Phenotypic and genotypic characteristics of Escherichia coli strains isolated during a longitudinal follow-up study of chronic urinary tract infections.

Worldwide, Urinary Tract Infections (UTIs) are an important health problem with many cases reported annually, women being the most affected. UTIs are relevant because they can become a recurrent condition, associated with different factors that contribute to the chronicity of the disease (cUTI). cUTI can be classified as persistent (peUTI) when the causative agent is the same each time the infection occurs or as reinfection (reUTI) when the associated microorganism is different. The purpose of this work was to characterize Escherichia coli isolates obtained in two prospective studies of patients with cUTI, to define which of them corresponded to peUTI and which to reUTI. A total of 394 isolates of E. coli were analyzed by agglutination with specific sera, antimicrobial susceptibility by diffusion disc test, and the phylogroups and presence of genes associated with virulence by PCR assays. Additionally, in some characterized strains adherence, invasiveness, and biofilm formation were analyzed by in vitro assays. The results showed that the peUTI strains belonged mainly to the classical UPEC serogroups (O25, O75, O6), were included in the B2 phylogroup, carried a great number of virulence genes, and were adherent, invasive, and biofilm-forming. Meanwhile, reUTI strains showed great diversity of serogroups, belonged mainly in the A phylogroup, and carried fewer virulence genes. Both peUTI and reUTI strains showed extensively drug-resistant (XDR) and multidrug-resistant (MDR) profiles in the antimicrobial susceptibility test. In conclusion, it appears that peUTIs are caused principally by classical UPEC strains, while reUTIs are caused by strains that appear to be a part of the common E. coli intestinal biota. Moreover, although both peUTI and reUTI strains presented different serotypes and phylogroups, their antimicrobial resistance profile (XDR and MDR) was similar, confirming the importance of regulating prophylactic treatments and seeking alternatives for the treatment and control of cUTI. Finally, it was possible to establish the features of the E. coli strains responsible for peUTI and reUTI which could be helpful to develop a fast diagnostic methodology.

In Vitro Effect of the Cell-Free Supernatant of the Lactobacillus casei Strain IMAU60214 against the Different Pathogenic Properties of Diarrheagenic Escherichia coli.

Enteroaggregative Escherichia coli (EAEC) and enterohemorrhagic E. coli (EHEC) are E. coli pathotypes associated with unmanageable diarrhea in children and adults. An alternative to the treatment of infections caused by these microorganisms is the use of the bacteria of the Lactobacillus genus; however, the beneficial effects on the intestinal mucosa are specific to the strain and species. The interest of this study consisted of analyzing the coaggregation properties of Lactobacillus casei IMAU60214, as well as the effect of cell-free supernatant (CSF) on growth and anti-cytotoxic activity in a cell model of the human intestinal epithelium for an agar diffusion assay (HT-29) and the inhibition of biofilm formation on plates of DEC strains of the EAEC and EHEC pathotypes. The results showed that L. casei IMAU60214 exhibits time-dependent coaggregation (35-40%) against EAEC and EHEC that is similar to the control E. coli ATCC 25922. The CSF showed antimicrobial activity (20-80%) against EAEC and EHEC depending on the concentration. In addition, the formation and dispersion of biofilms of the same strains decrease, and the proteolytic pre-treatment with catalase and/or proteinase K (1 mg/mL) of CSF reduces the antimicrobial effect. When evaluating the effect in HT-29 cells pre-treated with CFS on the toxic activity induced by the EAEC and EHEC strains, a reduction of between 30 and 40% was observed. The results show that L. casei IMAU60214 and its CSF have properties that interfere with some properties associated with the virulence of the EAEC and EHEC strains that cause intestinal infection, which supports their use for the control and prevention of infections caused by these bacteria.

Prospective Study in Children with Complicated Urinary Tract Infection Treated with Autologous Bacterial Lysates.

Antimicrobial bacteria resistance is an important problem in children with recurrent urinary tract infections (rUTI), thus it is crucial to search for alternative therapies. Autologous bacterial lysates (ABL) may be a potential treatment for rUTI. Twenty-seven children with rUTI were evaluated for one year, urine and stool cultures were performed, 10 colonies of each culture were selected and those identified as Escherichia coli were characterized by serology. For patients who presented ≥105 UFC/mL, an ABL was manufactured and administered orally (1 mL/day) for a month. Twelve children were monitored for ≥1-year, 218 urine and 11 stool samples were analyzed. E. coli (80.5%) was the main bacteria isolated from urine and feces (72%). E. coli of classical urinary serotypes (UPEC), O25:H4, O75:HNM, and O9:HNM were identified in patients with persistent urinary infection (pUTI). In 54% of patients treated with ABL, the absence of bacteria was observed in urine samples after 3 months of treatment, 42% of these remained without UTI between 10-12 months. It was observed that the use of ABL controlled the infection for almost 1 year in more than 60% of the children. We consider it necessary to develop a polyvalent immunogen for the treatment and control of rUTI.

Probiotic Properties and Immunomodulatory Activity of Lactobacillus Strains Isolated from Dairy Products.

Lactobacilli species are an effective biotherapeutic alternative against bacterial infections and intestinal inflammatory disorders. However, it is important to evaluate their beneficial properties, before considering them as probiotics for medical use. In this study we evaluated some probiotic properties of Lactobacillus rhamnosus GG, Lactobacillus rhamnosus KLSD, Lactobacillus helveticus IMAU70129, and Lactobacillus casei IMAU60214 previously isolated from dairy products and as control Lactobacillus casei Shirota. Experimental evaluations revealed that all strains expressed hydrophobicity (25-40%), auto-aggregation (55-60%), NaCl tolerance (1-4%), adhesion to Caco-2 cells (25-33%), partial inhibition on adherence of Escherichia coli ATCC 35218, Salmonella Typhimurium ATCC 14028, and Staphylococcus aureus ATCC 23219. Cell-free supernatants (CFS) of Lactobacilli also inhibit growth of these pathogens. In immunomodulatory properties a reduction of interleukin-8 (IL-8) and nitric oxide (NO) release was observed in assays with Caco-2 cells stimulated with interleukin-1ß (1 ng/mL), or lipopolysaccharide (0.1 µg/mL). On the other hand, the damage induced to Caco-2 cells with sodium dodecyl sulfate (SDS) was attenuated when the cultured cells were pretreated with L. rhamnosus KLDS, L. helveticus IMAU70129 and L. casei IMAU60214. These Lactobacilli possess probiotic properties determined by both an antagonistic activity on pathogenic bacteria and reduction in the inflammatory response of cells treated with SDS, a pro-inflammatory stimulant.

Effect and Analysis of Bacterial Lysates for the Treatment of Recurrent Urinary Tract Infections in Adults.

Urinary tract infection (UTI) is a relevant public health problem, economically and socially affecting the lives of patients. The increase of antimicrobial bacterial resistance significantly hinders the treatment of UTIs, raising the need to search for alternative therapies. Bacterial lysates (BL) obtained from Escherichia coli and other pathogens have been used to treat different infectious diseases with promising results. This work aims to evaluate the effect and composition of an autologous BL for the treatment and control of recurrent UTIs in adults. The results show remission in 70% of the patients within the first three months after the administration of BL, while the infection is maintained under control for 6-12 months. The analysis by liquid chromatography-mass spectrometry (LC-MS) of the BL fractions recognized by the sera of patients shows the presence of cytosolic proteins, fimbriae, OMPs, and LPS. Our study demonstrates that the autologous BL contributed to the treatment and control of recurrent UTIs in adults, and its composition shows that different surface components of E. coli are potential immunogens that could be used to create a polyvalent protective vaccine.

Characterization of auto-agglutinating and non-typeable uropathogenic Escherichia coli strains.

INTRODUCTION: Uropathogenic Escherichia coli (UPEC) are the main etiological agent of urinary tract infections (UTIs). Association between different serotypes and UTIs is known, however, some strains are incapable to be serotyped. The aim of this work was to study bthe phenotypical and genotypical characteristics of 113 non-typeable (NT) and auto-agglutinating (AA) E. coli strains, isolated from UTIs in children and adults. METHODOLOGY: The 113 UPEC strains were analyzed by PCR assays using specific primers to determine their serogroups, fimH, papC, iutA, sat, hlyCA and cnf1, virulence associated genes, and chuA, yjaA and TSPE4.C2 for phylogroup determination. Additionally, the diffusion disk method was performed to evaluate the antimicrobial resistance to 18 antimicrobial agents. RESULTS: Using the PCR assay, 63% (71) of the strains were genotyped showing O25 and O75 as the most common serogroups. The virulence genes fimH (86%) and iutA (74%) were the most prevalent, in relation to the phylogroups the commensal (A and B1) and virulent (B2 and D) showed similar frequencies (P > 0.05). The antimicrobial susceptibility test showed a high percentage (73%) of multidrug-resistant strains. CONCLUSIONS: The genotyping allowed identifying the serogroup in many of the strains that could not be typed by traditional serology. The strains carried virulence genes and were multidrug-resistant in both, commensal and virulent phylogroups. Our findings revealed that, in addition to the classical UPEC serogroups, there are pathogenic serogroups not reported yet.

Effects of static magnetic fields on the enteropathogenic Escherichia coli.

This study reports the effects of exposing cells of the prototypical enteropathogenic Escherichia coli (EPEC) strain E2348/69 to static magnetic fields (SMF) of varying intensities to observe their capacity to autoaggregate and the effect on cell adherence. The results showed that bacteria exposure over the course of 5 min to an intensity of 53 mT reduced autoaggregation by 28%. However, with intensities of up to 100 mT with the same exposure time, bacteria autoaggregation was reduced by approximately 50%; and after 30 min at the same intensity, it was indistinguishable from that observed in a non-autoaggregative strain. Furthermore, it was observed that SMF treatment also modified the typical localized adherence pattern of EPEC E2348/69. The observed effects are not related to bacteria damage. The above was confirmed because, after a 107 mT SMF treatment over the course of 30 min, cell viability and membrane permeability were the same to that observed in untreated controls. The obtained results suggest that the SMF effect on the E2348/69 EPEC strain alters the expression of the bundle-forming pilus (BFP), due to the fact that the same strain without the EPEC adherence factor plasmid that encodes the BFP operon was unable to autoaggregate. Electron microscopic analyses revealed structural differences between cells exposed to SMF with respect to untreated controls. In conclusion, the SMF treatment of 107 mT for 30 min reduced EPEC E2348/69 autoaggregation and modified its adherence pattern, with both events likely being associated with changes in BFP expression. Bioelectromagnetics. 38:570-578, 2017. © 2017 Wiley Periodicals, Inc.

Molecular analysis and distribution of multidrug-resistant Enterococcus faecium isolates belonging to clonal complex 17 in a tertiary care center in Mexico City.

BACKGROUND: Enterococcus faecium has recently emerged as a multidrug-resistant nosocomial pathogen involved in outbreaks worldwide. A high rate of resistance to different antibiotics has been associated with virulent clonal complex 17 isolates carrying the esp and hyl genes and the purK1 allele. RESULTS: Twelve clinical vancomycin-resistant Enterococcus faecium (VREF) isolates were obtained from pediatric patients at the Hospital Infantil de México Federico Gómez (HIMFG). Among these VREF isolates, 58.3% (7/12) were recovered from urine, while 41.7% (5/12) were recovered from the bloodstream. The VREF isolates showed a 100% rate of resistance to ampicillin, amoxicillin-clavulanate, ciprofloxacin, clindamycin, chloramphenicol, streptomycin, gentamicin, rifampicin, erythromycin and teicoplanin. In addition, 16.7% (2/12) of the isolates were resistant to linezolid, and 66.7% (8/12) were resistant to tetracycline and doxycycline. PCR analysis revealed the presence of the vanA gene in all 12 VREF isolates, esp in 83.3% (10/12) of the isolates and hyl in 50% (6/12) of the isolates. Phylogenetic analysis via molecular typing was performed using pulsed-field gel electrophoresis (PFGE) and demonstrated 44% similarity among the VREF isolates. MLST analysis identified four different sequence types (ST412, ST757, ST203 and ST612). CONCLUSION: This study provides the first report of multidrug-resistant VREF isolates belonging to clonal complex 17 from a tertiary care center in Mexico City. Multidrug resistance and genetic determinants of virulence confer advantages among VREF in the colonization of their host. Therefore, the prevention and control of the spread of nosocomial infections caused by VREF is crucial for identifying new emergent subclones that could be challenging to treat in subsequent years.

The hemorrhagic coli pilus (HCP) of Escherichia coli O157:H7 is an inducer of proinflammatory cytokine secretion in intestinal epithelial cells.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) O157:H7, the causative agent of hemorrhagic colitis and the hemolytic uremic syndrome (HUS), produces long bundles of type IV pili (TFP) called hemorrhagic coli pili (HCP). HCP are capable of mediating several phenomena associated with pathogenicity: i) adherence to human and bovine epithelial cells; ii) invasion of epithelial cells; iii) hemagglutination of rabbit erythrocytes; iv) biofilm formation; v) twitching motility; and vi) specific binding to laminin and fibronectin. HCP are composed of a 19 kDa pilin subunit (HcpA) encoded by the hcpA chromosomal gene (called prepilin peptidase-dependent gene [ppdD] in E. coli K-12). METHODOLOGY/PRINCIPAL FINDINGS: In this study we investigated the potential role of HCP of E. coli O157:H7 strain EDL933 in activating the release of pro- and anti-inflammatory cytokines from a variety of host epithelial cells. We found that purified HCP and a recombinant HcpA protein induced significant release of IL-8 and TNF-alpha, from cultured polarized intestinal cells (T84 and HT-29 cells) and non-intestinal HeLa cells. Levels of proinflammatory IL-8 and TNF-alpha, but not IL-2, IL6, or IL-10 cytokines, were increased in the presence of HCP and recombinant HcpA after 6 h of incubation with >or=50 ng/ml of protein, suggesting that stimulation of IL-8 and TNF-alpha are dose and time-dependent. In addition, we also demonstrated that flagella are potent inducers of cytokine production. Furthermore, MAPK activation kinetics studies showed that EHEC induces p38 phosphorylation under HCP-producing conditions, and ERK1/2 and JNK activation was detectable after 3 h of EHEC infection. HT-29 cells were stimulated with epidermal growth factor stimulation of HT-29 cells for 30 min leading to activation of three MAPKs. CONCLUSIONS/SIGNIFICANCE: The HcpA pilin monomer of the HCP produced by EHEC O157:H7 is a potent inducer of IL-8 and TNF-alpha release, an event which could play a significant role in the pathogenesis of hemorrhagic colitis caused by this pathogen.

The majority of enteroaggregative Escherichia coli strains produce the E. coli common pilus when adhering to cultured epithelial cells.

Enteroaggregative Escherichia coli (EAEC) have emerged as a significant worldwide cause of chronic diarrhea in the pediatric population and in HIV patients. The vast majority of EAEC strains do not produce the aggregative adherence fimbriae I-III (AAFs) so far reported and thus, what adherence factors are present in these strains remains unknown. Here, we investigated the prevalence of the chromosomal E. coli common pilus (ECP) genes and ECP production amongst 130 EAEC strains of diverse origin as well as the role of ECP in EAEC adherence. Through multiplex PCR analysis we found that 96% of EAEC strains contained the ecpA structural pilin gene whereas only 3.1% and 5.4% were positive for AAF fimbrial genes aggA or aafA, respectively. Among the ecpA(+) strains, 63% produced ECP when adhering to cultured epithelial cells. An ecpA mutant derived from prototypic strain 042 (AAF/II(+)) was not altered in adherence suggesting that the AAF/II, and not ECP, plays a major role in this strain. In contrast, strain 278-1 (AAF(-)) deleted of the ecpA gene was significantly reduced in adherence to cultured epithelial cells. In all, these data indicate a potential role of ECP in adherence for EAEC strains lacking the known AAFs and that in association with other adhesive determinants, ECP may contribute to their survival and persistence within the host and in the environment.