Bordetella Pertussis Isolates Research Articles

Macrolide-resistant Bordetella pertussis strain identified during an ongoing epidemic, Finland, January to October 2024.

Since April 2024, a pertussis epidemic has been ongoing in Finland with 2,215 notified cases by end October. Of them, 30.1% (n = 667) were aged 10-14 years. Of the 462 Bordetella pertussis isolates characterised, one was macrolide-resistant (minimum inhibitory concentration (MIC) of erythromycin, azithromycin and clarithromycin > 256 μg/mL). The resistant isolate was serotype FIM2, vaccine antigen pertactin-deficient and harboured ptxP3 allele. The emergence of macrolide-resistant B. pertussis in Europe is worrisome and its rapid identification is important.

Whole-genome comparison of two same-genotype macrolide-resistant Bordetella pertussis isolates collected in Japan.

The emergence of macrolide-resistant Bordetella pertussis (MRBP) is a significant problem because it reduces treatment options for pertussis and exacerbates the severity and spread of the disease. MRBP has been widely prevalent in mainland China since the 2010s and has been sporadically detected in other Asian countries. In Japan, two MRBP clinical strains were first isolated in Tokyo and Osaka between June and July 2018. The isolates BP616 in Osaka and BP625 in Tokyo harbored the same virulence-associated allelic genes (including ptxP1, ptxA1, prn1, fim3A, and fhaB3) and MT195 genotype and exhibited similar antimicrobial susceptibility profiles. However, despite their simultaneous occurrence, a distinguishable epidemiological link between these isolates could not be established. To gain further insight into the genetic relationship between these isolates in this study, we performed whole-genome analyses. Phylogenetic analysis based on genome-wide single-nucleotide polymorphisms revealed that the isolates belonged to one of the three clades of Chinese MRBP isolates, but there were 11 single-nucleotide polymorphism differences between BP616 and BP625. Genome structure analysis revealed two large inversions (202 and 523 kbp) and one small transposition (3.8 kbp) between the genomes. These findings indicate that the two Japanese MRBP isolates are closely related to Chinese MRBP isolates but are genomically distinct, suggesting that they were introduced into Japan from mainland China through different transmission routes.

Bordetella pertussis isolates in Finland after acellular vaccination: serotype change and biofilm formation

ObjectivesIn Finland, whole cell pertussis vaccine (wP) was introduced in 1952 and was replaced by acellular pertussis vaccine (aP) without fimbrial (FIM) antigen in 2005. We aimed to analyse the changes in serotypes of circulating Bordetella pertussis before and after acellular vaccination and to explore the relationship between biofilm formation and serotype diversity after the introduction of aP vaccine. MethodsSerotyping of 1399 B. pertussis isolates collected at the Finnish National Reference Laboratory for Pertussis and Diphtheria in Turku, Finland, from 1974 to 2023 was performed by slide agglutination or indirect ELISA. Of 278 isolates collected after 2005, 53 were selected, genotyped for fim3 and fim2 alleles, and tested for biofilm formation. The selection criteria included maintaining a relatively equal distribution of isolates per time interval, ensuring approximately a 50:50 ratio of FIM2 (N = 26) and FIM3 (N = 27) serotypes. The reference strain Tohama I was used as a control. ResultsDuring the wP era, the majority of circulating B. pertussis exhibited the FIM2 serotype. However, FIM3 strains have appeared since 1999 and become prevalent. After the implementation of aP vaccines, the distribution of serotypes has exhibited substantial variability. FIM3 isolates displayed an enhanced biofilm formation compared to FIM2 isolates (Geometric mean value (95% CI): 0.90 (0.79–1.03) vs. 0.75 (0.65–0.85); p < 0.05). Of the 27 FIM3 isolates, 8 harboured fim3-1 and 19 fim3-2 alleles. FIM3 isolates with fim3-2 allele were significantly associated with increased biofilm formation when compared to those with fim3-1 (1.07 (0.96–1.19) vs. 0.61 (0.52–0.72); p < 0.0001). ConclusionFollowing the implementation of aP vaccines, the distribution of serotypes in Finland has exhibited substantial variability. FIM3 isolates with the fim3-2 allele displayed an enhanced biofilm formation capability compared to FIM2 isolates.

Strain identity of Bordetella pertussis isolates from household members based on whole-genome sequencing.

We evaluated the genetic diversity of Bordetella pertussis, the causative agent of pertussis, within households by whole-genome sequencing. In pairwise comparisons of 23 isolates collected from 11 households, single-nucleotide polymorphism (SNP) analysis revealed extremely low SNP diversity (≤1 SNP) between isolate pairs: no SNPs were detected in 10 households and one SNP was obtained in the remaining household. This SNP was uncommon for B. pertussis and resulted in a nonsynonymous substitution (Ala303Thr) in nicotinate phosphoribosyltransferase. We demonstrated that the same strain is transmitted between household members and that B. pertussis is genomically stable during household transmission.

Genomic characterization of Bordetella pertussis in South Africa, 2015-2019.

Pertussis remains a public health concern in South Africa, with an increase in reported cases and outbreaks in recent years. Whole genome sequencing was performed on 32 Bordetella pertussis isolates sourced from three different surveillance programmes in South Africa between 2015 and 2019. Genome sequences were characterized using multilocus sequence typing, vaccine antigen genes (ptxP, ptxA, ptxB, prn and fimH) and overall genome structure. All isolates were sequence type 2 and harboured the pertussis toxin promoter allele ptxP3. The dominant genotype was ptxP3-ptxA1-ptxB2-prn2-fimH2 (31/32, 96.9 %), with no pertactin-deficient or other mutations in vaccine antigen genes identified. Amongst 21 isolates yielding closed genome assemblies, eight distinct genome structures were detected, with 61.9 % (13/21) of the isolates exhibiting three predominant structures. Increases in case numbers are probably not due to evolutionary changes in the genome but possibly due to other factors such as the cyclical nature of B. pertussis disease, waning immunity due to the use of acellular vaccines and/or population immunity gaps.

The whole-cell proteome shows the characteristics of macrolides-resistant Bordetella pertussis in China linked to the biofilm formation

The macrolides-resistant Bordetella pertussis (MR-Bp) isolates in China evolved from the ptxP1/fhaB3 allele and rapidly became predominant, suggestive of an adaptive transmission ability. This was different from the global prevalent ptxP3 strains, in which MR-Bp was rarely reported. The study aimed to determine the underlying mechanism responsible for fitness and resistance in these two strains. We identify proteomic differences between ptxP1/fhaB3 and ptxP3/fhaB1 strains using tandem mass tag (TMT)-based proteomics. We then performed in-depth bioinformatic analysis to determine differentially expressed genes (DEGs), followed by gene ontology (GO), and protein–protein interaction (PPI) network analysis. Further parallel reaction monitoring (PRM) analysis confirmed the expression of four target proteins. Finally, the crystal violet method was used to determine biofilm-forming ability. The results showed that the main significantly different proteins between the two represent isolates were related to biofilm formation. Furthermore, we have confirmed that ptxP1/fhaB3 showed hyperbiofilm formation in comparison with ptxP3/fhaB1. It is suggested that the resistance and adaptability of ptxP1/fhaB3 strains may be related to the formation of biofilm through proteomics. In a word, we determined the significantly different proteins between the ptxP1/fhaB3 and ptxP3/fhaB1 strains through whole-cell proteome, which were related to biofilm formation.

Five Days azithromycin is more efficient than teo DaysErythromycin for the Treatment of pertussis

Whooping cough (pertussis) is a highly contagious caused by Bordetella pertussis. During the last decade three Bordetella pertussis isolates have been found to be resistant to erythromycin, the azithromycin are used for treatment of pertussis. Sixty Child as 31 (5-day group) and 29 (10-day group) with pertussis in private clinic were evaluated for more efficiency of a 3-day course azithromycin in compares to a 10-day course erythromycin therapy. Treatment outcomes were stand on rates of bacteriological cure. Participants were randomized to receive 5 Days azithromycin (10 mg/kg/d) once a day or 10 Days Erythromycin 40 -30mgkg/ d in 4 divided doses, Bacteriological persistence did not happened for azithromycin ( 0% ) But for erythromycin ( one =3.44 %). Bacteriological failures occurred with azithromycin treatment (one =322%)while in Erythromycin (two ~~ =7.40 %) the estimates of bacterial cure were elevated for azithromyein 96.77% in compares for erythromycin 93.10% the bacterial cure rate time was 16.48 days for azithromycin less than for erythromycin 24.2625 days. The Variance in clinical manifestation duration for azithromycin 36.6 days less than for erythromycin 47.5 days for that our study recommended that 5-day azithromycin is more efficient than 10-day erythromycin in the treatment of pertussis in children.

A Cross-Sectional Study Revealing the Emergence of Erythromycin-Resistant Bordetella pertussis Carrying ptxP3 Alleles in China.

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.

Comparative genomics of Bordetella pertussis isolates from New Zealand, a country with an uncommonly high incidence of whooping cough.

Whooping cough, the respiratory disease caused by Bordetella pertussis , has undergone a wide-spread resurgence over the last several decades. Previously, we developed a pipeline to assemble the repetitive B. pertussis genome into closed sequences using hybrid nanopore and Illumina sequencing. Here, this sequencing pipeline was used to conduct a more high-throughput, longitudinal screen of 66 strains isolated between 1982 and 2018 in New Zealand. New Zealand has a higher incidence of whooping cough than many other countries; usually at least twice as many cases per 100000 people as the USA and UK and often even higher, despite similar rates of vaccine uptake. To the best of our knowledge, these strains are the first New Zealand B. pertussis isolates to be sequenced. The analyses here show that, on the whole, genomic trends in New Zealand B. pertussis isolates, such as changing allelic profile in vaccine-related genes and increasing pertactin deficiency, have paralleled those seen elsewhere in the world. At the same time, phylogenetic comparisons of the New Zealand isolates with global isolates suggest that a number of strains are circulating in New Zealand, which cluster separately from other global strains, but which are closely related to each other. The results of this study add to a growing body of knowledge regarding recent changes to the B. pertussis genome, and are the first genetic investigation into B. pertussis isolates from New Zealand.

Evolutionary genomics of recent clinical Bordetella pertussis isolates from Iran: wide circulation of multiple ptxP3 lineages and report of the first ptxP3 filamentous hemagglutinin-negative B. pertussis.

Here we investigated nationwide clinical Bordetella pertussis isolated during 2005-2017 from different provinces of Iran, a country with more than 50years whole cell vaccine immunisation history. Our results revealed the ongoing increase in the population of ptxP3/fim3-2 B. pertussis isolates in different provinces which were differentiated into nine clades. The largest clade (clade 8) which was previously found to be prevalent in Tehran was also prevalent across the country and clade 5 with ptxP3/prn9 genotype has also increased in frequency (14% of all ptxP3 isolates) in recent years. Furthermore, we detected the first ptxP3 B. pertussis isolates that does not express filamentous hemagglutinin (FhaB) as one of the major antigens of the pathogen and a key component of the acellular pertussis vaccine.

Molecular epidemiology of Bordetella pertussis and analysis of vaccine antigen genes from clinical isolates from Shenzhen, China

BackgroundAlthough pertussis cases globally have been controlled through the Expanded Programme on Immunization (EPI), the incidence of pertussis has increased significantly in recent years, with a “resurgence” of pertussis occurring in developed countries with high immunization coverage. Attracted by its fast-developing economy, the population of Shenzhen has reached 14 million and has become one of the top five largest cities by population size in China. The incidence of pertussis here was about 2.02/100,000, far exceeding that of the whole province and the whole country (both < 1/100,000). There are increasing numbers of reports demonstrating variation in Bordetella pertussis antigens and genes, which may be associated with the increased incidence. Fifty strains of Bordetella pertussis isolated from 387 suspected cases were collected in Shenzhen in 2018 for genotypic and molecular epidemiological analysis.MethodsThere were 387 suspected cases of pertussis enrolled at surveillance sites in Shenzhen from June to August 2018. Nasopharyngeal swabs from suspected pertussis cases were collected for bacterial culture and the identity of putative Bordetella pertussis isolates was confirmed by real-time PCR. The immunization history of each patient was taken. The acellular pertussis vaccine (APV) antigen genes for pertussis toxin (ptxA, ptxC), pertactin (prn) and fimbriae (fim2 and fim3) together with the pertussis toxin promoter region (ptxP) were analyzed by second-generation sequencing. Genetic and phylogenetic analysis was performed using sequences publicly available from GenBank, National Institutes of Health, Bethesda, MD, USA (https://www.ncbi.nlm.nih.gov/genbank/). The antimicrobial susceptibility was test by Kirby-Bauer disk diffusion.ResultsFifty strains of Bordetella pertussis were successfully isolated from nasopharyngeal swabs of 387 suspected cases, with a positivity rate of 16.79%, including 28 males and 22 females, accounting for 56.0% and 44.0% respectively. Thirty-eight of the 50 (76%) patients were found to be positive for B. pertussis by culture. Among the positive cases with a history of vaccination, 30 of 42 (71.4%) cases had an incomplete pertussis vaccination history according to the national recommendation. Three phylogenetic groups (PG1-PG3) were identified each containing a predominant genotype. The two vaccines strains, CS and Tohama I, were distantly related to these three groups. Thirty-one out of fifty (62%) isolates belonged to genotype PG1, with the allelic profile prn2/ptxC2/ptxP3/ptxA1/fim3-1/fim2-1. Eighteen out of fifty (36%) isolates contained the A2047G mutation and were highly resistant to erythromycin, and all belonged to genotype PG3 (prn1/ptxA1/ptxP1/ptxC1/fim3-1/fim2-1), which is closely related to the recent epidemic strains found in northern China.ConclusionsThe positive rate of cases under one-year-old was significantly higher than that of other age groups and should be monitored. The dominant antigen genotypes of 50 Shenzhen isolates are closely related to the epidemic strains in the United States, Australia and many countries in Europe. Despite high rates of immunization with APV, epidemics of pertussis have recently occurred in these countries. Therefore, genomic analysis of circulating isolates of B. pertussis should be continued, for it will benefit the control of whooping cough and development of improved vaccines and therapeutic strategies.

Effectiveness of experimental and commercial pertussis vaccines in the elimination of Bordetella pertussis isolates with different genetic profiles in murine model

The aim of this study was to compare the elimination of Bordetella pertussis clinical isolates, representing different genotypes in relation to alleles encoding virulence factors (MLST—multi-locus antigen sequence typing), MLVA type (multi-locus variable-number tandem repeat analysis) and PFGE group (pulsed-field gel electrophoresis) from the lungs of naive mice or mice were immunised with the commercial whole-cell pertussis vaccine, the acellular pertussis vaccine and the experimental whole-cell pertussis vaccine. Molecular data indicate that the resurgence of pertussis in populations with high vaccine coverage is associated with genomic adaptation of B. pertussis, to vaccine selection pressure. Pertactin-negative B. pertussis isolates were suspected to contribute to the reduced vaccine effectiveness. It was shown that one of the isolates used is PRN deficient. The mice were intranasally challenged with bacterial suspension containing approximately 5 × 10 7 CFU/ml B. pertussis. The immunogenicity of the tested vaccines against PT (pertussis toxin), PRN (pertactin), FHA (filamentous haemagglutinin) and FIM (fimbriae types 2 and 3) was examined. The commercial whole-cell and acellular pertussis vaccines induced an immunity effective at eliminating the genetically different B. pertussis isolates from the lungs. However, the elimination of the PRN-deficient isolate from the lungs of mice vaccinated with commercial vaccines was delayed as compared to the PRN ( +) isolate, suggesting phenotypic differences with the circulating isolates and vaccine strains. The most effective vaccine was the experimental vaccine with the composition identical to that of the strains used for infection.

Pertactin-deficient Bordetella pertussis isolates: evidence of increased circulation in Europe, 1998 to 2015.

IntroductionPertussis outbreaks have occurred in several industrialised countries using acellular pertussis vaccines (ACVs) since the 1990s. High prevalence of pertactin (PRN)-deficient Bordetella pertussis isolates has been found in these countries.AimsTo evaluate in Europe: (i) whether proportions of PRN-deficient strains increased in consecutive collections of B. pertussis clinical isolates; (ii) if the frequency of PRN-deficient strains in countries correlated with the time since ACV introduction; (iii) the presence of pertussis toxin (PT)-, filamentous haemagglutinin (FHA)- or fimbriae (Fim)-deficient isolates.Methods B. pertussis clinical isolates were obtained from different European countries during four periods (EUpert I–IV studies): 1998 to 2001 (n = 102), 2004 to 2005 (n = 154), 2007 to 2009 (n = 140) and 2012 to 2015 (n = 265). The isolates’ selection criteria remained unchanged in all periods. PRN, PT, FHA and Fim2 and Fim3 expression were assessed by ELISA.ResultsIn each period 1.0% (1/102), 1.9% (3/154), 6.4% (9/140) and 24.9% (66/265) of isolates were PRN-deficient. In EUpert IV, PRN-deficient isolates occurred in all countries sampled and in six countries their frequency was higher than in EUpert III (for Sweden and the United Kingdom, p < 0.0001 and p = 0.0155, respectively). Sweden and Italy which used ACVs since the mid 1990s had the highest frequencies (69%; 20/29 and 55%; 11/20, respectively) while Finland, where primary immunisations with ACV containing PRN dated from 2009 had the lowest (3.6%). Throughout the study, no PT- or FHA-deficient isolate and one Fim2/3-deficient was detected.ConclusionResults suggest that the longer the period since the introduction of ACVs containing PRN, the higher the frequency of circulating PRN-deficient isolates.

Pertussis epidemiology in Tunisian infants and children and characterization of Bordetella pertussis isolates: results of a 9-year surveillance study, 2007 to 2016.

Pertussis remains a public health concern in most countries. Our study aimed to prospectively explore the epidemiology of pertussis in the Tunis area of Tunisia between 2007 and 2016, and to characterize the virulence-associated genes of the collected Bordetella pertussis isolates. Infants and children hospitalized at the Children's Hospital of Tunis, Tunisia, between 2007 and 2016 for suspicion of pertussis were enrolled in the study. Culture and real-time PCR (qPCR) assays targeting IS481, IS1001, recA, H-IS1001 and ptxP were used to confirm the pertussis diagnosis. Phenotypic and genotypic characterization of recovered isolates was performed.Results/Key findings. A total of 1844 children were included in the study. Overall, 306 children (16.6 %) with Bordetella infection were confirmed by qPCR. Among them, 265 (86.6 %) were confirmed as having B. pertussis (IS481+, ptxP+, H-IS1001-), 18 (5.9 %) as having Bordetella parapertussis (IS481-, IS1001+) and 11 (3.6 %) as having Bordetella spp. (IS481+, ptxP-, H-IS1001-). No Bordetella holmesii (IS481+, IS1001-, H-IS1001+) was identified. The estimated pertussis incidence in the Tunis area was 134/100 000 in children aged less than 5 years. Two epidemic peaks were observed in 2009 and 2014. Ten B. pertussis isolates were cultured and characterized. Deficiency in pertactin expression was not observed, and genotyping of the isolates revealed a predominant allelic profile: ptxP3-ptxA1-prn2-fim2-1-fim3-2. This study demonstrated that pertussis is still present as a cyclical disease in Tunisia, despite high primo-vaccination coverage with a pertussis whole-cell vaccine. The predominant genotype of Tunisian B. pertussis isolates is similar to isolates circulating in countries using the acellular vaccine.

Pertussis Outbreak in a Primary School in China: Infection and Transmission of the Macrolide-resistant Bordetella pertussis.

A pertussis outbreak was studied in a primary school in Xi'an, China, in March 2016. The school consisted of 536 pupils 6-12 years of age who were divided into 12 classes of 6 grades (2 classes for each grade). The identified index case was an 11-year-old girl at class 2 of grade 5. Interview was conducted and nasopharyngeal swabs were taken from all pupils (N = 94) in the 2 classes of grade 5. Nasopharyngeal swabs were tested by both culture and polymerase chain reaction (PCR). Four culture- and 17 PCR-positive cases were identified in 94 pupils. Infection rate was significantly higher in class 2 compared with that in class 1 [37.0% (17/46) vs. 14.6% (7/48), χ(2) = 4.26, P < 0.05]. All Bordetella pertussis isolates were macrolide-resistant, harbored prn1/ptxP1/fim3-1 as previously reported and belonged to multilocus variable tandem repeat analysis type MLVA 195. Of the 17 DNAs positive for diagnostic PCR, 12 were also positive for 23S ribosomal RNA PCR. All the 12 DNAs had the A2047G mutation of 23S rRNA gene of B. pertussis. This study described a pertussis outbreak caused by macrolide-resistant B. pertussis in a primary school and indicated that close contact of index case causes the bacterial transmission.

Bordetella pertussis isolates vary in their interactions with human complement components

Whooping cough is a re-emerging respiratory tract infection. It has become clear that there is a need for better understanding of protective immune responses and variation between Bordetella pertussis strains to aid the development of improved vaccines. In order to survive in the host, B. pertussis has evolved mechanisms to evade complement-mediated killing, including the ability to bind complement-regulatory proteins. Here we evaluate the variation in interactions with the complement system among recently isolated strains. Isolates whose genomes appear highly similar and cluster together on a SNP-based dendrogram were found to vary significantly in resistance to complement-mediated killing and in the deposition of C3b/iC3b, C5b-9 and C1 esterase inhibitor (C1-INH). The key role of Vag8 as a receptor for C1-INH was confirmed and its expression was shown to vary in a panel of isolates. A Vag8 knockout mutant showed increased sensitivity to complement-mediated killing. Antibodies in convalescent sera blocked C1-INH binding to B. pertussis and may play an important role in natural immunity.

Genomic Survey of Bordetella pertussis Diversity, United States, 2000-2013.

We characterized 170 complete genome assemblies from clinical Bordetella pertussis isolates representing geographic and temporal diversity in the United States. These data capture genotypic shifts, including increased pertactin deficiency, occurring amid the current pertussis disease resurgence and provide a foundation for needed research to direct future public health control strategies.

Antimicrobial susceptibility testing of Finnish Bordetella pertussis isolates collected during 2006-2017.

Macrolides, such as azithromycin and erythromycin, are first-line drugs for the (prophylactic) treatment of pertussis. This study aimed to screen for macrolide-, quinolone- or trimethoprim/sulfamethoxazole (SXT)-resistant strains among Finnish Bordetella pertussis isolates. Antimicrobial susceptibility testing was performed on 148 B. pertussis strains isolated during 2006-2017. Isolates were analysed by allele-specific PCR for detection of the macrolide resistance-associated mutation A2047G in the 23S rRNA gene. The gyrA gene was sequenced for detection of the A260G mutation associated with quinolone resistance. For phenotyping, a random selection was made by selecting every third isolate (n=50) to determine the minimum inhibitory concentrations (MICs) for erythromycin and azithromycin by Etest and the inhibition zone size for nalidixic acid (NAL) and SXT by single disk diffusion assay. Neither the macrolide resistance-associated mutation A2047G nor the quinolone resistance-associated mutation A260G was detected in any of the B. pertussis isolates. MICs of azithromycin and erythromycin ranged between 0.016-0.19μg/mL and 0.016-0.25μg/mL, respectively. The size of the inhibition zone surrounding the NAL disk ranged between 22-27mm in diameter. The inhibition zone surrounding the SXT disk ranged between 24-37mm in diameter. No isolates resistant to any of the tested antimicrobials were identified. The allele-specific PCR is a simple and useful tool for screening B. pertussis resistance to macrolides. All Finnish isolates tested were susceptible to macrolides, quinolones and SXT.

Multi-locus variable-number tandem repeat analysis of Bordetella pertussis isolates circulating in Poland in the period 1959-2013.

Despite the long history of pertussis vaccination and high vaccination coverage in Poland and many other developed countries, pertussis incidence rates have increased substantially, making whooping cough one of the most prevalent vaccine-preventable diseases. Among the factors potentially involved in pertussis resurgence, the adaptation of the Bordetella pertussis population to country-specific vaccine-induced immunity through selection of non-vaccine-type strains still needs detailed studies. Multi-locus variable-number tandem repeat analysis (MLVA), also linked to MLST and PFGE profiling, was applied to trace the genetic changes in the B. pertussis population circulating in Poland in the period 1959-2013 versus country-specific vaccine strains. Generally, among 174 B. pertussis isolates, 31 MLVA types were detected, of which 11 were not described previously. The predominant MLVA types of recent isolates in Poland were different from those of the typical isolates circulating in other European countries. The MT27 type, currently predominant in Europe, was rarely seen and detected in only five isolates among all studied. The features of the vaccine strains used for production of the pertussis component of a national whole-cell diphtheria-tetanus-pertussis (DTP) vaccine, as studied by MLVA and MLST tools, were found to not match those observed in the currently circulating B. pertussis isolates in Poland. Differences traced by MLVA in relation to the MLST and PFGE profiling confirmed that the B. pertussis strain types currently observed elsewhere in Europe, even if appearing in Poland, were not able to successfully disseminate within a human population in Poland that has been vaccinated with a whole-cell pertussis vaccine not used in other countries.

Development of a qualitative assay for screening of Bordetella pertussis isolates for pertussis toxin production.

Bordetella pertussis infection has been increasing in the US, with reported cases reaching over 50,000 in 2012, a number last observed in the 1950s. Concurrently, B. pertussis lacking the pertactin protein, one of the immunogens included in the acellular vaccine formulations, has rapidly emerged since 2010, and has become the predominant circulating phenotype. Monitoring the production of the remaining acellular vaccine immunogens, such as pertussis toxin (Pt), is a critical next step. To date, methods for screening Pt have been either through genomic sequencing means or by conventional ELISAs. However, sequencing limits detection to the DNA level, missing potential disruptions in transcription or translation. Conventional ELISAs are beneficial for detecting the protein; however, they can often suffer from poor sensitivity and specificity. Here we describe a rapid, highly sensitive and specific electrochemiluminescent capture ELISA that can detect Pt production in prepared inactivated bacterial suspensions. Over 340 isolates were analyzed and analytical validation parameters, such as precision, reproducibility, and stability, were rigorously tested. Intra-plate and inter-plate variability measured at 9.8% and 11.5%, respectively. Refrigerated samples remained stable for two months and variability was unaffected (coefficient of variation was 12%). Interestingly, despite the intention of being a qualitative method, the assay was sensitive enough to detect a small, but statistically significant, difference in protein production between different pertussis promoter allelic groups of strains, ptxP1 and ptxP3. This technology has the ability to perform screening of multiple antigens at one time, thus, improving testing characteristics while minimizing costs, specimen volume, and testing time.