a Department of Pathology (Clinical Microbiology), Hershey Medical Center, 500 University Drive, Hershey, PA 17033, USA; b Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd, Kohoku-ku, Yokohama 222-8567, Japan; c Department of Pathology (Clinical Microbiology), Case Western Reserve University, Cleveland, OH 44106, USA; d Institute of Microbial Chemistry, Shinagawa-ku, Tokyo, 141-0021, Japan
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Abstract |
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Introduction |
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Materials and methods |
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We tested 148 clinical isolates of S. pneumoniae screened by optochin susceptibility and bile solubility collected during the past 5 years from the following countries: Bulgaria (n = 3), Canada (n = 9), Greece (n = 16), Poland (n = 1), Romania (n = 6), Slovenia (n = 8), South Africa (n = 2), Spain (n = 4) and the USA (n = 99).
Susceptibility testing
Agar dilution MICs of penicillin G (Sigma, St Louis, MO, USA), ceftriaxone (Sigma), erythromycin (Abbott Laboratories, Chicago, IL, USA), azithromycin (Pfizer, Groton, CT, USA) and clindamycin (Pharmacia & Upjohn, Kalamazoo, MI, USA) were determined.8 Each compound was obtained from its manufacturer. Standard quality control strains, including S. pneumoniae ATCC 49619, were included in each run. NCCLS susceptibility breakpoints9 were used to interpret MICs.
PCR primers
The sequences of the primers used for PCR are as follows: lytA: 5'-681CAACCGTACAGAATGAAGCGG701-3', 5'-999TTATTCGTGCAATACTCGTGCG978-3'; pbp1a: 5'-2256AAACAAGGTCGGACTCAACC2275-3', 5'-2450AT ATACATTGGTTTATAGTAAGTT2427-3'; pbp2x: 5'-1255CCAGGTTCCACTATGAAAGTG1275-3', 5'-1451ATC CCAACGTTACTTGAGTGT1431-3'; pbp2b: 5'-1566CCTA TATGGTCCAAACAGCCT1586-3', 5'-1693GGTCAATTC CTGTCGCAGTA1712-3'; mef(A): 5'-180CTGTATGGAG CTACCTGTCTGG199-3', 5'-581CCCAGCTTAGGTATACGTAC562-3'; erm(B): 5'-721CGTACCTTGGATATT CACCG740-3', 5'-944GTAAACAGTTGACGATATTCT CG922-3'.
The oligonucleotide primers for detection of three pbp genes were designed to amplify parts of the pbp1a, 2x and 2b genes only in susceptible strains. These parts were positioned in blocks of highly diverged sequences identified in the mosaic pbp genes of penicillin non-susceptible S. pneumoniae. Primer mixture A contained the primers for detecting lytA and pbp1a genes, primer mixture B contained the primers for detecting pbp2x and 2b genes, and primer mixture C contained the primers for detectin mef(A) and erm(B). Each primer mixture (100 µL), which contained 0.1 µM of each primer and 8 mM dNTPs, is available commercially in Japan (Wakunaga Pharmaceutical, Co., Ltd, Hiroshima, Japan). Before testing, 50 µL of 10x PCR buffer (50 mM KCl, 10 mM TrisHCl pH 8.4, 1.5 mM MgCl2), 20 U of Tth DNA polymerase (Toyobo, Co., Ltd, Osaka, Japan) and 348 µL of dH2O, were added to each primer mixture. Then, it was divided into 30 µL aliquots and stored at -30°C.
PCR condition
A single colony of S. pneumoniae grown on a blood agar plate was suspended in 30 µL of lysis solution. The composition of the lysis solution has been reported previously.7 The tubes with the lysis solution were set into a thermal cycler (GeneAmp 9700; PE Applied Biosystems, Foster City, CA, USA) and incubated at 60°C for 10 min and 94°C for 5 min. These lysates were then used as template DNA for PCR.
Next, 2 µL of the bacterial lysate was added to each of three tubes containing primer mixtures A, B and C. A positive and negative control was included in each run. PCR was performed with the thermal cycler for 30 cycles at 94°C for 15 s, 30 cycles at 53°C for 15 s and 30 cycles 72°C for 15 s. Following amplification, 10 µL of each of the three PCR products was electrophoresed on a 3% agarose gel (Agarose LE; Promega Co., Madison, WI, USA) for 40 min at 100 V.
For interpretation of PCR results, the three bands were seen on the agarose gel if the isolate did not have altered (abnormal) pbps, while one or more of these bands were not detected for strains with alterations in the pbps. Bands appeared on the gel if the isolate has the erm(B) and/or mef(A) gene. The positions of DNA fragments amplified from a positive control in this primer mix and three isolates tested are shown in the Figure.
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Results |
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PCR results and the MIC distribution of penicillin G and ceftriaxone are shown in the Table. Thirty-eight (86.4%, 95% CI 72.794.8) of the 44 penicillin G-susceptible isolates (MIC
0.06 mg/L) had unaltered pbps, while six isolates (13.6%, 95% CI 5.227.4) had either one or two alterations in pbps. Twenty-four of the 58 (41.4%, 95% CI 28.655.1) penicillin G-intermediate isolates had alterations in all three pbps, 17 isolates (32.1%, 95% CI 20.346.0) had two pbps altered and 20 isolates (34.5%, 95% CI 22.548.1) had one pbp altered. Of 47 penicillin G-resistant isolates (MIC
2 mg/L), 41 isolates (87.3%, 95% CI 74.395.2) had all three pbps altered, six isolates (12.8%, 95% CI 4.825.7) had altered pbp1a + 2x.
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All 84 pneumococcal isolates that were susceptible to erythromycin, azithromycin and clindamycin, were negative for both mef(A) and erm(B). MIC90s (MIC ranges) (mg/L) for these isolates were: 0.06 (<0.0080.12), erythromycin; 0.12 (0.0160.25), azithromycin; 0.06 (0.0160.12), clindamycin. mef(A) was found in 18 isolates with MIC90s (MIC ranges) (mg/L) of 8 (0.58), erythromycin; 8 (116), azithromycin; 0.12 (0.030.12), clindamycin. All 18 strains with mef(A) were susceptible to clindamycin.
erm(B) was found in 43 isolates for which the MIC90s were >64 mg/L of erythromycin (MIC range 2>64 mg/L), azithromycin (MIC range 8>64 mg/L) and clindamycin (MIC range 2>64 mg/L). Three isolates had both mef(A) and erm(B), and MICs for these isolates were >64 mg/L of erythromycin, azithromycin and clindamycin.
All 148 isolates were optochin sensitive, bile soluble and were positive for lytA by PCR. Positive and negative controls were detected correctly in each run.
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Discussion |
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Optochin susceptibility and bile solubility have been recommended for screening for S. pneumoniae; however, the lytA gene seemed to have good reliability for screening of pneumococci in this study. The specificity of the lytA gene for S. pneumoniae had already been tested when the primers were developed. Streptococcus oralis, Streptococcus mitis, Streptococcus salivarius, Staphylococcus aureus and Staphylococcus epidermidis were all lytA negative; only S. pneumoniae proved lytA positive (K. Ubukata, unpublished data).
Determination of the pbp genotype seemed to be useful in estimating the degree of penicillin G resistance in our study. Six penicillin G-susceptible isolates (MIC range 0.030.06 mg/L) had one or two altered pbp(s) by PCR. Similar patterns have been seen in a previous study.7 Antibiotic treatment of the latter strains, especially in meningitis, should be chosen carefully because these strains may not be detected by conventional oxacillin screening, but may also have altered pbps.
MICs of ceftriaxone for isolates with alteration of pbp1a + 2x or all three pbps were higher (0.5 mg/L) and those patterns of altered pbps might be useful in estimating isolates with higher MICs of this agent. However, 10 isolates for which ceftriaxone MICs were 0.060.25 mg/L also had these alterations. DNA sequencing of pbp1a, 2x and 2b for six of the latter isolates (penicillin G MICs 0.250.5 mg/L) showed that there was no mutation in the 370SerThrMetLys373 motif, which is the area of conserved amino acids in pbp1a. The primers for pbp1a were located c. 240 bp away from the SerThrMetLys region, and those isolates that had altered pbp2x + 2b had point mutations in the region of the pbp1a primers. These results indicate that the results of pbp1a PCR for those six isolates were false positive and re-design of pbp1a primers is necessary to detect isolates in those categories more accurately.
Macrolide resistance mechanisms, erm(B) and mef(A), were accurately detected and correlated with MICs of erythromycin, azithromycin and clindamycin. The PCR results in this study matched our previous results (P. C. Appelbaum, unpublished data), which had been been determined already using other primers described by Sutcliffe et al.10
In summary, the primers that we tested may be helpful for rapid screening of pneumococcal isolates from patients with severe systemic infection such as meningitis or life-threatening pneumonia, where rapid MIC results are necessary. It was also thought to be useful for detection of common macrolide resistance. However, more appropriate primers are required to detect and distinguish ß-lactam resistance accurately.
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Notes |
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References |
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Received 25 June 2001; returned 3 August 2001; revised 28 August 2001; accepted 4 September 2001