Antimicrobial resistant Helicobacter fennelliae isolated from non-diarrheal child stool sample in Battambang, Cambodia

Antimicrobial resistant Helicobacter fennelliae isolated from non-diarrheal child stool sample in... Helicobacter fennelliae (H. fennelliae) is associated with human gastroenteritis; however, H. fennelliae was isolated and confirmed by phenotypic and genotypic identification from a non-diarrheal child stool sample in Cambodia. Antimi- crobial susceptibility testing demonstrated that this isolate had a high minimal inhibitory concentration against mac- rolides and quinolones, which are first-line antibiotic treatment choices for Campylobacter infections. Consequently, macrolides and quinolones were likewise expected to be ineffective against Campylobacter-like organisms such as H. fennelliae. This isolate warranted further genetic characterization to better understand associated antibiotic resistance mechanisms. Resistant pathogens from asymptomatic diarrheal cases are likely underestimated, and as such colo- nized individuals may spread resistant organisms to local community members and the environment. Keywords: Helicobacter fennelliae, Non-diarrheal sample, Child, Cambodia, First-report Background chaperonin protein present in virtually all eubacteria, Helicobacter fennelliae (H. fennelliae) is a new Campy- some archaea, and in the plastids and mitochondria of lobacter species originally isolated from asymptomatic, eukaryotes. The utility of this target for bacterial spe - homosexual men with enteritis and proctitis in the past cies identification, detection, quantification, phyloge - few decades [1]. Like H. cinaedi, this species is classified netic analysis, and microbial community profiling was as enterohepatic Helicobacter that inhabits and causes well established [4]. Treatment recommendation guide- bacteremia in intestinal and hepatobiliary tracts of vari- lines are still not available for enterohepatic Helicobac- ous mammal and other species [2]. Additional evidence ter species. Various individual and combined antibiotic suggests that H. fennelliae was implicated as a contrib- regimens were successfully used in treating Helicobac- uting cause of human proctocolitis, gastroenteritis, ter infections; however, there is insufficient information and bacteremia, particularly in immunocompromised to determine resistance rates of H. fennelliae. The main individuals [2, 3]. This Helicobacter species is a fastidi- objective of this report is to describe phenotypic, geno- ous organism that is likely underestimated, and little typic, and antimicrobial susceptibility (AST) data from is known about routes of transmission other than evi- this H. fennelliae isolate from the stool of non-diarrheal dence indicates it is a zoonotic infection [2]. As a fas- child in Cambodia. tidious organism, molecular genotyping methods are recommended to identify Helicobacter species. Towards Methods that end, the groEL and hsp60 genes encode a 60  kDa A surveillance study to describe diarrhea etiologic agents in children and military personnel in Battambang, Cam- bodia has been conducted from 2014 until present. Both *Correspondence: woradeel.fsn@afrims.org diarrheal and non-diarrheal stool samples were observed Department of Enteric Diseases, Armed Forces Research Institute by microscopic examination for the presence of parasites, of Medical Sciences, 315/6 Rajvithi Road, Bangkok 10400, Thailand protozoa, and larvae. Samples were also assessed for the Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ruksasiri et al. Gut Pathog (2018) 10:18 Page 2 of 5 presence of Giardia, Cryptosporidium by enzyme-linked 1 min at 94 °C, 1 min at 46 °C, 1 min at 72 °C, and a final immunosorbent assay (ELISA), and for diarrheagenic E. extension at 72 °C for 10 min. The purified PCR products coli by polymerase chain reaction (PCR) [5]. Enteric path- were additionally differentiate Campylobacter species ogens, including Campylobacter species, were isolated from Helicobacter and Acrobacter species using primers and identified by traditional culture methods [6]. The sus - M13F-pUC (− 40) 5′-GTT TTC CCA GTC ACGAC-3′ and pected Campylobacter-like colonies were subcultured on M13R (− 20) 5′-GCGGA-TAA CAA TTT CAC ACAGG-3′. blood agar supplemented with 6% sodium formate and The result of partial cpn60 sequences (555 bp) was com - fumarate for 48–72 h at 37 °C under microaerobic condi- pared with the database in cpnDB (http://cpndb .cbr.nrc. tions (10% CO and 5% O ). The biochemical identifica - ca) [4]. The confirmed partial sequence was submitted 2 2 tions were included oxidase, catalase, indoxyl hydrolysis, to the National Center for Biotechnology Information hippurate hydrolysis, nitrate reduction, urease, hydrogen (NCBI) before constructing phylogenetic analysis by Bio- sulfide production, susceptibility to cephalothin and nali - Numerics software version 7.6 (Applied Maths, Belgium). dixic acid (30 µg disc) (BD, Spark, USA), oxygen and tem- perature tolerance test. According to no antimicrobial Results and discussion susceptibility recommendation guidelines, H. fennelliae A non-diarrheal stool sample of a young child who pre- resistance was determined using the minimal inhibitory sented to the hospital with fever and, cough was sub- concentration (MIC) by E test (Liofilchem, Roseto degli mitted for laboratory testing. The stool characteristic Abruzzi TE, Italy) against azithromycin (AZM), erythro- was loose without mucus, blood, RBCs, or WBCs. No mycin (ERY), nalidixic acid (NAL), ciprofloxacin (CIP), gastrointestinal parasites were detected microscopically levofloxacin (LEV), ceftriaxone (CRO), spectinomycin or by ELISA. Other enteric bacterial pathogens, includ- (SPT), and tetracycline (TET). C. jejuni ATCC 33560 was ing diarrheagenic E. coli, were not identified, except used as a quality control strain. for suspected colonies of a Campylobacter-like organ- Genomic DNA of suspected Campylobacter-like ism. The colonies characteristics which were presented colonies was extracted and subsequently confirmed as after 6  days incubation were thin, flat, film-like colony, belonging to the Campylobacter genus by screening for with a hypochlorite odor. Biochemical reactions of the the 16S rRNA gene [7]. To determine Campylobacter colony were positive for oxidase, catalase, and indoxyl species, the 15 primer sets of cpn60 target gene were used acetate hydrolysis. It was susceptible to cephalothin disk for verified species as described elsewhere [7, 8]. Sub- but resistant to nalidixic acid disk and could be grown sequently, the unknown Campylobacter species beyond at 42  °C under microaerobic conditions. Culture results 15 primer sets identification were further sequencing indicated that H. fennelliae grows well by supplementing analysis by amplifying cpn60 target gene with degenerate 6% sodium formate and fumarate in blood agar. This is primers H729 and H730 [4]. The sequences of degener - likely due to the fact that formate replaces hydrogen as ate primers were H729: 5′-CGC CAG GGT TTT CCC AGT the electron donor, and fumarate serves as the termi- CAC GAC GAIIIIGCIGGIGAYGGIACIACIAC-3′ and nal electron acceptor for hydrogen-required organism H730 5′-AGC GGA TAA CAA TTT CAC ACA GGA YKI- growth [9]. Notably, an absence of hydrogen, the low-cost YKITCICCRAAI CCIGGIGCYTT-3′. PCR amplification supplemented media, and a long incubation period are was carried out in a total volume of 50 µL containing 6 µL suggested to support growth of H. fennelliae. of genomic DNA template, 2.5 U AmpliTaq G old DNA The MIC results of H. fennelliae and C. jejuni ATCC polymerase (Applied Biosystems, Foster City, Calif.), 33560 were presented in Table  1. Results for this H. fen- 5 mM MgCl , 100 µM each of the dNTPs and 50 nM each nelliae isolate demonstrated high MICs to macrolides of degenerate primers [4]. The cycling conditions were and quinolones, consistent with previous studies [10, 11] performed at 94  °C for 5  min, followed by 28 cycles of and similar to H. cinaedi data [12]. Macrolides, generally Table 1 Determination the  minimal inhibitory concentration (MIC) results of  H. fennelliae and  C. jejuni ATCC 33560 against  azithromycin (AZM), erythromycin (ERY), nalidixic acid (NAL), ciprofloxacin (CIP), levofloxacin (LEV), tetracyclin (TET), ceftriaxone (CRO) and spectinomycin (SPT) Isolates MIC (µg/mL) AZM ERY NAL CIP LEV TET CRO SPT H. fennelliae ≥ 256 ≥ 256 ≥ 256 ≥ 32 3 0.125 0.125 4 C. jejuni ATCC33560 0.125 0.75 4 0.094 0.25 0.25 ≥ 32 0.5 Ruksasiri et al. Gut Pathog (2018) 10:18 Page 3 of 5 considered the drug of choice for Campylobacter treat- prevalence and antimicrobial resistant profile might be ment [1], may be clinically less effective for Campylo - considerably underestimated due to inadequate isola- bacter-like organism infections such as H. fennelliae tion and identification methods [14]. To the best of our and H. cinaedi. Little is known about the antimicrobial knowledge, this is the first report of a macrolide and resistance mechanisms of H. fennelliae. Mutations of quinolone resistant H. fennelliae identified in a young the gyrase and 23S rRNA genes may be responsible for Cambodian child asymptomatic for intestinal infection. decreased susceptibility to quinolones and macrolides, This isolate resembles H. fennelliae, which was previ - respectively [10]. However, decreased susceptibility to ously identified in a boy suffering gastroenteritis and is low MIC macrolide levels were mentioned in a previ- also isolated from dog specimens [15]. With the intro- ous study [13]. The H. fennelliae isolate from our study duction of the ‘Cape Town Protocol,’ H. fennelliae may exhibited a high MIC to macrolides, warranting further be isolated from stool and blood culture in an H -rich molecular characterization to explore other resistance microaerophilic atmosphere. Prior evidence indicated mechanisms. that Helicobacter species related to H. fennelliae were The genotyping confirmation of this H. fennelliae iso - isolated from blood of a young child suffering diarrhea late was performed by sequencing the cpn60 gene, and symptoms [16]. Nevertheless, the nucleotide sequences the result was submitted to NCBI under the accession of H. fennelliae obtained from blood and stool were not number MG696736. A phylogenetic tree analysis (Fig. 1) significantly different [17]. Unfortunately blood sam - divided Helicobacter and Campylobacter strains into ples were not available from the child in this study, so seven distinct groups (cut-off of 90%). The MG696736 that comparison was not achievable. H. fennelliae was entry was classified as group IV, which was 97.2% simi - predominantly isolated from children who presented lar to H. fennelliae ATCC 35684, whereas Campylobacter with diarrheal symptoms, although stools from asymp- species was classified as group VII, which is distinct from tomatic diarrheal children with asthma and/or failure the Helicobacter group VI (cut-off of 90%). to thrive (FTT) were also positive for H. fennelliae [16, H. fennelliae was suggested as a significant pathogen 17]. Another possible explanation of this H. fennelliae associated with human gastroenteritis; however, its finding in stool of asymptomatic diarrheal Cambodia Fig. 1 Neighbor-joining phylogenetic trees based on partial cpn60 gene sequences (555 bp) of Helicobacter strain (MG696736), as compared to cpn60 sequences of other Helicobacter, and Campylobacter strains in cpnDB database Ruksasiri et al. Gut Pathog (2018) 10:18 Page 4 of 5 be construed as official, or as reflecting true views of the Department of the child could relate to breastfeeding. Evidence suggests Army or the Department of Defense. that maternal milk contains a variety of functionally bioactive agents from her innate immune system [18], Availability of data and materials Data sharing not applicable to this article. as well as a mechanism to influence microbial changes in the infant’s gastrointestinal system [19]. As a result Consent for publication of widespread breastfeeding campaigns in the develop- Not applicable. ing world, this may play an important role in the level Ethics approval and consent to participate of asymptomatic carriage within a community [18, 20]. The study protocol was in accordance with ethical guideline of the ‘Code The association between asymptomatic carriage and of Federal Regulations, Title 32, Part 219: Protection of Human Subjects’ and was approved by the Review Board at National Ethics Committee for Health diarrheal pathogens such as Salmonella, E. coli O157 Research, Phnom Penh, Cambodia and Walter Reed Army Institute of Research and Campylobacter was previously reported in out- ( WRAIR), Silver Spring, MD, USA. breaks elsewhere [20]. Identification of an antibiotic Funding resistant H. fennelliae strain from an asymptomatic The study is supported by the Armed Forces Health Surveillance Branch diarrheal person would probably be transmitted into (AFHSB) and it’s GEIS (Global Emerging Infectious Disease Surveillance and local communities and environmental contamination. Response) Section. Hence, the public health significance of resistant patho - gens in human feces warrants effective monitoring to Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- prevent disease outbreaks. lished maps and institutional affiliations. In conclusion, phenotypic and genotypic assessments confirmed that H. fennelliae was isolated from a non- Received: 11 April 2018 Accepted: 19 May 2018 diarrheal stool sample of a Cambodian child suffering from fever with cough and convulsion. The supplement media, incubation atmosphere, and incubation period utilized permitted culture, isolation, and identification of H. fennelliae. The high MICs values against macrolides References 1. Totten PA, Fennell CL, Tenover FC, Wezenberg JM, Perine PL, Satamm WE, (AZM, ERY) and quinolones (NAL, CIP) indicated these et al. Campylobacter cinaedi (sp.nov.) and Campylobacter fennelliae (sp. are less effective against H. fennelliae. This isolate should Nov): two new Campylobacter species associated with enteric diseases in be further characterized to better understand associated homosexual men. J Infect Dis. 1985;151(1):131–9. 2. James HJ, Michael AP, Karen CC, Guido FMLL, Sandry SR, David WW. resistance mechanisms. Manual of clinical microbiology. Washington DC: American Society for Microbiology press; 2015. Authors’ contributions 3. O’Rourke JL, Grehan M, Lee A. Non-pylori Helicobacter species in humans. WL and SR participated in the conception and design of the study. SR, PW, and Gut. 2001;49(5):601–6. CS performed the laboratory work. NS was clinical coordinator and subject 4. Hill JE, Paccagnellla A, Law K, Melito PL, Woodward DL, Price L, et al. Iden- enrollment. WL and OS analyzed the data and wrote the manuscript. SL and tification of Campylobacter spp. and discrimination from Helicobacter and LC coordinated and fully supported this study in Cambodia. LB and JC con- Arcobacter spp. by direct sequencing of PCR-amplified cpn60 sequences tributed to the analysis and helped in writing the manuscript. All authors read and comparison to cpnDB, a chaperonin reference sequence database. J and approved the final manuscript. Med Microbiol. 2006;55(4):393–9. 5. Meng CY, Smith BL, Bodhidatta L, Richard SA, Vansith K, Thy B, et al. Etiol- Author details 1 ogy of diarrhea in young children and patterns of antibiotic resistance in Department of Enteric Diseases, Armed Forces Research Institute of Medical 2 Cambodia. Pediatr Infect Dis J. 2011;30(4):331–5. Sciences, 315/6 Rajvithi Road, Bangkok 10400, Thailand. Battambang Referral 6. Garcia LS. Clinical microbiology procedures handbook. 3rd ed, Washing- Hospital, PrekMohatep Village, SvayPor Commune, Battambang, Cambodia. 3 ton, D.C.: ASM Press; 2010. P. 3.8.1.1–3.8.2.16 and 3.17.1.1–3.18.2.1. Armed Forces Research Institute of Medical Sciences, 18.118 Street Sangkat 4 7. Bullman S, O’Leary J, Corcoran D, Sleator RD, Lucey B. Molecular-based Mettapheap Khan 7 Makara, Phnom Penh, Cambodia. Communicable Dis- detection of non-culturable and emerging campylobacteria in patients ease Control Department, Ministry of Health, 151-153, Kampuchea KromBlvd, presenting with gastroenteritis. Epidemiol Infect. 2012;140(4):684–8. Phnom Penh, Cambodia. 8. Chaban B, Musil KM, Himsworht CG, Hill JE. Development of cpn60-based Real-time quantitative PCR assays for the detection of 14 Campylobacter Acknowledgements species and application to screening of canine fecal samples. Appl Envi- We thank David Saunders, Brett E. Swierczewski, Carl J. Mason, SokVannara, ron Microbiol. 2009;75(10):3055–61. Koy Lenin and Prom Satharath for supervision of this surveillance study. We 9. Roop RM II, Smibert RM, Johnson JL, Krieg NR. Campylobacter mucosalis thank AFRIMS Enteric Diseases Department Staff, Bangkok, Thailand and Bat - (Lawson, Leaver, Pettigrew, and Rowland 1981) comb. nov.: emended tambang Referral Hospital & AFRIMS-CNM Staff, Battambang, Cambodia, for description. Int J Syst Bacteriol. 1985;35(1):189–92. their assistance and kind support. 10. Rimbara E, Mori S, Kim H, Matsui M, Suzuki S, Takahashi S, et al. Helicobac- ter cinaedi and Helicobacter fennelliae transmission in a hospital from 2008 Competing interests to 2012. J Clin Microbiol. 2013;51(7):2439. The authors declare that they have no competing interests. 11. Fujiya Y, Nagamatsu M, Tomida J, Kawamura Y, Yamamoto K, Mawa- tari M, et al. Successful treatment of recurrent Helicobacter fennelliae Authors’ disclaimers bacteraemia by selective digestive decontamination with kanamycin Material has been reviewed by the Walter Reed Army Institute of Research. in a lung cancer patient receiving chemotherapy. JMM Case Rep. There is no objection to its presentation and/or publication. The opinions or 2016;3(5):e005069. assertions contained herein are the private views of the author, and are not to Ruksasiri et al. Gut Pathog (2018) 10:18 Page 5 of 5 12. Kawamura Y, Tomida J, Morita Y, Fujii S, Okamoto T, Akaike T. Clinical and from paediatric patients in South Africa. J Pathog. 2011. https ://doi. bacteriological characteristics of Helicobacter cinaedi infection. J Infect org/10.4061/2011/21737 6. Chemother. 2014;20(9):517–26. 18. Morrow AL, Ruiz-Palacios GM, Altaye M, Jiang X, Guerrero ML, Meinzen- 13. Hsueh PR, Teng LJ, Hung CC, Chen YC, Yang PC, Ho SW, et al. Septic shock Derr JK, et al. Human milk oligosaccharides are associated with protec- due to Helicobacter fennelliae in a non-human immunodeficiency virus- tion against diarrhea in breast-fed infants. J Pediatr. 2004;145(3):297–303. infected heterosexual patient. J Clin Microbiol. 1999;37(6):2084–6. 19. Ogbo FA, Agho K, Ogeleka P, Woolfenden S, Page A, Eastwood J, 14. Lastovica AJ. Emerging Campylobacter spp.: the tip of the iceberg. Clin Global Child Health Research Interest Group. Infant feeding practices Microbiol Newsl. 2006;28(7):49–56. and diarrhoea in sub-Saharan African countries with high diarrhoea 15. Burnens AP, Stanley J, Schaad UB, Nicolet J. Novel Campylobacter-like mortality. PLoS ONE. 2017;12(2):e0171792. https ://doi.org/10.1371/journ organism resembling Helicobacter fennelliae isolated from a boy with al.pone.01717 92. gastroenteritis and from dogs. J Clin Microbiol. 1993;31(7):1916–7. 20. Quilliam RS, Cross P, Williams AP, Edwards-Jones G, Salmon RL, Rigby D, 16. Tee W, Hinds S, Montgomery J, Dyall-Smith ML. A probable new Helico- et al. Subclinical infection and asymptomatic carriage of gastrointestinal bacter species isolated from a patient with bacteremia. J Clin Microbiol. zoonoses: occupational exposure, environmental pathways, and the 2000;38(10):3846–8. anonymous spread of disease. Epidemiol Infect. 2013;141(10):2011–21. 17. Smuts HE, Lastovica AJ. Molecular characterization of the 16S rRNA Gene of Helicobacter fennelliae isolated from stools and blood cultures Ready to submit your research ? Choose BMC and benefit from: fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Gut Pathogens Springer Journals

Antimicrobial resistant Helicobacter fennelliae isolated from non-diarrheal child stool sample in Battambang, Cambodia

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Abstract

Helicobacter fennelliae (H. fennelliae) is associated with human gastroenteritis; however, H. fennelliae was isolated and confirmed by phenotypic and genotypic identification from a non-diarrheal child stool sample in Cambodia. Antimi- crobial susceptibility testing demonstrated that this isolate had a high minimal inhibitory concentration against mac- rolides and quinolones, which are first-line antibiotic treatment choices for Campylobacter infections. Consequently, macrolides and quinolones were likewise expected to be ineffective against Campylobacter-like organisms such as H. fennelliae. This isolate warranted further genetic characterization to better understand associated antibiotic resistance mechanisms. Resistant pathogens from asymptomatic diarrheal cases are likely underestimated, and as such colo- nized individuals may spread resistant organisms to local community members and the environment. Keywords: Helicobacter fennelliae, Non-diarrheal sample, Child, Cambodia, First-report Background chaperonin protein present in virtually all eubacteria, Helicobacter fennelliae (H. fennelliae) is a new Campy- some archaea, and in the plastids and mitochondria of lobacter species originally isolated from asymptomatic, eukaryotes. The utility of this target for bacterial spe - homosexual men with enteritis and proctitis in the past cies identification, detection, quantification, phyloge - few decades [1]. Like H. cinaedi, this species is classified netic analysis, and microbial community profiling was as enterohepatic Helicobacter that inhabits and causes well established [4]. Treatment recommendation guide- bacteremia in intestinal and hepatobiliary tracts of vari- lines are still not available for enterohepatic Helicobac- ous mammal and other species [2]. Additional evidence ter species. Various individual and combined antibiotic suggests that H. fennelliae was implicated as a contrib- regimens were successfully used in treating Helicobac- uting cause of human proctocolitis, gastroenteritis, ter infections; however, there is insufficient information and bacteremia, particularly in immunocompromised to determine resistance rates of H. fennelliae. The main individuals [2, 3]. This Helicobacter species is a fastidi- objective of this report is to describe phenotypic, geno- ous organism that is likely underestimated, and little typic, and antimicrobial susceptibility (AST) data from is known about routes of transmission other than evi- this H. fennelliae isolate from the stool of non-diarrheal dence indicates it is a zoonotic infection [2]. As a fas- child in Cambodia. tidious organism, molecular genotyping methods are recommended to identify Helicobacter species. Towards Methods that end, the groEL and hsp60 genes encode a 60  kDa A surveillance study to describe diarrhea etiologic agents in children and military personnel in Battambang, Cam- bodia has been conducted from 2014 until present. Both *Correspondence: woradeel.fsn@afrims.org diarrheal and non-diarrheal stool samples were observed Department of Enteric Diseases, Armed Forces Research Institute by microscopic examination for the presence of parasites, of Medical Sciences, 315/6 Rajvithi Road, Bangkok 10400, Thailand protozoa, and larvae. Samples were also assessed for the Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ruksasiri et al. Gut Pathog (2018) 10:18 Page 2 of 5 presence of Giardia, Cryptosporidium by enzyme-linked 1 min at 94 °C, 1 min at 46 °C, 1 min at 72 °C, and a final immunosorbent assay (ELISA), and for diarrheagenic E. extension at 72 °C for 10 min. The purified PCR products coli by polymerase chain reaction (PCR) [5]. Enteric path- were additionally differentiate Campylobacter species ogens, including Campylobacter species, were isolated from Helicobacter and Acrobacter species using primers and identified by traditional culture methods [6]. The sus - M13F-pUC (− 40) 5′-GTT TTC CCA GTC ACGAC-3′ and pected Campylobacter-like colonies were subcultured on M13R (− 20) 5′-GCGGA-TAA CAA TTT CAC ACAGG-3′. blood agar supplemented with 6% sodium formate and The result of partial cpn60 sequences (555 bp) was com - fumarate for 48–72 h at 37 °C under microaerobic condi- pared with the database in cpnDB (http://cpndb .cbr.nrc. tions (10% CO and 5% O ). The biochemical identifica - ca) [4]. The confirmed partial sequence was submitted 2 2 tions were included oxidase, catalase, indoxyl hydrolysis, to the National Center for Biotechnology Information hippurate hydrolysis, nitrate reduction, urease, hydrogen (NCBI) before constructing phylogenetic analysis by Bio- sulfide production, susceptibility to cephalothin and nali - Numerics software version 7.6 (Applied Maths, Belgium). dixic acid (30 µg disc) (BD, Spark, USA), oxygen and tem- perature tolerance test. According to no antimicrobial Results and discussion susceptibility recommendation guidelines, H. fennelliae A non-diarrheal stool sample of a young child who pre- resistance was determined using the minimal inhibitory sented to the hospital with fever and, cough was sub- concentration (MIC) by E test (Liofilchem, Roseto degli mitted for laboratory testing. The stool characteristic Abruzzi TE, Italy) against azithromycin (AZM), erythro- was loose without mucus, blood, RBCs, or WBCs. No mycin (ERY), nalidixic acid (NAL), ciprofloxacin (CIP), gastrointestinal parasites were detected microscopically levofloxacin (LEV), ceftriaxone (CRO), spectinomycin or by ELISA. Other enteric bacterial pathogens, includ- (SPT), and tetracycline (TET). C. jejuni ATCC 33560 was ing diarrheagenic E. coli, were not identified, except used as a quality control strain. for suspected colonies of a Campylobacter-like organ- Genomic DNA of suspected Campylobacter-like ism. The colonies characteristics which were presented colonies was extracted and subsequently confirmed as after 6  days incubation were thin, flat, film-like colony, belonging to the Campylobacter genus by screening for with a hypochlorite odor. Biochemical reactions of the the 16S rRNA gene [7]. To determine Campylobacter colony were positive for oxidase, catalase, and indoxyl species, the 15 primer sets of cpn60 target gene were used acetate hydrolysis. It was susceptible to cephalothin disk for verified species as described elsewhere [7, 8]. Sub- but resistant to nalidixic acid disk and could be grown sequently, the unknown Campylobacter species beyond at 42  °C under microaerobic conditions. Culture results 15 primer sets identification were further sequencing indicated that H. fennelliae grows well by supplementing analysis by amplifying cpn60 target gene with degenerate 6% sodium formate and fumarate in blood agar. This is primers H729 and H730 [4]. The sequences of degener - likely due to the fact that formate replaces hydrogen as ate primers were H729: 5′-CGC CAG GGT TTT CCC AGT the electron donor, and fumarate serves as the termi- CAC GAC GAIIIIGCIGGIGAYGGIACIACIAC-3′ and nal electron acceptor for hydrogen-required organism H730 5′-AGC GGA TAA CAA TTT CAC ACA GGA YKI- growth [9]. Notably, an absence of hydrogen, the low-cost YKITCICCRAAI CCIGGIGCYTT-3′. PCR amplification supplemented media, and a long incubation period are was carried out in a total volume of 50 µL containing 6 µL suggested to support growth of H. fennelliae. of genomic DNA template, 2.5 U AmpliTaq G old DNA The MIC results of H. fennelliae and C. jejuni ATCC polymerase (Applied Biosystems, Foster City, Calif.), 33560 were presented in Table  1. Results for this H. fen- 5 mM MgCl , 100 µM each of the dNTPs and 50 nM each nelliae isolate demonstrated high MICs to macrolides of degenerate primers [4]. The cycling conditions were and quinolones, consistent with previous studies [10, 11] performed at 94  °C for 5  min, followed by 28 cycles of and similar to H. cinaedi data [12]. Macrolides, generally Table 1 Determination the  minimal inhibitory concentration (MIC) results of  H. fennelliae and  C. jejuni ATCC 33560 against  azithromycin (AZM), erythromycin (ERY), nalidixic acid (NAL), ciprofloxacin (CIP), levofloxacin (LEV), tetracyclin (TET), ceftriaxone (CRO) and spectinomycin (SPT) Isolates MIC (µg/mL) AZM ERY NAL CIP LEV TET CRO SPT H. fennelliae ≥ 256 ≥ 256 ≥ 256 ≥ 32 3 0.125 0.125 4 C. jejuni ATCC33560 0.125 0.75 4 0.094 0.25 0.25 ≥ 32 0.5 Ruksasiri et al. Gut Pathog (2018) 10:18 Page 3 of 5 considered the drug of choice for Campylobacter treat- prevalence and antimicrobial resistant profile might be ment [1], may be clinically less effective for Campylo - considerably underestimated due to inadequate isola- bacter-like organism infections such as H. fennelliae tion and identification methods [14]. To the best of our and H. cinaedi. Little is known about the antimicrobial knowledge, this is the first report of a macrolide and resistance mechanisms of H. fennelliae. Mutations of quinolone resistant H. fennelliae identified in a young the gyrase and 23S rRNA genes may be responsible for Cambodian child asymptomatic for intestinal infection. decreased susceptibility to quinolones and macrolides, This isolate resembles H. fennelliae, which was previ - respectively [10]. However, decreased susceptibility to ously identified in a boy suffering gastroenteritis and is low MIC macrolide levels were mentioned in a previ- also isolated from dog specimens [15]. With the intro- ous study [13]. The H. fennelliae isolate from our study duction of the ‘Cape Town Protocol,’ H. fennelliae may exhibited a high MIC to macrolides, warranting further be isolated from stool and blood culture in an H -rich molecular characterization to explore other resistance microaerophilic atmosphere. Prior evidence indicated mechanisms. that Helicobacter species related to H. fennelliae were The genotyping confirmation of this H. fennelliae iso - isolated from blood of a young child suffering diarrhea late was performed by sequencing the cpn60 gene, and symptoms [16]. Nevertheless, the nucleotide sequences the result was submitted to NCBI under the accession of H. fennelliae obtained from blood and stool were not number MG696736. A phylogenetic tree analysis (Fig. 1) significantly different [17]. Unfortunately blood sam - divided Helicobacter and Campylobacter strains into ples were not available from the child in this study, so seven distinct groups (cut-off of 90%). The MG696736 that comparison was not achievable. H. fennelliae was entry was classified as group IV, which was 97.2% simi - predominantly isolated from children who presented lar to H. fennelliae ATCC 35684, whereas Campylobacter with diarrheal symptoms, although stools from asymp- species was classified as group VII, which is distinct from tomatic diarrheal children with asthma and/or failure the Helicobacter group VI (cut-off of 90%). to thrive (FTT) were also positive for H. fennelliae [16, H. fennelliae was suggested as a significant pathogen 17]. Another possible explanation of this H. fennelliae associated with human gastroenteritis; however, its finding in stool of asymptomatic diarrheal Cambodia Fig. 1 Neighbor-joining phylogenetic trees based on partial cpn60 gene sequences (555 bp) of Helicobacter strain (MG696736), as compared to cpn60 sequences of other Helicobacter, and Campylobacter strains in cpnDB database Ruksasiri et al. Gut Pathog (2018) 10:18 Page 4 of 5 be construed as official, or as reflecting true views of the Department of the child could relate to breastfeeding. Evidence suggests Army or the Department of Defense. that maternal milk contains a variety of functionally bioactive agents from her innate immune system [18], Availability of data and materials Data sharing not applicable to this article. as well as a mechanism to influence microbial changes in the infant’s gastrointestinal system [19]. As a result Consent for publication of widespread breastfeeding campaigns in the develop- Not applicable. ing world, this may play an important role in the level Ethics approval and consent to participate of asymptomatic carriage within a community [18, 20]. The study protocol was in accordance with ethical guideline of the ‘Code The association between asymptomatic carriage and of Federal Regulations, Title 32, Part 219: Protection of Human Subjects’ and was approved by the Review Board at National Ethics Committee for Health diarrheal pathogens such as Salmonella, E. coli O157 Research, Phnom Penh, Cambodia and Walter Reed Army Institute of Research and Campylobacter was previously reported in out- ( WRAIR), Silver Spring, MD, USA. breaks elsewhere [20]. Identification of an antibiotic Funding resistant H. fennelliae strain from an asymptomatic The study is supported by the Armed Forces Health Surveillance Branch diarrheal person would probably be transmitted into (AFHSB) and it’s GEIS (Global Emerging Infectious Disease Surveillance and local communities and environmental contamination. Response) Section. Hence, the public health significance of resistant patho - gens in human feces warrants effective monitoring to Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- prevent disease outbreaks. lished maps and institutional affiliations. In conclusion, phenotypic and genotypic assessments confirmed that H. fennelliae was isolated from a non- Received: 11 April 2018 Accepted: 19 May 2018 diarrheal stool sample of a Cambodian child suffering from fever with cough and convulsion. The supplement media, incubation atmosphere, and incubation period utilized permitted culture, isolation, and identification of H. fennelliae. The high MICs values against macrolides References 1. Totten PA, Fennell CL, Tenover FC, Wezenberg JM, Perine PL, Satamm WE, (AZM, ERY) and quinolones (NAL, CIP) indicated these et al. Campylobacter cinaedi (sp.nov.) and Campylobacter fennelliae (sp. are less effective against H. fennelliae. This isolate should Nov): two new Campylobacter species associated with enteric diseases in be further characterized to better understand associated homosexual men. J Infect Dis. 1985;151(1):131–9. 2. James HJ, Michael AP, Karen CC, Guido FMLL, Sandry SR, David WW. resistance mechanisms. Manual of clinical microbiology. Washington DC: American Society for Microbiology press; 2015. Authors’ contributions 3. O’Rourke JL, Grehan M, Lee A. Non-pylori Helicobacter species in humans. WL and SR participated in the conception and design of the study. SR, PW, and Gut. 2001;49(5):601–6. CS performed the laboratory work. NS was clinical coordinator and subject 4. Hill JE, Paccagnellla A, Law K, Melito PL, Woodward DL, Price L, et al. Iden- enrollment. WL and OS analyzed the data and wrote the manuscript. SL and tification of Campylobacter spp. and discrimination from Helicobacter and LC coordinated and fully supported this study in Cambodia. LB and JC con- Arcobacter spp. by direct sequencing of PCR-amplified cpn60 sequences tributed to the analysis and helped in writing the manuscript. All authors read and comparison to cpnDB, a chaperonin reference sequence database. J and approved the final manuscript. Med Microbiol. 2006;55(4):393–9. 5. Meng CY, Smith BL, Bodhidatta L, Richard SA, Vansith K, Thy B, et al. Etiol- Author details 1 ogy of diarrhea in young children and patterns of antibiotic resistance in Department of Enteric Diseases, Armed Forces Research Institute of Medical 2 Cambodia. Pediatr Infect Dis J. 2011;30(4):331–5. Sciences, 315/6 Rajvithi Road, Bangkok 10400, Thailand. Battambang Referral 6. Garcia LS. Clinical microbiology procedures handbook. 3rd ed, Washing- Hospital, PrekMohatep Village, SvayPor Commune, Battambang, Cambodia. 3 ton, D.C.: ASM Press; 2010. P. 3.8.1.1–3.8.2.16 and 3.17.1.1–3.18.2.1. Armed Forces Research Institute of Medical Sciences, 18.118 Street Sangkat 4 7. 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Campylobacter mucosalis thank AFRIMS Enteric Diseases Department Staff, Bangkok, Thailand and Bat - (Lawson, Leaver, Pettigrew, and Rowland 1981) comb. nov.: emended tambang Referral Hospital & AFRIMS-CNM Staff, Battambang, Cambodia, for description. Int J Syst Bacteriol. 1985;35(1):189–92. their assistance and kind support. 10. Rimbara E, Mori S, Kim H, Matsui M, Suzuki S, Takahashi S, et al. Helicobac- ter cinaedi and Helicobacter fennelliae transmission in a hospital from 2008 Competing interests to 2012. J Clin Microbiol. 2013;51(7):2439. The authors declare that they have no competing interests. 11. Fujiya Y, Nagamatsu M, Tomida J, Kawamura Y, Yamamoto K, Mawa- tari M, et al. Successful treatment of recurrent Helicobacter fennelliae Authors’ disclaimers bacteraemia by selective digestive decontamination with kanamycin Material has been reviewed by the Walter Reed Army Institute of Research. in a lung cancer patient receiving chemotherapy. JMM Case Rep. 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J Clin Microbiol. zoonoses: occupational exposure, environmental pathways, and the 2000;38(10):3846–8. anonymous spread of disease. Epidemiol Infect. 2013;141(10):2011–21. 17. Smuts HE, Lastovica AJ. Molecular characterization of the 16S rRNA Gene of Helicobacter fennelliae isolated from stools and blood cultures Ready to submit your research ? Choose BMC and benefit from: fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions

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Gut PathogensSpringer Journals

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