Direct Detection of Erythromycin-Resistant Bordetella pertussis in Clinical Specimens by PCR

Abstract

Resistance of Bordetella pertussis to erythromycin has been increasingly reported. We developed an allele-specific PCR method for rapid detection of erythromycin-resistant B. pertussis directly from nasopharyngeal (NP) swab samples submitted for diagnostic PCR. Based on the proven association of erythromycin resistance with the A2047G mutation in the 23S rRNA of B. pertussis, four primers, two of which were designed to be specific for either the wild-type or the mutant allele, were used in two different versions of the allele-specific PCR assay. The methods were verified with results obtained by PCR-based sequencing of 16 recent B. pertussis isolates and 100 NP swab samples submitted for diagnostic PCR. The detection limits of the two PCR assays ranged from 10 to 100 fg per reaction for both erythromycin-susceptible and -resistant B. pertussis. Two amplified fragments of each PCR, of 286 and 112 bp, respectively, were obtained from a mutant allele of the isolates and/or NP swab samples containing B. pertussis DNAs. For the wild-type allele, only a 286-bp fragment was visible when the allele-specific PCR assay 1 was performed. No amplification was found when a number of non-Bordetella bacterial pathogens and NP swab samples that did not contain the DNAs of B. pertussis were examined. This assay can serve as an alternative for PCR-based sequencing, especially for local laboratories in resource-poor countries. P ertussis has resurged in many countries. Macrolides, especially erythromycin, are considered the first-choice antibiotics for treatment of pertussis and postexposure prophylaxis (1). The first erythromycin-resistant Bordetella pertussis was discovered in 1994 (2). Although resistant strains are still rare, they have been reported in several countries, including China (3-7), where macrolide-resistant B. pertussis has become prevalent (7).B. pertussis is a fastidious bacterium. The culture positivity rate is low in immunized populations. The PCR method has been developed and used for diagnosis of pertussis in many laboratories all over the world. However, it is possible that resistant B. pertussis strains are missed due to failure of the culture and when culture is not performed (8).Several studies have proven the association between macrolide resistance and the A2047G mutation in the 23S rRNA of B. pertussis (3-7). Based on the molecular mechanism identified, methods have been developed for detection of the point mutation in 23S rRNA of B. pertussis in order to study the susceptibility to macrolides when B. pertussis isolates are available (3, 5). In a previous study, we developed a PCR-based sequencing method for identification of the A2047G mutation in cultured B. pertussis isolates and clinical nasopharyngeal (NP) specimens (7).Although the sequencing is widely used, it is not available or accessible everywhere because it takes longer to obtain the results in many of the local laboratories, especially in resource-poor countries. In this study, we aimed to develop a simple allele-specific PCR method for di...

Genome Structural Diversity among 31 Bordetella pertussis Isolates from Two Recent U.S. Whooping Cough Statewide Epidemics

“…Interestingly, a majority of SNPs in virulence-related genes were found in GC-rich regions ( Table 3 ). Additionally, no epidemic genomes harbored the 23S A2047G mutation known to produce erythromycin/azithromycin resistance ( 31 – 33 ).…”

Genomic epidemiology of erythromycin-resistant Bordetella pertussis in China

“…There are two known mechanisms for resistance to erythromycin: the acquisition of erythromycin-resistant methylase ( erm ) genes resulting in high-level resistance [7], and mutations in the 23S rRNA gene leading to structural changes that prevent the binding of erythromycin [2]. Previous studies of erythromycin-resistant B. pertussis isolates in China showed that all resistant isolates contained 23S rRNA gene mutations [5,8]. Moreover, these erythromycin-resistant isolates are also resistant to other macrolides [4,6].…”

The global prevalence ptxP3 lineage of Bordetella pertussis was rare in young children with the co-purified aPV vaccination: a 5 years retrospective study

“…Cause the A2047G of 23S rRNA was associated with erythromycin resistance, if the nucleotide position 2047 of the 23S rRNA was the wild type as adenine (A), we defined as an erythromycin sensitive B. pertussis infection. A mutation type as guanine (G) of site 2047 was taken for erythromycin resistance B. pertussis infection strain [11,12]. The allele of ptxP and fhaB was performed by DNAs of strains and/or NPs as previously reported when successful sequencing of 23S rRNA [13].…”