Multiclonal dissemination of ptxP3 allele macrolide-resistant Bordetella pertussis in Okinawa, Japan.

Objective To understand the epidemiological characteristics, genomic variations and macrolide resistance of Bordetella pertussis (B.pertussis)strains circulating in Shenzhen with clinical data analysis, genotype profiling, phylogenetic analysis and antimicrobial susceptibility test. Methods Clinical data of patients with pertussis in Shenzhen Children′s Hospital were collected from the electronic medical record system. Genome sequences of 31 B. pertussis isolates were analyzed with next-generation sequencing and de novo assembled. Multilocus sequence typing (MLST) was performed to identify their sequences types. Sequence alignment by BLASTn was used to identify virulence genotypes and mutations in 23S rRNA gene. A phylogenetic tree was constructed to analyze the relationships among them. E-test was used to identify macrolide resistance. Results All of the 31 B. pertussis strains were identified as sequence type-2 (ST-2) by MLST with diverse virulence genotypes. Two were prn-deficient strains. Based on the phylogenetic tree, all of the isolates were distant from vaccine strains. Nineteen isolates were resistant to erythromycin with A2047G mutation in 23S rRNA. Conclusions The virulence genotypes of B. pertussis strains in Shenzhen were diverse with increasing non-vaccine genotypes. Macrolide-resistant strains were prevalent. This study might provide reference for improving the prevention, management and vaccination strategy of pertussis. Key words: Bordetella pertussis; Next-generation sequencing; Genotype; Macrolide resistance

Macrolide resistance-associated 23S rRNA mutation in Mycoplasma genitalium, Japan.

Macrolide Resistance–associated 23S rRNA Mutation in<i>Mycoplasma genitalium</i>, Japan

BackgroundTo develop a TaqMan-based real-time allelic discrimination assay for the simultaneous detection of two point mutations (A2058G and A2059G) in the 23S rRNA gene of T pallidum that have been...

Objectives Mycoplasma genitalium (MG) is a sexually transmitted organism associated with non-gonococcal urethritis in men and several inflammatory reproductive tract syndromes in women. These infections are commonly treated with azithromycin....

Neisseria gonorrhoeae, the causative agent of gonorrhea, represents a major public health concern due to its increasing antimicrobial resistance. While often asymptomatic—particularly in extragenital infections—untreated cases can lead to severe complications and further transmission. Despite global efforts to monitor antimicrobial resistance, data on the molecular determinants underlying decreased susceptibility in N. gonorrhoeae remain scarce in Peru. This study aimed to detect mutations in the penA and 23S rRNA genes, which confer decreased susceptibility to cephalosporins and azithromycin resistance. We extracted DNA from 124 N. gonorrhoeae-positive clinical rectal specimens collected in Aptima Combo 2 transport tubes from MSM patients. These DNA samples were then screened using the Mismatch Amplification Mutation Assay-based real-time PCR (MAMA-qPCR) to identify mutations in the 23S rRNA and penA genes. Each sample underwent separate reactions to detect A2059G and C2611T mutations in the 23S rRNA gene, and 86 of these samples were further tested in individual qPCR assays for the penA D345 deletion (D345del) or G545S mutations. Sanger sequencing was performed on all DNA samples positive for 23S rRNA mutations by MAMA-qPCR assay, and on 27 DNA samples that yielded sufficient penA amplicons for additional sequencing. Using the MAMA-qPCR assay for the 23S rRNA gene, 64 of 124 samples amplified in the A2059G reaction: 2 (3.1%) carried the mutation, and 62 were classified as wild type. In the C2611T reaction, 42 of 124 samples amplified, and none of them carried the mutation. Using the MAMA-qPCR assay for the penA gene, we only analyzed 86 samples, as the remaining 38 samples had insufficient DNA yield. A total of 44 of the 86 samples amplified in the D345del reaction: 5 (11.4%) carried the D345del, and 39 were classified as wild type. In the G545S reaction, 4 (6.4%) carried the mutation, and 58 were classified as wild type. Finally, sequencing of the penA gene in the 27 samples revealed mutations related to decreased susceptibility to cephalosporins. This study identified genetic mutations conferring resistance to azithromycin and decreased susceptibility to cephalosporins, providing an overview of the circulating mutations conferring resistance in N. gonorrhoeae strains in Peru.

Analysis of prevailing pertussis strains from different regions in China after the application of acellular vaccines.

BackgroundPrevious limited studies have identified that Bordetella pertussis (B. pertussis) isolates circulating in China possess distinct molecular features and high rates of erythromycin-resistance (ER). Their evolution and potential impact on the prevention and control of global pertussis are worthy of attention.MethodsThe present cross-sectional study involved 311 non-duplicate and unrelated B. pertussis strains isolated from Chinese children from 2017 to 2019. Their antimicrobial susceptibilities were assessed using both E-test strips and Kirby-Bauer (KB) disk diffusion methods. Seven virulence-related genes (ptxA, ptxC, ptxP, prn, fim2, fim3, and tcfA2) and the A2047G mutation in the 23S rRNA gene were detected by PCR. Based on the susceptibilities and genotypes, 50 isolates were selected for multi-locus variable-number tandem-repeat analysis (MLVA) typing and whole-genome sequencing.ResultsA total of 311 B. pertussis strains were isolated from children with a median age of 4 months (interquartile range: 2–9 months). Strains carrying the ptxP1 allele were more frequent (84.9%, 264/311), were always ER (except for one strain), and were mainly related to ptxA1/ptxC1/prn1 alleles (99.6%, 263/264). The remaining 47 (15.1%) strains carried the ptxP3 allele, mainly harboring the ptxA1/ptxC2/prn2 alleles (93.6%, 44/47), and were sensitive to erythromycin (except for two strains). The two ER-ptxP3 isolates were first identified in China, belonged to MT27 and MT28 according to MLVA, and were classified into sub-lineage IVd by phylogenetic analysis of their genome sequences. This sub-lineage also includes many strains carrying the ptxP3 allele spreading in developed countries. For each tested antimicrobial, the susceptibilities judged by KB disks were consistent with those determined by E-test strips.ConclusionThe present results reveal that B. pertussis strains with the ptxP1-ER profile still dominate in China, and a few strains carrying the ptxP3 allele have acquired the A2047G mutation in the 23S rRNA gene and the ER phenotype. The surveillance of the drug susceptibility of B. pertussis is necessary for all countries, and the KB disk method can be adopted as a screening test.

A Fast Real-Time Polymerase Chain Reaction Method for Sensitive and Specific Detection of the Neisseria gonorrhoeae porA Pseudogene

Detection of clarithromycin-resistant Helicobacter pylori in clinical specimens by molecular methods: A review.

Identification of Theileria parva and Theileria sp. (buffalo) 18S rRNA gene sequence variants in the African Buffalo (Syncerus caffer) in southern Africa

Mycoplasma genitalium is a sexually transmitted organism commonly treated with azithromycin. However, macrolide resistance has been reported and is associated with point mutations in the 23S rRNA gene. To evaluate the prevalence of macrolide resistance in M. genitalium isolates from clinical specimens from France, we first used a previously reported high-resolution melting assay. Because susceptible and resistant M. genitalium isolates were hardly discriminated in M. genitalium-positive clinical specimens, we developed a new molecular assay for the rapid detection of macrolide resistance. An assay using real-time PCR based on fluorescence resonance energy transfer (FRET) coupled with melting curve analysis was designed. The assay was first validated on characterized macrolide-resistant M. genitalium isolates and then applied to 202 urogenital M. genitalium-positive specimens collected from 178 patients from France in 2011 and 2012. Resistant genotypes were confirmed by 23S rRNA gene sequencing. Among the 202 M. genitalium-positive specimens, 155 were amplified, demonstrating a sensitivity of 76.7%. A substitution in the 23S rRNA gene was found in 14.2% of the patient samples. Nine and six patients had M. genitalium isolates with a substitution at positions 2059 and 2058, respectively. In four cases, a mixed population of wild-type and mutated M. genitalium isolates was observed. The prevalence of M. genitalium macrolide resistance has been stable in France since its detection in 2006. Our FRET PCR assay is able to discriminate between wild-type and resistant genotypes directly from clinical specimens. This assay will allow clinicians to shorten the time to the initiation of effective disease treatment.

The Effect of Primer-Template Mismatches on the Detection and Quantification of Nucleic Acids Using the 5′ Nuclease Assay

Epidemiologic data from Asia have documented the rapid and extensive emergence of macrolide resistance in Mycoplasma pneumoniae. This drug resistance has also been documented in Europe and recently in the United States, but there is very little information currently available on its prevalence. A rapid technique to identify macrolide-resistant M. pneumoniae is needed to guide management of patients with community-acquired respiratory infections. Culture and Minimum Inhibitory Concentration testing identified macrolide-resistant M. pneumoniae infection in 2 seriously ill hospitalized children with community-acquired pneumonia. A portion of the 23S ribosomal RNA gene from 2 macrolide-resistant M. pneumoniae isolates from these children as well as 4 laboratory-induced macrolide-resistant strains was amplified by PCR, and the PCR products were sequenced to identify mutations associated with macrolide resistance. A real-time PCR assay was designed to identify 3 known mutations in the 23S rRNA gene associated with macrolide resistance and applied to the clinical specimens from which these isolates were obtained and to the bacterial isolates. : Macrolide-resistant M. pneumoniae from both children were found to carry an A2063G transition in the 23S rRNA gene previously identified in resistant isolates from China, Japan, France, and recently in an encephalitis outbreak in Rhode Island. Three laboratory-induced mutant strains had an A2064G mutation whereas the other one had an A2063G mutation. A real-time PCR assay successfully detected the macrolide-resistant M. pneumoniae directly in clinical specimens and discriminated them from wild-type isolates. Macrolide-resistant M. pneumoniae can be associated with prolonged severe respiratory infection in children. Real-time PCR offers a rapid method of diagnosing macrolide resistance in community-acquired respiratory infections due to M. pneumoniae.

Considering their abundance and broad distribution, non-extremophilic Crenarchaeota are likely to play important roles in global organic and inorganic matter cycles. The diversity and abundance of archaeal 16S rRNA and putative ammonia monooxygenase alpha-subunit (amoA) genes were comparatively analyzed to study genetic potential for nitrification of ammonia-oxidizing archaea (AOA) in the surface layers (0-1 cm) of four marine sediments of the East Sea, Korea. After analysis of a 16S rRNA gene clone library, we found various archaeal groups that include the crenarchaeotal group (CG) I.1a (54.8%) and CG I.1b (5.8%), both of which are known to harbor ammonia oxidizers. Notably, the 16S rRNA gene of CG I.1b has only previously been observed in terrestrial environments. The 16S rRNA gene sequence data revealed a distinct difference in archaeal community among sites of marine sediments. Most of the obtained amoA sequences were not closely related to those of the clones retrieved from estuarine sediments and marine water columns. Furthermore, clades of unique amoA sequences were likely to cluster according to sampling sites. Using real-time PCR, quantitative analysis of amoA copy numbers showed that the copy numbers of archaeal amoA ranged from 1.1x10(7) to 4.9x10(7) per gram of sediment and were more numerous than those of bacterial amoA, with ratios ranging from 11 to 28. In conclusion, diverse CG I.1a and CG I.1b AOA inhabit surface layers of marine sediments and AOA, and especially, CG I.1a are more numerous than other ammonia-oxidizing bacteria.