Characterization of Staphylococcus aureus Isolates from White-Brined Urfa Cheese

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01302-07 ◽

2008 ◽

Vol 52 (6) ◽

pp. 1940-1944 ◽

Cited By ~ 16

Author(s):

Keith Miller ◽

Alexander J. O'Neill ◽

Mark H. Wilcox ◽

Eileen Ingham ◽

Ian Chopra

Keyword(s):

Staphylococcus Aureus ◽

Homologous Recombination ◽

Antimicrobial Agents ◽

Suppressive Effect ◽

23S Rrna ◽

Rrna Gene ◽

Selection Window ◽

23S Rrna Gene ◽

Resistant Mutants ◽

Selection Of

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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PCR-RFLP detection of point mutations A2143G and A2142G in 23S rRNA gene conferring resistance to clarithromycin in Helicobacter pylori strains.

Acta Biochimica Polonica ◽

10.18388/abp.2014_1901 ◽

2014 ◽

Vol 61 (2) ◽

Cited By ~ 22

Author(s):

Karolina Klesiewicz ◽

Paweł Nowak ◽

Elżbieta Karczewska ◽

Iwona Skiba ◽

Izabela Wojtas-Bonior ◽

...

Keyword(s):

Helicobacter Pylori ◽

Point Mutations ◽

Rflp Analysis ◽

23S Rrna ◽

Rrna Gene ◽

Efficient Management ◽

Clarithromycin Resistance ◽

Pcr Rflp ◽

23S Rrna Gene ◽

H Pylori

The occurrence of clarithromycin resistance among Helicobacter pylori strains is a major cause of the treatment failure. Resistance to this drug is conferred by point mutations in 23S rRNA gene and the most prevalent mutations are A2143G and A2142G. The aim of the study was to evaluate the occurrence of A2143G and A2142G mutations in a group of H. pylori strains resistant to clarithromycin. The study included 21 clarithromycin-resistant H. pylori strains collected between 2006 and 2009 in southern Poland. Resistance to clarithromycin was quantitatively tested with the E-test to determine the minimal inhibitory concentration (MIC value). The point mutations of H. pylori isolates were detected by PCR followed by RFLP analysis. The MIC values for clarithromycin for the analyzed strains ranged from 1.5 mg/L to 64 mg/L. Nine H. pylori strains exhibited A2143G mutation and A2142G mutation was found in 9 isolates as well. The results of RFLP analysis of 3 clarithromycin-resistant strains were negative for both mutations. The average MIC values for A2143G and A2142G mutants were 6 and 30 mg/L, respectively. Frequencies of A2143G and A2142G mutations were the same in all isolates tested. Strains with A2143G mutation exhibited lower MIC values than A2142G mutants. Application of PCR-RFLP method for detection of clarithromycin resistance allows for better and more efficient management of H. pylori infections.

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Genotyping of Mycoplasma pneumoniaeClinical Isolates Reveals Eight P1 Subtypes within Two Genomic Groups

Journal of Clinical Microbiology ◽

10.1128/jcm.38.3.965-970.2000 ◽

2000 ◽

Vol 38 (3) ◽

pp. 965-970 ◽

Cited By ~ 32

Author(s):

J. Wendelien Dorigo-Zetsma ◽

Jacob Dankert ◽

Sebastian A. J. Zaat

Keyword(s):

Sequence Data ◽

Rflp Analysis ◽

Clinical Isolates ◽

23S Rrna ◽

Rrna Gene ◽

Intergenic Spacer Region ◽

Long Pcr ◽

23S Rrna Gene

Three methods for genotyping of Mycoplasma pneumoniaeclinical isolates were applied to 2 reference strains and 21 clinical isolates. By a modified restriction fragment length polymorphism (RFLP) analysis of PCR products of the M. pneumoniae cytadhesin P1 gene, 5 subtypes were discriminated among 13 P1 type 1 strains and 3 subtypes were discriminated among 8 P1 type 2 strains. Sequence analysis of the 16S-23S rRNA gene spacer region and part of the 23S rRNA gene revealed one nucleotide difference in the intergenic spacer region in 3 of the 21 isolates. In the 23S rRNA gene sequence of the 8 P1 type 2 strains an extra adenosine was present, but it was absent from the 13 P1 type 1 strains. On the basis of M. pneumoniae genome sequence data, primers were designed to amplify large interrepeat fragments by long PCR, and these fragments were subsequently analyzed by RFLP analysis. Only two types, long PCR types 1 and 2, could be discriminated among the M. pneumoniaeisolates. All P1 type 1 strains were assigned to long PCR type 1, and all P1 type 2 strains were assigned to long PCR type 2. These data obtained by three independent typing methods thus confirm the existence of two distinct M. pneumoniae genomic groups but expand the possibility of strain typing on the basis of variations within their P1 genes.

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Macrolide-Resistant Mycoplasma pneumoniae in Adults in Zhejiang, China

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04308-14 ◽

2014 ◽

Vol 59 (2) ◽

pp. 1048-1051 ◽

Cited By ~ 31

Author(s):

Zibo Zhou ◽

Xiangzhi Li ◽

Xiaojian Chen ◽

Fangjun Luo ◽

Changwang Pan ◽

...

Keyword(s):

Mycoplasma Pneumoniae ◽

Rflp Analysis ◽

Zhejiang Province ◽

23S Rrna ◽

Rrna Gene ◽

Type I ◽

Content Type ◽

23S Rrna Gene ◽

Resistant Strains ◽

Domain V

ABSTRACTMycoplasma pneumoniaeis a major pathogen causing community-acquired pneumoniae (CAP), which is generally treated with macrolides. In recent years, however, although macrolide-resistantM. pneumoniaehas been reported frequently, particularly in China, very little is known about the prevalence of macrolide-resistantM. pneumoniaeinfection in adults. In this study, we survey the macrolide-resistantM. pneumoniaein adults in Zhejiang province and characterize the mechanisms of resistance to macrolide. Six hundred fifty throat swab samples were collected from adult patients with CAP from January 2012 to August 2014. These samples were assayed by nested PCR and then cultivated forM. pneumoniae. All isolates were sequenced to determine the mutation in domain V of the 23S rRNA gene. The activities of 10 antibiotics against macrolide-resistantM. pneumoniaeisolates were also investigatedin vitro. Moreover, restriction fragment length polymorphism (RFLP) analysis of the amplified P1 gene was used to type 50 resistant strains. One hundred percent (71/71) ofM. pneumoniaestrains isolated from adults with CAP were resistant to erythromycin (MIC = 128 to >256 μg/ml), clarithromycin (MIC = 128 to >256 μg/ml), and azithromycin (MIC = 32 to >64 μg/ml). Furthermore, all macrolide-resistantM. pneumoniaestrains identified had an A2063G mutation in domain V of the 23S rRNA gene. Forty-six resistant strains (92.0%) were classified into type I strain on the basis of P1 gene PCR-RFLP analysis. According to these findings, it is suggested that macrolide-resistantM. pneumoniaeinfection is very prevalence among adults in Zhejiang province. Thus, there is necessary to perform the epidemiological monitoring of macrolide-resistantM. pneumoniaein the future.

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Trends towards Lower Antimicrobial Susceptibility and Characterization of Acquired Resistance among Clinical Isolates of Brachyspira hyodysenteriae in Spain

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01749-10 ◽

2011 ◽

Vol 55 (7) ◽

pp. 3330-3337 ◽

Cited By ~ 45

Author(s):

Álvaro Hidalgo ◽

Ana Carvajal ◽

Birte Vester ◽

Märit Pringle ◽

Germán Naharro ◽

...

Keyword(s):

Antimicrobial Susceptibility ◽

Molecular Mechanisms ◽

Tandem Repeats ◽

Point Mutations ◽

Clinical Isolates ◽

23S Rrna ◽

Rrna Gene ◽

Content Type ◽

Brachyspira Hyodysenteriae ◽

23S Rrna Gene

ABSTRACTThe antimicrobial susceptibility of clinical isolates ofBrachyspira hyodysenteriaein Spain was monitored, and the underlying molecular mechanisms of resistance were investigated. MICs of tylosin, tiamulin, valnemulin, lincomycin, and tylvalosin were determined for 87B. hyodysenteriaeisolates recovered from 2008 to 2009 by broth dilution. Domain V of the 23S rRNA gene and the ribosomal protein L3 gene were sequenced in 20 isolates for which the tiamulin MIC was ≥4 μg/ml, presenting decreased susceptibility, and in 18 tiamulin-susceptible isolates (MIC ≤ 0.125 μg/ml), and all isolates were typed by multiple-locus variable-number tandem repeats analysis. A comparison with antimicrobial susceptibility data from 2000 to 2007 showed an increase in pleuromutilin resistance over time, doubling the number of isolates with decreased susceptibility to tiamulin. No alteration in susceptibility was detected for lincomycin, and the MIC of tylosin remained high (MIC50> 128 μg/ml). The decreased susceptibility to tylosin and lincomycin can be explained by mutations at position A2058 of the 23S rRNA gene (Escherichia colinumbering). A2058T was the predominant mutation, but A2058G also was found together with a change of the neighboring base pair at positions 2057 to 2611. The role of additional point mutations in the vicinity of the peptidyl transferase center and mutations in the L3 at amino acids 148 and 149 and their possible involvement in antimicrobial susceptibility are considered. An association between G2032A and high levels of tiamulin and lincomycin MICs was found, suggesting an increasing importance of this mutation in antimicrobial resistance of clinical isolates ofB. hyodysenteriae.

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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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Klasterisasi Staphylococcus aureus Resisten Neutrofil Berdasar Assesory Gene Regulator

Jurnal Sain Veteriner ◽

10.22146/jsv.50653 ◽

2020 ◽

Vol 38 (2) ◽

pp. 95

Author(s):

Christin Marganingsih Santosa ◽

Fajar Budi Lestari ◽

Rini Widayanti ◽

Siti Isrina Oktavia Salasia

Keyword(s):

Staphylococcus Aureus ◽

Food Poisoning ◽

Pcr Amplification ◽

Aureus Strain ◽

23S Rrna ◽

Rrna Gene ◽

Polymorphonuclear Cells ◽

Biochemical Tests

Staphylococcus aureus is recognized worldwide as a major pathogen causing subclinical intramammary infections in dairy cows and food poisoning due to its ability to produce enterotoxin. The study aimed to identify enterotoxins of S. aureus and clustering the enterotoxins based on assessory gene regulator (agr). Virulence of S. aureus to the host was characterized based on the response of polymorphonuclear cells to the infection. Twelve S. aureus could be isolated from milk cows in central of dairy farming in Sumedang West Java. The identification of S. aureus was based on cultural and biochemical tests and an amplification of a specific section of the 23S rRNA gene. The sensitivity test against antibiotics revealed that some isolates of S. aureus were resistant to penicillin and methycillin. By PCR amplification one or more staphylococcal enterotoxin genes could be observed five genes in combinations of sea (216 bp), seb (478 bp), seh (375 bp), sei (576 bp), and sej (142 bp). Clustering of S. aureus based on the assesory gene regulator could be grouped into 4 clusters for agr1 (1 isolat), agr2 (2 isolates), in combination for agr1 and agr2 (1 isolate), and for non agr (2 isolates). Based on the response of polymorphonuclear cell in vitro and in vivo assays, revealed that S. aureus strain I-2 (agr1 cluster) and P1 (agr1+agr2 cluster) were more resistant to polymorphonuclear cells and could survive intracellularly, indicated that these strains could be used as proper candidates to develop dignostic tool based on agr against staphylococcal mastitis.

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