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.

Detection and characterization of class 1 integrons in Aeromonas spp. isolated from human diarrheic stool in Mexico.

We determined the presence of class 1 integrons related to the acquisition of resistance to antimicrobials in Aeromonas spp. isolated from individuals with diarrhea. Species were identified as A. caviae, A. hydrophila, A. veronii and A. media using PCR-RFLP of the 16S rDNA. Selected isolates were further characterized by ERIC-PCR. Resistance to chloramphenicol, aztreonam, tetracycline, trimethoprim/sulfamethoxazole, nalidixic acid and streptomycin, among others, was determined using the Kirby-Bauer method. Integrons were detected by PCR amplification of the 5' conserved, variable, and 3' conserved regions. Sequencing of the variable regions revealed class 1 integrons with cassettes encoding resistance to trimethoprim (dfrA12, dfrA15, dfrB4), streptomycin/spectinomycin (aadA2, aadA1), oxacillin (oxa2) and chloramphenicol (catB3, cmlA4). Others had an open reading frame (orfD) or no insert at all. To our knowledge, this is the first description of the occurrence of genes cmlA4 and dfrA15 in Aeromonas class 1 integrons. Not all the integron-linked cassettes conferred their associated resistances, which suggests the inactivity of some cassettes. Most integrons were chromosomally located. The presence of class 1 integrons similar to those found in a wide variety of bacterial genera from different origins, including environmental and fish-borne Aeromonas, confirms the stability and horizontal transfer of these genetic elements.

Bacteriological quality of bottled water obtained from Mexico City small water purification plants: Incidence and identification of potentially pathogenic nontuberculous mycobacteria species.

The aim of this study was to determine the bacteriological quality of bottled water samples obtained from small purification plants located in Mexico City and to identify potentially pathogenic nontuberculous mycobacteria (NTM) species found in these samples. All 111 samples analyzed were positive for aerobic mesophilic bacteria (AMB) and 46 (41.4%) did not comply with Mexico's Official Guidelines. Sixty-nine (62.1%) and 23 (20.7%) water samples were positive for total coliforms (TC) and fecal coliforms (FC), respectively. A total of 81 (72.9%) of the water samples exceeded the maximum allowed limit stipulated in the guideline. Thirty-three (29.7%) of the purified water samples were positive for NTM, being recovered a total of 40 isolates. These NTM isolates were identified using three molecular markers (hsp65, rrs and rpoB genes) which corresponded to the fast-growing mycobacteria M. chelonae (n = 12), M. porcinum (n = 8), M. senegalense (n = 5), M. abscessus (n = 4), M. septicum (n = 4), M. wolinskyi (n = 3), M. mucogenicum (n = 2), M. fortuitum (n = 1) and M. sp. (n = 1). In seven purified water samples, two different NTM species were isolated simultaneously. Overall, these results showed that most of the purified bottled water samples analyzed in this study had unsatisfactory microbiological quality and some harbored NTM associated with illness. Our data could hasten health authorities to intensify efforts in the routine monitoring of activities in the purified bottled water industry in order to supply safe and healthy water to the public.

Presence of Aeromonas spp in water from drinking-water- and wastewater-treatment plants in Mexico City.

The frequency of Aeromonas spp in three wastewater-treatment plants (WWTPs) and two drinking-water plants (DWPs) in México City was determined. Samples were taken throughout a year by the Moore's swab technique. A total of 144 samples were obtained from WWTPs and 96 from DWPs of both incoming and outflowing water. Aeromonas spp was isolated in 31% of the samples, from both kinds of sources. The technique used for the isolation of the pathogen was suitable for samples with high associate microbiota content and for those with a scarce microbial content. The presence of mesophilic-aerobic, coliform, and fecal-coliform organisms was investigated to determine whether there was any correlation with the presence of Aeromonas spp. Most samples from WWTP, which did not comply with the Mexican standards, had the pathogen, and some of the samples from the outflow of the DWP, which were within the limits set by the Mexican standards, also had Aeromonas spp. Most samples containing Aeromonas spp. had concentrations below 0.1 ppm residual chlorine, and the strains were resistant to 0.3 ppm, which supports the recommendation to increase the residual chlorine concentration to 0.5 to 1.0 ppm, as recommended by the Mexican standards.

Microbiological Quality of Ready-to-Eat Vegetables Collected in Mexico City: Occurrence of Aerobic-Mesophilic Bacteria, Fecal Coliforms, and Potentially Pathogenic Nontuberculous Mycobacteria.

The aims of this study were to evaluate the microbiological quality and the occurrence of nontuberculous mycobacteria (NTM) in a variety of salads and sprouts from supermarkets and street vendors in Mexico City. Aerobic-mesophilic bacteria (AMB) were present in 100% of RTE-salads samples; 59% of samples were outside guidelines range (>5.17 log10 CFU per g). Although fecal coliforms (FC) were present in 32% of samples, only 8% of them exceeded the permissible limit (100 MPN/g). Regarding the 100 RTE-sprouts, all samples were also positive for AMB and total coliforms (TC) and 69% for FC. Seven NTM species were recovered from 7 salad samples; they included three M. fortuitum, two M. chelonae, one M. mucogenicum, and one M. sp. Twelve RTE-sprouts samples harbored NTM, which were identified as M. porcinum (five), M. abscessus (two), M. gordonae (two), M. mucogenicum (two), and M. avium complex (one). Most RTE-salads and RTE-sprouts had unsatisfactory microbiological quality and some harbored NTM associated with illness. No correlation between the presence of coliforms and NTM was found. Overall, these results suggest that RTE-salads and RTE-sprouts might function as vehicles for NTM transmission in humans; hence, proper handling and treatment before consumption of such products might be recommendable.

Occurrence of potentially pathogenic nontuberculous mycobacteria in Mexican household potable water: a pilot study.

BACKGROUND: Nontuberculous mycobacteria (NTM) are environmental opportunistic pathogens found in natural and human-engineered waters, including drinking water distribution systems and household plumbing. This pilot study examined the frequency of occurrence of NTM in household potable water samples in Mexico City. Potable water samples were collected from the "main house faucet" and kitchen faucet. The presence of aerobic-mesophilic bacteria (AMB), total coliforms (TC), fecal coliforms (FC) and NTM species were determined. Mycobacteria species were identified by PCR restriction enzyme pattern analysis (PRA) of the 65-kDa heat shock protein gene (hsp65) and sequencing of the hypervariable region 2 (V2) of the 16S rRNA gene and of the rpoB gene. RESULTS: AMB (<100 CFU/ml) were present in 118 out of 120 samples; only two samples were outside guidelines ranges (>100 CFU/ml). TC and FC were detected in four and one samples, respectively. NTM species were recovered from 16% samples (19/120) and included M. mucogenicum (nine), M. porcinum (three), M. avium (three), M. gordonae (one), M. cosmeticum (one), M. fortuitum (one), and Mycobacterium sp (one). All household water samples that contained NTM complied with the standards required to grade the water as "good quality" potable water. CONCLUSION: Household potable water may be a potential source of NTM infection in Mexico City.

Pathogenic determinants of clinical Klebsiella pneumoniae strains associated with their persistence in the hospital environment

Background: Klebsiella pneumoniae is considered an opportunistic pathogen associated with nosocomial infections occurring mainly in pediatric patients, such as premature infants placed in intensive care units. The aim of this study was to characterize K. pneumoniae strains isolated from different clinical sources based on their resistance to antibiotics and the presence of virulence factors associated with their persistence in the hospital environment. Methods: Fifty clinical strains of K. pneumoniae isolated from urine, blood, catheters, and cerebrospinal fluid sources were characterized. Susceptibility testing of antibiotics was performed by the Kirby-Bauer method (Clinical Laboratory Standards Institute, 2010). The ability to form a biofilm was determined by the 96-well microplate method. Capsule and fimbrial structures were visualized by transmission electron microscopy (TEM). Adherence was evaluated on A549 and HT29 cells. Assessment for the presence and expression of the ecpA, fimH, and mrkA genes was performed by PCR and RT-PCR. Results: Clinical strains of K. pneumoniae were isolated from 48% of urine, 24% of blood, 18% of catheters, and 10% of cerebrospinal fluid. Ninety-two percent of the strains showed resistance to cefpodoxime, whereas few strains showed resistance to imipenem and meropenem (4 and 2%, respectively). The extended spectrum-type beta-lactamase (ESBL) phenotype was identified in 97% of the strains positive for resistance to third-generation cephalosporins. In addition, 88% of the strains were multidrug resistant. All strains were able to form biofilms. Capsule and fimbirial structures were visualized by TEM. Based on our adhesion assays, the A549 cell line was more permissive to K. pneumoniae strains than the HT-29 cell line. K. pneumoniae strains amplified and expressed ecpA (100/70%), fimH (98/2%), and mrkA (84/48%) genes, respectively. Conclusion: The K. pneumoniae strains exhibited features that allowed them to survive in the hospital environment (formation of biofilm) and resist antimicrobial therapy (multidrug resistant MDR strains). These strains also possessed a capsule, adhesive properties, and expression of genes encoding colonization factors that favor the selection and persistence of these strains in hospitals.

Elaboración de un manual de procedimientos para el estudo de brotes provocados por el consumos de alimentos / The desugning of a proceeding manual for evaluation of foodborne disease

Se elaboró un manual de procedimientos que contiene aspectos importantes que se descuidan en la investigación de brotes de enfermedades transmitidas por alimentos con la finalidad de mejorar la calidad del estudio de los mismos.Se probó el funcionamiento del manual, en un estudio cuasiexperimental, de asignación aleatoria de sujetos, de ensayo de campo.La brigada disponible en el momento del estudio, capacitada en el uso del manual propuesto, investigó un brote alimentario. El informe generado por la investigación fue comparado con el obtenido por otra brigada en un evento anterior que se presentó en la misma entidad federativa, después de asignar calificaciones a cada parte.Los promedios se analizaron por el método de "t" de Student para comparación de medias aritméticas con un nivel de confianza del 95 por ciento, y se determinó que sí existía diferencia significativa entre ambos.Se concluye que se mejora la calidad del estudio de los brotes alimentarios con el uso del manual de procedimientos propuesto.

Ausencia de vibrio parahaemolyticus en pescado crudo / Abscence of vibrio parahemolyticus in raw fish

Se analizaron 389 muestras de pescado colectadas por los Promotores de la Secretaria de Salud a lo largo de un año. Se analizaron 329 muestrs de pescado por la técnica de Nava y 60 muestras por la técnica de la FDA; no se obtuvieron aislamientos positivos de Bibrio parahaemolyticus aún en la época de verano en la cual se tiene la mayor incidencia del microorganismo de acuerdo a datos bibliográficos. Se señala que aunque el microorganismo pudo estar presente en los pescados analizados, debido al tamaño de la muestra analizada y al manejo inadecuado de los pescados desde la pesca hasta su venta se propicia un incremento de la flora asociada lo que dificulta el aislamiento de Vibrio parahaemolyticus por las técnicas utilizadas