The Mechanism of MRSA Drug Resistance and Its Detection

What is MRSA?

Staphylococcus aureus is a commonly seen and highly toxic bacterium in clinic. The infectious diseases caused by Staphylococcus aureus have been well controlled since the advent of penicillin in the 1940s. Yet, with the widespread use of penicillin, some Staphylococcus aureus has developed penicillinase which hydrolyzes the β-lactam ring and exhibits resistance to penicillin.

Therefore, a new type of semi-synthetic penicillin that resists penicillinase, i.e., methicillin, was developed. After its clinical use in 1959, it effectively controlled the infection of penicillinase-producing staphylococcus aureus strains. However, only two years later, Jevons in the UK was the first to find Methicillin-Resistant Staphylococcus Aureus (MRSA).

From its discovery to now, MRSA infection has almost spread worldwide, becoming one of the important pathogens for nosocomial infection. Therefore, carrying out the detection of MRSA is of great significance for controlling the prevalence of nosocomial infections and guiding clinical treatment.

Characteristics of MRSA

Heterogeneous Drug Resistance

Within a MRSA colony, there are two subgroups, sensitive and resistant bacteria, meaning only a small proportion of bacteria, about 10^-4 to 10^-7, in a MRSA strain are highly resistant to methicillin and can still survive under conditions of 50 μg/ml methicillin. The majority of bacteria in the colony are sensitive to methicillin and a large number of sensitive bacteria are killed within a few hours of using antibiotics, but a minority of resistant strains grow slowly and reproduce rapidly again within a few hours.

Broad-spectrum Drug Resistance

MRSA is not only resistant to methicillin, but also to all other β-lactam and cephalosporins with the same structure as methicillin. MRSA can also resist aminoglycosides, macrolides, tetracyclines, fluoroquinolones, sulfa drugs, and rifampicin through mechanisms such as changing the target sites of antibiotics, producing modifying enzymes, reducing membrane permeability, and producing a large amount of PABA. But it is sensitive to vancomycin.

Special Growth Characteristics

MRSA grows slowly and grows faster under conditions of 30°C, culture medium pH 7.0, and high osmotic pressure (40 g/L NaCl solution). At 30°C, heterogeneous resistant strains appear to be uniformly resistant and highly resistant, returning to heterogeneous resistance at 37°C. Uniformly resistant strains can be inhibited and appear sensitive at >37°C or pH<5.2. Increasing the concentration of NaCl, low temperature incubation, and extension of time can fully express the drug resistance in the sensitive subgroup of the heterogeneous resistant strain group, i.e., they can tolerate a higher concentration of methicillin, and have no effect on the resistant subgroup.

However, recent reports suggest that extending the culture time under high osmotic conditions can affect the detection of MRSA, because in high salt situations, after 48 hours of culture, methicillin-sensitive Staphylococcus aureus (MSSA) is easy to produce a large amount of β-lactamase, which can slowly hydrolyze methicillin, leading to bacterial growth and misrecognition as MSSA. Therefore, MRSA should be incubated in a high salt environment for 24 hours, and Methicillin-Resistant Coagulase-Negative Staphylococci (MRCNS), due to the smaller number of resistant subgroup bacteria than golden staphylococci, should be observed for 48 hours.

Mechanism of MRSA Drug Resistance

Innate Resistance

This is chromosomal mediated resistance, associated with the production of a type of penicillin binding protein (PBP) in the bacterium. Five types of PBP (1, 2, 3, 3' and 4) are produced, which have the function of synthesizing the bacterial cell wall. They have a high affinity for β-lactam antibiotics and can covalently bind to the active sites of β-lactam drugs, losing their activity and causing bacterial death.

However, MRSA produces a unique PBP, a PBP that has increased in molecular weight by 78 to 1000 Dalton, and is therefore called PBP2a or PBP2', because its electrophoretic mobility is between PBP2 and PBP3. PBP2a has a very low affinity for β-lactam antibiotics and is therefore rarely or not bound by β-lactam drugs. Bacteria can still grow in the presence of β-lactam antibiotics, showing resistance. The production of PBP2a is regulated by the chromosomal methicillin resistance gene (mec A). The fundamental difference between MRSA and MSSA lies in their different PBPs.

Acquired Resistance

This is plasmid-mediated resistance. Some strains show resistance by producing a large amount of β-lactamase through drug resistance factors, which slowly deactivates the enzyme-resistant penicillin, generally exhibiting high levels of resistance.

Typing of MRSA

Typing MRSA plays a crucial role in tracing the source of infection and examining the relationship between type and infection category and drug resistance. Bacteriophage typing, started earlier overseas, involves culturing the tested bacteria in broth at 35°C for 6 hours, smearing on a typing agar plate, and injecting 23 kinds of bacteriophages into the small squares in the agar plate after it dries, then incubating at 35°C for 6 hours before transferring to room temperature overnight to observe the results.

With 4 groups of 23 bacteriophages, MRSA can be divided into 4 groups, often with group Ⅰ being the most numerous, and reports also show group Ⅲ as the largest. Bacteriophage typing results are often unsatisfactory, with 29.3% of the strains are incapable of typing as confirmed by Japan's Komatsuzaki, and its repeatability is poor, making it unsuitable for epidemiological investigation. Plasmid profile typing is more reliable, and it can be divided into 18 types, accurately analyzing the correlation between strains, distinguishing epidemic strains from non-epidemic strains.

In China, MRSA widely carries plasmids with molecular weights of 1.6 MD, 1.8 MD, and 2.67 MD, and specialized plasmid bands may appear in different regions and hospitals. The Immunoblot typing method divides MRSA into 9 types, B and C being the most common, each type containing characteristic molecular bands, it is relatively stable.

Chromosomal restriction endonuclease analysis can identify specific sites and nucleotide sequences on the pathogen DNA chain, showing pathogen characteristics at the genetic level. MRSA can also be typed using serology, coagulase, resistance spectrum, and other methods. Southern blotting is also gradually being applied to MRSA typing.

Detection of MRSA

Due to the heterogeneity in drug resistance of MRSA, it brings some difficulties to its detection. The detection rate for MRSA is influenced by many factors such as incubation temperature, time, pH, NaCl concentration of the culture medium, amount of bacterial fluid, etc. Therefore, at present, there isn't a single best detection method.

Disc Diffusion Method (K-B Method)

The thickness of MH agar in the petri dish is 4mm, the bacterial fluid is adjusted to a 0.5 McFarland standard, smeared on the above petri dish. With 5 µg/piece of methicillin, incubation at 35 °C for 24 hours; inhibition zone of ≤11mm indicates resistance, ≥17mm indicates sensitivity. Since MRSA is usually also resistant to other beta-lactamase-resistant semi-synthesized penicillins, the National Committee for Clinical Laboratory Standards (NCCLS) recommends the use of oxacillin to replace methicillin for MRSA detection. The amount of oxacillin is 1 µg/piece: an inhibition zone of ≤10mm means drug resistance, ≥13mm means sensitivity, and 11-12mm means intermediate. Control strains are Staphylococcus aureus ATCC 29213 (resistant strain), Staphylococcus aureus ATCC 25923 (sensitive strain).

The biggest advantage of the Disc Diffusion Method is that it's fast, easy, and cheap, thus easily accepted by laboratory personnel. Under appropriate conditions such as the right antibiotic, incubation temperature, the concentration of bacterial fluid, thickness of the culture medium, etc., MRSA detection is feasible.

Broth Microdilution (MIC) Method

The Centers for Disease Control and Prevention (CDC) recommends the use of a MH broth culture medium adding NaCl up to a concentration of 20g/L, along with Ca and Mg ions. The antibiotic oxacillin is diluted in a series from 0.125 to 16 µg/ml, with a bacterial concentration of 104/mL and incubated at 35°C for 24 hours. If the MIC is <2 µg/ml, it is considered sensitive, while >4 µg/ml is seen as resistance. The detection rate can reach 95% with this method, but it's more tedious to operate.

Agar Dilution (MIC) Method

Using MH agar containing 20g/L NaCl, oxacillin is diluted in a series to 12 different concentrations and poured into petri dishes. The final concentration of oxacillin varies from 0.125 to 256 µg/ml. Then, bacterial suspension adjusted to 0.5 McFarland standard is spot-plated on the medium with different oxacillin concentrations, and the plates are incubated for 24 hours at 35°C. This method is suitable for detecting a large number of MRSA strains, and the results are straightforward with good repeatability. However, it is time-consuming and labor-intensive.

Agar Screening Method

This is a confirmatory test for MRSA recommended by NCCLS in 1997, which involves adding oxacillin (6 µg/ml) to MH culture base plus 40g/L NaCl. Then, bacterial suspension (0.5 McFarland standard) is spot-plated or streak-plated and incubated at 35°C for 24 hours. Even if there is only one colony grown on the plate, it is considered MRSA. The sensitivity of this method is 100%, often used as a standard to calibrate other methods. It is particularly suitable for the detection of Staphylococcus aureus with intermediate sensitivity.

Gradient Concentration (Etest) Method

This was introduced by the AB Biodisk company in 1988. On an MH agar plate with 20g/L NaCl, an oxacillin strip is attached. The bacterial fluid is adjusted to a McFarland standard of 0.5 to 1, and incubated at 35°C for 24 hours, then the MIC value is directly read. MIC <2µg/ml means sensitivity, >4µg/ml means resistance. The Etest method combines the advantages of the disc diffusion method and the broth dilution method. The long plastic strip contains a continuous gradient of oxacillin (0.016~256 µg/ml) which is exponentially varied, thus resulting in more accurate detection of MRSA with low or medium levels of resistance. Novak et al. reported that the results of the Etest and Alamar methods for the detection of 127 MRSA strains were highly correlated. Out of 127 MRSA strains detected by the Etest method, 93 strains had MIC >256 µg/ml, and 28 strains were between 6~256 µg/ml, with a detection rate up to 96%. The Etest method is accurate, reliable, and stable, but the downside is its high cost.

Automated Antimicrobial Susceptibility Testing

Currently, there are systems such as the Vitek system, ATB system, MicroScan system, Sensititre ARIS, etc. The bacterial suspension is diluted and injected into the drug sensitivity plate or holes, and the results are determined by detecting the turbidity of the bacterial fluid, the fluorescence intensity of the fluorescence indicator, or the hydrolysis reaction of the fluorescent substrate. Its advantages include fast processing time, but in the case of MRSA that grows slowly or expresses resistance slowly, it may not reach the detectable level within 3-4 hours, thus possibly resulting in missed diagnoses or false MRSA reports.

DNA Probe Hybridization

The above methods all test the MRSA's resistant phenotype. MRSA can be classified into categories 1, 2, 3, and 4, with resistance frequencies of 10^-7, 10^-4, 10^-3, and 10^-1 respectively. The conventional detection methods listed above generally do not pose a problem with types 3 and 4 MRSA, but can easily miss the detection of low-frequency types 1 and 2 MRSA. Therefore, for MRSA with low-level resistance or borderline resistance, molecular biology methods with high specificity should be chosen for detection.

DNA probe hybridization uses a specific mecA DNA fragment marked with Digoxin to hybridize with the suspected strain. Some scholars have reported that DNA probes only hybridize with MRSA DNA, and there are no hybridization bands with MSSA DNA. Its specificity is higher than the agar dilution method, and its sensitivity is higher than the broth dilition method. Moreover, it can be directly applied to clinical specimens without needing prior bacterial isolation and cultivation. However, probes are more expensive and have a shorter shelf-life.

PCR (Polymerase Chain Reaction) Technique

In the late 1980s, researchers abroad used Polymerase Chain Reaction (PCR) to detect the mecA gene of PBP2a. This method is based on designing a primer according to the DNA sequence of the mecA gene of Staphylococcus aureus TK 784. The DNA of the bacteria to be tested is then extracted and amplified under certain conditions. After agarose gel electrophoresis, bands are observed under UV light to check for the presence of a band identical to the positive control strain (Staphylococcus aureus ATCC 29213).

PCR has high sensitivity; as long as the tested bacteria have a trace of the gene, a positive result will appear. Therefore, it is often used as a reference method for detecting MRSA. Chen Xiushu's experiment showed that the resistance level of Staphylococcus aureus to oxacillin has a good correlation with the mecA gene. The mecA gene was detected in all 22 strains of bacteria with MIC >4 µg/ml. However, since PCR is very sensitive, false positives may occur due to laboratory contamination.

To enhance PCR's reliability, its amplified product should be subjected to probe hybridization or sequencing to improve specificity. However, some drug-resistant genes are silent genes and do not express the mecA gene product, which may lead to false resistance conclusions. Therefore, molecular biology methods are not 100% sensitive and specific. Moreover, the pre-processing operation of this method is complicated and requires certain equipment, so this test is only performed in doubtful or special situations.

Epidemiology of MRSA Infections

Since the discovery of MRSA in the UK in 1961, nosocomial infections caused by MRSA have been reported in Europe, America and parts of Asia. From the late 1960s to the 1980s, the MRSA infection rate greatly increased. According to NNIS reports in the United States, MRSA constituted 2.4% of total Staphylococcus aureus infections in 182 hospitals in 1975, a figure that had risen to 24.8% by 1991, particularly in teaching hospitals and central hospitals with over 500 beds.

These hospitals had higher chances of MRSA infections, antibiotic-resistant strains could either be brought into the hospital by infected patients or could develop within the hospital due to antibiotic misuse:

• - In Europe, by 1993, up to 60% of Staphylococcus aureus species isolated from the Intensive Care Units (ICUs) of 1,417 hospitals were MRSA.
• - In Japan, the rate of MRSA isolation reached 41% at the Kansai Medical University Hospital by 1993.
• - In China, MRSA was detected in the 1970s and the rate has been increasing annually. In 1978, only 5% out of 200 Staphylococcus aureus strains were MRSA in Shanghai, but by 1988, this figure rose to 24%, and in 1996, surged to 72%. In Tianjin, the MRSA isolation rate was 47% in 1988, while at the Beijing Medical University Affiliated Hospital, it was 58.3% in 1996. In Huaifang, Shandong, 198 strains of Staphylococcus aureus were isolated from nurseries of three hospitals in 1996, 112 of which were MRSA (56.5%). In 1992, the Tongji Medical University Affiliated Hospital in Wuhan had an MRSA isolation rate of 79.6%.

MRSA infections commonly occur in immunodeficient individuals, those with extensive burns, post-major surgery patients, long-term hospital residents, and elderly patients. MRSA is prone to cause epidemic and outbreak of infection. MRSA transmission primarily occurs through the hands of medical staff, spreading between patients and healthcare workers. Clothing and dressings can also carry MRSA, promoting its spread in hospitals. Once a patient is infected or carrying MRSA, the bacteria can persist on them for several months.

Treatment and Prevention of MRSA

Treatment of MRSA

Treating MRSA infection is one of the most difficult challenges in clinical practice due to its multi-drug resistance. Vancomycin is currently the only antibiotic with proven efficacy against MRSA. Over 30 years of use, no antibiotic-resistant strains have been found. Vancomycin is a tricyclic glycopeptide antibiotic that inhibits cell wall synthesis, disrupts cell membranes, and inhibits bacterial RNA synthesis.

In 1996, Japan reported the first case of a vancomycin-modestly sensitive Staphylococcus aureus (MIC = 8 μg/ml). In 1997, the United States isolated a vancomycin-moderately sensitive Staphylococcus aureus from a patient with peritonitis. According to NCCLS standards, senstivity to vancomycin is ≤4 μg/ml, moderate sensitivity is 8-16 μg/ml, and antibiotic resistance is ≥32 μg/ml. So far globally, no Staphylococcus aureus strain with MIC>32 μg/ml has been detected.

However, with the widespread use of vancomycin, it is expected that vancomycin-resistant MRSA will appear soon, hence the need to use vancomycin judiciously. Apart from vancomycin, new drugs such as wall teicoplanin and coumamycin have potent antimicrobial activity against MRSA. Furthermore, vancomycin can also be used in conjunction with fosfomycin, rifampin, aminoglycosides, and quinolones to improve treatment effects.

Prevention of MRSA

The first step is the rational use of antibiotics. Currently, the overuse of antibiotics in clinical settings has played a pivotal role in the spread of MRSA. Therefore, caution should be taken when choosing antibiotics to avoid the development of MRSA strains. For example, to prevent deep staphylococcal infection after major surgery, the use of first and second-generation cephalosporins (such as cefazolin, cefuroxime) is preferable. Though third-generation cephalosporins do not fare as well as the first generation against Staphylococcus aureus. Long-term use of third-generation cephalosporins correlates with the appearance of MRSA.

Early Detection of Carriers

Hospitals should intensify checks on new admissions and those susceptible to MRSA, especially patients in the burn units, ICUs, respiratory wards, hematology, and pediatrics departments. Meanwhile, accurate detection methods should be adopted in microbiology labs to promptly detect MRSA and inform clinical teams for infection control and isolation treatment.

Strengthening Disinfection

Medical staff should strictly disinfect their hands before and after examining patients. If possible, disposable masks, hats, gloves should be used, and medical supplies should be kept separate to prevent cross-infection within the hospital.

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