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Cruz A.,National Autonomous University of Mexico | Xicohtencatl-Cortes J.,Laboratorio Of Bacteriologia Intestinal | Gonzalez-Pedrajo B.,National Autonomous University of Mexico | Bobadilla M.,Instituto Nacional Of Ciencias Medicas Y Nutricion Salvador Zubiran | And 2 more authors.
Canadian Journal of Microbiology | Year: 2011

Cronobacter spp. (Enterobacter sakazakii) includes gram-negative opportunistic foodborne pathogens known as rare but important causes of life-threatening neonatal infections. However, the pathogenic mechanism is not yet clear. In this study, 43 isolates of Cronobacter, from human and nonhuman sources, were analyzed. A total of four clusters were identified and 32 DNA pulsotypes were observed by pulsed-field gel electrophoresis. In addition, 86% of the Cronobacter isolates were able to adhere to HEp-2 cells and 35% were invasive, Cronobacter sakazakii isolates being the most efficient. Twentysix percent of Cronobacter isolates were able to form biofilms, mainly those from nonhuman sources, such as Cronobacter dublinensis and Cronobacter malonaticus. Three putative virulence genes (siderophore-interacting protein (sip), type III hemolysin (hly), and plasminogen activator (cpa)) were identified by bioinformatic analysis and then detected by PCR. The sip gene was the most frequently detected (60%; 26/43), followed by the hly gene (37%; 16/43) and the cpa gene (28%; 12/43). The three genes were identified primarily in C. sakazakii. Our data show that Cronobacter species harbor different virulence traits.

Xicohtencatl-Cortes J.,Laboratorio Of Bacteriologia Intestinal | Saldana Z.,University of Florida | Deng W.,University of British Columbia | Castaneda E.,Autonomous University of Puebla | And 5 more authors.
Journal of Biological Chemistry | Year: 2010

We present a body of ultrastructural, biochemical, and genetic evidence that demonstrates the oligomerization of virulence-associated autotransporter proteins EspC or EspP produced by deadly human pathogens enterohemorrhagic and enteropathogenic Escherichia coli into novel macroscopic rope-like structures (>1 cm long). The rope-like structures showed high aggregation and insolubility, stability to anionic detergents and high temperature, and binding to Congo Red and thioflavin T dyes. These are properties also exhibited by human amyloidogenic proteins. These macroscopic ropes were not observed in cultures of nonpathogenic Escherichia coli or isogenic espP or espC deletion mutants of enterohemorrhagic or enteropathogenic Escherichia coli but were produced by an Escherichia coli K-12 strain carrying a plasmid expressing espP. Purified recombinant EspP monomers were able to self-assemble into macroscopic ropes upon incubation, suggesting that no other protein was required for assembly. The ropes bound to and showed cytopathic effects on cultured epithelial cells, served as a substratum for bacterial adherence and biofilm formation, and protected bacteria from antimicrobial compounds. We hypothesize that these ropes play a biologically significant role in the survival and pathogenic scheme of these organisms. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Saldana Z.,University of Florida | Sanchez E.,University of Costa Rica | Xicohtencatl-Cortes J.,Laboratorio Of Bacteriologia Intestinal | Puente J.L.,National Autonomous University of Mexico | Giron J.A.,University of Florida
Frontiers in Microbiology | Year: 2011

Shiga-toxigenic Escherichia coli (STEC) O157:H7 uses a myriad of surface adhesive appendages including pili, flagella, and the type 3 secretion system (T3SS) to adhere to and inflict damage to the human gut mucosa. Consumption of contaminated ground beef, milk, juices, water, or leafy greens has been associated with outbreaks of diarrheal disease in humans due to STEC. The aim of this study was to investigate which of the known STEC O157:H7 adherence factors mediate colonization of baby spinach leaves and where the bacteria reside within tainted leaves. We found that STEC O157:H7 colonizes baby spinach leaves through the coordinated production of curli, the E. coli common pilus, hemorrhagic coli type 4 pilus, flagella, and T3SS. Electron microscopy analysis of tainted leaves revealed STEC bacteria in the internal cavity of the stomata, in intercellular spaces, and within vascular tissue (xylem and phloem), where the bacteria were protected from the bactericidal effect of gentamicin, sodium hypochlorite or ozonated water treatments. We confirmed that the T3S escN mutant showed a reduced number of bacteria within the stomata suggesting that T3S is required for the successful colonization of leaves. In agreement, non-pathogenic E. coli K-12 strain DH5α transformed with a plasmid carrying the locus of enterocyte effacement (LEE) pathogenicity island, harboring the T3SS and effector genes, internalized into stomata more efficiently than without the LEE. This study highlights a role for pili, flagella, and T3SS in the interaction of STEC with spinach leaves. Colonization of plant stomata and internal tissues may constitute a strategy by which STEC survives in a nutrient-rich microenvironment protected from external foes and may be a potential source for human infection. © 2011 Saldaña, Sánchez, Xicohtencatl-Cortes, Puente and Girón.

Ochoa S.A.,Laboratorio Of Bacteriologia Intestinal | Lopez-Montiel F.,Laboratorio Of Bacteriologia Intestinal | Escalona G.,Laboratorio Of Bacteriologia Intestinal | Cruz-Cordova A.,Laboratorio Of Bacteriologia Intestinal | And 7 more authors.
Boletin Medico del Hospital Infantil de Mexico | Year: 2013

Background. In recent years, the worldwide emergence of multidrug-resistant strains of Pseudomonas aeruginosa has been observed. This opportunistic pathogen produces mechanisms of resistance to several antibiotics. The resistance to carbapenems in P. aeruginosa strains has been associated with bacterial biofilm formation, favored by the presence of exopolysaccharides (EPS) embedded in an extracellular matrix and to the production of type IV pili (T4P). We undertook this study to assess biofilm formation in clinical strains of P. aeruginosa resistant to carbapenems isolated at the Hospital Infantil de Mexico Federico Gomez (HIMFG) through quantification of total-reducing EPS and its association with the phenotypic expression of T4P. Methods. Antibiotic susceptibility tests were performed using the Kirby-Bauer method in 92 clinical isolates of P. aeruginosa; likewise, the minimum inhibitory concentration (MIC) was determined for imipenem (IMP) and meropenem (MEM) by the serial dilution method in agar plates with a Steers replicator. Production of metallo-β-lactamase (MBL) was determined by the disk diffusion method and synergism. Biofilm formation was performed in clinical isolates of P. aeruginosa resistant to carbapenems through the quantification of crystal violet, total sugar (anthrone), and reducing sugar (DNS), in addition to the phenotypic expression of T4P activity of twitching motility. The genetic diversity of strains forming biofilm and producing reducing sugars was evaluated by pulsed-field gel electrophoresis (PFGE).

Ochoa S.A.,Laboratorio Of Bacteriologia Intestinal | Escalona G.,Laboratorio Of Bacteriologia Intestinal | Cruz-Cordova A.,Laboratorio Of Bacteriologia Intestinal | Davila L.B.,Laboratorio Of Bacteriologia Intestinal | And 6 more authors.
BMC Microbiology | Year: 2013

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. © 2013 Ochoa et al.; licensee BioMed Central Ltd.

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