Study of antifungal agent caspofungin adsorption to laboratory materials.

Treatment of invasive fungal infections with Caspofungin is used as the first-line antifungal agents. The minimum inhibitory concentration value is a test which indicates the degree of sensitivity of a strain regarding a drug. However, no value of minimum inhibitory concentration for caspofungin is available because very variable value is obtained. In this work, we study the link with the adsorption phenomenon of CSF previously described in literature and the lack of minimum inhibitory concentration value. A systematic study of the impact of different parameters on CSF adsorption is reported. The effect of the nature of container material, the aqueous solution pH and the organic solvent proportion was studied. In addition, the possibility of using a coating agent to minimize the adsorption was assayed and evaluated. Results obtained showed the importance of the material used during the manipulation of CSF. The use of acidic pH aqueous solution or the addition of acetonitrile or methanol proportions (50 % and 70 %, respectively) were found efficient to avoid adsorption of CSF on glassware material, which is the relevant strategy for analytical samples of caspofungin. The treatment of HPLC glass vials and 96-well plates with N-(2-aminoethyl)-3-aminopropyltrimethoxysilane reduced the adsorption. The significant adsorption observed in this work especially with plastic materials, questions the results obtained before in different assays and explained the absence of MIC value.

Melanin and pyomelanin in Aspergillus fumigatus: from its genetics to host interaction.

Aspergillus fumigatus is a worldwide-distributed saprophytic fungus and the major cause of invasive aspergillosis. This fungus can produce two types of melanin-dihydroxynaphthalene melanin (DHN-melanin) and pyomelanin. These pigments are considered important resistance mechanisms to stress, as well as virulence factors. The aim of this review is to present the current knowledge of the genetic basis and metabolic pathways of melanin production, their activation, function, and interaction with the host immune system. The DHN-melanin pathway is encoded in a cluster that includes six genes (abr1, abr2, ayg1, arp1, arp2, and pksP/alb1 genes) whose encoded proteins seem to be the origin of the pigment in endosomes. These vesicles are secreted and the pigment is subsequently located in the wall of the conidium beneath the rodlet layer. Unlike DHN-melanin, pyomelanin does not have its own biosynthetic pathway but is related to the activation of the L-tyrosine/L-phenylalanine degradation pathway that includes a cluster of six genes (hppD, hmgX, hmgA, fahA, maiA, and hmgR). Its production is due to the polymerization of homogentisic acid and is linked to conidial germination. Despite the knowledge gained in recent years, further studies will be necessary to confirm the pathways that produce these pigments and their role in the virulence mechanisms of A. fumigatus.

Is there a direct relationship between stress biomarkers in oysters and the amount of metals in the sediments where they inhabit?

The effects exerted by metals in oysters are still a matter of debate and require more detailed studies. In this work we have investigated whether the health status of oysters are affected by the amount of metals present in the sediments of their habitat. Sediments and oysters were collected in the tidal part of the estuary of the Oka River (Basque Country), representative of other mesotidal, well mixed and short estuaries of the European Atlantic coast. The concentrations of 14 elements were determined in all the samples. Several biomarkers were also measured in the soft tissues of oysters. According to the concentrations found, the sediments were classified as non-toxic or slightly toxic. In good agreement, the histological alterations observed in oysters were not severe. Interestingly, in those sampling sites where the sediments showed relatively high metal concentrations, the metallic content in oysters was lower, and vice versa.

Rapid and specific detection of section Fumigati and Aspergillus fumigatus in human samples using a new multiplex real-time PCR.

Invasive aspergillosis is an opportunistic infection caused primarily by Aspergillus fumigatus. However, other common fungal pathogens belonging to section Fumigati are often misidentified as A. fumigatus. Thus, we have developed a multiplex real-time PCR (qPCR) assay with primers and specific TaqMan probes based on internal transcribed spacer regions or benA gene to discriminate, in less than 3 h, species of section Fumigati and, specifically, A. fumigatus. The multiplex qPCR showed a limit of detection of 20 and 50 fg of DNA for section Fumigati and A. fumigatus, respectively. Moreover, it enabled detection of a single germinated conidia. The inclusion of some PCR facilitators together with the dilution of samples makes it possible to completely avoid PCR inhibitions in all bronchoalveolar lavage (BAL) samples assayed. This technique may be a useful complementary tool in the diagnosis of invasive pulmonary aspergillosis caused by A. fumigatus using BAL fluid.

Treatment of acute antibody-mediated rejection: a single-center experience.

INTRODUCTION: Acute antibody-mediated rejection (AMR) leads to graft loss. The combination of plasmapheresis (PP), intravenous immunoglobulin (IVIG), and rituximab (RTX) has been reported to be effective therapy. PATIENTS AND METHODS: Between October 2005 and September 2009, 8 (4.7%) kidney transplant recipients developed AMR, diagnosed by severe acute rejection and extensive C4d staining in peritubular capillaries. RESULTS: All patients were treated with two to six sessions of PP with IVIG added after the last PP. In two patients, RTX was prescribed after PP and IVIG. Baseline immunosuppression was based on steroids, mycophenolate mofetil or azathioprine, and tacrolimus or cyclosporine or everolimus. The presence of subsequent significant decrease in anti-HLA class I antibodies was demonstrated in a highly sensitized patient before and after transplantation with PP treatment. An increase was observed before the diagnosis of AMR. After a mean follow-up of 10 months (range=1-23), patient and graft survivals were 100% and 50%, respectively. Three patients lost their transplants to AMR refractory to treatment and one patient, due to interstitial fibrosis and tubular atrophy at 23 months after AMR. Finally, four patients recovered renal function, showing a mean serum creatinine of 2.2±0.45 mg/dL. CONCLUSIONS: Early diagnosis and treatment with PP, IVIG, and RTX may resolve AMR. PP before and after transplantation in high-risk patients may result in anti-HLA class I and class II antibody removal from plasma and prevention of AMR.

Characterization of a monoclonal antibody directed against Salmonella enterica serovar Typhimurium and serovar [4,5,12:i:-].

Flagellar extracts of Salmonella enterica serovars expressing phase 2 H1 antigenic complex (H:1,2, H:1,5, H:1,6, and H:1,7) and a mutant flagellin obtained by site-directed mutagenesis of the fljB gene from serovar Typhimurium at codon 218, transforming threonine to alanine, expressed in Escherichia coli (fljB218(A)) were used to analyze the H1 antigenic complex. Cross-reactions were detected by Western blotting and dot blotting using commercial polyclonal antibodies against the different wild-type extracts and mutant FljB218(A). Therefore, we produced a monoclonal antibody (MAb), 23D4, isotyped as immunoglobulin M, against H:1,2 S. enterica serovar Typhimurium flagellin. The mutant flagellin was not recognized by this MAb. When a large number of phase 1 and phase 2 flagellin antigens of different serovars were used to characterize the 23D4 MAb, only extracts of serovars Typhimurium and [4,5,12:i:-] reacted. The protein composition of phase 1 and phase 2 extracts and highly purified H:1,2 flagellin from serovar Typhimurium strain LT2 and extract of strain 286 (serovar [4,5,12:i:-]), which reacted with the MAb, was studied. Phase 2 flagellin (FljB(H:1,2)) was detected in phase 1 and phase 2 flagellar heat extracts of serovar Typhimurium and was the single protein identified in all spots of purified H:1,2 flagellin. FliC, FlgK, and other proteins were detected in some immunoreactive spots and in the flagellar extract of serovar [4,5,12:i:-]. Immunoelectron microscopy of complete bacteria with 23D4 showed MAb attachment at the base of flagella, although the MAb failed to recognize the filament of flagella. Nevertheless, the results obtained by the other immunological tests (enzyme-linked immunosorbent assay, Western blotting, and dot blotting) indicate a reaction against flagellins. The epitopes could also be shared by other proteins on spots where FljB is not present, such as aminopeptidase B, isocitrate lyase, InvE, EF-TuA, enolase, DnaK, and others. In conclusion, MAb 23D4 can be useful for detection and diagnostic purposes of S. enterica serovar Typhimurium and serovar [4,5,12:i:-] and could be also helpful for epitope characterization of flagellum-associated antigens.

Comparison of tablet and disk diffusion methods for fluconazole and voriconazole in vitro activity testing against clinical yeast isolates.

We have compared a commercially available tablet diffusion method for the in vitro antifungal susceptibility testing of fluconazole (FCZ) and voriconazole (VCZ) with the disk diffusion method M44 (CLSI) with 282 clinical yeast isolates. The superior stability of antifungal agents in tablets can explain the differences for each category of susceptibility by both methods.Neo-Sensitabs tablets antifungal susceptibility testing showed an excellent correlation (0.98 for FCZ and 0.98 for VCZ at 24h and 0.96 for FCZ and 0.94 for VCZ at 48 h ), a reduced percentage of disagreements (4.6% and 8.2% for FCZ at 24h and 48 h respectively; 1.1% and 2.1% for VCZ at 24h and 48 h respectively) and the absence of statistically significant difference in comparison with the reference protocol for performing antifungal susceptibility testing with the agar diffusion method.

Selección e implantación de cepas de levadura del género Sccharomyces en la producción de vinos de la Denominación de Origen (DO) Chacolí de Vizcaya/Bizkaiko Txacolina / Selection and implantation of yeast strains of genus Saccharomyces at a winery regulated by Appellation Contrôlée "Chacolí de Vizcaya/Bizkaiko Txakolina"

El Chacolí de Vizcaya/Bizkaiko Txaxolina es un vino blanco característico del País Vaso con denominación de origen (BOPV 14/6/94). El objetivo del presente estudio fue seleccionar cepas de levaduras autóctonas para mejorar las condiciones de elaboración manteniendo las características propias de los vinos de esta región. Se aislaron levaduras identificadas como Saccharomyces bayanus durante las campañas 1996-1998 y se sometieron a un proceso selectivo en función de sus características enológicas y su comportamiento fermentativo. Tres de las cepas seleccionadas se inocularon a escala de bodega sobre mostos monovarietales de las dos variedades de uva aceptadas en esta denominación de origen, Hondarrabi Zuri y Folle Blanche. La implantación de las cepas inoculadas en las respectivas vinificaciones fue controlada mediante el análisis del polimorfismo de restricción ADN mitocondrial (REAmt) con la comparación con la enzima Alul, dada su especificidad, rapidez y sencillez tecnológica en comparación con otras técnicas de tipificación molecular utilizadas también en este estudio: cariotipificación cromosómica mediante electroforesis en campo pulsado, Random Amplified Polymorphic DNA-pCR (RAPD-pCR) y análisis del polimorfismo de restricción originado por la enzima Sfil de corte infrecuente (REA infrecuente). Este estudio ha demostrado que cepas con comportamientos fenotípicos diferentes presentan los mismos patrones de restricción con REAmt, pero pueden ser diferenciadas con otras técnicas aplicadas en este estudio, como RAPD-PCR, que pese a su baja reproductibilidad pueden ser una herramienta complementaria a REAmt. Nuestros resultados demuestran que, a pesar de utilizar cepas autóctonas seleccionadas, la inoculación de una caldo con una cepa no es garantía de que doina y dirija la fermentación del mosto, ya que una misma cepa puede imponerse o no dependiendo del tipo de mosto y de la campaña de que se trate. De las levaduras estudiadas, la cepa 2 fue la que mejores resultados proporcionó tanto en cata como en implantación, por lo que podría ser utilizada para la producción de Chacolí de Vizcaya con fines comerciales, sobre todo de mostos procedentes de Folle Blanche(AU)

The white wine Chacolía de Vizcaya/Bizkaiko Txakolina is characteristic from The Basque Country region and regulated under Appellation Contrôlée standards (BOPV 14/6/94). The objective of this study was the identification and selection of autochthonous yeast strains, to improve the conditions used to maintain the typical characteristics of this region wines. Yeasts identified as Saccharomyces bayanus isolated around these fields from 1996 to 1998, were subjected to a selective procedure based on enological characteristics and fermentative behaviour. Three of the selected strains were used to inoculate, at winery scale, two grape juice varieties accepted by the Appellation Contrôlée (Hondarrabi Zuri and Folle Blanche). The inoculated strains on the respective vinifications was followed by restriction fragment length polymorphism of mitochondrial DNA (REAmt) method with AluI enzyme, due to their specificity, short outcome, and technological simplicity compared with other molecular typing methods such as: chromosomal karyotyping analyzed by pulsed field gel electrophoresis, Random Amplified Polymorphic DNA-PCR (RAPD-PCR) and restriction fragment length polymorphism using the infrequently cutting enzyme SfiI (REA infrequent). This study demonstrated that strains with different phenotypic traits could show indistinguishable restriction patterns with REAmt, but could be discriminated using other typing methods such as RAPD-PCR, which although showing low reproducibility could be used as complementary to REAmt. Our results demonstrate that in spite of using autochthonous selected strains, the inoculation of musts with a particular strain do not guarantee its predominance and driving fermentation features. Of all yeast strains studied, strain no. 2 showed the best results in sensory testing and at the implantation process. Therefore, it could be used with commercial purposes for the production of Chacolí de Vizcaya/Bizkaiko Txakolina, especially when using musts from Folle Blanche(AU)

Kefir: a symbiotic yeasts-bacteria community with alleged healthy capabilities

El kéfir es una bebida láctea fermentada. Los gránulo de kéfir, comunidades de microorganismos que se agrupan en una matriz polisacárida denominada kefirano, son los responsables de esta fermentación. Estos gránulos son un ejemplo de simbiosis entre levaduras y bacterias y se han utilizado a través del tiempo para producir el kéfir, que es consumido por todo el mundo a pesar de su origen caucásico. En esa relación simbiótica, que son los gránulos de kéfir, se han aislado e identificado una amplia variedad de especies microbianas que comprenden levaduras y bacterias. El kéfir es un alimento prebiótico. Los prebióticos han demostrado ser beneficiosos para la salud, siendo de gran interés para la industria alimentaria en la actualidad. Según se afirma, el kéfir ha mostrado actividades antibacterianas, antifúngicas y antitumorales, entre otros atributos beneficiosos. Este trabajo incluye una revisión crítica de la composición microbiológica del kéfir junto con sus propiedades beneficiosas para la salud humana(AU)

Kefir is a fermented milk beverage. The milk fermentation is achieved by the of kefir grains, a cluster of microorganisms held together by a polysaccharide matrix named kefiran. Kefir grains are an example of symbiosis between yeast and bacteria. They have been used over years to produce kefir, a fermented beverage that is consumed all over the world, although its origin is Caucasian. A vast variety of different species of organisms forming the kefir grains, comprising yeast and bacteria, have been isolated and identified. Kefir is a probiotic food. Probiotics have shown to be beneficial to health, being presently of great interest to the food industry. Kefir has been accredited with antibacterial, antifungal and antitumoural activities among other beneficial attributes. This review includes a critical revision of the microbiological composition of kefir along with its beneficial properties to human health(AU)

Cáncer invasivo de cérvix en España (1995). Encuesta de la Sociedad Española de Ginecología y Obstetricia / Invasive cervical cancer in Spain (1995). Survey of the Spanish Society of Obstetrics and Gynecology

Objetivos: Conocer la atención al cáncer de cérvix en España mediante una encuesta retrospectiva sobre casos de 1995 realizada por la Sección de Ginecología Oncológica y Patología Mamaria de la SEGO. Sujetos y métodos: Contestaron a la encuesta 45 hospitales que informaron de 633 casos. El número de casos por hospital osciló entre 1 y 40, siendo la mediana de 11. Pudieron procesarse 578 fichas. Resultados: La edad media de las pacientes fue de 51,2 años. El 49,3% de los casos se diagnosticó en estadio I; el 27,5% en estadio II; el 16,2% en estadio III, y el 4,6% en estadio IV. El tratamiento más empleado fue quirúrgico, mediante la intervención de Wertheim. La supervivencia a los 5 años fue del 56,4%. Conclusiones: Los factores pronósticos identificados en la evolución del cáncer de cérvix fueron el estado ganglionar, la invasión parametrial, la afección del espacio linfovascular y el tipo histológico

Objectives: To ascertain the diagnosis, treatment and outcomes of cervical carcinomas in Spain through a retrospective survey of cases diagnosed in 1995 carried out by the Oncological Gynecology and Breast Disease Section of the Spanish Society of Obstetrics and Gynecology (SEGO). Subjects and methods: Forty-five hospitals responded, providing information on 633 patients with cervical carcinoma diagnosed in 1995. The number of cases per hospital ranged between one and 40, with a median of 11. A total of 578 forms were included. Results: The mean age at diagnosis was 51.2 years. A total of 49.3% of carcinomas were in stage I, 27.5% were in stage II, 16.2% were in stage III, and 4.6% were in stage IV. The most commonly used treatment was surgery (Wertheim’s intervention). Gross 5-year survival was 56.4%. Conclusions: The prognostic factors identified were affected lymph nodes, parametrial and lymphovascular invasion, and histological subtypes

Yeast associated with spontaneous fermentations of white wines from the "Txakoli de Bizkaia" region (Basque Country, North Spain).

The microbiota of eight spontaneous fermentation of white wine from different grape varieties and different wineries from the "Txakoli de Bizkaia" region (Basque country, North Spain), in 1996 and 1997 campaigns was studied. The yeast population was higher in grapes harvested in 1997, in which late summer and early autumn was warmer and drier. Eight species belonging to five genera were identified in total. The most frequent genera in grapes were Rhodotorula in 1996 and Kloeckera in 1997. Saccharomyces bayanus was the most frequent species during vigorous and final fermentation, and it was occasionally isolated from grapes and must. Only another Saccharomyces spp., i.e., S. kluyvery, was identified in some samples from 1997.

Radioquimioterapia en el tratamiento del carcinoma localmente avanzado o recurrente de vulva / Chemoradiation therapy in the treatment of locally advanced or recurrent vulvar carcinoma

Se trata a 3 pacientes con carcinomas localmente avanzados de vulva, y otras seis con carcinoma recurrente vulvar o inguinal mediante radioquimioterapia. Como agentes quimioterápicos se utilizan mitomicina C y 5-fluorouracilo. El tratamiento radioterápico alcanzó, por lo menos, 60 Gy. Todas las pacientes con carcinoma avanzado obtuvieron una respuesta clínica completa, y en el momento de la última revisión estaban vivas y sin evidencia de enfermedad. De las pacientes con recidiva, dos (33,3%) consiguieron una respuesta completa, dos (33,3%) una respuesta parcial (que posibilitó la cirugía de rescate), y dos (33,3%) no obtuvieron respuesta. De este grupo, el 66,6% estaban vivas y sin enfermedad. La radioquimioterapia es un excelente método de tratamiento del carcinoma vulvar localmente avanzado que evita cirugías mutilantes, y también es efectivo en el tratamiento de la recidiva tumoral (AU)

Comparative evaluation of three commercial software packages for analysis of DNA polymorphism patterns.

OBJECTIVE: In the present study we have compared three commercial software packages, GelCompar, Molecular Analyst Fingerprinting, and BioImage, to determine if the results generated by the programs were comparable and correlated adequately with visual interpretation of electrophoretic gels, in the analysis of several well characterized incidents of infections. METHODS: Infections caused by Pseudomonas aeruginosa, Candida dubliniensis, C. albicans, and serotypes of Salmonella were characterized by restriction endonuclease analysis, macrorestriction analysis of genomic DNA with pulsed-field gel electrophoresis, and random amplified polymorphic DNA. The genotypes were visually detected based on band presence or absence in the different gels. The similarity values of DNA profiles were computed using Dice coefficient and were presented in dendrograms by UPGMA. The concordance or agreement between the number of genotypes obtained and their clustering, using the computerized programs, was determined. RESULTS: In general, agreement in number of genotypes obtained visually and by using the commercial DNA analysis software was achieved, but discrepancies were also denoted between the systems. The concordance between the visual and the computerized analysis ranged from 72% to 100%. CONCLUSION: In our experience, although the programs evaluated in the present study performed acceptably well, such programs may be used as an aid in the analysis of complex banding patterns, and they do not provide an indisputably correct analysis in genotype definition.

Differences in the Candida albicans antigenic expression after heat shock and infection.

Heat-shock and infection induce changes in protein expression in C. albicans. To investigate if these alterations induce changes in antigenicity, we have compared the reactivity mediated by IgA antibodies of protein extracts from a strain of C. albicans and the same strain recovered from an infected animal, both at 24 degrees C and 37 degrees C. The antigenic variability was detected mainly in antigens recognized by salivary IgA. Antigens of 223, 205, 180 and 140 kDa were over-expressed in both strains at 37 degrees C, indicating that variations due to heat shock were present before and after infection. The antigens were characterized as mannoproteins located at the outer side of the cell wall. An antigen of 61 kDa was also detected in which the expression decreased significantly after infection This was independent of heat shock.

Suitability of PCR fingerprinting, infrequent-restriction-site PCR, and pulsed-field gel electrophoresis, combined with computerized gel analysis, in library typing of Salmonella enterica serovar enteritidis.

Strains of Salmonella enterica (n = 212) of different serovars and phage types were used to establish a library typing computerized system for serovar Enteritidis on the basis of PCR fingerprinting, infrequent-restriction-site PCR (IRS-PCR), or pulsed-field gel electrophoresis (PFGE). The rate of PCR fingerprinting interassay and intercenter reproducibility was low and was only increased when DNA samples were extracted at the same time and amplified with the same reaction mixtures. Reproducibility of IRS-PCR technique reached 100%, but discrimination was low (D = 0.52). The PFGE procedure showed an intercenter reproducibility value of 93.3%. The high reproducibility of PFGE combined with the previously determined high discrimination directed its use for library typing. The use of PFGE with enzymes XbaI, BlnI, and SpeI for library typing of serovar Enteritidis was assessed with GelCompar 4.0 software. Three computer libraries of PFGE DNA profiles were constructed, and their ability to recognize new DNA profiles was analyzed. The results obtained pointed out that the combination of PFGE with computerized analysis could be suitable in long-term epidemiological comparison and surveillance of Salmonella serovar Enteritidis, specially if the prevalence of genetic events that could be responsible for changes in PFGE profiles in this serovar was low.

Genotypic characterisation by PFGE of Salmonella enterica serotype Enteritidis phage types 1, 4, 6, and 8 isolated from animal and human sources in three European countries.

A total of 101 strains of Salmonella Enteritidis phage types (PT) 1, 4, 6, and 8 from Denmark, England and Spain were studied by PFGE to elucidate genetic relationships among strains isolated from animal, human and environmental sources between 1983 and 1997. Analysis with Xba I, Bln I and Spe I enzymes showed that the power of discrimination of this method was increased by the combination of the three enzymes (D=0.802), subdividing the strains into 28 genomic groups or genotypes. Many of the PT1, PT4, and PT6 strains from the three countries shared the same PFGE combination profile A1-A1-A1, confirming the close relationship among these phage types and the protracted spread of a single clone over a large geographical area. In general, strains from Denmark showed more variation in their PFGE profiles than those from England and Spain. PT4 strains exhibited genetic homogeneity in the three countries independently of their sources and period of isolation. Spe I gave the highest index of discrimination among PT6 strains as evidenced by a variety of PFGE profiles. The data clearly confirmed that PT8 strains isolated in the three countries were of a unique clonal origin, and the PFGE combination profile A10-A10-A1 was predominant and specific for this phage type. It is concluded that PFGE, in combination with phage typing, represents a suitable tool for the epidemiological typing of Salmonella Enteritidis strains which could be used for investigations or surveillance of the international spread of these clones.

Characterization of Candida albicans antigenic determinants by two-dimensional polyacrylamide gel electrophoresis and enhanced chemiluminescence.

The use of a two-dimensional polyacrylamide gel electrophoresis joined with Western blotting allowed us to investigate the reactivities of antibodies present in sera from mice and humans to antigens of Candida albicans blastoconidia. The analysis of the antibody response in the two models studied and the comparison between the antibody response in infected and noninfected individuals showed that the infection by C. albicans produces changes in the antibody response which may be of relevance in the serodiagnosis of invasive candidiasis. These changes include the induction of antibodies against new antigens, the disappearance of antibodies against a group of antigens and variations in the reactivity of antibodies directed to a different group of antigens. The technique used resolved the isoforms of several antigens including enolase. It is concluded that the antibody response in humans and mice with candidiasis is not homogeneously directed to all the isoforms of an antigen.

Typing of Salmonella enteritidis of different phage types of PCR fingerprinting.

Fifty-nine isolates of Salmonella spp. were typed by PCR fingerprinting using three single primers: ERIC2, M13 and OPS-19. First, their discrimination power in a group of nine different serotypes were studied and considerable differences in the band patterns were obtained. Further, a panel of 51 isolates of Salmonella enteritidis with eight different phage types were analysed with the three primers. The discriminating power increased by combining the patterns of the three primers, and in this case it was possible to distinguish between some phage types of Salm. enteritidis, but not all of them were differentiated.

Phage typing combined with pulsed-field gel electrophoresis and random amplified polymorphic DNA increases discrimination in the epidemiological analysis of Salmonella enteritidis strains.

Phage typing (PT) combined with pulsed-field gel electrophoresis (PFGE) and a random amplified polymorphic DNA (RAPD) fingerprinting method was used to characterize Salmonella enteritidis strains. Twenty-four epidemiologically unrelated isolates, sampled from diverse ecological niches and fifteen isolates from four well-defined outbreaks of foodborne gastroenteritis, were studied. Seven phage types, with a predominance of PT 4 (63% of isolates), were observed when analysing the epidemiologically unrelated group. PT 4 was detected in all of the ecological niches studied, including food and fecally polluted river and beach water. The discriminatory power for phage typing, the average probability that the typing system will assign a different type to two unrelated strains randomly sampled in the microbial population, was 0.62. Ten PFGE pattern types were obtained with Xba I restriction endonuclease enzyme among the unrelated isolates; thirteen isolates belonged to PFGE pattern type 1 and the rest of the PFGE types were assigned to one or two isolates. The Dice coefficient clustered the similarities of the PFGE patterns between 80-100%. PFGE showed a discriminatory power of 0.72. Five clearly distinct RAPD patterns were observed with the OPS-19 oligonucleotide, but the discrimination obtained was low (0.46). The combination of the three typing methods increased the number of types to seventeen, giving high discrimination (0.92). Seven of the isolates recovered from various ecological niches belonged to the combination PT 4/PFGE 1/RAPD A and other combinations were unique or included only two strains. The four epidemiologically well-defined foodborne outbreaks were associated with the PT 4 phage type. In two of the outbreaks, other phage types (PT 7a and RDNC) were also observed in two isolates. Most of the isolates belonging to the foodborne outbreaks had an identical PFGE pattern (PFGE pattern type 1), but a difference in a restriction band was observed in an isolate belonging to an outbreak. Two RAPD patterns were observed in the outbreaks; RAPD pattern type A was detected in three of the four outbreaks. When the combined typing method was applied to the study, high concordance was observed and most of the outbreak strains belonged to the combination PT 4/PFGE 1/RAPD A. It is concluded that the combination of phage type with PFGE and RAPD provides a powerful discriminatory tool for the epidemiological analysis of unrelated and related strains of S. enteritidis.