Strengthening Bordetella pertussis genomic surveillance by direct sequencing of residual positive specimens.

Whole-genome sequencing (WGS) of microbial pathogens recovered from patients with infectious disease facilitates high-resolution strain characterization and molecular epidemiology. However, increasing reliance on culture-independent methods to diagnose infectious diseases has resulted in few isolates available for WGS. Here, we report a novel culture-independent approach to genome characterization of Bordetella pertussis, the causative agent of pertussis and a paradigm for insufficient genomic surveillance due to limited culture of clinical isolates. Sequencing libraries constructed directly from residual pertussis-positive diagnostic nasopharyngeal specimens were hybridized with biotinylated RNA "baits" targeting B. pertussis fragments within complex mixtures that contained high concentrations of host and microbial background DNA. Recovery of B. pertussis genome sequence data was evaluated with mock and pooled negative clinical specimens spiked with reducing concentrations of either purified DNA or inactivated cells. Targeted enrichment increased the yield of B. pertussis sequencing reads up to 90% while simultaneously decreasing host reads to less than 10%. Filtered sequencing reads provided sufficient genome coverage to perform characterization via whole-genome single nucleotide polymorphisms and whole-genome multilocus sequencing typing. Moreover, these data were concordant with sequenced isolates recovered from the same specimens such that phylogenetic reconstructions from either consistently clustered the same putatively linked cases. The optimized protocol is suitable for nasopharyngeal specimens with diagnostic IS481 Ct < 35 and >10 ng DNA. Routine implementation of these methods could strengthen surveillance and study of pertussis resurgence by capturing additional cases with genomic characterization.

Combined transcriptomic and ChIPseq analyses of the Bordetella pertussis RisA regulon.

The regulation of Bordetella pertussis virulence is mediated by the two-component system BvgA/S, which activates the transcription of virulence-activated genes (vags). In the avirulent phase, the vags are not expressed, but instead, virulence-repressed genes (vrgs) are expressed, under the control of another two-component system, RisA/K. Here, we combined transcriptomic and chromatin immunoprecipitation sequencing (ChIPseq) data to examine the RisA/K regulon. We performed RNAseq analyses of RisA-deficient and RisA-phosphoablative B. pertussis mutants cultivated in virulent and avirulent conditions. We confirmed that the expression of most vrgs is regulated by phosphorylated RisA. However, the expression of some, including those involved in flagellum biosynthesis and chemotaxis, requires RisA independently of phosphorylation. Many RisA-regulated genes encode proteins with regulatory functions, suggesting multiple RisA regulation cascades. By ChIPseq analyses, we identified 430 RisA-binding sites, 208 within promoter regions, 201 within open reading frames, and 21 in non-coding regions. RisA binding was demonstrated in the promoter regions of most vrgs and, surprisingly, of some vags, as well as for other genes not identified as vags or vrgs. Unexpectedly, many genes, including some vags, like prn, brpL, bipA, and cyaA, contain a BvgA-binding site and a RisA-binding site, which increases the complexity of the RisAK/BvgAS network in B. pertussis virulence regulation.IMPORTANCEThe expression of virulence-activated genes (vags) of Bordetella pertussis, the etiological agent of whooping cough, is under the transcriptional control of the two-component system BvgA/S, which allows the bacterium to switch between virulent and avirulent phases. In addition, the more recently identified two-component system RisA/K is required for the expression of B. pertussis genes, collectively named vrgs, that are repressed during the virulent phase but activated during the avirulent phase. We have characterized the RisA/K regulon by combined transcriptomic and chromatin immunoprecipitation sequencing analyses. We identified more than 400 RisA-binding sites. Many of them are localized in promoter regions, especially vrgs, but some were found within open reading frames and in non-coding regions. Surprisingly, RisA-binding sites were also found in promoter regions of some vags, illustrating the previously underappreciated complexity of virulence regulation in B. pertussis.

[Impact of vaccination on the evolution of Bordetella pertussis]. / Impact de la vaccination sur l'évolution de Bordetella pertussis.

Vaccines against pertussis, or whooping cough, have been commercialized and used in most countries worldwide for decades. The history of these vaccines is distinctive, marked by the transition from whole-cell vaccines to acellular vaccines in many developed countries over the last two decades. This particular history has had a significant impact on the evolution of Bordetella pertussis, the etiological agent of whooping cough. Both genetic and phenotypic changes appeared, with the emergence of novel alleles for antigens targeted by the vaccines and changes in the expression of these antigens. The main consequence of these changes is the resurgence of whooping cough in many countries and the appearance of strains capable of evading vaccine-induced immunity. The emergence of novel strains under vaccine pressure underscores the importance of considering biological evolution in the conception of new vaccines and vaccine strategies.

Title: Impact de la vaccination sur l'évolution de Bordetella pertussis. Abstract: Les vaccins contre la coqueluche sont commercialisés et utilisés dans la plupart des pays du monde depuis déjà plusieurs décennies. L'histoire des vaccins anticoquelucheux est particulière, les traditionnels vaccins cellulaires ayant été remplacés par des vaccins acellulaires dans de nombreux pays développés au cours des deux décennies passées. Selon leur composition, ces vaccins ont un effet important sur l'évolution de l'agent étiologique de la coqueluche, la bactérie Bordetella pertussis. Cette évolution se traduit par des changements génétiques et phénotypiques chez la bactérie, et a eu comme conséquences la résurgence de la coqueluche dans de nombreux pays ainsi que l'apparition de nouvelles souches échappant aux vaccins. L'émergence de nouvelles souches par pression vaccinale illustre ainsi l'importance de la prise en compte de la biologie de l'évolution de la bactérie dans l'élaboration de nouveaux vaccins.

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.

OBJECTIVES: In 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. METHODS: Serotyping 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. RESULTS: During 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). CONCLUSION: Following 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.

Novel vaccine candidates of Bordetella pertussis identified by reverse vaccinology.

Whooping cough is a disease caused by Bordetella pertussis, whose morbidity has increased, motivating the improvement of current vaccines. Reverse vaccinology is a strategy that helps identify proteins with good characteristics fast and with fewer resources. In this work, we applied reverse vaccinology to study the B. pertussis proteome and pangenome with several in-silico tools. We analyzed the B. pertussis Tohama I proteome with NERVE software and compared 234 proteins with B. parapertussis, B. bronchiseptica, and B. holmessi. VaxiJen was used to calculate an antigenicity value; our threshold was 0.6, selecting 84 proteins. The candidates were depurated and grouped in eight family proteins to select representative candidates, according to bibliographic information and their immunological response predicted with ABCpred, Bcepred, IgPred, and C-ImmSim. Additionally, a pangenome study was conducted with 603 B. pertussis strains and PanRV software, identifying 3421 core proteins that were analyzed to select the best candidates. Finally, we selected 15 proteins from the proteome study and seven proteins from the pangenome analysis as good vaccine candidates.

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.

Genome-based prediction of cross-protective, HLA-DR-presented epitopes as putative vaccine antigens for multiple Bordetella species.

IMPORTANCE: Pertussis, caused by Bordetella pertussis, can cause debilitating respiratory symptoms, so whole-cell pertussis vaccines (wPVs) were introduced in the 1940s. However, reactogenicity of wPV necessitated the development of acellular pertussis vaccines (aPVs) that were introduced in the 1990s. Since then, until the COVID-19 pandemic began, reported pertussis incidence was increasing, suggesting that aPVs do not induce long-lasting immunity and may not effectively prevent transmission. Additionally, aPVs do not provide protection against other Bordetella species that are observed during outbreaks. The significance of this work is in determining potential new vaccine antigens for multiple Bordetella species that are predicted to elicit long-term immune responses. Genome-based approaches have aided the development of novel vaccines; here, these methods identified Bordetella vaccine candidates that may be cross-protective and predicted to induce strong memory responses. These targets can lead to an improved vaccine with a strong safety profile while also strengthening the longevity of the immune response.

RpoN (sigma factor 54) contributes to bacterial fitness during tracheal colonization of Bordetella bronchiseptica.

The Gram-negative pathogenic bacterium Bordetella bronchiseptica is a respiratory pathogen closely related to Bordetella pertussis, the causative agent of whooping cough. Despite sharing homologous virulence factors, B. bronchiseptica infects a broad range of mammalian hosts, including some experimental animals, whereas B. pertussis is strictly adapted to humans. Therefore, B. bronchiseptica is often used as a representative model to explore the pathogenicity of Bordetella in infection experiments with laboratory animals. Although Bordetella virulence factors, including toxins and adhesins have been studied well, our recent study implied that unknown virulence factors are involved in tracheal colonization and infection. Here, we investigated bacterial genes contributing to tracheal colonization by high-throughput transposon sequencing (Tn-seq). After the screening, we picked up 151 candidate genes of various functions and found that a rpoN-deficient mutant strain was defective in tracheal colonization when co-inoculated with the wild-type strain. rpoN encodes σ54 , a sigma factor that regulates the transcription of various genes, implying its contribution to various bacterial activities. In fact, we found RpoN of B. bronchiseptica is involved in bacterial motility and initial biofilm formation. From these results, we propose that RpoN supports bacterial colonization by regulating various bacteriological functions.

Biosynthesis of the Inner Core of Bordetella pertussis Lipopolysaccharides: Effect of Mutations on LPS Structure, Cell Division, and Toll-like Receptor 4 Activation.

Previously developed whole-cell vaccines against Bordetella pertussis, the causative agent of whooping cough, appeared to be too reactogenic due to their endotoxin content. Reduction in endotoxicity can generally be achieved through structural modifications in the lipid A moiety of lipopolysaccharides (LPS). In this study, we found that dephosphorylation of lipid A in B. pertussis through the heterologous production of the phosphatase LpxE from Francisella novicida did, unexpectedly, not affect Toll-like receptor 4 (TLR4)-stimulating activity. We then focused on the inner core of LPS, whose synthesis has so far not been studied in B. pertussis. The kdtA and kdkA genes, responsible for the incorporation of a single 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residue in the inner core and its phosphorylation, respectively, appeared to be essential. However, the Kdo-bound phosphate could be replaced by a second Kdo after the heterologous production of Escherichia coli kdtA. This structural change in the inner core affected outer-core and lipid A structures and also bacterial physiology, as reflected in cell filamentation and a switch in virulence phase. Furthermore, the eptB gene responsible for the non-stoichiometric substitution of Kdo-bound phosphate with phosphoethanolamine was identified and inactivated. Interestingly, the constructed inner-core modifications affected TLR4-stimulating activity. Whereas endotoxicity studies generally focus on the lipid A moiety, our data demonstrate that structural changes in the inner core can also affect TLR4-stimulating activity.

Molecular Evolution and Increasing Macrolide Resistance of Bordetella pertussis, Shanghai, China, 2016-2022.

Resurgence and spread of macrolide-resistant Bordetella pertussis (MRBP) threaten global public health. We collected 283 B. pertussis isolates during 2016-2022 in Shanghai, China, and conducted 23S rRNA gene A2047G mutation detection, multilocus variable-number tandem-repeat analysis, and virulence genotyping analysis. We performed whole-genome sequencing on representative strains. We detected pertussis primarily in infants (0-1 years of age) before 2020 and older children (>5-10 years of age) after 2020. The major genotypes were ptxP1/prn1/fhaB3/ptxA1/ptxC1/fim2-1/fim3-1 (48.7%) and ptxP3/prn2/fhaB1/ptxA1/ptxC2/fim2-1/fim3-1 (47.7%). MRBP increased remarkably from 2016 (36.4%) to 2022 (97.2%). All MRBPs before 2020 harbored ptxP1, and 51.4% belonged to multilocus variable-number tandem-repeat analysis type (MT) 195, whereas ptxP3-MRBP increased from 0% before 2020 to 66.7% after 2020, and all belonged to MT28. MT28 ptxP3-MRBP emerged only after 2020 and replaced the resident MT195 ptxP1-MRBP, revealing that 2020 was a watershed in the transformation of MRBP.

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.

Domination of an emerging erythromycin-resistant ptxP3 Bordetella pertussis clone in Shanghai, China.

The number of cases of pertussis has been increasing since 2014 in China, with high prevalence of macrolide resistance in ptxP1 isolates and low prevalence in ptxP3 isolates. This study aimed to investigate the dynamic changes in the B. pertussis population from paediatric patients and household contacts in Shanghai between 2018 and 2022. Clinical data of laboratory-confirmed cases of pertussis were analysed, while isolates recovered from hospitalized children and household contacts were characterized by antimicrobial susceptibility testing, whole-genome sequencing, vaccine antigen gene typing and phylogenetical analysis. Among 640 laboratory-confirmed cases, 340 (53.1%) were fully vaccinated with DTaP and 114 (17.8%) were hospitalized for treatment. The frequency of erythromycin resistance in the 103 B. pertussis isolates from inpatients (n=73) and household contacts (n=30) was 78.6% (81/103), increasing from 65% (13/20) in 2018 to 100% (26/26) in 2022. The proportion of ptxP3 isolates increased from 35% (7/20) in 2018 to 100% (26/26) in 2022. Based on genomic analysis, a novel ptxP3 clone (MT28-Shanghai) belonging to sublineage IVd was discovered and dominated in 2021-2022, which was characterized with ptxP3, erythromycin resistance and prn150. Twelve (11.7%) predicted pertactin-deficient isolates were found; of these, nine were ptxP3 isolates and three were ptxP1 isolates. A complete shift from ptxP1 to ptxP3 in Shanghai, China, which may have been accelerated by the domination of a novel erythromycin-resistant MT28 clone, challenges the pertussis vaccines used at present in China.

Development of a droplet digital PCR for pertussis toxin locus copy number determination in a genetically-modified Bordetella pertussis strain.

To improve pertussis toxin (PT) yield in B. pertussis strains for vaccine production a genetically-engineered strain (gdPT 191-134 strain) with a second copy of the genetically detoxified PT (gdPT) locus was developed. The consistency of the production and genetic stability of the strain when used for vaccine production must be established. We developed two simplex ddPCR assays with PCR systems for ptxA, the target gene present in two copies, and pgm, the reference gene present as a single copy. The ddPCR assay had sufficient precision to discriminate the copy number of the PT locus accurately in two B. pertussis strains: one copy in the parent, non-genetically-engineered strain and two copies in the gdPT 191-134 strain. Using the ddPCR assays, we were able to show that the ratio of the ptxA to pgm genes decreased during serial culture passages, due to the loss of PT locus, which in turn, resulted in lower levels of PT production over time. We were then able to assess culture conditions that improved the stability of the double locus, as shown by non-significant reduction in gdPT toxin yield.

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.

Whole-Genome Analysis of Bordetella pertussis MT27 Isolates from School-Associated Outbreaks: Single-Nucleotide Polymorphism Diversity and Threshold of the Outbreak Strains.

Bordetella pertussis, the causative agent of whooping cough, can cause pertussis outbreaks in humans, especially in school-aged children. Here, we performed whole-genome sequencing of 51 B. pertussis isolates (epidemic strain MT27) collected from patients infected during 6 school-associated outbreaks lasting less than 4 months. We compared their genetic diversity with that of 28 sporadic isolates (non-outbreak MT27 isolates) based on single-nucleotide polymorphisms (SNPs). Our temporal SNP diversity analysis revealed a mean SNP accumulation rate (time-weighted average) of 0.21 SNPs/genome/year during the outbreaks. The outbreak isolates showed a mean of 0.74 SNP differences (median, 0; range, 0 to 5) between 238 isolate pairs, whereas the sporadic isolates had a mean of 16.12 SNP differences (median, 17; range 0 to 36) between 378 isolate pairs. A low SNP diversity was observed in the outbreak isolates. Receiver operating characteristic analysis demonstrated that the optimal cutoff value to distinguish between the outbreak and sporadic isolates was 3 SNPs (Youden's index of 0.90 with a true-positive rate of 0.97 and a false-positive rate of 0.07). Based on these results, we propose an epidemiological threshold of ≤3 SNPs per genome as a reliable marker of B. pertussis strain identity during pertussis outbreaks that span less than 4 months. IMPORTANCE Bordetella pertussis is a highly infectious bacterium that easily causes pertussis outbreaks in humans, especially in school-aged children. In detection and investigation of outbreaks, excluding non-outbreak isolates is important for understanding the bacterial transmission routes. Currently, whole-genome sequencing is widely used for outbreak investigations, and the genetic relatedness of outbreak isolates is assessed based on differences in the number of single-nucleotide polymorphisms (SNPs) in the genomes of different isolates. The optimal SNP threshold defining strain identity has been proposed for many bacterial pathogens, but not for B. pertussis. In this study, we performed whole-genome sequencing of 51 B. pertussis outbreak isolates and identified a genetic threshold of ≤3 SNPs per genome as a marker defining the strain identity during pertussis outbreaks. This study provides a useful marker for identifying and analyzing pertussis outbreaks and can serve as a basis for future epidemiological studies on pertussis.

Immune responses and protection against Streptococcus pneumoniae elicited by recombinant Bordetella pertussis adenylate cyclase (CyaA) carrying fragments of pneumococcal surface protein A, PspA.

Streptococcus pneumoniae is a common agent of important human diseases such as otitis media, pneumonia, meningitis and sepsis. Current available vaccines that target capsular polysaccharides induce protection against invasive disease and nasopharyngeal colonization in children, yet their efficacy is limited to the serotypes included in the formulations. The virulence factor Pneumococcal Surface Protein A (PspA) interacts with host immune system and helps the bacteria to evade phagocytosis. Due to its essential role in virulence, PspA is an important vaccine candidate. Here we have tested a delivery system based on the adenylate cyclase toxin of Bordetella pertussis (CyaA) to induce immune responses against PspA in mice. CyaA was engineered to express fragments of the N-terminal region of PspAs from clades 2 and 4 (A2 and A4) and the resulting proteins were used in immunization experiments in mice. The recombinant CyaA-A2 and CyaA-A4 proteins were able to induce high levels of anti-PspA antibodies that reacted with pneumococcal strains expressing either PspA2 or PspA4. Moreover, reactivity of the antibodies against pneumococcal strains that express PspAs from clades 3 and 5 (PspA3 and PspA5) was also observed. A formulation containing CyaA-A2 and CyaA-A4 was able to protect mice against invasive pneumococcal challenges with isolates that express PspA2, PspA4 or PspA5. Moreover, a CyaA-A2-A4 fusion protein induced antibodies at similar levels and with similar reactivity as the formulation containing both proteins, and protected mice against the invasive challenge. Our results indicate that CyaA-PspA proteins are good candidates to induce broad protection against pneumococcal isolates.