Juan Manuel Bello-López

Inability of some Aeromonas hydrophila strains to act as recipients of plasmid pRAS1 in conjugal transfer experiments.

Plasmids belonging to the IncU incompatibility group are mobile genetic elements isolated frequently from Aeromonas spp. These plasmids share structural and functional characteristics and often carry Class-1 integrons bearing antibiotic resistance genes. In this work the ability of two IncU plasmids, pAr-32 and pRAS1 to establish in different A. hydrophila strains after conjugal transfer was studied. In vitro transfer frequencies on solid surface ranged from 10−1 to 10−6 for pAr-32 and from 10−3 to 10−5 for pRAS1. While carrying out these experiments we detected four strains unable to acquire plasmid pRAS1, indicating that the genetic background of recipients affects the establishment of the plasmid. We explored the possible reasons why these strains failed to yield transconjugants after mating experiments using A. salmonicida 718 as a donor. Factors included donor cell recognition, incompatibility, surface exclusion and restriction of incoming DNA. We found that none of these factors could explain the refractivity of non-receptive A. hydrophila strains to yield transconjugants. Although we do not know the reasons of this refractivity, we may speculate that these isolates lack a product necessary to replicate or stabilize plasmid pRAS1. Alternatively, these strains could contain a product that impedes plasmid establishment.

In vivo transfer of plasmid pRAS1 between Aeromonas salmonicida and Aeromonas hydrophila in artificially infected Cyprinus carpio L.

This study investigated the possible in vivo transfer of plasmid pRAS1 between Aeromonas salmonicida and A. hydrophila inhabiting two different organs of Cyprinus carpio L. To distinguish transconjugants from naturally occurring antibiotic resistant bacteria, twelve luminescent transposon-tagged A. hydrophila strains using mini Tn5luxCDABEKm2 transposon were generated. In conjugal transfer experiments, fish were conditioned with the donor bacteria and subsequently immersed in water containing the recipient strain. Bacteria were recovered from gills and intestines and isolated by growth on selective plates. Transconjugants were identified by their resistance to the pRAS1 encoded antimicrobials and by light emission. In vivo transfer frequencies ranged between 10(-3) and 10(-6) and were somewhat lower in intestines, compared to gills. Transfer frequencies were also smaller relative to those obtained in vitro. The minimal amount of donor and recipient bacteria needed to yield detectable transconjugants in vivo was 1 x 10(4) CFU mL(-1). Implications of this plasmid transfer in natural settings and its possible consequences to human health are discussed.

Molecular characterization of microbial contaminants isolated from Umbilical Cord Blood Units for transplant

Disposal of Umbilical Cord Blood Units due to microbial contamination is a major problem in Cord Blood Banks worldwide as it reduces the number of units available for transplantation. Additionally, economic losses are generated as result of resources and infrastructure used to obtain such units. Umbilical Cord Blood Units that showed initial microbial contamination were subject to strains isolation, identification, and characterization by sequencing the 16S rRNA gene and Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR). Moreover, tests of antimicrobial resistance/sensitivity and phenotypic activities that may play an important role in microbial infection were performed. Microbial contamination was detected in 120 Umbilical Cord Blood Units (2.31%) in the period from 2003 to 2013. The most frequently isolated strains were Enterococcus faecium, followed by Staphylococcus epidermidis, Escherichia coli, Enterococcus faecalis, Staphylococcus haemoliticus, Klebsiella pneumoniae, Enterococcus durans, Lactobacillus helveticus, Enterococcus hiriae and Roseomonas genomospecies 5. The ERIC-PCR assays revealed a wide genetic diversity in some strains although belonging to the same genus and specie, indicating different sources of contamination. Broad-spectrum penicillins, third generation cephalosporins, aminoglycosides, and fluoroquinolones showed lower inhibitory activity on the tested strains. All strains were proteolytic, 67.69% were amylase-positive, 27.6% hemolysis-positive, and 34.71% nuclease-positive. The most common sources of contamination were: vaginal flora, digestive tract, and skin flora, highlighting the need for staff training in good manufacturing practices in collection SCU since all contaminants identified are part of the microbial flora of the donors. Implications and consequences in the therapeutic use of Umbilical Cord Blood Units for transplantation contaminated by multiresistant bacteria in immunocompromised patients are discussed.

Propagation capacity of bacterial contaminants in platelet concentrates using a luciferase reporter system

INTRODUCTION Currently the use of molecular tools and techniques of Genetic Engineering in the study of microbial behavior in blood components has replaced the employment of classical methods of microbiology. This work focuses on the use of a novel lux reporter system for monitoring the contaminating propagation capacity of bacteria present in platelet concentrates under standard storage conditions in the blood bank. METHODS A miniTn5 promotor probe carrying the lux operon from Photorhabdus luminiscens (pUTminiTn5luxCDABEKm2) was used to construct four bacterial bioluminescent mutants: Escherichia coli, Salmonella typhi, Proteus mirabilis and Pseudomonas aeruginosa. Luminescent mutants were used for contamination tests with 20 CFU in platelet concentrates bags and were stored under standard storage conditions in the blood bank (100 rpm at 22 °C). The measurements of luminous activity and optical density were used to monitor bacterial proliferation during 7 days (168 h). RESULTS During the exponential growth phase (log) of bacterial strains, a lineal correlation between luminous activity vs biomass was observed (R(2) = 0.985, 0.976, 0.981) for E. coli::Tn5luxCDABEKm2, P. mirabilis::Tn5luxCDABEKm2 and P. auriginosa::Tn5luxCDABEKm2, respectively. The above indicates that metabolic activity (production of ATP) is directly related to biomass in this phase of microbial growth. While conducting experiments, the inability to propagate S. typhi::Tn5luxCDABEKm2 was detected. We can speculate that platelet concentrates contain specific components that prevent the propagation of S. typhi. CONCLUSION The use of luxCDABE system for the quantification of luminous activity is a rapid and sensitive alternative to study the propagation and auto-sterilization of bacterial contaminants in platelet concentrates.

Changes in the Bacterial Community Structure of Remediated Anthracene-Contaminated Soils

Mixing soil or adding earthworms (Eisenia fetida (Savigny, 1826)) accelerated the removal of anthracene, a polycyclic aromatic hydrocarbon, from a pasture and an arable soil, while a non-ionic surfactant (Surfynol® 485) inhibited the removal of the contaminant compared to the untreated soil. It was unclear if the treatments affected the soil bacterial community and consequently the removal of anthracene. Therefore, the bacterial community structure was monitored by means of 454 pyrosequencing of the 16S rRNA gene in the pasture and arable soil mixed weekly, amended with Surfynol® 485, E. fetida or organic material that served as food for the earthworms for 56 days. In both soils, the removal of anthracene was in the order: mixing soil weekly (100%) > earthworms applied (92%) > organic material applied (77%) > untreated soil (57%) > surfactant applied (34%) after 56 days. There was no clear link between removal of anthracene from soil and changes in the bacterial community structure. On the one hand, application of earthworms removed most of the contaminant from the arable soil and had a strong effect on the bacterial community structure, i.e. a decrease in the relative abundance of the Acidobacteria, Chloroflexi and Gemmatimonadetes, and an increase in that of the Proteobacteria compared to the unamended soil. Mixing the soil weekly removed all anthracene from the arable soil, but had little or no effect on the bacterial community structure. On the other hand, application of the surfactant inhibited the removal of anthracene from the arable soil compared to the untreated soil, but had a strong effect on the bacterial community structure, i.e. a decrease in the relative abundance of Cytophagia (Bacteroidetes), Chloroflexi, Gemmatimonadetes and Planctomycetes and an increase in that of the Flavobacteria (Bacteroidetes) and Proteobacteria. Additionally, the removal of anthracene was similar in the different treatments of both the arable and pasture soil, but the effect of application of carrot residue, earthworms or the surfactant on the bacterial community structure was more accentuated in the arable soil than in the pasture soil. It was found that removal of anthracene was not linked to changes in the bacterial community structure.

Structural Diversity of Class 1 Integrons in Multiresistant Strains of Escherichia coli Isolated from Patients in a Hospital in Mexico City.

AbstractSince a decade, Escherichia coli has been considered an important nosocomial pathogen due to the high number of isolates multiresistant to antimicrobials reported worldwide. In clinical and environmental strains, transposons, plasmids, and integrons are currently considered the principal genetic elements responsible for the acquisition of antibiotic resistance through horizontal transfer. The objective of this research was to correlate the resistance to antibiotics of E. coli clinical strains with the presence class I integrons. In the present study, one hundred E. coli strains were isolated and tested for susceptibility and resistance to antimicrobials. Class 1 integrons were detected by PCR, and the arrangement of gene cassettes was determined by sequencing. Twenty two strains were found to carry Class 1 integrons. Sequence analysis of the variable regions revealed the presence of several gene cassettes, such as dihydrofolate reductases (dfr2d, dfrA17, and dhfrXVb), adenylyl transferases (aadA2, addA5, and addA22), and chloramphenicol efflux pump (cmlA), and oxacillinase (blaOXA–1). The dfrA17–addA5 arrangement prevailed upon other integrons in the study. This is the first report of the presence of the dfr2d and dhfrXVb–aadA2 cassette arrangements in a Class 1 integrons from clinical strains of E. coli. In most of the strains, it was found a direct relationship between genetic arrangements and resistance phenotypes. Four integrons were detected in plasmids that might be involved in the resistance genes transfer to other bacteria of clinical importance. Our results confirm the presence of Class 1 integrons and their essential role in the dissemination of resistance cassettes among E. coli strains.

Bactericidal effect of γ-radiation with 137Cesium in platelet concentrates

INTRODUCTION γ-radiation is a method that was originally designed for inactivation of T lymphocytes in blood and blood components in order to prevent transfusion associated-graft versus host disease (TA-GVHD). Klebsiella pneumoniae, Escherichia coli, Enterococcus faecium and Staphylococcus epidermidis strains are important pathogens in blood banks since they have been related to post-transfusional sepsis. This study was conducted to demonstrate that γ-radiation is effective in reducing the viability of bacteria in platelet concentrates (PC). MATERIAL AND METHODS Klebsiella pneumoniae, E. coli, E. faecium and S. epidermidis strains were adjusted at 101 to 106 CFU/ml and used in artificial contamination assays in PC. Contaminated platelet concentrates were subjected to γ-radiation with doses of 2500 cGy in a 137Cesium irradiator. The average of surviving bacteria at different bacterial concentrations, logarithmic reduction values (LRV) and bacterial death after γ-radiation percentage was calculated. RESULTS Escherichia coli and K. pneumoniae were eliminated in 101 to 103 CFU; in contrast with 104 to 106 CFU, the LRV were 2.4, 2.6 and 2.6 for E. coli and 3.3, 2.7 and 3.0 for K. pneumoniae strains at 104, 105 and 106 CFU respectively. For Gram-positive strains, 101 CFU in PC, the inactivation post γ-radiation was not completed. Logarithmic reduction values post γ-radiation were 0.8 to 1.2 for E. faecium and S. epidermidis strains respectively. CONCLUSION γ-radiation cannot be an alternative for the inactivation of pathogens in PC, because of the bacterial concentration and pathogen nature - being resistant to γ-radiation, the Gram-positive bacteria.

Changes in the incidence of intestinal giardiosis in Mexican population during five years (2011-2015)

Giardiosis is a parasitic disease caused by the protozoan Giardia intestinalis, which is distributed worldwide. Most of the data on the prevalence of giardiosis in Mexico comes from research, but it is also necessary to study the data provided by the Mexican Health Ministry and issued by the General Directorate of Epidemiology. The aim of this work was analyse the national surveillance data for human giardiosis in order to update the epidemiological data of this disease in Mexico. A retrospective observational analysis of giardiosis (from January 2011 to December 2015) was performed in the annual reports emitted by the GDE in Mexico. The cases were classified by year, state, age group, gender and seasons of the year. During the period of 2011–2015, a reduction of 38.51% was observed in the total number of new cases of giardiosis reported in the whole country. The states of Sinaloa, Yucatan, and Chiapas presented the highest number of new cases reported during the analysed period. Giardiosis rates were always higher among women in all age groups, but the maximum incidence was observed in both sexes in the age group of 1–4 years old (the most susceptible group). On the other hand, the number of cases increased dramatically in southern states during warmer months. Giardiosis is influenced by ambient temperature changes along the year, although this study suggests that tends to decrease in all the analysed states and could be related to the overall improvement of hygienic practices within the Mexican population

HLA frequency in candidates to transplant without compatible cord blood at the National Center of Blood Transfusion (Mexico)

INTRODUCTION Umbilical Cord Blood Units (UCBU) for transplantation, are a therapeutic possibility for patients with a wide range of oncohaematological diseases and other immunologic disorders. The search of compatible donors for bone marrow transplantation is increasingly difficult for patients of mixed ethnicity. The aim of this work was determine the HLA frequency of candidates for transplantation without compatible UCBU at the National Center of from Blood Transfusion (NCBT) - Mexico. MATERIAL AND METHODS A retrospective analysis of candidates to transplant without compatible UCBU was performed in the archives from 2003 to 2016 at the NCBT. HLA class I and II genotyping of candidates was performed by medium resolution methods: Sequence Specific Primer and/or Specific Sequence Oligonucleotide. HLA frequencies were obtained by including individuals without any particular bias to a phenotype and strict statistical and genetic analysis of populations were done. The database in www.allelefrequencies.net was used in order to identify the ethnic origin of the most frequent alleles. RESULTS Three hundred and sixty-four candidates without compatible UCBU for transplantation were identified. The most frequent haplotype HLA I and II were: HLA-A*02/02, 02/24, 24/24, 02/68, 01/24 and 24/68; HLA-B*39/39, 35/51, 44/44, 44/40, 35/40 and 35/35; HLA-DRB1*04/04, 13/07, 04/13, 13/13 and 03/11. The ethnic origins of the analyzed data were represented in most cases by Amerindians, Caucasics, Orientals, Asians, Arabs and Africans. CONCLUSION This work shows the existence of a broad genetic diversity of candidates for transplantation with UCBU, making it difficult to find compatible units considering donors only from the capital.