Graciela Castro-Escarpulli

Characterisation of Aeromonas spp. isolated from frozen fish intended for human consumption in Mexico

A total of 82 strains of presumptive Aeromonas spp. were identified biochemically and genetically (16S rDNA-RFLP). The strains were isolated from 250 samples of frozen fish (Tilapia, Oreochromis niloticus niloticus) purchased in local markets in Mexico City. In the present study, we detected the presence of several genes encoding for putative virulence factors and phenotypic activities that may play an important role in bacterial infection. In addition, we studied the antimicrobial patterns of those strains. Molecular identification demonstrated that the prevalent species in frozen fish were Aeromonas salmonicida (67.5%) and Aeromonas bestiarum (20.9%), accounting for 88.3% of the isolates, while the other strains belonged to the species Aeromonas veronii (5.2%), Aeromonas encheleia (3.9%) and Aeromonas hydrophila (2.6%). Detection by polymerase chain reaction (PCR) of genes encoding putative virulence factors common in Aeromonas, such as aerolysin/hemolysin, lipases including the glycerophospholipid-cholesterol acyltransferase (GCAT), serine protease and DNases, revealed that they were all common in these strains. Our results showed that first generation quinolones and second and third generation cephalosporins were the drugs with the best antimicrobial effect against Aeromonas spp. In Mexico, there have been few studies on Aeromonas and its putative virulence factors. The present work therefore highlights an important incidence of Aeromonas spp., with virulence potential and antimicrobial resistance, isolated from frozen fish intended for human consumption in Mexico City.

Complete Type III Secretion System of a Mesophilic Aeromonas hydrophila Strain

ABSTRACT We have investigated the existence and genetic organization of a functional type III secretion system (TTSS) in a mesophilic Aeromonas strain by initially using the Aeromonas hydrophila strain AH-3. We report for the first time the complete TTSS DNA sequence of an Aeromonas strain that comprises 35 genes organized in a similar disposition as that in Pseudomonas aeruginosa. Using several gene probes, we also determined the presence of a TTSS in clinical or environmental strains of different Aeromonas species: A. hydrophila, A. veronii, and A. caviae. By using one of the TTSS genes (ascV), we were able to obtain a defined insertion mutant in strain AH-3 (AH-3AscV), which showed reduced toxicity and virulence in comparison with the wild-type strain. Complementation of the mutant strain with a plasmid vector carrying ascV was fully able to restore the wild-type toxicity and virulence.

Virulence potential and genetic diversity of Aeromonas caviae, Aeromonas veronii, and Aeromonas hydrophila clinical isolates from Mexico and Spain: a comparative study

A comparative study of 109 Aeromonas clinical isolates belonging to the 3 species most frequently isolated from patients with diarrhea in Mexico and Spain was performed to investigate the distribution of 3 prominent toxin genes and the gene encoding flagellin of lateral flagella; 4 well-established virulence factors in the genus Aeromonas. The aerolysin-hemolysin toxin genes were the most prevalent, being present in 89% of the total isolates. The ast toxin gene was conspicuously absent from the Aeromonas caviae and Aeromonas veronii groups but was present in 91% of the Aeromonas hydrophila isolates. Both the alt toxin gene and the lafA flagellin gene also had a low incidence in A. caviae and A. veronii. Differences in the prevalence of alt and lafA were observed between isolates from Mexico and Spain, confirming genus heterogeneity according to geographic location. Carriage of multiple toxin genes was primarily restricted to A. hydrophila isolates, suggesting that A. caviae and A. veronii isolates circulating in Mexico and Spain possess a limited array of virulence genes. Enterobacterial repetitive intergenetic consensus - polymerase chain reaction showed that the Aeromonas populations sampled lack dominant clones and were genetically heterogeneous, with A. caviae being the most diverse species. Further surveys of virulence determinants in genetically heterogeneous populations of Aeromonas isolates circulating worldwide are required to enhance the understanding of their capacity to cause disease.

A DNA probe specific for Aeromonas colonies

Members of the genus Aeromonas are important enteropathogens. Commercial identification systems are often unable to correctly identify Aeromonas strains and misidentification as Vibrio spp. is common. A digoxigenin-DNA probe based on a 237 bp of the glycerophospholipid-cholesterol acyltransferase gene has been tested in a colony hybridization assay. The probe hybridized with all Aeromonas species tested (n = 16) but not with strains of other enteropathogenic bacteria (n = 20). The probe allowed the unequivocal identification of Aeromonas in primary isolation media within 36 h.

Lactobacillus species isolated from vaginal secretions of healthy and bacterial vaginosis-intermediate Mexican women: a prospective study

BackgroundLactobacillus jensenii, L. iners, L. crispatus and L. gasseri are the most frequently occurring lactobacilli in the vagina. However, the native species vary widely according to the studied population. The present study was performed to genetically determine the identity of Lactobacillus strains present in the vaginal discharge of healthy and bacterial vaginosis (BV) intermediate Mexican women.MethodsIn a prospective study, 31 strains preliminarily identified as Lactobacillus species were isolated from 21 samples collected from 105 non-pregnant Mexican women. The samples were classified into groups according to the Nugent score criteria proposed for detection of BV: normal (N), intermediate (I) and bacterial vaginosis (BV). We examined the isolates using culture-based methods as well as molecular analysis of the V1–V3 regions of the 16S rRNA gene. Enterobacterial repetitive intergenic consensus (ERIC) sequence analysis was performed to reject clones.ResultsClinical isolates (25/31) were classified into four groups based on sequencing and analysis of the 16S rRNA gene: L. acidophilus (14/25), L. reuteri (6/25), L. casei (4/25) and L. buchneri (1/25). The remaining six isolates were presumptively identified as Enterococcus species. Within the L. acidophilus group, L. gasseri was the most frequently isolated species, followed by L. jensenii and L. crispatus. L. fermentum, L. rhamnosus and L. brevis were also isolated, and were placed in the L. reuteri, L. casei and L. buchneri groups, respectively. ERIC profile analysis showed intraspecific variability amongst the L. gasseri and L. fermentum species.ConclusionsThese findings agree with previous studies showing that L. crispatus, L. gasseri and L. jensenii are consistently present in the healthy vaginal ecosystem. Additional species or phylotypes were detected in the vaginal microbiota of the non-pregnant Mexican (Hispanic-mestizo) population, and thus, these results further our understanding of vaginal lactobacilli colonisation and richness in this particular population.

Highly specific and efficient primers for in-house multiplex PCR detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum

BackgroundAlthough sophisticated methodologies are available, the use of endpoint polymerase chain reaction (PCR) to detect 16S rDNA genes remains a good approach for estimating the incidence and prevalence of specific infections and for monitoring infections. Considering the importance of the early diagnosis of sexually transmitted infections (STIs), the development of a sensitive and affordable method for identifying pathogens in clinical samples is needed. Highly specific and efficient primers for a multiplex polymerase chain reaction (m-PCR) system were designed in silico to detect the 16S rDNA genes of four bacteria that cause genital infections, and the PCR method was developed.MethodsThe Genosensor Probe Designer (GPD) (version 1.0a) software was initially used to design highly specific and efficient primers for in-house m-PCR. Single-locus PCR reactions were performed and standardised, and then primers for each locus in turn were added individually in subsequent amplifications until m-PCR was achieved. Amplicons of the expected size were obtained from each of the four bacterial gene fragments. Finally, the analytical specificity and limits of detection were tested.ResultsBecause they did not amplify any product from non-STI tested species, the primers were specific. The detection limits for the Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum primer sets were 5.12 × 105, 3.9 × 103, 61.19 × 106 and 6.37 × 105 copies of a DNA template, respectively.ConclusionsThe methodology designed and standardised here could be applied satisfactorily for the simultaneous or individual detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum. This method is at least as efficient as other previously described methods; however, this method is more affordable for low-income countries.

The outer membrane vesicles: Secretion system type zero

Gram‐negative bacteria have mechanisms through which they can colonize and survive in different environments, such as the secretion systems types (1‐6) that have been widely studied and characterized. Nowadays, some authors have proposed extracellular structures, such as the outer membrane vesicles (OMVs), to be considered as an additional and independent secretion system. The OMVs are spherical particles of 50‐250 nm in diameter; they originate in the outer membrane, and therefore they have a very similar composition to the latter. These particles can transport an important variety of biomolecules: enzymes, toxins, antigenic determinants and even nucleic acids. Thus, it is of great interest to collect data describing the advantages of the transport of biomolecules through the OMVs and, thus, determine their role as a potential secretion system.

Phenotypical characteristics, genetic identification, and antimicrobial sensitivity of Aeromonas species isolated from farmed rainbow trout (Onchorynchus mykiss) in Mexico

In the present study, Aeromonas isolates from diseased and healthy farmed rainbow trout (Oncorhynchus mykiss) in Mexico, were characterized phenotypically and identified to species level by using 16S rDNA RFLP-PCR. A total of 50 isolates were included in the study and 10 Aeromonas species identified. The species A. veronii biovar sobria (22%), A. hydrophila (20%) and A. bestiarum (20%) were the most predominant. All isolates (100%) were resistant to cephalothin.

Aetiology and frequency of cervico-vaginal infections among Mexican women

There are major concerns worldwide regarding sexually transmitted infections (STI), bacterial vaginosis (BV), and candidiasis as a major cause of morbidity as they result in significant health and economic consequences, particularly in developing countries. This study was intended to obtain information about the prevalence of these pathologies in women considered to be at low risk using both traditional and in-house NAAT methods. Cervical and vaginal samples were collected all volunteers signed an informed consent form and completed a survey. BV, trichomoniasis, candidiasis, genital mycoplasmas colonization and Chlamydia trachomatis or Neisseria gonorrhoeae cervicitis were diagnosed. Candidiasis and abnormal vaginal flora associated with BV were very frequent. The high colonization with micoplasmas was detected. C. trachomatis cervicitis was found in 10.67% from which a third of the cases were of asymptomatic woman. No cases of gonorrhoea or trichomoniasis were diagnosed. In house NAAT’s used seems to viable tools for the cheap and reliable test for the diagnosis of gonorrhoea and chlamydial infections. Increased awareness of the importance of protected sexual intercourse is imperative to prevent the transmission of sexually transmitted. Further studies for a comprehensive understanding of the rates of these infections in Mexican women are necessary and should be an impulse for make community-based assessment of STI and RTI.

An overview of the infection of CMV, HSV 1/2 and EBV in Mexican patients with glioblastoma multiforme

Several risk factors are involved in glioblastoma, including cytomegalovirus (CMV). This research was carried out to determine the rate of CMV infection, as well as HSV 1/2 and EBV in brain tissue, in patients with glioblastomamultiforme (GBM). The tissues were tested using immunohistochemistry, PCR, in situ hybridization and real-time PCR. At least, one HHV was detected in 21/29 (72%) patients as follows: single infections with HSV-1/2 in 4/21 (19%), EBV in 6/21 (28.6%) and CMV in 1/21 (4.8%). Mixed viral infection, HSV-1/2 and EBV were detected in 4/21 patients (19%), CMV and EBV in 5/21 (23.8%), and HSV-1/2, EBV, and CMV in 1/21. The CMV viral load ranged from 3×102 to 4.33×105 genome/100ng of tissue. Genotype based on CMV gB was 3/7 where 2/3 was gB1 and 1/3 gB4. HSV, EBV and CMV were frequently found in brain tissues, more in mix in a population reported as highly seropositive.