https://doi.org/10.5812/jjm.83711

Distribution of Virulence and Antimicrobial Resistance Genes in Phylogenetic Groups of Escherichia coli Strains Isolated from Mexican Patients with Urinary Infection

authors:

avatar Juan Carlos Bravata-Alcantara ORCID 1 , avatar Juan Manuel Bello-Lopez ORCID 2 , avatar Iliana Alejandra Cortes-Ortiz ORCID 3 , avatar Juan Jose Mendez-Velazquez ORCID 4 , avatar Brandon Aviles-Soto ORCID 5 , avatar Laura Itzel Quintas-Granados 6 , avatar Sonia del Carmen Chavez-Ocaña 1 , avatar Cecilia Rosel-Pech ORCID 7 , avatar Juan Antonio Gonzalez-Barrios 8 , avatar Monica Sierra Martinez ORCID 1 , *

Genetics and Molecular Diagnosis Laboratory, Juárez de Mexico Hospital, Mexico City, Mexico
Research Unit in Microbiology and Toxicology, Juárez de Mexico Hospital, Mexico City, Mexico
Bacteriology Laboratory, Juárez de Mexico Hospital, Mexico City, Mexico
Zaragoza School of Higher Studies, National Autonomous University of Mexico, Mexico City, Mexico
Technological University of Tecámac, Tecámac, Mexico
Mexiquense del Bicentenario University, Higher Studies Unit of Tultitlán, Mexico City, Mexico
Medical Research Unit in Immunology and Infectious Diseases, Hospital of Infectology Daniel Méndez Hernández, la Raza National Medical Center, Mexico City, Mexico
Laboratory of Genomic Medicine, Regional Hospital, ISSSTE, Mexico City, Mexico
Jundishapur Journal of Microbiology: Vol.12, issue 3; e83711
published online: February 27, 2019
article type: Research Article
received: August 29, 2018
revised: January 9, 2019
accepted: January 16, 2019

How To Cite Bravata-Alcantara J C , Bello-Lopez J M, Cortes-Ortiz I A, Mendez-Velazquez J J, Aviles-Soto B, et al. Distribution of Virulence and Antimicrobial Resistance Genes in Phylogenetic Groups of Escherichia coli Strains Isolated from Mexican Patients with Urinary Infection. Jundishapur J Microbiol. 2019;12(3):e83711. https://doi.org/10.5812/jjm.83711.

Abstract

Background:

The causative primary agent of urinary tract infections (UTI) is uropathogenic Escherichia coli (UPEC); however, commensal Escherichia coli (CEC) has been also implicated in the development of UTI. Due to the recent emergence of virulent and resistant strains, it is necessary to have evidence to demonstrate that UPEC and CEC are isolated from patients with UTI and subsequently they have the genes to be considered pathogenic.

Objectives:

To determine the distribution of resistance and virulence genes in UPEC and CEC strains isolated from the patients with UTI (hospitalized and ambulatory).

Methods:

One hundred seven E. coli strains were genotyped according to Clermont protocol and were subjected to PCR assays in order to detect resistance and virulence genes. In addition, the antimicrobial test in solid media was performed to determinate the correlation “pheno/genotype”.

Results:

Genotyping analysis showed that group B2 (42.05%) was the most predominant, strains followed by A (27.1%), D (24.29%), and finally, B1 (6.54%). β-lactams, carbapenems, aminoglycosides, and nitrofurans showed the best activity. The virulence gene frequencies were: fimH (92/85.98%), iutA (68/63.55%), traT (66/61.68%), papC (36/33.64%), and cnf1 (15/14.02%) while resistance genes were: blaCTX-M(41/38.32%), blaOXA (49/45.79%), and blaSHV (2/1.87%). The associations fimH/blaCTX, fimH/blaOXA, traT/blaCTX, traT/blaOXA, iutA/blaCTX, and iutA/blaOXA showed higher incidence while the associations with cnf1 and papC genes were low.

Conclusions:

Uropathogenic E. coli has the necessary genetic elements (virulence and resistance) to be considered the main pathogen causing UTI in the Mexican population. Additionally, to our knowledge, there are no studies in our country demonstrating that the CEC isolated from the patients with UTI contains genetic elements of virulence and resistance that allow them to be potentially pathogenic.

1. Background

Urinary tract infections (UTI) are the most common bacterial infections worldwide, affecting 150 million people every year and can be involved in any structure of the urinary system such as the urethra, bladder, ureters, and kidney. They are considered the Major cause of ≈ 10 million healthcare visits each year (1). Both genders might be affected by UTI; however, the female population is the most susceptible to be infected due to the closeness of anal and vaginal sites that can result in at least one UTI episode during their lives (2). The epidemiological data for 2016 by the Mexican Health Department showed that the female gender is the main population affected by urinary tract infections (3). These infections caused serious consequences such as high morbidity in a vulnerable population, pyelonephritis, renal damage, preterm birth, and complications by irrational use of antimicrobials (4).

High prevalence, recurrence, and severe health effects resulted in high costs of treatment; therefore, UTI is considered a public health problem (5). Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterococcus faecalis, and Staphylococcus saprophyticus are the main pathogens causing UTI (6, 7). Moreover, uropathogenic E. coli (UPEC) has been responsible for more than 85% of UTIs (8). Uropathogenic E. coli pathogenesis begins with periurethral contamination, followed by urethral colonization and subsequent bacterial migration to the bladder (1). Several virulence factors e.g. pili associated-adhesins, including FIC, P, S, type I pili, and PapC have been recognized as receptors in the bladder epithelium and are involved in the colonization event (9-17). Also, host cell colonization and invasion are mediated by the FimH adhesin, localized in the tip of type I fimbriae, due to its capability to bind to mannosylated receptors in the bladder epithelium (18).

Moreover, UPEC synthetizes the cytotoxic necrotizing factor type 1 (CNF1) to increase its capability to access deeper tissue and to persist in the lower urinary tract (19). In recent years, the effectiveness of UTI treatment has become difficult due to the emergence of multidrug-resistant pathogens. In fact, the wide-spectrum β-lactamases (ESBL) are responsible for multidrug-resistance in a large number of genera and species of the Enterobacteriaceae family. Uropathogenic E. coli carries blaSHV and blaTEM genes that are involved in antimicrobial resistance and have been mainly reported as conjugative plasmids (20). Also, blaCTX-M gene encodes an enzyme that hydrolyzes cefotaxime and ceftazidime, and its importance in pathogen resistance has been demonstrated (21). Furthermore, E. coli can produce a carbapenemase (encoded in blaOXA gene) allowing bacteria to confer resistance to carbapenems (22).

2. Objectives

This study was conducted in order to know the genetic distribution and relation of virulence and antimicrobial resistance genes in E. coli strain. In other words, the aim of this work was to identify virulence and resistance genes in UPEC and CEC isolated from patients with UTI in hospitalized and ambulatory patients in Mexico.

3. Methods

3.1. Strains Origin, Identification, and Antimicrobial Resistance Assay

One hundred seven E. coli strains were isolated from ambulatory and hospitalized diagnosed patients with UTI at the “Hospital Juárez de México” (HJM), from April 2016 to January 2017. Urine samples (obtained in the second urine streams) were streaked on Mac Conkey agar and were incubated aerobically at 37°C for 24 - 48 hours. Typical colonies of E. coli were selected and purified on LB agar and were cultured in LB-broth and frozen in glycerol at -70°C. The identification of strains and the antimicrobial susceptibility were performed by using BD Phoenix™ automated identification and susceptibility testing system according to the manufacturer’s protocols.

Antimicrobial susceptibility was performed for 16 antimicrobial agents (according to the microorganism identified “Gram-negative”), by using amoxicillin/clavulanic acid (AMC, 20/10 µg), ampicillin (AM, 10 µg), cefoxitin (FOX, 30 µg), piperacillin/tazobactam (TZP, 100/10 µg), ertapenem (ETP, 10 µg), imipenem (IPM, 10 µg), meropenem (MEM, 10 µg), cefazolin (CZ, 30 µg), cefepime (FEP, 30 µg), cefotaxime (CTX, 30 µg), ceftriaxone (CRO, 30 µg), amikacin (AN, 30 µg), gentamicin (GM, 10 µg), levofloxacin (LVX, 5 µg), nitrofurantoin (FM, 300 µg), and trimethoprim/sulfamethoxazole (SXT, 23.75/1.25 µg). Antibiotic resistance was calculated and represented in percentage (%). Antimicrobial resistance to different antibiotics was confirmed using the disk diffusion method on Mueller-Hilton agar plates according to the guidelines set by “The Clinical and Laboratory Standards Institute (CLSI 100-S21)”. Escherichia coli ATCC 25922 was used as the control.

3.2. Bacterial Phylogenetic Assignment

All the amplification reactions were performed in a SEEAMP™ PCR system (Seegene Inc. Korea). DNA was extracted by using the Qiagen Mini kit (Qiagen, Courtaboeuf, France). PCR reactions were carried out in a total volume of 20 µL with 200 ng of DNA, 10 pmol of each primers pair, 10 mM dNTP’s, 1.5 mM MgCl2, 1X PCR Taq buffer, and 0.25 U of Taq DNA polymerase (Invitrogen). Phylogenetic assignment was performed by using the protocol described by Clermont et al., with modifications (23, 24). The assignment of the criteria for the E. coli phylogenetic analysis was carried out by the amplification of 4 genes (chuA, yjaA, TSPE4.C2, and arpA) corresponding to one of the four phylogenetic groups (A, B1, B2, and D, respectively). Primers for PCR amplification assays are shown in Table 1. Specificity of the primers, a bioinformatic analysis was performed by using MFEprimer-2.0 server available at http://biocompute.bmi.ac.cn/CZlab/MFEprimer-2.0 (25). PCR multiplex conditions for gene fragments were performed as follows: 4 minutes at 94°C followed by up to 30 cycles of 94°C for 5 seconds, 59°C for 20 seconds, 72°C for 1 minutes, and finally, 72°C for 5 minutes. E. coli LMM36-ULA (chuA+, yjaA+) and LMM32-ULA (TSPE4.C2+) strains were used as positive controls.

Table 1.

Primers Used in This Study

GeneCategorySequenceaSize, bp
ForwardReverse
Phylogenetic assignment
chuAHemin uptake system5’-TGCCGCCAGTACCAAAGACA-3’5’-GACGAACCAACGGTCAGGAT-3’279
yjaAUnknow5’-TGAAGTGTCAGGAGACGCTG-3’5’-ATGGAGAATGCGTTCCTCAAC-3’211
TSPE4.C2Anonymous DNA fragment5’-GAGTAATGTCGGGGCATTCA-3’5’-CGCGCCAACAAAGTATTACG-3’152
arpAAnkyrin-like regulatory protein5’-AACGCTATTCGCCAGCTTGC-3’5’-TCTCCCCATACCGTACGCTA-3’400
Virulence factors
traTSerum resistance associated5’-GGTGTGGTGCGATGAGCACAG-3’5’-CACGGTTCAGCCATCCCTGAG-3’290
fimHAdhesin5’-TCGAGAACGGATAAGCCGTGG-3’5’-GCAGTCACCTGCCCTCCGGTA-3’508
iutAAerobactin siderophore5’-GGCTGGACATCATGGGAACTGG-3’5’-CGTCGGGAACGGGTAGAATCG-3’300
papCPyelonephritis associated pili5’-ATATCCTTTCTGCAGGGATGCAATA-3’5’-GTGGCAGTATGAGTAATGACCGTT-3’200
cnf1Cytotoxic necrotizing factor 15’-TCACGGGAATGAACTTATCACCC-3’5’-GTGACATGGCAAAATGATTACAGC-3’498
Antimicrobial resistance
blaCTX-MAntimicrobial resistance5’-GACGATGTCACTGGCTGAGCTTAGC-3’5’-AGCCGCCGACGCTAATACA-3’499
blaOXA5’-GGCACCAGATTCAACTTTCAAG-3’5’-GACCCCAAGTTTCCTGTAAGTG-3’564
blaSHV5’-GATGAACGCTTTCCCATGATG-3’5’-CGCTGTTATCGCTCATGGTAA-3’214

aPrimers design was performed according to previous reports (26-30).

3.3. Virulence and Resistance Genes Detection

Virulence genes fimH, traT, cnf1, iutA, and papC were detected by multiplex PCR in a volume of 20 µL with minor modifications (Table 1). PCR conditions for virulence genes were: 4 minutes at 94°C followed by up to 30 cycles of 94°C for 1 minutes, 59°C for 10 seconds, 72°C for 30 seconds, and finally, 72°C for 5 minutes. Furthermore, antibiotics resistance genes (blaCTX-M, blaOXA, and blaSHV) were amplified under the following conditions: 4 minutes at 94°C followed by up to 30 cycles of 94°C for 1 minute, 59°C for 10 seconds, 72°C for 30 seconds, and finally, 72°C for 5 minutes. PCR products were analyzed by electrophoresis in agarose gels (2%) for 1 hour at 60 V/cm and visualized with ethidium bromide and documented by using the Image Lab software. For all agarose gels, the Olerup SSP® DNA Size Marker was used as a molecular length marker (Sweden).

3.4. Statistical Analyses

χ2 and Fisher tests were carried out to compare the populations of ambulatory and hospitalized patients positive to E. coli in urine culture and their relationship with other variables (gender and origin). For statistical analysis of distribution related to the virulence and antimicrobial resistance genes, the following issues were considered: Null hypothesis (H0) = independent variable (presence of detected genes in strains), alternative hypothesis (H1) = dependent variable (detected genes related to the strain or type of gene). Considering P > 0.05, the null hypothesis is accepted (significant) and the alternative hypothesis is rejected. As in all cases, the results were greater than 0.05; therefore, it was concluded that the results were significantly different at 95% confidence, indicating that there was no relationship between strains and the presence of detected genes.

4. Results

4.1. Description of the Study Population

A total of 107 Mexican patients with urinary tract infection by E. coli were treated from April 2016 to January 2017. The patients attending at HJM were included after obtaining their written informed consent. The financial support for the research was provided by the HJM. All patients were classified by gender and origin (ambulatory and hospitalized). The majority of the patients (71/66.35%) were female patients in comparison to 36 (33.64%) male patients. Age ranges of the study population were 24 to 56 and 23 to 34 years old for male and female, respectively. The proportions for ambulatory and hospitalized patients per gender were: 52.11% (n = 37) versus 47.88% (n = 34), and 52.77% (n = 19) versus 47.22% (n = 17) in female and male patients, respectively. Statistical analysis showed that no significant differences were found between hospitalized and ambulatory patient groups (P > 0.05).

4.2. Antimicrobial Resistance Assay

Escherichia coli strains showed differences in susceptibility and resistance patterns to the antimicrobials tested. These results showed that some β-lactams (cefoxitin, piperacillin/tazobactam) carbapenems, aminoglycosides, and nitrofurans were the antimicrobials with the best antimicrobial activity against all strains tested. Penicillins such as amoxicillin/clavulanic acid, and ampicillin, cephalosporins, quinolones, and inhibitors of folate showed lower inhibitory activity on the tested strains. Resistance patterns for all E. coli strains are shown in Figure 1.

Antimicrobial resistance profile of Escherichia coli strains isolated of mexican patients with urinary infection. Antibiotics tested: Amoxicillin/clavulanic acid (AMC), ampicillin (AM), cefoxitin (FOX), piperacillin/tazobactam (TZP), ertapenem (ETP), imipenem (IPM), meropenem (MEM), cefazolin (CZ), cefepime (FEP), cefotaxime (CTX), ceftriaxone (CRO), amikacin (AN), gentamicin (GM), levofloxacin (LVX), nitrofurantoin (FM), trimethoprim/sulfamethoxazole (SXT).
Antimicrobial resistance profile of Escherichia coli strains isolated of mexican patients with urinary infection. Antibiotics tested: Amoxicillin/clavulanic acid (AMC), ampicillin (AM), cefoxitin (FOX), piperacillin/tazobactam (TZP), ertapenem (ETP), imipenem (IPM), meropenem (MEM), cefazolin (CZ), cefepime (FEP), cefotaxime (CTX), ceftriaxone (CRO), amikacin (AN), gentamicin (GM), levofloxacin (LVX), nitrofurantoin (FM), trimethoprim/sulfamethoxazole (SXT).
Figure 1.

Antimicrobial resistance profile of Escherichia coli strains isolated of mexican patients with urinary infection. Antibiotics tested: Amoxicillin/clavulanic acid (AMC), ampicillin (AM), cefoxitin (FOX), piperacillin/tazobactam (TZP), ertapenem (ETP), imipenem (IPM), meropenem (MEM), cefazolin (CZ), cefepime (FEP), cefotaxime (CTX), ceftriaxone (CRO), amikacin (AN), gentamicin (GM), levofloxacin (LVX), nitrofurantoin (FM), trimethoprim/sulfamethoxazole (SXT).

4.3. Bacterial Phylogenetic Assignment

The phylogenetic group B2 (42.05%) was the most predominant, followed by group A (27.1%), group D (24.29%), and finally, group B1 (6.54%). No statistically significant differences were observed between the phylogenetic groups identified by the type of the patient (ambulatory and hospitalized). The distribution of the phylogenetic groups of E. coli strains is shown in Table 2.

Table 2.

Phylogenetic Group’s Distribution of Escherichia coli Strains Isolated of Hospitalized and Ambulatory Mexican Patients with Urinary Infectiona

Phylogenetic GroupStrains OriginTotal
HospitalizedAmbulatory
A11 (37.93)18 (62.06)29 (27.10)
B14 (57.14)3 (42.85)7 (6.54)
B223 (51.11)22 (48.88)45 (42.05)
D13 (50.00)13 (50.00)26 (24.29)
Total51 (47.66)56 (52.33)107 (100)

aValues are expressed as No. (%).

4.4. Virulence Genes Distribution by Phylogenetic Group and Origin

All strains were subjected to a multiplex amplification by PCR in order to detect virulence and antimicrobial resistance genes. The frequencies of virulence genes identified in the E. coli strains were: fimH (92/85.98%), iutA (68/63.55%), traT (66/61.68%), papC (36/33.64%), and cnf1 (15/14.02%) (Figure 2A and B). Eight strains (7.47%) carrying all virulence genes were identified; these strains belonged to the phylogenetic groups B2 (n = 7) and D (n = 1) (Table 3). The proportion of the detection genes was calculated as the number of positive E. coli strains between total strains from the phylogenetic group. The proportion of the identified virulence genes was 1.7 and 1.4 for phylogenetic groups A and B1, respectively and no statistically significant difference was found in the virulence gene frequency from strains that belonged to these phylogenetic groups (P > 0.5).

Table 3.

Virulence and Antimicrobial Resistance Genes Distribution by Phylogenetic Groups and Origin (Hospitalized and Ambulatory) in Escherichia coli Strains Isolated of Mexican Patients with Urinary Infectiona

Phylogenetic Group E. coli/ OriginStrains, No.Virulence GenesResistance Genes
fimHiutAtraTpapCcnf1blaCTX-MblaOXAblaSHV
A
Ambulatory1816 (88.9)b7 (38.9)10 (55.6)0 (0.00)0 (0.00)10 (55.6)b5 (27.8)b1 (5.6)b
Hospitalized115 (45.5)4 (36.4)8 (72.7)0 (0.00)0 (0.00)5 (45.5)2 (18.2)0 (0.00)
B1
Ambulatory32 (66.7)0 (0.00)0 (0.00)0 (0.00)0 (0.00)0 (0.00)0 (0.00)0 (0.00)
Hospitalized43 (75.0)2 (50.0)b2 (50.0)b1 (25.0)b0 (0.00)2 (50.0)b1 (25.0)b0 (0.00)
B2
Ambulatory2221 (95.5)18 (81.8)8 (36.4)13 (59.1)7 (31.8)9 (40.9)9 (40.9)0 (0.00)
Hospitalized2322 (95.7)22 (95.7)20 (87.0)b17 (73.9)7 (30.4)22 (8.7)b19 (82.6)b1 (4.3)b
D
Ambulatory1312 (92.3)5 (38.5)9 (69.2)3 (23.1)0 (0.00)5 (38.5)6 (46.2)0 (0.00)
Hospitalized1311 (84.6)10 (76.9)b9 (69.2)2 (15.4)1 (7.7)b8 (61.5)7 (53.8)0 (0.00)

aValues are expressed as No. (%).

bSignificant difference between origin strains (ambulatory and hospitalized) (P > 0.05).

A, electrophoresis of virulence genes (fimH (508 bp) and traT (290 bp)) by duplex PCR of Escherichia coli strains. Lane M: 1 Kb DNA marker; lane 1: Negative control; lane 2: Positive control; lane 3 - 8: 30, 40, 87, 64, 125 and 192 E. coli strains. B, electrophoresis of virulence genes (iutA (300 bp), papC (200), cnf1 (498 bp)) by triplex PCR of E. coli strains. Lane M: 1 Kb DNA marker; lane 9: Negative control; lane 10: Positive control; lane 11 - 16: 17, 32, 96, 98, 95, 52 E. coli strains. C, electrophoresis of antibiotic resistance genes (blaCTX-M(499 bp), blaOXA (564 bp), and blaSHV (214 bp)) by triplex PCR of E. coli strains. Lane M: 1 Kb DNA marker; lane 17: Negative control; lane 18: Positive control; lane 19 - 24: 40, 84, 114, 200, 16, 132 E. coli strains
A, electrophoresis of virulence genes (fimH (508 bp) and traT (290 bp)) by duplex PCR of Escherichia coli strains. Lane M: 1 Kb DNA marker; lane 1: Negative control; lane 2: Positive control; lane 3 - 8: 30, 40, 87, 64, 125 and 192 E. coli strains. B, electrophoresis of virulence genes (iutA (300 bp), papC (200), cnf1 (498 bp)) by triplex PCR of E. coli strains. Lane M: 1 Kb DNA marker; lane 9: Negative control; lane 10: Positive control; lane 11 - 16: 17, 32, 96, 98, 95, 52 E. coli strains. C, electrophoresis of antibiotic resistance genes (blaCTX-M(499 bp), blaOXA (564 bp), and blaSHV (214 bp)) by triplex PCR of E. coli strains. Lane M: 1 Kb DNA marker; lane 17: Negative control; lane 18: Positive control; lane 19 - 24: 40, 84, 114, 200, 16, 132 E. coli strains
Figure 2.

A, electrophoresis of virulence genes (fimH (508 bp) and traT (290 bp)) by duplex PCR of Escherichia coli strains. Lane M: 1 Kb DNA marker; lane 1: Negative control; lane 2: Positive control; lane 3 - 8: 30, 40, 87, 64, 125 and 192 E. coli strains. B, electrophoresis of virulence genes (iutA (300 bp), papC (200), cnf1 (498 bp)) by triplex PCR of E. coli strains. Lane M: 1 Kb DNA marker; lane 9: Negative control; lane 10: Positive control; lane 11 - 16: 17, 32, 96, 98, 95, 52 E. coli strains. C, electrophoresis of antibiotic resistance genes (blaCTX-M(499 bp), blaOXA (564 bp), and blaSHV (214 bp)) by triplex PCR of E. coli strains. Lane M: 1 Kb DNA marker; lane 17: Negative control; lane 18: Positive control; lane 19 - 24: 40, 84, 114, 200, 16, 132 E. coli strains

In contrast, the highest frequency rates were found in the phylogenetic groups B2 and D; proportions were 3.5 and 2.4, respectively. Representing two-fold and 2.5 higher than A (P < 0.05) and B1 (P < 0.001) phylogenetic groups (for group B2). The proportion of virulence genes from the phylogenetic group D was 1.7-fold higher than group B1 (P < 0.05). Significant differences were identified (P < 0.05) in the majority of genes detected in hospitalized patients; iutA, traT, and papC in group B1, traT in group B2, and fimH and cnf1 in group D. Finally, virulence factor fimH presented significant differences in the ambulatory patients in group A.

4.5. Antimicrobial Resistance Genes Distribution by Phylogenetic Groups

The frequencies of the virulence genes identified in E. coli strains were: blaCTX-M(41/38.32%), blaOXA (49/45.79%), and blaSHV (2/1.87%) is shown in Figure 2C. One strain (0.93%) carrying all antimicrobial resistance genes was identified; this strain belonged to the phylogenetic group B2. A summary of antimicrobial resistance genes distribution by phylogenetic groups detected in all strains is shown in Table 3. Proportion of antimicrobial resistance genes was 0.8 and 0.4 for phylogenetic groups A and B1, respectively. The frequencies of antimicrobial resistance genes were 1.3 and 1.0 in the phylogenetic groups B2 and D, respectively. The frequency of antimicrobial resistance in the phylogenetic group B2 was 1.3, representing 1.6-fold and 3.2-fold higher than the phylogenetic groups A (0.8) and B1 (0.4), respectively. Significant differences were identified (P <0.05) in the majority of genes detected in ambulatory patients; blaCTX-M, blaOXA, and blaSHV in group A and B2 and blaCTX-Mand blaOXA in group B1 of hospitalized patients.

4.6. The Relationship Between Virulence and Antimicrobial Resistance Genes in E. coli Strains

The relationship between virulence and antimicrobial resistance genes was evaluated in all E. coli strains. The genetic associations: fimH/blaCTX, fimH/blaOXA, traT/blaCTX, traT/blaOXA, iutA/blaCTX, and iutA/blaOXA showed higher incidence, while the associations with genes cnf1 and papC showed lower incidence in the strains evaluated. Finally, there was no association of the virulence gene cnf1 with the blaSHV resistance gene. The relationship between virulence and antimicrobial resistance genes in E. coli strains is shown in Table 4.

Table 4.

The Relationship Between Virulence and Antimicrobial Resistance Genes in Escherichia coli Strains Isolated of Mexican Patients with Urinary Infectiona

Resistance GenesVirulence Genes
fimHtraTcnf1papCiutA
blaCTX54 (50.5)b42 (39.3)b6 (5.6)26 (24.3)61 (57.0)b
blaSHV2 (1.9)2 (1.9)0 (0.0)1 (0.9)1 (0.9)
blaOXA47 (43.9)b35 (32.7)b6 (5.6)27 (25.2)46 (43.0)b

aValues are expressed as No. (%).

bCorrelation with significant difference (P > 0.05).

5. Discussion

Urinary tract infections are mainly caused by Gram-negative and facultative anaerobic bacilli such as UPEC (31). In this work, we reported the detection of the main phylogenetic groups of E. coli in the patients (ambulatory and hospitalized) with UTI and the presence of virulence and antimicrobial resistance genes. Data in this work agree with previous reports that indicate that female gender is the most susceptible population to develop UTI. The highest frequency is attributed to this gender due to the anatomy of the vaginal and anorectal region. It has been reported that 80% of the UTIs is caused by UPEC that are different from CEC strains that normally inhabit the gastrointestinal tract (32, 33). Phylogenetic analysis of E. coli strains was divided into four mains groups (A, B1 B2, and D) (34, 35).

The results suggest that most of UTI in Mexican population were mainly caused by E. coli of the phylogenetic group B2. Furthermore, ambulatory and hospitalized patients were infected by E. coli phylogenetic groups A, B1, B2, or D with the same frequency (regardless of the origin of the patient). Virulent and resistant strains are mainly included in the phylogenetic groups B2 and D, whereas commensal ones belong to group A. In previous reports, it was indicated that virulent E. coli strains belonged to B2 and D groups, whereas isolates that belonged to group A were CEC strains and could acquire genes of virulence and antimicrobial resistance by horizontal gene transfer; therefore, they can cause infection (36). Interestingly, we found virulence genes (fimH, iutA, and traT) in CEC (A and B1 groups); the high frequency of fimH in the isolates confirms the importance of the participation of this gene in the initial stage of the infection, since this protein recognizes receptors of the bladder cells; a previous report indicated that mannose-binding type 1 pili, codified in fimH gene, is an important virulence factor involved in the establishment of UTI (37).

On the contrary, the IutA protein is the most important receptor during the infection that contributes to bacterial colonization; this gene had a high frequency detected in pathogenic strains isolated from hospitalized patients. The same phenomenon was observed for the traT gene. The papC and cnf1 genes encoded for a P-fimbriae and cytotoxic necrotizing factor type 1, respectively; these genes were detected in low frequencies in strains belonging to the phylogenetic group A. The absence of virulence genes in CEC is a typical group characteristic; however, the study of the strains that presented these genes (papC gene) is interesting to elucidate the reason why they have these genes to be non-pathogenic strains. The importance of the study of the papC gene in pathogenic and non-pathogenic isolates acquires relevance since this gene is associated with adherence; therefore, its detection provides necessary information about the capacity of these strains to cause infections in the high urinary tract (38).

The simultaneous detection of the papC genes, cnf1 in 31 strains, indicate the possible presence of the pathogenicity island IIJ96 (39), since the presence of these genes is strongly associated with strains that produce UTI cases. However, it is necessary to perform the detection of the hlyA gene, which encodes a hemolysin. The frequency of detection of this genetic binomial in our work was high, compared to other works previously reported (40-42). Virulence and resistance genes were more frequent in strains isolated from hospitalized patients compared to ambulatory patients. We hypothesized that in hospitalized patients, the use of antibiotics in the nosocomial environment might allow bacteria to resist the transfer of genetic material between bacteria by conjugation, transformation or transduction.

Recent work indicates that urine is a rich source of bacteriophages that could participate in the transfer process of DNA (43). Due to the detection of antimicrobial resistance genes in CEC strains, we can speculate that these strains could have participated as receptors in processes of genetic material transfer containing resistance or virulence genes. Since E. coli has a broad phylogenetic diversity, it has a high degree of genome plasticity, with gene losses and gains, through horizontal transfer of DNA (44). Therefore, the gain of genes might allow the commensal bacteria to cause infection due to the acquisition of new genetic material. The differences identified in the phenotype of resistance to antibiotics in the tested strains revealed that the antibiotics of the aminoglycoside and carbapenems families could be the drugs of choice for the treatment of UTI in the population studied, since the strains tested showed greater sensitivity.

The beta-lactam antibiotics, cephalosporins, nitrofurantoins, and inhibitors of folate metabolism, showed little activity against the strains tested. Correlation between the detection of the bla genes and the phenotype against the antibiotics tested was observed. The analysis of the relationship between virulence and resistance genes showed that the blaCTX and blaOXA genes were strongly associated with the presence of the fimH, traT, and iutA genes. The virulence/resistance relationship with the blaSHV gene could not be detected due to the low frequency of this gene in all the isolates. It is clear that the increased virulence and the emergence of antibiotic resistance often arise simultaneously; however, its genetic connection has been little studied, thus it is interesting to know the existence of a possible synergism between these two characteristics in pathogens of clinical interest. The results presented in the current work demonstrated the wide diversity in the distribution of virulence and antibiotic resistance genes between the UPEC and CEC strains.

5.1. Conclusions

In conclusion, the phylogenetic group, the virulence factors, and the susceptibility to antibiotics of E. coli causing UTI infections varied significantly among the Mexican populations, In this regard, the predominant phylogenetic groups causing UTI with virulence and resistance properties were the B2 and D.

References

  • 1.

    Herzer PJ, Inouye S, Inouye M, Whittam TS. Phylogenetic distribution of branched RNA-linked multicopy single-stranded DNA among natural isolates of Escherichia coli. J Bacteriol. 1990;172(11):6175-81. [PubMed ID: 1699928]. [PubMed Central ID: PMC526797].

  • 2.

    Flores-Mireles AL, Walker JN, Caparon M, Hultgren SJ. Urinary tract infections: Epidemiology, mechanisms of infection and treatment options. Nat Rev Microbiol. 2015;13(5):269-84. [PubMed ID: 25853778]. [PubMed Central ID: PMC4457377]. https://doi.org/10.1038/nrmicro3432.

  • 3.

    Kostakioti M, Hultgren SJ, Hadjifrangiskou M. Molecular blueprint of uropathogenic Escherichia coli virulence provides clues toward the development of anti-virulence therapeutics. Virulence. 2012;3(7):592-4. [PubMed ID: 23154288]. [PubMed Central ID: PMC3545937]. https://doi.org/10.4161/viru.22364.

  • 4.

    Sistema Nacional de Vigilancia Epidemiológica (SINAVE). Anuarios de Morbilidad: Infecciones de vías urinarias. 2016. Available from: http://www.epidemiologia.salud.gob.mx/anuario/20160/incidencia/incidencia_casos_nuevos_enfermedad_grupo_edad.pdf.

  • 5.

    Bjorkman I, Berg J, Viberg N, Stalsby Lundborg C. Awareness of antibiotic resistance and antibiotic prescribing in UTI treatment: A qualitative study among primary care physicians in Sweden. Scand J Prim Health Care. 2013;31(1):50-5. [PubMed ID: 23281925]. [PubMed Central ID: PMC3587295]. https://doi.org/10.3109/02813432.2012.751695.

  • 6.

    Vranic SM, Zatric N, Rebic V, Aljicevic M, Abdulzaimovic A. The most frequent isolates from outpatients with urinary tract infection. Mater Sociomed. 2017;29(1):17-20. [PubMed ID: 28484348]. [PubMed Central ID: PMC5402357]. https://doi.org/10.5455/msm.2017.29.17-20.

  • 7.

    Chabros L, Mlynarczyk G, Sawicka-Grzelak A, Swoboda-Kopec E, Kuthan R, de Walthoffen SW, et al. Comparison of bacteria isolated from urinary tract infections in patients on transplant versus urologic wards. Transplant Proc. 2011;43(8):3125-7. [PubMed ID: 21996243]. https://doi.org/10.1016/j.transproceed.2011.09.004.

  • 8.

    Momtaz H, Karimian A, Madani M, Safarpoor Dehkordi F, Ranjbar R, Sarshar M, et al. Uropathogenic Escherichia coli in Iran: Serogroup distributions, virulence factors and antimicrobial resistance properties. Ann Clin Microbiol Antimicrob. 2013;12:8. [PubMed ID: 23627669]. [PubMed Central ID: PMC3651382]. https://doi.org/10.1186/1476-0711-12-8.

  • 9.

    Nielubowicz GR, Mobley HL. Host-pathogen interactions in urinary tract infection. Nat Rev Urol. 2010;7(8):430-41. [PubMed ID: 20647992]. https://doi.org/10.1038/nrurol.2010.101.

  • 10.

    Wright KJ, Hultgren SJ. Sticky fibers and uropathogenesis: Bacterial adhesins in the urinary tract. Future Microbiol. 2006;1(1):75-87. [PubMed ID: 17661687]. https://doi.org/10.2217/17460913.1.1.75.

  • 11.

    Hemmatinezhad B, Khamesipour F, Mohammadi M, Safarpoor Dehkordi F, Mashak Z. Microbiological investigation of o-serogroups, virulence factors and antimicrobial resistance properties of shiga toxin-producing escherichia coliisolated from ostrich, turkey and quail meats. J Food Saf. 2015;35(4):491-500. https://doi.org/10.1111/jfs.12199.

  • 12.

    Ranjbar R, Safarpoor Dehkordi F, Sakhaei Shahreza MH, Rahimi E. Prevalence, identification of virulence factors, O-serogroups and antibiotic resistance properties of Shiga-toxin producing Escherichia coli strains isolated from raw milk and traditional dairy products. Antimicrob Resist Infect Control. 2018;7:53. [PubMed ID: 29686859]. [PubMed Central ID: PMC5902837]. https://doi.org/10.1186/s13756-018-0345-x.

  • 13.

    Momtaz H, Dehkordi FS, Hosseini MJ, Sarshar M, Heidari M. Serogroups, virulence genes and antibiotic resistance in Shiga toxin-producing Escherichia coli isolated from diarrheic and non-diarrheic pediatric patients in Iran. Gut Pathog. 2013;5(1):39. [PubMed ID: 24330673]. [PubMed Central ID: PMC3866933]. https://doi.org/10.1186/1757-4749-5-39.

  • 14.

    Dehkordi FS, Yazdani F, Mozafari J, Valizadeh Y. Virulence factors, serogroups and antimicrobial resistance properties of Escherichia coli strains in fermented dairy products. BMC Res Notes. 2014;7:217. [PubMed ID: 24708594]. [PubMed Central ID: PMC3983858]. https://doi.org/10.1186/1756-0500-7-217.

  • 15.

    Dormanesh B, Safarpoor Dehkordi F, Hosseini S, Momtaz H, Mirnejad R, Hoseini MJ, et al. Virulence factors and o-serogroups profiles of uropathogenic Escherichia coli isolated from Iranian pediatric patients. Iran Red Crescent Med J. 2014;16(2). e14627. [PubMed ID: 24719745]. [PubMed Central ID: PMC3965878]. https://doi.org/10.5812/ircmj.14627.

  • 16.

    Shahrani M, Dehkordi FS, Momtaz H. Characterization of Escherichia coli virulence genes, pathotypes and antibiotic resistance properties in diarrheic calves in Iran. Biol Res. 2014;47:28. [PubMed ID: 25052999]. [PubMed Central ID: PMC4105491]. https://doi.org/10.1186/0717-6287-47-28.

  • 17.

    Ranjbar R, Masoudimanesh M, Dehkordi FS, Jonaidi-Jafari N, Rahimi E. Shiga (Vero)-toxin producing Escherichia coli isolated from the hospital foods; virulence factors, o-serogroups and antimicrobial resistance properties. Antimicrob Resist Infect Control. 2017;6:4. [PubMed ID: 28074125]. [PubMed Central ID: PMC5219770]. https://doi.org/10.1186/s13756-016-0163-y.

  • 18.

    Nagamatsu K, Hannan TJ, Guest RL, Kostakioti M, Hadjifrangiskou M, Binkley J, et al. Dysregulation of Escherichia coli alpha-hemolysin expression alters the course of acute and persistent urinary tract infection. Proc Natl Acad Sci U S A. 2015;112(8):E871-80. [PubMed ID: 25675528]. [PubMed Central ID: PMC4345586]. https://doi.org/10.1073/pnas.1500374112.

  • 19.

    Guiton PS, Cusumano CK, Kline KA, Dodson KW, Han Z, Janetka JW, et al. Combinatorial small-molecule therapy prevents uropathogenic Escherichia coli catheter-associated urinary tract infections in mice. Antimicrob Agents Chemother. 2012;56(9):4738-45. [PubMed ID: 22733070]. [PubMed Central ID: PMC3421856]. https://doi.org/10.1128/AAC.00447-12.

  • 20.

    Rippere-Lampe KE, O'Brien AD, Conran R, Lockman HA. Mutation of the gene encoding cytotoxic necrotizing factor type 1 (cnf(1)) attenuates the virulence of uropathogenic Escherichia coli. Infect Immun. 2001;69(6):3954-64. [PubMed ID: 11349064]. [PubMed Central ID: PMC98434]. https://doi.org/10.1128/IAI.69.6.3954-3964.2001.

  • 21.

    Koshesh M, Mansouri S, Hashemizadeh Z, Kalantar-Neyestanaki D. Identification of extended-spectrum β-lactamase genes and ampc-β-lactamase in clinical isolates of escherichia coli recovered from patients with urinary tract infections in Kerman, Iran. Arch Pediatr Infect Dis. 2016;5(2). e37968. https://doi.org/10.5812/pedinfect.37968.

  • 22.

    Woodford N, Ward ME, Kaufmann ME, Turton J, Fagan EJ, James D, et al. Community and hospital spread of Escherichia coli producing CTX-M extended-spectrum beta-lactamases in the UK. J Antimicrob Chemother. 2004;54(4):735-43. [PubMed ID: 15347638]. https://doi.org/10.1093/jac/dkh424.

  • 23.

    Clermont O, Bonacorsi S, Bingen E. Rapid and simple determination of the Escherichia coli phylogenetic group. Appl Environ Microbiol. 2000;66(10):4555-8. [PubMed ID: 11010916]. [PubMed Central ID: PMC92342]. https://doi.org/10.1128/AEM.66.10.4555-4558.2000.

  • 24.

    Clermont O, Christenson JK, Denamur E, Gordon DM. The clermont Escherichia coli phylo-typing method revisited: Improvement of specificity and detection of new phylo-groups. Environ Microbiol Rep. 2013;5(1):58-65. [PubMed ID: 23757131]. https://doi.org/10.1111/1758-2229.12019.

  • 25.

    Qu W, Zhou Y, Zhang Y, Lu Y, Wang X, Zhao D, et al. MFEprimer-2.0: A fast thermodynamics-based program for checking PCR primer specificity. Nucleic Acids Res. 2012;40(Web Server issue):W205-8. [PubMed ID: 22689644]. [PubMed Central ID: PMC3394324]. https://doi.org/10.1093/nar/gks552.

  • 26.

    Tartof SY, Solberg OD, Riley LW. Genotypic analyses of uropathogenic Escherichia coli based on fimH single nucleotide polymorphisms (SNPs). J Med Microbiol. 2007;56(Pt 10):1363-9. [PubMed ID: 17893175]. https://doi.org/10.1099/jmm.0.47262-0.

  • 27.

    Johnson JR, Stell AL. Extended virulence genotypes of Escherichia coli strains from patients with urosepsis in relation to phylogeny and host compromise. J Infect Dis. 2000;181(1):261-72. [PubMed ID: 10608775]. https://doi.org/10.1086/315217.

  • 28.

    Magoue CL, Melin P, Gangoue-Pieboji J, Okomo Assoumou MC, Boreux R, De Mol P. Prevalence and spread of extended-spectrum beta-lactamase-producing Enterobacteriaceae in Ngaoundere, Cameroon. Clin Microbiol Infect. 2013;19(9):E416-20. [PubMed ID: 23647948]. https://doi.org/10.1111/1469-0691.12239.

  • 29.

    Hassan SA, Shobrak MY. Detection of genes mediating beta-lactamase production in isolates of enterobacteria recovered from wild pets in Saudi Arabia. Vet World. 2015;8(12):1400-4. [PubMed ID: 27047051]. [PubMed Central ID: PMC4774817]. https://doi.org/10.14202/vetworld.2015.1400-1404.

  • 30.

    Kim J, Jeon S, Rhie H. Rapid detection of extended spectrum β-lactamase (ESBL) for Enterobacteriaceae by use of a multiplex PCR-based method. Infect Chemother. 2009;41(3):181-4. https://doi.org/10.3947/ic.2009.41.3.181.

  • 31.

    Foxman B. The epidemiology of urinary tract infection. Nat Rev Urol. 2010;7(12):653-60. [PubMed ID: 21139641]. https://doi.org/10.1038/nrurol.2010.190.

  • 32.

    Stamm WE. Scientific and clinical challenges in the management of urinary tract infections. Am J Med. 2002;113 Suppl 1A:1S-4S. [PubMed ID: 12113865]. https://doi.org/10.1016/S0002-9343(02)01053-7.

  • 33.

    Neamati F, Firoozeh F, Saffari M, Zibaei M. Virulence genes and antimicrobial resistance pattern in uropathogenic Escherichia coli isolated from hospitalized patients in Kashan, Iran. Jundishapur J Microbiol. 2015;8(2). e17514. [PubMed ID: 25825647]. [PubMed Central ID: PMC4376973]. https://doi.org/10.5812/jjm.17514.

  • 34.

    Orsi RH, Stoppe NC, Sato MI, Ottoboni LM. Identification of Escherichia coli from groups A, B1, B2 and D in drinking water in Brazil. J Water Health. 2007;5(2):323-7. [PubMed ID: 17674580]. https://doi.org/10.2166/wh.2007.028.

  • 35.

    Boyd EF, Hartl DL. Chromosomal regions specific to pathogenic isolates of Escherichia coli have a phylogenetically clustered distribution. J Bacteriol. 1998;180(5):1159-65. [PubMed ID: 9495754]. [PubMed Central ID: PMC107003].

  • 36.

    Bingen E, Picard B, Brahimi N, Mathy S, Desjardins P, Elion J, et al. Phylogenetic analysis of Escherichia coli strains causing neonatal meningitis suggests horizontal gene transfer from a predominant pool of highly virulent B2 group strains. J Infect Dis. 1998;177(3):642-50. [PubMed ID: 9498443]. https://doi.org/10.1086/514217.

  • 37.

    Bouckaert J, Berglund J, Schembri M, De Genst E, Cools L, Wuhrer M, et al. Receptor binding studies disclose a novel class of high-affinity inhibitors of the Escherichia coli FimH adhesin. Mol Microbiol. 2005;55(2):441-55. [PubMed ID: 15659162]. https://doi.org/10.1111/j.1365-2958.2004.04415.x.

  • 38.

    Marklund BI, Tennent JM, Garcia E, Hamers A, Baga M, Lindberg F, et al. Horizontal gene transfer of the Escherichia coli pap and prs pili operons as a mechanism for the development of tissue-specific adhesive properties. Mol Microbiol. 1992;6(16):2225-42. [PubMed ID: 1357526]. https://doi.org/10.1111/j.1365-2958.1992.tb01399.x.

  • 39.

    Bidet P, Bonacorsi S, Clermont O, De Montille C, Brahimi N, Bingen E. Multiple insertional events, restricted by the genetic background, have led to acquisition of pathogenicity island IIJ96-like domains among Escherichia coli strains of different clinical origins. Infect Immun. 2005;73(7):4081-7. [PubMed ID: 15972496]. [PubMed Central ID: PMC1168593]. https://doi.org/10.1128/IAI.73.7.4081-4087.2005.

  • 40.

    Robino L, Scavone P, Araujo L, Algorta G, Zunino P, Pirez MC, et al. Intracellular bacteria in the pathogenesis of Escherichia coli urinary tract infection in children. Clin Infect Dis. 2014;59(11):e158-64. [PubMed ID: 25091303]. [PubMed Central ID: PMC4650771]. https://doi.org/10.1093/cid/ciu634.

  • 41.

    Najafi A, Hasanpour M, Askary A, Aziemzadeh M, Hashemi N. Distribution of pathogenicity island markers and virulence factors in new phylogenetic groups of uropathogenic Escherichia coli isolates. Folia Microbiol (Praha). 2018;63(3):335-43. [PubMed ID: 29199378]. https://doi.org/10.1007/s12223-017-0570-3.

  • 42.

    Lopez-Banda DA, Carrillo-Casas EM, Leyva-Leyva M, Orozco-Hoyuela G, Manjarrez-Hernandez AH, Arroyo-Escalante S, et al. Identification of virulence factors genes in Escherichia coli isolates from women with urinary tract infection in Mexico. Biomed Res Int. 2014;2014:959206. [PubMed ID: 24895634]. [PubMed Central ID: PMC4026957]. https://doi.org/10.1155/2014/959206.

  • 43.

    Stone L. Microbiota: Bacteriophage diversity in the urinary microbiome. Nat Rev Urol. 2018;15(3):135. [PubMed ID: 29460923]. https://doi.org/10.1038/nrurol.2018.23.

  • 44.

    Baharoglu Z, Garriss G, Mazel D. Multiple pathways of genome plasticity leading to development of antibiotic resistance. Antibiotics (Basel). 2013;2(2):288-315. [PubMed ID: 27029305]. [PubMed Central ID: PMC4790341]. https://doi.org/10.3390/antibiotics2020288.