Genomic characterization of a multidrug-resistant uropathogenic Escherichia coli and evaluation of Echeveria plant extracts as antibacterials.

Uropathogenic Escherichia coli (UPEC) is the most common bacterial agent associated with urinary tract infections, threatening public health systems with elevated medical costs and high morbidity rates. The successful establishment of the infection is associated with virulence factors encoded in its genome, in addition to antibacterial resistance genes, which could limit the treatment and resolution of the infection. In this sense, plant extracts from the genus Echeveria have traditionally been used to treat diverse infectious diseases. However, little is known about the effects of these extracts on bacteria and their potential mechanisms of action. This study aims to sequence a multidrug-resistant UPEC isolate (UTI-U7) and assess the multilocus sequence typing (MLST), virulence factors, antimicrobial resistance profile, genes, serotype, and plasmid content. Antimicrobial susceptibility profiling was performed using the Kirby-Bauer disk diffusion. The antibacterial and anti-adherent effects of the methanol extracts (ME) of Echeveria (E. craigiana, E. kimnachii, and E. subrigida) against UTI-U7 were determined. The isolate was characterized as an O25:H4-B2-ST2279-CH40 subclone and had resistant determinants to aminoglycosides, ß-lactams, fluoroquinolones/quinolones, amphenicols, and tetracyclines, which matched with the antimicrobial resistance profile. The virulence genes identified encode adherence factors, iron uptake, protectins/serum resistance, and toxins. Identified plasmids belonged to the IncF group (IncFIA, IncFIB, and IncFII), alongside several prophage-like elements. After an extensive genome analysis that confirmed the pathogenic status of UTI-U7 isolate, Echeveria extracts were tested to determine their antibacterial effects; as an extract, E. subrigida (MIC, 5 mg/mL) displayed the best inhibitory effect. However, the adherence between UTI-U7 and HeLa cells was unaffected by the ME of the E. subrigida extract.

Occurrence of Giardia duodenalis in Cats from Queretaro and the Risk to Public Health.

Giardia is a protozoan that affects humans as well as a wide range of domestic species. It is distributed worldwide, and the highest frequency is seen in developing countries. Due to the potential for domestic cats to be carriers of this parasite and subsequently transmit the infection to humans, it is important to know the risk of transmission. For this reason, the objective of this study was to determine the frequency of this parasite in the cat population of the city of Santiago de Queretaro, Mexico, and identify the assemblages present to determine the role this host plays in public health, this being the first study of its type to be performed in the country. This was a cross-sectional study during which 200 fecal samples were collected from cats of both sexes and varying ages and strata of origin. The samples were analyzed by microscopy following the flotation technique, having obtained a general frequency of 25%. Giardia cysts were found at higher frequency in pasty stools. The assemblages found were zoonotic, specifically assemblage A, which suggests that the cat poses an important risk for the dissemination of the parasite to humans, making it an important public health problem.

Bacteriostatic effect of Echeveria extracts on diarrheagenic E. coli pathotypes and non-cytotoxicity on human Caco-2 cells.

INTRODUCTION: Diarrheagenic Escherichia coli pathotypes are important aetiological agents of diarrhoeal illness among children from less developed areas, worldwide. Diarrheagenic E. coli pathotypes strains are increasingly becoming drug resistant, thus effective and accessible therapeutic alternatives are required for their treatment; herbal extracts may be a potential alternative. AIMS: to evaluate Echeveria craigiana, E. kimnachii, and E. subrigida methanol extracts antibacterial effect on six diarrheagenic E. coli reference strains and on human colorectal adenocarcinoma cells viability and cytokine production. METHODOLOGY: Diarrheagenic E. coli pathotypes reference strains: typical enteropathogenic E2348/69, enterotoxigenic H10407, enterohaemorrhagic O157:H7/EDL933, enteroinvasive E11, diffusely adherent C18451-A, and enteroaggregative 042 E. coli. E craigiana, E. kimnachii, and E. subrigida leaves, collected at Sinaloa, Mexico, were freeze-dried and macerated in methanol solvent. Antibacterial activity was determined by a novel method developed in our laboratory, bacterial oxygen consumption by polarographic oxygen electrode technique and membrane integrity by two methods (live/dead and protein leakage assays). Colorectal adenocarcinoma cells viability by MTT assay and cytokine production using a Cytometric Bead Array kit. RESULTS: Extracts concentrations of 100 µg/mL and 5-hour incubation, reduced more than 93% the growth of all diarrheagenic E. coli pathotypes tested strains and significantly decreased bacterial oxygen consumption, like bacteriostatic antibiotics. After 24-hour incubation methanol extracts had a differential antibacterial effect on each diarrheagenic E. coli pathotypes strain. Echeveria extracts did not have any effect on viability and cytokine production of colorectal adenocarcinoma cells. CONCLUSIONS: Echeveria methanol extracts have a bacteriostatic effect on all diarrheagenic E. coli pathotypes strains, thus potentially they could be used as antibacterial agents on diarrheagenic E. coli pathotypes-contaminated products and on patients with diarrheagenic E. coli pathotypes infections.

Prevalence and Zoonotic Potential of Giardia intestinalis in Dogs of the Central Region of Mexico.

Giardia intestinalis is a protozoan of worldwide distribution capable of infecting a large number of species, including humans and domestic animals. Dogs represent a risk to public health due to cross-infections by the zoonotic assemblages. However, there is little information concerning the prevalence and frequency of this parasite and its assemblages in dogs of the central region of Mexico, thus this study aimed to contribute to this matter. A total of 402 feces samples from dogs of different settings (shelter, breeding establishments, domestic and stray) were obtained and direct coproparasitoscopic examination by flotation revealed a prevalence of 25%. PCR was performed for amplification of the ß-Giardin gene, to which 24 samples were positive. Assemblages were obtained through RFLP analysis, using enzymes Hae III to obtain the main genotypes (A-G), and Hha I to subtype assemblage A. All 24 samples were genotyped as assemblage A, with 83% as AI and 17% as AII. Thus, these findings confirm that dogs in the central region of Mexico are a risk for zoonotic transmission of this parasite, emphasizing the importance of a much needed control of the disease in this species.

CD40 Ligand Deficient C57BL/6 Mouse Is a Potential Surrogate Model of Human X-Linked Hyper IgM (X-HIGM) Syndrome for Characterizing Immune Responses against Pathogens.

Individuals with X-HIGM syndrome fail to express functional CD40 ligand; consequently they cannot mount effective protective antibody responses against pathogenic bacteria. We evaluated, compared, and characterized the humoral immune response of wild type (WT) and C57-CD40L deficient (C57-CD40L(-/-)) mice infected with Citrobacter rodentium. Basal serum isotype levels were similar for IgM and IgG3 among mice, while total IgG and IgG2b concentrations were significantly lower in C57-CD40L(-/-) mice compared with WT. Essentially IgG1 and IgG2c levels were detectable only in WT mice. C57-CD40L(-/-) animals, orally inoculated with 2 × 10(9) CFU, presented several clinical manifestations since the second week of infection and eventually died. In contrast at this time point no clinical manifestations were observed among C57-CD40L(-/-) mice infected with 1 × 10(7) CFU. Infection was subclinical in WT mice inoculated with either bacterial dose. The serum samples from infected mice (1 × 10(7) CFU), collected at day 14 after infection, had similar C. rodentium-specific IgM titres. Although C57-CD40L(-/-) animals had lower IgG and IgG2b titres than WT mice, C57-CD40L(-/-) mice sera displayed complement-mediated bactericidal activity against C. rodentium. C. rodentium-infected C57-CD40L(-/-) mice are capable of producing antibodies that are protective. C57-CD40L(-/-) mouse is a useful surrogate model of X-HIGM syndrome for studying immune responses elicited against pathogens.

Enteroaggregative coli: A Pathogen Bridging the North and South.

Enteroaggregative Escherichia coli (EAEC) is a heterogeneous emerging enteric pathogen. Identified during the 1980's when EAEC strains where isolated from cases of acute and persistent diarrhea among infants from developing countries and of traveler's diarrhea. Subsequently, EAEC strains were linked with foodborne outbreaks and diarrhea illness in adults and children from industrialized countries, HIV-infected subjects and stunting of malnourished poor children. Nowadays, EAEC is increasingly recognized as a major cause of acute diarrhea in children recurring hospitalization and of traveler's diarrhea worldwide. EAEC strains defining phenotype is the aggregative adherence (AA) pattern on epithelial cells. AggR a transcriptional regulator of several EAEC virulence genes has been a key factor in both understanding EAEC pathogenesis and defining typical EAEC (tEAEC) strains. EAEC virulence genes distribution among these strains is highly variable. Present challenges are the identification of key virulence genes and how they coordinately function in the setting of enteric disease.

Persistent bloody diarrhoea without fever associated with diffusely adherent Escherichia coli in a young child.

Diffusely adherent Escherichia coli (DAEC) is thought to cause diarrhoea in children, and so too are other diarrhoeagenic E. coli (DEC); however, the evidence base is inconclusive. DEC pathotypes are differentiated on the basis of their pathogenic features, and thus cannot be quickly identified on selective culture media. Molecular techniques, not readily available in most clinical laboratories, are required to differentiate DEC strains from non-pathogenic E. coli in the stool flora. We report a case of persistent bloody diarrhoea, without fever, in a previously healthy 21-month infant from whom we isolated five DAEC strains. The child's stools movements were loose, with gross blood and mucus; fresh mount analysis revealed numerous faecal leukocytes and erythrocytes. Response to antimicrobial treatment with trimethoprim-sulfamethoxazole was poor despite susceptibility in vitro. Although the patient improved with azithromycin, blood was present in the patient's stools for over 30 days. The severe diarrhoea in this patient might be explained by the fact that these DAEC isolates harboured a siderophore receptor, which allows the bacteria to use iron derived from haem compounds that promote its multiplication. The isolates also induced in vitro secretion of several immunomodulatory cytokines that may account for the patient's loose stools and faecal leukocytes. DAEC may play a greater role than suspected in afebrile children with bloody diarrhoea.

C57-CD40 ligand deficient mice: a potential model for enterotoxigenic Escherichia coli (H10407) colonization.

Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrheal disease in humans, calves and pigs. In humans, these infections mainly occur in developing countries leading to a high diarrheal morbidity and infant mortality and to travellers' diarrhea. ETEC strains constitute a phenotypically and genetically diverse pathotype with as common characteristics the production of heat-labile (LT) and/or heat-stable enterotoxins (ST) as well as of one or more fimbrial colonization factors. Despite the global importance of these pathogens, a broadly ETEC protective vaccine is not yet available, partially due to the lack of a suitable animal model for human ETEC. Such model would allow to test more ETEC molecules as potential vaccine candidates. The C57-CD40 ligand deficient (C57-cd40l(-/-)) mouse has been successfully used to develop infection models of intestinal pathogens, but little is known about its humoral immune response. Therefore, the aims of this study were to characterize the humoral immune response of C57 and C57-cd40l(-/-) mice and to determine the persistence of ETEC H10407 and two of its variants after oral inoculation. The serum IgM, IgG and IgA and faecal IgG and IgA concentrations, of twelve mice per mouse strain (C57 and C57-cd40l(-/-)), were determined by ELISA. All serum immunoglobulins and the faecal IgG concentration were significantly lower in C57-cd40l(-/-) than in C57 mice. In contrast the faecal IgA concentration was significantly higher in the C57-cd40l(-/-) mice. This high intestinal IgA concentration might be a compensatory T cell-independent production of IgA production. Both mouse strains were orally inoculated with 5×10(8) ETEC H10407 (LT(+), ST-colonization factor antigen I (CFA/I)(+)) and ETEC in animal faeces was established by culture followed by st and lt loci identification by PCR until day 14 post infection. Most C57 mice eliminated the strain within 3 days whereas infection remained in C57-cd40l(-/-) mice until day 14. Subsequently both mouse strains were inoculated with ETEC H10407 variants and followed up until day 113. Likewise C57 mice eliminated both ETEC variants within 4 days. All C57-cd40l(-/-) mice had eliminated the LT(-) variant at day 31, whereas the ST-CFA/I(-) variant remained in mice stools until day 113. These observations suggest that C57-cd40l(-/-) mice are permissive for ETEC H10407 colonization.