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BACKGROUND: Despite of medical advances, the number of patients suffering on non-healing chronic wounds is still increasing. This fact is attended by physical and emotional distress and an economic load. The majority of chronic wounds are infected of harmful microbials in a protecting extracellular matrix. These biofilms inhibit wound healing. Biofilm-growing bacteria developed unique survival properties, which still challenge the appropriate wound therapy. The present in-vitro biofilm models are not suitable for translational research. By means of a novel in-vivo like human plasma biofilm model (hpBIOM), this study systematically analysed the influence of 3 probiotics on the survival of five clinically relevant pathogenic microorganisms. METHODS: Human plasma was used to produce the innovate biofilm. Pathogenic microorganisms were administered to the plasma. By stimulating the production of a fibrin scaffold, stable coagula-like discs with integrated pathogens were produced. The five clinically relevant pathogens P. aeruginosa, S. aureus, S. epidermidis, E. faecium and C. albicans were challenged to the probiotics L. plantarum, B. lactis and S. cerevisiae. The probiotics were administered on top of the biofilm and the survival was quantified after 4 h and 24 h of incubation. For statistics, two-way ANOVA with post-hoc Tukey's HSD test was applied. P-value > 0.05 was considered to be significant. RESULTS: SEM micrographs depicted the pathogens on the surface of the fibrin scaffold, arranged in close proximity and produced the glycocalyx. The application of probiotics induced different growth-reducing capacities towards the pathogens. B. lactis and S. cerevisiae showed slight bacteria-reducing properties. The survival of C. albicans was not affected at all. The most antimicrobial activity was detected after the treatment with L. plantarum. CONCLUSIONS: This study successfully reproduced a novel human biofilm model, which provides a human wound milieu and individual immune competence. The success of bacteriotherapy is dependent on the strain combination, the number of probiotics and the activity of the immune cells. The eradicating effect of L. plantarum on P. aeruginosa should be emphasized.
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Biopelículas , Plasma/microbiología , Probióticos/uso terapéutico , Candida albicans , Enterococcus faecium , Humanos , Pseudomonas aeruginosa , Saccharomyces cerevisiae , Staphylococcus aureus , Investigación Biomédica Traslacional , Cicatrización de HeridasRESUMEN
BACKGROUND: Carbapenemase-producing gram-negative bacteria are increasing globally and have been associated with outbreaks in hospital settings. Thus, the accurate detection of these bacteria in infections is mandatory for administering the adequate therapy and infection control measures. This study aimed to establish and evaluate a multiplex real-time PCR assay for the simultaneous detection of carbapenemase gene variants in gram-negative rods and to compare the performance with a commercial RT-PCR assay (Check-Direct CPE). METHODS: 116 carbapenem-resistant Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii isolates were genotyped for carbapenemase genes by PCR and sequencing. The defined isolates were used for the validation of the in-house RT-PCR by use of designed primer pairs and probes. RESULTS: Among the carbapenem-resistant isolates the genes bla KPC, bla VIM, bla NDM or bla OXA were detected. Both RT-PCR assays detected all bla KPC, bla VIM and bla NDM in the isolates. The in-house RT-PCR detected 53 of 67 (79.0%) whereas the commercial assay detected only 29 (43.3%) of the OXA genes. The in-house sufficiently distinguished the most prevalent OXA types (23-like and 48-like) in the melting curve analysis and direct detection of the genes from positive blood culture vials. CONCLUSION: The Check-Direct CPE and the in-house RT-PCR assay detected the carbapenem resistance from solid culture isolates. Moreover, the in-house assay enabled the identification of carbapenemase genes directly from positive blood-culture vials. However, we observed insufficient detection of various OXA genes in both assays. Nevertheless, the in-house RT-PCR detected the majority of the OXA type genes in Enterobacteriaceae and A. baumannii.
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Proteínas Bacterianas/genética , Enterobacteriaceae/enzimología , Bacterias Gramnegativas/enzimología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , beta-Lactamasas/genética , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Enterobacteriaceae/efectos de los fármacos , Enterobacteriaceae/genética , Enterobacteriaceae/aislamiento & purificación , Infecciones por Enterobacteriaceae/microbiología , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/genética , Bacterias Gramnegativas/aislamiento & purificación , Infecciones por Bacterias Gramnegativas/microbiología , Humanos , Pruebas de Sensibilidad Microbiana , Reacción en Cadena en Tiempo Real de la Polimerasa/economía , beta-Lactamasas/metabolismoRESUMEN
BACKGROUND: Antibiotic resistance in bacteria leads to massive health problems. Incidence of carbapenem and multidrug resistance in Gram-negative bacteria are increasing globally and turn out to be a very urgent challenge in health care. Resistant bacteria play an important clinical role during hospital outbreaks as well as in sepsis. Rapid diagnostic tests are necessary to provide immediate information for antimicrobial treatment and infection control measures. METHODS: Our mass spectrometry-based assay was validated with 63 carbapenemase-producing Gram-negative bacterial isolates, and 35 carbapenem-resistant Gram-negative species with no carbapenemase production. These were analyzed from solid culture media and positive blood culture vials. After 4 h of incubation the carbapenemase products were analyzed with the MALDI-TOF MS. All the isolates were genotyped for carbapenemase genes by PCR and sequencing. RESULTS: For culture isolates the concordance of hydrolysis assay to genetic results was 98 % for OXA variants, KPC, VIM, IMP, GIM, and NDM. In contrast, only 14 of 29 Acinetobacter baumannii isolates carrying the OXA and NDM genes could be identified from blood culture. However, from blood culture vials our method allowed the detection of carbapenemases in 98 % of Pseudomonas and Enterobacteriaceae isolates harboring different genes. CONCLUSIONS: This MALDI-TOF MS-based assay permitted the detection of carbapenemases either from solid culture media (98-100 %) or blood culture vials (96 %) for all non-A. baumannii isolates within 4 h. In case of A. baumannii isolates the assay was highly sensitive for the detection of carbapenemases directly from solid culture media.
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Bacterias/enzimología , Bacterias/aislamiento & purificación , Infecciones Bacterianas/microbiología , Proteínas Bacterianas/análisis , Sangre/microbiología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , beta-Lactamasas/análisis , Bacterias/química , Bacterias/clasificación , Infecciones Bacterianas/sangre , Proteínas Bacterianas/metabolismo , Humanos , beta-Lactamasas/metabolismoRESUMEN
Antibiotic resistance is an unsolved healthcare problem with increasing impact on patient management in the last years. In particular, multidrug resistance among Gram-negative bacterial strains has become the most pressing challenge. In order to deliver the most efficacious antimicrobial therapy with minimum delay, rapid diagnostic tests are required in order to detect multidrug resistant pathogens early during infection. In line with these efforts, we have developed a mass spectrometry-based assay for the rapid determination of ampicillin and cefotaxime resistance. The assay quantifies beta-lactamase activities towards ampicillin and cefotaxime within a turnaround time of 150 min, which is substantially faster than classical susceptibility testing.
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Antibacterianos/metabolismo , Cefotaxima/metabolismo , Cromatografía Liquida/métodos , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Espectrometría de Masas/métodos , beta-Lactamasas/análisis , Ampicilina/metabolismo , Técnicas Bacteriológicas/métodos , Humanos , Factores de Tiempo , Resistencia betalactámicaRESUMEN
The aim of this review is to describe the biology of human adenovirus (HAdV), the clinical and epidemiological characteristics of adenoviral epidemic keratoconjunctivitis and to present a practical update on its diagnosis, treatment, and prophylaxis. There are two well-defined adenoviral keratoconjunctivitis clinical syndromes: epidemic keratoconjunctivitis (EKC) and pharyngoconjunctival fever (PCF), which are caused by different HAdV serotypes. The exact incidence of adenoviral conjunctivitis is still poorly known. However, cases are more frequent during warmer months. The virus is endemic in the general population, and frequently causes severe disease in immunocompromised patients, especially the pediatric patients. Contagion is possible through direct contact or fomites, and the virus is extremely resistant to different physical and chemical agents. The clinical signs or symptoms of conjunctival infection are similar to any other conjunctivitis, with a higher incidence of pseudomembranes. In the cornea, adenoviral infection may lead to keratitis nummularis. Diagnosis is mainly clinical, but its etiology can be confirmed using cell cultures, antigen detection, polymerase chain reaction or immunochromatography. Multiple treatments have been tried for this disease, but none of them seem to be completely effective. Prevention is the most reliable and recommended strategy to control this contagious infection.
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For a long time, Staphylococcus epidermidis, as a member of the coagulase-negative staphylococci, was considered as part of the physiological skin flora of the human being with no pathogenic significance. Today, we know that S. epidermidis is one of the most prevalent causes for implant-associated and nosocomial infections. We performed pheno- and genotypic analysis (ica, IS256, SCCmec types, agr groups) of biofilm formation in 200 isolates. Fifty percent were genetically ica-positive and produced biofilm. Among all studied isolates, agr II and III and SCCmec type I were the most prevalent, whereas within the selected multi-resistant isolates (29%), agr I and III and SCCmec type II dominated. SCCmec type I and mecA-negative S. epidermidis isolates were associated with agr II. The majority of the blood culture and biopsy isolates were assigned to agr III and SCCmec type I, whereas agr II was predominantly detected in mecA-negative S. epidermidis isolated from catheter and implant materials. MLST analysis revealed the major clonal lineages of ST2, ST5, ST10, and ST242 (total 13 STs). ST2 isolates from blood cultures were icaA/D-positive and harbored SCCmec types II and III and IS256, whereas the icaA/D- and IS256-positive ST23 isolates were assigned to SCCmec types I and IV.
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Extended-spectrum beta-lactamases (ESBLs), which confer resistance to third generation cephalosporins, demonstrate how rapidly resistance mechanisms can evolve. These enzymes were discovered in few isolates in the late 1980s. They have now reached significant levels in hospitals and the community worldwide. Different ESBL enzyme variants (TEM, SHV, CTX-M) have been identified. They now substantially impair the utility of the later generation of cephalosporins and narrow the therapy options for patients with Gram-negative infections. Recently, reports on the wide dissemination of ESBL-producing bacteria in newborns, livestock, and in food accumulated. Meanwhile the worldwide dissemination of particular ESBL variants in epidemiologically successful Enterobacteriaceae strains has occurred.
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Antibacterianos/uso terapéutico , Infecciones Bacterianas/tratamiento farmacológico , Proteínas Bacterianas/metabolismo , Cefalosporinas/uso terapéutico , Farmacorresistencia Bacteriana Múltiple , beta-Lactamasas/metabolismo , Animales , Antibacterianos/efectos adversos , Infecciones Bacterianas/microbiología , Infecciones Bacterianas/prevención & control , Cefalosporinas/efectos adversos , Infección Hospitalaria/tratamiento farmacológico , Infección Hospitalaria/microbiología , Desinfección , Enterobacteriaceae/efectos de los fármacos , Infecciones por Enterobacteriaceae/tratamiento farmacológico , Infecciones por Enterobacteriaceae/microbiología , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Infecciones por Bacterias Gramnegativas/microbiología , Humanos , Recién NacidoRESUMEN
The aim of this study was to determine the prevalence of bacterial carriage in the anterior nares of two different patient cohorts, aged 5-15 years. By use of a sensitive enrichment broth, Gram-positive and -negative bacteria were cultured from the two cohorts of each 100 patients at the Referral Clinic in Eritrea and at a German University Hospital. In the German cohort, 27% of the patients were positive either for Gram-negative (n=5) or -positive bacteria, including Staphylococcus aureus (n=8; MRSA (n=2)), Staphylococcus epidermidis (n=12), Corynebacterium spp. (n=4), and Streptococcus pyogenes (n=1). In comparison, the Eritrean cohort revealed 33% bacterial carriers in the anterior nares. Among the identified species were S. aureus (n=2), S. epidermidis (n=13), Streptococcus haemolyticus (n=9), and Gram-negative rods including Klebsiella pneumoniae/oxytoca (n=5), Enterobacter agglomerans (n=4), Escherichia coli (n=2), and Pseudomonas aeruginosa (n=1). Noteworthy, none of the Eritrean patients were positive for MRSA. In both cohorts there was no co-occurrence of Gram-positive and -negative bacteria in the anterior nares. However, we observed in two subjects of the Eritrean cohort co-colonization with S. epidermidis and S. haemolyticus. The occurrence of Gram-negative bacteria was less significant by age in the German cohort, whereas in the Eritrean cohort Gram-negative bacteria were more frequently detected in carriers aged 5-9 years. Continued surveillance of S. aureus and Gram-negative bacteria carriage deserves further attention and might help to determine future trends in the characteristics of nasal carriage, subsequent incidence of infections, and the potential effectiveness of targeted population based intervention.
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Of 109 clinical Escherichia coli isolates from two major tertiary hospitals in Lagos (University Teaching Hospital and the National Orthopaedic Hospital Igbobi), 14 (12.8%) extended-spectrum beta-lactamse (ESBL) producers were characterized using PCR and sequencing, ERIC-PCR and multilocus sequence typing. All ESBL-producing isolates encoded only the CTX-M-15 gene. Clonal group ST131 (35.7%) was the predominant ST, followed by ST617 (28.6%). Isolated cases of other sequence types were also observed. Plasmid-mediated quinolone resistance genes qnrA, qnrB1 and aac-(6')-lb-cr were detected among these ESBL isolates of different clonal groups. This is the first description of the clonality of CTX-M-15-producing E. coli from Nigeria. The presence of diverse clonal lineages shows the continuing potential for genetic diversification and emergence of new epidemic strains.
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Infecciones por Escherichia coli/epidemiología , Infecciones por Escherichia coli/microbiología , Escherichia coli/clasificación , Escherichia coli/aislamiento & purificación , Tipificación de Secuencias Multilocus , Reacción en Cadena de la Polimerasa , beta-Lactamasas/metabolismo , Antibacterianos/farmacología , Análisis por Conglomerados , ADN Bacteriano/química , ADN Bacteriano/genética , Farmacorresistencia Bacteriana , Escherichia coli/enzimología , Genotipo , Hospitales , Humanos , Epidemiología Molecular , Nigeria/epidemiología , Plásmidos , Quinolonas/farmacología , beta-Lactamas/farmacologíaRESUMEN
Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are responsible for rising health care costs and have a high attribution to mortality. Reliable and rapid detection of MRSA carriage is essential. Real-time PCR allows an early detection of MRSA colonization within 2 h. By using the BD GeneOhm-MRSA assay we analysed directly swabs of different sampling sites and compared the assay with culture method. One thousand one hundred and sixty samples from 129 patients in Magdeburg were examined. Of the samples, 8 (0.69%) or 1117 (96.3%) were tested equally positive or negative by both methods whereas 16 (1.38%) specimens were MRSA positive only by the GeneOhm-MRSA assay and 6 (0.52%) were MRSA positive only by culture method. Thirteen samples (1.12%), which are culture negative, were unresolved by the GeneOhm-MRSA. With regard to the patients, seven were detected as MRSA carriers only by the GeneOhm-MRSA while one patient was tested positive for MRSA only by culture. Assuming 100% correct results by the culture method, sensitivity and specificity of GeneOhm-MRSA assay could be calculated as 84.4% and 96.1% for nasal swabs, 78.7% and 96.9% for all swabs under study, and 94.8% and 99.5% when focussed on patients. PPV and NPV were 70.3% and 98% for all specimens together, respectively. BD GeneOhm-MRSA assay is a sensitive test for the detection of MRSA colonization from swab specimens without the need for an initial culture, but should always be performed in parallel to the culture method for comparison reasons. Furthermore, our results indicate that in addition swabs taken from different body sites were successfully analysed by the BD GeneOhm-MRSA assay. However, we conclude that the PCR assay might not be a preferred tool for screening in haematologic patients with low MRSA rate; for screening haematologic patients, the culture method is sufficient enough.
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Phenotypic, genotypic, and toxin gene analyses have not yet been done all in one for the Nigerian Staphylococcus aureus population. This study provides a comprehensive overview of the molecular epidemiology and genetic diversity of S. aureus strains at the largest university clinic in Ibadan, Nigeria. From 1,300 patients' clinical samples collected at the University Teaching Hospital in Ibadan, Nigeria, during a 1-year-surveillance in 2007, 346 nonduplicate S. aureus isolates were obtained. All isolates underwent antibiotic susceptibility testing, toxin gene analysis, multilocus sequence typing, agr group typing, and spa typing. For methicillin (meticillin)-resistant S. aureus (MRSA), staphylococcal cassette chromosome mec (SCCmec) typing was also performed. Of the 346 isolates, 20.23% were methicillin resistant. Thirty-three patients' isolates (47.15%) fulfilled the definition criteria for community-associated MRSA (CA-MRSA) according to a review of the medical charts. The majority of MRSA strains analyzed were isolated from surgical or pediatric patients. The commonest types of MRSA infection identified were surgical-site infections (>70%), whereas those for CA-MRSA were conjunctivitis and otitis (19 patients [57.6%]) and accidental skin and subcutaneous tissue infections (14 patients [42.4%]). The methicillin-susceptible S. aureus strains (ST1, ST5, ST15, ST7, ST8, ST25, ST30, ST72, ST80, ST121, and ST508) were heterogeneous by phenotypic and genotypic analyses. The first report of a Panton-Valentine leukocidin-positive ST88 strain (agr III, SCCmec IV) in Nigeria, as well as genetic analyses of this strain, is presented in this study. The ST88 strain was resistant to trimethoprim-sulfamethoxazole as well as to penicillin and oxacillin. CA-MRSA infections are increasing rapidly among young patients with ophthalmologic and auricular infections. Urban regions with populations of lower socioeconomic status and evidence of overcrowding appear to be at high risk for the emergence of this clone.
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Antibacterianos/farmacología , Infecciones Comunitarias Adquiridas/epidemiología , Infecciones Comunitarias Adquiridas/microbiología , Staphylococcus aureus Resistente a Meticilina/clasificación , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Infecciones Estafilocócicas/epidemiología , Infecciones Estafilocócicas/microbiología , Adolescente , Adulto , Toxinas Bacterianas/genética , Técnicas de Tipificación Bacteriana , Niño , Preescolar , Análisis por Conglomerados , Conjuntivitis/microbiología , Dermatoglifia del ADN , Variación Genética , Genotipo , Hospitales , Humanos , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Epidemiología Molecular , Nigeria/epidemiología , Otitis/microbiología , Análisis de Secuencia de ADN , Infecciones de los Tejidos Blandos/microbiología , Infecciones Cutáneas Estafilocócicas/microbiología , Infección de la Herida Quirúrgica/microbiología , Población Urbana , Adulto JovenRESUMEN
Aspergillus peritonitis is a rare life-threatening complication of peritoneal dialysis (PD). We report a case of symptomatic Neosartorya pseudofischeri peritonitis in a 60-year-old woman treated by continuous ambulatory peritoneal dialysis (CAPD) for 13 months, who performed peritoneal exchanges independently. This is believed to be the first published case of N. pseudofischeri in an elderly patient. Comprehensive treatment included early removal of the PD catheter and the use of voriconazole (200 mg Vfend twice daily) for a period of 5 weeks. This case supports the need for more effective prophylaxis and treatment of non-Candida fungal infections in CAPD patients. Our conclusions from this case and a review of the literature are that infection with this fungus can cause substantial morbidity and is best treated with prompt catheter removal, aggressive antifungal therapy with voriconazole or amphotericin B, and vigilant observation for complications. Our report describes for what is believed to be the first time the administration of voriconazole to treat a Neosartorya peritonitis case.
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Antifúngicos/uso terapéutico , Fallo Renal Crónico/terapia , Micosis/tratamiento farmacológico , Neosartorya , Diálisis Peritoneal/efectos adversos , Peritonitis/microbiología , Pirimidinas/uso terapéutico , Triazoles/uso terapéutico , Femenino , Humanos , Fallo Renal Crónico/complicaciones , Persona de Mediana Edad , Micosis/diagnóstico , Peritonitis/tratamiento farmacológico , Resultado del Tratamiento , VoriconazolRESUMEN
Nowadays, influenza antigen detection test kits are used most frequently to detect influenza A or B virus to establish the diagnosis of influenza rapidly and initiate appropriate therapy. This study was conducted to evaluate the performance of the actim Influenza A&B test (Medix Biochemica). Overall, 473 respiratory specimens were analysed in the actim Influenza A&B test and the results were compared with those from an RT-PCR assay; 461 of these samples originated from paediatric patients aged 7 weeks to 6.5 years either with influenza-related symptoms or from the intensive care unit, and 12 samples originated from adults with underlying lung or haematological diseases. Diagnosis of influenza A or B virus could be established using the actim Influenza A&B test (9/473 samples for influenza A virus and 6/473 for influenza B virus). RT-PCR revealed 23 patients with influenza virus (13/473 for influenza A virus and 10/473 for influenza B virus). The sensitivity and specificity of the actim Influenza A&B test were 65 and 100 % compared with the RT-PCR assay. However, 32 external quality assessment samples containing seven different strains of influenza A subtypes H1N1 and H3N2 and the avian H5N1 were detected correctly by the actim Influenza A&B test. No cross-reactivity to a range of bacterial, fungal and other viral pathogens was observed. In conclusion, the actim Influenza A&B test is reliable for positive results due to its high specificity. Nevertheless, negative results from this test need to be confirmed by a more sensitive assay because of the low sensitivity observed with diagnostic samples.
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Antígenos Virales/aislamiento & purificación , Virus de la Influenza A/aislamiento & purificación , Virus de la Influenza B/aislamiento & purificación , Gripe Humana/diagnóstico , Adolescente , Adulto , Anticuerpos Monoclonales , Niño , Preescolar , Reacciones Cruzadas , ADN Complementario/aislamiento & purificación , ADN Viral/aislamiento & purificación , Humanos , Lactante , Virus de la Influenza A/genética , Virus de la Influenza A/inmunología , Virus de la Influenza B/genética , Virus de la Influenza B/inmunología , Gripe Humana/virología , Persona de Mediana Edad , Estudios Prospectivos , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Adulto JovenRESUMEN
The analysis of 16S rRNA gene sequences has been the technique generally used to study the evolution and taxonomy of staphylococci. However, the results of this method do not correspond to the results of polyphasic taxonomy, and the related species cannot always be distinguished from each other. Thus, new phylogenetic markers for Staphylococcus spp. are needed. We partially sequenced the gap gene (approximately 931 bp), which encodes the glyceraldehyde-3-phosphate dehydrogenase, for 27 Staphylococcus species. The partial sequences had 24.3 to 96% interspecies homology and were useful in the identification of staphylococcal species (F. Layer, B. Ghebremedhin, W. König, and B. König, J. Microbiol. Methods 70:542-549, 2007). The DNA sequence similarities of the partial staphylococcal gap sequences were found to be lower than those of 16S rRNA (approximately 97%), rpoB (approximately 86%), hsp60 (approximately 82%), and sodA (approximately 78%). Phylogenetically derived trees revealed four statistically supported groups: S. hyicus/S. intermedius, S. sciuri, S. haemolyticus/S. simulans, and S. aureus/epidermidis. The branching of S. auricularis, S. cohnii subsp. cohnii, and the heterogeneous S. saprophyticus group, comprising S. saprophyticus subsp. saprophyticus and S. equorum subsp. equorum, was not reliable. Thus, the phylogenetic analysis based on the gap gene sequences revealed similarities between the dendrograms based on other gene sequences (e.g., the S. hyicus/S. intermedius and S. sciuri groups) as well as differences, e.g., the grouping of S. arlettae and S. kloosii in the gap-based tree. From our results, we propose the partial sequencing of the gap gene as an alternative molecular tool for the taxonomical analysis of Staphylococcus species and for decreasing the possibility of misidentification.
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Proteínas Bacterianas/genética , Técnicas de Tipificación Bacteriana , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Staphylococcus/clasificación , Staphylococcus/genética , Chaperonina 60/genética , ADN Bacteriano/análisis , ARN Polimerasas Dirigidas por ADN/genética , Genes de ARNr , Humanos , Datos de Secuencia Molecular , Filogenia , Especificidad de la Especie , Superóxido Dismutasa/genéticaRESUMEN
Classical phenotypic and biochemical testing do not lead to correct identification of the distinct Staphylococcus species. Therefore, the aim of our study was to develop a method for the reliable and accurate determination of distinct Staphylococcus species. In the present study, the 931-934-bp partial sequences of the glyceraldehyde-3-phosphate dehydrogenase-encoding (gap) gene of 28 validly described Staphylococcus species were amplified and sequenced. By using the respective sequence information we performed a terminal-restriction fragment length polymorphism (T-RFLP) analysis. For T-RFLP the partial gap gene was amplified with double-fluorescently labelled primers and digested with the restriction enzymes DdeI, BspHI and TaqI. Distinctive T-RFLP patterns were rendered by the use of capillary electrophoresis with laser-induced fluorescence detection. This molecular method allowed us to identify all 28 Staphylococcus species with high specificity. This was validated by analysis of 34 Staphylococcus epidermidis and 28 Staphylococcus haemolyticus isolates. These results demonstrate the feasibility and applicability of the T-RFLP method based on the partial gap gene sequences for rapid and accurate species identification.
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Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Staphylococcus/clasificación , Secuencia de Bases , Cartilla de ADN , Genes Bacterianos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Longitud del Fragmento de Restricción , Mapeo Restrictivo/métodos , Staphylococcus/enzimología , Staphylococcus/genéticaRESUMEN
Emergence of the meticillin-resistant Staphylococcus aureus (MRSA) Barnim epidemic strain (ST22-MRSA-IV) was demonstrated recently at University Hospital in Magdeburg, Germany. To aid the study of transmission events, it is important to have an epidemiological typing method with the ability to distinguish among MRSA isolates. The aim of this study was to determine the ability of phenotypic and genotypic methods to type ST22-MRSA-IV strains within a hospital for microevolution events. Forty-two ST22-MRSA-IV strains collected from 2002 to 2005 were analysed using antimicrobial testing, toxin gene analysis, PFGE, spa typing, fluorescent amplified fragment length polymorphism (fAFLP) and determination of staphylococcal interspersed repeat units (SIRUs). Four different antimicrobial patterns were observed. The majority of the isolates (n=31) were resistant towards erythromycin, ciprofloxacin and clindamycin, in addition to penicillin and oxacillin. All strains harboured the sec gene and showed a homogeneous profile of toxin genes. One isolate was typed as spa t022, two as spa t474 and the remainder belonged to spa type t032. PFGE yielded eight profiles and SIRU typing resulted in six different patterns. The fAFLP technique subdivided the individual PFGE profiles, but the grouping of isolates differed from that obtained by PFGE or SIRU typing. These results showed a diversity of ST22-MRSA-IV strains within a narrow clinical setting, indicating microevolution of the Barnim MRSA clone. The ability to distinguish among MRSA strains within an endemic setting will lead to a greater understanding of the transmission of MRSA and is necessary to be able to control the spread of various clones.
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Técnicas de Tipificación Bacteriana/métodos , Infección Hospitalaria/microbiología , Resistencia a la Meticilina , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/clasificación , Staphylococcus aureus/efectos de los fármacos , Adenosina Trifosfatasas/genética , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Toxinas Bacterianas/análisis , Análisis por Conglomerados , Infección Hospitalaria/epidemiología , Dermatoglifia del ADN , ADN Bacteriano/genética , Electroforesis en Gel de Campo Pulsado , Enfermedades Endémicas , Genotipo , Alemania/epidemiología , Hospitales Universitarios , Humanos , Secuencias Repetitivas Esparcidas/genética , Proteínas de Transporte de Membrana/genética , Pruebas de Sensibilidad Microbiana , Fenotipo , Canales de Translocación SEC , Proteína SecA , Infecciones Estafilocócicas/epidemiología , Staphylococcus aureus/genética , Staphylococcus aureus/aislamiento & purificaciónRESUMEN
Coagulase-negative staphylococci (CNS) play a predominant role in nosocomial infections. Rapid, reliable identification of these organisms is essential for accurate diagnosis and prompt effective treatment of these infections. Quite recently, the VITEK 2 g-positive (gram-positive [GP]) identification card (bioMérieux) has been redesigned for greater accuracy in the identification of gram-positive cocci. We compared the BD Phoenix (Becton Dickinson) and VITEK 2 (bioMérieux) automated microbiology systems, using their respective update version cards, and the API ID32 STAPH test. The glyceraldehyde-3-phosphate dehydrogenase (gap) gene-based T-RFLP (terminal restriction fragment length polymorphism) method was used for verifying the results. In total, 86 clinical isolates of CNS and 27 reference strains were analyzed. The results show that for identification of CNS, the automated identification methods using the newest VITEK 2 and BD Phoenix identification cards are comparable. However, API ID32 STAPH revealed more correct results compared to both automated microbiology systems. Despite the increased performance of the phenotypic automated identification systems compared to the former versions, molecular methods, e.g., the gap-based T-RFLP method, still show superior accuracy in identifying Staphylococcus species other than Staphylococcus aureus.
Asunto(s)
Técnicas de Tipificación Bacteriana/métodos , Polimorfismo de Longitud del Fragmento de Restricción , Infecciones Estafilocócicas/diagnóstico , Infecciones Estafilocócicas/microbiología , Staphylococcus/clasificación , Staphylococcus/aislamiento & purificación , ADN Bacteriano/genética , Gliceraldehído 3-Fosfato Deshidrogenasa (NADP+)/genética , Humanos , Sensibilidad y Especificidad , Staphylococcus/genética , Staphylococcus/metabolismoRESUMEN
Recently, we demonstrated rapid dissemination of different methicillin-resistant Staphylococcus aureus (MRSA) clones at the Institute for Microbiology at the University of Magdeburg (B. Ghebremedhin, W. König, and B. König, Eur. J. Clin. Microbiol. Infect. Dis. 24:388-398, 2005). The majority of them harbored the readily transmissible mec cassette type IV. Thus, theoretically, methicillin-susceptible Staphylococcus aureus (MSSA) might capture the mecA gene from circulating MRSA, or MRSA strains might catch mobile toxin genes from MSSA. Therefore, we characterized MSSA strains circulating at the University Hospital in Magdeburg. Among a total of 84 MSSA strains under study, about 40% possessed the tst (toxic shock syndrome toxin) gene and up to four additional enterotoxin genes. tst-positive MSSA strains belonged to all known agr groups (I to IV) and to 14 different spa types (t008, t012, t015, t019, t024, t056, t065, t127, t133, t162, t271, t287, t399, and t400), and they were classified by multilocus sequence typing (MLST) as ST1, ST8, ST30, ST39, ST45, ST101, ST121, ST395, and ST426. In contrast, simultaneously circulating MRSA strains (n = 24) harbored in general two or three genes of the enterotoxin gene cluster, and the tst-positive MRSA isolates belonged to the well-known epidemic types ST22, ST45, and ST228 and were classified as spa types t001, t028, and t032. From our results, one may conclude that the pool of circulating MSSA strains is an important parameter with regard to the epidemiology of hospital- and community-acquired MRSA clones and their potential virulence.
Asunto(s)
Antibacterianos/farmacología , Hospitales Universitarios , Resistencia a la Meticilina/genética , Meticilina/farmacología , Staphylococcus aureus/efectos de los fármacos , Toxinas Bacterianas/genética , Enfermedades Transmisibles Emergentes/microbiología , Enterotoxinas/genética , Transferencia de Gen Horizontal , Alemania , Humanos , Pruebas de Sensibilidad Microbiana , Análisis de Secuencia de ADN , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/clasificación , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidad , Superantígenos/genéticaRESUMEN
Heterogeneous methicillin-resistant Staphylococcus aureus (MRSA) strains, including community-acquired MRSA strains, have been observed in Central Europe. The purpose of this study was to characterize by molecular methods MRSA isolated during the period 2002-2003 at the Otto-von-Guericke University Hospital in Magdeburg, Germany, and at a nearby chronic care facility. Strains were analyzed for their resistance phenotype. Selected isolates were typed by multilocus sequence typing (MLST), by a multiplex polymerase chain reaction (PCR) for the staphylococcal cassette chromosome mec (SCCmec), by an allele-specific PCR for the staphylococcal accessory gene regulator (agr), and by PCR for the presence of toxin genes (sea-sej, tsst-1, hlgA, C, and B, lukE/D, and luk-pvl). Of the 2,731 S. aureus isolates studied, 199 (7.3%) were MRSA, with a prevalence of 21.6%, 19.6%, and 12% in the department of dermatology, the chronic care facility, and the intensive care units. Six different sequence types (ST247, ST228, ST22, ST22a, ST225, and ST45) were observed. Of these, ST22, ST22a, and ST45 dominated (>50%) in the department of dermatology and the chronic care facility. Strains with these sequence types were usually not resistant to gentamicin and were associated with agr group I, the SCCmec type IV element, and the presence of the sec and sed toxin genes. ST228 strains were found mainly in the intensive care units and had a broader resistance phenotype and were associated with agr group II and the SCCmec type I element. All luk-pvl-positive MRSA isolates (n=8) belonged to agr group I and were typed as ST22 or ST45 and contained the SCCmec type I (n=1), type III (n=1), or type IV (n=6) element. The main observations of this study are in concordance with previously reported findings showing dissemination of MRSA in Central Europe. Through the multitude of applied methods, the data from this study contribute to a more precise knowledge about the heterogeneity of MRSA in a clinical setting. Rapid dissemination of MRSA clones at a university hospital was demonstrated, indicating that dissemination may depend on the environmental conditions within the individual departments.