Molecular cloning and mapping of 16S-23S rRNA gene complexes of Staphylococcus aureus.

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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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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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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Novel Ribosomal Mutations in Staphylococcus aureus Strains Identified through Selection with the Oxazolidinones Linezolid and Torezolid (TR-700)

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00871-09 ◽

2009 ◽

Vol 53 (12) ◽

pp. 5265-5274 ◽

Cited By ~ 118

Author(s):

Jeffrey B. Locke ◽

Mark Hilgers ◽

Karen Joy Shaw

Keyword(s):

Staphylococcus Aureus ◽

Ribosomal Proteins ◽

Spontaneous Mutation ◽

Gene Mutations ◽

Serial Passage ◽

23S Rrna ◽

Rrna Gene ◽

Genes Encoding ◽

23S Rrna Gene ◽

Domain V

ABSTRACT TR-700 (torezolid), the active moiety of the novel oxazolidinone phosphate prodrug TR-701, is highly potent against gram-positive pathogens, including strains resistant to linezolid (LZD). Here we investigated the potential of Staphylococcus aureus strains ATCC 29213 (methicillin-susceptible S. aureus [MSSA]) and ATCC 33591 (methicillin-resistant S. aureus [MRSA]) to develop resistance to TR-700. The spontaneous frequencies of mutation of MSSA 29213 and MRSA 33591 resulting in reduced susceptibility to TR-700 at 2× the MIC were 1.1 × 10−10 and 1.9 × 10−10, respectively. These values are ∼16-fold lower than the corresponding LZD spontaneous mutation frequencies of both strains. Following 30 serial passages in the presence of TR-700, the MIC for MSSA 29213 remained constant (0.5 μg/ml) while increasing eightfold (0.25 to 2.0 μg/ml) for MRSA 33591. Serial passage of MSSA 29213 and MRSA 33591 in LZD resulted in 64- and 32-fold increases in LZD resistance (2 to 128 μg/ml and 1 to 32 μg/ml, respectively). Domain V 23S rRNA gene mutations (Escherichia coli numbering) found in TR-700-selected mutants included T2500A and a novel coupled T2571C/G2576T mutation, while LZD-selected mutants included G2447T, T2500A, and G2576T. We also identified mutations correlating with decreased susceptibility to TR-700 and LZD in the rplC and rplD genes, encoding the 50S ribosomal proteins L3 and L4, respectively. L3 mutations included Gly152Asp, Gly155Arg, Gly155Arg/Met169Leu, and ΔPhe127-His146. The only L4 mutation detected was Lys68Gln. TR-700 maintained a fourfold or greater potency advantage over LZD against all strains with ribosomal mutations. These data bring to light a variety of novel and less-characterized mutations associated with S. aureus resistance to oxazolidinones and demonstrate the low resistance potential of torezolid.

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Molecular pathways to high-level azithromycin resistance in Neisseria gonorrhoeae

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkab084 ◽

2021 ◽

Cited By ~ 1

Author(s):

J G E Laumen ◽

S S Manoharan-Basil ◽

E Verhoeven ◽

S Abdellati ◽

I De Baetselier ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

Ribosomal Proteins ◽

Efflux Pump ◽

23S Rrna ◽

Rrna Gene ◽

23S Rrna Gene ◽

Evolution Of Resistance ◽

High Level ◽

Azithromycin Resistance ◽

Level Resistance

Abstract Background The prevalence of azithromycin resistance in Neisseria gonorrhoeae is increasing in numerous populations worldwide. Objectives To characterize the genetic pathways leading to high-level azithromycin resistance. Methods A customized morbidostat was used to subject two N. gonorrhoeae reference strains (WHO-F and WHO-X) to dynamically sustained azithromycin pressure. We tracked stepwise evolution of resistance by whole genome sequencing. Results Within 26 days, all cultures evolved high-level azithromycin resistance. Typically, the first step towards resistance was found in transitory mutations in genes rplD, rplV and rpmH (encoding the ribosomal proteins L4, L22 and L34 respectively), followed by mutations in the MtrCDE-encoded efflux pump and the 23S rRNA gene. Low- to high-level resistance was associated with mutations in the ribosomal proteins and MtrCDE efflux pump. However, high-level resistance was consistently associated with mutations in the 23S ribosomal RNA, mainly the well-known A2059G and C2611T mutations, but also at position A2058G. Conclusions This study enabled us to track previously reported mutations and identify novel mutations in ribosomal proteins (L4, L22 and L34) that may play a role in the genesis of azithromycin resistance in N. gonorrhoeae.

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Molecular characterization of a ceftriaxone-resistant Neisseria gonorrhoeae strain found in Switzerland: a case report

Annals of Clinical Microbiology and Antimicrobials ◽

10.1186/s12941-021-00456-5 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Konrad Egli ◽

Anna Roditscheff ◽

Ursula Flückiger ◽

Martin Risch ◽

Lorenz Risch ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

23S Rrna ◽

Rrna Gene ◽

Limited Information ◽

Specific Pcr ◽

Appropriate Treatment ◽

23S Rrna Gene ◽

Allele Type ◽

Ciprofloxacin Resistance

Abstract Background The resistance of Neisseria gonorrhoeae to ceftriaxone is unusual in Switzerland. The underlying genotype responsible for resistance is suspected to be novel. Generally, resistance in Neisseria gonorrhoeae (Ng) involves a comprehensive set of genes with many different mutations leading to resistance to different β-lactams and fluoroquinolones. Case presentation A patient had a positive result from specific PCR for Ng. We routinely culture all clinical specimens with a positive NG-PCR. In this particular case, we isolated a strain with resistance to ceftriaxone in Switzerland. A total of seven different genes (penA, ponA, porinB, mtr, gyrA, parC, 23S rRNA gene) in this strain were partially sequenced for comparison with phenotypic susceptibility testing. Interestingly, two different mutations in the porinB gene were observed, and data on this gene are limited. Information on the identified allele type of the penA gene is very limited as well. Three different mutations of parC and gyrA that correlate with ciprofloxacin resistance were found. The combination of ceftriaxone and ciprofloxacin resistance makes an appropriate treatment difficult to obtain due to multidrug resistance. Conclusion The combined results for all genes show the appearance of new mutations in central Europe either due to worldwide spread or the emergence of new genetic combinations of mutations.

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Molecular Basis of Macrolide Resistance inCampylobacterStrains Isolated from Poultry in South Korea

BioMed Research International ◽

10.1155/2018/4526576 ◽

2018 ◽

Vol 2018 ◽

pp. 1-9 ◽

Cited By ~ 3

Author(s):

Bai Wei ◽

Min Kang

Keyword(s):

Molecular Mechanisms ◽

Macrolide Resistance ◽

23S Rrna ◽

Rrna Gene ◽

Campylobacter Coli ◽

Erythromycin Resistance ◽

23S Rrna Gene ◽

Ribosomal Protein L22 ◽

Resistant Strains ◽

Azithromycin Resistance

We investigated the molecular mechanisms underlying macrolide resistance in 38 strains ofCampylobacterisolated from poultry. Twenty-seven strains were resistant to azithromycin and erythromycin, five showed intermediate azithromycin resistance and erythromycin susceptibility, and six showed azithromycin resistance and erythromycin susceptibility. FourCampylobacter jejuniand sixCampylobacter colistrains had azithromycin MICs which were 8–16 and 2–8-fold greater than those of erythromycin, respectively. The A2075G mutation in the 23S rRNA gene was detected in 11 resistant strains with MICs ranging from 64 to ≥ 512μg/mL. Mutations including V137A, V137S, and a six-amino acid insertion (114-VAKKAP-115) in ribosomal protein L22 were detected in theC. jejunistrains. Erythromycin ribosome methylase B-erm(B) was not detected in any strain. All strains except three showed increased susceptibility to erythromycin with twofold to 256-fold MIC change in the presence of phenylalanine arginine ß-naphthylamide (PAßN); the effects of PAßN on azithromycin MICs were limited in comparison to those on erythromycin MICs, and 13 strains showed no azithromycin MIC change in the presence of PAßN. Differences between azithromycin and erythromycin resistance and macrolide resistance phenotypes and genotypes were observed even in highly resistant strains. Further studies are required to better understand macrolide resistance inCampylobacter.

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Prevalence and Antimicrobial Resistance of Campylobacter spp. Isolated from Poultry Carcasses in Poland

Journal of Food Protection ◽

10.4315/0362-028x.jfp-13-035 ◽

2013 ◽

Vol 76 (8) ◽

pp. 1451-1455 ◽

Cited By ~ 23

Author(s):

KINGA WIECZOREK ◽

IWONA KANIA ◽

JACEK OSEK

Keyword(s):

Campylobacter Jejuni ◽

23S Rrna ◽

Rrna Gene ◽

Campylobacter Coli ◽

Genetic Determinants ◽

Gyra Gene ◽

23S Rrna Gene ◽

Pcr Method ◽

Almost All ◽

Campylobacter Spp

The purpose of the present study was to determine the prevalence of Campylobacter in poultry carcasses at slaughter in Poland. For the isolated strains, resistance to selected antibiotics and the associated genetic determinants were identified. A total of 498 Campylobacter isolates were obtained from 802 poultry samples during the 2-year study period. Strains were identified to species with the PCR method; 53.6% of the strains were Campylobacter jejuni and 46.4% were Campylobacter coli. A high percentage of the tested Campylobacter strains were resistant to ciprofloxacin and nalidixic acid (74.1 and 73.5%, respectively) followed by tetracycline (47.4%) and streptomycin (20.5%). Only one C. jejuni and two C. coli isolates were resistant to gentamicin. Seventy-nine (15.9%) of the 498 strains were resistant to three or more classes of antibiotics examined. Higher levels of resistance, irrespective of the antimicrobial agent tested, were found within the C. coli group. Almost all strains resistant to quinolones (99.5%) and to tetracycline (99.6%) carried the Thr-86-to-Ile mutation in the gyrA gene and possessed the tet(O) marker, respectively. All isolates resistant to erythromycin had the A2075G mutation in the 23S rRNA gene. These results reveal that poultry carcasses in Poland are a reservoir of potentially pathogenic and antimicrobial-resistant Campylobacter strains for humans, which may pose a public health risk.

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