Identification of virulence factors genes in Escherichia coli isolates from women with urinary tract infection

E coli isolates (108) from Mexican women, clinically diagnosed with urinary tract infection, were screened to identify virulence genes, phylogenetic groups, and antibiotic resistance. Isolates were identified by MicroScan4 system; additionally, the minimum inhibitory concentration (MIC) was assessed. The phylogenetic groups and 16 virulence genes encoding adhesins, toxins, siderophores, lipopolysaccharide (LPS), and invasins were identified by PCR. Phylogenetic groups distribution was as follows: B1 9.3%, A 30.6%, B2 55.6%, and D 4.6%. Virulence genes prevalence was ecp 98.1%, fimH 86.1%, traT 77.8%, sfa/focDE 74.1%, papC 62%, iutA 48.1%, fyuA 44.4%, focG 2.8%, sfaS 1.9%, hlyA 7.4%, cnf-1 6.5%, cdt-B 0.9%, cvaC 2.8%, ibeA 2.8%, and rfc 0.9%. Regarding antimicrobial resistance it was above 50% to ampicillin/sulbactam, ampicillin, piperacillin, trimethoprim/sulfamethoxazole, ciprofloxacin, and levofloxacin. Uropathogenic E. coli clustered mainly in the pathogenic phylogenetic group B2. The isolates showed a high presence of siderophores and adhesion genes and a low presence of genes encoding toxins. The high frequency of papC gene suggests that these isolates have the ability to colonize the kidneys. High resistance to drugs considered as first choice treatment such as trimethoprim/sulfamethoxazole and fluoroquinolones was consistently observed

Nocardia transvalensis Disseminated Infection in an Immunocompromised Patient with Idiopathic Thrombocytopenic Purpura

Nocardia transvalensis complex includes a wide range of microorganisms with specific antimicrobial resistance patterns. N. transvalensis is an unusual Nocardia species. However, it must be differentiated due to its natural resistance to aminoglycosides while other Nocardia species are susceptible. The present report describes a Nocardia species involved in an uncommon clinical case of a patient with idiopathic thrombocytopenic purpura and pulmonary nocardiosis. Microbiological and molecular techniques based on the sequencing of the 16S rRNA gene allowed diagnosis of Nocardia transvalensis sensu stricto. The successful treatment was based on trimethoprim-sulfamethoxazole and other drugs. We conclude that molecular identification of Nocardia species is a valuable technique to guide good treatment and prognosis and recommend its use for daily bases diagnosis

Frecuencia de M. hyopneumoniae, M. hyorhinis y M. hyosynoviae en muestras nasales y de pulmón de cerdos con síntomas de neumonía enzoótica porcina

M. hyopneumoniae, M. hyorhinis and M. hyosynoviae are genetically related species of the genus Mycoplasma that affect pig production. The objective of this work was the isolation and identification by PCR of M. hyopneumoniae, M. hyorhinis and M. hyosynoviae from nasal swabs and lung samples of pigs from different regions of Mexico in order to determine the frequency of these species and to evaluate PCR as a diagnostic tool for PEP. Pigs aged 4 to 8 weeks with clinical diagnosis of PEP were included. Lung samples and nasal swabs were obtained for the isolation of the Mycoplasma in liquid Friis medium and identified by species-specific PCR based on the 16S rRNA subunit. Isolation was achieved in 37.11 % (36/97) of the samples. The three Mycoplasma species were identified in lung and nasal swab samples. Mycoplasma co-infection was identified in 27.77 % (10/36). The bacterial genera associated with Mycoplasma infections were E. coli, Bordetella, Enterobacter, SCN, Corynebacterium, Pasteurella, Streptococcus, Shigella and Klebsiella. Mixed infection was present in 26 nasal swabs (45.61 %) and absent in the lungs. It was concluded that the frequency of  Mycoplasma on  production  farms  was higher  than  expected (40.27 %). It was also identified other Mycoplasma species involved in the development of PEP. Therefore, surveillance through isolation and molecular techniques can be of great help to breeding stock providers, as well as for removing Mycoplasma from pig farms.M. hyopneumoniae, M. hyorhinis y M. hyopsynoviae son especies genéticamente relacionadas del género Mycoplasma que afectan la producción porcina. El objetivo de este trabajo fue el aislamiento e identificación por PCR de M. hyopneumoniae, M. hyorhinis y M. hyosynoviae a partir de hisopos nasales y muestras de pulmón de cerdos de diferentes regiones mexicanas para determinar la frecuencia de estas especies y evaluar la PCR como herramienta diagnóstica para NEP. Se incluyeron cerdos de 4 a 8 semanas con diagnóstico clínico de NEP. Se obtuvieron muestras de pulmón e hisopos nasales para el aislamiento de Mycoplasma en medio Friis líquido y se identificaron mediante la PCR especie-específica basada en la subunidad 16S rRNA. El aislamiento se logró en 37.11 % (36/97) muestras. Las tres especies de Mycoplasma se identificaron en muestras de pulmón e hisopos nasales. La co-infección por Mycoplasma se identificó en el 27.77 % (10/36). Los géneros bacterianos asociados a las infecciones por Mycoplasma fueron E. coli, Bordetella, Enterobacter, SCN, Corynebacterium, Pasteurella, Streptococcus, Shigella y Kebsiella. La infección mixta estuvo presente en 26 hisopos nasales (45.61 %) y ausente en pulmones. Se concluyó que la frecuencia de Mycoplasma en las fincas de producción fue mayor a la esperada (40.27 %). También se identificaron otras especies de Mycoplasma involucradas en el desarrollo de la NEP. Por lo tanto, la vigilancia asistida por el aislamiento y las técnicas moleculares pueden ser de gran ayuda para la eliminación de Mycoplasma de las explotaciones porcinas y para los proveedores de pie de cría

Production of the Escherichia coli common pilus by uropathogenic E. coli is associated with adherence to HeLa and HTB-4 cells and invasion of mouse bladder urothelium.

Uropathogenic Escherichia coli (UPEC) strains cause urinary tract infections and employ type 1 and P pili in colonization of the bladder and kidney, respectively. Most intestinal and extra-intestinal E. coli strains produce a pilus called E. coli common pilus (ECP) involved in cell adherence and biofilm formation. However, the contribution of ECP to the interaction of UPEC with uroepithelial cells remains to be elucidated. Here, we report that prototypic UPEC strains CFT073 and F11 mutated in the major pilin structural gene ecpA are significantly deficient in adherence to cultured HeLa (cervix) and HTB-4 (bladder) epithelial cells in vitro as compared to their parental strains. Complementation of the ecpA mutant restored adherence to wild-type levels. UPEC strains produce ECP upon growth in Luria-Bertani broth or DMEM tissue culture medium preferentially at 26°C, during incubation with cultured epithelial cells in vitro at 37°C, and upon colonization of mouse bladder urothelium ex vivo. ECP was demonstrated on and inside exfoliated bladder epithelial cells present in the urine of urinary tract infection patients. The ability of the CFT073 ecpA mutant to invade the mouse tissue was significantly reduced. The presence of ECP correlated with the architecture of the biofilms produced by UPEC strains on inert surfaces. These data suggest that ECP can potentially be produced in the bladder environment and contribute to the adhesive and invasive capabilities of UPEC during its interaction with the host bladder. We propose that along with other known adhesins, ECP plays a synergistic role in the multi-step infection of the urinary tract

Identification of Virulence Factors Genes in Escherichia coli Isolates from Women with Urinary Tract Infection in Mexico

E coli isolates (108) from Mexican women, clinically diagnosed with urinary tract infection, were screened to identify virulence genes, phylogenetic groups, and antibiotic resistance. Isolates were identified by MicroScan4 system; additionally, the minimum inhibitory concentration (MIC) was assessed. The phylogenetic groups and 16 virulence genes encoding adhesins, toxins, siderophores, lipopolysaccharide (LPS), and invasins were identified by PCR. Phylogenetic groups distribution was as follows: B1 9.3%, A 30.6%, B2 55.6%, and D 4.6%. Virulence genes prevalence was ecp 98.1%, fimH 86.1%, traT 77.8%, sfa/focDE 74.1%, papC 62%, iutA 48.1%, fyuA 44.4%, focG 2.8%, sfaS 1.9%, hlyA 7.4%, cnf-1 6.5%, cdt-B 0.9%, cvaC 2.8%, ibeA 2.8%, and rfc 0.9%. Regarding antimicrobial resistance it was above 50% to ampicillin/sulbactam, ampicillin, piperacillin, trimethoprim/sulfamethoxazole, ciprofloxacin, and levofloxacin. Uropathogenic E. coli clustered mainly in the pathogenic phylogenetic group B2. The isolates showed a high presence of siderophores and adhesion genes and a low presence of genes encoding toxins. The high frequency of papC gene suggests that these isolates have the ability to colonize the kidneys. High resistance to drugs considered as first choice treatment such as trimethoprim/sulfamethoxazole and fluoroquinolones was consistently observed

Primers used in this study.

<p>Primers used in this study.</p

Demonstration of ECP on UPEC strains.

<p>(A) CFT073 displays peritrichous pili after growth in LB at 26°C with shaking and after incubation with pre-immune serum followed by negative staining and TEM observation. (B–D) Immunogold labeling of ECP (indicated by arrows) produced by F11 (B), CFT073 (C), and UTI89 (D) strains grown in LB at 26°C with anti-ECP antibodies and rabbit anti-IgG gold conjugate. (E) Magnification of labeled ECP on CFT073. Scale bars represent 500 nm. (F–H) Detection of ECP (green) on UPEC strains [F11 (F), CFT073 (G), UTI89 (H)] after 2 h of incubation with HeLa cells at 37°C and reacted with anti-ECP antibodies and Alexa-Fluor 488-conjugated secondary antibody. Cellular and bacterial DNA was stained with propidium iodide (red). Panel I is the negative control CFT073 incubated with preimmune serum. Images were taken with a 60× objective. The insets in panels F and G are high magnifications of the areas within the corresponding panels and show a fibrillar network (green) between bacteria formed by ECP.</p

Role of ECP in adherence of UPEC to HeLa and HTB-4 cells.

<p>(A) Comparison of adherence levels of wild-type strain CFT073 and isogenic <i>ecpA</i> mutant to HeLa and HTB-4 cells at 37°C, as determined by plating out serial dilutions of adhering bacteria. (B) Comparison of invasion capabilities of HeLa and HTB-4 cells by the indicated UPEC strains as determined by the gentamycin-protection assay. For panels A and B standard deviations are represented by error bars and represent the average of all the results obtained from the 3 experiments performed. *, Statistically significant with respect to the wild-type strain (<i>p</i><0.05). (C) Giemsa staining of HeLa cell monolayers infected for 2 h with UPEC CFT073 strains showing reduction of adherence in the isogenic <i>ecpA</i> mutant. Images were taken with a 60× objective.</p

Optimal conditions for induction of ECP.

<p>Production of ECP in CFT073 bacteria grown in LB or DMEM at 26°C or 37°C was measured by flow cytometry using primary anti-EcpA antibodies and Alexa-Fluor 488 conjugate. In addition, levels of ECP production were determined in the presence of HeLa cells at 37°C. Standard deviations are represented by error bars and represent the average of all the results obtained from the 3 experiments performed.</p