Prevalence and molecular characterization of pertactin-deficient Bordetella pertussis in the United States.

Pertussis has shown a striking resurgence in the United States, with a return to record numbers of reported cases as last observed in the 1950s. Bordetella pertussis isolates lacking pertactin, a key antigen component of the acellular pertussis vaccine, have been observed, suggesting that B. pertussis is losing pertactin in response to vaccine immunity. Screening of 1,300 isolates from outbreak and surveillance studies (historical isolates collected from 1935 up to 2009, isolates from the 2010 California pertussis outbreak, U.S. isolates from routine surveillance between 2010-2012, and isolates from the 2012 Washington pertussis outbreak) by conventional PCR and later by Western blotting and prn sequencing analyses ultimately identified 306 pertactin-deficient isolates. Of these pertactin-deficient strains, 276 were identified as having an IS481 in the prn gene (prnIS481 positive). The first prnIS481-positive isolate was found in 1994, and the next prnIS481-positive isolates were not detected until 2010. The prevalence of pertactin-deficient isolates increased substantially to more than 50% of collected isolates in 2012. Sequence analysis of pertactin-deficient isolates revealed various types of mutations in the prn gene, including two deletions, single nucleotide substitutions resulting in a stop codon, an inversion in the promoter, and a single nucleotide insertion resulting in a frameshift mutation. All but one mutation type were found in prn2 alleles. CDC 013 was a predominant pulsed-field gel electrophoresis (PFGE) profile in the pertactin-positive isolates (203/994) but was found in only 5% (16/306) of the pertactin-deficient isolates. Interestingly, PFGE profiles CDC 002 and CDC 237 represented 55% (167/306) of the identified pertactin-deficient isolates. These results indicate that there has been a recent dramatic increase in pertactin-deficient B. pertussis isolates throughout the United States.

Identification of a mutation associated with erythromycin resistance in Bordetella pertussis: implications for surveillance of antimicrobial resistance.

Erythromycin treatment failures and in vitro resistance of Bordetella pertussis have been reported on several occasions in the past few years, but the mechanism of resistance has not been described. One potential mechanism, genetic modification of the erythromycin-binding site on the 23S rRNA of the 50S ribosomal subunit, has been observed in other bacteria. To explore this possibility, we amplified the portion of the 23S rRNA gene encoding the central loop of domain V. DNA sequencing and restriction fragment length polymorphism of the PCR products showed that each of the four erythromycin-resistant B. pertussis strains tested contained an A-to-G transition mutation at position 2058 (Escherichia coli numbering) of the 23S rRNA gene. The mutation was not found in seven erythromycin-susceptible isolates tested. Two of the resistant isolates were heterozygous, containing at least one mutant copy and one wild-type copy of the 23S rRNA gene. These results indicate that erythromycin resistance in these strains is likely due to a mutation of the erythromycin-binding site in the 23S rRNA gene. Identification of the resistance mechanism will facilitate development of molecular susceptibility testing methods that can be used directly on clinical specimens in the absence of an isolate.

Bordetella pertussis isolates with a heterogeneous phenotype for erythromycin resistance.

Erythromycin is currently being used for both prophylaxis and treatment of pertussis infections. Erythromycin resistance was first recognized in Bordetella pertussis in Arizona in 1994, and since then, three additional resistant isolates have been identified in the United States. To better assess the potential public health impact of erythromycin-resistant B. pertussis, we used the disk diffusion assay to evaluate the frequency of erythromycin resistance among 1,030 recently circulating U.S. isolates and found the rate of occurrence to be <1%. We also describe a novel heterogeneous phenotype, with erythromycin-resistant colonies appearing only after a 7-day incubation period. To optimize patient management, we recommend that clinicians be alert to potential treatment failures and that laboratorians use a 7-day incubation period when screening for resistance. Our ongoing national surveillance will continue to monitor for resistant B. pertussis isolates and their potential association with changing pertussis epidemiology.

Molecular epidemiology of Bordetella pertussis by pulsed-field gel electrophoresis profile: Cincinnati, 1989-1996.

Reported cases of pertussis have increased in the United States, with peaks occurring every few years. Bordetella pertussis isolates collected in Cincinnati from 1989 to 1996 were analyzed with pulsed-field gel electrophoresis (PFGE), to evaluate trends. Among 496 isolates, 30 PFGE profiles were identified; 32% were CYXXI-010, the profile that predominated each year. Eighteen profiles (198 strains) were identified in 1989-1992, 20 profiles (197 strains) were identified during the 1993 epidemic, and 11 profiles (101 strains) were identified in 1994-1996. From 1989 to 1996, among 42 patients, isolates from household members in 17 (89%) of 19 households had concordant PFGE profiles. There was no association between PFGE profile and seasonality, age, and hospitalization or pneumonia in infants <1 year old. The 1993 epidemic was associated primarily with an increased prevalence of PFGE profiles that circulated before and after 1993, which suggests that the epidemic was due to factors other than the emergence of a novel B. pertussis strain.

Polymorphism in Bordetella pertussis pertactin and pertussis toxin virulence factors in the United States, 1935-1999.

To elucidate the potential role of the etiologic agent in recent increases of pertussis incidence in the United States, we studied the polymorphism in pertactin and pertussis toxin, which are Bordetella pertussis proteins important for pathogenesis and immunity. We sequenced regions of their genes (prn and ptx) in 152 B. pertussis strains isolated from 1935 through 1999 and identified 2 prn sequences: prn1 (old), observed continuously since 1935, and prn2 (new), not recognized until 1981 but seen in 97% of tested isolates in 1999. There were 3 ptx S1 subunit sequences: ptxS1D (old) was identified in 3 strains (1935 and 1939); ptxS1B (old) represented 87% of the strains recovered during 1935-1974; and ptxS1A (new) was the most prevalent during 1975-1987 and 1989-1999 (64% and 78%, respectively). Potential association between vaccination and the observed shift from old to new types requires further study. Our results provide the basis for prospectively monitoring for changes among circulating B. pertussis that might have epidemiologic relevance.

Molecular epidemiology of Bordetella pertussis by DNA fingerprinting with pulsed-filled gel electrophoresis, 1989-1996

Pertussis is an endemic disease in the United States of America, with epidemics occurring every three to four years. In Cincinnati, Bordetella pertussis isolates collected from 1989 to 1996 were analysed by genomic subtyping with pulsed-field gel electrophoresis (PFGE) to evaluate the B pertussis population before, during and after a large epidemic of epidemiologically relevant changes. Among the 496 B pertussis isolates, 31 PFGE profiles were identified; 32 percent of isolates were CYXXI-010 and this profile predominated in each year. Nineteen, 20 and 12 PFGE profiles were identified in the pre-epidemic period (n=198), during the epidemic (n = 197) and in the post-epidemic period (n = 101), resulting in genotypic diversities of 0.82, 0.83 and 0.76 respectively. From 1989 to 1996, among 19 households clusters of 42 patients, 17 (89 percent) households had concordant PFGE profiles among isolates from household members. There was no association between PFGE type and seasonality, age, hospitalisation or pneumonia in infants. The 1993 epidemic was primarily associated with increased prevalence of B pertussis PFGE profiles that circulated before and after the epidemic, suggesting increased susceptibility to pertussis rather than a novel strain as a cause of the outbreak.(Au)

Evidence for transmission of pertussis in schools, Massachusetts, 1996: epidemiologic data supported by pulsed-field gel electrophoresis studies.

In 1996, 18 of 20 pertussis outbreaks reported in Massachusetts occurred in schools. Pertussis surveillance data were reviewed and a retrospective cohort study was conducted in a high school that experienced an outbreak. Bordetella pertussis isolates from 9 school cases and from 58 cases statewide were examined by use of pulsed-field gel electrophoresis (PFGE). Statewide incidence rates were highest among children aged <1 year, 10-14 years, and 15-19 years (106, 117, and 104 cases per 100,000, respectively). Among 34 confirmed and 20 probable cases at the school, 61% had cough onset within 8 weeks of school opening. Five different PFGE types were identified among the 58 B. pertussis isolates from throughout the state. All 9 isolates from the affected high school were the same PFGE type. School-aged children may play an important role in pertussis epidemics. Consideration should be given to use of acellular pertussis vaccines among school-aged children.

Analysis of Bordetella pertussis isolates from an epidemic by pulsed-field gel electrophoresis.

We examined genetic variation among 78 clinical isolates of Bordetella pertussis, including 54 strains recovered during a 1986 pertussis epidemic. A total of 16 pulsed-field gel electrophoresis (PFGE) profiles, generated with each of three different enzymes (XbaI, SpeI, and DraI), were obtained from the epidemic and sporadic isolates included in the study. Indistinguishable profiles were seen among strains unrelated temporally or geographically, as well as among strains isolated sporadically from the same geographic areas. All isolates from the epidemic had indistinguishable PFGE profiles. The PFGE pattern of the epidemic strains was shared with only 1 of 25 strains isolated independently of the outbreak. This isolate was cultured from a specimen from a laboratory scientist who had been working with the epidemic strains, further implicating the usefulness of PFGE for the epidemiologic study of clinical strains of B. pertussis. Differences in PFGE profiles for single epidemic strains occurred occasionally upon repeated passage on agar medium, suggesting that subculturing of initial isolates should be minimized before pulsed-field analysis.

Comparison of Legionella pneumophila isolates by arbitrarily primed PCR and pulsed-field gel electrophoresis: analysis from seven epidemic investigations.

Arbitrarily primed PCR (AP-PCR) and pulsed-field gel electrophoresis (PFGE) subtyping were applied to clinical and environmental isolates from seven unrelated outbreaks of Legionnaires' disease. The patterns observed with each method matched patient isolates and the epidemiologically linked source of disease for each of the seven outbreaks. PFGE allowed more discrimination among various isolates, although AP-PCR usually gave comparable results. With both methods, certain patterns appeared to predominate in the comparison of the seven outbreaks. Of five clinical isolates not associated with the outbreaks, three gave profiles distinct from those observed in the outbreaks by both methods. This suggests that there are at least two predominant subtypes of Legionella pneumophila serogroup 1 associated with outbreaks. Investigations of outbreaks of legionellosis should employ either PFGE or AP-PCR in addition to monoclonal antibody analysis.

Pertussis infection among adults during the 1993 outbreak in Chicago.

To evaluate the role of adults in the transmission of pertussis during an epidemic, persons presenting with unexplained cough to ambulatory care clinics were evaluated for evidence of pertussis infection. Nasopharyngeal specimens for culture and serum samples for IgG and IgA antibodies to filamentous hemagglutinin and pertussis toxin antigens of Bordetella pertussis were obtained. Thirty-eight adults were enrolled in the study; 10 (26%) had serologic evidence of B. pertussis infection. Clinical findings were not significantly different among persons with and without evidence of pertussis infection. Pertussis should be considered in the differential diagnosis of persistent cough in all age groups. Future use of new acellular pertussis vaccines in adults may substantially impact the control of the infection.

Viability of Bordetella pertussis in four suspending solutions at three temperatures.

We studied the survival of Bordetella pertussis in four suspending solutions (Casamino Acids broth, deionized water, phosphate-buffered saline, and serum inositol), subjected to three storage temperatures (4, -20, and -70 degrees C) and two freezing methods (direct freezing and fast-freezing in an ethanol-dry-ice bath). Recovery rates were higher for longer periods for suspensions stored at -70 degrees C than those stored at -20 or 4 degrees C. Serum inositol showed the highest recovery rates for all experimental conditions, followed by Casamino Acids, deionized water, and phosphate-buffered saline. Cell viability was significantly reduced in phosphate-buffered saline suspensions fast-frozen before storage. These results identify optimal conditions for storing B. pertussis cells and are applicable to the collection, transport, and storage of aspirated nasopharyngeal samples for use in the laboratory diagnosis of pertussis.

Rapid immunodot technique for identifying Bordetella pertussis.

We developed and evaluated a rapid test with monoclonal antibodies to identify cultures of Bordetella pertussis. Samples of 5 microliters of cells suspended in formalin-saline were dried onto a nitrocellulose disk. The disk was placed in a filtration device, and 5-microliters volumes of murine monoclonal antibody directed against B. pertussis lipooligosaccharide and peroxidase conjugate were added consecutively, with washing after each addition. The disk was removed and immersed in peroxidase substrate solution. All of 66 B. pertussis isolates confirmed by direct fluorescent-antibody assay were correctly identified by using four different monoclonal antibodies. One of the monoclonal antibodies did not react with over 20 bacterial species tested, including other Bordetella, Acinetobacter, Haemophilus, Moraxella, Mycobacterium, Neisseria, and Staphylococcus spp. This technique detected > or = 2 micrograms of lipooligosaccharide per ml or > or = 5 x 10(8) B. pertussis cells per ml. This rapid procedure used small amounts of reagents, needed less equipment, and was less subjective and more specific than the direct fluorescent-antibody assay.

Replica plating of colonies from Listeria-selective agars to blood agar to improve the isolation of Listeria monocytogenes from foods.

Bacterial colonies from Listeria-selective agars were replica plated to sheep blood agar to screen for beta-hemolysis. By using the replica plating method to test for the beta-hemolytic characteristic of all the colonies growing on Listeria-selective agars instead of picking 3 to 10 suspected colonies for further testing, we recovered Listeria monocytogenes from 59 of 142 Listeria-selective agar plates which contained colonies of hemolytic and nonhemolytic Listeria species and were negative when tested by conventional colony picks.

Evaluation of three newly developed direct plating media to enumerate Listeria monocytogenes in foods.

LiCl-phenylethanol-moxalactam Agar (LPMA), ARS Modified McBride Agar, and Modified Vogel Johnson Agar were compared with previously tested plating media in the enumeration of Listeria monocytogenes from pasteurized whole milk, chocolate ice cream mix, Brie cheese, and raw cabbage. LPMA was most suitable for analyzing Brie cheese and cabbage. Gum base-nalidixic acid-tryptone-soya medium (previously tested) was most suitable for analyzing milk and chocolate ice cream mix.