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- Rivista
- eISSN
- 1875-855X
- Prima pubblicazione
- 01 Jun 2007
- Frequenza di pubblicazione
- 6 volte all'anno
- Lingue
- Inglese
To evaluate the sensitivity and specificity pattern of various phenotypic methods used in screening
Clinical isolates of
Out of 50
The combination of two phenotypic methods, CFD and oxacillin MIC, can be used for the detection of MRSA in clinical laboratories.
Keywords
- cefoxitin
- heterogenous population
The existence of a BORSA has made the detection of MRSA more complicated. BORSA isolates are characterized as small populations that exhibit an intermittent MIC ranging from 4 to 8 μg/mL even in the absence of
A total of 50 non-repetitive,
The antibiotic susceptibility test was performed by disk diffusion test using Mueller–Hinton agar (Hi-Media, India), following the CLSI guidelines [10]. Susceptibility test was performed using the following antibiotic disks: oxacillin (1 μg), cefotaxine (30 μg), cefoxitin (30 μg), methicillin (5 μg), penicillin (10 μg), clindamycin (2 μg), cefepime (30 μg), ampicillin (10 μg), erythromycin (15 μg), gentamycin (10 μg), ciprofloxacin (5 μg), tetracycline (30 μg), and rifampicin (5 μg). ATCC 25923 was used as a quality control strain.
The oxacillin agar screen test was performed in accordance with the CLSI guidelines to detect MRSA. The test involves spot inoculation of 0.5 McFarland standard suspension of each test isolates in Mueller–Hinton agar plates, containing 4% of NaCl and 6 μg/mL of oxacillin, and the plate was incubated at 37ºC for 24 h. Those isolates that showed any visible growth was considered as MRSA and the result obtained was quality controlled by ATCC 43300 [11, 12].
Minimum inhibitory concentration (MIC) for
All the isolates were subjected to CFD test using a 30 μg cefoxitin disk (Hi-Media, India). A bacterial suspension of 0.5 McFarland standard was prepared; the bacterial lawn was made on Muller–Hinton agar plate; and the cefoxitin disk was placed. Plates were incubated at 37°C for 16–18 h and zone diameters were measured. ATCC 25923 and ATCC 43300 were used as negative and positive quality control strains, respectively [15].
Penicillin solution (10,000 IU) of 100 μL was distributed in 96-well microtiter plates. The staphylococcal isolates were suspended in wells to obtain a final concentration of 109 cells/mL (McFarland equivalent of 4). Ten microliters of 1% freshly prepared starch indicator was added in each well and thoroughly mixed. The titer plates were incubated at room temperature for 30–60 min. After the incubation, one drop of iodine reagent was dispensed in each well and the test was performed in triplicates. The rapid decolorization of blue color within 10 min was regarded as positive for β-lactamase production and the prolonged existence of blue color was regarded as negative. ATCC 25923 and ATCC 29213 were used as negative and positive quality control strains, respectively [16].
Chromosomal DNA was extracted using phenol:chloroform method [17, 18] and it was used as a template DNA in polymerase chain reaction (PCR) for the detection of
All data analysis was performed using MedCalc Statistical Software version 19.1 (MedCalc Software Inc., Mariakerke, Belgium). Comparisons between four phenotypic methods with
A total of 50 staphylococcal isolates were used in this study, and the isolates were collected from pus (n = 31), urine (n = 2), sputum (n = 2), wound swab (n = 2), blood (n = 1), umbilical swab (n = 1), bronchial fluid (n = 1), catheter tip (n = 1), and some unknown (n = 9). All the isolates were coagulase positive and confirmed to be
All the isolates studied were observed to be resistant to ≥ 3 antibiotics, so they were categorized as multidrug resistant (MDR). The antibiotic resistance pattern was found to be highly resistant to methicillin (100%), penicillin (98%), ampicillin (96%), cefotaxime (80%) ciprofloxacin (78%), erythromycin (72%), cefepime (66%), cefoxitin (64%), oxacillin (56%), gentamycin (40%), tetracycline (8%), and rifampicin (8%) whereas clindamycin (4%) is the least resistant antibiotic. The resistance pattern of various antibiotic disks is shown in
Figure 1
The percentage of resistance (dark-gray bars) and susceptibility (light-gray bars) observed for 13 antibiotics by disk diffusion test
In this study, four phenotypic methods that are used routinely in laboratories for the detection of MRSA were evaluated for their accuracy. The presence of
Result of genotypic and phenotypic methods of 50
| SA1 | + | Growth | 128 (R) | Resistant | Resistant | Positive |
| SA2 | − | No growth | 1 (S) | Susceptible | Susceptible | Negative |
| SA3 | + | Growth | >128 (R) | Resistant | Resistant | Positive |
| SA4 | + | Growth | 64 (R) | Resistant | Resistant | Positive |
| SA5 | + | No growth | 8 (R) | Resistant | Resistant | Positive |
| SA6 | − | No growth | 8 (R) | Susceptible | Resistant | Positive |
| SA7 | + | Growth | >128 (R) | Resistant | Resistant | Positive |
| SA8 | − | Growth | 64 (R) | Resistant | Resistant | Positive |
| SA9 | + | Growth | 128 (R) | Resistant | Resistant | Positive |
| SA10 | + | Growth | 36 (R) | Resistant | Resistant | Positive |
| SA11 | + | Growth | >128 (R) | Resistant | Resistant | Positive |
| SA12 | + | No growth | 128 (R) | Resistant | Resistant | Positive |
| SA13 | + | Growth | >128 (R) | Resistant | Resistant | Positive |
| SA14 | + | Growth | >128 (R) | Resistant | Resistant | Positive |
| SA15 | + | Growth | 4 (R) | Resistant | Susceptible | Positive |
| SA16 | + | Growth | 8 (R) | Resistant | Susceptible | Positive |
| SA17 | + | Growth | 64 (R) | Resistant | Resistant | Positive |
| SA18 | + | Growth | >128 (R) | Resistant | Resistant | Positive |
| SA19 | − | No growth | 32 (R) | Resistant | Resistant | Positive |
| SA20 | − | Growth | >128 (R) | Resistant | Resistant | Positive |
| SA21 | + | Growth | >128 (R) | Resistant | Resistant | Positive |
| SA22 | − | Growth | 8 (R) | Susceptible | Resistant | Positive |
| SA23 | − | Growth | >128 (R) | Resistant | Resistant | Positive |
| SA24 | + | Growth | >128 (R) | Resistant | Resistant | Positive |
| SA25 | − | No growth | 1 (S) | Susceptible | Susceptible | Positive |
| SA26 | + | Growth | >128 (R) | Resistant | Resistant | Positive |
| SA27 | − | No growth | 1 (S) | Susceptible | Resistant | Positive |
| SA28 | − | Growth | 1 (S) | Susceptible | Susceptible | Positive |
| SA29 | − | Growth | >128 (R) | Resistant | Susceptible | Positive |
| SA30 | − | No growth | 64 (R) | Susceptible | Susceptible | Positive |
| SA31 | − | Growth | >128 (R) | Resistant | Resistant | Positive |
| SA32 | − | Growth | 32 (R) | Resistant | Resistant | Positive |
| SA33 | − | Growth | 64 (R) | Resistant | Resistant | Positive |
| SA34 | − | Growth | 2 (S) | Susceptible | Susceptible | Positive |
| SA35 | − | No growth | 1 (S) | Susceptible | Susceptible | Positive |
| SA36 | − | No growth | 0.5 (S) | Susceptible | Susceptible | Positive |
| SA37 | + | Growth | 8 (R) | Resistant | Resistant | Positive |
| SA38 | − | No growth | 1 (S) | Susceptible | Susceptible | Positive |
| SA39 | − | Growth | 64 (R) | Resistant | Resistant | Positive |
| SA40 | + | No growth | 16 (R) | Resistant | Susceptible | Positive |
| SA41 | − | No growth | 2 (S) | Susceptible | Susceptible | Positive |
| SA42 | − | No growth | 1 (S) | Susceptible | Susceptible | Positive |
| SA43 | − | Growth | 1 (S) | Susceptible | Susceptible | Positive |
| SA44 | − | No growth | 0.5 (S) | Susceptible | Susceptible | Positive |
| SA45 | − | Growth | 16 (R) | Resistant | Resistant | Positive |
| SA46 | − | Growth | 8 (R) | Resistant | Resistant | Positive |
| SA47 | − | No growth | 0.5 (S) | Susceptible | Susceptible | Positive |
| SA48 | + | Growth | 8 (R) | Resistant | Resistant | Positive |
| SA49 | − | No growth | 2 (S) | Susceptible | Susceptible | Positive |
| SA50 | − | Growth | 2 (S) | Susceptible | Resistant | Positive |
OSA, oxacillin screen agar; MIC, minimum inhibitory concentration; R, resistance; S, susceptible; +, positive; −, negative.
Out of 50 isolates screened for
Those isolates showing positive for
Isolates showing non-
Isolates with non-
In order to statistically determine the accuracy of four phenotypic studies taken into consideration, sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were calculated with the aid of MedCalc Statistical Software version 19.1, which showed that CFD had a high AUC of 0.810, 100% sensitivity, and 78% accuracy. The AUC and sensitivity for MIC were slightly equivalent to CFD, whereas the AUC and sensitivity for OD and OSA were below average. ROCs for four phenotypic methods calculated are shown in
Figure 2
Receiver operating characteristics (ROCs) for (
Comparison of phenotypic methods for the detection on methicillin resistance in
| 18 | 3 | 14 | 15 | 86 | 48 | 54.54 | 83.35 | 64 | |
| 21 | 0 | 15 | 14 | 100 | 52 | 60 | 100 | 72 | |
| 18 | 3 | 15 | 14 | 86 | 52 | 56.25 | 83.3 | 66 | |
| 21 | 0 | 18 | 11 | 100 | 62 | 65.6 | 100 | 78 | |
Correlation coefficient by Pearson's correlation coefficient method for comparison of phenotypic methods
| MIC+CFD | 0.8729 |
| MIC+OD | 0.6910 |
| OD+CFD | 0.6528 |
| CFD+OSA | 0.6052 |
| MIC+OSA | 0.5431 |
| OD+OSA | 0.5172 |
SN, sensitivity; SP, specificity; PPV, positive predicted value; NPV, negative predicted value; AUC, area under the curve; OSA, oxacillin screen agar test; MIC, minimal inhibitory concentration; OD, oxacillin disk diffusion; CFD, cefoxitin disk diffusion.
MIC, minimal inhibitory concentration; CFD, cefoxitin disk diffusion; OD, oxacillin disk diffusion; OSA, oxacillin screen agar test.
The infections caused by
Earlier Adhikari et al. had reported 100% sensitivity in CFD and oxacillin MIC method. CFD and oxacillin MIC method showed non-
The other two phenotypic methods in this study showed a low reliability, where OSA test could detect MRSA only in 18
A recent study by Basset et al. showed that the presence of
In the present study, 98% of the isolates were β-lactamase producers estimated by both penicillin disk diffusion test and iodometric method. Globally, β-lactamase rate for staphylococci lies from 56% to 93% [28]. Penicillin can be preferred as a treatment for MSSA infections which are penicillinase non-producers [29]. In our study, one MSSA showed susceptible to penicillin antibiotic and negative for β-lactamase production. All the isolates showed resistance to methicillin in disk diffusion test, including one penicillinase nonproducer. The rate of MRSA in our study was found to be 42%, which is high in comparison with a report given by Kogan et al. This illustrates that rate of the MRSA infection is escalating day by day [30, 31] and simple phenotypic MRSA detection methods such as CFD and oxacillin MIC can be preferred in routine clinical laboratories. In spite of various phenotypic and genotypic methods available for detecting methicillin resistance in
This study aims to bring to notice that there is a high change that few MRSA strains are undetected due to the lack of improper expression of resistant proteins that require inducers. Of all phenotypic methods studied earlier, the high accuracy was found to be shown by CFD test which is easy and labor-intensive, whereas genotypic screening and oxacillin MIC methods had an advantage in effective detection of BORSA populations. Even though the detection of BORSA was unpredictable, infection due to BORSA in an individual can be treated effectively by β-lactamase inhibitors. Thus this study concludes that screening of MRSA has become complicated in spite of various phenotypic methods available, and the existence of non-
Figure 1
Figure 2
Result of genotypic and phenotypic methods of 50 S. aureus
| SA1 | + | Growth | 128 (R) | Resistant | Resistant | Positive |
| SA2 | − | No growth | 1 (S) | Susceptible | Susceptible | Negative |
| SA3 | + | Growth | >128 (R) | Resistant | Resistant | Positive |
| SA4 | + | Growth | 64 (R) | Resistant | Resistant | Positive |
| SA5 | + | No growth | 8 (R) | Resistant | Resistant | Positive |
| SA6 | − | No growth | 8 (R) | Susceptible | Resistant | Positive |
| SA7 | + | Growth | >128 (R) | Resistant | Resistant | Positive |
| SA8 | − | Growth | 64 (R) | Resistant | Resistant | Positive |
| SA9 | + | Growth | 128 (R) | Resistant | Resistant | Positive |
| SA10 | + | Growth | 36 (R) | Resistant | Resistant | Positive |
| SA11 | + | Growth | >128 (R) | Resistant | Resistant | Positive |
| SA12 | + | No growth | 128 (R) | Resistant | Resistant | Positive |
| SA13 | + | Growth | >128 (R) | Resistant | Resistant | Positive |
| SA14 | + | Growth | >128 (R) | Resistant | Resistant | Positive |
| SA15 | + | Growth | 4 (R) | Resistant | Susceptible | Positive |
| SA16 | + | Growth | 8 (R) | Resistant | Susceptible | Positive |
| SA17 | + | Growth | 64 (R) | Resistant | Resistant | Positive |
| SA18 | + | Growth | >128 (R) | Resistant | Resistant | Positive |
| SA19 | − | No growth | 32 (R) | Resistant | Resistant | Positive |
| SA20 | − | Growth | >128 (R) | Resistant | Resistant | Positive |
| SA21 | + | Growth | >128 (R) | Resistant | Resistant | Positive |
| SA22 | − | Growth | 8 (R) | Susceptible | Resistant | Positive |
| SA23 | − | Growth | >128 (R) | Resistant | Resistant | Positive |
| SA24 | + | Growth | >128 (R) | Resistant | Resistant | Positive |
| SA25 | − | No growth | 1 (S) | Susceptible | Susceptible | Positive |
| SA26 | + | Growth | >128 (R) | Resistant | Resistant | Positive |
| SA27 | − | No growth | 1 (S) | Susceptible | Resistant | Positive |
| SA28 | − | Growth | 1 (S) | Susceptible | Susceptible | Positive |
| SA29 | − | Growth | >128 (R) | Resistant | Susceptible | Positive |
| SA30 | − | No growth | 64 (R) | Susceptible | Susceptible | Positive |
| SA31 | − | Growth | >128 (R) | Resistant | Resistant | Positive |
| SA32 | − | Growth | 32 (R) | Resistant | Resistant | Positive |
| SA33 | − | Growth | 64 (R) | Resistant | Resistant | Positive |
| SA34 | − | Growth | 2 (S) | Susceptible | Susceptible | Positive |
| SA35 | − | No growth | 1 (S) | Susceptible | Susceptible | Positive |
| SA36 | − | No growth | 0.5 (S) | Susceptible | Susceptible | Positive |
| SA37 | + | Growth | 8 (R) | Resistant | Resistant | Positive |
| SA38 | − | No growth | 1 (S) | Susceptible | Susceptible | Positive |
| SA39 | − | Growth | 64 (R) | Resistant | Resistant | Positive |
| SA40 | + | No growth | 16 (R) | Resistant | Susceptible | Positive |
| SA41 | − | No growth | 2 (S) | Susceptible | Susceptible | Positive |
| SA42 | − | No growth | 1 (S) | Susceptible | Susceptible | Positive |
| SA43 | − | Growth | 1 (S) | Susceptible | Susceptible | Positive |
| SA44 | − | No growth | 0.5 (S) | Susceptible | Susceptible | Positive |
| SA45 | − | Growth | 16 (R) | Resistant | Resistant | Positive |
| SA46 | − | Growth | 8 (R) | Resistant | Resistant | Positive |
| SA47 | − | No growth | 0.5 (S) | Susceptible | Susceptible | Positive |
| SA48 | + | Growth | 8 (R) | Resistant | Resistant | Positive |
| SA49 | − | No growth | 2 (S) | Susceptible | Susceptible | Positive |
| SA50 | − | Growth | 2 (S) | Susceptible | Resistant | Positive |
Correlation coefficient by Pearson's correlation coefficient method for comparison of phenotypic methods
| MIC+CFD | 0.8729 |
| MIC+OD | 0.6910 |
| OD+CFD | 0.6528 |
| CFD+OSA | 0.6052 |
| MIC+OSA | 0.5431 |
| OD+OSA | 0.5172 |
Comparison of phenotypic methods for the detection on methicillin resistance in S. aureus
| 18 | 3 | 14 | 15 | 86 | 48 | 54.54 | 83.35 | 64 | |
| 21 | 0 | 15 | 14 | 100 | 52 | 60 | 100 | 72 | |
| 18 | 3 | 15 | 14 | 86 | 52 | 56.25 | 83.3 | 66 | |
| 21 | 0 | 18 | 11 | 100 | 62 | 65.6 | 100 | 78 | |
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