Diagnostic Microbiology and Infectious Disease 80 (2014) 204–206

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Clinical Studies

Distribution of Campylobacter jejuni capsular types, 2007–2012, Philadelphia, PA Charles Garrigan a, Abora Ettela a, Frédéric Poly b, Patricia Guerry b, Irving Nachamkin a,⁎ a b

Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Naval Medical Research Center, Silver Spring, MD, USA

a r t i c l e

i n f o

Article history: Received 23 May 2014 Received in revised form 28 July 2014 Accepted 1 August 2014 Available online 13 August 2014

a b s t r a c t The distribution of Campylobacter jejuni capsular serotypes in the Philadelphia region from 2007 to 2012 was determined using molecular methods. Compared with the last U.S. survey in 1990, there does not appear to be a major shift in circulating capsular types. © 2014 Elsevier Inc. All rights reserved.

Keywords: Campylobacter Serotyping Molecular epidemiology Gastroenteritis Vaccines

Campylobacter jejuni infection is the leading cause of bacterial gastroenteritis in the United States with over 1 million cases each year (Scallan et al., 2011). Typical symptoms of uncomplicated campylobacteriosis are watery diarrhea and abdominal pain and, in uncomplicated cases, is generally a self-limited illness. Guillain–Barré syndrome (GBS) is a well-known post-infectious complication of C. jejuni infection (Willison, 2005). The Penner heat stable (HS) serotyping scheme has been used as a common method to classify and study the epidemiology of Campylobacter since the early 1980s (Penner and Hennessy, 1980). The polysaccharide capsule (CPS) expressed by Campylobacter was identified to be the primary serodeterminent of the Penner scheme with lipooligosaccharide also being a factor for some serotypes (Karlyshev et al., 2000, Poly et al., 2011). HS serotyping is time and labor intensive with repeat testing often required due to cross-reacting serotype complexes (Nicholson and Patton, 1993). The cost and complexity of producing the required antisera have also limited its usefulness (Poly et al., 2011). In their investigation into developing a conjugate CPS vaccine for C. jejuni and in lieu of classical serotyping, Poly et al. (2011) developed a multiplex PCR method for rapid determination of CPS type. A general epidemiological study of C. jejuni HS serotypes in the United States has not been performed since the 1993 study by Patton et al. (1993). With an interest in circulating capsular types in the United States toward development of a Campylobacter vaccine and, in addition, the prevalence of C. jejuni CPS types related to GBS, such as ⁎ Corresponding author. Tel.: +1-215-662-3435; fax: +1-215-349-5090. E-mail address: [email protected] (I. Nachamkin). http://dx.doi.org/10.1016/j.diagmicrobio.2014.08.002 0732-8893/© 2014 Elsevier Inc. All rights reserved.

HS19, prompted us to analyze strains using CPS molecular typing of C. jejuni isolated in the Philadelphia region over a multi-year period. C. jejuni isolated from stool samples of patients seen within the University of Pennsylvania Health System from 2007 through 2012 were included in the study. The study was approved by the Institutional Review Board of the University of Pennsylvania. Stool samples were processed in the Clinical Microbiology Laboratory at the Hospital of the University of Pennsylvania by plating on cefoperazone–vancomycin– amphotericin selective agar (BD Diagnostics, Sparks, MD) and incubating plates at 42 °C in a microaerobic environment for at 72 hours. Isolates were initially identified as C. jejuni based on growth at 42 °C, gram stain, and positive for hippurate hydrolysis (Fitzgerald and Nachamkin, 2011). A total of 170 of 284 (60%) available C. jejuni isolates were tested using CPS molecular typing. The other isolates were either non-viable or could not be found in storage. The number of isolates tested by year was 2007 (n = 13), 2008 (n = 35), 2009 (n = 21), 2010 (n = 34), 2011 (n = 38), and 2012 (n = 29). The average age of patients from whom the isolates were obtained was 41.6 (range 17–90) for female patients and 42.7 (range 19–83) for male patients. There were slightly more female patients in the group (ratio 1.12). Approximately 40% of patients were from an inpatient setting; approximately 60% were outpatients. Approximately 15% of isolates were from patients with travel-related infections. Isolates were grown overnight on Brucella blood agar plates at 37 °C, 5% O2 and 10% CO2. Genomic DNA extractions were performed using the Qiagen DNeasy Blood and Tissue kit. Isolates were confirmed as C. jejuni through PCR detection of the hipO gene (Linton et al., 1997). CPS typing was performed using a multiplex PCR method as initially described by Poly et al. (2011) from the Naval Medical

C. Garrigan et al. / Diagnostic Microbiology and Infectious Disease 80 (2014) 204–206

Research Center (NMRC). Version 6.0 of the NMRC method was used, which utilizes 4 multiplexed mixes designated alpha, beta, gamma, and delta (Poly et al., in preparation). This is an expanded version of the original by introducing 23 new primer sets. These primer mixes contain a total of 38 primer sets able to detect 37 CPS type variations. The alpha mix is able to detect the CPS types HS2, HS3 complex, HS4 complex A (HS4cA), HS6/HS7, HS10, HS15, HS19, HS33/HS35, HS41, HS53, and HS63. HS4cA includes HS13. The beta mix can detect HS1/ HS1(44), HS4 complex B (HS4cB), HS5/HS31, HS8/HS17, HS12, HS21, HS23/HS36, HS27, HS42, and HS57. HS4cB includes HS16 and HS64. The gamma mix detects HS9, HS18, HS22, HS29, HS37, HS44, and HS45. The gamma mix also contains primers for the lpxA gene to serve as an internal control for C. jejuni. The delta mix detects HS11, HS32, HS38, HS40, HS52, HS55, HS58, and HS60. Primer mixes were prepared to a concentration of 25 ng/μL of each primer. Reactions contained 1 μL of primer mix, 3 mmol/L MgCl2, 0.4 mmol/L dNTPs, 1X AmpliTaq Gold PCR buffer, 1.25 units Amplitaq Gold polymerase, and 40 ng of DNA template in a final reaction volume of 50 μL. The cycling conditions were 95 °C for 5 min, and then 30 cycles of 95 °C for 40 s, 56 °C for 60 s, 72 °C for 45 s, and a final extension of 72 °C for 3 min. Five microliters of PCR products was run on 2.3% agarose gels run in parallel with a 100-bp DNA Ladder (New England BioLabs, N3231). Gels were stained with ethidium bromide and viewed on a UV-transilluminator. CPS type is determined by the size of the PCR amplicon. PCR products range in size from 62 bp to 941 bp. Routine quality control of the 4 primer mixes was performed using control DNA samples provided by the NMRC. In addition, we further validated the primer mixes using 21 C. jejuni isolates in our collection that had previously been HS serotyped by Patton et al. (1993). These isolates represented a random selection of clinical strains that had been previously serotyped, and all results were concordant with the molecular CPS typing system. CPS types were identified for 165 of the 170 C. jejuni isolates. Five isolates did not produce a PCR amplicon correlating to a CPS type. These 5 isolates may correspond to serotypes that were not described in the original Penner typing system. The capsule locus is one of the most variable regions of the C. jejuni chromosome. Such variability arises from duplication, deletion, fusion, and exchange of capsular genes (Karlyshev et al., 2005). Such low percentage of non-typable strains can be expected with this system. Twenty-eight different CPS types were detected (Table 1). Fortyone (24.9%) of the isolates were positive for HS4cA and/or HS4cB. HS2 was the second most common CPS type with 22 isolates (13.3%). HS4cA/HS4cB and HS2 were the only CPS types present in the 70th percentile of typable isolates in each of the 6 years analyzed. Overall, HS4cA/HS4cB, HS2, HS4cA, HS3c, HS53, HS21, HS8/HS17, HS1/HS1 (44), HS55, HS18, and HS42 encompassed 122 (73.8%) of the 165 isolates typed. Three isolates were typed as HS19. One hundred and fifty-five of the typed isolates had previously been tested for susceptibility to ciprofloxacin and erythromycin. Fiftysix (36.1%) of the isolates were resistant to ciprofloxacin, and 3 isolates (1.9%) were resistant to erythromycin. There was no association between CPS type and resistance pattern. Overall, resistance rates varied by year for all C. jejuni isolates from the laboratory ranging from 20.0% to 45.5% resistance for ciprofloxacin, with no trends observed. For 2011 and 2012, as an example, the rate of ciprofloxacin resistance in C. jejuni was 37.3% and 37.0%, respectively. Travel-related isolates were more likely to be fluoroquinolone resistant (64%) versus non–travel-related isolates (30.3%). In a previous 1989–90 U.S. survey of Campylobacter circulating serotypes, 298 Campylobacter isolates from FoodNet sites were analyzed (Patton et al., 1993). In comparing our regional data with circulating strains in the United States from that survey, HS4cA/B, HS13, and HS16 were the most common types in the previous study as well as the current analysis, making up over 20% of isolates in both data sets. Comparing isolates that account for approximately 70% of

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serotypes in both studies (i.e., 70th percentile) (Fig. 1), there were a similar CPS type distributions for the common types suggesting that there hasn't been a major shift in circulating types in over 20 years. Of note, there were several less common serotypes detected in our current study that were not common in the 1989–90 survey and vice versa. An analysis of global CPS types by Pike et al. (2013) found the HS4 complex, HS2, and HS1(44) serotypes to dominate. This is consistent with our results with HS4cA/HS4cB and HS2 being the 2 most common and HS1/HS1(44) within the 70th percentile for the Philadelphia area. Campylobacteriosis is a worldwide disease and 1 of the most common bacterial causes of gastrointestinal disease with significant medical and economic burden (Molbak and Havelaar, 2008). Several groups are working toward development of relevant vaccine strategies, and the current study will aid in proper development of a multivalent vaccine strategy (Guerry et al., 2012). Acknowledgments Work at NMRC was funded by Navy Work Unit 6000.RAD1.DA3. A0308. The opinions and assertions contained herein are the private ones of the authors and do not reflect the official policy of the Department of Navy, Department of Defense, nor the U.S. government. PG is an employee of the U.S. government, and this work was prepared as part of her official duties. Title 17 USC 105 provides that “Copyright protection under this title is not available for any work of the U.S. government.” Title 17 USC 101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. government as part of that person's official duties.

Table 1 C. jejuni CPS type distribution of 165 isolates collected in Philadelphia from 2007 through 2012. CPS type

Frequency

% of total

Cumulative %

Hospitalizeda (n)

Travel relatedb (n)

HS4cA/HS4cB HS2 HS4cA HS3c HS53 HS21 HS8/HS17 HS1/HS1(44) HS55 HS18 HS42 HS4cB HS10 HS15 HS23/HS36 HS44 HS6/HS7 HS19 HS57 HS5/HS31 HS4cA/HS53 HS6/HS7/HS4cB HS9 HS12 HS15/HS5/HS31 HS27 HS37 HS45/HS5/HS31

24 22 12 11 10 9 8 7 7 6 6 5 5 5 5 4 3 3 3 2 1 1 1 1 1 1 1 1

14.5 13.3 7.3 6.7 6.1 5.5 4.8 4.2 4.2 3.6 3.6 3.0 3.0 3.0 3.0 2.4 1.8 1.8 1.8 1.2 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6

14.5 27.9 35.2 41.8 47.9 53.3 58.2 62.4 66.7 70.3 73.9 77.0 80.0 83.0 86.1 88.5 90.3 92.1 93.9 95.2 95.8 96.4 97.0 97.6 98.2 98.8 99.4 100.0

8 8 9 2 4 5 1 4 4 4 1 1 2 1 2 0 3 0 1 0 0 0 1 0 0 0 0 0

2 6 0 2 1 1 4 1 0 1 2 0 2 1 1 0 0 0 1 0 0 0 0 0 0 0 0 0

a b

Admitted to hospital or seen in emergency room. Sixty charts were either not available or travel not specifically noted.

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Fig. 1. 70th percentile of isolate CPS types in Philadelphia compared to the 70th percentile of circulating serotypes in the United States, 1989–90 from Patton et al. (1993).

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Distribution of Campylobacter jejuni capsular types, 2007-2012, Philadelphia, PA.

The distribution of Campylobacter jejuni capsular serotypes in the Philadelphia region from 2007 to 2012 was determined using molecular methods. Compa...
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