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615800.9806Nitric oxide stress resistance in Porphyromonas gingivalis is mediated by a putative hydroxylamine reductase. Porphyromonas gingivalis, the causative agent of adult periodontitis, must maintain nitric oxide (NO) homeostasis and surmount nitric oxide stress from host immune responses or other oral bacteria to survive in the periodontal pocket. To determine the involvement of a putative hydroxylamine reductase (PG0893) and a putative nitrite reductase-related protein (PG2213) in P. gingivalis W83 NO stress resistance, genes encoding those proteins were inactivated by allelic exchange mutagenesis. The isogenic mutants P. gingivalis FLL455 (PG0893ermF) and FLL456 (PG2213ermF) were black pigmented and showed growth rates and gingipain and hemolytic activities similar to those of the wild-type strain. P. gingivalis FLL455 was more sensitive to NO than the wild type. Complementation of P. gingivalis FLL455 with the wild-type gene restored the level of NO sensitivity to a level similar to that of the parent strain. P. gingivalis FLL455 and FLL456 showed sensitivity to oxidative stress similar to that of the wild-type strain. DNA microarray analysis showed that PG0893 and PG2213 were upregulated 1.4- and 2-fold, respectively, in cells exposed to NO. In addition, 178 genes were upregulated and 201 genes downregulated more than 2-fold. The majority of these modulated genes were hypothetical or of unknown function. PG1181, predicted to encode a transcriptional regulator, was upregulated 76-fold. Transcriptome in silico analysis of the microarray data showed major metabolomic variations in key pathways. Collectively, these findings indicate that PG0893 and several other genes may play an important role in P. gingivalis NO stress resistance.201222247513
999710.9798RNAi screen of DAF-16/FOXO target genes in C. elegans links pathogenesis and dauer formation. The DAF-16/FOXO transcription factor is the major downstream output of the insulin/IGF1R signaling pathway controlling C. elegans dauer larva development and aging. To identify novel downstream genes affecting dauer formation, we used RNAi to screen candidate genes previously identified to be regulated by DAF-16. We used a sensitized genetic background [eri-1(mg366); sdf-9(m708)], which enhances both RNAi efficiency and constitutive dauer formation (Daf-c). Among 513 RNAi clones screened, 21 displayed a synthetic Daf-c (SynDaf) phenotype with sdf-9. One of these genes, srh-100, was previously identified to be SynDaf, but twenty have not previously been associated with dauer formation. Two of the latter genes, lys-1 and cpr-1, are known to participate in innate immunity and six more are predicted to do so, suggesting that the immune response may contribute to the dauer decision. Indeed, we show that two of these genes, lys-1 and clc-1, are required for normal resistance to Staphylococcus aureus. clc-1 is predicted to function in epithelial cohesion. Dauer formation exhibited by daf-8(m85), sdf-9(m708), and the wild-type N2 (at 27°C) were all enhanced by exposure to pathogenic bacteria, while not enhanced in a daf-22(m130) background. We conclude that knockdown of the genes required for proper pathogen resistance increases pathogenic infection, leading to increased dauer formation in our screen. We propose that dauer larva formation is a behavioral response to pathogens mediated by increased dauer pheromone production.201021209831
616620.9792Intraperitoneal infection with Salmonella abortusovis is partially controlled by a gene closely linked with the Ity gene. The aim of the present study was to determine whether the Ity gene, which controls the resistance to S. typhimurium infection in mice, also governs the resistance to S. abortusovis, a serotype specific for goat and sheep. During either i.v. or i.p. infection, BALB/c mice (Itys) were not able to control the growth of S. abortusovis and eventually died from infection. In contrast CBA (Ityr) or (C.CB)F1 (Ityr/s) mice were able to control the growth of these bacteria. Using congenic C.D2 Ityr mice, we found that the gene controlling resistance to S. abortusovis was tightly linked to the Ity gene on chromosome 1. Furthermore, in the spleen and the liver of backcross BALB/c x (CBA x BALB/c) mice, the S. abortusovis resistance phenotype cosegregated with the two alleles of the Len-1 gene, a gene tightly linked to the Ity gene. By contrast, in these backcross mice, the level of infection of the peritoneal cavity, the site of inoculation, did not correlated with the Len-1 phenotype of the animal. These results provide evidence that after i.p. inoculation the control of S. abortusovis growth in the spleen and the liver is controlled by the Ity gene, but also suggest that additional gene(s) regulate the number of bacteria at the site of inoculation.19921544222
241930.9789Presence and antibiotic resistance of Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens in children. BACKGROUND/AIMS: Only limited information exists about the prevalence in children of pathogens associated with periodontitis. The aim of the present study was to determine by culture whether 8-11-year-old children carry Porphyromonas gingivalis, Prevotella intermedia, and/or P. nigrescens in samples from the gingiva and/or the buccal mucosa taken before, and after caries treatment and oral hygiene instruction. A second aim was to assess the proportion of subjects who had gram-negative anaerobes carrying the tet(Q) and erm(F) genes, suggesting antibiotic resistance to tetracycline or erythromycin. METHOD: A total of 150 children provided gingival and buccal swab bacterial samples that were cultured for P. gingivalis, P. intermedia, and P. nigrescens. The species was verified using DNA-DNA hybridization with species-specific probes made from the variable region of the 16S rRNA sequences. Antibiotic-resistant genes, tet(Q) and erm(F), were identified using specific DNA-DNA hybridization with specific DNA probes. RESULTS: A total of 116 isolates of black-pigmented bacteria were cultured from 47 (31%) of 150 children. Five isolates were identified as P. gingivalis, 29 as P. intermedia, 33 as P. nigrescens, and 49 as other species. In general, the bacteria were not culturable at more than one time period. We found that 55% of these 47 children harbored black pigmented bacteria that carried either one or both of the two antibiotic-resistant genes studied (tet(Q), and erm(F)). CONCLUSION: The present study demonstrated that children not exposed to regular dental treatment carry bacteria outside the gingival sulcus that have been associated with periodontitis, and that standard treatment procedures may not clear the presence of the putative pathogens. In addition, antibiotic-resistant genes are common in identifiable gram-negative anaerobes, including putative pathogens.200212445225
247740.9789Evaluation of targeted next-generation sequencing for microbiological diagnosis of acute lower respiratory infection. PURPOSE: To evaluate the performance of targeted next-generation sequencing (tNGS) in pathogen detection in acute lower respiratory infection. METHODS: The retrospective study was conducted between July 2023 and May 2024 at the Yantai Yuhuangding Hospital. Patients with acute lower respiratory infections were included. Qualified sputum or bronchoalveolar lavage fluid samples were collected for tNGS and conventional microbiological tests(CMTs), including culture, staining, polymerase chain reaction (PCR), and reverse transcription-PCR (RT-PCR). The time required and cost were counted. RESULTS: A total of 968 patients were enrolled. Study analysis discovered 1,019 strains of bacteria, 259 strains of fungi, 302 strains of viruses, 76 strains of Mycoplasma pneumoniae, and two strains of Chlamydia psittaci using tNGS. In addition, tNGS also identified 39 mecA, four KPC, 19 NDM, and two OXA-48 genes. The positive rates for bacteria, fungi, viruses, mycoplasma, and chlamydia obtained using tNGS were significantly higher than those determined using traditional methods. Among them, tNGS showed high consistence with mycobacterium DNA test, influenza A (H1N1) virus nucleic acid test and COVID-19 nucleic acid test. Poor consistency between drug resistance genes and bacterial resistance phenotypes was found. In addition, tNGS also had advantages over traditional methods in terms of detection time and cost. CONCLUSION: Compared to traditional methods, tNGS had higher sensitivity in detecting bacteria, fungi, viruses, and other pathogens in acute lower respiratory infection, and also had the advantages of timeliness and cost-effectiveness, making it a promising method for guiding clinical diagnosis.202540901079
509550.9787Quantitative real-time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQ gene and total bacteria. Accurate quantification of bacterial species in dental plaque is needed for microbiological diagnosis of periodontal diseases. The present study was designed to assess the sensitivity, specificity and quantitativity of the real-time PCR using the GeneAmp Sequence Detection System with two fluorescence chemistries. TaqMan probe with reporter and quencher dye, and SYBR Green dye were used for sources of the fluorescence. Primers and probes were designed for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia and total bacteria based on the nucleotide sequences of the respective 16S ribosomal RNA genes. Since spread of antibiotic resistance genes is one of the crucial problems in periodontal therapy, quantitative detection of tetQ gene, which confers resistance to tetracycline, was included in the examination. The detection of P. gingivalis, P. intermedia and A. actinomycetemcomitans was linear over a range of 10-10(7) cells (10-10(7) copies for tetQ gene), while the quantitative range for total bacteria was 10(2)-10(7) cells. Species-specific amplifications were observed for the three periodontal bacteria, and there was no significant difference between the TaqMan and SYBR Green chemistry in their specificity, quantitativity and sensitivity. The SYBR Green assay, which was simpler than TaqMan assay in its manipulations, was applied to the clinical plaque samples. The plaque samples were obtained from eight patients (eight periodontal pockets) before and 1 week after the local drug delivery of minocycline. Although the number of P. gingivalis, P. intermedia and A. actinomycetemcomitans markedly decreased after the antibiotic therapy in most cases, higher copy numbers of the tetQ gene were detectable. The real-time PCR demonstrated sufficient sensitivity, specificity and quantitativity to be a powerful tool for microbiological examination in periodontal disease, and the quantitative monitoring of antibiotic resistance gene accompanied with the antibiotic therapy should be included in the examination.200314557000
579560.9785Direct identification of Gram-positive bacteria and resistance determinants from blood cultures using a microarray-based nucleic acid assay: in-depth analysis of microarray data for undetermined results. BACKGROUND: The Verigene Gram-Positive Blood Culture (BC-GP) nucleic acid assay (Nanosphere, Inc., Northbrook, IL, USA) is a newly developed microarray-based test with which 12 Gram-positive bacterial genes and three resistance determinants can be detected using blood culture broths. We evaluated the performance of this assay and investigated the signal characteristics of the microarray images. METHODS: At the evaluation stage, we tested 80 blood cultures that were positive for various bacteria (68 bacteria covered and 12 not covered by the BC-GP panel) collected from the blood of 36 patients and 44 spiked samples. In instances where the automated system failed and errors were called, we manually inspected microarray images, measured the signal intensities of target spots, and reclassified the results. RESULTS: With the manual analysis of the microarray images of 14 samples for which error calls were reported, we could obtain correct identification results for 12 samples without the need for retesting, because strong signals in the target spots were clearly discriminable from background noise. With our interpretation strategy, we could obtain 97.1% sensitivity and 100% specificity for bacterial identification by using the BC-GP assay. The two unidentified bacteria were viridans group streptococci, which produced weaker target signals. During the application stage, among 25 consecutive samples positive for Gram-positive bacteria, we identified two specimens with error calls as Streptococcus spp. by using manual analysis. CONCLUSIONS: With help of the manual review of the microarray images, the BC-GP assay could successfully identify species and resistance markers for many clinically important Gram-positive bacteria.201525536666
579870.9784Rapid identification of bacteria, mecA and van genes from blood cultures. The Genotype technology, a quick molecular genetic assay based on DNA multiplex amplification with biotinylated primers followed by hybridization to membrane bound probes, complies with the requirements for a fast diagnosis of sepsis. We evaluated the new Genotype BC Gram-negative and Gram-positive test kits (Hain Life Science, Germany) which respectively allow for the identification of 15 species of Gram-negative (GN) rods, and the identification of 17 Gram-positive (GP) bacteria species together with the determination of methicillin and vancomycin resistance (mecA and van genes). The study was performed on 60 positive blood cultures from BacT/ALERT bottles (aerobic, anaerobic and pediatric bottles). First, a Gram stain was carried out to select between Genotype BC GP or GN test, then identification were performed by the Genotype BC tests and by biochemical conventional tests after subculture and phenotypic susceptibility determination. The operating procedure was very easy to carry out and required a small amount of starting material (5 to 10 microL of blood culture). The results were available within 4.5 hours. For all the blood cultures, the Genotype BC results correlated with the biochemical identification and phenotypic antibiotics susceptibility. According to our results, this DNA strip technology based assay can easily be incorporated into routine diagnosis.200717913394
243780.9783Periodontal pathogens and tetracycline resistance genes in subgingival biofilm of periodontally healthy and diseased Dominican adults. OBJECTIVE: The objective of this study was to compare the periodontopathogen prevalence and tetracycline resistance genes in Dominican patients with different periodontal conditions. METHODS: Seventy-seven samples were collected from healthy, gingivitis, chronic (CP) and aggressive (AgP) periodontitis patients. Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Prevotella intermedia, Parvimonas micra, Eikenella corrodens and Dialister pneumosintes and 11 resistance genes were studied by PCR. P. gingivalis fimA genotype was determined. RESULTS: In healthy patients, P. micra and P. intermedia were the most and least frequently detected, respectively. T. forsythia and E. corrodens appeared in 100% of gingivitis patients. Red complex, D. pneumosintes and E. corrodens were significantly more prevalent in CP compared to healthy patients. F. nucleatum and T. denticola were detected more frequently in AgP. A. actinomycetemcomitans was the most rarely observed in all groups. The fimA II genotype was the most prevalent in periodontitis patients. Seven tetracycline-resistant genes were detected. tet(Q), tet(32) and tet(W) showed the greatest prevalence. tet(32) was significantly more prevalent in CP than in healthy patients. CONCLUSIONS: Red complex bacteria and D. pneumosintes were significantly the most prevalent species among periodontitis patients. T. forsythia was the most frequently detected in this population. To our knowledge, this is the first study describing the tet(32) gene in subgingival biofilm from healthy and periodontally diseased subjects. CLINICAL RELEVANCE: This study contributes to the knowledge on the subgingival microbiota and its resistance genes of a scarcely studied world region. Knowing the prevalence of resistance genes could impact on their clinical prescription and could raise awareness to the appropriate use of antibiotics.201626121972
873790.9783Role of Biosynthetic Gene Cluster BGC3 in the Cariogenic Virulence of Streptococcus mutans. OBJECTIVE: To investigate the role of the biosynthetic gene cluster BGC3 of Streptococcus mutans (S. mutans) in the process of dental caries. METHODS: BGC3 and ∆BGC3 S. mutans strains were constructed and their growth curves were evaluated. Acid production capacity was assessed by evaluating pH reduction levels over identical culture periods. The survival of bacteria in phosphate citrate buffer solution (pH 3.0) was quantified. The expression levels of virulence genes (atpF, gtfC, gtfD, spaP, vicR and ftf) were analysed using the qPCR. Co-culture experiments were conducted to evaluate bacterial adaptability. Bacterial viability was determined by microscopical examination of live/dead staining. RESULTS: Deletion of BGC3 did not significantly impact S. mutans growth or acid production in biofilms. The ∆BGC3 strain exhibited enhanced acid resistance and higher expression levels of virulence genes compared to the wild type. In addition, ∆BGC3 exhibited superior bacterial viability in the co-culture system. CONCLUSION: BGC3 affected the acid resistance and expression of caries-related genes in S. mutans. The BGC3 knockout strain exhibited a more robust survival capability than the wild-type strain.202540162656
2475100.9783Examination of single and multiple mutations involved in resistance to quinolones in Staphylococcus aureus by a combination of PCR and denaturing high-performance liquid chromatography (DHPLC). Detection of DNA sequence variation is fundamental to the identification of the genomic basis of phenotypic variability. Denaturing high-performance liquid chromatography (DHPLC) is a novel technique that has been used to detect mutations in human DNA. We report on the first study to use this technique as a tool to detect mutations in genes encoding antibiotic resistance in bacteria. Three methicillin-sensitive and three methicillin-resistant clinical Staphylococcus aureus isolates, susceptible to ciprofloxacin (MIC Leu, Ser-112-->Pro, Glu-88-->Lys in GyrA, Glu-84-->Val, Ser-80-->Phe in GrlA, Pro-456-->Ser in GyrB and Glu-422-->Asp, Pro-451-->Ser, Asp-432-->Gly in GrlB. Mutations could be rapidly and reproducibly identified from the PCR products using DHPLC, producing specific peak patterns that correlate with genotypes. This system facilitates the detection of resistance alleles, providing a rapid (5 min per sample), economic (96 sample per run) and reliable technique for characterizing antibiotic resistance in bacteria.200212407120
625110.9782Identification of hopanoid biosynthesis genes involved in polymyxin resistance in Burkholderia multivorans. A major challenge to clinical therapy of Burkholderia cepacia complex (Bcc) pulmonary infections is their innate resistance to a broad range of antimicrobials, including polycationic agents such as aminoglycosides, polymyxins, and cationic peptides. To identify genetic loci associated with this phenotype, a transposon mutant library was constructed in B. multivorans ATCC 17616 and screened for increased susceptibility to polymyxin B. Compared to the parent strain, mutant 26D7 exhibited 8- and 16-fold increases in susceptibility to polymyxin B and colistin, respectively. Genetic analysis of mutant 26D7 indicated that the transposon inserted into open reading frame (ORF) Bmul_2133, part of a putative hopanoid biosynthesis gene cluster. A strain with a mutation in another ORF in this cluster, Bmul_2134, was constructed and named RMI19. Mutant RMI19 also had increased polymyxin susceptibility. Hopanoids are analogues of eukaryotic sterols involved in membrane stability and barrier function. Strains with mutations in Bmul_2133 and Bmul_2134 showed increased permeability to 1-N-phenylnaphthylamine in the presence of increasing concentrations of polymyxin, suggesting that the putative hopanoid biosynthesis genes are involved in stabilizing outer membrane permeability, contributing to polymyxin resistance. Results from a dansyl-polymyxin binding assay demonstrated that polymyxin B does not bind well to the parent or mutant strains, suggesting that Bmul_2133 and Bmul_2134 contribute to polymyxin B resistance by a mechanism that is independent of lipopolysaccharide (LPS) binding. Through this work, we propose a role for hopanoid biosynthesis as part of the multiple antimicrobial resistance phenotype in Bcc bacteria.201222006009
8732120.9781RNA-Seq identification of candidate defense genes targeted by endophytic Bacillus cereus-mediated induced systemic resistance against Meloidogyne incognita in tomato. BACKGROUND: The endophytic bacteria Bacillus cereus BCM2 has shown great potential as a defense against the parasitic nematode Meloidogyne incognita. Here, we studied endophytic bacteria-mediated plant defense against M. incognita and searched for defense-related candidate genes using RNA-Seq. RESULTS: The induced systemic resistance of BCM2 against M. incognita was tested using the split-root method. Pre-inoculated BCM2 on the inducer side was associated with a dramatic reduction in galls and egg masses on the responder side, but inoculated BCM2 alone did not produce the same effect. In order to investigate which plant defense-related genes are specifically activated by BCM2, four RNA samples from tomato roots were sequenced, and four high-quality total clean bases were obtained, ranging from 6.64 to 6.75 Gb, with an average of 21 558 total genes. The 34 candidate defense-related genes were identified by pair-wise comparison among libraries, representing the targets for BCM2 priming resistance against M. incognita. Functional characterization revealed that the plant-pathogen interaction pathway (ID: ko04626) was significantly enriched for BCM2-mediated M. incognita resistance. CONCLUSION: This study demonstrates that B. cereus BCM2 maintains a harmonious host-microbe relationship with tomato, but appeared to prime the plant, resulting in more vigorous defense response toward the infection nematode. © 2018 Society of Chemical Industry.201829737595
5228130.9780Mycobacterium bolletii respiratory infections. Contrary to other species in the Mycobacterium chelonae-abscessus complex, we reidentified M. bolletii strains isolated from 4 respiratory patients and found these strains to be uniformly resistant to clarithromycin. No mutations previously associated with macrolide resistance in bacteria were detected in either the 23S rDNA or the genes encoding riboproteins L4 and L22.200919193279
1476140.9780Evaluation of the BioFire FilmArray Pneumonia Panel for rapid detection of respiratory bacterial pathogens and antibiotic resistance genes in sputum and endotracheal aspirate specimens. OBJECTIVES: The performance of the investigational-use-only version of the BioFire FilmArray Pneumonia Panel (FA-Pneumo), a high-order nested multiplex PCR, was evaluated for the detection of typical respiratory bacterial pathogens and antibiotic resistance genes in sputa and endotracheal aspirate (ETA) specimens. METHODS: Thirty-one sputa and 69 ETA specimens were analyzed. The diagnostic performance of FA-Pneumo was assessed using routine microbiological methods as the reference standard. RESULTS: Overall sensitivity and specificity for organism detection using FA-Pneumo were 98.5% and 76.5%, respectively. The sensitivity for each pathogen was 100%, except for Klebsiella aerogenes, and the range of specificity was 83.3-99.0%. FA-Pneumo detected antimicrobial resistance genes in 17 out of 18 specimens (94.4%) that were resistant by antimicrobial susceptibility testing. FA-Pneumo additionally detected 25 resistance genes in 22 specimens, and sequencing for the presence of resistance genes confirmed the majority of these results (20/25, 80%). Semi-quantitative analysis of bacterial nucleic acid amounts by FA-Pneumo revealed that 88.2% of the identified bacteria (67/76) with ≥10(6) copies/ml also gave culture-positive results with significant amounts of bacteria. CONCLUSIONS: FA-Pneumo is a rapid test with high sensitivity for the detection of bacteria and antimicrobial resistance genes in sputum and ETA specimens and could aid in determining antibiotic therapy.202032179139
1484150.9780Use of a commercial PCR-based line blot method for identification of bacterial pathogens and the mecA and van genes from BacTAlert blood culture bottles. In this study, the PCR-based DNA strip assay GenoType BC for the identification of bacteria and the resistance genes mecA, vanA, vanB, vanC1, and vanC2/3 directly from positive BacTAlert blood culture bottles was evaluated in a multicenter study. Of a total of 511 positive blood cultures, correct identification percentages for Gram-negative bacteria, Gram-positive bacteria, and the mecA gene were 96.1%, 89.9%, and 92.9%, respectively. Results were available 4 h after growth detection.201222075585
5094160.9780A duplex one-step recombinase aided PCR assay for the rapid and sensitive detection of the isoniazid resistance genes katG and inhA in Mycobacterium tuberculosis. OBJECTIVES: Drug resistance in tuberculosis seriously affects the eradication of tuberculosis, and isoniazid resistance is the second most commonly observed drug resistance in patients with tuberculosis. Timely and accurate detection of isoniazid resistance is critical to the treatment of tuberculosis. METHODS: A duplex one-step recombinase-aided PCR (DO-RAP) assay was developed for the rapid and sensitive detection of the katG Ser315Thr and inhA-15 (C-T) mutations in Mycobacterium tuberculosis, which are the most common isoniazid-resistant mutations. Quantitative recombinant plasmids were used to evaluate the sensitivity of DO-RAP, and 91 Mycobacterium tuberculosis strains with different genotypes, as well as 5 common respiratory tract bacteria, were used to evaluate the specificity of DO-RAP. A total of 78 sputum specimens were simultaneously detected using DO-RAP, quantitative PCR (qPCR) and sanger sequencing of nested PCR products. Sanger sequencing results were used as the standard to verify the clinical performance of DO-RAP. RESULTS: The reaction time of DO-RAP was less than 1 h. The sensitivity of DO-RAP was 2 copies/reaction, which was 10 times higher than qPCR. The sensitivity of DO-RAP for detecting heterogenous resistance was 5%. There was no cross-reactivity between the isoniazid wild-type gene, drug-resistant mutant genes, and other common respiratory tract bacteria. Compared with Sanger sequencing, the sensitivity, specificity, PPV and NPV of DO-RAP were all 100%. There were 7 specimens with gray zone or negative qPCR results but positive DO-RAP test results. CONCLUSION: The DO-RAP can be adopted in ordinary qPCR equipment for the rapid, highly sensitive and specific detection of the isoniazid resistance genes of Mycobacterium tuberculosis.202540182291
5229170.9779Paradoxical High-Level Spiramycin Resistance and Erythromycin Susceptibility due to 23S rRNA Mutation in Streptococcus constellatus. Objectives: The aim of the study was to characterize phenotypically and genotypically an uncommon mechanism of resistance to macrolides, lincosamides, and streptogramins (MLS) in a Streptococcus milleri group clinical isolate. Materials and Methods: The isolate UCN96 was recovered from an osteoradionecrosis wound, and was identified using the matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and the partial sequencing of the sodA gene. Antimicrobial susceptibility testing were carried out by the disk diffusion method and minimal inhibitory concentrations (MICs) were determined by the broth microdilution technique. PCR screening was performed for MLS resistance genes described in Gram-positive bacteria. Specific mutations in the ribosomal proteins L3-, L4-, and L22-encoding genes were also screened and those in domain V of the 23S rRNA gene (rrl). The number of mutated copies of the rrl gene was determined using amplification-refractory mutation system quantitative-polymerase chain reaction (qPCR) analysis. Results: The clinical isolate UCN96 was unambiguously identified as Streptococcus constellatus. It was susceptible to all macrolides and lincosamides (ML) antibiotics except spiramycin (MIC >256 mg/L) while it was also resistant to streptogramins. Screening for all acquired resistance genes was negative and no mutation was found in genes coding for L3, L4, and L22 ribosomal proteins. Of interest, a single mutation, A2062C (according to Escherichia coli numbering), was detected in the domain V of 23S rRNA. Conclusion: Mutations at the position 2062 of 23S rRNA have been detected once in Streptococcus pneumoniae, and not yet in other Streptococcus spp. This mechanism is very likely uncommon in Gram-positive bacteria because different copies of 23S rRNA operons should be mutated for development of such a resistance pattern.202032031922
5222180.9778Resistance to macrolides by ribosomal mutation in clinical isolates of Turicella otitidis. The genetic basis of erythromycin resistance in Turicella otitidis, a coryneform bacteria associated with otitis, was studied in five macrolide-resistant clinical isolates. Macrolide resistance genes were searched for by polymerase chain reaction (PCR). Genes for domain V of 23S rRNA (rrl) as well as rplD (L4 protein) and rplV (L22 protein) genes were characterised, amplified by PCR from total genomic DNA and sequenced. In the resistant isolates, cross-resistance to macrolides and clindamycin was associated with mutations at positions 2058 and/or 2059 (Escherichia coli numbering). Three isolates displayed A2058 mutations, one isolate had an A2059G mutation whereas another one contained mutations at positions 2058 and 2059. Southern blot experiments revealed that T. otitidis had three copies of the rrl gene. In conclusion, resistance to macrolides in T. otitidis is due, at least in part, to mutations in the rrl gene.200919414240
6162190.9778The resistance of BALB/cJ mice to Yersinia pestis maps to the major histocompatibility complex of chromosome 17. Yersinia pestis, the causative agent of plague, has been well studied at the molecular and genetic levels, but little is known about the role that host genes play in combating this highly lethal pathogen. We challenged several inbred strains of mice with Y. pestis and found that BALB/cJ mice are highly resistant compared to susceptible strains such as C57BL/6J. This resistance was observed only in BALB/cJ mice and not in other BALB/c substrains. Compared to C57BL/6J mice, the BALB/cJ strain exhibited reduced bacterial burden in the spleen and liver early after infection as well as lower levels of serum interleukin-6. These differences were evident 24 h postinfection and became more pronounced with time. Although a significant influx of neutrophils in the spleen and liver was exhibited in both strains, occlusive fibrinous thrombi resulting in necrosis of the surrounding tissue was observed only in C57BL/6J mice. In an effort to identify the gene(s) responsible for resistance, we measured total splenic bacteria in 95 F(2) mice 48 h postinfection and performed quantitative trait locus mapping using 58 microsatellite markers spaced throughout the genome. This analysis revealed a single nonrecessive plague resistance locus, designated prl1 (plague resistance locus 1), which coincides with the major histocompatibility complex of chromosome 17. A second screen of 95 backcrossed mice verified that this locus confers resistance to Y. pestis early in infection. Finally, eighth generation backcrossed mice harboring prl1 were found to maintain resistance in the susceptible C57BL/6J background. These results identify a novel genetic locus in BALB/cJ mice that confers resistance to Y. pestis.200818573896