Uropathogenic E. coli and Hybrid Pathotypes in Mexican Women with Urinary Tract Infections: A Comprehensive Molecular and Phenotypic Overview.

Uropathogenic Escherichia coli (UPEC) is the main cause of urinary tract infections (UTIs) and carries virulence and resistance factors often found in mobilizable genetic elements, such as plasmids or pathogenicity islands (PAIs). UPEC is part of the extraintestinal pathogenic E. coli (ExPEC), but hybrid strains possessing both diarrheagenic E. coli (DEC) and ExPEC traits, termed "hypervirulent", present a significant health threat. This study assessed the prevalence of UPEC PAIs, ExPEC sequence types (ST), DEC genes, carbapenemase and extended-spectrum ß-lactamase (ESBL) phenotypes, resistance genotypes, and plasmids in 40 clinical isolates of UPEC. Results showed that 72.5% of isolates had PAIs, mainly PAI IV536 (53%). ESBL phenotypes were found in 65% of ß-lactam-resistant isolates, with 100% of carbapenem-resistant isolates producing carbapenemase. The predominant ESBL gene was blaCTX-M-2 (60%), and the most common resistance gene in fluoroquinolone and aminoglycoside-resistant isolates was aac(6')Ib (93%). Plasmids were present in 57% of isolates, and 70% belonged to the ST131 clonal group. Molecular markers for DEC pathotypes were detected in 20 isolates, with 60% classified as hybrid pathotypes. These findings indicate significant pathogenic potential and the presence of hybrid pathotypes in E. coli UTI clinical isolates in the Mexican population.

Draft genome of three uropathogenic Escherichia coli strains.

Uropathogenic Escherichia coli (UPEC) remains the main etiological agent of urinary tract infections affecting females and males. The draft genome sequence of three strains of UPEC isolated from senior citizens and pregnant women in the state of Puebla, Mexico, is reported here.

Draft genome sequence of a triple hybrid Escherichia coli strain isolated from a healthy donor feces.

Herein is reported the draft genome sequence of a triple hybrid Escherichia coli strain isolated from a healthy donor feces. The assembly is 5.2 Mbp, composed of 247 contigs, with a N50 of 77, 241 bp, presenting a GC content of 50.8%.

Uropathogenic Escherichia coli in Mexico, an Overview of Virulence and Resistance Determinants: Systematic Review and Meta-analysis.

BACKGROUND: Urinary tract infections (UTI) are one of the most common pathologies in Mexico and the majority are caused by uropathogenic Escherichia coli (UPEC). UPEC possesses virulence and resistance determinants that promote UTI development and affect diagnosis and treatment. This study aims to systematically review published reports of virulence genes, antibiotic resistance, and phylogenetic groups prevalent in clinical isolates of UPEC in the Mexican population. METHODS: Systematic review with meta-analysis was performed following PRISMA guidelines. Articles in both English and Spanish were included. Total prevalence with a 95% confidence interval of each characteristic was calculated. Heterogeneity between studies and geographical areas was assessed by the Cochran Q test (Q), I-square (I2), and H-square (H2). Egger's test was used for risk of bias in publications and asymmetry evaluations. RESULTS: Forty-two articles were analyzed. The most prevalent virulence genes were ecp (97.25%; n = 364) and fimH (82.34%; n = 1,422), which are associated with lower UTI, followed by papGII (40.98%; n = 810), fliC (38.87%; n = 319), hlyA (23.55%; n = 1,521), responsible for with upper UTI. More than 78.13% (n = 1,893) of the isolates were classified as multidrug-resistant, with a higher prevalence of resistance to those antibiotics that are implemented in the basic regimen in Mexico. The most frequently reported Extended Spectrum ß-Lactamase (ESBL) was CTX-M-1 (55.61%; n = 392), and the predominant phylogroup was B2 (35.94%; n = 1,725). CONCLUSION: UPEC strains are responsible for a large portion of both lower and upper UTI in Mexico, and their multi-drug resistance drastically reduces the number of therapeutic options available.

Biosynthesis of Silver Nanoparticles Using Seasonal Samples of Sonoran Desert Propolis: Evaluation of Its Antibacterial Activity against Clinical Isolates of Multi-Drug Resistant Bacteria.

Multi-drug resistant (MDR) bacteria have gained importance as a health problem worldwide, and novel antibacterial agents are needed to combat them. Silver nanoparticles (AgNPs) have been studied as a potent antimicrobial agent, capable of countering MDR bacteria; nevertheless, their conventional synthesis methods can produce cytotoxicity and environmental hazards. Biosynthesis of silver nanoparticles has emerged as an alternative to reduce the cytotoxic and environmental problems derived from their chemical synthesis, using natural products as a reducing and stabilizing agent. Sonoran Desert propolis (SP) is a poplar-type propolis rich in polyphenolic compounds with remarkable biological activities, such as being antioxidant, antiproliferative, and antimicrobial, and is a suitable candidate for synthesis of AgNPs. In this study, we synthesized AgNPs using SP methanolic extract (SP-AgNPs) and evaluated the reduction capacity of their seasonal samples and main chemical constituents. Their cytotoxicity against mammalian cell lines and antibacterial activity against multi-drug resistant bacteria were assessed. Quercetin and galangin showed the best-reduction capacity for synthesizing AgNPs, as well as the seasonal sample from winter (SPw-AgNPs). The SPw-AgNPs had a mean size of around 16.5 ± 5.3 nm, were stable in different culture media, and the presence of propolis constituents was confirmed by FT-IR and HPLC assays. The SPw-AgNPs were non-cytotoxic to ARPE-19 and HeLa cell lines and presented remarkable antibacterial and antibiofilm activity against multi-drug resistant clinical isolates, with E. coli 34 and ATCC 25922 being the most susceptible (MBC = 25 µg/mL), followed by E. coli 2, 29, 37 and PNG (MBC = 50 µg/mL), and finally E. coli 37 and S. aureus ATCC 25923 (MBC = 100 µg/mL). These results demonstrated the efficacy of SP as a reducing and stabilizing agent for synthesis of AgNPs and their capacity as an antibacterial agent.

Characterization of Diarreaghenic Escherichia coli Strains Isolated from Healthy Donors, including a Triple Hybrid Strain.

Escherichia coli is a well-recognized inhabitant of the animal and human gut. Its presence represents an essential component of the microbiome. There are six pathogenic variants of E. coli associated with diarrheal processes, known as pathotypes. These harbor genetic determinants that allow them to be classified as such. In this work, we report the presence of diarrheagenic pathotypes of E. coli strains isolated from healthy donors. Ninety E. coli strains were analyzed, of which forty-six (51%) harbored virulence markers specifics for diarrheagenic pathotypes, including four hybrids (one of them with genetic determinants of three DEC pathotypes). We also identified phylogenetic groups with a higher prevalence of B2 (45.6%) and A (17.8%). In addition, resistance to sulfonamides (100%), and aminoglycosides (100%) was found in 100% of the strains, with a lower prevalence of resistance to cefotaxime (13.3%), ceftriaxone (12.2%), fosfomycin (10%), and meropenem (0%). All analyzed strains were classified as multidrug resistant. Virulence genes were also investigated, which led us to propose three new virotypes. Among the virulence traits observed, the ability to form biofilms stands out, which was superior to that of the E. coli and Staphylococcus aureus strains used as positive controls.

Mechanisms of Inhibition of Quorum Sensing as an Alternative for the Control of E. coli and Salmonella.

Quorum sensing (QS) is a process of cell-cell communication for bacteria such as E. coli and Salmonella that cause foodborne diseases, with the production, release, and detection of autoinducer (AI) molecules that participate in the regulation of virulence genes. All of these proteins are useful in coordinating collective behavior, the expression of virulence factors, and the pathogenicity of Gram-negative bacteria. In this work, we review the natural or synthetic inhibitor molecules of QS that inactivate the autoinducer and block QS regulatory proteins in E. coli and Salmonella. Furthermore, we describe mechanisms of QS inhibitors (QSIs) that act as competitive inhibitors, being a useful tool for preventing virulence gene expression through the downregulation of AI-2 production pathways and the disruption of signal uptake. In addition, we showed that QSIs have negative regulatory activity of genes related to bacterial biofilm formation on clinical artifacts, which confirms the therapeutic potential of QSIs in the control of infectious pathogens. Finally, we discuss resistance to QSIs, the design of next-generation QSIs, and how these molecules can be leveraged to provide a new antivirulence therapy to combat diseases caused by E. coli or Salmonella.

Bacterial Morphotypes as Important Trait for Uropathogenic E. coli Diagnostic; a Virulence-Phenotype-Phylogeny Study.

Urinary tract infections (UTIs) belong to the most common pathologies in Mexico and are mainly caused by Uropathogenic Escherichia coli (UPEC). UPEC possesses a wide diversity of virulence factors that allow it to carry out its pathogenesis mechanism in the urinary tract (UT). The development of morphotypes in UT represents an important feature of UPEC because it is associated with complications in diagnosis of UTI. The aim of this study was to determine the presence of bacterial morphotypes, virulence genes, virulence phenotypes, antibiotic resistant, and phylogenetic groups in clinical isolates of UPEC obtained from women in Sonora, Mexico. Forty UPEC isolates were obtained, and urine morphotypes were observed in 65% of the urine samples from where E. coli was isolated. Phylogenetic group B2 was the most prevalent. The most frequent virulence genes were fimH (100%), fliCD (90%), and sfaD/focC (72%). Biofilm formation (100%) and motility (98%) were the most prevalent phenotypes. Clinical isolates showed high resistance to aminoglycosides and ß-lactams antibiotics. These data suggest that the search for morphotypes in urine sediment must be incorporated in the urinalysis procedure and also that clinical isolates of UPEC in this study can cause upper, lower, and recurrent UTI.

Antibiotic resistance mechanisms in Acinetobacter spp. strains isolated from patients in a paediatric hospital in Mexico.

OBJECTIVES: The aim of this study was to identify Acinetobacter spp. strains from paediatric patients, to determine their genetic relationship, to detect antibiotic resistance genes and to evaluate the role of efflux pumps in antibiotic resistance. METHODS: A total of 54 non-duplicate, non-consecutive Acinetobacter spp. isolates were collected from paediatric patients. Their genetic relationship, antibiotic resistance profile, efflux pump activity, antibiotic resistance genes and plasmid profile were determined. RESULTS: The isolates were identified as 24 Acinetobacter haemolyticus, 24 Acinetobacter calcoaceticus-baumannii (Acb) complex and 1 strain each of Acinetobacter junii, Acinetobacter radioresistens, Acinetobacter indicus, Acinetobacter lwoffii, Acinetobacter ursingii and Acinetobacter venetianus. The 24 A. haemolyticus were considered genetically unrelated. One strain was resistant to carbapenems, two to cephalosporins, two to ciprofloxacin and sixteen to aminoglycosides. The antibiotic resistance genes blaOXA-214 (29%), blaOXA-215 (4%), blaOXA-264 (8%), blaOXA-265 (29%), blaNDM-1 (4%), aac(6')-Ig (38%) and the novel variants blaOXA-575 (13%), blaTEM-229 (75%), aac(6')-Iga (4%), aac(6')-Igb (13%) and aac(6')-Igc (42%) were detected. Among 24 Acb complex, 5 were multidrug-resistant, carbapenem-resistant strains carrying blaOXA-51 and blaOXA-23; they were genetically related and had the same plasmid profile. Other species were susceptible. In some strains of A. haemolyticus and Acb complex, the role of RND efflux pumps was evidenced by a decrease in the MICs for cefotaxime, amikacin and ciprofloxacin in the presence of an efflux pump inhibitor. CONCLUSIONS: This study identified isolates of A. haemolyticus carrying new ß-lactamase variants and shows for the first time the contribution of efflux pumps to antibiotic resistance in this species.

Virulence and Resistance Determinants of Uropathogenic Escherichia coli Strains Isolated from Pregnant and Non-Pregnant Women from Two States in Mexico.

BACKGROUND/PURPOSE: Uropathogenic E. coli (UPEC) is the main cause of urinary tract infection (UTI) and it is known that pregnant women have a higher risk for UTI. UPEC has a variety of virulence and antibiotic resistance factors that facilitate its pathogenic success and it is crucial to know which are the susceptibility patterns, Extended-Spectrum-ß-Lactamase (ESBL) production, virulence genes, pathogenicity islands (PAI), phylogenetic groups and serotypes among strains isolated from pregnant and non-pregnant women. METHODS: One hundred fifty UPEC strains were isolated from pregnant and non-pregnant women from two different Mexican states (Sonora and Puebla). Strains were analyzed using the Kirby-Bauer method for the determination of antibiotic susceptibility and ESBL. Virulence genes, PAIs and phylogenetic groups were determined using a multiplex PCR. Strains were serotyped by an agglutination assay. Blood agar and CAS agar were used for phenotypic assays. RESULTS: 92.7% of UPEC strains showed multidrug-resistant (MDR), 6.7% extremely-resistant (XDR) and 0.6% pandrug-resistant (PDR). The highest resistance was determined to be for ß-lactam antibiotics (>72% in both states) and 44.5% of the UPEC strains were ESBL+. The predominant virulence genes found were fimH (100%), iucD (85%) and iha (60%). The strains isolated from pregnant women from Puebla presented a large percentage of genes associated with upper urinary tract infections. PAIs were found in 51% and 68% of the strains from Sonora and Puebla, respectively. All the strains were siderophores producers and 41.5% produced hemolysis. The serotypes found were diverse and belonged to phylogroups A, B2 and C. CONCLUSION: The UPEC strains from this study are MDR with tendency to XDR or PDR, they can cause upper UTIs and are serotypically and phylogenetically diverse, which supports the need to develop new strategies for UTI treatment in pregnant and non-pregnant Mexican women.

Adherent/invasive Escherichia coli (AIEC) isolates from asymptomatic people: new E. coli ST131 O25:H4/H30-Rx virotypes.

BACKGROUND: The widespread Escherichia coli clone ST131 implicated in multidrug-resistant infections has been recently reported, the majority belonging to O25:H4 serotype and classified into five main virotypes in accordance with the virulence genes carried. METHODS: Pathogenicity Islands I and II (PAI-I and PAI-II) were determined using conventional PCR protocols from a set of four E. coli CTXR ST131 O25:H4/H30-Rx strains collected from healthy donors' stool. The virulence genes patterns were also analyzed and compared them with the virotypes reported previously; then adherence, invasion, macrophage survival and biofilm formation assays were evaluated and AIEC pathotype genetic determinants were investigated. FINDINGS: Non-reported virulence patterns were found in our isolates, two of them carried satA, papA, papGII genes and the two-remaining isolates carried cnfI, iroN, satA, papA, papGII genes, and none of them belonged to classical ST131 virotypes, suggesting an endemic distribution of virulence genes and two new virotypes. The presence of PAI-I and PAI-II of Uropathogenic E. coli was determined in three of the four strains, furthermore adherence and invasion assays demonstrated higher degrees of attachment/invasion compared with the control strains. We also amplified intI1, insA and insB genes in all four samples. INTERPRETATION: The results indicate that these strains own non-reported virotypes suggesting endemic distribution of virulence genes, our four strains also belong to an AIEC pathotype, being this the first report of AIEC in México and the association of AIEC with healthy donors.

Characterization of antimicrobial resistance mechanisms in carbapenem-resistant Pseudomonas aeruginosa carrying IMP variants recovered from a Mexican Hospital.

PURPOSE: Pseudomonas aeruginosa infections in hospitals constitute an important problem due to the increasing multidrug resistance (MDR) and carbapenems resistance. The knowledge of resistance mechanisms in Pseudomonas strains is an important issue for an adequate antimicrobial treatment. Therefore, the objective was to investigate other antimicrobial resistance mechanisms in MDR P. aeruginosa strains carrying bla IMP, make a partial plasmids characterization, and determine if modifications in oprD gene affect the expression of the OprD protein. METHODOLOGY: Susceptibility testing was performed by Kirby Baüer and by Minimum Inhibitory Concentration (presence/absence of efflux pump inhibitor); molecular typing by Pulsed-field gel electrophoresis (PFGE), resistance genotyping and integrons by PCR and sequencing; OprD expression by Western blot; plasmid characterization by MOB Typing Technique, molecular size by PFGE-S1; and bla IMP location by Southern blot. RESULTS: Among the 59 studied P. aeruginosa isolates, 41 multidrug resistance and carbapenems resistance isolates were detected and classified in 38 different PFGE patterns. Thirteen strains carried bla IMP; 16 bla GES and four carried both genes. This study centered on the 17 strains har-boring bla IMP. New variants of ß-lactamases were identified (bla GES-32, bla IMP-56, bla IMP-62) inside of new arrangements of class 1 integrons. The presence of bla IMP gene was detected in two plasmids in the same strain. The participation of the OprD protein and efflux pumps in the resistance to carbapenems and quinolones is shown. No expression of the porin OprD due to stop codon or IS in the gene was found. CONCLUSIONS: This study shows the participation of different resistance mechanisms, which are reflected in the levels of MIC to carbapenems. This is the first report of the presence of three new variants of ß-lactamases inside of new arrangements of class 1 integrons, as well as the presence of two plasmids carrying bla IMP in the same P. aeruginosa strain isolated in a Mexican hospital.

Environmental regulation of the long polar fimbriae 2 of enterohemorrhagic Escherichia coli O157:H7.

The molecular mechanisms controlling expression of the long polar fimbriae 2 (Lpf2) of enterohemorrhagic Escherichia coli (EHEC) O157:H7 were evaluated. Primer extension was used to locate the lpfA2 transcriptional start site in EHEC strain EDL933 at 171 bp upstream of the lpfA2 start codon. Semi-quantitative RT-PCR demonstrated that the highest lpfA2 expression occurs between an OD600 of 1.0 and 1.2 in DMEM at pH 6.5 and 37 °C. The level of lpfA2 transcription at OD600 1.2 and pH 6.5 was four times greater than that at pH 7.2. Although lpfA2 expression was decreased under iron-depleted conditions, its expression was increased in a ferric-uptake-regulator (Fur) mutant strain. The lpfA2 transcript was 0.7 and 2 times more abundant in wt EHEC grown in DMEM pH 6.5 plus iron and MacConkey broth at 25 °C, respectively, than in DMEM at pH 6.5. The lpf2 expression in DMEM pH 6.5 plus iron and bile salts was 2.7 times more abundant than baseline conditions. Further, transcription in the EDL933∆fur was 0.6 and 0.8 times higher as compared with the wt strain grown in DMEM pH 6.5 plus iron and MacConkey broth, respectively. Electrophoretic mobility shift assays showed that purified Fur interacts with the lpf2 regulatory region, indicating that Fur repression is exerted by direct binding to the promoter region. In summary, we demonstrated that the EHEC lpf2 operon is regulated in response to temperature, pH, bile salts and iron, during the exponential phase of growth, and is controlled by Fur.

Clinical implications of enteroadherent Escherichia coli.

Pathogenic Escherichia coli that colonize the small intestine primarily cause gastrointestinal illness in infants and travelers. The main categories of pathogenic E. coli that colonize the epithelial lining of the small intestine are enterotoxigenic E. coli, enteropathogenic E. coli, and enteroaggregative E. coli. These organisms accomplish their pathogenic process by a complex, coordinated multistage strategy, including nonintimate adherence mediated by various adhesins. These so called "enteroadherent E. coli" categories subsequently produce toxins or effector proteins that are either secreted to the milieu or injected to the host cell. Finally, destruction of the intestinal microvilli results from the intimate adherence or the toxic effect exerted over the epithelia, resulting in water secretion and diarrhea. In this review, we summarize the current state of knowledge regarding these enteroadherent E. coli strains and the present clinical understanding of how these organisms colonize the human intestine and cause disease.

Regulatory control of the Escherichia coli O157:H7 lpf1 operon by H-NS and Ler.

Long polar fimbriae 1 (Lpf1) of Escherichia coli O157:H7 is a tightly regulated adhesin, with H-NS silencing the transcriptional expression of the lpf1 operon while Ler (locus of enterocyte effacement-encoded regulator) acts as an antisilencer. We mapped the minimal regulatory region of lpf1 required for H-NS- and Ler-mediated regulation and found that it is 79% AT rich. Three putative sites for H-NS binding were identified. Two of them, named silencer regulatory sequence 1 (SRS1) and SRS2, are located on a region that covers both of the lpf1 promoters (P1 and P2). The third putative H-NS binding site is located within the lpfA1 gene in a region extending from +258 bp to +545 bp downstream of ATG; however, this site does not seem to play a role in lpfA1 regulation under the conditions tested in this work. Ler was also found to interact with Ler binding sites (LBSs). Ler binding site 1 (LBS1) and LBS2 are located upstream of the two promoters. LBS1 overlaps SRS1, while LBS3 overlaps the P1 promoter and SRS2. Based on the experimental data, we propose that H-NS silences lpf1 expression by binding to both of the SRSs on the promoter region, forming an SRS-H-NS complex that prevents RNA polymerase-mediated transcription. A model of the regulation of the lpfA1 operon of E. coli O157:H7 by H-NS and Ler is discussed.

Contribution of the Ler- and H-NS-regulated long polar fimbriae of Escherichia coli O157:H7 during binding to tissue-cultured cells.

The expression of the long polar fimbriae (LPF) of enterohemorrhagic Escherichia coli (EHEC) O157:H7 is controlled by a tightly regulated process, and, therefore, the role of these fimbriae during binding to epithelial cells has been difficult to establish. We recently found that histone-like nucleoid-structuring protein (H-NS) binds to the regulatory sequence of the E. coli O157:H7 lpf1 operon and "silences" its transcription, while Ler inhibits the action of the H-NS protein and allows lpf1 to be expressed. In the present study, we determined how the deregulated expression of LPF affects binding of EHEC O157:H7 to tissue-cultured cells, correlating the adherence phenotype with lpf1 expression. We tested the adherence properties of EHEC hns mutant and found that this strain adhered 2.8-fold better than the wild type. In contrast, the EHEC ler mutant adhered 2.1-fold less than the wild type. The EHEC hns ler mutant constitutively expressed the lpf genes, and, therefore, we observed that the double mutant adhered 5.6-fold times better than the wild type. Disruption of lpfA in the EHEC hns and hns ler mutants or the addition of anti-LpfA serum caused a reduction in adhesion, demonstrating that the increased adherence was due to the expression of LPF. Immunogold-labeling electron microscopy showed that LPF is present on the surface of EHEC lpfA(+) strains. Furthermore, we showed that EHEC expressing LPF agglutinates red blood cells from different species and that the agglutination was blocked by the addition of anti-LpfA serum. Overall, our data confirmed that expression of LPF is a tightly regulated process and, for the first time, demonstrated that these fimbriae are associated with adherence and hemagglutination phenotypes in EHEC O157:H7.

Ler and H-NS, regulators controlling expression of the long polar fimbriae of Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 colonizes the human intestine and is responsible for diarrheal outbreaks worldwide. Previously we showed that EHEC produces long polar fimbriae (LPF) and that maximum expression is observed during the exponential phase of growth at 37 degrees C and pH 6.5. In this study, we analyzed the roles of several regulators in the expression of LPF using the beta-galactosidase reporter system, and we found that H-NS functions as a transcriptional silencer while Ler functions as an antisilencer of LPF expression. Interestingly, deletion of the hns and ler genes in EHEC caused constitutive expression of the fusion reporter protein. Semiquantitative reverse transcription (RT)-PCR was also used to analyze LPF expression in the EHEC ler or hns mutant strain. The hns mutant exhibited an increase in lpf mRNA expression, while expression in the ler mutant was decreased, compared to that in the wild-type strain. Using primer extension analysis, we identified two potential transcriptional start sites within the regulatory region of lpf and located consensus hexamers of -10 (CAAGAT) and -35 (TTCAAA), which are commonly found in sigma(70)-dependent promoters. Further, we determined whether H-NS and Ler interact directly with the lpf promoter region by using purified His-tagged proteins and electrophoretic mobility shift assays. Our data are the first to show direct binding interactions between the H-NS and Ler proteins within the regulatory sequence of the lpf operon. Based on the electrophoretic mobility shift assay, RT-PCR, primer extension, and beta-galactosidase assay results, we concluded that the E. coli O157:H7 lpf operon possesses a promoter dependent on sigma(70), that H-NS binds to the regulatory sequence of lpfA and "silences" the transcription of lpf, and that Ler binds to the regulatory sequence and inhibits the action of the H-NS protein.