Combinational detection of autolysin and penicillin-binding protein 2B genes of Streptococcus pneumoniae by PCR

Abstract

PCR was used to identify penicillin resistance in 1,062 clinical isolates of Streptococcus pneumoniae. Three sets of primers were designed to amplify (i) a 240-bp fragment of the penicillin-binding protein (PBP) 2B gene (pbp2b) of penicillin-susceptible S. pneumoniae (PSSP), (ii) a 215-bp fragment of the class A mutations of the pbp2b gene present in penicillin-resistant S. pneumoniae, and (iii) a 286-bp fragment of the class B mutation. In addition, a set of primers that amplify 273 bp of the autolysin (lytA) gene was applied in combination with the above to identify S. pneumoniae. Of 621 isolates for which MICs of penicillin were Ϲ0.06 g/ml, 614 (98.9%) were ascertained as having DNA fragments amplified by the PSSP primers. Of 441 isolates for which MICs of penicillin were м0.125 g/ml, a class A mutation was detected in only 8 (1.8%), a class B mutation was detected in 310 (70.3%), and neither class A nor class B mutations were found in the remaining 123 (27.9%). However, when analysis was limited to isolates for which MICs of penicillin were м1.0 g/ml, 247 isolates (89.8%) of 275 were found to possess a class B mutation. When PBPs were analyzed in 12 isolates with unclear mutations of the pbp2b gene by using [ 3 H]benzylpenicillin, low affinity to PBP 2B was observed in them all. These findings suggest that a pbp2b mutation other than class A or class B is present in these isolates. These results also indicate that it may be possible to identify PSSP and penicillin-resistant S. pneumoniae by applying PCR using a combination of primers to detect the susceptible pbp2b gene, resistant pbp2b gene mutations, and the lytA gene.

A reação em cadeia da polimerase na detecção da resistência à penicilina em Streptococcus pneumoniae

“…These results are in accordance with A reação em cadeia da polimerase na detecção da resistência à penicilina em Streptococcus pneumoniae those of other studies, which have demonstrated that isolated mutations in the pbp2x genome result in a low level of resistance to penicillin, whereas high resistance to penicillin requires genetic alterations in pbp1a and pbp2b as well (26)(27)(28)(29)(30) . Ubukata et al (19) used PCR to identify mutations in the pbp2b genes of 1062 clinical samples of S. pneumoniae and found a correlation between such mutations and penicillin MICs in 98.9% of the sensitive strains and in 70.3% of the resistant strains.…”

“…S. pneumoniae strains isolated in culture media were simultaneously submitted to PCR for detection of the lytA gene as described by Ubukata et al (19) in order to confirm bacterial identification and mutations in pbp1a, pbp2b and pbp2x, in accordance with the protocol introduced by Jalal et al (20) and subsequently described herein. Isolated S. pneumoniae colonies were removed from the culture medium through scraping with a platinum spatula and placed into a tube with 50 ml of distilled water for extraction of bacterial DNA.…”

“…These tests are carried out in laboratories using methods such as the agar dilution, which is recommended by the National Committee for Clinical Laboratory Standards. The main drawback in the use of these methods, however, is the long time it takes to obtain results, usually over 48 hours (18) , whereas polymerase chain reaction (PCR) detects mutations responsible for pneumococcus resistance to penicillin in a much shorter time, within approximately 8 hours (19) .…”

Limiting the Spread of Resistant Pneumococci: Biological and Epidemiologic Evidence for the Effectiveness of Alternative Interventions

“…PCR-based methods can provide rapid assessment of pneumococcal resistance profiles and information on the relatedness of various resistant isolates. To identify pneumococcal isolates and distinguish among penicillin-resistant and -sensitive strains, PCR protocols using oligonucleotides specific to sensitive and nonsusceptible penicillin-binding protein (PBP) gene allele sequences have been developed (36,37,89). Although potentially a powerful approach, these methods have not yet been tested using a defined strain collection that includes major internationally dispersed clones.…”

Alterations in penicillin binding protein gene of Streptococcus pneumoniae and their correlation with susceptibility patterns

“…Thr-371 substitution in pbp 1a was found to be associated with Pen-G r [12] and substitution within or adjacent to the conserved amino acid motif of pbp 2x was reported [13]. Together with gene mutations in pbp2b, mutations in pbp1a and pbp2x were also detected by PCR [14,15].…”