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Evaluation of LAMP Assay Using Phenotypic Tests and Conventional PCR for Detection of nuc and mecA genes Among Clinical Isolates of Staphylococcus spp.

ABSTRACT: INTRODUCTION:The purpose of this study is to develop a nuc and mecA gene specific Loop-mediated isothermal Amplification (LAMP) assay for rapid identification and detection of methicillin resistant Staphylococcus aureus among clinical isolates. MATERIALS AND METHODS:A total of 100 (70 from pus and 30 from blood), clinical isolates of Staphylococcus spp were screened for the nuc gene to differentiate between S.aureus and Coagulase negative Staphylococci (CONS) by a nuc gene specific LAMP assay. The isolates were also screened for the presence of the mec Agene by the mecA specific LAMP assay. The results were compared with the phenotypic identification and methicillin resistance by Vitek-2 system (bioMérieux, Marcy l'Etoile, France) and conventional PCR. RESULTS:Among 100 Staphylococcus isolates, there were 82 (82%) Staphylococcus aureus isolates and 18 (18%) coagulase negative Staphylococcus as detected by the Vitek 2, conventional PCR and the LAMP assay using the nuc gene. The mecA gene was detected by the LAMP assay in 56(56%) isolates (44 Methicillin resistant Staphylococcus aureus (MRSA) and 12 Methicillin resistant coagulase negative Staphylococcus (MRCONS), which were also identified by the Vitek 2 and conventional PCR as methicillin resistant. The results of the LAMP assay were available within 90min as compared to the Vitek 2 results (18- 24hours) and conventional PCR (3-4 hours). CONCLUSION:The present study proved that LAMP assay can be used for the simultaneous differentiation of Staphylococcal spp and detection of methicillin resistance.

SUBMITTER: Sudhaharan S

PROVIDER: S-EPMC4576532 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Evaluation of LAMP Assay Using Phenotypic Tests and Conventional PCR for Detection of nuc and mecA genes Among Clinical Isolates of Staphylococcus spp.

Sudhaharan Sukanya S Vanjari Lavanya L Mamidi Neeraja N Ede Nagapriyanka N Vemu Lakshmi L

Journal of clinical and diagnostic research : JCDR 20150801 8

<h4>Introduction</h4>The purpose of this study is to develop a nuc and mecA gene specific Loop-mediated isothermal Amplification (LAMP) assay for rapid identification and detection of methicillin resistant Staphylococcus aureus among clinical isolates.<h4>Materials and methods</h4>A total of 100 (70 from pus and 30 from blood), clinical isolates of Staphylococcus spp were screened for the nuc gene to differentiate between S.aureus and Coagulase negative Staphylococci (CONS) by a nuc gene specifi ...[more]

PMID: 26435941

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Project description:BACKGROUND:The increase and spread of virulent-outbreak associated, methicillin and vancomycin resistant (MRSA/VRSA) Staphylococcus aureus require a better understanding of the resistance and virulence patterns of circulating and emerging strains globally. This study sought to establish the resistance phenotype, and strains of 32 non-duplicate clinical MRSA and MSSA S. aureus isolates from four Kenyan hospitals, identify their resistance and virulence genes and determine the genetic relationships of MRSA with global strains. METHODS:Antimicrobial susceptibility profiles were determined on a Vitek 2, genomic DNA sequenced on an Illumina Miseq and isolates typed in-silico. Resistance and virulence genes were identified using ARIBA and phylogenies generated using RAxML. RESULTS:The MRSA isolates were 100% susceptible to vancomycin, teicoplanin, linezolid, and tigecycline. Nine distinct CC, 12 ST and 15 spa types including the novel t17826 and STs (4705, 4707) were identified with CC8 and CC152 predominating. MRSA isolates distributed across 3 CCs; CC5-ST39 (1), CC8 - ST241 (4), a novel CC8-ST4705 (1), ST8 (1) and CC152 (1). There was > 90% phenotype-genotype concordance with key resistance genes identified only among MRSA isolates: gyrA, rpoB, and parC mutations, mecA, ant (4')-lb, aph (3')-IIIa, ermA, sat-4, fusA, mphC and msrA. Kenyan MRSA isolates were genetically diverse and most closely related to Tanzanian and UK isolates. There was a significant correlation between map, hlgA, selk, selq and cap8d virulence genes and severe infections. CONCLUSION:The findings showed a heterogeneous S. aureus population with novel strain types. Though limited by the low number of isolates, this study begins to fill gaps and expand our knowledge of S. aureus epidemiology while uncovering interesting patterns of distribution of strain types which should be further explored. Although last-line treatments are still effective, the potential for outbreaks of both virulent and resistant strains remain, requiring sustained surveillance of S. aureus populations.

Project description:Canine superficial pyoderma (CSP) is a bacterial infection secondary to several skin diseases of the dog. Staphylococcus pseudintermedius, which is a commensal bacterium of the dog's skin, is the leading agent found in dogs affected by CSP, which can progress to deep pyoderma. It is also of clinical significance because S. pseudintermedius strains carry antimicrobial resistance genes, mainly the mecA gene. In this descriptive longitudinal study, molecular characterization of bacterial isolates from dogs affected by CSP was performed in addition to phenotyping, antimicrobial profiling, and assessment of resistance carriage status. Fifty dogs (24 females and 26 males) attending the CES University Veterinary Teaching Hospital were included in the study. CSP was confirmed according to clinical signs and cytological examination. Swabs were taken from active skin lesions for bacterial culture, and phenotyping and antimicrobial resistance profiles were assessed using API-Staph phenotyping and the Kirby-Bauer method, respectively. We also performed molecular detection and characterization of the mecA and nuc encoding gene of coagulase-positive Staphylococci. The mecA gene frequency was established by qPCR amplification of a 131bp gene fragment. Data were evaluated by descriptive statistics. Erythema, peeling, pruritus, and alopecia were the predominant symptoms (72, 56, and 46%, respectively). We isolated bacteria compatible with Staphylococcus species from all samples tested. API phenotyping showed 83.1 to 97.8% compatibility with S. pseudintermedius. PCR-genotyping resulted in 15, 3, and 1 isolates positive for S. pseudintermedius, S. aureus, and S. schleiferi, respectively. Isolated strains showed high susceptibility to Imipenem, Ampicillin/Sulbactam, and Rifampicin (100, 94, and 92%, respectively). The highest resistance was against Vancomycin and Trimethoprim/Sulfamethoxazole (98 and 74%, respectively). S. pseudintermedius, S. aureus, and S. schleiferi isolates were cloned and shared 96% sequence homology. Finally, we found 62% carriage status of the mecA gene in isolates of CSP patients, although only 36% of the isolates were methicillin-resistant. Identification of three Staphylococcus species causing CSP, high-level resistance against conventional antimicrobials, and carriage of the mecA gene highlight the importance of performing molecular characterization of bacteria causing dermatological conditions in dogs.

Dataset Information

Evaluation of LAMP Assay Using Phenotypic Tests and Conventional PCR for Detection of nuc and mecA genes Among Clinical Isolates of Staphylococcus spp.

Publications

Evaluation of LAMP Assay Using Phenotypic Tests and Conventional PCR for Detection of nuc and mecA genes Among Clinical Isolates of Staphylococcus spp.

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