RESUMEN
Staphylococcus pseudintermedius is an opportunistic pathogen in dogs. Four housekeeping genes with allelic polymorphisms were identified and used to develop an expanded multilocus sequence typing (MLST) scheme. The new seven-locus technique shows S. pseudintermedius to have greater genetic diversity than previous methods and discriminates more isolates based upon host origin.
Asunto(s)
Técnicas de Tipificación Bacteriana/métodos , Tipificación de Secuencias Multilocus/métodos , Staphylococcus/clasificación , Staphylococcus/genética , Animales , Enfermedades de los Perros/microbiología , Perros , Variación Genética , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/veterinaria , Staphylococcus/aislamiento & purificaciónRESUMEN
BACKGROUND: The Netherlands is one of the most densely populated countries in the world, with extensive livestock of pigs. In 2005, the emergence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) was a fact, with a relatively high MRSA colonisation among pig farmers. These MRSA isolates mostly belonged to sequence type 398 (ST398). Compared to hospital-associated MRSA (HA-MRSA), severe infections due to LA-MRSA and transmission between individuals are still relatively rare. Therefore, LA-MRSA may warrant less stringent containment measures than HA-MRSA in hospital settings. RESULTS: The aim of this study was to develop a rapid diagnostic tool to distinguish LA-MRSA from non-LA-MRSA in aid of infection control. Here, we show that ST398 strains can be readily detected with real-time polymerase chain reaction (PCR). Analysis of a large panel of related and unrelated microorganisms confirmed that the real-time ST398 PCR (ST398-qPCR) assay does not cross-react with other microorganisms or with non-LA-S. aureus strains. ST398-qPCR analysis of MRSA isolates collected in 2010, 2011 and 2012 at the Jeroen Bosch Hospital (n = 275) showed that an average of 78 % of MRSA belonged to sequence type ST398. CONCLUSION: We conclude that the ST398 real-time PCR is a reliable assay to detect LA-S. aureus and anticipate that the use of this assay can prevent the unnecessary closing of hospital wards, which may lead to substantial savings for the health care system.
Asunto(s)
Staphylococcus aureus Resistente a Meticilina/clasificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Infecciones Estafilocócicas/diagnóstico , Animales , Técnicas de Tipificación Bacteriana , Infección Hospitalaria/diagnóstico , Infección Hospitalaria/transmisión , Reacciones Cruzadas , ADN Bacteriano/análisis , Humanos , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Países Bajos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Porcinos/microbiologíaRESUMEN
OBJECTIVES: Fast and adequate detection of extended-spectrum beta-lactamases (ESBLs) is crucial for infection control measures and the choice of antimicrobial therapy. The aim of this study was to develop and evaluate a novel ESBL assay using ligation-mediated amplification combined with microarray analysis to detect the most prevalent ESBLs in Enterobacteriaceae: TEM, SHV and CTX-M. METHODS: Analysis of the Lahey database revealed that the vast majority of TEM and SHV ESBLs differ from non-ESBL variants in three amino acid positions. TEM ESBLs have at least one of the following amino acid substitutions: R164S/H/C, G238D/N/S and E104K. In SHV ESBLs, one or more of the following substitutions is observed: D179A/N/G, G238S/A and E240K. Oligonucleotide probes were designed to detect these substitutions, covering 95% of ESBL TEM variants and 77% of ESBL SHV variants. In addition, probes were designed to distinguish between CTX-M groups 1, 2, 9 and 8/25. For evaluation of the assay, 212 Enterobacteriaceae isolates with various beta-lactamases were included (n = 106 ESBL positive). RESULTS: The sensitivity of the microarray was 101/106 (95%; 95% CI 89%-98%), and the specificity 100% (95% CI 97%-100%) using molecular characterization of ESBLs by PCR and sequencing as reference. Assay performance time was 8 h for 36 isolates. CONCLUSIONS: This novel commercially available DNA microarray system may offer an attractive option for rapid and accurate detection of CTX-M, TEM and SHV ESBL genes in Enterobacteriaceae in the clinical laboratory.
Asunto(s)
Proteínas Bacterianas/genética , Técnicas Bacteriológicas/métodos , Enterobacteriaceae/enzimología , Reacción en Cadena de la Ligasa/métodos , Análisis por Micromatrices/métodos , beta-Lactamasas/genética , ADN Bacteriano/genética , Enterobacteriaceae/efectos de los fármacos , Enterobacteriaceae/genética , Infecciones por Enterobacteriaceae/microbiología , Humanos , Sondas de Oligonucleótidos/genética , Sensibilidad y Especificidad , Resistencia betalactámicaAsunto(s)
Enfermedades de los Perros/transmisión , Resistencia a la Meticilina , Infecciones Estafilocócicas/transmisión , Infecciones Estafilocócicas/veterinaria , Staphylococcus intermedius/efectos de los fármacos , Zoonosis , Animales , Recuento de Colonia Microbiana/veterinaria , Enfermedades de los Perros/tratamiento farmacológico , Enfermedades de los Perros/epidemiología , Enfermedades de los Perros/microbiología , Perros , Genotipo , Países Bajos/epidemiología , Factores de Riesgo , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/epidemiología , Staphylococcus intermedius/genéticaRESUMEN
Campylobacter fetus is a species of gram-negative bacteria whose primary reservoir is the gastrointestinal tracts of cattle and sheep. Human infections are rare, though often invasive and sometimes fatal. In this paper, we studied an outbreak of six patients with a C. fetus infection and outlined their disease histories. In each case we were able to identify factors that led to a reduced resistance, including pre-existing illnesses and old age. Because of the unusually high number of patients that presented in a time period of only five months, the Community Health Services were commissioned to identify the source of infection. Using whole genome sequencing, we showed that 5 out of 6 patients belonged to the same cluster. This One Health approach resulted in the conclusion that the infection originated from unpasteurized sheep's milk processed into unripened cheese. Finally, various measures were put into place to prevent any further outbreaks.
Asunto(s)
Infecciones por Campylobacter/epidemiología , Campylobacter fetus/aislamiento & purificación , Queso/microbiología , Anciano , Animales , Brotes de Enfermedades , Humanos , Huésped Inmunocomprometido , Masculino , Leche/microbiología , Países Bajos/epidemiología , OvinosRESUMEN
A real-time PCR assay with a DNA purification and inhibition control (internal control; IC) was developed to detect Chlamydophila psittaci DNA in human clinical samples. Novel C. psittaci-specific primers targeting the ompA gene were developed. The IC DNA contained the same primer-binding sites and had the same length and nucleotide content as the C. psittaci DNA amplicon, but had a shuffled probe-binding region. The lower limit of detection was 80 target copies/PCR, corresponding to 6,250 copies/mL in a clinical sample. Specificity was tested using reference strains of 30 bacterial species. No amplification was observed from any of these samples. Respiratory samples from eight patients were positive with this PCR. Six of these patients were confirmed as positive for C. psittaci with serological testing. Two patients had increasing antibody titres, but did not fulfil criteria proposed previously for serologically proven Chlamydia spp. infection. The real-time PCR described in this paper is a sensitive, specific and rapid method to detect C. psittaci DNA in human clinical respiratory samples.
Asunto(s)
Chlamydophila psittaci/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Psitacosis/diagnóstico , Psitacosis/microbiología , Animales , Líquido del Lavado Bronquioalveolar/microbiología , Cartilla de ADN/química , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Faringe/microbiología , Reacción en Cadena de la Polimerasa/instrumentación , Sensibilidad y Especificidad , Esputo/microbiologíaRESUMEN
The molecular diversity of protein D of nonencapsulated Haemophilus influenzae strains isolated from persistently infected patients with chronic bronchitis was studied by sequencing the hpd gene of four independently obtained isolates. The nucleotide (nt) sequences of the hpd genes of two strains were identical. The other two hpd sequences showed nt substitutions which were mostly synonymous. As a consequence the deduced amino acid (aa) sequences differed from the consensus sequence only by a few aa. No changes in the hpd genes were observed among the four variants of the four strains persisting in chronic bronchitis patients for 9, 11, 8 and 3 months, respectively, although variation in their major outer membrane proteins P2 and P5 occurred. We conclude that the hpd gene is conserved during chronic infections of nonencapsulated H. influenzae.
Asunto(s)
Proteínas Bacterianas , Bronquitis/microbiología , Proteínas Portadoras/genética , Variación Genética , Infecciones por Haemophilus/microbiología , Haemophilus influenzae/genética , Inmunoglobulina D , Lipoproteínas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Enfermedad Crónica , ADN Complementario , Genes Bacterianos , Haemophilus influenzae/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Conformación Proteica , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
Non-capsulate strains of Haemophilus influenzae were genotyped by analysis of variable DNA segments obtained by amplification of genomic DNA with the polymerase chain reaction (PCR fingerprinting). Discrete fragments of 100-2000 bp were obtained. The reproducibility of the procedure was assessed by comparing: (i) the fingerprints of 16 colonies of a single H. influenzae strain; (ii) isolates obtained from individual sputum samples (a total of 57 H. influenzae isolates from three cystic fibrosis patients); and (iii) 17 isolates collected during an outbreak of H. influenzae infection in a local pulmonary rehabilitation centre. The discriminatory power of the method was demonstrated by showing that the PCR fingerprints of eight unrelated H. influenzae strains from sputum samples of patients with chronic obstructive pulmonary disease (COPD) and 32 strains from cystic fibrosis patients were all different. These 40 isolates also differed with respect to their restriction fragment length polymorphisms (RFLP) and major outer-membrane protein (MOMP) composition. Twelve MOMP antigenic strain variants from sputum samples of five COPD patients had identical PCR fingerprints and RFLPs. It was concluded that PCR fingerprinting is a reliable and reproducible method for genotyping non-capsulate strains of H. influenzae. The discriminatory power of PCR fingerprinting was similar to that of RFLP analysis, but the results of PCR fingerprinting were easier to interpret.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/análisis , Dermatoglifia del ADN , ADN Bacteriano/análisis , Haemophilus influenzae/clasificación , Polimorfismo de Longitud del Fragmento de Restricción , Secuencia de Bases , Secuencia de Consenso , Fibrosis Quística/microbiología , Cartilla de ADN/química , Genotipo , Infecciones por Haemophilus/microbiología , Haemophilus influenzae/química , Haemophilus influenzae/genética , Humanos , Enfermedades Pulmonares Obstructivas/microbiología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Secuencias Repetitivas de Ácidos Nucleicos , Reproducibilidad de los Resultados , Esputo/microbiologíaRESUMEN
Our purpose was to determine the dynamics of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) carriage and its determinants in persons working at pig farms, in order to identify targets for interventions. This prospective cohort study surveyed 49 pig farms in the Netherlands on six sampling dates in 1 year (2010-11). Nasal and oropharyngeal swabs were collected, as well as environmental surface samples from stables and house. Of 110 pig farmers, 38% were persistent MRSA nasal carriers. The average cross-sectional MRSA prevalence was 63%. Methicillin-susceptible S. aureus (MSSA) nasal carriage was associated with fewer MRSA acquisitions (prevalence rate (PR) = 0.47, p 0.02). In multivariate analysis, an age of 40-49 years (PR = 2.13, p 0.01), a working week of ≥40 h (PR=1.89, p 0.01), giving birth assistance to sows (PR=2.26, p 0.03), removing manure of finisher pigs (PR=0.48, p 0.02), and wearing a facemask (PR = 0.13, p 0.02) were significantly related with persistent MRSA nasal carriage. A higher MRSA exposure in stables was associated with MRSA in pig farmers (p <0.0001). This study describes a very high prevalence of LA-MRSA carriage in pig farmers, reflecting extensive exposure during work. We identified the possible protective effects of MSSA carriage and of continuously wearing a facemask during work.
Asunto(s)
Portador Sano/microbiología , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Infecciones Estafilocócicas/epidemiología , Infecciones Estafilocócicas/transmisión , Adolescente , Adulto , Anciano , Animales , Estudios Transversales , Femenino , Humanos , Ganado/microbiología , Masculino , Persona de Mediana Edad , Boca/microbiología , Países Bajos , Nariz/microbiología , Estudios Prospectivos , Factores de Riesgo , Infecciones Estafilocócicas/microbiología , Sus scrofa , Adulto JovenRESUMEN
A real-time quantitative PCR (qPCR) for detection of the apxIVA gene of Actinobacillus pleuropneumoniae was validated using pure cultures of A. pleuropneumoniae and tonsillar and nasal swabs from experimentally inoculated Caesarean-derived/colostrum-deprived piglets and naturally infected conventional pigs. The analytical sensitivity was 5colony forming units/reaction. In comparison with selective bacterial examination using tonsillar samples from inoculated animals, the diagnostic sensitivity of the qPCR was 0.98 and the diagnostic specificity was 1.0. The qPCR showed consistent results in repeatedly sampled conventional pigs. Tonsillar brush samples and apxIVA qPCR analysis may be useful for further epidemiological studies and monitoring for A. pleuropneumoniae.
Asunto(s)
Infecciones por Actinobacillus/veterinaria , Actinobacillus pleuropneumoniae/genética , Proteínas Bacterianas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Enfermedades de los Porcinos/diagnóstico , Infecciones por Actinobacillus/diagnóstico , Infecciones por Actinobacillus/microbiología , Actinobacillus pleuropneumoniae/aislamiento & purificación , Animales , Cesárea/veterinaria , Calostro/microbiología , Nariz/microbiología , Tonsila Palatina/microbiología , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , Porcinos , Enfermedades de los Porcinos/microbiologíaRESUMEN
The objective of this study was to investigate the prevalence of methicillin-resistant Staphylococcus pseudintermedius (MRSP) in people, pets and the environment in households with a pet with a clinical MRSP-infection within the past year. Personnel and the environment at veterinary clinics were also screened. Nasal swabs (humans), nasal and perineal swabs (pets) and environmental wipes were examined using selective culturing. Twenty households were enrolled; 10/20 index cases still had clinical signs of infection at the start of the study and all were MRSP-positive. Of the remaining 10 index cases five were MRSP-positive in nasal and/or perineal samples. Five of 14 (36%) contact dogs and four of 13 (31%) contact cats were found MRSP-positive. In the households with an index case with clinical signs of infection 6/7 (86%) contact animals were MRSP-positive. MRSP was cultured from 2/45 (4%) human nasal samples. Domestic contamination was widespread as positive samples were found in 70% of the households and 44% of all environmental samples were MRSP-positive. In all but one of these MRSP-positive households the index case was still MRSP positive. Among the personnel in veterinary clinics 4/141 (3%) were MRSP-positive. MRSP was cultured from 31/200 environmental samples in 7/13 clinics at the first sampling and in 3/6 clinics the environment remained MRSP-positive after cleaning and disinfection indicating that current cleaning procedures often were unable to eliminate MRSP. These results show that transmission of MRSP between infected or colonized dogs and cats and healthy people does occur but is relatively uncommon, while transmission to contact pets occurs frequently, especially when the index case still has clinical signs of MRSP-infection.
Asunto(s)
Enfermedades de los Gatos/microbiología , Enfermedades de los Perros/transmisión , Resistencia a la Meticilina , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/veterinaria , Staphylococcus/efectos de los fármacos , Animales , Enfermedades de los Gatos/epidemiología , Gatos , Enfermedades de los Perros/epidemiología , Enfermedades de los Perros/microbiología , Perros , Microbiología Ambiental , Hospitales Veterinarios , Humanos , Mascotas , Infecciones Estafilocócicas/epidemiología , Infecciones Estafilocócicas/transmisión , Staphylococcus/aislamiento & purificaciónRESUMEN
Psittacosis, caused by Chlamydophila psittaci, is a well described but sporadically occurring clinical entity, which mainly presents as community-acquired pneumonia. Diagnosis used to be relatively difficult. However, new molecular techniques, such as real-time polymerase chain reaction, increased detection of cases. Furthermore, genotyping of the ompA gene can be used as a tool to trace the possible source of an outbreak or to link a specific bird to a particular patient.
Asunto(s)
Psitacosis/diagnóstico , Secuencia de Aminoácidos , Animales , Técnicas de Tipificación Bacteriana , Aves/microbiología , Chlamydophila psittaci/clasificación , Chlamydophila psittaci/genética , Genoma Bacteriano , Humanos , Datos de Secuencia Molecular , Países Bajos/epidemiología , Psitacosis/complicaciones , Psitacosis/epidemiología , Salud PúblicaRESUMEN
A Luminex suspension array, which had been developed for identification of Cryptococcus neoformans and Cryptococcus gattii isolates, was tested by genotyping a set of 58 mostly clinical isolates. All genotypes of C. neoformans and C. gattii were included. In addition, cerebrospinal fluid (CSF) obtained from patients with cryptococcal meningitis was used to investigate the feasibility of the technique for identification of the infecting strain. The suspension array correctly identified haploid isolates in all cases. Furthermore, hybrid isolates possessing two alleles of the Luminex probe region could be identified as hybrids. In CSF specimens, the genotype of the cryptococcal strains responsible for infection could be identified after optimization of the PCR conditions. However, further optimization of the DNA extraction protocol is needed to enhance the usability of the method in clinical practice.
Asunto(s)
Cryptococcus neoformans/clasificación , Cryptococcus/clasificación , Citometría de Flujo/métodos , Técnicas de Tipificación Micológica , Reacción en Cadena de la Polimerasa/métodos , Adulto , Anciano , Líquido Cefalorraquídeo/microbiología , Criptococosis/microbiología , Cryptococcus/genética , Cryptococcus/aislamiento & purificación , Cryptococcus neoformans/genética , Cryptococcus neoformans/aislamiento & purificación , ADN de Hongos/análisis , ADN de Hongos/aislamiento & purificación , Femenino , Genotipo , Humanos , Masculino , Meningitis Criptocócica/microbiología , Microesferas , Persona de Mediana Edad , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Especificidad de la Especie , SuspensionesRESUMEN
Three hundred sixty Enterobacteriaceae and nonfermenting gram-negative bacilli, isolated during one week in May 2004 at five hospitals in Amsterdam, The Netherlands, were evaluated for the presence of extended-spectrum beta-lactamases (ESBLs). A prevalence of 7.8% was found, in contrast to the 1% observed in 1997. CTX-M ESBLs dominated, and four types were identified in 18 isolates.
Asunto(s)
Proteínas Bacterianas/análisis , Infecciones por Enterobacteriaceae/microbiología , Enterobacteriaceae/enzimología , beta-Lactamasas/análisis , Antibacterianos/farmacología , Enterobacteriaceae/efectos de los fármacos , Enterobacteriaceae/aislamiento & purificación , Hospitales , Humanos , Pruebas de Sensibilidad Microbiana , Países BajosRESUMEN
For epidemiological studies of Campylobacter infections, molecular typing methods that can differentiate campylobacters at the strain level are needed. In this study we used a recently developed genotyping method, amplified fragment length polymorphism (AFLP), which is based on selective amplification of restriction fragments of chromosomal DNA, for genetic typing of Campylobacter jejuni and Campylobacter coli strains derived from humans and poultry. We developed an automated AFLP fingerprinting method in which restriction endonucleases HindIII and HhaI were used in combination with one set of selective PCR primers. This method resulted in evenly distributed band patterns for amplified fragments ranging from 50 to 500 bp long. The discriminatory power of AFLP was assessed with a C. jejuni strain, an isogenic flagellin mutant, and distinct C. jejuni strains having known pulsed-field gel electrophoresis and fla PCR-restriction fragment length polymorphism genotypes. Unrelated C. jejuni strains produced heterogeneous patterns, whereas genetically related strains produced similar AFLP patterns. Twenty-five Campylobacter strains obtained from poultry farms in The Netherlands grouped in three C. jejuni clusters that were separate from a C. coli cluster. The band patterns of 10 C. jejuni strains isolated from humans were heterogeneous, and most of these strains grouped with poultry strains. Our results show that AFLP analysis can distinguish genetically unrelated strains from genetically related strains of Campylobacter species. However, desirable genetically related strains can be differentiated by using other genotyping methods. We concluded that automated AFLP analysis is an attractive tool which can be used as a primary method for subtyping large numbers of Campylobacter strains and is extremely useful for epidemiological investigations.
Asunto(s)
Infecciones por Campylobacter/microbiología , Campylobacter/clasificación , Campylobacter/genética , Dermatoglifia del ADN/métodos , Aves de Corral/microbiología , Animales , Técnicas de Tipificación Bacteriana , Campylobacter/aislamiento & purificación , Infecciones por Campylobacter/epidemiología , Campylobacter coli/genética , Campylobacter coli/aislamiento & purificación , Campylobacter jejuni/genética , Campylobacter jejuni/aislamiento & purificación , Cartilla de ADN/genética , Desoxirribonucleasa HindIII/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Electroforesis en Gel de Campo Pulsado , Genotipo , Reacción en Cadena de la Polimerasa/métodos , Reproducibilidad de los ResultadosRESUMEN
The sequence of the gene encoding major outer membrane protein (MOMP) P2 of antigenic variants of non-encapsulated Haemophilus influenzae isolated from persistently infected chronic bronchitis patients was analysed. Antigenic drift was shown to result from single base changes in the P2 gene, all generating amino acid changes in the surface-exposed loops of MOMP P2, predominantly in loop 6. Similar single base changes were observed in H. influenzae persistently present in a subcutaneous cage implanted in rabbits, as well as in a spontaneous H. influenzae mutant that had survived MOMP P2 specific monoclonal-antibody-dependent bactericidal killing in vitro. We hypothesize that accumulation of point mutations under the selection pressure of immunity is a mechanism of antigenic drift of a surface-exposed protein during persistent H. influenzae infection.
Asunto(s)
Variación Antigénica/genética , Proteínas de la Membrana Bacteriana Externa/genética , Bronquitis/microbiología , Frecuencia de los Genes , Haemophilus influenzae/genética , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/genética , Cápsulas Bacterianas , Proteínas de la Membrana Bacteriana Externa/inmunología , Secuencia de Bases , Enfermedad Crónica , Cámaras de Difusión de Cultivos , Modelos Animales de Enfermedad , Genes Bacterianos/genética , Haemophilus influenzae/inmunología , Humanos , Datos de Secuencia Molecular , Mutación Puntual , Reacción en Cadena de la Polimerasa , Conejos , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Factores de TiempoRESUMEN
Thirty-five Finnish Campylobacter jejuni strains with five SmaI/SacII pulsed-field gel electrophoresis (PFGE) genotypes selected among human and chicken isolates from 1997 and 1998 were used for comparison of their PFGE patterns, amplified fragment length polymorphism (AFLP) patterns, HaeIII ribotypes, and heat-stable (HS) serotypes. The discriminatory power of PFGE, AFLP, and ribotyping with HaeIII were shown to be at the same level for this selected set of strains, and these methods assigned the strains into the same groups. The PFGE and AFLP patterns within a genotype were highly similar, indicating genetic relatedness. The same HS serotypes were distributed among different genotypes, and different serotypes were identified within one genotype. HS serotype 12 was only associated with the combined genotype G1 (PFGE-AFLP-ribotype). These studies using polyphasic genotyping methods suggested that common Finnish C. jejuni genotypes form genetic lineages which colonize both humans and chickens.
Asunto(s)
Técnicas de Tipificación Bacteriana , Infecciones por Campylobacter/microbiología , Campylobacter jejuni/clasificación , Campylobacter jejuni/genética , Animales , Pollos , Electroforesis en Gel de Campo Pulsado , Finlandia , Genotipo , Humanos , Polimorfismo de Longitud del Fragmento de Restricción , Enfermedades de las Aves de Corral/microbiología , Ribotipificación , SerotipificaciónRESUMEN
The genetic stability of selected epidemiologically linked strains of Campylobacter jejuni during outbreak situations was investigated by using subtyping techniques. Strains isolated from geographically related chicken flock outbreaks in 1998 and from a human outbreak in 1981 were investigated. There was little similarity in the strains obtained from the different chicken flock outbreaks; however, the strains from each of three chicken outbreaks, including strains isolated from various environments, were identical as determined by fla typing, amplified fragment length polymorphism (AFLP) analysis, and pulsed-field gel electrophoresis, which confirmed the genetic stability of these strains during the short time courses of chicken flock outbreaks. The human outbreak samples were compared with strain 81116, which originated from the same outbreak but has since undergone innumerable laboratory passages. Two main AFLP profiles were recognized from this outbreak, which confirmed the serotyping results obtained at the time of the outbreak. The major type isolated from this outbreak (serotype P6:L6) was exemplified by strain 81116. Despite the long existence of strain 81116 as a laboratory strain, the AFLP profile of this strain was identical to the profiles of all the other historical P6:L6 strains from the outbreak, indicating that the genotype has remained stable for almost 20 years. Interestingly, the AFLP profiles of the P6:L6 group of strains from the human outbreak and the strains from one of the recent chicken outbreaks were also identical. This similarity suggests that some clones of C. jejuni remain genetically stable in completely different environments over long periods of time and considerable geographical distances.
Asunto(s)
Infecciones por Campylobacter/epidemiología , Campylobacter jejuni/clasificación , Campylobacter jejuni/genética , Brotes de Enfermedades , Enfermedades de las Aves de Corral/microbiología , Animales , Técnicas de Tipificación Bacteriana , Infecciones por Campylobacter/microbiología , Infecciones por Campylobacter/veterinaria , ADN Bacteriano/análisis , Electroforesis en Gel de Campo Pulsado , Flagelina/genética , Humanos , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Aves de Corral , Enfermedades de las Aves de Corral/epidemiologíaRESUMEN
The pCloDF13-encoded bacteriocin release protein (BRP) is a lipoprotein which is synthesized as a precursor with an amino-terminal signal peptide that appears to be stable after cleavage. The role of the stable signal peptide in the functioning of the BRP was studied with respect to the release of cloacin DF13, 'lysis' and leakage of periplasmic proteins. The BRP gene fragment encoding the stable signal peptide was replaced by a fragment encoding the unstable peptide of the murein lipoprotein (Lpp). The resulting hybrid protein was normally acylated and processed by signal peptidase II, leaving no stable signal peptide in the cells. Expression of the hybrid protein did not result in the specific release of cloacin DF13, whereas 'lysis' and the release of periplasmic enzymes were unaffected. These results indicated a role for the stable BRP signal peptide in the translocation of cloacin DF13 across the cytoplasmic membrane.
Asunto(s)
Proteínas Bacterianas/metabolismo , Bacteriocinas/metabolismo , Proteínas de Escherichia coli , Señales de Clasificación de Proteína/metabolismo , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/genética , Secuencia de Bases , Clonación Molecular , ADN Bacteriano , Escherichia coli/genética , Datos de Secuencia Molecular , Mutagénesis , Procesamiento Proteico-Postraduccional , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
During persistence of nonencapsulated Haemophilus influenzae in the respiratory tracts of patients with chronic bronchitis, the major outer membrane proteins (MOMPs) P2 and P5 show antigenic drift. The hypothesis that appearance of antigenic variants is the consequence of antibody-dependent selection was tested in a rabbit model. Persistence of H. influenzae d1 was achieved in subcutaneous tissue cages for up to 948 days. During persistence in the rabbits, similar changes in MOMP P2 of H. influenzae occurred, as observed in isolates from chronic bronchitis patients. In rabbits vaccinated with strain d3 and in nonvaccinated rabbits, antigenic drift occurred later than in rabbits vaccinated with strain d1. High titers of antibodies against H. influenzae were measured in tissue cage fluid and serum. Vaccination of the rabbits with H. influenzae d1 or d3, an antigenic variant of strain d1, resulted neither in eradication of H. influenzae d1 nor in increased antibody titers in serum and tissue cage fluid. The sera of nonvaccinated rabbits during persistence had no strain d1-specific bactericidal activity in the presence of complement. Vaccination with H. influenzae d1 induced serum bactericidal activity against strain d1 in the presence of complement. However, a variant of strain d1 appearing in the tissue cages was not killed by this serum bactericidal activity. We conclude that immunological pressure leads to the selection of MOMP variants of H. influenzae and that these variants escape the antibody-mediated strain-specific bactericidal activity against H. influenzae.