Sergio Vaca

Two or more enteropathogens are associated with diarrhoea in Mexican children

BackgroundDiarrhoeal diseases constitute a major public health problem, particularly in the developing world, where the rate of mortality and morbidity is very high. The purpose of this study was to conduct a 2 years and 3 months study in order to determine the prevalence of five enteropathogen diarrheogenic agents in Mexico City.MethodsFaecal samples were obtained from 300 Mexican children diagnosed as positive for diarrhoea, aged > 2 to < 12 years old, and from 80 children matched for age but with no symptoms of the disease (control group). Two multiplex PCR were used to detect Escherichia coli, Salmonella spp., and Shigella spp. In addition, the two protozoan parasites Entamoeba histolytica/Entamoeba dispar and Giardia intestinalis were detected by conventional methods.ResultsAll diarrhoeal samples were positive for one or more enteropathogens. The most common enteropathogens in diarrhoeal samples were E. histolytica/E. dispar (70.3%), Salmonella (ohio 28.3%; typhimurium 16.3%; infantis 8%; anatum 0.6%; Newport 0.3%), G. intestinalis (33%), E. coli (ETEC 13.3%; EPEC 9.3%; VTEC 8.6%; EIEC 1%) and Shigella spp. (flexneri 1.6%, sonnei 1%). Infections by two (24%) three (16%) and four (12%) pathogens were observed.ConclusionThis study revealed that 52% of the patients were infected by more than one enteropathogen, notably E. histolitica/E. dispar and Salmonella ohio. These results are useful for clinicians to improve the empiric treatment used in such cases.

Frequency of vacA, cagA and babA2 virulence markers in Helicobacter pylori strains isolated from Mexican patients with chronic gastritis

BackgroundHelicobacter pylori has been strongly associated with chronic gastritis, peptic and duodenal ulcers, and it is a risk factor for gastric cancer. Three major virulence factors of H. pylori have been described: the vacuolating toxin (VacA), the cytotoxin-associated gene product (CagA) and the adhesion protein BabA2. Since considerable geographic diversity in the prevalence of H. pylori virulence factors has been reported, the aim of this work was to establish the H. pylori and vacA, cagA and babA2 gene status in 238 adult patients, from a marginal urban area of Mexico, with chronic gastritis.MethodsH. pylori was identified in cultures of gastric biopsies by nested PCR. vacA and cagA genes were detected by multiplex PCR, whereas babA2 gene was identified by conventional PCR.ResultsH. pylori-positive biopsies were 143 (60.1%). All H. pylori strains were vacA+; 39.2% were cagA+; 13.3% were cagA+babA2+ and 8.4% were babA2+. Mexican strains examined possessed the vacA s1, m1 (43.4%), s1, m2 (24.5%), s2, m1 (20.3%) and s2, m2 (11.9%) genotypes.ConclusionThese results show that the Mexican patients suffering chronic gastritis we have studied had a high incidence of infection by H. pylori. Forty four percent (63/143) of the H. pylori strains analyzed in this work may be considered as highly virulent since they possessed two or three of the virulence markers analyzed: vacA s1 cagA babA2 (9.8%, 14/143), vacA s1 babA2 (4.9%, 7/143), and vacA s1 cagA (29.4%, 42/143). However, a statistically significant correlation was not observed between vacAs1, cagA and babA2 virulence markers (χ2 test; P > 0.05).

Frequency and expression of ALS and HWP1 genotypes in Candida albicans strains isolated from Mexican patients suffering from vaginal candidosis.

To detect the frequency and expression of eight ALS (agglutinin‐like sequence) genes and the HWP1 genotype in a group of Candida albicans strains isolated from Mexican women suffering from vaginal candidosis. A group of 264 women (age 15–57 years) with vaginal infections were evaluated. C. albicans was identified by PCR amplification of the rRNA internal transcribed spacer regions ITS1 and ITS2. The ALS and HWP1 genes were identified by conventional PCR, and their expression levels were determined by real‐time PCR after growing C. albicans strains in reconstituted human vaginal epithelium (RHVE). C. albicans was identified in 50 women (18.9%). The genotypic frequencies were ALS1 100%, ALS2 60%, ALS3 36%, ALS4 54%, ALS5 70%, ALS6 56%, ALS7 64%, ALS9 66% and HWP1 92%. The most frequently expressed genes in the strains harbouring all of the genes were ALS4 (100%), ALS1 (87.5%), ALS2 (87.5%), ALS3 (87.5%), ALS5 (87.5%), ALS7 (87.5%) and HWP1 (75.0%). Nineteen per cent of the vaginal infections were caused by C. albicans, and a high proportion of the strains carried genes encoding proteins involved in adhesion to epithelia. The ALS and HWP1 genes were expressed in RHVE, suggesting that the Als and Hwp1 proteins play an important role in the pathogenesis of the infection.

Outer membrane vesicles of Pasteurella multocida contain virulence factors.

Pasteurella multocida (Pm) is a gram‐negative bacterium able to infect different animal species, including human beings. This bacterium causes economic losses to the livestock industry because of its high morbidity and mortality in animals. In this work, we report the characterization of outer membrane vesicles (OMVs) released into the culture medium by different Pm serogroups. Purified OMVs in the range of 50–300 nm were observed by electron microscopy. Serum obtained from chickens infected with Pm recognized several proteins from Pm OMVs. Additionally, rabbit antiserum directed against a secreted protease from Actinobacillus pleuropneumoniae recognized a similar protein in the Pm OVMs, suggesting that OMVs from these bacterial species contain common immunogenic proteins. OmpA, a multifunctional protein, was identified in OMVs from different Pm serogroups, and its concentration was twofold higher in OMVs from Pm serogroups B and D than in OMVs from other serogroups. Three outer membrane proteins were also identified: OmpH, OmpW, and transferrin‐binding protein. Three bands of 65, 110, and 250 kDa with proteolytic activity were detected in Pm OMVs of serogroups A and E. Additionally, β‐lactamase activity was detected only in OMVs from Pm 12945 Ampr (serogroup A). Pm OMVs may be involved in different aspects of disease pathogenesis.

Virulence factors, antibiotic resistance phenotypes and O-serogroups of Escherichia coli strains isolated from community-acquired urinary tract infection patients in Mexico.

BACKGROUND/PURPOSE Uropathogenic Escherichia coli (UPEC) strains isolated from patients with community-acquired urinary tract infections (UTIs) were assessed to determine the prevalence of virulence genes, antibiotic resistance, and the O-serogroup of the strains. METHODS Consenting patients with community-acquired UTI were enrolled at Unidad Médica Familiar Number 64 (Instituto Mexicano del Seguro Social, Estado de Mexico, Mexico) and 321 urine samples were collected. Polymerase chain reaction (PCR) was used to assess 24 virulence genes and 14 O-serogroups. The Kirby-Bauer method was used to evaluate the antibiotic susceptibility of the isolated strains to 12 commonly used antibiotics. RESULTS A total of 194 strains were identified as E. coli using standard biochemical tests, followed by PCR amplification of 16S ribosomal RNA gene. Only 58.2% of the strains belonged to the assessed 14 O-serogroups. The serogroups O25, O15, O8, and O75 were present in 20.6%, 17%, 6.1%, and 4.6% of strains, respectively. The most frequently occurring virulence genes among UPEC strains included kpsMT (92.2% strains), usp (87.1%), irp2 (79.3%), iha (64.9%), fim (61.3%), set (36%), astA (33.5%), pap (24.7%), and papGII (21.1%). In addition, 97% of the strains were multi-drug resistant (coresistance to 3-11 antibiotics). CONCLUSION The isolated UPEC strains predominantly belonged to three serogroups (O25, O15, and O8), harboured numerous virulence genes, and are multiresistant to antibiotics. The findings of this study could be used to orient UTI treatment strategies and in epidemiological studies in Mexico.

Gallibacterium elongation factor-Tu possesses amyloid-like protein characteristics, participates in cell adhesion, and is present in biofilms

Gallibacterium, which is a bacterial pathogen in chickens, can form biofilms. Amyloid proteins present in biofilms bind Congo red dye. The aim of this study was to characterize the cell-surface amyloid-like protein expressed in biofilms formed by Gallibacterium strains and determine the relationship between this protein and curli, which is an amyloid protein that is commonly expressed by members of the Enterobacteriaceae family. The presence of amyloid-like proteins in outer membrane protein samples from three strains of G. anatis and one strain of Gallibacterium genomospecies 2 was evaluated. A protein identified as elongation factor-Tu (EF-Tu) by mass spectrometric analysis and in silico analysis was obtained from the G. anatis strain F149T. This protein bound Congo red dye, cross-reacted with anti-curli polyclonal serum, exhibited polymerizing properties and was present in biofilms. This protein also reacted with pooled serum from chickens that were experimentally infected with G. anatis, indicating the in vivo immunogenicity of this protein. The recombinant EF-Tu purified protein, which was prepared from G. anatis 12656-12, polymerizes under in vitro conditions, forms filaments and interacts with fibronectin and fibrinogen, all of which suggest that this protein functions as an adhesin. In summary, EF-Tu from G. anatis presents amyloid characteristics, is present in biofilms and could be relevant for the pathogenesis of G. anatis.

Implementation of a novel in vitro model of infection of reconstituted human epithelium for expression of virulence genes in methicillin-resistant Staphylococcus aureus strains isolated from catheter-related infections in Mexico

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) are clinically relevant pathogens that cause severe catheter-related nosocomial infections driven by several virulence factors.MethodsWe implemented a novel model of infection in vitro of reconstituted human epithelium (RHE) to analyze the expression patterns of virulence genes in 21 MRSA strains isolated from catheter-related infections in Mexican patients undergoing haemodialysis. We also determined the phenotypic and genotypic co-occurrence of antibiotic- and disinfectant-resistance traits in the S. aureus strains, which were also analysed by pulsed-field-gel electrophoresis (PFGE).ResultsIn this study, MRSA strains isolated from haemodialysis catheter-related infections expressed virulence markers that mediate adhesion to, and invasion of, RHE. The most frequent pattern of expression (present in 47.6% of the strains) was as follows: fnbA, fnbB, spa, clfA, clfB, cna, bbp, ebps, eap, sdrC, sdrD, sdrE, efb, icaA, and agr. Seventy-one percent of the strains harboured the antibiotic- and disinfectant-resistance genes ermA, ermB, tet(M), tet(K), blaZ, qacA, qacB, and qacC. PFGE of the isolated MRSA revealed three identical strains and two pairs of identical strains. The strains with identical PFGE patterns showed the same phenotypes and genotypes, including the same spa type (t895), suggesting hospital personnel manipulating the haemodialysis equipment could be the source of catheter contamination.ConclusionThese findings help define the prevalence of MRSA virulence factors in catheter-related infections. Some of the products of the expressed genes that we detected in this work may serve as potential antigens for inclusion in a vaccine for the prevention of MRSA-catheter-related infections.

High virulence and antifungal resistance in clinical strains of Candida albicans

Antifungal resistance and virulence properties of Candida albicans are a growing health problem worldwide. To study the expression of virulence and azole resistance genes in 39 clinical strains of C. albicans, we used a model of infection of human vaginal epithelial cells with C. albicans strains isolated from Mexican women with vulvovaginal candidiasis (VVC). The strains were identified by PCR amplification of the ITS1 and ITS2 regions of rRNA. The detection and expression of virulence genes and azole resistance genes MDR1 and CDR1 were performed using PCR and RT-PCR, respectively. All strains were sensitive to nystatin and 38 (97.4%) and 37 (94.9%) were resistant to ketoconazole and fluconazole, respectively. ALS1, SAP4–SAP6, LIP1, LIP2, LIP4, LIP6, LIP7, LIP9, LIP10, and PLB1-PLB2 were present in all strains; SAP1 was identified in 37 (94.8%) isolates, HWP1 in 35 (89.7%), ALS3 in 14 (35.8%), and CDR1 in 26 (66.6%). In nearly all of the strains, ALS1, HWP1, SAP4–SAP6, LIP1–LIP10, PLB1, and PLB2 were expressed, whereas CDR1 was expressed in 20 (51.3%) and ALS3 in 14 (35.8%). In our in vitro model of infection with C. albicans, the clinical strains showed different expression profiles of virulence genes in association with the azole resistance gene CDR1. The results indicate that the strains that infect Mexican patients suffering from VVC are highly virulent and virtually all are insensitive to azoles.

Mannheimia haemolytica OmpP2-like is an amyloid-like protein, forms filaments, takes part in cell adhesion and is part of biofilms

AbstractMannheimia haemolytica causes respiratory disease in cattle. Amyloid proteins are a major component of biofilms; they aid in adhesion and confer resistance against several environmental insults. The amyloid protein curli is highly resistant to protease digestion and physical and chemical denaturation and binds Congo red (CR) dye. The purpose of this study was to characterize an approximately 50-kDa CR-binding amyloid-like protein (ALP) expressed by M. haemolytica. This protein resisted boiling and formic acid digestion and was recognized by a polyclonal anti-Escherichia coli curli serum, suggesting its relationship with amyloid proteins. Immunolabeling and transmission electron microscopy showed that antibodies bound long, thin fibers attached to the bacterial surface. Mass spectrometry analysis indicated that these fibers are M. haemolytica OmpP2-like proteins. The purified protein formed filaments in vitro, and antiserum against it reacted positively with biofilms. An in silico analysis of its amino acid sequence indicated it has auto-aggregation properties and eight amyloid peptides. Rabbit polyclonal antibodies generated against this ALP diminished the adhesion of ATCC 31612 and BA1 M. haemolytica strains to A549 human epithelial cells, indicating its participation in cell adhesion. ALP expressed by M. haemolytica may be important in its pathogenicity and ability to form biofilms.

Expression of enterotoxin-coding genes in methicillin-resistant Staphylococcus aureus strains isolated from Mexican haemodialysis patients

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) causes severe catheter-related infections in haemodialysis patients ranging from local-site infections and septic thrombophlebitis to bacteraemia but the associated virulence factors and exotoxins remain unclear.FindingsWe employed an in vitro infection model using reconstituted human epithelium (RHE) to analyse the expression profiles of 4 virulence genes and 12 exotoxin-coding virulence genes in 21 MRSA strains isolated from catheter-related infections in 21 Mexican patients undergoing haemodialysis.All 21 strains (100%) expressed the seg, seh, sei, eta, etb, or hla genes coding staphylococcal toxins. Eleven MRSA strains (52.3%) expressed the sea gene coding staphylococcal enterotoxin A, and two strains (9.5%) expressed the v8 gene coding serine protease. The tst, chp, and arcA genes coding toxic shock syndrome toxin 1, chemotaxis inhibitory protein, and arginine deiminase, respectively, were expressed in separate single strains (4.7%). The most frequent expression profile (42.8% of the strains) comprised seg, seh, sei, eta, etb, and hla.ConclusionIt is likely that the SEG, SEH, SEI, ETA, ETB, and Hla toxins may play a role in MRSA catheter-related infections. Consideration of these toxins in the development of a vaccine or as targets for monoclonal antibody therapy could provide an improved therapeutic strategy for the treatment of catheter-related infections in haemodialysis patients.