Identification and characterization of class 1 integrons among multidrug-resistant uropathogenic Escherichia coli strains in Mexico.

This study aimed to identify and characterize integrons among multidrug-resistant (MDR) uropathogenic Escherichia coli (UPEC) from outpatients in Mexico City, Mexico. PCR assays were used to screen for the presence of class 1, 2 and 3 integrons, whose PCR products were sequenced to identify the inserted gene cassettes within the variable regions. Out of 83 tested strains, 53 (63.9%) were positive for the presence of class 1 integrons, whereas no integrons were detected in the remaining strains, regardless of their classes. Most of the strains carrying the intI1 gene belonged to the extraintestinal B2 (41.5%) and commensal A (32.1%) phylogroups, and to a lesser extent, the extraintestinal D (20.8%) and commensal B1 (5.7%) phylogroups. Moreover, 8 different gene cassette arrangements were detected, with dfrA17 and aadA5 being the most common (32.1% of the class 1 integron-positive strains), which confer resistance to trimethoprim/sulfamethoxazole and aminoglycosides, respectively. Our results suggest that class 1 integrons are widely distributed among MDR-UPEC strains in Mexico, which may directly or indirectly contribute to the selection of MDR strains. These findings are important for a better understanding of the factors and mechanisms that promote multidrug resistance among UPEC strains.

Cost-effective flow-through nanohole array-based biosensing platform for the label-free detection of uropathogenic E. coli in real time.

Rapid, inexpensive and sensitive detection of uropathogenic Escherichia coli (UPEC), a common cause of ascending urinary tract infections (UTIs) including cystitis and pyelonephritis, is critical given the increasing number of cases and its recurrence worldwide. In this paper, we present a label-free nanoplasmonic sensing platform, built with off-the-shelf optical and electronic components, which can detect intact UPEC at concentrations lower than the physiological limit for UTI diagnosis, in real time. The sensing platform consists of a red LED light source, lens assembly, CMOS detector, Raspberry Pi interface in conjugation with a metallic flow-through nanohole array-based sensor. Detection is achieved exploiting nanoplasmonic phenomena from the nanohole arrays through surface plasmon resonance imaging (SPRi) technique. The platform has a bulk sensitivity of 212 pixel intensity unit (PIU)/refractive index unit (RIU), and a resolution in the order of 10-6 RIU. We demonstrate capture and detection of UPEC with a detection limit of ~100 CFU/ml - a concentration well below the threshold limit for UTI diagnosis in clinical samples. We also demonstrate detection of UPEC in spiked human urine samples for two different concentrations of bacteria. This work is particularly relevant for point-of-care applications, especially for regions around the world where accessibility to medical facilities is heavily dependent upon economy, and availability.

Characterization of Escherichia coli causing community acquired urinary tract infections in Mexico City.

The O25-ST131 clone was identified within 169 uropathogenic Escherichia coli (UPEC) strains. The 44.8% of the 29 O25-ST131 clones detected were positive to least to one extended-spectrum ß-lactamase gene. The phylogroup D was mainly found. The O25-ST131 clone appeared to be associated with community-acquired UTI in Mexico City.

Escherichia coli clonal group A among uropathogenic infections in Mexico City.

Escherichia coli clonal group A (CGA) causes urinary tract and other extra-intestinal infections in humans. CGA is an important cause of trimethoprim/sulfamethoxazole (SXT) resistance in extra-intestinal pathogens. We examined the extent to which resistance in this area is related to CGA dissemination of E. coli from urinary tract infections (UTIs) in Mexico City. The virulence backgrounds of the isolates were also characterized. In this study, the frequency of resistance to SXT used for UTI treatment was high (56-65 %), and CGA isolates accounted for 9 of the 78 SXT-resistant isolates (11.5 %). Although all CGA isolates were found to be multidrug resistant (MDR), none of them were extended-spectrum ß-lactamase-producing organisms. The prevalence of CGA among the 45 MDR isolates that we identified was 20 %, indicating that this clonal group moderately contributes to the antibiotic resistance of uropathogenic E. coli isolates in this region. Most of the nine CGA isolates carried transferable, large-size plasmids of approximately 80 to 100 kb, which were able to transfer antimicrobial resistance to E. coli J53 in mating assays. CGA isolates mainly belonged to phylogenetic groups F and D. We found no association between antimicrobial resistance and virulence-associated genes: the median virulence scores of CGA isolates were slightly higher (4.6) than those of non-CGA isolates, whether they were susceptible (3.7) or resistant (3.5) to SXT. Our results indicate that CGA is not a major contributor to the high level of resistance to SXT in this region but, instead, seems to be an important constituent of MDR isolates from UTIs.

Identification of secretory immunoglobulin A antibody targets from human milk in cultured cells infected with enteropathogenic Escherichia coli (EPEC).

Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system (T3SS) to inject effectors into host cells and alter cellular physiology. The aim of the present study was to identify targets of human secretory immunoglobulin A (sIgA) antibodies from the proteins delivered by EPEC into HEp-2 cells after infection. Bacterial proteins delivered into EPEC-infected cells were obtained in sub-cellular fractions (cytoplasmic, membrane, and cytoskeleton) and probed with sIgA antibodies from human milk and analyzed by Western blotting. These sIgA antibodies reacted with Tir and EspB in the cytoplasmic and membrane fractions, and with intimin in the membrane fraction mainly. The sIgA also identified an EPEC surface-associated Heat-shock protein 70 (Hsp70) in HEp-2 cells infected with EPEC. Purified Hsp70 from EPEC was able to bind to HEp-2 cells, suggesting adhesive properties in this protein. EspC secreted to the medium reacted strongly with the sIgA antibodies. An EPEC 115 kDa protein, unrelated to the EspC protein, was detected in the cytoplasm of infected HEp-2 cells, suggesting that this is a new protein translocated by EPEC. The results suggest that there is a strong host antibody response to Tir and intimin, which are essential proteins for attaching and effacing (A/E) pathogen mediated disease.

Drug resistance, serotypes, and phylogenetic groups among uropathogenic Escherichia coli including O25-ST131 in Mexico City.

INTRODUCTION: The increasing prevalence of uropathogenic Escherichia coli (UPEC) strains resistant to multiple antibiotics complicates the treatment of urinary tract infections (UTIs). This study aimed to analyze the antimicrobial resistance, serotypes, and phylogenetic groups among strains of E. coli isolated from outpatients with UTIs in Mexico City. METHODOLOGY: A total of 119 E. coli isolates were recovered from urine samples from outpatients with clinical diagnosis of uncomplicated UTIs from 2004 to 2007. The serotype was assessed by agglutination in microtiter plates; susceptibility to antimicrobials was determined by the disk diffusion method. Clone O25-ST131 and phylogenetic groups of E. coli strains were tested by methods based on PCR multiplex. RESULTS: The predominant serotype was O25:H4 (21.2%). Resistance to antibiotics was ampicillin (83.7%); piperacillin (53.8%); the fluoroquinolone group (55.5-60.6%), and trimethoprim/sulfamethoxazole (TMP/SMX) (56.4%). Additionally, 36 (30.2%) isolates were multidrug-resistant and 13 of these 36 strains were identified as E. coli O25-ST131 clone by an allele-specific PCR-based assay. Phylogenetic analysis showed that 15 of 17 isolates with serotype O25:H4 belonged to group B2. CONCLUSIONS: This is the first report that establishes the presence in Mexico of the O25-ST131 clonal group of E. coli, which has been associated with multidrug-resistance and with high virulence potential. The spread of this clone in Mexico should be monitored closely. We found a correlation between serotype O25:H4 and multidrug resistance in UPEC strains. Our results indicate that the use of ampicillin, fluoroquinolones, and TMP/SMX should be reviewed when selecting empirical therapy for UTIs.

Antibody response in mice immunized by mucosal routes with formalin-inactivated enteropathogenic Escherichia coli (EPEC) strains.

Secretory and systemic antibody response in mice against enteropathogenic Escherichia coli (EPEC) was evaluated. Groups of mice were immunized with formalin inactivated EPEC 0127:H6 strain by intranasal, peroral, intragastric and intrarectal route, with and without cholera toxin (CT) used as mucosal adjuvant. Mice immunized subcutaneously and a non treated control group were included. Other groups of mice were immunized intranasally with different EPEC strains and a non pathogenic E. coli K12 strain. Antibody response tested by ELISA assay showed that specific anti EPEC 0127:H6 antibodies were induced in serum by intranasal, subcutaneous and intragastric routes. A strong increase of antibody response against EPEC 0127:H6 strain was observed in saliva after intranasal delivery, while a lower response was detected by peroral and intrarectal immunization. Only the intranasal route increased IgA anti EPEC 0127:H6 antibody titers in feces. Specific and cross reactive antibodies to EPEC 0127:H6 were seen in mice immunized intranasally with different EPEC strains. Some control mice showed a background of anti EPEC 0127:H6 antibodies in feces. CT had a negative effect as adjuvant. We showed that nasal mucosa rendered the strongest antibody response in serum and secretions. These results might contribute to optimize the protective effect of enteric vaccines against infections associated to EPEC.