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Salmonella enterica subspecies enterica serovar Choleraesuis (S. Choleraesuis) is a nontyphoidal Salmonella pathogen that causes swine paratyphoids. S. Choleraesuis is a zoonotic pathogen transmitted to humans via contaminated food and causes sepsis. Here, we report a rare case of pyelonephritis caused by S. Choleraesuis in a Japanese patient with a carcinoma of unknown primary origin. On the day of admission, the patient was diagnosed with pyelonephritis associated with ureteral stent obstruction. He had no history of raw pork consumption or gastrointestinal symptoms. Gram-negative rods were isolated from urine and blood cultures, identified as Salmonella enterica subsp. enterica using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The serological typing results were O7: -: 1 and 5; however, the serotypes could not be determined. The isolate was identified as S. Choleraesuis using multilocus sequence typing, nucleotide sequence analysis of the fliC gene, and biochemical examination. Four days after a 14-day course of intravenous piperacillin-tazobactam (9 g/day), the patient showed relapse of the condition. Subsequently, the patient was treated with intravenous ceftriaxone (2 g/day) and oral amoxicillin (1000 mg/day) for 14 days each; recurrence was not observed. This novel case of pyelonephritis with bacteremia was caused by S. Choleraesuis in Japan. Conventional testing methods could not identify the serotypes; however, the case highlights the importance of adopting advanced diagnostic techniques based on molecular biology to ensure accurate pathogen identification.
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INTRODUCTION: To understand the in-vivo dynamics in pneumococci, investigation into the carriage in patients with invasive pneumococcal disease (IPD) is extremely important. METHODS: To clarify genomic and morphological differences between pneumococcal strains simultaneously isolated from different sites in a patient with IPD, we conducted comparative analyses of two strains. A capsular strain isolated from the blood and a non-capsular strain isolated from the sputum of a patient with IPD were used. RESULTS: The strain isolated from blood was serotype 24B with capsule. The strain isolated from sputum with capsular type 24 genes was non-encapsulated, and genomic analysis revealed an insertion region in the wcxK gene. Its biofilm-forming capacity was higher than that of the capsular strain, as was that of the pspK-positive true non-encapsulated strain. Furthermore, observing the microbe using transmission electron microscopy revealed that the strain isolated from sputum lacked a capsule, like the pspK-positive true non-encapsulated strain. CONCLUSIONS: Our analysis of the two strains isolated from the blood and sputum of a patient with IPD showed one possible in-vivo morphological change in Streptococcus pneumoniae.
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We report a case of bacteremia caused by Bacillus subtilis variant natto after a gastrointestinal perforation in a patient in Japan. Genotypic and phenotypic studies of biotin identified B. subtilis var. natto. This case and 3 others in Japan may have been caused by consuming natto (fermented soybeans).
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Bacteriemia , Alimentos de Soja , Bacillus subtilis/genética , Bacteriemia/diagnóstico , Humanos , JapónRESUMEN
Cryptococcus neoformans, basidiomycetous pathogenic yeast, is basically an environmental fungus and, therefore, challenged by ever changing environments. In this study, we focused on how C. neoformans responds to stress caused by cadmium that is one of high-risk pollutants. By tracking phenotypes of the resistance or sensitivity to cadmium, we undertook forward and reverse genetic studies to identify genes involved in cadmium metabolism in C. neoformans. We found that the main route of Cd2+ influx is through Mn2+ ion transporter, Smf1, which is an ortholog of Nramp (natural resistance-associated macrophage protein 1) of mouse. We found that serotype A strains are generally more resistant to cadmium than serotype D strains and that cadmium resistance of H99, a representative of serotype A strains, was found to be due to a partial defect in SMF1. We found that calcium channel has a subsidiary role for cadmium uptake. We also showed that Pca1 (P-type-ATPase) functions as an extrusion pump for cadmium. We examined the effects of some metals on cadmium toxicity and suggested (i) that Ca2+ and Zn2+ could exert their protective function against Cd2+ via restoring cadmium-inhibited cellular processes and (ii) that Mg2+ and Mn2+ could have antagonistic roles in an unknown Smf1-independent Cd2+ uptake system. We proposed a model for Cd2+-response of C. neoformans, which will serve as a platform for understanding how this organism copes with the toxic metal.
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Criptococosis , Cryptococcus neoformans , Cadmio/toxicidad , Criptococosis/microbiología , Cryptococcus neoformans/genéticaRESUMEN
After the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13), serotype replacement has occurred in Japan, and serotype 24 has become the most common serotype in paediatric invasive pneumococcal disease (IPD). To understand the characteristics of serotype 24-IPD in Japanese children in the post-PCV13 era, we conducted a retrospective study in children aged ≤15 years from 2010 to 2020 using a database of paediatric IPD surveillance in Chiba prefecture, Japan. We identified a total of 357 IPD cases and collected clinical information on 225 cases (24: 32 cases, non-24: 193 cases). Compared with the non-serotype 24-IPD, serotype 24-IPD was independently related to be <2 years of age [odds ratio (OR) 3.91, 95% confidence interval (CI) 1.47-10.44; P = 0.0064] and bacteremia (OR 2.28, 95% CI 1.01-5.13; P = 0.0475), as a result of the multivariate regression analysis. We also conducted a bacterial analysis, and the isolates of serotype 24-IPD had tendencies of PCG-susceptible (24: 100.0%, non-24: 61.3%; P < 0.0001) and macrolide-resistance (24: 100.0%, non-24: 87.3%; P = 0.0490). Their multilocus sequence typing was mostly ST2572 and the variants, which were unique to Japan. This tendency might have been a result of the progress made in the Japanese PCV13 immunisation programme.
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Infecciones Neumocócicas , Streptococcus pneumoniae , Niño , Preescolar , Humanos , Incidencia , Lactante , Japón/epidemiología , Infecciones Neumocócicas/microbiología , Vacunas Neumococicas , Estudios Retrospectivos , SerogrupoRESUMEN
Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections have increased worldwide in people without underlying diseases. CA-MRSA can often cause serious bacterial infections, especially skin and soft tissue infections (SSTI). Here, we describe a case of severe subcutaneous abscess due to Panton-Valentine leucocidin (PVL)-positive CA-MRSA in an infant without underlying diseases. A 4-month-old girl presented with a 4-day history of fever, with extensive redness and swelling of the lumbar region and buttocks. She was diagnosed with extensive subcutaneous abscess of the lumbar region and buttocks. Surgical drainage was performed, and a substantial volume of pus was drained. MRSA was detected in the pus on culture. Antibiotic therapy that covered MRSA was also administered for 3 weeks, and the abscess healed. As it was a severe SSTI due to MRSA, analysis of MRSA revealed PVL-positive MRSA. This patient had no underlying disease or history of antibiotic administration, and as MRSA was present in the nasopharyngeal cavity, it was considered a case of CA-MRSA. Furthermore, the prevalence of PVL-positive CA-MRSA in MRSA isolated from patients with SSTI has also increased in Japan. The Infectious Diseases Society of America recommends surgical intervention and empirical antibiotic therapy for MRSA-complicated SSTI cases in an era of CA-MRSA. Pediatricians must strongly consider the possibility of MRSA in children with severe SSTIs.
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Infecciones Comunitarias Adquiridas , Staphylococcus aureus Resistente a Meticilina , Infecciones de los Tejidos Blandos , Infecciones Estafilocócicas , Niño , Lactante , Femenino , Humanos , Leucocidinas , Absceso , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/epidemiología , Infecciones Estafilocócicas/microbiología , Infecciones de los Tejidos Blandos/epidemiología , Infecciones de los Tejidos Blandos/microbiología , Antibacterianos/uso terapéuticoRESUMEN
INTRODUCTION: In 2010, oral fluoroquinolone tosufloxacin (TFX) granules were released as the first oral respiratory quinolone for children in Japan. METHODS: To investigate the recent trend of H. influenzae strains with low susceptibility to quinolones in children, we analyzed the gene sequences of quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC, and parE of 23 clinical isolates from 15 patients aged <15 years with an MIC of ≥0.5 µg/mL for TFX from 2010 to 2018. RESULTS: Amino acid substitutions were observed in both GyrA and ParC in 13 strains (81%, 13/16), except two strains with a TFX MIC of 0.5 µg/mL with amino acid substitution in only GyrA and one strain with a TFX MIC of 1 µg/mL with no amino acid substitution. Four ST422 strains were observed in 2018, the detection age range was wide (0-7 years), and the residential city was varied. A total of 3/15 patients had a clear history of TFX treatment. CONCLUSIONS: Even for the strain with an MIC of 0.5 µg/mL for TFX, it is highly possible that it harbors a mutation in gyrA, which is the first step toward quinolone resistance, and it may also harbor mutations in both gyrA and parC. Furthermore, several specific sequence type quinolone-resistant H. influenzae strains, particularly ST422, may be widespread among children in Japan. It is necessary to investigate changes in resistance both at the MIC and gene levels. The continuous monitoring of strains and the use of antimicrobial drugs in treatment should be carefully observed.
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Haemophilus influenzae , Quinolonas , Sustitución de Aminoácidos , Niño , Preescolar , Girasa de ADN/genética , Topoisomerasa de ADN IV/genética , Fluoroquinolonas/farmacología , Haemophilus influenzae/genética , Humanos , Lactante , Recién Nacido , Japón , Pruebas de Sensibilidad Microbiana , MutaciónRESUMEN
INTRODUCTION: Neisseria lactamica is a commensal bacterium of the upper respiratory tract in humans and is closely related to Neisseria meningitidis. N. lactamica colonization may contribute to preventing N. meningitidis colonization and invasive meningococcal disease. However, the transference of antimicrobial resistance genes from N. lactamica to N. meningitidis has been reported. METHODS: In this study, we aimed to identify N. lactamica using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and performed multilocus sequence typing of seven N. lactamica strains isolated from Japanese children. We also analyzed the antimicrobial susceptibility of these strains and the mutations in their antimicrobial resistance genes (penA, gyrA, and parC). RESULTS: All the N. lactamica strains could be identified using MALDI-TOF MS. All strains were of different sequence types (STs), including five new STs. Five strains had intermediate susceptibility, two were resistant to ampicillin, and all had five out of the five known PBP2 mutations. Six strains were resistant to levofloxacin. Among the quinolone-resistant strains, three had GyrA mutations, and three had both ParC and GyrA mutations. CONCLUSIONS: N. lactamica STs may vary in Japanese children, and penicillin- and quinolone-resistant strains may be prevalent. We should pay attention not only to the drug resistance of N. meningitidis but also to the drug susceptibility of N. lactamica whose drug-resistance genes may transfer to N. meningitidis.
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Infecciones Meningocócicas , Neisseria lactamica , Neisseria meningitidis , Niño , Humanos , Japón/epidemiología , Neisseria lactamica/genética , Neisseria meningitidis/genética , Sistema RespiratorioRESUMEN
Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has become a pathogen of major importance in pediatric patients. CA-MRSA can cause skin and soft tissue infection in children and young active adults with no predisposing factors, and life-threatening infections such as meningitis or necrotizing pneumonia have been reported. We report here a case of CA-MRSA meningitis complicated by acute left middle cerebral artery (MCA) infarction and necrotizing pneumonia in a previously healthy 1-month-old Vietnamese boy. He was firstly treated with vancomycin, but changed to linezolid because of persistent fever and low vancomycin trough level. He recovered successfully with residual right-sided hemiparesis. The mode of transmission of CA-MRSA and the mechanism of cerebral infarction (thrombotic or embolic) were unknown. The isolate was genotyped as staphylococcal cassette chromosome (SCC) mec type V with a novel sequence type (ST) 5959 harboring the Panton-Valentine leukocidin (PVL) gene. ST 5959 is a double locus variant of ST 59, which is a major PVL-positive CA-MRSA strain isolated in invasive disease in Asian countries. This case report may serve as a warning about the dissemination of PVL-positive CA-MRSA in and around Japan, with the possibility of causing serious life-threatening disease. The potential of linezolid for the treatment of MRSA meningitis as one of the alternative MRSA therapeutic drugs is also discussed.
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Infecciones Comunitarias Adquiridas , Meningitis , Staphylococcus aureus Resistente a Meticilina , Infecciones Estafilocócicas , Adulto , Asia , Toxinas Bacterianas , Infarto Cerebral/complicaciones , Infarto Cerebral/tratamiento farmacológico , Niño , Infecciones Comunitarias Adquiridas/tratamiento farmacológico , Exotoxinas/genética , Humanos , Lactante , Japón , Leucocidinas/genética , Masculino , Staphylococcus aureus Resistente a Meticilina/genética , Infecciones Estafilocócicas/complicaciones , Infecciones Estafilocócicas/tratamiento farmacológicoRESUMEN
The non-encapsulated Streptococcus pneumoniae (NESp) has emerged and increased in the clinical setting. The majority of NESp strains have been isolated from the nasopharynxes of healthy carriers and from respiratory specimens of patients with otitis media. NESp strains were shown to be more effective than encapsulated counterparts at forming biofilms. Therefore, NESp should become one of the leading causes of emerging refractory respiratory disease after the introduction of pneumococcal conjugate vaccines. We report the first case of multidrug-resistant - including fluoroquinolone-resistant - NESp isolated from the intrabronchial aspirate of a patient with pneumonia. Drug-resistant NESp infections can possibly emerge as a clinical problem and thus the continuous monitoring of NESp infections is of utmost importance.
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Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Neumonía Neumocócica/tratamiento farmacológico , Streptococcus pneumoniae/efectos de los fármacos , Adolescente , Ampicilina/farmacología , Ampicilina/uso terapéutico , Antibacterianos/uso terapéutico , Cefalosporinas/uso terapéutico , Quimioterapia Combinada , Femenino , Humanos , Pruebas de Sensibilidad Microbiana , Miopatías Estructurales Congénitas , Oftalmoplejía , Neumonía Neumocócica/diagnóstico , Neumonía Neumocócica/microbiología , Canal Liberador de Calcio Receptor de Rianodina/deficiencia , Esputo/microbiología , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/aislamiento & purificación , Sulbactam/farmacología , Sulbactam/uso terapéutico , CefozopránRESUMEN
Although the pneumococcal conjugate vaccine (PCV) has decreased the incidence of invasive pneumococcal disease (IPD) in children, cases of IPD caused by non-PCV serotypes have been increasing. Here, we report two cases of bacterial meningitis caused by meropenem-resistant Streptococcus pneumoniae; in both the cases, 13-valent PCV (PCV13) had been administered. The isolated S. pneumoniae strains were non-PCV13 serotype 35B and resistant to penicillin G, cefotaxime, and meropenem. In addition, multilocus sequence typing (MLST) revealed the sequence type (ST) to be 558. In case 1, a 6-month-old girl recovered without sequelae after antibiotic therapy comprising cefotaxime and vancomycin, whereas in case 2, a 9-month-old boy was treated with an empirical treatment comprising ceftriaxone and vancomycin administration. However, maintaining the blood concentration of vancomycin within the effective range was difficult, due to which the antibiotics were changed to panipenem/betamipron. During the treatment, he presented with seizures, which were effectively controlled with antiepileptic drugs. The rate of incidence of penicillin-susceptible IPD has been substantially increasing after the introduction of PCV. However, an upsurge in IPD cases due to multidrug-resistant (MDR) serotype 35B has been reported in countries where PCV13 was introduced before introducing in Japan. Moreover, an increase in the proportion of MDR serotype 35B and decrease in the susceptibility to broad-spectrum antimicrobials, including meropenem, have been reported. Hence, the number of meningitis cases caused by MDR serotype 35B/ST558 may increase in the future.
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Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Meningitis Neumocócica/tratamiento farmacológico , Meropenem/farmacología , Streptococcus pneumoniae/genética , Antibacterianos/uso terapéutico , Cefotaxima/farmacología , Cefotaxima/uso terapéutico , Femenino , Humanos , Lactante , Masculino , Meningitis Neumocócica/sangre , Meningitis Neumocócica/diagnóstico , Meningitis Neumocócica/microbiología , Meropenem/uso terapéutico , Pruebas de Sensibilidad Microbiana , Tipificación de Secuencias Multilocus , Vacunas Neumococicas/administración & dosificación , Serotipificación , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/aislamiento & purificación , Tienamicinas/farmacología , Tienamicinas/uso terapéutico , Resultado del Tratamiento , beta-Alanina/análogos & derivados , beta-Alanina/farmacología , beta-Alanina/uso terapéuticoRESUMEN
The prevalence of nonencapsulated Streptococcus pneumoniae (NESp) has increased with the introduction of pneumococcal conjugate vaccines in children; however, the bacteriological characteristics of NESp have not been sufficiently clarified. In this study, NESp strains isolated from the nasopharyngeal carriage of children from four nursery schools in Japan were analyzed for molecular type, antibiotic susceptibility, and biofilm productivity. A total of 152 putative S. pneumoniae strains were identified by optochin-susceptibility analysis, of which 21 were not serotypeable by slide agglutination, quellung reaction, or multiplex PCR. Among these 21 strains, three were lytA-negative and, therefore, not S. pneumoniae. The remaining 18 strains were positive for lytA, ply, pspK, and bile solubility and were confirmed as NESp. Therefore, the isolation rate of NESp in the S. pneumoniae strains in this study was 12.0% (18/149). Molecular-typing analyses classified five strains as two existing sequence types (STs; ST7502 and ST7786), and 13 strains formed four novel STs. Horizontal spread was suspected, because strains with the same ST were often isolated from the same nursery school. The NESp isolates were generally susceptible to most antimicrobials, with the exception of macrolides; however, all isolates possessed more than one abnormal penicillin-binding protein gene. Furthermore, NESp strains were more effective than encapsulated counterparts at forming biofilms, which showed obvious differences in morphology. These data indicated that NESp strains should be continuously monitored as emerging respiratory pathogens.
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Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Infecciones Neumocócicas/terapia , Vacunas Neumococicas/uso terapéutico , Streptococcus pneumoniae/aislamiento & purificación , Antibacterianos/uso terapéutico , Farmacorresistencia Bacteriana/genética , Humanos , Lactante , Recién Nacido , Japón/epidemiología , Pruebas de Sensibilidad Microbiana , Tipificación Molecular/métodos , Mutación , Mucosa Nasal/microbiología , Proteínas de Unión a las Penicilinas/genética , Infecciones Neumocócicas/epidemiología , Infecciones Neumocócicas/microbiología , Prevalencia , Serotipificación , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/inmunología , Factores de Virulencia/genéticaRESUMEN
Chronic granulomatous disease (CGD) is a type of primary immunodeficiency disease, which increases susceptibility to recurrent bacterial and fungal infections. Sputum and bronchoalveolar lavage fluid are often obtained using bronchoscopy from adult patients for pathogenic diagnosis, although this approach is much more invasive for infants. We report the case of a 2-month-old boy with CGD, in which gastric aspirate culture was used to diagnose fungal pneumonia. Rasamsonia piperina was isolated from the gastric aspirate, and the patient was successfully treated with micafungin based on the drug susceptibility test results for the fungal isolate. The acid tolerance test revealed that R. piperina could grow at pH 2, indicating high acid resistance. Although we can only report our experience with a single case, gastric aspirate culture may be a useful tool for detecting fungal respiratory pathogens in children with primary immunodeficiency. Detecting these pathogens may help improve outcomes, as early diagnosis and appropriate treatment are extremely important for immunocompromised patients with respiratory infections.
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Ascomicetos/fisiología , Síndromes de Inmunodeficiencia/congénito , Síndromes de Inmunodeficiencia/diagnóstico , Micosis/microbiología , Neumonía/complicaciones , Neumonía/microbiología , Estómago/patología , Humanos , Concentración de Iones de Hidrógeno , Síndromes de Inmunodeficiencia/complicaciones , Lactante , Neumonía/diagnóstico por imagen , Succión , Tomografía Computarizada por Rayos XRESUMEN
A novel stereoisomer of eushearilide, 23-demethyleushearilide, was synthesized, and the structure-activity relationships of this compound along with known eushearilide stereoisomers were investigated in order to design novel lead compounds for the treatment of fungal infections. It was discovered that all of these congeners, together with the natural product, exhibited a wide range of antimicrobial activity against not only fungi but also against bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE).
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Macrólidos/síntesis química , Macrólidos/farmacología , Fosforilcolina/análogos & derivados , Antiinfecciosos/síntesis química , Antiinfecciosos/química , Antiinfecciosos/farmacología , Macrólidos/química , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Fosforilcolina/síntesis química , Fosforilcolina/química , Fosforilcolina/farmacología , Estereoisomerismo , Resistencia a la Vancomicina , Enterococos Resistentes a la Vancomicina/efectos de los fármacosRESUMEN
We elucidated a unique feature of sulfur metabolism in Cryptococcus neoformans. C. neoformans produces cysteine solely by the O-acetylserine pathway that consists of serine-O-acetyl transferase and cysteine synthase. We designated the gene encoding the former enzyme CYS2 (locus tag CNE02740) and the latter enzyme CYS1 (locus tag CNL05880). The cys1Δmutant strain was found to be avirulent in a murine infection model. Methionine practically does not support growth of the cys1Δ strain, and cysteine does not serve as a methionine source, indicating that the transsulfuration pathway does not contribute to sulfur amino acid synthesis in C. neoformans. Among the genes encoding enzymes catalyzing the reactions from homoserine to methionine, the gene corresponding to the Saccharomyces cerevisiae MET17 encoding O-acetylhomoserine sulfhydrylase (Met17p) had remained to be identified in C. neoformans. By genetic analysis of Met- mutants obtained by Agrobacterium tumefaciens-mediated mutagenesis, we concluded that Cnc01220, most similar to Str2p (36% identity), cystathionine-γ-synthase, in the Saccharomyces genome, is the C. neoformans version of O-acetylhomoserine sulfhydrylase. We designated CNC01220 as MET17. The C. neoformans met3Δ mutant defective in the first step of the sulfate assimilation pathway, sulfate adenylyltransferase, barely uses methionine as a sulfur source, whereas it uses cysteine efficiently. The poor utilization of methionine by the met3Δ mutant is most probably due to the absence of the transsulfuration pathway, causing an incapability of C. neoformans to produce cysteine and hydrogen sulfide from methionine. When cysteine is used as a sulfur source, methionine is likely produced de novo by using hydrogen sulfide derived from cysteine via an unidentified pathway. Altogether, the unique features of sulfur amino acid metabolism in C. neoformans will make this fungus a valuable experimental system to develop anti-fungal agents and to investigate physiology of hydrogen sulfide.
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Aminoácidos Sulfúricos/biosíntesis , Cryptococcus neoformans/metabolismo , Agrobacterium tumefaciens/genética , Animales , Cryptococcus neoformans/genética , Cryptococcus neoformans/patogenicidad , Cisteína/metabolismo , Cisteína Sintasa/genética , Genoma Fúngico , Sulfuro de Hidrógeno/metabolismo , Masculino , Metionina/metabolismo , Ratones Endogámicos ICR , Modelos Animales , Mutagénesis , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Serina/análogos & derivados , Serina/metabolismo , Azufre/metabolismo , VirulenciaRESUMEN
Haemophilus influenzae type b (Hib) conjugate vaccines have led to dramatic reductions in Hib disease among young children worldwide. Nontypeable H. influenzae (NTHi) is now the major cause of invasive H. influenzae infections. We investigated the clinical characteristics of invasive NTHi diseases among children in Japan, to clarify the pathogenicity of isolated NTHi strains. The mortality rate was 10.7%, with deaths occurring mainly among children with underlying comorbidities. Biotypes II and III were the most common, and most strains (64.3%) had multiple amino acid substitutions at the Asp-350, Ser-357, Ser-385, and/or Met-377 sites of penicillin-binding protein 3. Two strains were ß-lactamase positive and ampicillin-clavulanate resistant. Biofilm indices varied widely, and IS1016 was detected in 10.7% of the strains tested. Moreover, there was wide variation in the characteristics of invasive NTHi strains. NTHi strains, showing great genetic diversity, are responsible for most invasive H. influenzae infections in children in the postvaccine era. Continuous monitoring of NTHi strains responsible for invasive diseases in children is important to detect changes in the epidemiology of invasive H. influenzae infections in the postvaccine era.
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Infecciones por Haemophilus/microbiología , Haemophilus influenzae/clasificación , Haemophilus influenzae/fisiología , Antibacterianos/farmacología , Adhesión Bacteriana/genética , Técnicas de Tipificación Bacteriana , Biopelículas/crecimiento & desarrollo , Niño , Preescolar , Elementos Transponibles de ADN , Farmacorresistencia Bacteriana/genética , Variación Genética , Genoma Bacteriano/genética , Infecciones por Haemophilus/epidemiología , Infecciones por Haemophilus/mortalidad , Infecciones por Haemophilus/fisiopatología , Haemophilus influenzae/efectos de los fármacos , Haemophilus influenzae/genética , Humanos , Lactante , Recién Nacido , Japón/epidemiología , Pruebas de Sensibilidad Microbiana , Análisis de Secuencia de ADNRESUMEN
Haemophilus influenzae is a major pathogenic bacteria causing invasive disease, which is classified into six capsular serotypes (a-f) and non-typeable (NT) strains. Capsular serotyping of H. influenzae is traditionally determined by serological methods and more recently by PCR methods. However, these methods are time-consuming and expensive. In the present study, matrix-associated laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was evaluated as an alternative method for capsular serotyping of H. influenzae clinical strains. We created an in-house database of all six serotypes and NT H. influenzae strains using the main spectrum creation standard method set to the default parameters in MADI-TOF MS. We evaluated the performance of the in-house database using 79 clinical strains already identified by PCR and 58 prospectively collected clinical strains. Measurements were performed using the Bruker MALDI BioTyper system. The peak list was matched against the reference library using the integrated pattern algorithm of the software. The best-matched spectrum was considered the serotyping result. All 137 test strains were correctly identified as H. influenzae using MALDI-TOF MS. The sensitivity and specificity for identification for type b, type e, and type f capsular serotypes and NT H. influenzae using MALDI-TOF MS were 100%/94.3%, 94.7%/97.9%, 97.4%/97.9%, and 85.5%/99.2%, respectively. Our findings indicate that MALDI-TOF MS is a useful alternative method for capsular serotyping of H. influenzae strains. This method is faster and more cost-effective than traditional methods and will therefore be useful for routine applications in clinical laboratories.
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Cápsulas Bacterianas/inmunología , Infecciones por Haemophilus/microbiología , Haemophilus influenzae/clasificación , Serotipificación/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , ADN Bacteriano/genética , Infecciones por Haemophilus/diagnóstico , Haemophilus influenzae/genética , Haemophilus influenzae/inmunología , Humanos , Sensibilidad y Especificidad , Programas InformáticosRESUMEN
It is well known that 5-fluoroorotic acid (5-FOA)-resistant mutants isolated from wild-type Cryptococcus neoformans are exclusively either ura3 or ura5 mutants. Unexpectedly, many of the 5-FOA-resistant mutants isolated in our selective regime were Ura+. We identified CNM00460 as the gene responsible for these mutations. Cnm00460 belongs to the nucleobase cation symporter 1/purine-related transporter (NCS1/PRT) super family of fungal transporters, representative members of which are uracil transporter, uridine transporter and allantoin transporter of Saccharomyces cerevisiae. Since the CNM00460 gene turned out to be involved in utilization of orotic acid, most probably as transporter, we designated this gene Orotic Acid Transporter 1 (OAT1). This is the first report of orotic acid transporter in this family. C. neoformans has four members of the NCS1/PRT family, including Cnm00460, Cnm02550, Cnj00690, and Cnn02280. Since the cnm02550∆ strain showed resistance to 5-fluorouridine, we concluded that CNM02550 encodes uridine permease and designated it URidine Permease 1 (URP1). We found that oat1 mutants were sensitive to 5-FOA in the medium containing proline as nitrogen source. A mutation in the GAT1 gene, a positive transcriptional regulator of genes under the control of nitrogen metabolite repression, in the genetic background of oat1 conferred the phenotype of weak resistance to 5-FOA even in the medium using proline as nitrogen source. Thus, we proposed the existence of another orotic acid utilization system (tentatively designated OAT2) whose expression is under the control of nitrogen metabolite repression at least in part. We found that the OAT1 gene is necessary for full pathogenic activity of C. neoformans var. neoformans.
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Transporte Biológico/genética , Cryptococcus neoformans/genética , Proteínas de Transporte de Membrana/genética , Cryptococcus neoformans/efectos de los fármacos , Cryptococcus neoformans/patogenicidad , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Mutación , Nitrógeno/metabolismo , Ácido Orótico/análogos & derivados , Ácido Orótico/farmacología , Uracilo/metabolismoRESUMEN
We constructed deletion mutants of Cryptococcus neoformans var neoformans (serotype D) genes encoding late ergosterol biosynthetic pathway enzymes and found that the mutations enhanced susceptibility to various drugs including micafungin, one of the echinocandins, to which wild-type Cryptococcus strains show no susceptibility. Furthermore, through isolation of a mutant resistant to micafungin from a micafungin-sensitive erg mutant and genetic analysis of it, we found that the responsible mutation occurred in the hotspot 2 of FKS1 encoding ß-1, 3-glucan synthase, indicating that micafungin inhibited the growth of the erg mutant via inhibiting Fks1 activity. Addition of ergosterol to the culture of the erg mutants recovered the resistance to micafungin, suggesting that the presence of ergosterol in membrane inhibits the accession of micafungin to its target. We found that a loss of one of genes encoding subunits of v-ATPase, VPH1, made Cryptococcus cells sensitive to micafungin. Our observation that the erg2 vph1 double mutant was more sensitive to micafungin than either single mutant suggests that these two genes act differently in becoming resistant to micafungin. The erg mutants allowed us to study the physiological significance of ß-1, 3-glucan synthesis in C. neoformans; the inhibition of ß-1, 3-glucan synthesis induced cell death and changes in cellular morphology. By observing the erg mutant cells recovering from the growth inhibition imposed by micafungin, we recognized ß-1, 3-glucan synthesis would suppress filamentous growth in C. neoformans.
Asunto(s)
Cryptococcus neoformans/genética , Farmacorresistencia Fúngica/genética , Equinocandinas/farmacología , Regulación Fúngica de la Expresión Génica , Glucosiltransferasas/genética , Lipopéptidos/farmacología , ATPasas de Translocación de Protón Vacuolares/genética , Antifúngicos/farmacología , Cryptococcus neoformans/efectos de los fármacos , Cryptococcus neoformans/enzimología , Cryptococcus neoformans/crecimiento & desarrollo , Ergosterol/biosíntesis , Ergosterol/farmacología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ingeniería Genética , Glucosiltransferasas/deficiencia , Micafungina , Pruebas de Sensibilidad Microbiana , Mutación , Subunidades de Proteína/deficiencia , Subunidades de Proteína/genética , ATPasas de Translocación de Protón Vacuolares/deficienciaRESUMEN
Tosufloxacin (TFLX) is a fluoroquinolone antimicrobial agent. TFLX granules for children were initially released in Japan in 2010 to treat otitis media and pneumonia caused by drug-resistant bacteria, e.g. penicillin-resistant Streptococcus pneumoniae and beta-lactamase-negative, ampicillin-resistant Haemophilus influenzae. The evolution of bacterial resistance since TFLX approval is not known. To clarify the influence of quinolones administered to children since their approval, we examined the resistance mechanism of TFLX-resistant S. pneumoniae isolated from paediatric patients as well as patient clinical characteristics. TFLX-resistant strains (MIC ≥ 2 mg/L) were detected among clinical isolates of S. pneumoniae derived from children (≤15 years old) between 2010 and 2014. These strains were characterised based on quinolone resistance-determining regions (QRDRs), i.e. gyrA, gyrB, parC, and parE. In addition, the antimicrobial susceptibility, serotype, and multilocus sequence type of strains were determined, pulsed-field gel electrophoresis was performed, and patient clinical characteristics based on medical records were assessed for cases with underling TFLX-resistant strains. Among 1168 S. pneumoniae isolates, two TFLX-resistant strains were detected from respiratory specimens obtained from paediatric patients with frequent exposure to TFLX. Both strains had mutations in the QRDRs of gyrA and parC. One case exhibited gradual changes in the QRDR during the clinical course. This is the first study of quinolone-resistant S. pneumoniae isolated from children, including clinical data, in Japan. These data may help prevent increases in infections of quinolone-resistant S. pneumoniae in children; specifically, the results emphasise the importance of administering fluoroquinolones only in appropriate cases.