Detection Of Methicillin-resistant Staphylococci Research Articles

Evaluation of in-house cefoxitin screening broth to determine methicillin-resistant staphylococci

Methicillin-resistant staphylococci (MRS), including methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative staphylococci (MRCoNS), have a global impact as a public health threat contributing significantly to morbidity, mortality, and socio-economic costs. Accurate and rapid detection of MRS results in effective antimicrobial therapy, immediate patient isolation, dissemination control, and appropriate disinfection measures. Herein, we developed an in-house cefoxitin screening broth and compared it to the cefoxitin disk diffusion method and polymerase chain reaction (PCR) for the detection of MRS. Verification of this screening broth on 52 MRSA, 37 MRCoNS, 44 methicillin-susceptible S. aureus (MSSA), and 11 MSCoNS revealed greater validity for MRSA/MSSA than for MRCoNS/MSCoNS. The kappa coefficient of 0.87 was superior for determination of MRSA and MSSA, whereas it was 0.54, which was considered poor, for determination of MRCoNS and MSCoNS. Application of this assay to screen MRSA should be useful in clinical laboratories and hospital infection-control units.

Direct detection of methicillin-resistant in Staphylococcus spp. in positive blood culture by isothermal recombinase polymerase amplification combined with lateral flow dipstick assay.

Methicillin-resistant staphylococci (MRS) are important antimicrobial-resistant pathogens in sepsis. Conventional blood cultures take 24-72h. The polymerase chain reaction (PCR)-based methods give faster results (2-3h) but need expensive thermal cyclers. We therefore developed an isothermal recombinase polymerase amplification (RPA) combined with lateral flow dipstick (LFD) assay for rapid detection of MRS in spiked blood culture samples. Fifty-six clinical isolates including 38 mecA-carrying staphylococci and 18 non-mecA-carrying organisms as confirmed by PCR methods were studied. RPA primer set and probe specific for mecA gene (encoding penicillin-binding protein 2a) were designed. RPA reaction was carried out under isothermal condition (45°C) within 20min and read by LFD in 5min. The RPA-LFD provided 92.1% (35/38) sensitivity for identifying MRS in positive blood culture samples, and no cross-amplification was found (100% specificity). This test failed to detect three mecA-carrying S.sciuri isolates. The detection limits of RPA-LFD method for identifying MRS were equal to those of PCR method. The RPA-LFD is simple, fast, and user-friendly. This method could detect the mecA gene directly from the positive blood culture samples without requirement for special equipment. This method would be useful for appropriate antibiotic therapy and infection control, particularly in a low-resource setting.

Direct Detection of Methicillin Resistant Staphylococci: Comparison of Phenotypic Methods with Multiplex PCR and Direct Susceptibility Testing

Bloodstream infections are augean diseases characterized by a high morbidity and mortality, related with the lag in administration of the first adequate anti-infectious agent. Clinical and epidemiological data guide the clinicians towards empirical anti-infectious treatments whose effectualness remains questionable especially in the present day milieu of multidrug-resistant organisms. Early microbiological evidence of the causative agent is hierophantic of antimicrobial stewardship. We evaluated three rapid methods for the direct identification of S. aureus from 720 positive blood cultures in comparison to Vitek 2 automated microbial identification system. Early distinction S. aureus from CoNS was attempted with Direct Tube Coagulase. Multiplex PCR assay was used to separate MRSA from MSSA by the presence of mecA gene. Direct antibiotic susceptibility (DST) from positive blood culture bottles was performed in an endeavour to reduce time and compared to the routine Vitek 2 compact antimicrobial susceptibility. For Direct tube coagulase at 4 hrs of incubation sensitivity was 79.6% while specificity was 100% however, on overnight incubation sensitivity increased to 97.9% with 100% specificity. The positive predictive value and negative predictive values were 100% and 96.9% respectively when compared to Vitek 2 identification of staphylococcal species. By DST Categorical agreement of 95% was seen for coagulase negative staphylococci microorganism-antibiotic combinations. Categorical agreement of 89.7% was seen for S. aureus microorganism-antibiotic combinations. Direct multiplex PCR testing did not misidentify any S. aureus isolate compared to Vitek 2. In 12 methicillin resistant CoNS isolates mecA gene was not detected by PCR. In total 13(3.5%) strains of staphylococci identified by DST as methicillin resistant were not identified by PCR analysis. Each of the tests has positive qualities and all may have a place in a Gram positive cocci algorithm for testing blood cultures depending on the laboratory setting, workload volume and staffing. However, rapid detection methods are a pressing priority.

Molecular detection of methicillin resistant Staphylococcus aureus harbouring β- lactamase resistance genes isolated from different sources of infections

Background and objective: The detection and investigation of methicillin resistance staphylococci specifically S. aureus in clinical microbiology setting is very helpful both for informing the appropriate treatment of individual patients and also for the surveillance of these organisms. This study aimed at the rapid molecular detection of methicillin resistant staphylococci harbouring β-lactamase gene and determination of the efficiency of m-PCR through comparison with uniplex PCR. Methods: Standard microbiological techniques were applied for the determination of the presence of methicillin resistant S. aureus in samples recovered from different body sites of patients who attended Al-Kadhumyhia Teaching and Baghdad Teaching Hospitals. The resulting methicillin resistant S. aureus (MRSA) isolates were subjected to uni and multiplex PCR amplifications for detecting the existence of mec A gene and β-lactamase (TEM) resistance gene. Results: Half of the cases involved were found to be caused by MRSA. All the tested isolates showed positive amplification bands for the presence of mec A gene and only 48.8% of these harbored TEM gene. The obtained results revealed high sensitivity of universal bacterial and TEM primers expressed as 97.6% and 100% respectively, while the sensitivity of mec A primer was limited to 60%. Conclusion: The phenotypic identification of MRSA revealed a higher incidence rate and that different molecular techniques can yield conflicting results and it can also be concluded that resistant due to beta- lactamase production can be a crucial factor added to the previously settled methicillin resistant due to mec A gene.

Detection of methicillin-resistant staphylococci by biosensor assay consisting of nanoscale films on optical fiber long-period gratings.

Methicillin-resistance among Staphylococcus species is a major health problem in hospitals, communities, and animals. There is a need for culture-free diagnostic assays that can be carried out rapidly, and maintain a high degree of sensitivity and specificity. To address this need an ionic self-assembled multilayer (ISAM) film was deposited on the surface of a long-period grating (LPG) optical fiber by immersion alternately in poly-allylamine hydrochloride and in poly-1-[p-(3'-carboxy-4'-hydroxyphenylazo) benzenesulfonamido]-1,2-ethandiyl (PCBS), resulting in terminal carboxyl groups on the LPG-ISAM. The terminal carboxyl groups were covalently conjugated to monoclonal antibodies (MAb) specific to penicillin-binding-protein 2a of methicillin resistant (MR) staphylococci. After exposure of the LPG-ISAM to 10(2) colony forming units (CFU)/ml of MR S. aureus (MRSA) for 50 min., light transmission was reduced by 19.7%. In contrast, after exposure to 10(6) CFU/ml of methicillin-sensitive S. aureus (MSSA) attenuation of light transmission was less than 1.8%. Exposure of the LPG-ISAM to extracts of liver, lungs, or spleen from mice infected with MRSA attenuated light transmission by 11.7-73.5%. In contrast, exposure of the biosensor to extracts from MSSA-infected mice resulted in 5.6% or less attenuation of light transmission. When the sensor was tested with 36 strains of MR staphylococci, 15 strains of methicillin-sensitive staphylococci, 10 strains of heterologous genera (all at 10(4) CFU/ml), or tissue samples from mice infected with MRSA, there was complete agreement between MR and non-MR bacteria determined by antibiotic susceptibility testing and the biosensor assay when the cutoff value for attenuation of light transmission was 6.3%. Thus, the biosensor described has the potential to detect MR staphylococci in clinical samples with a high degree of sensitivity and specificity.

Isolation and Detection of Methicillin-Resistant Staphylococci in Healthy Broilers in Nsukka Southeast, Nigeria

This study was conducted to isolate and detect methicillin-resistant staphylococci (MRS) in healthy broilers in Nsukka Southeast, Nigeria and determine the antibiogram of the isolates. Cloacal and skin swabs were collected from each of 101 randomly sampled broilers meant for slaughter. The samples were processed for isolation and identification of methicillin-resistant Staphylococcus species, following standard methods. Confirmation of methicillin-resistance by the isolates was done using penicillin binding protein 2a (PBP2a) kit. Phenotypic resistance of the isolates to antimicrobial agents was determined using disc diffusion method. Out of 202 samples processed, 200 (99.01%) yielded positive growth of staphylococci on oxacillin-supplemented oxacillin-resistance staphylococcal agar base (ORSAB). A total of 200 methicillin-resistant staphylococcal isolates were obtained. Of these, 91 (45.5%) were identified as methicillin-resistant coagulase-positive Staphylococcus (MRCoPS), while 109 (54.5%) were identified as methicillin-resistant coagulase-negative Staphylococcus species (MRCoNS). Out of the 91 MRCoPS, 53 (58.2%) were identified as methicillin-resistant Staphylococcus aureus (MRSA). Resistance of the isolates was 99.5% to erythromycin and chloramphenicol, 100% to oxacillin, 76.5% to gentamicin, 96.5% to clindamycin, 92.5% to ciprofloxacin, 99% to sulphamethoxazole/trimethoprim and tetracycline, and 98.5% to streptomycin and cefoxitin. All the isolates were multidrug resistant. This study has shown that healthy broilers reared and slaughtered in Nsukka Southeast, Nigeria harbour multidrug-resistant MRS and thus serve as their reservoirs.

Isolation and Detection of Methicillin-Resistant Staphylococci in Healthy Broilers in Nsukka Southeast, Nigeria

Isolation and Detection of Methicillin-Resistant Staphylococci in Healthy Broilers in Nsukka Southeast, Nigeria

Evaluation of rapid mecA gene detection versus standard culture in staphylococcal chronic prosthetic joint infections

In case of periprosthetic joint infections, the antibiotic treatment administered intraoperatively entails consequences on bacterial ecology with potential secondary effects. This study evaluates the rapid detection of methicillin-resistant staphylococci (MRS) by Xpert® technology directly on intraoperative samples. Xpert® technology was compared to conventional culture for 104 clinical specimens performed on 30 patients. The performance of the test expressed in terms of sensitivity, specificity, positive predictive value, and negative predictive value was, respectively, 87.1%, 100%, 100%, and 94.5% for the 104 specimens, and 92.3%, 100%, 100%, and 94.4% for the 30 patients. With the rapid detection of MRS, the use of vancomycin was limited for 17 of these 30 patients. In conclusion, this technique would allow the implementation of first-line antibiotic treatment adapted to the presence of MRS or not within approximately 1 h and would strongly reduce the use of broad-spectrum antibiotics.

Rapid detection of methicillin-resistant staphylococci by real-time PCR directly from positive blood culture bottles

For the rapid detection of methicillin-resistant staphylococci directly from blood cultures containing gram-positive cocci in clusters, we implemented a real-time (LightCycler) polymerase chain reaction (PCR) specific for the Staphylococcus aureus nuc gene encoding nuclease and the mecA gene encoding methicillin resistance. For the 475 positive blood cultures tested, the assay turned out to have 100% sensitivity and 100% specificity for the identification of methicillin-susceptible (n = 108) and methicillin-resistant (n = 34) S. aureus. When coagulase-negative staphylococci (CoNS) were included, the overall sensitivity for the detection of methicillin resistance was 93% and the specificity was 99%. Real-time PCR for nuc and mecA from blood culture bottles with staphylococci yields therefore a rapid (2-3 h) identification of S. aureus and CoNS including methicillin resistance.

Presence of mec Genes and Overproduction of Beta-Lactamase in the Expression of Low-Level Methicillin Resistance among Staphylococci

Detection of methicillin-resistant staphylococci is critical for the management of infected patients in the hospital. A total of 55 nonreplicated clinical isolates of staphylococci (31 Staphylococcus aureus and 24 coagulase-negative staphylococci; CNS) collected during a one-year period and expressing low-level resistance to methicillin (oxacillin MIC of 2–4 mg/l for S. aureus and 0.5–4 mg/l for CNS) were studied. mec determinants and overproduction of β-lactamase were investigated and pulsed-field gel electrophoresis (PFGE) was applied as a typing method. Twenty-four S. aureus isolates and 19 CNS carried the mecA gene. The presence of mecR1/mecI and blaR1/blaI genes correlated with the expression of low-level methicillin resistance in CNS. Four mecA-negative isolates (2 S. aureus and 2 CNS) overproduced β-lactamase. PFGE revealed the presence of 2 major clonal types in mecA-positive S. aureus isolates, and 3 in CNS. Low-level methicillin resistance of staphylococci is correlated with the presence of the mecA gene and overproduction of β-lactamase.

Detection of methicillin-resistant staphylococci by using the polymerase chain reaction.

A polymerase chain reaction (PCR)-based test was developed for the detection of mecA in staphylococci. To facilitate this process, a rapid cell lysis procedure was established for the release of DNA from staphylococcal strains. Primers based on the DNA sequence of the mecA gene from Staphylococcus aureus were used in PCRs to screen for the presence of this gene in a total of 98 staphylococcal isolates. Fifty-one isolates were mecA positive (17 S. aureus strains and 34 coagulase-negative staphylococci including S. epidermidis, S. haemolyticus, and S. simulans). Results obtained with PCRs were generally consistent with those of standard microbiological assays. PCRs designed to detect the femA gene (factor essential for methicillin resistance) revealed the presence of the gene in all S. aureus strains examined regardless of the susceptibility profiles of the strains to methicillin. In contrast, femA could not be detected in coagulase-negative staphylococci by PCR with the same primers. Low-stringency hybridization suggested the presence of a gene structurally related to femA in S. epidermidis and other coagulase-negative staphylococci examined.