Delayed Development of Linezolid Resistance in Staphylococcus aureus following Exposure to Low Levels of Antimicrobial Agents

ABSTRACT The development of resistance to linezolid (LZD) in gram-positive bacteria depends on the mutation of a single 23S rRNA gene, followed by homologous recombination and gene conversion of the other alleles. We sought to inhibit this process in Staphylococcus aureus using a range of antibacterial agents, including some that suppress recombination. A model for the rapid selection of LZD resistance was developed which allowed the selection of LZD-resistant mutants with G2576T mutations in all five copies of the 23S rRNA gene following only 5 days of subculture. The emergence of LZD-resistant isolates was delayed by exposing cultures to low concentrations of various classes of antibiotics. All antibiotic classes were effective in delaying the selection of LZD-resistant mutants and, with the exception of fusidic acid (FUS) and rifampin (RIF), prolonged the selection window from 5 to ∼15 days. Inhibitors of DNA processing were no more effective than any other class of antibiotics at suppressing resistance development. However, the unrelated antimicrobials FUS and RIF were particularly effective at preventing the emergence of LZD resistance, prolonging the selection window from 5 to 25 days. The enhanced suppressive effect of FUS and RIF on the development of LZD resistance was lost in a recA-deficient host, suggesting that these drugs affect recA-dependent recombination. Furthermore, FUS and RIF were shown to be effective inhibitors of homologous recombination of a plasmid into the staphylococcal chromosome. We suggest that RIF or FUS in combination with LZD may have a role in preventing the emergence of LZD resistance.

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Spontaneous Erythromycin Resistance Mutation in a 23S rRNA Gene, rrlA, of the Extreme Thermophile Thermus thermophilus IB-21

Journal of Bacteriology ◽

10.1128/jb.183.14.4382-4385.2001 ◽

2001 ◽

Vol 183 (14) ◽

pp. 4382-4385 ◽

Cited By ~ 9

Author(s):

Steven T. Gregory ◽

Jamie H. D. Cate ◽

Albert E. Dahlberg

Keyword(s):

Genetic Manipulation ◽

Thermus Thermophilus ◽

Resistance Mutation ◽

Rrna Genes ◽

23S Rrna ◽

Rrna Gene ◽

Erythromycin Resistance ◽

23S Rrna Gene ◽

Resistant Mutants ◽

23S Rrna Genes

ABSTRACT Spontaneous, erythromycin-resistant mutants of Thermus thermophilus IB-21 were isolated and found to carry the mutation A2058G in one of two 23S rRNA operons. The heterozygosity of these mutants indicates that A2058G confers a dominant or codominant phenotype in this organism. This mutation provides a valuable tool for the genetic manipulation of the 23S rRNA genes ofThermus.

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PCR-Based Rapid Identification System Using Bridged Nucleic Acids for Detection of Clarithromycin-Resistant Mycobacterium avium-M. intracellulare Complex Isolates

Journal of Clinical Microbiology ◽

10.1128/jcm.02954-15 ◽

2016 ◽

Vol 54 (3) ◽

pp. 699-704 ◽

Cited By ~ 3

Author(s):

Takashi Hirama ◽

Ayako Shiono ◽

Hiroshi Egashira ◽

Etsuko Kishi ◽

Koichi Hagiwara ◽

...

Keyword(s):

Mycobacterium Avium ◽

Detection System ◽

Point Mutations ◽

23S Rrna ◽

Clinical Samples ◽

Identification System ◽

Rrna Gene ◽

Clarithromycin Resistance ◽

23S Rrna Gene ◽

Resistant Mutants

The nontuberculous mycobacteria (NTM) cause miscellaneous disorders in humans, especially in the lungs, which present with a variety of radiological features. To date, knowledge of the pathogenic role of theMycobacterium avium-intracellularecomplex (MAC) in the human lung and the definitive criteria for initiating multidrug therapy are still lacking. However, there is little doubt that clarithromycin is the most efficacious drug among the various treatment regimens for lung NTM. In this study, with the use of a bridged nucleic acid (BNA) probe a detection system based on a real-time PCR (BNA-PCR) for the identification of the point mutations at position 2058 or 2059 in domain V of the 23S rRNA gene responsible for clarithromycin resistance was developed and has been assessed using MAC isolates from clinical samples. Out of 199 respiratory specimens, the drug susceptibility test demonstrated 12 strains resistant to clarithromycin, while the BNA-PCR showed 8 strains carrying the point mutation at position 2058 or 2059 of the 23S rRNA gene. This system revealed that there were mycobacterial strains resistant to clarithromycin which do not carry previously identified resistance genes. This paper documents a novel system for detecting clarithromycin-resistant strains and demonstrates that although these mutations are tacitly assumed to account for >90% of the reported resistant mutants, there is a significant fraction of resistant mutants that do not harbor these mutations. Therefore, unknown mechanisms affecting clarithromycin resistance remain to be elucidated.

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Relationship between the severity of acne vulgaris and antimicrobial resistance of bacteria isolated from acne lesions in a hospital in Japan

Journal of Medical Microbiology ◽

10.1099/jmm.0.067611-0 ◽

2014 ◽

Vol 63 (5) ◽

pp. 721-728 ◽

Cited By ~ 41

Author(s):

Keisuke Nakase ◽

Hidemasa Nakaminami ◽

Yuko Takenaka ◽

Nobukazu Hayashi ◽

Makoto Kawashima ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Antimicrobial Agents ◽

Acne Vulgaris ◽

Normal Skin ◽

Resistance Mechanisms ◽

University Hospital ◽

23S Rrna ◽

Rrna Gene ◽

Severe Acne ◽

23S Rrna Gene

Propionibacterium acnes and Staphylococcus epidermidis are normal skin inhabitants that are frequently isolated from lesions caused by acne, and these micro-organisms are considered to contribute to the inflammation of acne. In the present study, we examined the antimicrobial susceptibilities and resistance mechanisms of P. acnes and S. epidermidis isolated from patients with acne vulgaris in a university hospital in Japan from 2009 to 2010. Additionally, we analysed the relationship between the antimicrobial resistance of P. acnes and the severity of acne vulgaris. Some P. acnes strains (18.8 %; 13/69) were resistant to clindamycin. All strains had a mutation in the 23S rRNA gene, except for one strain that expressed erm(X) encoding a 23S rRNA methylase. Tetracycline-resistant P. acnes strains were found to represent 4.3 % (3/69) of the strains, and this resistance was caused by a mutation in the 16S rRNA gene. Furthermore, three strains with reduced susceptibility to nadifloxacin (MIC = 16 µg ml−1) were detected. When analysing the correlation between the antimicrobial resistance of P. acnes and S. epidermidis, more than 80 % of the patients who carried clindamycin-resistant P. acnes also carried clindamycin-resistant S. epidermidis. However, no epidemic strain that exhibited antimicrobial resistance was detected in the P. acnes strains when analysed by PFGE. Therefore, our results suggest that the antimicrobial resistance of P. acnes is closely related to antimicrobial therapy. Additionally, those P. acnes strains tended to be frequently found in severe acne patients rather than in mild acne patients. Consequently, the data support a relationship between using antimicrobial agents and the emergence of antimicrobial resistance.

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Linezolid and Tiamulin Cross-Resistance in Staphylococcus aureus Mediated by Point Mutations in the Peptidyl Transferase Center

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01015-07 ◽

2008 ◽

Vol 52 (5) ◽

pp. 1737-1742 ◽

Cited By ~ 50

Author(s):

Keith Miller ◽

Colin J. Dunsmore ◽

Colin W. G. Fishwick ◽

Ian Chopra

Keyword(s):

Staphylococcus Aureus ◽

Point Mutations ◽

Single Copy ◽

23S Rrna ◽

Cross Resistance ◽

Limiting Factor ◽

Peptidyl Transferase ◽

Inhibit Protein Synthesis ◽

Resistant Mutants ◽

Selection Of

ABSTRACT Oxazolidinone and pleuromutilin antibiotics are currently used in the treatment of staphylococcal infections. Although both antibiotics inhibit protein synthesis and have overlapping binding regions on 23S rRNA, the potential for cross-resistance between the two classes through target site mutations has not been thoroughly examined. Mutants of Staphylococcus aureus resistant to linezolid were selected and found to exhibit cross-resistance to tiamulin, a member of the pleuromutilin class of antibiotics. However, resistance was unidirectional because mutants of S. aureus selected for resistance to tiamulin did not exhibit cross-resistance to linezolid. This contrasts with the recently described PhLOPSA phenotype, which confers resistance to both oxazolidinones and pleuromutilins. The genotypes responsible for the phenotypes we observed were examined. Selection with tiamulin resulted in recovery of mutants with changes in the single-copy rplC gene (Gly155Arg, Ser158Leu, or Arg149Ser), whereas selection with linezolid led to recovery of mutants with changes (G2576U in 23S rRNA) in all five copies of the multicopy operon rrn. In contrast, cross-resistance to linezolid was exhibited by tiamulin-resistant mutants generated in a single-copy rrn knockout strains of Escherichia coli, illustrating that the copy number of 23S rRNA is the limiting factor in the selection of 23S rRNA tiamulin-resistant mutants. The interactions of linezolid and tiamulin with the ribosome were modeled to seek explanations for resistance to both classes in the 23S rRNA mutants and the lack of cross-resistance between tiamulin and linezolid following mutation in rplC.

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Linezolid Resistance in Staphylococcus aureus: Gene Dosage Effect, Stability, Fitness Costs, and Cross-Resistances

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01098-07 ◽

2008 ◽

Vol 52 (4) ◽

pp. 1570-1572 ◽

Cited By ~ 88

Author(s):

Silke Besier ◽

Albrecht Ludwig ◽

Johannes Zander ◽

Volker Brade ◽

Thomas A. Wichelhaus

Keyword(s):

Staphylococcus Aureus ◽

Gene Dosage ◽

Single Point ◽

23S Rrna ◽

Cross Resistance ◽

Single Point Mutation ◽

Rrna Gene ◽

Gene Dosage Effect ◽

Linezolid Resistance ◽

23S Rrna Gene

ABSTRACT Linezolid resistance in Staphylococcus aureus is typically associated with mutations in the 23S rRNA gene. Here we show that the accumulation of a single point mutation, G2576T, in the different copies of this gene causes stepwise increases in resistance, impairment of the biological fitness, and cross-resistance to quinupristin-dalfopristin and chloramphenicol.

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Improved Multiplex Polymerase Chain Reaction for Rapid Staphylococcus Aureus Detection in Meat and Milk Matrices

Nova Biotechnologica et Chimica ◽

10.1515/nbec-2016-0007 ◽

2016 ◽

Vol 15 (1) ◽

pp. 65-76

Author(s):

Zuzana Šramková ◽

Barbora Vidová ◽

Andrej Godány

Keyword(s):

Staphylococcus Aureus ◽

Polymerase Chain Reaction ◽

Food Poisoning ◽

Detection Sensitivity ◽

23S Rrna ◽

Rrna Gene ◽

Chain Reaction ◽

23S Rrna Gene ◽

Polymerase Chain ◽

Toxic Shock Syndrome Toxin

Abstract Staphylococcal food poisoning represents one of the most frequently occurring intoxications, caused by staphylococcal enterotoxins (SE-s) and staphylococcal enterotoxin-like proteins (SEl-s). Therefore, there is a need for rapid, sensitive and specific detection method for this human pathogen and its toxin genes in food matrices. The present work is focused on Staphylococcus aureus detection by a nonaplex polymerase chain reaction, which targets the 23S rRNA gene for identification of S. aureus at the species level, genes for classical SE-s (SEA, SEC, SED), new SE-s (SEH, SEI), SEl-s (SEK, SEL) and tsst-1 gene (toxic shock syndrome toxin). Primers were properly designed to avoid undesirable interactions and to create a reliably identifiable profile of amplicons when visualized in agarose gel. According to obtained results, this approach is able to reach the detection sensitivity of 12 colony forming units from milk and meat matrices without prior culturing and DNA extraction.

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Characterization of Staphylococcus aureus Isolates from White-Brined Urfa Cheese

Journal of Food Protection ◽

10.4315/0362-028x.jfp-11-179 ◽

2011 ◽

Vol 74 (11) ◽

pp. 1788-1796 ◽

Cited By ~ 9

Author(s):

KURSAT KAV ◽

RAMAZAN COL ◽

MUSTAFA ARDIC

Keyword(s):

Staphylococcus Aureus ◽

Tandem Repeats ◽

Genetic Relatedness ◽

Food Poisoning ◽

Rflp Analysis ◽

23S Rrna ◽

Enzyme Linked Immunosorbent Assay ◽

Toxin Gene ◽

Rrna Gene ◽

23S Rrna Gene

The aim of this study was to investigate the presence of Staphylococcus aureus and staphylococcal enterotoxin (SE) genes in Urfa cheese samples and to characterize the enterotoxigenic potential of these isolates. From a total of 127 Urfa cheese samples, 53 isolates (from 41.7% of the samples) were identified by a species-specific PCR assay as S. aureus. Of these isolates, 40 (75.5%) gave positive PCR results for the 3′ end of the coa gene. The coa-positive S. aureus strains were characterized for their population levels and enterotoxigenic properties, including slime factor, β-lactamase, antibiotic susceptibilities, production of the classical SEs (SEA through SEE), in both cheese and liquid cultures by enzyme-linked immunosorbent assay (ELISA) and for the presence of specific genes, including classical SE genes (sea through see), mecA, femA, and spa, by PCR. The genetic relatedness among the coa-positive S. aureus isolates was investigated by PCR-based restriction fragment length polymorphism (RFLP) analysis and the 23S rRNA gene spacer. The 23S rRNA gene spacer and coa RFLP analysis using AluI and Hin6I revealed 14 different patterns. SEB, SEC, and SEA and SEE were detected by ELISA in three cheese samples. Fourteen S. aureus strains harbored enterotoxin genes sea through see, and three strains carried multiple toxin genes. The most commonly detected toxin gene was sec (25% of tested strains). Of the 40 analyzed S. aureus strains, 3 (7.5%) were mecA positive. Based on tandem repeats, four coa and spa types were identified. The results of this study indicate that S. aureus and SEs are present at significant levels in Urfa cheese. These toxins can cause staphylococcal food poisoning, creating a serious hazard for public health.

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Mutational and Transcriptomic Changes Involved in the Development of Macrolide Resistance in Campylobacter jejuni

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01927-12 ◽

2012 ◽

Vol 57 (3) ◽

pp. 1369-1378 ◽

Cited By ~ 26

Author(s):

Haihong Hao ◽

Zonghui Yuan ◽

Zhangqi Shen ◽

Jing Han ◽

Orhan Sahin ◽

...

Keyword(s):

Campylobacter Jejuni ◽

Dna Microarray ◽

Ribosomal Proteins ◽

Macrolide Resistance ◽

23S Rrna ◽

Rrna Gene ◽

Content Type ◽

23S Rrna Gene ◽

Resistant Mutants ◽

Antibiotic Efflux

ABSTRACTMacrolide antibiotics are important for clinical treatment of infections caused byCampylobacter jejuni. Development of resistance to this class of antibiotics inCampylobacteris a complex process, and the dynamic molecular changes involved in this process remain poorly defined. Multiple lineages of macrolide-resistant mutants were selected by stepwise exposure ofC. jejunito escalating doses of erythromycin or tylosin. Mutations in target genes were determined by DNA sequencing, and the dynamic changes in the expression of antibiotic efflux transporters and the transcriptome ofC. jejuniwere examined by real-time reverse transcription-PCR, immunoblotting, and DNA microarray analysis. Multiple types of mutations in ribosomal proteins L4 and L22 occurred early during stepwise selection. On the contrary, the mutations in the 23S rRNA gene, mediating high resistance to macrolides, were observed only in the late-stage mutants. Upregulation of antibiotic efflux genes was observed in the intermediately resistant mutants, and the magnitude of upregulation declined with the occurrence of mutations in the 23S rRNA gene. DNA microarray analysis revealed the differential expression of 265 genes, most of which occurred in the intermediate mutant, including the upregulation of genes encoding ribosomal proteins and the downregulation of genes involved in energy metabolism and motility. These results indicate (i) that mutations in L4 and L22 along with temporal overexpression of antibiotic efflux genes precede and may facilitate the development of high-level macrolide resistance and (ii) that the development of macrolide resistance affects the pathways important for physiology and metabolism inC. jejuni, providing an explanation for the reduced fitness of macrolide-resistantCampylobacter.

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Molecular Detection of Multidrug Resistant Staphylococcus aureus Isolated from Bovine Mastitis Milk in Bangladesh

Veterinary Sciences ◽

10.3390/vetsci7020036 ◽

2020 ◽

Vol 7 (2) ◽

pp. 36

Author(s):

Md. Salauddin ◽

Mir Rowshan Akter ◽

Md. Khaled Hossain ◽

K. H. M. Nazmul Hussain Nazir ◽

Ayman Noreddin ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Bovine Mastitis ◽

Multidrug Resistant ◽

Health Concern ◽

Diffusion Method ◽

23S Rrna ◽

Nucleotide Sequence Analysis ◽

Rrna Gene ◽

Milk Samples ◽

23S Rrna Gene

The current study was conducted to isolate and identify multidrug-resistant Staphylococcus aureus (MDR-SA) from mastitis milk samples and to determine their antimicrobial susceptibility pattern. A total of 48 bovine mastitis (BM) milk samples were collected from different parts of the Rangpur division, Bangladesh. After the collection of milk samples, mastitis was confirmed using the California mastitis test. Isolation and identification of Staphylococcus aureus were performed using conventional cultural and biochemical tests as well as using molecular methods of PCR. Nucleotide sequence analysis of the 23S rRNA gene of Staphylococcus aureus was determined. The antibiogram of the isolated bacteria was conducted using the disc diffusion method. Phylogenetic analysis of 23S rRNA was done using MEGA 7, ClustalW multiple sequence alignment, and NCBI-BLAST tools, where the sequence of the isolate showed 98% to 99% identity. Antibiogram test using 15 antimicrobial agents showed that all of the Staphylococcus aureus isolates were classified as multidrug-resistant (MDR). It was found that the isolates were resistant to tetracycline, novobiocin, methicillin, vancomycin, and cephradine, and the isolates were sensitive to ciprofloxacin, azithromycin, norfloxacin, levofloxacin, gentamicin, and amoxicillin. The detection of MDR-SA in mastitis milk is alarming and represents a great public health concern. The findings of the present study help identify Staphylococcus aureus at the molecular level using 23S rRNA gene sequencing and will help select the appropriate and effective antimicrobial agent to control BM in the northern part of Bangladesh.

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Helicobacter pylori isolated from patients who later failed H. pylori eradication triple therapy readily develop resistance to clarithromycin

Journal of Medical Microbiology ◽

10.1099/jmm.0.46316-0 ◽

2006 ◽

Vol 55 (6) ◽

pp. 737-740 ◽

Cited By ~ 17

Author(s):

Intetsu Kobayashi ◽

Takeshi Saika ◽

Hiroe Muraoka ◽

Kazunari Murakami ◽

Toshio Fujioka

Keyword(s):

Helicobacter Pylori ◽

Proton Pump Inhibitor ◽

Eradication Therapy ◽

23S Rrna ◽

Rrna Gene ◽

Triple Combination ◽

Clarithromycin Resistance ◽

23S Rrna Gene ◽

H Pylori ◽

Selection Of

In this study, the ease of selection of clarithromycin resistance was investigated in clarithromycin-susceptible Helicobacter pylori strains isolated from patients with H. pylori infection prior to the administration of triple-combination eradication therapy (clarithromycin plus amoxicillin plus a proton pump inhibitor). Clarithromycin-susceptible strains isolated from ten patients in whom the eradication therapy was successful and from six patients in whom the eradication therapy was unsuccessful were exposed serially to subinhibitory concentrations of clarithromycin. The number of transfers required for the MICs of the strains to increase by 8- and 32-fold were 6.6 and 7.2, respectively, in the successful eradication group, and as few as 2.4 and 1.5, respectively, in the unsuccessful eradication group. The number of transfers required for the A2142G or A2143G point mutation of the 23S rRNA gene to be detected in the strains were 5 and 8, respectively, for the strains in the successful eradication group, and 1 and 2, respectively, for the strains in the unsuccessful eradication group. These results suggest that patients in the unsuccessful eradication group were infected with strains of H. pylori that readily became resistant to clarithromycin on exposure to the drug.

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