of the indiscriminate and irrational use of beta-lactam drugs in practically all economical worldwide activities, especially in Mexico City. The gut bacteriome express beta-lactam resistome in healthy conditions is integrated by multiple beta-lactamases gen families, such as blaTEM, blaSHV, blaKPC, blaCYM, blaBIL, blaCFX, blaCYM, blaBIL, and blaP, which altogether confer resistant, multi-resistant, and pan-resistant characteristics against beta-lactam antibiotics.