TECHNICAL FIELD

The present invention relates to a method for identifying methicillin-resistantStaphylococcus aureus(MRSA), especially a method for distinguishing methicillin-resistantStaphylococcus aureusthrough a peptide biomarker.

BACKGROUND OF THE INVENTION

According to statistical results from 2000 to 2015, the annual antibiotic dose in 76 countries has increased from 21.1 billion doses of antibiotics to 34.8 billion doses of antibiotics, showing that the rate of antibiotic use has risen by 65% over the past 15 years. At the same time, the global human use of antibiotics has reached 42 billion doses in 2015 and it is estimated that by 2030, the daily antibiotic dose will increase by 200% to 128 billion doses per day. The statistical clinical results show that the abuse of antibiotics mainly occurs in the use of the wrong dose (19.9%), the frequency of use (18.9%), and the repeated treatment (18.1%). The wrong dose and repeated treatment may be the use of ineffective antibiotics by the antidrug bacteria.

The mechanisms of drug resistance in bacteria are controlled by drug resistance genes. Some of the drug resistance genes are inherently possessed by bacteria, and some are produced by passing plastids or jumpers to other bacteria. In an environment containing antibiotics, non-resistant bacteria will be eliminated, and the ones that survive are resistant bacteria. In other words, the abuse of antibiotics will cause more and more antibiotic-resistant bacteria, especially in areas where a lot of antibiotics are used, a significant increase in the proportion of resistant bacteria can be observed. For example, methicillin-resistanceStaphylococcus aureus(MRSA) is a special bacterium that is resistant to methicillin and other antibiotics. This bacterium has extremely infectious, so it is classified as one of the super bacteria.

Staphylococcus aureus(S. aureus) can be found in human respiratory tract and on the skin. In general,S. aureusdoes not cause symptoms, but the bacteria occasionally cause diseases, including skin, wounds, urethra, lungs, blood infections and food poisoning. The use of antibiotics can effectively inhibit most of the infections ofStaphylococcus aureus, but not inhibit the drug-resistantStaphylococcus aureus, which is resistant to methicillin and other antibiotics, such as oxacillin, oxacillin, penicillin, amoxicillin and cephalosporins. Therefore, the rapid and accurate diagnosis of drug-resistantS. aureusis urgently needed and extremely important for the subsequent use of antibiotics.

The diagnosis and treatment of microbial infections are completely different from cancer. Comprehensive judgment of clinical examination and immediate integration of clinical data are very important for the diagnosis and treatment of microbial infections. The infectious diseases such as sepsis, meningitis, pneumonia, urinary tract infection are all clinically manifested as acute symptoms. Therefore, it is necessary to correctly diagnose the pathogen of the infection and determine its antibiotic resistance in the shortest time. In clinical practice, when a patient has fever and other indicator of inflammatory reactions, indicating that the patient has the possibility of infection, the culture of microorganisms and empirical therapy of antibiotics are often carried out to control the patient's infection. Furthermore, the identification of microorganisms and antibiotic susceptibility tests can provide clinically correct use of antibiotics to improve patient survival. At the same time, it can also reduce the waste of medical expenses, the environmental pollution and destruction of antibiotics caused by the abuse of antibiotics.

The gold standard for clinical diagnosis of infectious diseases is mainly based on the results of laboratory microbial culture and identification. The current process is to inoculate the processed sample into a suitable medium for overnight culture after receiving the sample. After that, it still takes an additional 24 hours to perform traditional biochemical identification to determine the type of bacteria, and to conduct antibiotic drug susceptibility testing. Therefore, the complete microbial culture identification report needs to take 3-5 days to obtain. At present, to speed up the process of bacterial identification, many medical centers use matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a method of bacterial identification, and use a drug susceptibility test (test time is about 24 hours) to confirm whether the bacteria are resistant to specific antibiotics.

Methods for MRSA identification is to use polymerase chain reaction or gene sequencing targeting the drug resistance genes to determine whether the strain is drug resistant. However, compared with the MALDI-TOF MS identification method, the gene sequencing method requires higher costs, high training requirements for operators, and relatively long time consumption. Therefore, MALDI-TOF MS has emerged as a potential tool for microbial identification and diagnosis in the clinical microbial identification. There have been many prior disclosures using MALDI-TOF MS as the related technology for MRSA identification. In the past, related technology disclosures were mainly performed on known strains (including the mass spectrum of drug-resistant or drug-sensitiveStaphylococcus aureus) to analyze and compare to find novel biomarkers to identify MRSA. In addition, analyzing the mass spectra obtained through a mass spectrometer analysis by commercial software can directly identify bacterial species and further to predict whether they are drug-resistant strains. However, this prediction result is often different from the actual clinical test results obtained by clinical patients. Therefore, more accurate biomarkers for the diagnosis of MRSA are urgently needed for the correct diagnosis of clinical patient infections and the determination of antibiotic treatment.

SUMMARY OF THE INVENTION

The present invention relates to a method for identifying methicillin-resistantStaphylococcus aureus, comprising: (a) providing a bacterial sample; (b) depositing the bacterial sample on a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) mass spectrometer target plate; (c) acquiring a MALDI-TOF mass spectrum of the bacterial sample; and (d) determining the presence of a mass signal at m/z of 6580-6600 in the MALDI-TOF mass spectrum, and identifying the bacterial sample which comprises methicillin-resistantStaphylococcus aureus.

In one aspect, the present invention relates to a peptide for identifying methicillin-resistantStaphylococcus aureusconsists of SEQ NO ID:5.

In another aspect, the present invention relates to a method for identifying methicillin-resistantStaphylococcus aureus, comprising: (a) providing a bacterial sample; and (b) determining the presence of a peptide which consists of SEQ NO ID:5, and identifying the bacterial sample which comprises methicillin-resistantStaphylococcus aureus.

