Next Article in Journal
In Silico Profiling of Clinical Phenotypes for Human Targets Using Adverse Event Data
Previous Article in Journal
Mathematical Analysis and Treatment for a Delayed Hepatitis B Viral Infection Model with the Adaptive Immune Response and DNA-Containing Capsids
Article Menu
Issue 4 (December) cover image

Export Article

High-Throughput 2018, 7(4), 36; https://doi.org/10.3390/ht7040036

Article
Characterization of Gene Families Encoding Beta-Lactamases of Gram-Negative Rods Isolated from Ready-to-Eat Vegetables in Mexico City
1
CICSA Facultad de Ciencias de la Salud Universidad Anáhuac Mexico Campus Norte, Huixquilucan, Estado de Mexico 52786, Mexico
2
Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de Mexico. Coyoacán, Ciudad de Mexico 04510, Mexico
3
Laboratorio de Investigaciones Químico-Clínicas, Facultad de Ciencias Químicas, Benemerita Universidad Autónoma de Puebla, San Manuel, Ciudad Universitaria, Puebla 72570, Mexico
4
Laboratorio de Medicina Genómica, Hospital Regional “Primero de Octubre”, ISSSTE, Av. Instituto Politécnico Nacional 1669, Lindavista, Gustavo A. Madero, Ciudad de Mexico 07300, Mexico
5
División de Investigación en Salud, Unidad Médica de Alta Especialidad (UMAE), Hospital de Especialidades, Centro Médico Nacional General de División “Manuel Ávila Camacho”, Instituto Mexicano del Seguro Social (IMSS), Puebla 72090, Mexico
6
Departamento de Fisiología Biofísica y Neurociencias, Centro de Investigación y Estudios Avanzados, Av. Instituto Politécnico Nacional 2508, San Pedro Zacatenco, Gustavo A. Madero, Ciudad de Mexico 07360, Mexico
*
Author to whom correspondence should be addressed.
Received: 18 September 2018 / Accepted: 7 November 2018 / Published: 23 November 2018

Abstract

:
Beta-lactam resistant bacteria, which are commonly resident in tertiary hospitals, have emerged as a worldwide health problem because of ready-to-eat vegetable intake. We aimed to characterize the genes that provide resistance to beta-lactam antibiotics in Enterobacteriaceae, isolated from five commercial salad brands for human consumption in Mexico City. In total, twenty-five samples were collected, grown in blood agar plates, and the bacteria were biochemistry identified and antimicrobial susceptibility testing was done. The carried family genes were identified by endpoint PCR and the specific genes were confirmed with whole genome sequencing (WGS) by Next Generation Sequencing (NGS). Twelve positive cultures were identified and their microbiological distribution was as follows: 8.3% for Enterobacter aerogene (n = 1), 8.3% for Serratia fonticola (n = 1), 16.7% for Serratia marcesens (n = 2), 16.7% for Klebsiella pneumoniae (n = 2), and 50% (n = 6) for Enterobacter cloacae. The endpoint PCR results showed 11 colonies positive for blaBIL (91.7%), 11 for blaSHV (91.7%), 11 for blaCTX (97.7%), 12 for blaDHA (100%), four for blaVIM (33.3%), two for blaOXA (16.7%), two for blaIMP (16.7%), one for blaKPC (8.3%), and one for blaTEM (8.3%) gen; all samples were negative for blaROB, blaCMY, blaP, blaCFX and blaLAP gene. The sequencing analysis revealed a specific genotype for Enterobacter cloacae (blaSHV-12, blaCTX-M-15, blaDHA-1, blaKPC-2); Serratia marcescens (blaSHV-1, blaCTX-M-3, blaDHA-1, blaVIM-2); Klebsiella pneumoniae (blaSHV-12, blaCTX-M-15, blaDHA-1); Serratia fonticola (blaSHV-12, blaVIM-1, blaDHA-1); and, Enterobacter aerogene (blaSHV-1, blaCTX-M-1, blaDHA-1, blaVIM-2, blaOXA-9). Our results indicate that beta-lactam-resistant bacteria have acquired integrons with a different number of genes that provide pan-resistance to beta-lactam antibiotics, including penicillins, oxacillins, cefalosporins, monobactams, carbapenems, and imipenems.
Keywords:
Enterobacteriaceae; antibiotics; beta-lactamases; beta-lactam resistome; whole genome sequencing

1. Introduction

The intake of ready-to-eat salad greens from commercial sources is a worldwide health practice that might be a potential cause of gastrointestinal (GI) disease. Because of traditional agricultural techniques, fresh foods, such as fruits and vegetables, are usually contaminated with a great deal of diverse bacteria [1]. Frequently, these foods are eaten raw or without an appropriate sanitization process [2]. Food safety comprises the conditions and practices that preserve the quality and purity of food in order to prevent illness by its intake [3]. The intake of food and water with poor hygiene quality can cause GI illnesses [4]. It is estimated that around 2195 children die every day because of infectious diarrhea, with a mortality rate greater than that associated with AIDS, malaria, and measles together [5]. GI infections are the second cause of death in children younger than five years-old worldwide [5]. This situation becomes even more critical when dealing with multiresistant Enterobactrias [6]. Beta-lactam antibiotics are widely used as an election treatment for GI infections in pediatric patients. However, the increasing number of beta-lactam-resistant Enterobacteriaceae in the last two decades has dramatically decreased the availability of effective antibiotics [7].
The enzymatic degradation of beta-lactam rings by beta-lactamases is the most effective resistance mechanism that bacteria have for fighting back against the antibacterial effect of beta-lactam antibiotics [8]. The genes encoding the beta-lactamases have been spread widely between different bacterial populations, which are mainly due to their mobile genetic elements, such as carrier plasmids and transposons. The organization of integrons, carriers of multiple gene-encoding beta-lactamases that form multi-drug resistance cassettes, represents another mechanism that contributes to the increase of positive bacterial populations to the beta-lactamases [9]. Beta-lactamases (bla) are classified depending on their structure or resemblance in the amino acid sequence (Classes A-D) [10] or according to their function (Groups I-IV) [11]. There are two types of beta-lactamases: serine-beta-lactamases and metallo-beta-lactamases [12]. Classes A, C, and D are serin-beta-lactamases. All members of these groups share the same mechanism of action and have a characteristic serine in the active site. This serine is deprotonated to produce a nucleophile serine that can induce a nucleophilic attack in the beta-lactam ring, generating an acyl-enzyme intermediate which is easily hydrolyzed on a general basis [9]. The members of class B are metallo-beta-lactamases. These enzymes perform a zinc-dependent nucleophilic attack [9,12]. The class A or group II enzymes hydrolyze penicillin and cephalosporin. Several gene families of these beta-lactamases have been identified, such as blaTEM, blaSHV, blaCTX, blaCFX, blaLAP, blaROB, and blaKPC [13]; however, some bacteria have shown carbapenem resistance [14]. The members of class B of beta-lactamases hydrolyze carbapenems and the genes encoding them are blaIMP (Imipenem-resistant) and blaVIM (Verona Integron-encoded Metallo type beta-lactamases) [15]. Class C beta-lactamases belonging to group I have cefalosporinase activity. AmpC beta-lactamase [16] was the first isolated enzyme from this class. Later, isolations were named according to the antibiotic in which they showed inhibitory action. For example, blaCMY has activity against cephamycin [17], blaFOX against cefoxitin, blaMOX against moxalactam, and blaLAT against latamoxef. Other C group beta-lactamases were named according to the place of isolation, such as blaMIR-1 (Miriam Hospital in Providence, R.I.) or blaDHA (Dhahran hospital in Saudi Arabia). The C blaBIL beta-lactamase owes its name to the patient from which it was isolated (Bilal) [18]. Class D beta-lactamases have oxacillinase activity and they are encoded by blaOXA gene [19]. A worldwide increase of bacterial strains having blaOXA with weak resistance to carbapenems has been reported [14,20]. The increase of bacterial strains resistant to beta-lactams has been reported not only in clinical isolations, but also from diverse food sources, including vegetables [21,22].
Recently, the Mexican health system has been promoting the intake of vegetables, fruits, and herbs to prevent and reduce obesity, which is a health problem in Mexico City [23]. This lofty promotion entails another health risk, because the great majority of those products is grown by traditional agriculture techniques in Mexico and they have been related to the high prevalence of GI disease [24]. Therefore, this work aimed to identify the gene families coding for beta-lactamases in Enterobacteriaceae isolated from vegetable samples from salads sold in Mexico City, labelled as suitable for human consumption. Our results show the high probability of the contamination of fresh vegetables with pathogenic multi-resistant Enterobacteriaceae and they provide insight into the beta-lactam pan-resistance mechanism of Enterobacteriaceae.

2. Material and Methods

2.1. Bioinformatics Analysis and Primer Design

We obtained the beta-lactamase integrons and DNA sequences from the National Center for Biotechnology Information. We used all the sequences of beta-lactamase families reported in order to determine the conserved sequence in each gene family encoding beta-lactamases. The integrons and DNA alignments were made with ClustalW software (V2.1, Conway Institute UCD, Dublin, Ireland) [25] according to DNA loss model parameters using a gap of 0.05 [26,27]. The phylogenetic trees were edited with the FigTree software (V1.4.2 Institute of Evolutionary Biology, Ashworth Laboratories, Kings Buldingg, Edimburgh, UK), and the phylogenetic analysis was made according to the procedure that was described by Martinez-Perez et al., 2009 [28]. We used the conserved sequence identified in the alignment for all gene to design specific and degenerate primers by using PerPrimer Software (V1.1.21, Owen J. Marshall, Melbourne, Australia) [29] following the next criteria, length (18–25 bp), Tm (60–62 °C), GC (40–60%), ∆T° (1 °C), Amplicons (83–230 bp) to run all the endpoint PCRs reactions in the same plate.

2.2. Sampling

Under sterile conditions, samples of 2 cm2 were collected from the vegetables of five different salad brands, labelled as suitable for human consumption and sold in marketplaces in Mexico City. The samples were cultured in BHI (blood and heart infusion; Becton Dickinson; Franklin Lakes, NJ, USA) incubated at 37 °C for 24 h.

2.3. Bacterial Isolation

After its incubation, 10 μL from the BHI were taken and subcultured in MacConkey agar (Becton Dickinson; Franklin Lakes, NJ, USA), and then incubated at 37 °C for 24 h. The colonies obtained were subcultured in blood agar and incubated at 37 °C for 24 h.

2.4. Bacterial Identification

The bacterial identification was made from the pure colonies that were obtained from blood agar using Gram staining and oxidase, Indol and biochemistry tests. The BD BBL Gram Stain Kit (Becton Dickinson; Franklin Lakes, NJ, USA) was used. Oxidase and Indol tests were carried out by using BBL DrySlide Oxidase kit (Becton Dickinson, Franklin Lakes, NJ, USA) and BBL DrySlide Indole kit (Becton Dickinson; Franklin Lakes, NJ, USA) according to the manufacturer’s specifications. The bacterial identification was performed using the BBL Crystal Enteric/Nonfermenter ID system (Becton Dickinson; Franklin Lakes, NJ, USA). Briefly, bacterial suspensions were made depositing two or three medium-sized colonies (2 to 3 mm) in BBL Crystal Inoculum Broth (Becton Dickinson; Franklin Lakes, NJ, USA). That inoculum was adjusted to a Mac Farland 1.0 scale (Expected CFU/mL 3.0 × 108). To adjust the scale, a CrystalSpec Nephelometer (Becton Dickinson; Franklin Lakes, NJ, USA) was used. The inoculum was deposited in BBL Crystal Enteric/Nonfermenter ID Kit plates, incubated at 37 °C for 18 h, without CO2 and 40 to 60% humidity. Finally, the plates were read using a BBL Crystal AutoReader (Becton Dickinson; Franklin Lakes, NJ, USA) and the results were analyzed with the BBL Crystal MIND Software (Becton Dickinson; Franklin Lakes, NJ, USA).

2.5. Antimicrobial Susceptibility Testing

Antimicrobial susceptibility testing was carried out by the Kirby–Bauer method under the Clinical and Laboratory Standards Institute protocol (NCCLS standards). The pure colonies obtained from blood agar were resuspended in bacterial suspensions were made depositing two or three medium-sized colonies (2 to 3 mm) in BBL Crystal Inoculum Broth (Becton Dickinson; Franklin Lakes, NJ, USA). The obtained inoculum was adjusted while using a Mac Farland 0.5 reading (Expected CFU/mL 1.5 × 108), and cultured in Müeller Hinton 150 × 15 mm2 media BD BBL (Becton Dickinson Franklin Lakes, NJ, USA). The antibiotic discs were applied with the Sensi-Disc Designer Dispenser System. The antibiotics panel was conformed by ampicillin (10 µg), ampicillin/sulbactam (10/10 µg), mezlocillin (75 µg), carbenicillin (100 µg), piperacillin/tazobactam (100/10 µg), cefazolin (30 µg), cefaclor (30 µg), cefepime (30 µg), cefoperazone (75 µg), and cefotetan (30 µg) from Becton Dickinson (Franklin Lakes, NJ, USA)

2.6. DNA Extraction

2.6.1. Crude Extract

Aliquots of 5 mL of Luria–Bertani broth (LB) were inoculated with the isolated bacteria 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 (RT). The bacterial pellet was resuspended in 1 mL of RNase and DNase-free deionized water and then heated at 94 °C for 10 min, followed by thermal shock in ice. Finally, the sample of lysed bacterial was kept at −80 °C until use.

2.6.2. Genomic DNA Extraction

Samples of 500 µL of overnight bacterial culture was subjected to genomic DNA isolation by using an RTP pathogen kit (Invitek, Berlin, Germany) following the manufacturer’s instructions. Briefly, a bacterial pellet was collected by centrifugation at 13,000 rpm for 5 min, suspended with 400 µL of resuspension buffer, and placed into an extraction L tube to be incubated at 37 °C for 10 min and switched to 95 °C for 10 min. Then, 400 µL of binding buffer were added and loaded onto the RTA spin filter set, incubated for one minute, and centrifuged at 9300× g for one minute, then the column was replaced into a new collector tube, loaded with 500 µL of wash R1 buffer, and centrifuged at 9300× g for a min. Again, the column was replaced into a new collector tube, loaded with 700 µL of wash R2 buffer and centrifuged at 9300× g for one minute; then, the column was replaced into a new collector tube and centrifuged at 12,000× g for 4 min. Finally, the column was replaced into a new 1.5 mL Eppendorf tube and 60 µL of elution buffer prewarmed to 80 °C was added, incubated for 3 min at RT, and centrifuged at 9300× g for 2 min. The eluted DNA solution was quantified by absorbance and its integrity was verified by 2%-agarose gel electrophoresis. The sample was kept at −20 °C until use.

2.7. Plasmid DNA Extraction

Samples of overnight bacterial culture (100 mL) were subjected to a plasmid DNA isolation by using PureLink HiPure Plasmid Midiprep Kit (Invitrogen; Carlsbad, CA, USA) following the manufacturer’s instructions. Briefly, the bacterial culture was harvested by centrifugation at 4000× g for 10 min, then 10 mL of resuspension buffer (R3) and 10 mL of lysis buffer (L7) were added to the bacterial pellet, and incubated at RT for 5 min. Then, 10 mL of precipitation buffer (N3) was added and centrifuged at 12,000× g for 10 min at RT. The supernatant was placed onto a pre-equilibrated maxi-column using 30 mL equilibrium buffer (EQ1) and eluted by gravity. Then, 60 mL of wash buffer (W8) was added, eluted by gravidity, and the eluent was discarded. Finally, 15 mL of elution buffer (E4) was added, eluted by gravity, and the eluent was added with 10.5 mL of isopropanol and centrifuged at 12,000× g for 30 min at 4 °C. The DNA pellet was washed with 5 mL of 70% ethanol and centrifuged at 12,000× g for 5 min at 4 °C. The DNA pellet was dried at RT for 10 min and finally resuspended in 250 µL of sterile RNase- and DNase-free water. The Plasmid DNA concentration was quantified by digital spectrophotometry (NanoDrop 8000, Thermo Fisher Scientific; Wilmington, DE, USA) and its integrity was verified by electrophoresis in 2% agarose gel, and the plasmid DNA sample was kept at −20 °C until use.

2.8. Endpoint PCR

The endpoint PCR to identify the β-Lactamase gene family was made using the primers sense and antisense described in Table 1. Amplification was made in 25 μL of the reaction mixture containing 2.5 μL of 10× PCR buffer (100 mM TRIS·HCI, 15 mM MgCl2, and 500 mM KCl, pH 8.3), 200 nM each dNTP, 10 μM each primer, 1 U Taq DNA polymerase (Invitrogen; Carlsbad, CA, USA), and 2 μL of crude extract or 10 ng of DNA (genomic or plasmid). The PCR conditions were 94 °C for 5 min, then 35 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, and finally 72 °C for 10 min. The PCR products were analyzed in 2% agarose gel electrophoresis and the image was digitized in a Gel Logistic 2200 Digital Imaging System (Carestream, New Haven, CT, USA).

2.9. Whole Genome Sequencing (WGS) and Genotype Analysis

The WGS was performed from indexed libraries that were prepared using a standard Illumina Nextera XT DNA Sample Preparation Kit (FC-131-1096) for small genomes and was 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) [30]. 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) [31] 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. Petesburg State University, Russia) [32].
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) [33]. The beta-lactamase genes were identified by the comparative analysis while using the Basic Local Alignment Search Tool (BLAST, NCBI-NIH, Bethesda, MD, USA) [34].

3. Results

The bioinformatics analysis included 1007 complete sequences of different genes encoding beta-lactamases reported in the Genbank, of which 936 sequences were used for primer design after discarding 71 repeated sequences (Table 1).
From the aligned sequences, we constructed fourteen phylogenetic (Figure 1), which allowed the identification of the more conserved regions for each family of beta-lactamase genes. These conserved sequences were used to design all primers that were used in the endpoint PCR reactions (Table 2) in order to identify the gene families of beta-lactamases that were carried by the contaminated bacteria.
From the sampling of five different salad brands sold in Mexico City and labelled as suitable for human consumption, 12 different Enterobacteriaceae strains from five bacterial species were isolated. The distribution of the identified bacteria was 8.3% of Enterobacter aerogenes (n = 1), 8.3% of Serratia fonticola (n = 1), 16.7% of Serratia marcescens (n = 2), 16.7% Klebsiella pneumoniae (n = 2), and 50% (n = 6) of the samples were identified as Enterobacter cloacae (Figure 2, Table 3). All of these data were confirmed by whole genome sequencing using NGS (Table 4)
In the antimicrobial susceptibility testing, 100% of the isolated Enterobacteriaceae strains showed resistance to ampicillin and carbenicillin, and 8.3% (n = 1) showed resistance to mezlocillin. The antibiotic resistance towards cephalosporins was as follows: 75% (n = 9) were resistant to cefazolin, 67% (n = 8) to cefaclor, 8.3% (n = 1) to cefotetan and 8.3% (n = 1) to cefoperazone showing medium resistance, and 100% were sensitive to cefepime. As to the beta-lactam and beta-lactamase inhibitor combination (sulbactam or tazobactam), 33% (n = 4) showed resistance and 33% (n = 4) medium resistance to ampicillin + sulbactam, and 100% were sensitive to piperacillin + tazobactam.
The end point PCR was able to identify the gene families with the phenotype that was responsible for beta-lactamase resistance (Figure 3) and showed that 91.7% (n = 11) of characterized Endobacteriaceae strains were positive to blaBIL, 91.7% (n = 11) to blaSHV, 83.3% (n = 10) to blaCTX, 75% (n = 9) for blaDHA, 33.3% (n = 4) to blaVIM, 16.7% (n = 2) to blaOXA, 16.7% (n = 2) to blaIMP, 8.3% (n = 1) to blaKPC and 8.3% (n = 1) to blaTEM (Table 4). All identified strains were negative for gene families to encode blaROB, blaCMY, blaP, blaCFX and blaLAP; these data were also confirmed by whole genome sequencing of isolated strains (Table 5 and Table 6).
The specific gene expression pattern was analyzed, identifying the following genotypes: Enterobacter cloacae (blaSHV-12, blaCTX-M-15, blaDHA-1, blaKPC-2); Serratia marcescens (blaSHV-1, blaCTX-M-3, blaDHA-1, blaVIM-2); Klebsiella pneumoniae (blaSHV-12, blaCTX-M-15, blaDHA-1); Serratia fonticola (blaSHV-12, blaVIM-1, blaDHA-1) and Enterobacter aerogene (blaSHV-1, blaCTX-M-1, blaDHA-1, blaVIM-2, blaOXA-9). However, in the NGS analysis, all of the bacterial strains tested were negative to blaBIL (Table 7).
The strains that were isolated from ready-to-eat vegetable salads showed a broad pattern of resistance to beta-lactam antibiotic. The analysis of the different families of gene responsible for this resistance pattern showed a mixed genotype of multiple gene families encoding beta-lactamase and were specific for each strain, conformed from three (blaVIM + blaSHV + blaBIL) to six (blaOXA + blaVIM + blaSHV + blaCTX + blaDHA + blaBIL) gene families of beta-lactamases (Table 4).

4. Discussion

The implementation of food safety measures has been shown to have a great cost–benefit impact in the developing world, decreasing healthcare costs by $25.5 USD for each $1.0 USD invested in hygiene measures [35]. To contribute to this condition, both government organisms and some food industry enterprises have implemented new technologies that are dedicated to providing products with a high quality and safety for human consumption. An example is the non-traditional agriculture techniques for growing hydroponic vegetables suitable for immediate consumption by humans as salads. Some studies have revealed that human beings are exposed to a wide range of bacterial species due to the consumption of fresh fruits and raw vegetables. The diversity of contaminant organisms has been shown to depend on the vegetables consumed and the kind of agricultural technique used for the food production [36]. There are reports of multi-resistant Enterobacteriaceae (evaluated by the Kirby–Bauer method) isolated from vegetables for human consumption [37]. This is why the intake of raw green salads frequently leads to GI disease outbreaks, especially in developing countries [37].
One molecular mechanism by which bacteria acquire resistance is the production of beta-lactamase enzymes that annihilate the bactericidal effect of beta-lactam drugs. Some Enterobacteriaceae, which are multi-resistant to a wide range of beta-lactam drugs that produce extended-spectrum beta-lactamases, classified as ESBL-producing Enterobacteriaceae, represent a major threat to human health. Usually, ESBL-producing Enterobacteriaceae are resistant to a wide variety of penicillins and cephalosporins, including broad-spectrum cephalosporins. The USA Health Agency reported 140,000 annual infections caused by Enterobacteriaceae during 2013, and 18% of these cases were resistant to beta-lactams that are associated to ESBL enzyme production, 12% of Enterobacteriaceae infections were caused by Klebsiella, and 6% by Escherichia coli, both of which are ESBL-producing. It has been estimated that multi-resistant ESBL-producing Enterobacteriaceae cause 1700 annual deaths in the USA. Patients with bacteremia caused by ESBL-producing Enterobacteriaceae have a rate of mortality 57% higher than that of patients with bacteremia caused by non-resistant Enterobacteriaceae [38,39]. Due to the severity of ESBL-producing Enterobacteriaceae infections and to the limited therapeutic options, the indicated medical treatment consists of the use of beta-lactam antibiotics from the Carbapenem family. However, recent reports have shown that Carbapenemase-producing Enterobacteriaceae (CPE) have spread all over the world during the last years—a situation that complicates their treatment. It has been estimated that 9300 infection cases are caused by carbapenem-resistant Enterobacteriaceae (CRE) each year, of which 7900 are associated to CRE–Klebsiella and 1400 to CRE–Escherichia coli. These infections are responsible for more than 600 deaths annually [36,37,38,39].
In this study, we isolated Enterobacteriaceae with high pathogenic potential from ready-to-eat vegetables that are sold in supermarkets located in Mexico City. The isolated Enterobacteriaceae were Enterobacter cloacae, Enterobacter aerogenes, Serratia fonticola, Serratia marcescens, and Klebsiella pneumoniae. All of the organisms isolated showed multi-resistance to beta-lactam antibiotics, tested by the Kirby–Bauer method. The antibiogram results showed resistance to penicillin and cephalosporin. Ampicillin is considered to be a broad-spectrum penicillin due to its use in gastrointestinal infections for some Enterobacteriaceae [40]. However, our study showed that all Enterobacteriaceae isolated were resistant to ampicillin. Carbenicillin is also an antibiotic in the treatment of Enterobacteriaceae infections, specifically against Pseudmonas spp. [41]. Our study also showed that the Enterobacteriaceae isolated were resistant to carbenicillin. The mezlocillin is mainly used against both Pseudomonas spp. and Klebsiella spp. [42] and, with the exception of one strain, all the Enterobacteriaceae isolated were sensitive to this antibiotic. In addition, 75% and 67% of all Enterobacteriaceae isolated were resistant to the antibiotic effect of cefazolin and cefaclor, respectively, and 99% showed medium sensitivity. All Enterobacteriaceae isolated were resistant to cefoperazone. The sensitivity to cefepime was not tested. Finally, 66% of isolated Enterobacteriaceae showed different degrees of resistance to ampicillin and beta-lactamase inhibitor combination, while 33% were resistant to sulbactam and 33% showed medium sensitivity. These data show—by the Kirby–Bauer method—that the isolated Enterobacteriaceae have a multi-resistance pattern to beta-lactam antibiotics, including penicillins and cephalosporins, and even were resistant to beta-lactamase inhibitors. These results are in agreement with work done on nitrogen-fixing legumes, where the pattern of multi-resistance was evaluated by a disk diffusion method for β-lactam antibiotics, with reported resistance to Amoxicillin, Ampicillin, Cefadroxil, Ceftriaxone, Oxacillin, and Vancomycin [43]. The food contamination of ready-to-eat salads distributed freely in Mexico City has the potential to become a trigger vector of an epidemic of gastrointestinal infection that is caused by multi-resistant Enterobacteriaceae, which could compromise the lives of patients at risk by their difficult treatment.
Our results with bacterial lysates or plasmid coincided with the identification of the gene families encoding beta-lactamases that are involved in the multi-resistance pattern to beta-lactam antibiotics. We demonstrated that 92% of all Enterobacteriaceae isolated carry at least one member of the beta-lactamase gene family that is associated with cephalosporin resistance, and that 100% were positive to the blaDHA gene family, which is responsible for cephalosporin resistance [44]. In addition, 91.7% of the Enterobacteriaceae isolated were positive to the blaSHV gene family associated with penicillin and cephalosporin resistance, also related to two grand phenomena of resistance: first, to ESBL-producing Enterobacteriaceae and resistance to beta-lactamase inhibitors [8,45,46]. The spectrum of ESBL-producing Enterobacteriaceae includes penicillin, broad-spectrum cephalosporins, and monobactams [46]. We demonstrated that 16.3% of the Enterobacteriaceae isolated carry a blaCTX gene family member, causing resistance to penicillin and broad spectrum cephalosporins, and this also was associated to ESBL-producing Enterobacteriaceae. We found that 8.3% of all strains carry the blaTEM gene family; in previous works, associates to ESBL-producing Enterobacteriaceae have been found [8]. The blaVIM gene family was present in 33.3% of Enterobacteriaceae isolated, blaIMP were in 16.7%, blaOXA in 16.7%, and blaKPC were positives in 8.3%, all of which have been associated with resistance to carbapenem [41,47,48]. These results indicate that the beta-lactam-resistant Enterobacteriaceae that were isolated from our samples of ready-to-eat vegetables have accumulated integrons with a different number of genes that confer a great variety of beta-lactam antibiotic resistance, including penicillins, oxacillins, cephalosporins, monobactams, carbapenems, and imipenems. However, the presence of a single family of gene-encoding beta-lactamase does not explain by itself the pan-resistance to beta-lactams. This is why the analysis of genotype was crucial to explain this phenomenon. In this study, the beta-lactam resistome drugs identified at least one member of the gene families encoding beta-lactam drugs. The resistome evidenced in different gram-negative Enterobacteriaceae isolated from ready-to-eat vegetables is integrated at least by one extended-spectrum beta-lactamase in combination with a specific beta-lactamase as cephalosporinases, cefotaximases, imipenemases, oxacillinases or carbapenemases. This paper reports the presence of beta-lactam resistome in ready-to-eat salads, sold in supermarkets in Mexico City, contaminated with carrier enterobacteria of resistance genes to last line beta-lactam antibiotics exclusively used in hospital patients.

5. Conclusions

All of these results together demonstrate that ready-to-eat vegetables for human intake in Mexico City are contaminated with potentially pathogenic multi-resistant Enterobacteriaceae; their intake might lead to GI diseases with a high difficulty of treatment. Therefore, food processing under strict safety measures and quality standards must be followed by food industries and supervised by the Health National Agency in order to avoid GI disease outbreaks. In addition, our results provide insight into the beta-lactam pan-resistance mechanism of Enterobacteriaceae. Our methodological approach can be implemented by food enterprises as a routine safety control of food processing.

Author Contributions

R.V.-L. and S.S.-G. conducted the microbiological studies and conventional antibiograms; B.A.L.-C. and M.R.T.-B. participated in sample collections and molecular biology analysis; T.G.-G. conducted the NGS sequencing; D.M.-F. and E.G.-C. provided support for the project design and valuable discussions; and J.A.G.-B. conceived the project, analyzed the data, and wrote the paper. All of the authors read and made comments on the manuscript during its drafting. All of the authors read and approved the final manuscript.

Funding

This work was supported by the National Council for Science and Technology (CONACYT) Mexico through the Sectoral Fund for Research in Health and Social Security (FOSISS), grant MOD.ORD./2012/SSA/IMSS/ISSSTE-CONACYT/000000000181384.

Acknowledgments

The authors thankfully acknowledge Samuel Gabriel Horta Mendoza (Head of Hospital Regional “Primero de Octubre”) and Tomás Barrientos (Faculty of Health Sciences University Anahuac) for providing the necessary laboratory facilities for the study.

Conflicts of Interest

The authors declare that they have no competing interests.

References

  1. Nguyen-the, C.; Carlin, F. The microbiology of minimally processed fresh fruits and vegetables. Crit. Rev. Food Sci. Nutr. 1994, 34, 371–401. [Google Scholar] [CrossRef] [PubMed]
  2. Sammarco, M.L.; Ripabelli, G.; Grasso, G.M. Consumer attitude and awareness towards food-related hygienic hazards. J. Food Saf. 1997, 17, 215–221. [Google Scholar] [CrossRef]
  3. Heit, J. Food Safety. University of Maryland Medical Center, 2015. Available online: http://umm.edu/health/medical/ency/articles/food-safety (accessed on 10 January 2018).
  4. Harris, L.J.; Farber, J.N.; Beuchat, L.R.; Parish, M.E.; Suslow, T.V.; Garrett, E.H.; Busta, F.F. Outbreaks associated with fresh produce: Incidence, growth, and survival of pathogens in fresh and fresh-cut produce. Compr. Rev. Food Sci. 2003, 2, 78–141. [Google Scholar] [CrossRef]
  5. Liu, L.; Johnson, H.L.; Cousens, S.; Perin, J.; Scott, S.; Lawn, J.E.; Rudan, I.; Campbell, H.; Cibulskis, R.; Li, M.; et al. Child Health Epidemiology Reference Group of WHO and UNICEF. Global, regional, and national causes of child mortality: An updated systematic analysis for 2010 with time trends since 2000. Lancet 2012, 379, 2151–2156. [Google Scholar] [CrossRef]
  6. Buys, H.; Muloiwa, R.; Bamford, C.; Eley, B. Klebsiella pneumoniae bloodstream infections at a South African children’s hospital 2006–2011, a cross-sectional study. BMC Infect. Dis. 2016, 16, 570. [Google Scholar] [CrossRef] [PubMed][Green Version]
  7. Caltagirone, M.; Nucleo, E.; Spalla, M.; Zara, F.; Novazzi, F.; Marchetti, V.M.; Piazza, A.; Bitar, I.; De Cicco, M.; Paolucci, S.; et al. Occurrence of Extended Spectrum β-Lactamases, KPC-Type, and MCR-1.2-Producing Enterobacteriaceae from Wells, River Water, and Wastewater Treatment Plants in Oltrepò Pavese Area, Northern Italy. Front. Microbiol. 2017, 8, 2232. [Google Scholar] [CrossRef] [PubMed]
  8. Kpoda, D.S.; Ajayi, A.; Somda, M.; Traore, O.; Guessennd, N.; Ouattara, A.S.; Sangare, L.; Traore, A.S.; Dosso, M. Distribution of resistance genes encoding ESBLs in Enterobacteriaceae isolated from biological samples in health centers in Ouagadougou, Burkina Faso. BMC Res. Notes 2018, 11, 471. [Google Scholar] [CrossRef] [PubMed]
  9. Chen, C.M.; Ke, S.C.; Li, C.R.; Wu, Y.C.; Chen, T.H.; Lai, C.H.; Wu, X.X.; Wu, L.T. High Diversity of Antimicrobial Resistance Genes, Class 1 Integrons, and Genotypes of Multidrug-Resistant Escherichia coli in Beef Carcasses. Microb. Drug Resist. 2017, 23, 915–924. [Google Scholar] [CrossRef] [PubMed]
  10. Bush, K.; Jacoby, G.A. Updated functional classification of beta-lactamases. Antimicrob. Agents Chemother. 2010, 54, 969–976. [Google Scholar] [CrossRef] [PubMed]
  11. Bush, K. The ABCD’s of β-lactamase nomenclature. J. Infect. Chemother. 2013, 19, 549–559. [Google Scholar] [CrossRef] [PubMed]
  12. Salahuddin, P.; Kumar, A.; Khan, A.U. Structure, Function of Serine and Metallo-β-lactamases and their Inhibitors. Curr. Protein Pept. Sci. 2018, 19, 130–144. [Google Scholar] [CrossRef] [PubMed]
  13. Bush, K. Past and Present Perspectives on β-Lactamases. Antimicrob. Agents Chemother. 2018, 62. [Google Scholar] [CrossRef] [PubMed]
  14. Jovetic, S.; Zhu, Y.; Marcone, G.L.; Marinelli, F.; Tramper, J. β-Lactam and glycopeptide antibiotics: First and last line of defence? Trends Biotechnol. 2010, 28, 596–604. [Google Scholar] [CrossRef] [PubMed]
  15. Wilke, M.S.; Lovering, A.L.; Strynadka, N.C.J. b-Lactam antibiotic resistance: A current structural perspective. Curr. Opin. Microbiol. 2005, 8, 525–533. [Google Scholar] [CrossRef] [PubMed]
  16. Barnaud, G.; Arlet, G.; Danglot, C.; Philippon, A. Cloning and sequencing of the gene encoding the AmpC beta-lactamase of Morganella morganii. FEMS Microbiol. Lett. 1997, 148, 15–20. [Google Scholar] [CrossRef]
  17. Bush, K.; Jacoby, G.A.; Medeiros, A.A. A functional classification scheme for beta-lactamases and its correlation with molecular structure. Antimicrob. Agents Chemother. 1995, 39, 1211–1233. [Google Scholar] [CrossRef] [PubMed][Green Version]
  18. Bebrone, C. Metallo-beta-lactamases (classification, activity, genetic organization, structure, zinc coordination) and their superfamily. Biochem. Pharmacol. 2007, 74, 1686–1701. [Google Scholar] [CrossRef] [PubMed]
  19. Parker, A.C.; Smith, C.J. Genetic and biochemical analysis of a novel Ambler class A beta-lactamase responsible for cefoxitin resistance in Bacteroides species. Antimicrob. Agents Chemother. 1993, 37, 1028–1036. [Google Scholar] [CrossRef] [PubMed]
  20. Queenan, A.M.; Bush, K. Carbapenemases: The versatile beta-lactamases. Clin. Microbiol. Rev. 2007, 20, 440–458. [Google Scholar] [CrossRef] [PubMed]
  21. Walsh, T.R.; Toleman, M.A.; Poirel, L.; Nordmann, P. Metallo-beta-lactamases: The quiet before the storm? Clin. Microbiol. Rev. 2005, 18, 306–325. [Google Scholar] [CrossRef] [PubMed]
  22. Jacoby, G.A. AmpC β-Lactamases. Clin. Microbiol. Rev. 2009, 22, 161–182. [Google Scholar] [CrossRef] [PubMed]
  23. DiBonaventura, M.D.; Meincke, H.; Le Lay, A.; Fournier, J.; Bakker, E.; Ehrenreich, A. Obesity in Mexico: Prevalence, comorbidities, associations with patient outcomes, and treatment experiences. Diabetes Metab. Syndr. Obes. 2017, 11, 1–10. [Google Scholar] [CrossRef] [PubMed]
  24. Dhroove, G.; Saps, M.; Garcia-Bueno, C.; Leyva Jiménez, A.; Rodriguez-Reynosa, L.; Velasco-Benítez, C.A. Prevalence of functional gastrointestinal disorders in Mexican schoolchildren. Rev. Gastroenterol. Mex. 2017, 82, 13–18. [Google Scholar] [CrossRef] [PubMed]
  25. Larkin, M.A.; Blackshields, G.; Brown, N.P.; Chenna, R.; McGettigan, P.A.; McWilliam, H.; Valentin, F.; Wallace, I.M.; Wilm, A.; Lopez, R.; et al. Clustal W and Clustal X version 2.0. Bioinformatics 2007, 23, 2947–2948. [Google Scholar] [CrossRef] [PubMed][Green Version]
  26. Thompson, J.D.; Higgins, D.G.; Gibson, T.J. CLUSTAL W: Improving the sensitivity of progressive multiple sequence weighting, position specific gap penalties and weighting matrix chose. Nucl. Acids Res. 2014, 22, 4673–4680. [Google Scholar] [CrossRef]
  27. Martínez-Pérez, F.; Becerra, A.; Valdés, J.; Zinker, S.; Aréchiga, H. A possible molecular ancesor for mollussk APGWamide, insect adipokinetic hormone, and crustacean red pigment concentrating hormone. J. Mol. Evol. 2002, 54, 703–714. [Google Scholar] [CrossRef] [PubMed]
  28. Martínez-Pérez, F.; Bendena, W.G.; Chang, B.S.; Tobe, S.S. FGLamide Allatostatin gene in Arthropoda: Introns early or late? Peptides 2009, 30, 1241–1248. [Google Scholar] [CrossRef] [PubMed]
  29. Marshall, O.J. PerlPrimer: Cross-platform, graphical primer design for standard, bisulphite and real-time PCR. Bioinformatics 2004, 20, 2471–2472. [Google Scholar] [CrossRef] [PubMed]
  30. FastQC. Available online: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ (accessed on 10 September 2018).
  31. BWA-MEM. Available online: http://sourceforge.net/p/bio-bwa/mailman/message/30894287/ (accessed on 10 September 2018).
  32. SAMtools. Available online: http://www.codoncode.com/aligner/ (accessed on 10 September 2018).
  33. Kraken Taxonomic Sequence Classification System. Available online: https://ccb.jhu.edu/software/kraken/ (accessed on 10 September 2018).
  34. Basic Local Alignment Search Tool. Available online: https://blast.ncbi.nlm.nih.gov/Blast.cgi/ (accessed on 10 September 2018).
  35. Hutton, G.; Haller, L.; Bartram, J. Global cost-benefit analysis of water supply and sanitation interventions. J. Water Health 2007, 5, 481–502. [Google Scholar] [CrossRef] [PubMed][Green Version]
  36. Boehme, S.; Werner, G.; Klare, I.; Reissbrodt, R.; Witte, W. Occurrence of antibiotic resistant enterobacteria in agricultural foodstuffs. Mol. Nutr. Food Res. 2004, 48, 522–531. [Google Scholar] [CrossRef] [PubMed]
  37. Hamilton-Miller, J.M.; Shah, S. Identity and antibiotic susceptibility of enterobacterial flora of salad vegetables. Int. J. Antimicrob. Agents 2001, 18, 81–83. [Google Scholar] [CrossRef]
  38. Schmiedel, J.; Falgenhauer, L.; Domann, E.; Bauerfeind, R.; Prenger-Berninghoff, E.; Imirzalioglu, C.; Chakraborty, T. Multiresistant extended-spectrum beta-lactamase-producing Enterobacteriaceae from humans, companion animals and horses in central Hesse, Germany. BMC Microbiol. 2014, 14, 187. [Google Scholar] [CrossRef] [PubMed]
  39. Philippon, A.; Arlet, G.; Jacoby, G.A. Plasmid-determined AmpC-type beta-lactamases. Antimicrob. Agents Chemother. 2002, 46, 1–11. [Google Scholar] [CrossRef] [PubMed]
  40. Gilman, R.H.; Koster, F.; Islam, S.; McLaughlin, J.; Rahaman, M.M. Randomized trial of high- and low-dose ampicillin therapy for treatment of severe dysentery due to Shigella dysenteriae type 1. Antimicrob. Agents Chemother. 1980, 17, 402–405. [Google Scholar] [CrossRef] [PubMed]
  41. Rezaei, F.; Saderi, H.; Boroumandi, S.; Faghihzadeh, S. Relation between Resistance to Antipseudomonal β-Lactams and ampC and mexC Genes of Pseudomonas aeruginosa. Iran. J. Pathol. 2016, 11, 47–53. [Google Scholar] [PubMed]
  42. Lauretti, L.; Riccio, M.L.; Mazzariol, A.; Cornaglia, G.; Amicosante, G.; Fontana, R.; Rossolini, G.M. Cloning and characterization of blaVIM, a new integron-borne metallo-beta-lactamase gene from a Pseudomonas aeruginosa clinical isolate. Antimicrob. Agents Chemother. 1999, 43, 1584–1590. [Google Scholar] [CrossRef] [PubMed]
  43. Rangel, W.M.; de Oliveira Longatti, S.M.; Ferreira, P.A.A.; Bonaldi, D.S.; Guimarães, A.A.; Thijs, S.; Weyens, N.; Vangronsveld, J.; Moreira, F.M.S. Leguminosae native nodulating bacteria from a gold mine As-contaminated soil: Multi-resistance to trace elements, and possible role in plant growth and mineral nutrition. Int. J. Phytoremediat. 2017, 19, 925–936. [Google Scholar] [CrossRef] [PubMed]
  44. Sievert, D.M.; Ricks, P.; Edwards, J.R.; Schneider, A.; Patel, J.; Srinivasan, A.; Kallen, A.; Limbago, B.; Fridkin, S. National Healthcare Safety Network (NHSN) Team and Participating NHSN Facilities. Antimicrobial-resistant pathogens associated with healthcare-associated infections: Summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2009–2010. Infect. Control Hosp. Epidemiol. 2013, 34, 1–14. [Google Scholar] [CrossRef] [PubMed]
  45. Centers for Disease Control and Prevention (CDC). Vital Signs: Carbapenem-resistant Enterobacteriaceae. MMWR Morb. Mortal. Wkly. Rep. 2013, 62, 165–170. Available online: https://www.cdc.gov/mmwr/preview/mmwrhtml/mm6209a3.htm (accessed on 10 September 2018). [Google Scholar]
  46. Ye, Q.; Wu, Q.; Zhang, S.; Zhang, J.; Yang, G.; Wang, J.; Xue, L.; Chen, M. Characterization of Extended-Spectrum β-Lactamase-Producing Enterobacteriaceae from Retail Food in China. Front. Microbiol. 2018, 9, 1709. [Google Scholar] [CrossRef] [PubMed]
  47. Laolerd, W.; Akeda, Y.; Preeyanon, L.; Ratthawongjirakul, P.; Santanirand, P. Carbapenemase-Producing Carbapenem-Resistant Enterobacteriaceae from Bangkok, Thailand, and Their Detection by the Carba NP and Modified Carbapenem Inactivation Method Tests. Microb. Drug Resist. 2018, 24, 1006–1011. [Google Scholar] [CrossRef] [PubMed]
  48. Walther-Rasmussen, J.; Høiby, N. OXA-type carbapenemases. J. Antimicrob. Chemother. 2006, 57, 373–383. [Google Scholar] [CrossRef] [PubMed][Green Version]
Figure 1. Phylogenetic analysis of the gene family that confer beta-lactam resistance: all sequences were downloaded from NCBI GeneBankt and aligned in ClustalW. The phylogenetic trees were edited in FigTree v 1.4.0. All software was running in a Debian (Stretch) machine.
Figure 1. Phylogenetic analysis of the gene family that confer beta-lactam resistance: all sequences were downloaded from NCBI GeneBankt and aligned in ClustalW. The phylogenetic trees were edited in FigTree v 1.4.0. All software was running in a Debian (Stretch) machine.
High throughput 07 00036 g001
Figure 2. Bacterial frequency. The graph shows the percentage of different bacteria species isolated from sample ready-to-eat salad greens sold in supermarkets placed in Mexico City.
Figure 2. Bacterial frequency. The graph shows the percentage of different bacteria species isolated from sample ready-to-eat salad greens sold in supermarkets placed in Mexico City.
High throughput 07 00036 g002
Figure 3. End point polymerase chain reaction for the identification of beta-lactamase gene families. 3% agarosa gel electrophoresis showing the positives amplicons for gene families encoding beta-lactamase, MW = marker (100 bp), blaVIM (133 bp), blaSHV (106 and 141), blaP (204 bp), blaIMP (183 and 192 bp), blaCTX (116, 83 or 226 bp), blaCMY (123 or 160 bp), blaDHA (200, 147 or 117), blaTEM (213), blaCFX (177 bp), blaBIL (128 bp), blaKPC (117 bp), blaOXA (114 bp), blaROB (126 bp), and blaLAP (126 bp).
Figure 3. End point polymerase chain reaction for the identification of beta-lactamase gene families. 3% agarosa gel electrophoresis showing the positives amplicons for gene families encoding beta-lactamase, MW = marker (100 bp), blaVIM (133 bp), blaSHV (106 and 141), blaP (204 bp), blaIMP (183 and 192 bp), blaCTX (116, 83 or 226 bp), blaCMY (123 or 160 bp), blaDHA (200, 147 or 117), blaTEM (213), blaCFX (177 bp), blaBIL (128 bp), blaKPC (117 bp), blaOXA (114 bp), blaROB (126 bp), and blaLAP (126 bp).
High throughput 07 00036 g003
Table 1. Gene families encoding beta-lactamases used in the phylogenetic analysis and primers design.
Table 1. Gene families encoding beta-lactamases used in the phylogenetic analysis and primers design.
Gen FamilySequencesDiscardedAnalyzedDiscarded (%)Analyzed (%)
blaOXA32293132.8097.20
blaVIM1862915713.2471.68
blaSHV12931262.3297.67
blaTEM1029938.8291.18
blaIMP1005955.0095.00
blaROB161156.2593.75
blaKPC332316.0693.94
blaCTX312296.4593.55
blaCMY285232.8797.13
blaDHA211204.7695.24
blaP161156.2593.75
blaCFX91811.1188.89
blaLAP103730.0070.00
blaLAT3030.00100.00
blaBIL1010.00100.00
Table 2. Primers sequences. Each primer set* was designed to identify all the members of beta-lactamase gene family reported in the GenBank during the 2015 year.
Table 2. Primers sequences. Each primer set* was designed to identify all the members of beta-lactamase gene family reported in the GenBank during the 2015 year.
Gene FamilyPrimer NamePrimer Sequence (5′ to 3′)Tm (°C)PositionAmplicon (bp)
blaOXABlaOXA-FWGGTTTCGGTAATGCTGAAATTGG61.18214–236114
BlaOXA-RWGCTGTGTATGTGCTAATTGGGA61.19327–306
blaVIMBlaVIM-FWCGACAGTCARCGAAATTCC61.39105–123133
BlaVIM-RWCAATGGTCTSATTGTCCGTG61.34238–219
blaSHVBlaSHV-FW1CGTAGGCATGATAGAAATGGATC61.04133–155106
BlaSHV-RW1CGCAGAGCACTACTTTAAAGG61.33239–218
BlaSHV-FW2GCCTCATTCAGTTCCGTTTC61.62399–418141
BlaSHV-RW2CCATTACCATGAGCGATAACAG61.22540–518
blaTEMBlaTEM-FWGCCAACTTACTTCTGACAACG61.801699–1719213
BlaTEM-RWCGTTTGGAATGGCTTCATTC60.131912–1892
blaIMPBlaIMP-FW1GGAATAGARTGGCTTAAYTCTCG60.92319–332183
BlaIMP-RW1CYASTASGTTATCTKGAGTGTG62.45502–480
BlaIMP-FW2GGTGGAATAGARTGGCTTAAYTC61.11316–339192
BlaIMP-RW2CCAAACCACTACGTTATCTKGAG61.29508–485
blaROBBlaROB-FWCCAACATCGTGGAAAGTGTAG61.27718–739126
BlaROB-RWGTAAATTGCGTACTCATGATTGC60.90844–821
blaKPCBlaKPC-FWGCTAAACTCGAACAGGACTTTG61.79100–121117
BlaKPC-RWCTTGAATGAGCTGCACAGTG61.90216–197
blaCTXBlaCTX-FW1GATACCGCAGATAATACGCAG60.79161–181116
BlaCTX-RW1CGTTTTGCGTTTCACTCTG60.28276–258
BlaCTX-FW2GCTGATTCTGGTCACTTACTTC61.02789–81083
BlaCTX-RW2CGCCGACGCTAATACATC60.69855–872
BlaCTX-FW3CTGCTTAACTACAATCCSATTGC62.17314–336226
BlaCTX-RW3GGAATGGCGGTATTKAGC60.86539–522
blaCMYBlaCMY-FW1GTTTGAGCTAGGATCGGTTAG60.25337–357123
BlaCMY-RW1CTGTTTGCCTGTCAGTTCTG61.48460–441
BlaCMY-FW2GAACGAAGGCTACGTAGCT61.71213–231160
BlaCMY-RW2CTGAAACGTGATTCGATCATCA61.08372–351
blaDHABlaDHA-FW1GCATATTGATCTGCATATCTCCAC61.60399–422200
BlaDHA-RW1GCTGCTGTAACTGTTCTGC61.62598–580
BlaDHA-FW2GCGGATCTGCTGAATTTCTATC61.54464–485147
BlaDHA-RW2GCAGTCAGCAACTGCTCATAC61.05610–591
BlaDHA-FW3GTAAGATTCCGCATCAAGCTG61.74430–450117
BlaDHA-RW3GGGTTATCTCACACCTTTATTACTG61.08546–522
blaPBlaP-FWGGAGAATATTGGGATTACAATGGC61.74271–294204
BlaP-RWCGCATCATCGAGTGTGATTG61.80474–455
blaCFXBlaCFX-FWCCAGTCATATCATTGACAGTGAG60.86437–459177
BlaCFX-RWGACATTTCCTCTTCCGTATAAGC61.16613–591
blaLAPBlaLAP-FWAGGGCTTGAACAACTTGAAC61.07249–268126
BlaLAP-RWGTAATGGCAGCATTGCATAAC60.59374–354
blaBILBlaBIL-FWGCCGATATCGTTAATCGCAC61.65100–119128
BlaBIL-RWGTTATTGGCGATATCGGCTTTA60.98227–206
* All primers were designed in PerlPrimer v1.1.21 running under Debian 8 OS.
Table 3. Characteristics of whole genome sequencing of the bacteria isolated form of roads cultured from ready-to-eat vegetables.
Table 3. Characteristics of whole genome sequencing of the bacteria isolated form of roads cultured from ready-to-eat vegetables.
BacteriaCDSNumber of Sequence ContigsAssembled Genome Size (bp)Reported Genomic Size (bp)Genomic Size Difference
Enterobacter cloacae454514844,982,1764,772,910209,266
Serratia marcescens659688895,681,2105,241,455439,755
Klebsiella pneumoniae50715835,479,1735,315,120164,053
Serratia fonticola59459166,483,0436,000,511482,532
Enterobacter aerogenes454514845,578,7245,280,350298,374
Table 4. Frequency of beta-lactamase gene families identified by end point PCR in roads cultured from ready-to-eat vegetables.
Table 4. Frequency of beta-lactamase gene families identified by end point PCR in roads cultured from ready-to-eat vegetables.
Gen FamilynFrequency
blaOXA216.7
blaVIM433.3
blaSHV1191.6
blaTEM18.3
blaIMP216.6
blaKPC18.3
blaCTX1083.3
blaDHA975.0
blaBIL1191.6
blaROB00.0
blaCMY00.0
blaP00.0
blaCFX00.0
blaLAP00.0
Table 5. Beta-lactamase gen families identified by end point PCR in roads cultured from ready-to-eat vegetables.
Table 5. Beta-lactamase gen families identified by end point PCR in roads cultured from ready-to-eat vegetables.
BacteriaCarried Beta-Lactamases Gene FamiliesN
blaSHVblaCTXblaDHAblaBILblaKPCblaVIMblaIMPblaOXAblaTEM
Klebsiella pneumoniae+++1
Klebsiella pneumoniae+++++1
Enterobacter cloacae+++1
Enterobacter cloacae++++1
Enterobacter cloacae++++2
Enterobacter cloacae+++++1
Enterobacter cloacae+++++1
Enterobacter aerogene++++++1
Serratia fonticola++++1
Serratia marcescens+++1
Serratia marcescens+++++1
Table 6. Metagenomic identification of the isolated bacteria form of roads cultured from ready-to-eat vegetables.
Table 6. Metagenomic identification of the isolated bacteria form of roads cultured from ready-to-eat vegetables.
NGS CharacteristicsEnterobacter CloacaeSerratia MarcescensKlebsiella PneumoniaeSerratia [1 Fonticola]Enterobacter Aerogenes
Total Reads4,714,939241,0721,523,8213,848,5551,331,424
Classified Reads4,541,943 (96.33%)183,689 (76.20%)1,434,672 (94.15%)3,680,364 (95.63%)1,200,101 (90.14%)
Domain4,438,740 (94.14%)182,241 (75.60%)1,434,058 (94.11%)3,679,637 (95.61%)1,198,484 (90.02%)
Phylum4,401,268 (93.35%)179,018 (74.26%)1,427,130 (93.65%)3,678,453 (95.58%)1,193,110 (89.61%)
Class4,397,652 (93.27%)177,491 (73.63%)1,425,587 (93.55%)3,619,438 (94.05%)1,190,902 (89.45%)
Order4,394,905 (93.21%)178,775 (74.16%)1,419,060 (93.13%)3,609,215 (93.78%)1,188,531 (89.27%)
Family4,395,029 (93.21)178,735 (74.14%)1,419,175 (93.13%)3,603,190 (93.62%)1,188,519 (89.27%)
Gender1,730,480 (36.70%)176,087 (73.04%)1,348,898 (88.52%)3,600,628 (93.56%)1,141,985 (85.77%)
Species1,685,267 (35.74%)170,381 (70.68%)1,219,856 (80.05%)3,572,513 (92.83%)1,123,080 (84.35%)
Table 7. Genotype of the beta-lactama antibiotic resistome identified by next generation sequencing in roads cultured from ready-to-eat vegetables.
Table 7. Genotype of the beta-lactama antibiotic resistome identified by next generation sequencing in roads cultured from ready-to-eat vegetables.
BacteriaBeta-Lactamases Families
SHVCTXDHABILKPC/VIM/IMPOXA
Enterobacter cloacaeblaSHV-12blaCTX-M-15blaDHA-1blaBIL ***blaKPC-2
Serratia marcescensblaSHV-1blaCTX-M-3blaDHA-1blaBIL ***blaVIM-2
Klebsiella pneumoniaeblaSHV-12blaCTX-M-15blaDHA-1blaBIL ***blaIMP ***
Serratia fonticolablaSHV-12blaVIM-1blaDHA-1blaBIL ***
Enterobacter aerogeneblaSHV-1blaCTX-M-1blaDHA-1blaBIL ***blaVIM-2blaOXA-9
*** No member of this gene family was found in the bioinformatic analysis of WGS.

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
High-Throughput EISSN 2571-5135 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
Back to Top