RESUMO
Peptides can be converted to highly active compounds by introducing appropriate substituents on the suitable amino acid residue. Although modifiable residues in peptides can be systematically identified by peptide scanning methodologies, there is no practical method for optimization at the "scanned" position. With the purpose of using derivatives not only for scanning but also as a starting point for further chemical functionalization, we herein report the "scanning and direct derivatization" strategy through chemoselective acylation of embedded threonine residues by a serine/threonine ligation (STL) with the help of in situ screening chemistry. We have applied this strategy to the optimization of the polymyxin antibiotics, which were selected as a model system to highlight the power of the rapid derivatization of active scanning derivatives. Using this approach, we explored the structure-activity relationships of the polymyxins and successfully prepared derivatives with activity against polymyxin-resistant bacteria and those with Pseudomonas aeruginosa selective antibacterial activity. This strategy opens up efficient structural exploration and further optimization of peptide sequences.
Assuntos
Antibacterianos , Polimixinas , Polimixinas/farmacologia , Polimixinas/química , Antibacterianos/farmacologia , Antibacterianos/química , Bactérias , Relação Estrutura-Atividade , Treonina , Testes de Sensibilidade MicrobianaRESUMO
BACKGROUND: Colistin (CST) is a last-line drug for multidrug-resistant Gram-negative bacterial infections. CST-heteroresistant Enterobacter cloacae complex (ECC) has been isolated. However, integrated analysis of epidemiology and resistance mechanisms based on the complete ECC species identification has not been performed. METHODS: Clinical isolates identified as "E. cloacae complex" by MALDI-TOF MS Biotyper Compass in a university hospital in Japan were analyzed. Minimum inhibitory concentrations of CST were determined by the broth microdilution method. The population analysis profiling (PAP) was performed for detecting the heteroresistant phenotype. The heat shock protein 60 (hsp60) cluster was determined from its partial nucleotide sequence. From the data of whole-genome sequencing, average nucleotide identity (ANI) for determining ECC species, multilocus sequence type, core genome single-nucleotide-polymorphism-based phylogenetic analysis were performed. phoPQ-, eptA-, and arnT-deleted mutants were established to evaluate the mechanism underlying colistin heteroresistance. The arnT mRNA expression levels were determined by reverse transcription quantitative PCR. RESULTS: Thirty-eight CST-resistant isolates, all of which exhibited the heteroresistant phenotype by PAP, were found from 138 ECC clinical isolates (27.5%). The prevalence of CST-resistant isolates did not significantly differ among the origin of specimens (29.0%, 27.8%, and 20.2% for respiratory, urine, and blood specimens, respectively). hsp60 clusters, core genome phylogeny, and ANI revealed that the CST-heteroresistant isolates were found in all or most of Enterobacter roggenkampii (hsp60 cluster IV), Enterobacter kobei (cluster II), Enterobacter chuandaensis (clusters III and IX), and Enterobacter cloacae subspecies (clusters XI and XII). No heteroresistant isolates were found in Enterobacter hormaechei subspecies (clusters VIII, VI, and III) and Enterobacter ludwigii (cluster V). CST-induced mRNA upregulation of arnT, which encodes 4-amino-4-deoxy-L-arabinose transferase, was observed in the CST-heteroresistant isolates, and it is mediated by phoPQ pathway. Isolates possessing mcr-9 and mcr-10 (3.6% and 5.6% of total ECC isolates, respectively) exhibited similar CST susceptibility and PAP compared with mcr-negative isolates. CONCLUSIONS: Significant prevalence (approximately 28%) of CST heteroresistance is observed in ECC clinical isolates, and they are accumulated in specific species and lineages. Heteroresistance is occurred by upregulation of arnT mRNA induced by CST. Acquisition of mcr genes contributes less to CST resistance in ECC.
Assuntos
Colistina , Infecções por Enterobacteriaceae , Humanos , Colistina/farmacologia , Antibacterianos/farmacologia , Enterobacter cloacae , Prevalência , Filogenia , Infecções por Enterobacteriaceae/epidemiologia , Infecções por Enterobacteriaceae/microbiologia , Nucleotídeos , Testes de Sensibilidade MicrobianaRESUMO
BACKGROUND: Eradication treatment for Helicobacter pylori gastritis is covered by national health insurance since 2013 in Japan. However, eradication failure due to the increase of antimicrobial resistance has become a serious problem. The present study aims to establish a reference panel of Japanese H. pylori strains for antimicrobial susceptibility testing. METHOD: A total of 28 strains were collected from 4 medical facilities in Japan. Antimicrobial susceptibility tests (ASTs) to clarithromycin (CLR), amoxicillin (AMX), and metronidazole (MNZ), were used to select standard reference strains. Complete genome sequences were also determined. RESULTS: Three H. pylori strains (JSHR3, JSHR6 and JSHR31) were selected as standard reference strains by the Japanese Society for Helicobacter Research (JSHR). The minimum inhibitory concentrations (MICs) of the antibiotics against these 3 strains by agar dilution method with Brucella-based horse-serum-containing agar medium were as follows: JSHR3 (CLR 16 µg/ml, AMX 0.032 µg/ml and MNZ 4 µg/ml), JSHR6 (CLR 0.016 µg/ml, AMX 0.032 µg/ml and MNZ 4 µg/ml), and JSHR31 (CLR 16 µg/ml, AMX 1 µg/ml and MNZ 64 µg/ml). CONCLUSIONS: A reference panel of H. pylori JSHR strains was established. The panel consisted of JSHR6, which was antibiotic-susceptible, JSHR3, which was CLR-resistant, and JSHR31, which was multi-resistant. This reference panel will be essential for standardized ASTs before the optimal drugs are selected for eradication treatment.
Assuntos
Infecções por Helicobacter , Helicobacter pylori , Ágar/farmacologia , Ágar/uso terapêutico , Amoxicilina/uso terapêutico , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Claritromicina/uso terapêutico , Farmacorresistência Bacteriana , Infecções por Helicobacter/tratamento farmacológico , Helicobacter pylori/genética , Humanos , Metronidazol/uso terapêutico , Testes de Sensibilidade MicrobianaRESUMO
BACKGROUND: Polymyxin E (colistin) is a last-resort antibiotic to treat infections caused by carbapenemase-producing Enterobacteriaceae (CPE). However, reports of CPEs resistant to colistin have been increasing, and the mcr genes are emerging as resistance mechanisms. Among them, plasmid-mediate mcr-9 is known to be associated with colistin resistance, whereas reports on chromosomal mcr-9 and its association with colistin resistance in humans are few. CASE PRESENTATION: We identified Enterobacter asburiae harboring mcr-9 and blaIMP-60 in the pleural fluid of a patient with empyema. The long-read sequencing technique revealed that these genes were located on its chromosome. Despite the lack of exposure to colistin, the organism showed microcolonies in the inhibition circle in the E-test and disk diffusion test. Antibiotic susceptibility testing by broth microdilution confirmed its resistance to colistin. CONCLUSION: Our case report showed that mcr-9 can be present not only on plasmids but also on the chromosome in E. asburiae, and that the presence of mcr-9 on its chromosome may influence its susceptibility to colistin.
Assuntos
Empiema , Proteínas de Escherichia coli , Humanos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Cromossomos , Colistina/farmacologia , Farmacorresistência Bacteriana/genética , Enterobacter , Proteínas de Escherichia coli/genética , Japão , Testes de Sensibilidade Microbiana , PlasmídeosRESUMO
Muraymycins and caprazamycins are strong inhibitors of MraY, which is responsible for peptidoglycan biosynthesis. Although they are promising antibacterial agents with a novel mode of action, their chemical structures are rather complex. This study investigated the simplification of these natural products by structure-based drug design, synthesis, and biological evaluation. We developed a simplified rigid scaffold with an arylalkyne moiety, which shows sub-micromolar MraY inhibitory activity. The scaffold is suitable for further investigating the structure-activity relationship by virtue of our synthetic strategy, where the substituent of interest is installed in the last stage of synthesis. This scaffold shows the potential for further use in optimizing MraY inhibitory and antibacterial activities.
RESUMO
Intrauterine infection is one of the most important causes of maternal death. In perinatal emergency, we often miss an opportunity to obtain culture specimens. In this study, we tried to examine whether we investigated whether bacteria causing infection can be detected from a formalin-fixed paraffin-embedded (FFPE) placental specimen. We examined the placenta from a maternal invasive infection that resulted in infectious abortion at 18 weeks of gestation. The case was diagnosed by acute fever and abdominal pain, and the patient was cured after 3 weeks of intensive antimicrobial treatment. Four Streptococcus pyogenes strains were isolated from vaginal fluid and blood cultures of the patient. All of the strain types were emm1/ST28. We amplified the V1-V2 region of 16S rRNA from an FFPE placental specimen and sequencing was performed using a next-generation sequencer (NGS). Taxonomic analysis was then performed for sequenced data. We succeeded in detecting causative pathogens from the FFPE placenta: 69.1% of the predominantly identified bacteria were S. pyogenes and other small populations of bacteria were detected. Our results revealed the utility of NGS for 16S rRNA analysis of an FFPE placenta. This method may reveal previous perinatal invasive infections of unknown origin retrospectively.
Assuntos
Placenta , Streptococcus pyogenes , Feminino , Formaldeído , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Inclusão em Parafina , Gravidez , RNA Ribossômico 16S/genética , Estudos Retrospectivos , Streptococcus pyogenes/genéticaRESUMO
Haemophilus influenzae is a pathogenic bacterium that causes respiratory and otolaryngological infections. The increasing prevalence of ß-lactamase-negative high-level ampicillin-resistant H. influenzae (high-BLNAR) is a clinical concern. Fluoroquinolones are alternative agents to ß-lactams. However, the emergence and increasing prevalence of fluoroquinolone-resistant H. influenzae have been reported. The current risk of fluoroquinolone resistance in H. influenzae (especially in high-BLNAR) has not yet been evaluated. Here, we examined the development of fluoroquinolone resistance in fluoroquinolone-susceptible clinical H. influenzae isolates in vitro during passaging in the presence of moxifloxacin (from 0.03 to 128 mg/liter). Twenty-nine isolates were examined. Seventeen isolates (58.6%) showed reduced moxifloxacin susceptibility, and 10 of these 17 isolates (34.5% of all isolates) exceeded the Clinical and Laboratory Standards Institute breakpoint for moxifloxacin (MIC of >1 mg/liter) after repeat cultivation on moxifloxacin-containing agar. Seven of these ten isolates were high-BLNAR and represented multiple lineages. We identified 56 novel mutations in 45 genes induced during the development of fluoroquinolone resistance, except the defined quinolone resistance-determining regions (Ser84Leu and Asp88Tyr/Gly/Asn in GyrA and Gly82Asp, Ser84Arg, and Glu88Lys in ParC). Glu153Leu and ΔGlu606 in GyrA, Ser467Tyr and Glu469Asp in GyrB, and ompP2 mutations were novel mutations contributing to fluoroquinolone resistance in H. influenzae In conclusion, H. influenzae clinical isolates from multiple lineages can acquire fluoroquinolone resistance by multiple novel mutations. The higher rate of derivation of fluoroquinolone-resistant H. influenzae from high-BLNAR than ß-lactamase-negative ampicillin-susceptible isolates (P = 0.01) raises the possibility of the emergence and spread of fluoroquinolone-resistant high-BLNAR in the clinical setting.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Fluoroquinolonas/farmacologia , Haemophilus influenzae/efeitos dos fármacos , Haemophilus influenzae/genética , Mutação/genética , Farmacorresistência Bacteriana Múltipla , Genes Bacterianos/genética , Genoma Bacteriano , Haemophilus influenzae/crescimento & desenvolvimento , Humanos , Testes de Sensibilidade MicrobianaRESUMO
BACKGROUND: Treatment of VRE is of clinical concern. While certain numbers of vanD-type VRE have been isolated, only two vanD5-harbouring Enterococcus faecium isolates have been reported in Canada and Japan. METHODS: We report the isolation of vanD5-type E. faecium and the first ever determination of the whole-genome sequence to investigate the possible mechanisms of the acquisition of the vanD5 gene cluster in E. faecium. RESULTS: Two vanD5-harbouring vancomycin-resistant E. faecium were isolated from the skin (SMVRE19) and faeces (SMVRE20) of a patient with a skin ulcer in Japan. The isolates exhibited vancomycin and teicoplanin MIC values of 128 mg/L, whilst the previous isolates of vanD5-harbouring E. faecium were only resistant to vancomycin. SMVRE19 and SMVRE20 were clones related to ST18, which is also seen in vanA- and vanB-type VRE. These isolates harboured an insertion element, ISEfm1, in the ddl gene, similar to a previously described teicoplanin-resistant vanD3-type E. faecium. The vanD5 gene cluster was integrated into the SMVRE20 chromosome as a part of a large genomic island (approximately 127 kb), similar to other recently spreading vanD variants in the Netherlands. The genomic island shared the greatest similarity with a part of the Blautia coccoides genome sequence, except for the region surrounding the vanD gene cluster. CONCLUSIONS: This study reports that emergence of vancomycin- and teicoplanin-resistant vanD5-type E. faecium occurred via acquisition of the vanD5 cluster and ISEfm1 insertion into ddl. Considering the genetic similarity between the various VRE strains, the current study should serve as a warning against the spread of vanD5-type VRE.
Assuntos
Enterococcus faecium , Infecções por Bactérias Gram-Positivas , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Canadá , Clostridiales , Enterococcus faecium/genética , Ilhas Genômicas , Infecções por Bactérias Gram-Positivas/epidemiologia , Humanos , Japão , Testes de Sensibilidade Microbiana , Países Baixos , Teicoplanina/farmacologia , Vancomicina/farmacologiaRESUMO
Campylobacter upsaliensis is an enteropathogenic bacterium in animals, and is also rarely isolated from humans, where it can cause enteritis and bacteremia. This report describes the first case of isolation of C. upsaliensis from an infected giant hepatic cyst. This bacterium could not be cultured from abscess punctuate in a usual Campylobacter-selection medium (charcoal cefoperazone deoxycholate agar medium), because of high concentration of cefoperazone as a selection agent. It could not identified by matrix-assisted laser desorption ionization-time of flight mass spectrum. Rather, it was identified as C. upsaliensis by whole genome sequencing, including by multilocus sequence typing.
Assuntos
Infecções por Campylobacter/diagnóstico , Campylobacter upsaliensis/isolamento & purificação , Cistos/diagnóstico , Abscesso Hepático/diagnóstico , Idoso , Antibacterianos/administração & dosagem , Infecções por Campylobacter/microbiologia , Infecções por Campylobacter/terapia , Campylobacter upsaliensis/genética , Catéteres , Cefoperazona/administração & dosagem , Cistos/microbiologia , Cistos/terapia , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Quimioterapia Combinada , Humanos , Fígado/diagnóstico por imagem , Fígado/microbiologia , Abscesso Hepático/microbiologia , Abscesso Hepático/terapia , Masculino , Tipagem de Sequências Multilocus , Paracentese/instrumentação , Sulbactam/administração & dosagem , Tomografia Computadorizada por Raios X , Resultado do TratamentoRESUMO
INTRODUCTION: Eradication of asymptomatic bacteriuria (ASB) before urological procedures is important to reduce the risk for infectious complications after surgery. However, the appropriate regimen for antimicrobial treatment has not been fully determined. We experienced continuous (over 10 months) isolation of extended spectrum ß-lactamase (ESBL)-producing fluoroquinolone-resistant Escherichia coli from urine of an asymptomatic patient. The four isolates obtained (SMESC1 to 4) were international high-risk clones of O25b:H4-ST131-H30R, and originated from one strain, as revealed by the whole genome sequences. Although the patient received meropenem (MEPM) and fosfomycin (FOM), to which the strains were susceptible before the urological procedures, they could not be eradicated. METHODS: To explore the reason for the continuous isolation even after MEPM and FOM administration, antimicrobial killing of adherent and/or intracellular bacterial communities (IBC) formed by coculture of the E. coli cells and T24 bladder epithelial cells were examined. RESULTS: FOM and levofloxacin did not decrease viable E. coli cells compared with gentamicin. MEPM partly decreased them, and sitafloxacin (STFX) decreased them most potently. These observations indicate that E. coli can survive in the urinary tract under antimicrobial administration, and some antimicrobials such as FOM and MEPM cannot eradicate E. coli in uroepithelial cells. Adhesion on urinary epithelial cells and/or IBC formation might result in continuous isolation from the urinary tract and recurrence of ASB and urinary tract infections. CONCLUSIONS: The present study suggests that STFX is a promising optional agent for the eradication of ESBL-producing fluoroquinolone-resistant E. coli in the urinary tract before urological procedures.
Assuntos
Infecções por Escherichia coli , Infecções Urinárias , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Escherichia coli , Infecções por Escherichia coli/tratamento farmacológico , Fluoroquinolonas/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Infecções Urinárias/tratamento farmacológico , beta-Lactamases/genéticaRESUMO
Escherichia coli (E. coli) causes urinary tract infections, pneumonia, surgical site infections, and bloodstream infections and is the important pathogen for both community-acquired and healthcare-associated infections. To investigate the clonality of E. coli is important for infection control and prevention. We aimed to investigate the clonality of clinical E. coli isolates using Cica Geneus E. coli polymerase chain reaction (PCR)-based open-reading frame typing (POT) KIT and clarify the clinical usefulness of this kit. About 124 E. coli isolates obtained from inpatients at Sapporo Medical University Hospital were used. The POT method was used to classify 124 clinical isolates into 87 POT numbers. In addition to the clonality, it was possible to obtain additional information that 20 of the 124 isolates were extended-spectrum ß-lactamase (ESBL) producing E. coli (5 isolates of CTX-M-1 group and 15 isolates of CTX-M-9 group) and 13 were sequence type (ST) 131 clone. Furthermore, when these ESBL-producing 20 isolates were compared with pulsed-field gel electrophoresis (PFGE) or multilocus sequence typing (MLST), Simpson's index of diversity was 0.968 in POT method, 0.979 in PFGE, and 0.584 in MLST. POT method had an analytical power similar to that of PFGE. In conclusion, attention should be paid to the difference in the interpretation of the results between the POT method and the PFGE, but POT method may be useful to timely monitor the spread of E. coli in medical facilities.
Assuntos
Infecções por Escherichia coli/microbiologia , Escherichia coli/genética , Fases de Leitura Aberta/genética , Reação em Cadeia da Polimerase/métodos , Infecção Hospitalar , Eletroforese em Gel de Campo Pulsado/métodos , Escherichia coli/classificação , Genes Bacterianos/genética , Humanos , Tipagem de Sequências Multilocus/métodosRESUMO
The present study aimed to elucidate the prevalence of Clostridioides difficile in Japanese retail food products. For this purpose, retail food samples (242 fresh vegetables and 266 retail meat samples: 89 chicken meat; 28 chicken liver; 200 pork meat; 24 pig liver; 127 beef meat) were collected from 14 supermarkets between 2015 and 2019. C. difficile was isolated from eight (3.3%) fresh vegetable, six (6.7%) chicken meat, one (3.6%) chicken liver, one (0.5%) pork meat, and two (1.6%) beef meat samples; it was not isolated from pig liver. Of these isolates, 35% were toxigenic. All isolates were typable by PCR ribotyping and were resolved into 12 PCR ribotypes. Among these isolates, ribotype 014, which is distributed worldwide including in Japanese clinical cases, was detected among vegetable isolates. Therefore, although the C. difficile contamination rate in Japanese retail foods was low, these sources can be contaminated and could transmit these bacteria to humans.
Assuntos
Clostridioides difficile/classificação , Clostridioides difficile/isolamento & purificação , Infecções por Clostridium/epidemiologia , Infecções por Clostridium/microbiologia , Contaminação de Alimentos , Carne/microbiologia , Verduras/microbiologia , Animais , Antibacterianos/farmacologia , Clostridioides difficile/efeitos dos fármacos , Infecções por Clostridium/transmissão , Humanos , Japão/epidemiologia , Testes de Sensibilidade Microbiana , Filogenia , Reação em Cadeia da Polimerase , Prevalência , Vigilância em Saúde Pública , RibotipagemRESUMO
ß-Lactam-resistant Haemophilus influenzae is a clinical concern. A high prevalence (>40%) of ß-lactamase-negative high-level ampicillin-resistant H. influenzae (high-BLNAR) isolates in Japan has been reported. However, the reasons for the expansion are unknown. High-BLNAR strains possess an amino acid substitution, either Asn526Lys (group III) or Arg517His (group III-like) in addition to Ser385Thr, in penicillin-binding protein 3 (PBP3). To determine the current prevalence of high-BLNAR strains and the mechanisms behind their expansion in Japan, their prevalence, PBP3 types, multilocus sequence types, and susceptibilities to quinolones approved in Japan as alternatives were determined. Sixty percent of H. influenzae clinical isolates (62/104 isolates) were ß-lactamase-negative ampicillin-resistant H. influenzae (BLNAR) strains. Among BLNAR isolates, 92% (57/62 isolates) were high-BLNAR strains. Most isolates were classified as belonging to group III, which contained many genotypes (11 PBP3 types and 25 sequence types). These results indicated that the expansion of high-BLNAR isolates was multiclonal and such strains are still predominant in Japanese clinical settings. One high-BLNAR isolate harbored the novel amino acid substitution Asn526Met in addition to Ser385Thr in PBP3, suggesting a new group (group IV). No quinolone-resistant H. influenzae isolates were identified. The MICs for the quinolones (moxifloxacin, garenoxacin, and tosufloxacin) were similar to that for levofloxacin, whereas sitafloxacin exhibited a lower MIC. However, we obtained 4 H. influenzae isolates with decreased quinolone susceptibility with the amino acid substitution Ser84Leu in GyrA, and 3 of those isolates were high-BLNAR isolates. In summary, this study shows that multiclonal high-BLNAR strains predominate in a Japanese university hospital. Isolates remain sensitive to quinolones, but vigilance is required to prevent the development of fluoroquinolone resistance in high-BLNAR strains.
Assuntos
Resistência a Ampicilina/genética , Ampicilina/farmacologia , Antibacterianos/farmacologia , Haemophilus influenzae/efeitos dos fármacos , Haemophilus influenzae/genética , Quinolonas/farmacologia , beta-Lactamases/genética , Substituição de Aminoácidos/genética , Haemophilus influenzae/isolamento & purificação , Humanos , Japão , Testes de Sensibilidade MicrobianaRESUMO
Colistin is a last-line drug for multidrug-resistant Gram-negative bacteria. We previously reported four plasmid-mediated colistin resistance (mcr) gene-negative colistin-resistant Escherichia coli clinical isolates, including the major pathogenic and fluoroquinolone-resistant strains O25b:H4-ST131-H30Rx (isolates SRE34 and SRE44; MIC for colistin = 16 mg/liter), non-x (SME296; MIC = 8 mg/liter), and O18-ST416 (SME222; MIC = 4 mg/liter). In this study, we investigated the colistin resistance mechanism and identified novel amino acid substitutions or deletions in the PmrAB two-component system that activates eptA (encoding a phosphoethanolamine transferase) and arnT (encoding an undecaprenyl phosphate-alpha-4-amino-4-deoxy-l-arabinose arabinosyl transferase) in all colistin-resistant isolates. SRE34 possessed deletion Δ27-45 (LISVFWLWHESTEQIQLFE) in PmrB, SRE44 possessed substitution L105P in PmrA, and both SME222 and SME296 included substitution G206D in PmrB. Matrix-assisted laser desorption ionization-time of flight mass spectrometry revealed that lipid A is modified with phosphoethanolamine in all four isolates. Deletion of pmrAB decreased colistin MICs to 0.5 mg/liter and lowered eptA and arnT expression. Chromosomal replacement of mutated pmrA or pmrB in colistin-susceptible O25b:H4-ST131 strain SME98 (colistin MIC = 0.5 mg/liter) increased the colistin MIC to that of the respective parent colistin-resistant isolate. In addition, SME98 mutants in which pmrAB was replaced with mutated pmrAB showed no significant differences in bacterial growth and competition culture from the parent strain, except for the mutant with L105P in PmrA, whose growth was significantly suppressed in the presence of the parent strain. In conclusion, some O25b:H4-ST131 strains appear to acquire colistin resistance via phosphoethanolamine modification of lipid A through amino acid changes in PmrAB, and the amino acid changes in PmrB do not influence bacterial growth.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Colistina/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Fatores de Transcrição/genética , Substituição de Aminoácidos/genética , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/genética , Hexosiltransferases/biossíntese , Humanos , Lipídeo A/metabolismo , Testes de Sensibilidade Microbiana , Deleção de Sequência/genéticaRESUMO
The plaque-forming assay is the standard technique for determining viral titer, and a critical measurement for investigating viral replication. However, this assay is highly dependent on experimental technique and conditions. In the case of human respiratory syncytial virus (RSV) in particular, it can be difficult to objectively confirm the accuracy of plaque-forming assay because the plaques made by RSV are often small and unclear. In recent studies, RT-qPCR methods have emerged as a supportive procedure for assessment of viral titer, yielding highly sensitive and reproducible results. In this report, we compare the viral replication, as determined by plaque-forming assay, and the copy numbers of RSV genes NS1, NS2, N, and F, as determined by RT-qPCR. Two real-time PCR systems, SYBR Green and TaqMan probe, gave highly similar results for measurement of copy numbers of RSV N genes of virus subgroups A. We determined the RSV gene copy numbers in the culture cell supernatant and cell lysate measured at various multiplicities of infection. We found that copy number of the RSV N gene in the culture supernatant and cell lysate was highly correlated with plaque-forming units. In conclusion, RT-qPCR measurement of RSV gene copy number was highly dependent on viral titer, and the detailed comparison between each gene copy number and virus titer should be useful and supportive in confirming RSV plaque-forming assay and virus dynamics. The technique may also be used to estimate the amount of RSV present in clinical specimens.
Assuntos
Dosagem de Genes , Reação em Cadeia da Polimerase em Tempo Real/métodos , Vírus Sincicial Respiratório Humano/genética , Carga Viral/métodos , Humanos , RNA Viral/genética , Sensibilidade e Especificidade , Proteínas não Estruturais Virais/genética , Ensaio de Placa Viral/métodos , Proteínas Virais/genética , Virologia/métodos , Replicação ViralRESUMO
Multidrug-resistant Streptococcus pneumoniae strains were isolated from blood and sputum of a patient with disseminated intravascular coagulation in Sapporo city, Japan. These antibiograms were only susceptible to vancomycin, linezolid, daptomycin, some carbapenems, and some fluoroquinolones. Identical antibiograms, serotypes (19F), and sequence types (ST10017) suggested a shared origin of these isolates. Only one ST10017 strain has been isolated in the same city in Japan previously (2014), and the 2014 isolate is still susceptible to macrolides. The whole genome of the blood-derived isolate was sequenced. The strain harbored resistance mutations in parC, gyrA, pbp1a, pbp2a, pbp2b, and pbp2x, and harbored the resistance genes, ermB and tetM. The nucleotide sequences of parC and pbp2x genes of strain MDRSPN001 were clearly different from those of other S. pneumoniae strains and were similar to those of oral streptococci strains. These findings suggest that strain MDRSPN001 has been rapidly and drastically evolving multidrug resistance by gene replacement and accumulation of genes originating from other strains, such as oral streptococci, Streptococcus mitis.
Assuntos
Antibacterianos/farmacologia , Coagulação Intravascular Disseminada/tratamento farmacológico , Farmacorresistência Bacteriana Múltipla/genética , Infecções Pneumocócicas/tratamento farmacológico , Streptococcus pneumoniae/genética , Antibacterianos/uso terapêutico , Coagulação Intravascular Disseminada/diagnóstico por imagem , Coagulação Intravascular Disseminada/microbiologia , Feminino , Genoma Bacteriano/genética , Humanos , Japão , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Infecções Pneumocócicas/diagnóstico por imagem , Infecções Pneumocócicas/microbiologia , Sorogrupo , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/isolamento & purificação , Tomografia Computadorizada por Raios X , Sequenciamento Completo do GenomaRESUMO
Tigecycline (TGC) is a last-line drug for multidrug-resistant Enterobacteriaceae We investigated the mechanism(s) underlying TGC nonsusceptibility (TGC resistant/intermediate) in Escherichia coli clinical isolates. The MIC of TGC was determined for 277 fluoroquinolone-susceptible isolates (ciprofloxacin [CIP] MIC, <0.125 mg/liter) and 194 fluoroquinolone-resistant isolates (CIP MIC, >2 mg/liter). The MIC50 and MIC90 for TGC in fluoroquinolone-resistant isolates were 2-fold higher than those in fluoroquinolone-susceptible isolates (MIC50, 0.5 mg/liter versus 0.25 mg/liter; MIC90, 1 mg/liter versus 0.5 mg/liter, respectively). Two fluoroquinolone-resistant isolates (O25b:H4-ST131-H30R and O125:H37-ST48) were TGC resistant (MICs of 4 and 16 mg/liter, respectively), and four other isolates of O25b:H4-ST131-H30R and an isolate of O1-ST648 showed an intermediate interpretation (MIC, 2 mg/liter). No TGC-resistant/intermediate strains were found among the fluoroquinolone-susceptible isolates. The TGC-resistant/intermediate isolates expressed higher levels of acrA and acrB and had lower intracellular TGC concentrations than susceptible isolates, and they possessed mutations in acrR and/or marR The MICs of acrAB-deficient mutants were markedly lower (0.25 mg/liter) than those of the parental strain. After continuous stepwise exposure to CIP in vitro, six of eight TGC-susceptible isolates had reduced TGC susceptibility. Two of them acquired TGC resistance (TGC MIC, 4 mg/liter) and exhibited expression of acrA and acrB and mutations in acrR and/or marR In conclusion, a population of fluoroquinolone-resistant E. coli isolates, including major extraintestinal pathogenic lineages O25b:H4-ST131-H30R and O1-ST648, showed reduced susceptibility to TGC due to overexpression of the efflux pump AcrAB-TolC, leading to decreased intracellular concentrations of the antibiotics that may be associated with the development of fluoroquinolone resistance.
Assuntos
Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Fluoroquinolonas/farmacologia , Minociclina/análogos & derivados , Adulto , Idoso , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Feminino , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Japão , Lipoproteínas/genética , Masculino , Proteínas de Membrana Transportadoras/genética , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Minociclina/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Mutação , TigeciclinaRESUMO
Macrolide antibiotics have immunomodulatory activities, including suppression of cytokine production, cell adhesion molecule expression, and mucin production. These immunomodulatory activities improve the symptoms of respiratory diseases associated with chronic inflammation. However, the underlying molecular mechanism(s) is not well understood yet. To address this, we prepared clarithromycin (CAM)-conjugated Sepharose and examined bound cellular proteins by proteome analysis. We identified mitochondrial proteins 4-nitrophenylphosphatase domain and non-neuronal synaptosomal associated protein 25-like protein homolog (NIP-SNAP)-1 and -2 and very long-chain acyl-CoA dehydrogenase (VLCAD) as CAM-binding proteins. Production of proinflammatory cytokines (IL-8 and IL-6) induced by lipopolysaccharides (LPSs) and Pam3-CSK4 in human epithelial cell lines BEAS-2B and T24 were suppressed by knockdown of NIP-SNAP-1 or -2, and partly by knockdown of VLCAD. Also, knockdown of NIP-SNAP-1 or -2 in various cell lines suppressed LPS-induced expression of IL-8 and IL-6 mRNA and NF-κB activity. Thus, CAM suppresses NF-κB-mediated proinflammatory cytokine production by interacting with mitochondrial proteins, NIP-SNAP-1 and -2.
Assuntos
Claritromicina/farmacologia , Citocinas/biossíntese , Fatores Imunológicos/farmacologia , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Acil-CoA Desidrogenase de Cadeia Longa/antagonistas & inibidores , Acil-CoA Desidrogenase de Cadeia Longa/genética , Acil-CoA Desidrogenase de Cadeia Longa/metabolismo , Citocinas/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Lipopolissacarídeos/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , NF-kappa B/metabolismo , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Ligação Proteica , Proteínas/antagonistas & inibidores , Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Receptores Toll-Like/agonistasRESUMO
NIP-SNAP-1 and -2 are ubiquitous proteins thought to be associated with maintenance of mitochondrial function, neuronal transmission, and autophagy. However, their physiological functions remain largely unknown. To elucidate their functional importance, we screened for proteins that interact with NIP-SNAP-1 and -2, resulting in identification of HSP60 and P62/SQSTM1 as binding proteins. NIP-SNAP-1 and -2 localized in the mitochondrial inner membrane space, whereas HSP60 localized in the matrix. Native gel electrophoresis and filter trap assays revealed that human HSP60 prevented aggregation of newly synthesized NIP-SNAP-2 in an in vitro translation system. Moreover, expression levels of NIP-SNAP-1 and -2 in cells were decreased by knockdown of HSP60, but not HSP10. These findings indicate that HSP60 promotes folding and maintains the stability of NIP-SNAP-1 and -2.
Assuntos
Chaperonina 60/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Linhagem Celular , Chaperonina 10/antagonistas & inibidores , Chaperonina 10/genética , Chaperonina 10/metabolismo , Chaperonina 60/antagonistas & inibidores , Chaperonina 60/genética , Técnicas de Silenciamento de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/química , Proteínas de Membrana/genética , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Ligação Proteica , Dobramento de Proteína , Mapas de Interação de Proteínas , Estabilidade Proteica , Proteínas/química , Proteínas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína Sequestossoma-1/metabolismoRESUMO
Erythromycin, tylosin and tilmicosin are approved for use in cattle in Japan, the latter two being used to treat Mycoplasma bovis infection. In this study, 58 M. bovis isolates obtained from Japanese dairy calves all exhibited reduced susceptibility to these macrolides, this widespread reduced susceptibility being attributable to a few dominant lineages. All 58 isolates contained the G748A variant in both the rrl3 and rrl4 alleles of 23S rRNA, whereas a reference strain (PG45) did not. G748 localizes in the central loop of domain II (from C744 to A753) of 23S rRNA, which participates in binding to mycinose, a sugar residue present in both tylosin and tilmicosin. A number of in vitro-selected mutants derived from M. bovis PG45 showed reduced susceptibility to tylosin and tilmicosin and contained a nucleotide insertion within the central loop of domain II of rrl3 (U747-G748Ins_CU/GU or A743-U744Ins_UA), suggesting that mutations around G748 confer this reduced susceptibility phenotype. However, other Mycoplasma species containing G748A were susceptible to tylosin and tilmicosin. Sequence comparison with Escherichia coli revealed that M. bovis PG45 and isolates harbored five nucleotide alterations (U744C, G745A, U746C, A752C and A753G) in the central loop of domain II of 23S rRNA, whereas other Mycoplasma species lacked at least two of these five nucleotide alterations. It was therefore concluded that G748 mutations in combination with species-specific nucleotide alterations in the central loop of domain II of 23S rRNA are likely sufficient to reduce susceptibility of M. bovis to tylosin and tilmicosin.