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1.
Euro Surveill ; 25(2)2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31964459

RESUMEN

BackgroundMycoplasma pneumoniae is a leading cause of community-acquired pneumonia, with large epidemics previously described to occur every 4 to 7 years.AimTo better understand the diagnostic methods used to detect M. pneumoniae; to better understand M. pneumoniae testing and surveillance in use; to identify epidemics; to determine detection number per age group, age demographics for positive detections, concurrence of epidemics and annual peaks across geographical areas; and to determine the effect of geographical location on the timing of epidemics.MethodsA questionnaire was sent in May 2016 to Mycoplasma experts with national or regional responsibility within the ESCMID Study Group for Mycoplasma and Chlamydia Infections in 17 countries across Europe and Israel, retrospectively requesting details on M. pneumoniae-positive samples from January 2011 to April 2016. The Moving Epidemic Method was used to determine epidemic periods and effect of country latitude across the countries for the five periods under investigation.ResultsRepresentatives from 12 countries provided data on M. pneumoniae infections, accounting for 95,666 positive samples. Two laboratories initiated routine macrolide resistance testing since 2013. Between 2011 and 2016, three epidemics were identified: 2011/12, 2014/15 and 2015/16. The distribution of patient ages for M. pneumoniae-positive samples showed three patterns. During epidemic years, an association between country latitude and calendar week when epidemic periods began was noted.ConclusionsAn association between epidemics and latitude was observed. Differences were noted in the age distribution of positive cases and detection methods used and practice. A lack of macrolide resistance monitoring was noted.


Asunto(s)
Infecciones Comunitarias Adquiridas/epidemiología , Epidemias , Mycoplasma pneumoniae/aislamiento & purificación , Neumonía por Mycoplasma/epidemiología , Distribución por Edad , Antibacterianos/uso terapéutico , Infecciones Comunitarias Adquiridas/diagnóstico , Infecciones Comunitarias Adquiridas/microbiología , Farmacorresistencia Bacteriana/efectos de los fármacos , Correo Electrónico , Europa (Continente)/epidemiología , Femenino , Humanos , Israel/epidemiología , Macrólidos/farmacología , Mycoplasma pneumoniae/efectos de los fármacos , Técnicas de Amplificación de Ácido Nucleico , Neumonía por Mycoplasma/diagnóstico , Neumonía por Mycoplasma/tratamiento farmacológico , Estudios Retrospectivos , Encuestas y Cuestionarios
2.
Antimicrob Agents Chemother ; 60(1): 52-6, 2016 01.
Artículo en Inglés | MEDLINE | ID: mdl-26459899

RESUMEN

Ureaplasma spp. are associated with numerous clinical sequelae with treatment options being limited due to patient and pathogen factors. This report examines the prevalence and mechanisms of antibiotic resistance among clinical strains isolated from 95 neonates, 32 women attending a sexual health clinic, and 3 patients under investigation for immunological disorders, between 2007 and 2013 in England and Wales. MICs were determined by using broth microdilution assays, and a subset of isolates were compared using the broth microdilution method and the Mycoplasma IST2 assay. The underlying molecular mechanisms for resistance were determined for all resistant isolates. Three isolates carried the tet(M) tetracycline resistance gene (2.3%; confidence interval [CI], 0.49 to 6.86%); two isolates were ciprofloxacin resistant (1.5%; CI, 0.07 to 5.79%) but sensitive to levofloxacin and moxifloxacin, while no resistance was seen to any macrolides tested. The MIC values for chloramphenicol were universally low (2 µg/ml), while inherently high-level MIC values for gentamicin were seen (44 to 66 µg/ml). The Mycoplasma IST2 assay identified a number of false positives for ciprofloxacin resistance, as the method does not conform to international testing guidelines. While antibiotic resistance among Ureaplasma isolates remains low, continued surveillance is essential to monitor trends and threats from importation of resistant clones.


Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Ureaplasma/efectos de los fármacos , Cloranfenicol/farmacología , Ciprofloxacina/farmacología , Inglaterra/epidemiología , Monitoreo Epidemiológico , Fluoroquinolonas/farmacología , Gentamicinas/farmacología , Humanos , Lactante , Recién Nacido , Levofloxacino/farmacología , Macrólidos/farmacología , Pruebas de Sensibilidad Microbiana , Moxifloxacino , Estudios Retrospectivos , Tetraciclina/farmacología , Ureaplasma/genética , Ureaplasma/crecimiento & desarrollo , Ureaplasma/aislamiento & purificación , Infecciones por Ureaplasma/epidemiología , Infecciones por Ureaplasma/microbiología , Gales/epidemiología
3.
J Immunol ; 192(4): 1787-95, 2014 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-24446515

RESUMEN

Neutrophil dysfunction, resulting in inefficient bacterial clearance, is a feature of several serious medical conditions, including cystic fibrosis (CF) and sepsis. Poorly controlled neutrophil serine protease (NSP) activity and complement activation have been implicated in this phenomenon. The capacity for excess NSP secretion and complement activation to influence the expression and function of the important neutrophil-activating receptor C5aR was investigated. Purified NSPs cathepsin G (CG), neutrophil elastase (NE), and proteinase 3 cleaved C5aR to a 26- to 27-kDa membrane-bound fragment, thereby inactivating its C5a-induced signaling ability. In a supernatant transfer assay, NSPs released from neutrophils in response to C5a induced the cleavage of the C5aR on unstimulated cells. Stimulation of myeolomonocytic U937 cells and purified neutrophils with C5a resulted in downregulation of the C5aR on these cells, which, in the case of U937 cells, was largely caused by NSP-mediated cleavage of C5aR, but in the case of neutrophils, intracellular degradation was likely the main mediator in addition to a small role for NSPs. CG and NE in bronchoalveolar lavage fluid from CF patients both contributed to C5aR cleavage. We propose two converging models for C5a- and NSP-mediated neutrophil dysfunction whereby C5aR cleavage is induced by NSPs, secreted in response to: 1) excess C5a generation or other stimuli; or 2) necrosis. The consequent impairment of C5aR activity contributes to suboptimal local neutrophil priming and bacterial clearance. NSP inhibitors with specificity for both CG and NE may aid the treatment of pathologies associated with neutrophil dysfunction including sepsis and CF.


Asunto(s)
Activación de Complemento/inmunología , Neutrófilos/inmunología , Receptor de Anafilatoxina C5a/metabolismo , Serina Proteasas/metabolismo , Líquido del Lavado Bronquioalveolar/citología , Catepsina G/metabolismo , Línea Celular , Niño , Humanos , Elastasa de Leucocito/metabolismo , Mieloblastina/metabolismo , Activación Neutrófila/inmunología , Transducción de Señal/inmunología , Células U937
4.
J Clin Microbiol ; 53(10): 3195-203, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26202118

RESUMEN

Mycoplasma pneumoniae is a major human respiratory pathogen causing both upper and lower respiratory disease in humans of all ages, and it can also result in other serious extrapulmonary sequelae. A multilocus sequence typing (MLST) scheme for M. pneumoniae was developed based on the sequences of eight housekeeping genes (ppa, pgm, gyrB, gmk, glyA, atpA, arcC, and adk) and applied to 55 M. pneumoniae clinical isolates and the two type strains M129 and FH. A total of 12 sequence types (STs) resulted for 57 M. pneumoniae isolates tested, with a discriminatory index of 0.21 STs per isolate. The MLST loci used in this scheme were shown to be stable in 10 strains following 10 sequential subculture passages. Phylogenetic analysis of concatenated sequences of the eight loci indicated two distinct genetic clusters that were directly linked to multilocus variable-number tandem repeat analysis (MLVA) type. Genetic MLST clustering was confirmed by genomic sequence analysis, indicating that the MLST scheme developed in this study is representative of the genome. Furthermore, this MLST scheme was shown to be more discriminatory than both MLVA and P1 typing for the M. pneumoniae isolates examined, providing a method for further and more detailed analysis of observed epidemic peaks of M. pneumoniae infection. This scheme is supported by a public Web-based database (http://pubmlst.org/mpneumoniae).


Asunto(s)
Tipificación de Secuencias Multilocus/métodos , Mycoplasma pneumoniae/clasificación , Mycoplasma pneumoniae/genética , Análisis por Conglomerados , Genes Esenciales , Inestabilidad Genómica , Genotipo , Humanos , Infecciones por Mycoplasma/microbiología , Mycoplasma pneumoniae/aislamiento & purificación , Filogenia
5.
Antimicrob Agents Chemother ; 58(11): 6581-91, 2014 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-25155606

RESUMEN

Treatment of intrauterine infection is likely key to preventing a significant proportion of preterm deliveries before 32 weeks of gestation. Azithromycin (AZ) may be an effective antimicrobial in pregnancy; however, few gestation age-approriate data are available to inform the design of AZ-based treatment regimens in early pregnancy. We aimed to determine whether a single intra-amniotic AZ dose or repeated maternal intravenous (i.v.) AZ doses would safely yield therapeutic levels of AZ in an 80-day-gestation (term is 150 days) ovine fetus. Fifty sheep carrying single pregnancies at 80 days gestation were randomized to receive either: (i) a single intra-amniotic AZ administration or (ii) maternal intravenous AZ administration every 12 h. Amniotic fluid, maternal plasma, and fetal AZ concentrations were determined over a 5-day treatment regimen. Markers of liver injury and amniotic fluid inflammation were measured to assess fetal injury in response to drug exposure. A single intra-amniotic administration yielded significant AZ accumulation in the amniotic fluid and fetal lung. In contrast, repeated maternal intravenous administrations achieved high levels of AZ accumulation in the fetal lung and liver and a statistically significant increase in the fetal plasma drug concentration at 120 h. There was no evidence of fetal injury in response to drug exposure. These data suggest that (i) repeated maternal i.v. AZ dosing yields substantial fetal tissue uptake, although fetal plasma drug levels remain low; (ii) transfer of AZ from the amniotic fluid is less than transplacental transfer; and (iii) exposure to high concentrations of AZ did not elicit overt changes in fetal white blood cell counts, amniotic fluid monocyte chemoattractant protein 1 concentrations, or hepatotoxicity, all consistent with an absence of fetal injury.


Asunto(s)
Antibacterianos/farmacocinética , Azitromicina/farmacocinética , Intercambio Materno-Fetal , Complicaciones Infecciosas del Embarazo/tratamiento farmacológico , Líquido Amniótico/química , Animales , Antibacterianos/sangre , Antibacterianos/líquido cefalorraquídeo , Antibacterianos/uso terapéutico , Azitromicina/sangre , Azitromicina/líquido cefalorraquídeo , Azitromicina/uso terapéutico , Femenino , Circulación Placentaria , Embarazo , Complicaciones Infecciosas del Embarazo/microbiología , Segundo Trimestre del Embarazo , Nacimiento Prematuro/prevención & control , Distribución Aleatoria , Ovinos
6.
J Clin Microbiol ; 52(2): 599-606, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24478494

RESUMEN

Ureaplasma sp. infection in neonates and adults underlies a variety of disease pathologies. Of the two human Ureaplasma spp., Ureaplasma parvum is clinically the most common. We have developed a high-resolution melt (HRM) PCR assay for the differentiation of the four serovars of U. parvum in a single step. Currently U. parvum strains are separated into four serovars by sequencing the promoter and coding region of the multiple-banded antigen (MBA) gene. We designed primers to conserved sequences within this region for PCR amplification and HRM analysis to generate reproducible and distinct melt profiles that distinguish clonal representatives of serovars 1, 3, 6, and 14. Furthermore, our HRM PCR assay could classify DNA extracted from 74 known (MBA-sequenced) test strains with 100% accuracy. Importantly, HRM PCR was also able to identify U. parvum serovars directly from 16 clinical swabs. HRM PCR performed with DNA consisting of mixtures of combined known serovars yielded profiles that were easily distinguished from those for single-serovar controls. These profiles mirrored clinical samples that contained mixed serovars. Unfortunately, melt curve analysis software is not yet robust enough to identify the composition of mixed serovar samples, only that more than one serovar is present. HRM PCR provides a single-step, rapid, cost-effective means to differentiate the four serovars of U. parvum that did not amplify any of the known 10 serovars of Ureaplasma urealyticum tested in parallel. Choice of reaction reagents was found to be crucial to allow sufficient sensitivity to differentiate U. parvum serovars directly from clinical swabs rather than requiring cell enrichment using microbial culture techniques.


Asunto(s)
ADN Bacteriano/genética , Tipificación Molecular/métodos , Reacción en Cadena de la Polimerasa/métodos , Infecciones por Ureaplasma/diagnóstico , Infecciones por Ureaplasma/microbiología , Ureaplasma/clasificación , Ureaplasma/aislamiento & purificación , Adulto , Análisis Costo-Beneficio , Femenino , Genotipo , Humanos , Recién Nacido , Masculino , Tipificación Molecular/economía , Reacción en Cadena de la Polimerasa/economía , Embarazo , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Tiempo , Temperatura de Transición , Ureaplasma/genética
7.
Int J Med Microbiol ; 304(8): 1218-25, 2014 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-25444567

RESUMEN

While transposon mutagenesis has been successfully used for Mycoplasma spp. to disrupt and determine non-essential genes, previous attempts with Ureaplasma spp. have been unsuccessful. Using a polyethylene glycol-transformation enhancing protocol, we were able to transform three separate serovars of Ureaplasma parvum with a Tn4001-based mini-transposon plasmid containing a gentamicin resistance selection marker. Despite the large degree of homology between Ureaplasma parvum and Ureaplasma urealyticum, all attempts to transform the latter in parallel failed, with the exception of a single clinical U. urealyticum isolate. PCR probing and sequencing were used to confirm transposon insertion into the bacterial genome and identify disrupted genes. Transformation of prototype serovar 3 consistently resulted in transfer only of sequence between the mini-transposon inverted repeats, but some strains showed additional sequence transfer. Transposon insertion occurred randomly in the genome resulting in unique disruption of genes UU047, UU390, UU440, UU450, UU520, UU526, UU582 for single clones from a panel of screened clones. An intergenic insertion between genes UU187 and UU188 was also characterised. Two phenotypic alterations were observed in the mutated strains: Disruption of a DEAD-box RNA helicase (UU582) altered growth kinetics, while the U. urealyticum strain lost resistance to serum attack coincident with disruption of gene UUR10_137 and loss of expression of a 41 kDa protein. Transposon mutagenesis was used successfully to insert single copies of a mini-transposon into the genome and disrupt genes leading to phenotypic changes in Ureaplasma parvum strains. This method can now be used to deliver exogenous genes for expression and determine essential genes for Ureaplasma parvum replication in culture and experimental models.


Asunto(s)
Elementos Transponibles de ADN , Técnicas de Inactivación de Genes , Genética Microbiana/métodos , Mutagénesis Insercional/métodos , Ureaplasma/genética , Plásmidos , Transformación Bacteriana
8.
Am J Obstet Gynecol ; 211(2): 134.e1-9, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24589547

RESUMEN

OBJECTIVE: Ureaplasma spp are the most commonly isolated microorganisms in association with preterm birth. Maternal erythromycin administration is a standard treatment for preterm prelabor rupture of membranes. There is little evidence of its effectiveness in eradicating Ureaplasma spp from the intrauterine cavity and fetus. We used a sheep model of intrauterine Ureaplasma spp infection to investigate the efficacy of repeated maternal intramuscular and intraamniotic erythromycin treatment to eradicate such an infection. STUDY DESIGN: Thirty ewes with singleton pregnancies received an intraamniotic injection of 10(7) color change units of erythromycin-sensitive Ureaplasma parvum serovar 3 at 55 days' gestation. At 116 days' gestation, 28 ewes with viable fetuses were randomized to receive (1) intraamniotic and maternal intramuscular saline solution treatment (n = 8), (2) single intraamniotic and repeated maternal intramuscular erythromycin treatment (n = 10), or (3) single maternal intramuscular and repeated intraamniotic erythromycin treatment (n = 10). Fetuses were surgically delivered at 125 days' gestation. Treatment efficacy was assessed by culture, quantitative polymerase chain reaction, and histopathologic evaluation. RESULTS: Animals treated with intraamniotic erythromycin had significantly less viable U parvum serovar 3 in the amniotic fluid at delivery. However, neither combination of maternal intramuscular and intraamniotic erythromycin treatment successfully cleared U parvum serovar 3 from the amniotic fluid or fetal tissues. Three de novo erythromycin-resistant U parvum isolates were identified in erythromycin-treated animals. CONCLUSION: Erythromycin treatment, given both to the ewe and into the amniotic cavity, fails to eradicate intrauterine and fetal U parvum serovar 3 infection and may lead to development of erythromycin resistant U parvum.


Asunto(s)
Antibacterianos/administración & dosificación , Eritromicina/administración & dosificación , Complicaciones Infecciosas del Embarazo/tratamiento farmacológico , Infecciones por Ureaplasma/tratamiento farmacológico , Amnios , Líquido Amniótico/microbiología , Animales , Corion/metabolismo , Corion/microbiología , Corion/patología , ADN Bacteriano/sangre , Esquema de Medicación , Femenino , Inyecciones , Inyecciones Intramusculares , Interleucinas/metabolismo , Hígado/metabolismo , Pulmón/metabolismo , Modelos Animales , Embarazo , ARN Bacteriano/sangre , Distribución Aleatoria , Proteína Amiloide A Sérica/metabolismo , Ovinos , Piel/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Ureaplasma/genética , Ureaplasma/aislamiento & purificación
9.
J Virol ; 85(24): 13409-19, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21976640

RESUMEN

Coevolution of virus and host is a process that emerges in persistent virus infections. Here we studied the coevolutionary development of coxsackievirus B3 (CVB3) and cardiac myocytes representing the major target cells of CVB3 in the heart in a newly established persistently CVB3-infected murine cardiac myocyte cell line, HL-1(CVB3). CVB3 persistence in HL-1(CVB3) cells represented a typical carrier-state infection with high levels (10(6) to 10(8) PFU/ml) of infectious virus produced from only a small proportion (approximately 10%) of infected cells. CVB3 persistence was characterized by the evolution of a CVB3 variant (CVB3-HL1) that displayed strongly increased cytotoxicity in the naive HL-1 cell line and showed increased replication rates in cultured primary cardiac myocytes of mouse, rat, and naive HL-1 cells in vitro, whereas it was unable to establish murine cardiac infection in vivo. Resistance of HL-1(CVB3) cells to CVB3-HL1 was associated with reduction of coxsackievirus and adenovirus receptor (CAR) expression. Decreasing host cell CAR expression was partially overcome by the CVB3-HL1 variant through CAR-independent entry into resistant cells. Moreover, CVB3-HL1 conserved the ability to infect cells via CAR. The employment of a soluble CAR variant resulted in the complete cure of HL-1(CVB3) cells with respect to the adapted virus. In conclusion, this is the first report of a CVB3 carrier-state infection in a cardiomyocyte cell line, revealing natural coevolution of CAR downregulation with CAR-independent viral entry in resistant host cells as an important mechanism of induction of CVB3 persistence.


Asunto(s)
Evolución Biológica , Enterovirus Humano B/crecimiento & desarrollo , Enterovirus Humano B/genética , Miocitos Cardíacos/virología , Animales , Supervivencia Celular , Células Cultivadas , Infecciones por Coxsackievirus/patología , Infecciones por Coxsackievirus/virología , Enterovirus Humano B/patogenicidad , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratas , Virulencia
10.
Circulation ; 120(23): 2358-66, 2009 Dec 08.
Artículo en Inglés | MEDLINE | ID: mdl-19933937

RESUMEN

BACKGROUND: Group B coxsackieviruses (CVBs) are the prototypical agents of acute myocarditis and chronic dilated cardiomyopathy, but an effective targeted therapy is still not available. Here, we analyze the therapeutic potential of a soluble (s) virus receptor molecule against CVB3 myocarditis using a gene therapy approach. METHODS AND RESULTS: We generated an inducible adenoviral vector (AdG12) for strict drug-dependent delivery of sCAR-Fc, a fusion protein composed of the coxsackievirus-adenovirus receptor (CAR) extracellular domains and the carboxyl terminus of human IgG1-Fc. Decoy receptor expression was strictly doxycycline dependent, with no expression in the absence of an inducer. CVB3 infection of HeLa cells was efficiently blocked by supernatant from AdG12-transduced cells, but only in the presence of doxycycline. After liver-specific transfer, AdG12 (plus doxycycline) significantly improved cardiac contractility and diastolic relaxation compared with a control vector in CVB3-infected mice if sCAR-Fc was induced before infection (left ventricular pressure 59+/-3.8 versus 45.4+/-2.7 mm Hg, median 59 versus 45.8 mm Hg, P<0.01; dP/dt(max) 3645.1+/-443.6 versus 2057.9+/-490.2 mm Hg/s, median 3526.6 versus 2072 mm Hg/s, P<0.01; and dP/dt(min) -2125.5+/-330.5 versus -1310.2+/-330.3 mm Hg/s, median -2083.7 versus -1295.9 mm Hg/s, P<0.01) and improved contractility if induced concomitantly with infection (left ventricular pressure 76.4+/-19.2 versus 56.8+/-10.3 mm Hg, median 74.8 versus 54.4 mm Hg, P<0.05; dP/dt(max) 5214.2+/-1786.2 versus 3011.6+/-918.3 mm Hg/s, median 5182.1 versus 3106.6 mm Hg/s, P<0.05), respectively. Importantly, hemodynamics of animals treated with AdG12 (plus doxycycline) were similar to uninfected controls. Preinfection induction of sCAR-Fc completely blocked and concomitant induction strongly reduced cardiac CVB3 infection, myocardial injury, and inflammation. CONCLUSIONS: AdG12-mediated sCAR-Fc delivery prevents cardiac dysfunction in CVB3 myocarditis under prophylactic and therapeutic conditions.


Asunto(s)
Infecciones por Coxsackievirus/prevención & control , Regulación Viral de la Expresión Génica , Miocarditis/prevención & control , Receptores Virales/biosíntesis , Receptores Virales/genética , Enfermedad Aguda , Animales , Cardiomiopatías/genética , Cardiomiopatías/prevención & control , Cardiomiopatías/virología , Infecciones por Coxsackievirus/genética , Terapia Genética/métodos , Células HeLa , Humanos , Ratones , Ratones Endogámicos BALB C , Miocarditis/genética , Miocarditis/virología , Receptores Virales/administración & dosificación
11.
Thorax ; 65(3): 246-51, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20335295

RESUMEN

BACKGROUND: A proteolytic imbalance has been implicated in the development of "classical" chronic lung disease of prematurity (CLD). However, in "new" CLD this pattern has changed. This study examines the longitudinal relationship between neutrophil proteinases and their inhibitors in ventilated preterm infants and their relationship to microbial colonisation. METHODS: Serial bronchoalveolar lavage fluid was obtained from ventilated newborn preterm infants. Neutrophil elastase (NE) activity, cell counts, metalloproteinase (MMP)-9, MMP-9/TIMP-1 complex, SerpinB1 concentration and percentage of SerpinB1 and alpha(1)-antitrypsin (AAT) in complex with elastase were measured. The presence of microbial genes was examined using PCR for 16S rRNA genes. RESULTS: Statistically more infants who developed CLD had NE activity in at least one sample (10/20) compared with infants with resolved respiratory distress syndrome (RDS) (2/17). However, NE activity was present in a minority of samples, occurring as episodic peaks. Peak levels of MMP-9, MMP-9/TIMP-1 complex, percentage of AAT and SerpinB1 in complex and cell counts were all statistically greater in infants developing CLD than in infants with resolved RDS. Peak values frequently occurred as episodic spikes and strong temporal relationships were noted between all markers. The peak values for all variables were significantly correlated to each other. The presence of bacterial 16S rRNA genes was associated with the development of CLD and with elevated elastase and MMP-9. CONCLUSION: NE activity and MMP-9 appear to be important in the development of "new" CLD with both proteinase and inhibitor concentrations increasing episodically, possibly in response to postnatal infection.


Asunto(s)
Displasia Broncopulmonar/enzimología , Péptido Hidrolasas/metabolismo , Inhibidores de Proteasas/metabolismo , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/microbiología , Displasia Broncopulmonar/microbiología , Recuento de Células , Femenino , Genes Bacterianos , Humanos , Recién Nacido , Recien Nacido Prematuro , Elastasa de Leucocito/metabolismo , Estudios Longitudinales , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , ARN Ribosómico 16S/genética , Respiración Artificial , Serpinas/metabolismo , alfa 1-Antitripsina/metabolismo
12.
Antimicrob Agents Chemother ; 53(5): 2020-7, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19273669

RESUMEN

Antibiotic resistance determination of Ureaplasma spp. (Ureaplasma parvum and Ureaplasma urealyticum) usually requires predetermination of bacterial titer, followed by antibiotic interrogation using a set bacterial input. This 96-well method allows simultaneous quantification of bacteria in the presence and absence of antibiotics. A method for determining precise MICs and a method for screening against multiple antibiotics using breakpoint thresholds are detailed. Of the 61 Ureaplasma-positive clinical isolates screened, one (1.6%) was resistant to erythromycin (MIC, >64 mg/liter) and clarithromycin (MIC, 4 mg/liter), one to ciprofloxacin (1.6%), and one to tetracycline/doxycycline (1.6%). Five isolates were also consistently found to have an elevated MIC of 8 mg/liter for erythromycin, but this may not represent true antibiotic resistance, as no mutations were found in the 23S rRNA operons or ribosome-associated L4 and L22 proteins for these strains. However, two amino acids (R66Q67) were deleted from the L4 protein of the erythromycin-/clarithromycin-resistant strain. The tetM genetic element was detected in the tetracycline-resistant clinical isolate as well as in the positive control Vancouver strain serotype 9. The tetM gene was also found in a fully tetracycline-susceptible Ureaplasma clinical isolate, and no mutations were found in the coding region that would explain its failure to mediate tetracycline resistance. An amino acid substitution (D82N) was found in the ParC subunit of the ciprofloxacin-resistant isolate, adjacent to the S83L mutation reported by other investigators in many ciprofloxacin-resistant Ureaplasma isolates. It is now possible to detect antibiotic resistance in Ureaplasma within 48 h of positive culture without prior knowledge of bacterial load, identifying them for further molecular analysis.


Asunto(s)
Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana/genética , Enfermedades del Prematuro/microbiología , Mutación Puntual , Infecciones por Ureaplasma/microbiología , Ureaplasma/efectos de los fármacos , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Líquido del Lavado Bronquioalveolar/microbiología , Ciprofloxacina/farmacología , Claritromicina/farmacología , Medios de Cultivo , Eritromicina/farmacología , Humanos , Recién Nacido , Pruebas de Sensibilidad Microbiana/métodos , Pruebas de Sensibilidad Microbiana/normas , Reacción en Cadena de la Polimerasa/métodos , Análisis de Secuencia de ADN , Especificidad de la Especie , Tetraciclina/farmacología , Ureaplasma/clasificación , Ureaplasma/genética , Ureaplasma/crecimiento & desarrollo
13.
J Antimicrob Chemother ; 64(3): 529-38, 2009 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-19567408

RESUMEN

OBJECTIVES: To determine the role of amino acid substitutions in Ureaplasma GyrA, GyrB, ParC and ParE proteins in mediating fluoroquinolone resistance. METHODS: Nucleic acid sequences from gyrA, gyrB, parC and parE genes from all 14 Ureaplasma serovars were aligned. Full genome sequences for serovars 1, 3-7, 9 and 11-14 were available from the National Center for Biotechnology Information database and we sequenced the full topoisomerase genes from ciprofloxacin-susceptible reference strains of serovars 2, 8 and 10. Phylogenetic trees were constructed to analyse nucleotide sequence similarity. Deduced amino acid sequences were compared with all 33 previously reported fluoroquinolone-resistant strains to clarify true fluoroquinolone-resistance-associated substitutions. RESULTS: Non-resistance-associated polymorphisms were identified in GyrA (39), GyrB (26), ParC (107) and ParE (34) proteins. Phylogenetic analysis demonstrated species clustering for all genes, except parE in which serovars 4, 12, 10 and 13 formed a separate cluster more similar to Ureaplasma parvum than the remaining Ureaplasma urealyticum serovars. Examination of all previously reported fluoroquinolone-resistant strains found that one-third of identified residue substitutions could be attributed to normal species polymorphism; therefore, the mechanism of resistance for these strains is still undetermined. In particular, Glu or Asp at position 112 in GyrA and Ala or Thr at 125/136 in ParC were substitutions identified when U. urealyticum strain sequences were previously aligned with the published serovar 3 genome sequence. CONCLUSION: Combining analysis of the recently available Ureaplasma genomes with sequences from the additional serovars has enabled us to clarify which substitutions found by previous investigators could potentially be responsible for fluoroquinolone resistance.


Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana , Fluoroquinolonas/farmacología , Mutación Missense , Ureaplasma urealyticum/genética , Ureaplasma/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Análisis por Conglomerados , Girasa de ADN/genética , Análisis Mutacional de ADN , Topoisomerasa de ADN IV/genética , ADN Bacteriano/química , ADN Bacteriano/genética , Genotipo , Humanos , Recién Nacido , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia , Estados Unidos , Ureaplasma/efectos de los fármacos , Infecciones por Ureaplasma/microbiología , Ureaplasma urealyticum/efectos de los fármacos
14.
Immunology ; 123(2): 228-38, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17764451

RESUMEN

Complement is an essential part of the innate immune system, which clears pathogens without requirement for previous exposure, although it also greatly enhances the efficacy and response of the cellular and humoral immune systems. Kaposi's sarcoma-associated herpesvirus (KSHV) is the most recently identified human herpesvirus and the likely aetiological agent of Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease. We previously reported that the KSHV complement control protein (KCP) was expressed on infected cells and virions, and could inhibit complement through decay-accelerating activity (DAA) of the classical C3 convertase and cofactor activity (CFA) for factor I (FI)-mediated degradation of C4b and C3b, as well as acting as an attachment factor for binding to heparan sulphate on permissive cells. Here, we determined the ability of a panel of monoclonal anti-KCP antibodies to block KCP functions relative to their recognized epitopes, as determined through binding to recombinant KCP containing large (entire domain) or small (2-3 amino acid residue) alterations. One antibody recognizing complement control protein (CCP) domain 1 blocked heparin binding, DAA and C4b CFA, but was poor at blocking C3b CFA, while a second antibody recognizing CCP4 blocked C3b CFA and 80% DAA, but not C4b CFA or heparan sulphate binding. Two antibodies recognizing CCP2 and CCP3 were capable of blocking C3b and C4b CFA and heparan sulphate binding, but only one could inhibit DAA. These results show that, while KCP is a multifunctional protein, these activities do not completely overlap and can be isolated through incubation with monoclonal antibodies.


Asunto(s)
Antígenos CD55/inmunología , Herpesvirus Humano 8/inmunología , Proteínas Virales/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Antitrombina III/inmunología , Unión Competitiva/inmunología , Células CHO , C3 Convertasa de la Vía Clásica del Complemento/inmunología , Complemento C3b/inmunología , Complemento C4b/inmunología , Cricetinae , Cricetulus , Mapeo Epitopo/métodos , Heparitina Sulfato/metabolismo , Ratones , Ratones Endogámicos BALB C
15.
J Immunol Methods ; 336(2): 175-82, 2008 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-18504046

RESUMEN

Four commercially available monoclonal antibodies (clones NP57, 256-3K1, 39A and 203) were characterised for their ability to block human neutrophil elastase (HNE) activity; capture free purified HNE or neutralised HNE in complex with alpha-1-antitrypsin (AAT); detect HNE and HNE-AAT by Western blot analysis; and detect intracellular HNE by flow cytometry. The ability to block small substrate cleavage by HNE ranged from 0% (265-3K1) to 15-18% (39A and 203) to 100% (NP57). All antibodies had the ability to capture free HNE with varying degrees of sensitivity, but HNE neutralisation by AAT resulted in complete loss of detection (NP57) to 2-4-fold decreased detection (39A and 203) to a 8-fold increase in detection (265-3K1). None of the monoclonal antibodies could detect 200 ng of free HNE, or HNE in complex with AAT, by Western blot analysis, which was easily detected by polyclonal antibodies. NP57 and 265-3K1 gave 10-fold higher fluorescence when detecting intracellular HNE than 39A and 203, and intracellular fluorescence decreased by 10-28% following maximal stimulation of purified neutrophils with fMLP and cytochalasin B (compared to 40% release determined by functional assay). However, for sub-maximal stimulation of neutrophils intracellular anti-HNE antibody binding increased, likely due to increased accessibility following redistribution of enzyme, indicating that measuring residual intracellular HNE as an index of release is a less reliable method than directly measuring extracellular HNE.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Inmunoensayo/métodos , Elastasa de Leucocito/inmunología , Elastasa de Leucocito/metabolismo , Neutrófilos/enzimología , alfa 1-Antitripsina/metabolismo , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Elastasa de Leucocito/antagonistas & inhibidores , Pruebas de Neutralización , Sensibilidad y Especificidad
16.
Mol Immunol ; 44(1-3): 11-22, 2007 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-16905191

RESUMEN

The complement system is an important part of innate immunity providing immediate protection against pathogens without a need for previous exposure, as well as priming the adaptive immune response through opsonisation, leukocyte recruitment and enhancing humoral immune responses. Its importance is not only shown through recurring fulminant infections in individuals with complement component deficiencies, but also through the many complement evasion strategies discovered for a wide range of infectious microbes (including acquisition of endogenous host complement inhibitors and expression of own homologues). Knowledge of these mechanisms at a molecular level may aid development of vaccines and novel therapeutic strategies. Here, we review the structure-function studies of the membrane-bound complement inhibitor KCP that is expressed on the surface of Kaposi's sarcoma-associated herpesvirus (KSHV) virions and infected cells. KCP accelerates the decay of classical C3 convertase and induces the degradation of activated complement factors C4b and C3b by a serine proteinase, factor I. Molecular modeling and site-directed mutagenesis have identified sites on the surface of KCP required for complement inhibition and support the hypothesis that KCP has evolved to mimic the structure and function of endogenous human inhibitors. KCP additionally enhances virion binding to permissive cells through a heparin/heparan sulfate-binding site located at the N-terminus of the protein.


Asunto(s)
Convertasas de Complemento C3-C5/antagonistas & inhibidores , Herpesvirus Humano 8/patogenicidad , Proteínas Virales/fisiología , Aminoácidos/análisis , Heparina/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Inmunidad Innata , Proteínas Virales/análisis , Proteínas Virales/química
17.
Mol Immunol ; 43(10): 1665-75, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16442624

RESUMEN

The Kaposi's sarcoma-associated herpesvirus (KSHV) complement control protein (KCP) inhibits the human complement system, and is similar in structure and function to endogenous complement inhibitors. Other inhibitors such as C4b-binding protein and factor H, as well as the viral homologue vaccinia virus complement control protein are known to bind heparin and, for the two latter, also to glycosaminoglycans at the surface of cells. We report here that KCP also binds to heparin at physiological ionic strength. With help of site directed mutagenesis, positively charged amino acids in the two N-terminal complement control protein (CCP) domains 1-2 were found to be necessary for heparin binding. In silico molecular docking of heparin to KCP confirmed the experimental data, and further explored the heparin binding site, enabling us to present a model of the KCP-heparin interaction. Furthermore, the docking analysis also yielded insights of the KCP structure, by indicating that the angle between CCP domains 1-2 during the initial binding of heparin is more extended than in the model we have previously presented. We also found that KCP binds to heparan sulfate and weakly to glycosaminoglycans at the surface of cells. This might indicate that KCP at the surface of viral particles aids in the primary attachment to the target cells, which is known to involve binding to heparan sulfate. Therefore, the present study contributes to the knowledge of heparin-protein interactions in general as well as to the understanding of the biology of KSHV.


Asunto(s)
Aminoácidos Básicos/metabolismo , Membrana Celular/metabolismo , Heparina/metabolismo , Proteínas Virales/metabolismo , Aminoácidos Básicos/genética , Animales , Células CHO , Membrana Celular/química , Proteína de Unión al Complemento C4b/antagonistas & inhibidores , Proteína de Unión al Complemento C4b/metabolismo , Cricetinae , Cricetulus , Glicosaminoglicanos/análisis , Glicosaminoglicanos/metabolismo , Heparina/farmacología , Heparitina Sulfato/metabolismo , Humanos , Mutagénesis Sitio-Dirigida , Mutación , Estructura Terciaria de Proteína , Proteínas Virales/genética
18.
Front Microbiol ; 7: 157, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26909073

RESUMEN

Investigations of patients with suspected Mycoplasma pneumoniae infection have been undertaken in England since the early 1970s. M. pneumoniae is a respiratory pathogen that is a common cause of pneumonia and may cause serious sequelae such as encephalitis and has been documented in children with persistent cough. The pathogen is found in all age groups, with higher prevalence in children aged 5-14 years. In England, recurrent epidemic periods have occurred at ~4-yearly intervals. In addition, low-level sporadic infection occurs with seasonal peaks from December to February. Voluntarily reports from regional laboratories and hospitals in England from 1975 to 2015 were collated by Public Health England for epidemiological analysis. Further data pertaining cases of note and specimens submitted to Public Health England from 2005 to 2015 for confirmation, molecular typing is included.

19.
Pediatrics ; 138(2)2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27418415

RESUMEN

Isolation of Ureaplasma spp. from preterm neonates and the association with development of bronchopulmonary dysplasia has been previously investigated. However, few studies have contrasted the nature of infection in twins. In this article, we report that dizygotic twins (1 girl, 1 boy) born at 24 weeks gestation both yielded culturable Ureaplasma from endotracheal secretions. The samples were part of a serial blind collection cohort of ventilated premature neonates, and analysis of repeat cultures showed stable, separate infections over a period of 17 and 21 days, respectively. Immunoblot and probe-specific quantitative polymerase chain reaction analysis determined that Twin 1 was solely infected with Ureaplasma parvum (specifically, serovar 6 by gene sequencing), whereas Twin 2 was solely infected with Ureaplasma urealyticum (specifically, genotype A- serovars 2, 5, and 8 by gene sequencing). Immunoblot analysis found that the major surface antigen (multiple-banded antigen) altered relative mass for both strains during the course of infection. Quantitative polymerase chain reaction analysis of extracted endotracheal aspirates confirmed no evidence of mixed infection for either twin. Failure of sentinel ventilated preterm infants on the same ward to acquire Ureaplasma infection after the first week of birth suggests no cot-to-cot transfer of Ureaplasma infection occurred. This study demonstrated not only a contrasting clinical outcome for a set of twins infected with 2 separate species of Ureaplasma, but also the first real-time demonstration of multiple-banded antigen alteration and evolution of Ureaplasma over the course of a clinical infection.


Asunto(s)
Secreciones Corporales/microbiología , Enfermedades en Gemelos/microbiología , Tráquea/microbiología , Infecciones por Ureaplasma/microbiología , Ureaplasma/aislamiento & purificación , Femenino , Humanos , Recién Nacido , Recien Nacido Prematuro , Masculino , Ureaplasma/clasificación
20.
Int J Antimicrob Agents ; 46(3): 319-24, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26141231

RESUMEN

There is a strong association between vaginal and/or amniotic fluid Ureaplasma spp. colonisation and risk of preterm birth. The novel fluoroketolide antibiotic solithromycin (CEM-101) is active against Ureaplasma spp. in vitro. Evidence from ex vivo and in vivo models suggests that, unlike most macrolide antibiotics, solithromycin readily crosses the placenta. Solithromycin metabolism varies according to species; in pregnant sheep, the bioactive metabolites CEM-214 and N-acetyl-CEM-101 (NAc-CEM-101) have been shown to accumulate in the amniotic cavity following maternal solithromycin administration, potentially contributing to its antimicrobial effects. To determine the antimicrobial activity of these metabolites against Ureaplasma spp., the effects of solithromycin, CEM-214, NAc-CEM-101 and the comparator azithromycin were tested on a collection of 100 clinical Ureaplasma spp. isolates from the UK and Australia using a modified 96-well broth microdilution method. MIC90 values observed for the combined cohort were: solithromycin, 0.125 mg/L; CEM-214, 0.5mg/L; NAc-CEM-101, 0.5mg/L; and azithromycin, 2mg/L. Solithromycin showed 34-fold greater activity against Ureaplasma spp. isolates than azithromycin, whilst CEM-214 and NAc-CEM-101 possessed ca. 22% and 17% of the activity of solithromycin, respectively, significantly greater than that of azithromycin. One bacterial isolate showed resistance to azithromycin (MIC=16 mg/L) but had a much lower MIC for solithromycin (MIC=0.25mg/L). In conclusion, the metabolites of solithromycin had reduced, but still potent, activity against 100 clinical Ureaplasma spp. isolates in vitro. This may be important in some instances such as pregnancy, however studies to determine levels of the metabolites in these settings are required.


Asunto(s)
Antibacterianos/farmacología , Azitromicina/farmacología , Macrólidos/farmacología , Triazoles/farmacología , Ureaplasma/efectos de los fármacos , Australia , Farmacorresistencia Bacteriana , Femenino , Humanos , Recién Nacido , Pruebas de Sensibilidad Microbiana , Embarazo , Reino Unido , Ureaplasma/aislamiento & purificación , Infecciones por Ureaplasma/microbiología
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