Bordetella pertussis strain lacking pertactin and pertussis toxin.
Prodromal pertussis symptoms developed on March 4, 2013, in an 11-month-old white, non-Hispanic infant from New York State while the family was traveling outside the state. Cough reportedly began on March 14, 2013, and 12 days later (March 26) he was brought to his healthcare provider (HCP) with symptoms consistent with pertussis.
Since the child's birth, the diagnosing HCP had seen the child only once; no visits to other HCPs were known. Per parental report, the case-patient was experiencing paroxysmal cough, apnea, and posttussive vomiting. No thoracic radiograph was obtained. A 5-day course of oral azithromycin was prescribed; the parent reported that the infant received treatment for 3 consecutive days, beginning March 26, 2013. The infant was not reported to have any pertussis-associated complications (seizures, pneumonia, or encephalopathy) and had only light coughing as of April 11, 2013.
The infant was unvaccinated because the parents refused administration of all vaccines. Three siblings, ages 12, 10, and 8 years, lived with the infant and were undervaccinated; they had received 2, 1, and 3 doses, respectively, of pertussis-containing vaccines. No coughing illness was reported among the siblings. The mother reported that she received Tdap vaccine during her pregnancy with the case-patient, but receipt of vaccine could not be verified.
A nasopharyngeal swab specimen was collected from the infant on March 26, 2013, for testing at a commercial laboratory. The isolate was also forwarded to New York State's public health laboratory, the Wadsworth Center, where it was found to be positive for B. pertussis by PCR targeting IS481 and BP283 (5). Both laboratories yielded positive culture results for B. pertussis. No other testing was performed.
The Wadsworth Center forwarded the isolate, designated I979, to the Centers for Disease Control and Prevention (CDC; Atlanta, Georgia, USA) for confirmatory identification and molecular typing as part of the Enhanced Pertussis Surveillance program (6). PCR amplification of the gene encoding the first subunit of Pt (ptxA) was unsuccessful while the CDC multitarget real-time PCR diagnostic assay was performed (7). Amplification of the promoter region (ptxP) and ptxA was also unsuccessful during multilocus sequence typing targeting acellular vaccine component genes ptxA, ptxP, prn, and fim3 (8).
Further characterization of I979 and French strain FR3749 (4) was undertaken by multilocus sequence typing, multilocus variable-number tandem-repeat analysis, pulsed-field gel electrophoresis (9), and whole-genome sequencing. Long sequencing reads were obtained with the Pacific Biosciences RS II (Menlo Park, CA, USA) at >120 x coverage and assembled de novo into a single contig by using HGAP v3 and Quiver v1 (Pacific Biosciences). Assembly structure was confirmed with a genome optical map after restriction digestion with Kpnl (OpGen, Gaithersburg, MD, USA). The final sequence was polished with short reads obtained with Illumina MiSeq and CLC Genomics Workbench v7.5.1 (QIAGEN, Valencia, CA, USA) with >90 x coverage. Completed genomes were submitted to the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) with GenBank accession nos. CP010965 (FR3749) and CP010966 (I979). Basic genome metrics are listed in Table 1.
Prn production was determined by ELISA (2,11). Pt production was examined through Western blot analysis of cultures grown in cyclodextrin-modified StainerScholte liquid medium to optical density (OD) 600 nm = 0.1 (12). Proteins precipitated with trichloroacetic acid were washed, reduced, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Pt detection is described in Figure 1.
I979 and FR3749 share the same multilocus variablenumber tandem-repeat analysis type 27 andprn-2 genotype, the most common recent types (3,13). I979 and FR3749 are fim3-1 andfim3-2, respectively. Thefim3 locus has fluctuated between these 2 alleles recently (3,8). Pulsed-field gel electrophoresis indicated that I979 displays profile CDC306, and FR3749 displays CDC046. I979 and FR3749 both lacked Pt production, as shown by Western blot (Figure 1). I979 also failed to produce Prn, whereas FR3749 was positive for Prn production by ELISA, within the range of negative (OD 0.3-0.6) and positive (OD 1.2-1.6) controls.
Comparison of assembled I979 and FR3749 genomes with that of Tohama I (GenBank accession no. NC_002929.2) (10) indicated that the entire ptx/ptl operon is missing as the result of a putative deletion spanning 28,040 bp (Figure 2). Both genomes contain a conserved, truncated IS481 immediately upstream of the deletion and a single IS481 (FR3749) or 2 tandem IS481 sequences (I979) immediately downstream (Figure 2). Within Tohama I, the region absent from I979 and FR3749 encodes 30 predicted genes bound by 2 NCATGN motifs, the target sequence for IS481 insertion (Table 2). The insertion element IS1002 is located within the 3' end of this region, and shares a GCATGG motif with IS481 immediately downstream. The 3' deletion boundary is between IS1002 and IS481 (Figure 2). Whole-genome alignment, using progressiveMauve (14), of I979 and FR3749 with Tohama I revealed structural variation through genomic rearrangements and inversions. In particular, I979 and FR3749 genomes differ by a single, large inversion, the coordinates of which correspond to 2 conserved insertions of IS481 in opposing orientations (online Technical Appendix Figure). I979 and FR3749 differ by 31 single nucleotide polymorphisms, each differing from Tohama I by 204 and 173 single nucleotide polymorphisms, respectively. FR3749 contains wild-type prn at position 1613, whereas I979 prn contains an IS481 insertion, the most common cause of Prn-deficiency, at position 1613 (2,11).
B. pertussis strain I979, identified in our study, is both Pm- and Pt-deficient. Loss of Pt in B. pertussis is a rare occurrence; only 2 isolates have been documented in 8 years. Both I979 and FR3749 were isolated from unvaccinated infants (11 months and 3 months old, respectively), who exhibited typical pertussis symptoms, although FR3749 had difficulty colonizing and multiplying in respiratory tracts of adult mice (4). B. pertussis isolates with deletions at other sites across the genome, including part or all of prn, were reported previously (4,15). During the past 5 years, US B. pertussis isolates have become nearly 100% Prn-deficient (2,3) (unpub. data), and Prn-deficient isolates have been obtained from vaccinated persons (11). The loss of Pt may represent a higher fitness cost to B. pertussis than the loss of Prn. In addition, the possibility that only the Pt-deficient isolates were recovered from patients who were co-infected with wild-type and mutant B. pertussis cannot be discarded. Further testing in models to understand the clinical relevance of Prn- and Ptdeficient strains in vaccinated and unvaccinated persons is warranted.
Although incidence of combined Pt- and Prn-deficiency in B. pertussis is rare, any increased mutation in these or other acellular vaccine immunogens may have serious implications for the efficacy of current vaccines. Global epidemiologic, culture-based, and molecular-based monitoring of B. pertussis is critical for understanding current trends of the disease it causes.
Members of the CDC Pertussis Working Group: Dhwani Batra, Kanwar Bedi, Katherine E. Bowden, Pamela K. Cassiday, Marsenia J. Harrison, Owen Herzegh, Kristen Knipe, Vladimir Loparev, Lucia C. Pawloski, Yanhui Peng, Lori A. Rowe, Scott Sammons, Mili Sheth.
Margaret M. Williams, Kathryn A. Sen, Michael R. Weigand, Tami H. Skoff, Victoria A. Cunningham, Tanya A. Halse, M. Lucia Tondella, CDC Pertussis Working Group 
 Members of the CDC Pertussis Working Group are listed at the end of this article.
Author affiliations: Centers for Disease Control and Prevention, Atlanta, Georgia, USA (M.M. Williams, M.R. Weigand, T.H. Skoff, M.L. Tondella); New York State Department of Health, Albany, New York, USA (K.A. Sen); Livingston County Health Department, Mt. Morris, New York, USA (V.A. Cunningham); Wadsworth Center, Albany (T.A. Halse)
B. pertussis isolate I979 was contributed by the state of New York through participation in the Enhanced Pertussis Surveillance of the Emerging Infections Program Network. In particular, we thank Kathy Root, Kimberlee Musser, and Tammy Quinlan for providing clinical data and isolate I979 and for performing pertussis PCR testing. We also thank Nicole Guiso, Institut Pasteur, Paris, France, for sharing isolate FR3749.
Funding for genome sequencing was provided internally by CDC as part of its Advanced Molecular Detection Initiative.
Dr. Williams is a research scientist in the Pertussis and Diphtheria Laboratory, Division of Bacterial Diseases, National Center for Immunization and Respiratory Diseases, CDC, Atlanta, Georgia, USA. She specializes in molecular characterization of bacterial pathogens, with a current focus on pertussis genomics.
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Address for correspondence: Margaret M. Williams, Centers for Disease Control and Prevention, 1600 Clifton Rd, Atlanta, GA 30329-4027, USA; email: MWilliams7@cdc.gov
Table 1. Characterization of Bordetella pertussis strains I979 and FR3749 in comparison to strain Tohama I * GenBank No. prn Strain accession no. Length, bp IS481 type Prn Reference FR3749 CP010965 4,079,396 249 2 + This study I979 CP010966 4,082,551 252 2 - This study Tohama I NC_002929.2 4,086,189 238 1 + (10) * Prn, pertactin; + positive; -, negative. Table 2. Genes in Bordetella pertussis vaccine strain Tohama I deleted from B. pertussis I979 and FR3749 genomes, including the entire ptx/ptl operon * Protein ID, GenBank accession no. Gene Product NP 882281.1 Hypothetical protein NP 882282.1 ptxA Pertussis toxin subunit 1 NP 882283.1 ptxB Pertussis toxin subunit 2 NP 882284.1 ptxD Pertussis toxin subunit 4 NP 882285.1 ptxE Pertussis toxin subunit 5 NP 882286.1 ptxC Pertussis toxin subunit 3 NP 882287.1 ptlA Type IV secretion system protein PtlA NP 882288.1 ptlB Type IV secretion system protein PtlB NP 882289.1 ptlC Type IV secretion system protein PtlC NP 882290.1 ptlD Type IV secretion system protein PtlD NP 882291.1 ptlI Type IV secretion system protein PtlI NP 882292.1 ptlE Type IV secretion system protein PtlE NP 882293.1 ptlF Type IV secretion system protein PtlF NP 882294.1 ptlG Type IV secretion system protein PtlG NP 882295.1 ptlH Type IV secretion system protein PtlH tRNA-Asn NP 882296.1 Membrane protein NP 882297.1 AraC family transcriptional regulator NP 882298.1 Hypothetical protein NP 882299.1 Membrane protein NP 882300.1 Hypothetical protein NP 882301.1 Peptide ABC transporter substrate binding protein NP 882302.1 Transport system permease NP 882303.1 Transport system permease Pseudogene NP 882304.1 IS481 Transposase NP 882305.1 argJ Bifunctional ornithine acetyltransferase/ N-acetylglutamate synthase NP 882306.1 Hypothetical protein NP 882307.1 Hypothetical protein NP 882308.1 IS 1002 Transposase * ID, identification; ptx, pertussis toxin gene; ptl, pertussis toxin liberation gene.
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|Author:||Williams, Margaret M.; Sen, Kathryn A.; Weigand, Michael R.; Skoff, Tami H.; Cunningham, Victoria A.|
|Publication:||Emerging Infectious Diseases|
|Date:||Feb 1, 2016|
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