Resistance to β-lactams and distribution of β-lactam resistance genes in subgingival microbiota from Spanish patients with periodontitis

Abstract

Objectives

The aim of this study was to analyze the distribution of β-lactamase genes and the multidrug resistance profiles in β-lactam-resistant subgingival bacteria from patients with periodontitis.

Materials and methods

Subgingival samples were obtained from 130 Spanish patients with generalized periodontitis stage III or IV. Samples were grown on agar plates with amoxicillin or cefotaxime and incubated in anaerobic and microaerophilic conditions. Isolates were identified to the species level by the sequencing of their 16S rRNA gene. A screening for the following β-lactamase genes was performed by the polymerase chain reaction (PCR) technique: blaTEM, blaSHV, blaCTX-M, blaCfxA, blaCepA, blaCblA, and blaampC. Additionally, multidrug resistance to tetracycline, chloramphenicol, streptomycin, erythromycin, and kanamycin was assessed, growing the isolates on agar plates with breakpoint concentrations of each antimicrobial.

Results

β-lactam-resistant isolates were found in 83% of the patients. Seven hundred and thirty-seven isolates from 35 different genera were obtained, with Prevotella and Streptococcus being the most identified genera. blaCfxA was the gene most detected, being observed in 24.8% of the isolates, followed by blaTEM (12.9%). Most of the isolates (81.3%) were multidrug-resistant.

Conclusions

This study shows that β-lactam resistance is widespread among Spanish patients with periodontitis. Furthermore, it suggests that the subgingival commensal microbiota might be a reservoir of multidrug resistance and β-lactamase genes.

Clinical relevance

Most of the samples yielded β-lactam-resistant isolates, and 4 different groups of bla genes were detected among the isolates. Most of the isolates were also multidrug-resistant. The results show that, although β-lactams may still be effective, their future might be hindered by the presence of β-lactam-resistant bacteria and the presence of transferable bla genes.

This is a preview of subscription content, log in to check access.

References

  1. 1.

    Meyle J (2000) Chapple I (2015) molecular aspects of the pathogenesis of periodontitis. Periodontol 69:7–17. https://doi.org/10.1111/prd.12104

    Article  Google Scholar 

  2. 2.

    Herrera D, Sanz M, Jepsen S, Needleman I, Roldán S (2002) A systematic review on the effect of systemic antimicrobials as an adjunct to scaling and root planing in periodontitis patients. J Clin Periodontol 29:136–159. https://doi.org/10.1034/j.1600-051X.29.s3.8.x

    Article  PubMed  Google Scholar 

  3. 3.

    Matesanz-Pérez P, García-Gargallo M, Figuero E, Bascones-Martínez A, Sanz M, Herrera D (2013) A systematic review on the effects of local antimicrobials as adjuncts to subgingival debridement, compared with subgingival debridement alone, in the treatment of chronic periodontitis. J Clin Periodontol 40:227–241. https://doi.org/10.1111/jcpe.12026

    Article  PubMed  Google Scholar 

  4. 4.

    Rabelo CC, Feres M, Gonçalves C, Faver M, Tu Y-K, Chambrone L (2015) Systemic antibiotics in the treatment of aggressive periodontitis. A systematic review and a Bayesian Network meta-analysis. J Clin Periodontol 42:647–657. https://doi.org/10.1111/jcpe.12427

    Article  PubMed  Google Scholar 

  5. 5.

    Pretzl B, Sälzer S, Ehmke B, Schlagenhauf U, Dannewitz B, Dommisch H, Eickholz P, Jockel-Schneider Y (2019) Administration of systemic antibiotics during non-surgical periodontal therapy—a consensus report. Clin Oral Investig 23:3073–3085. https://doi.org/10.1007/s00784-018-2727-0

    Article  PubMed  Google Scholar 

  6. 6.

    Handal T, Olsen I, Walker CB, Caugant DA (2004) β-Lactamase production and antimicrobial susceptibility of subgingival bacteria from refractory periodontitis. Oral Microbiol Immunol 19:303–308. https://doi.org/10.1111/j.1399-302x.2004.00159.x

    Article  PubMed  Google Scholar 

  7. 7.

    Jacoby GA (2009) AmpC β-Lactamases. Clin Microbiol Rev 22:161–182. https://doi.org/10.1128/CMR.00036-08

    Article  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Perez F, Endimiani A, Hujer K, Bonomo R (2007) The continuing challenge of ESBLs. Curr Opin Pharmacol 7:459–469. https://doi.org/10.1016/j.coph.2007.08.003

    Article  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Danziger LH, Pendland SL (1995) Bacterial resistance to β-lactam antibiotics. Am J Heal Pharm 52:S3–S8. https://doi.org/10.1093/ajhp/52.6_Suppl_2.S3

    Article  Google Scholar 

  10. 10.

    Lee JH, Bae IK, Hee Lee S (2012) New definitions of extended-spectrum β-lactamase conferring worldwide emerging antibiotic resistance. Med Res Rev 32:216–232. https://doi.org/10.1002/med.20210

    Article  PubMed  Google Scholar 

  11. 11.

    Turner PJ (2005) Extended-spectrum β-lactamases. Clin Infect Dis 41:S273–S275. https://doi.org/10.1086/430789

    Article  PubMed  Google Scholar 

  12. 12.

    Handal T, Giraud-Morin C, Caugant DA, Madinier I, Olsen I, Foose T (2005) Chromosome- and plasmid-encoded β-lactamases in Capnocytophaga spp. Antimicrob Agents Chemother 49:3940–3943. https://doi.org/10.1128/AAC.49.9.3940-3943.2005

    Article  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Handal T, Olsen I, Walker CB, Caugant DA (2005) Detection and characterization of β-lactamase genes in subgingival bacteria from patients with refractory periodontitis. FEMS Microbiol Lett 242:319–324. https://doi.org/10.1016/j.femsle.2004.11.023

    Article  PubMed  Google Scholar 

  14. 14.

    Kim S-M, Kim HC, Lee S-WS (2011) Characterization of antibiotic resistance determinants in oral biofilms. J Microbiol 49:595–602. https://doi.org/10.1007/s12275-011-0519-1

    Article  PubMed  Google Scholar 

  15. 15.

    Søraas A, Olsen I, Sundsfjord A, Handal T, Bjørang O, Jenum PA (2014) Extended-spectrum beta-lactamase-producing bacteria are not detected in supragingival plaque samples from human fecal carriers of ESBL-producing Enterobacteriaceae. J Oral Microbiol 6:24026. https://doi.org/10.3402/jom.v6.24026

    Article  Google Scholar 

  16. 16.

    Olsen I, Tribble GD, Fiehn NE, Wang BY (2013) Bacterial sex in dental plaque. J Oral Microbiol 5:1. https://doi.org/10.3402/jom.v5i0.20736

    Article  Google Scholar 

  17. 17.

    Roberts AP, Mullany P (2010) Oral biofilms: a reservoir of transferable, bacterial, antimicrobial resistance. Expert Rev Anti-Infect Ther 8:1441–1450. https://doi.org/10.1586/eri.10.106

    Article  PubMed  Google Scholar 

  18. 18.

    ECDC (European Centre for Disease Prevention and Control), EFSA (European Food Safety Authority), and EMA (European Medicines Agency) (2017) ECDC/EFSA/EMA second joint report on the integrated analysis of the consumption of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from humans and food-producing animals - joint interagency antimicrobial consumption and resistance analysis (JIACRA) report. EFSA J 15:4872–5007. https://doi.org/10.2903/j.efsa.2017.4872

    Article  Google Scholar 

  19. 19.

    Armitage GC (1999) Development of a classification system for periodontal diseases and conditions. Ann Periodontol 4:1–6. https://doi.org/10.1902/annals.1999.4.1.1

    Article  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Caton JG, Armitage G, Berglundh T, Chapple ILC, Jepsen S, Kornman KS, Mealey BL, Papanou PN, Sanz M, Tonetti MS (2018) A new classification scheme for periodontal and peri-implant diseases and conditions - introduction and key changes from the 1999 classification. J Clin Periodontol 45:S1–S8. https://doi.org/10.1111/jcpe.12935

    Article  PubMed  Google Scholar 

  21. 21.

    Silness J, Löe H (1964) Periodontal disease in pregnancy II. Correlation between oral hygiene and periodontal condition. Acta Odontol Scand 22:121–135. https://doi.org/10.3109/00016356408993968

    Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Loesche WJ, Syed SA, Stoll J (1987) Trypsin-like activity in subgingival plaque. J Periodontol 58:266–273. https://doi.org/10.1902/jop.1987.58.4.266

    Article  PubMed  Google Scholar 

  23. 23.

    CLSI (2017) Performance standards for antimicrobial susceptibility testing (27th ed). Wayne, PA

  24. 24.

    The European Committee on Antimicrobial Susceptibility Testing (EUCAST) (2018) Breakpoint tables for interpretation of MICs and zone diameters, version 8.0. http://www.eucast.org/clinical_breakpoints

  25. 25.

    Shigematsu T, Hayashi M, Kikuchi I, Ueno S, Masaki H, Fujii T (2009) A culture-dependent bacterial community structure analysis based on liquid cultivation and its application to a marine environment. FEMS Microbiol Lett 293:240–247. https://doi.org/10.1111/j.1574-6968.2009.01536.x

    Article  PubMed  Google Scholar 

  26. 26.

    Fosse T, Madinier I, Hannoun L, Giraud-Morin C, Hitzig C, Charbit Y, Ourang S (2002) High prevalence of cfxA β-lactamase in aminopenicillin-resistant Prevotella strains isolated from periodontal pockets. Oral Microbiol Immunol 17:85–88. https://doi.org/10.1046/j.0902-0055.2001.00096.x

    Article  PubMed  Google Scholar 

  27. 27.

    Bou G, Martinez-Beltran J (2000) Cloning, nucleotide sequencing, and analysis of the gene encoding an AmpC β-lactamase in Acinetobacter baumannii. Antimicrob Agents Chemother 44:428–432. https://doi.org/10.1128/AAC.44.2.428-432.2000

    Article  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Danel F, Hall LM, Gur D, Livermore DM (1995) OXA-14, another extended-spectrum variant of OXA-10 (PSE-2) β-lactamase from Pseudomonas aeruginosa. Antimicrob Agents Chemother 39:1881–1884. https://doi.org/10.1128/AAC.39.8.1881

    Article  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Paterson DL, Hujer KM, Hujer AM, Yeiser B, Bonomo MD, Rice LB, Bonomo RA, International Klebsiella Study Group (2003) Extended-spectrum β-lactamases in Klebsiella pneumoniae bloodstream isolates from seven countries: dominance and widespread prevalence of SHV- and CTX-M-type β-lactamases. Antimicrob Agents Chemother 47:3554–3560. https://doi.org/10.1128/AAC.47.11.3554-3560.2003

  30. 30.

    Boyd DA, Tyler S, Christianson S, McGeer A, Muller MP, Willey BM, Bryce E, Gardam M, Nordmann P, Mulvey MR, Canadian Nosocomial Infection Surveillance Program (2004) Complete nucleotide sequence of a 92-Kilobase plasmid harboring the CTX-M-15 extended-spectrum Beta-lactamase involved in an outbreak in long-term-care facilities in Toronto, Canada. Antimicrob Agents Chemother 48:3758–3764. https://doi.org/10.1128/AAC.48.10.3758-3764.2004

    Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Monstein H-J, Östholm-Balkhed Å, Nilsson MV, Nilsson M, Dornbusch K, Nilsson LE (2007) Multiplex PCR amplification assay for the detection of blaSHV, blaTEM and blaCTX-M genes in Enterobacteriaceae. APMIS 115:1400–1408. https://doi.org/10.1111/j.1600-0463.2007.00722.x

    Article  PubMed  Google Scholar 

  32. 32.

    Slots J (2012) Low-cost periodontal therapy. Periodontol 2000(60):110–137. https://doi.org/10.1111/j.1600-0757.2011.00429.x

    Article  Google Scholar 

  33. 33.

    Segura-Egea JJ, Velasco-Ortega E, Torres-Lagares D, Velasco-Ponferrada MC, Monsalve-Guil L, Llamas-Carreras JM (2010) Pattern of antibiotic prescription in the management of endodontic infections amongst Spanish oral surgeons. Int Endod J 43:342–350. https://doi.org/10.1111/j.1365-2591.2010.01691.x

    Article  PubMed  Google Scholar 

  34. 34.

    Paterson DL, Bonomo RA (2005) Extended-spectrum β-lactamases: a clinical update. Clin Microbiol Rev 18:657–686. https://doi.org/10.1128/CMR.18.4.657-686.2005

    Article  PubMed  PubMed Central  Google Scholar 

  35. 35.

    D’Andrea MM, Arena F, Pallecchi L, Rossolini GM (2013) CTX-M-type β-lactamases: a successful story of antibiotic resistance. Int J Med Microbiol 303:305–317. https://doi.org/10.1016/j.ijmm.2013.02.008

    Article  PubMed  Google Scholar 

  36. 36.

    Van Winkelhoff AJ, Herrera Gonzales D, Winkel EG, Dellemijn-Kippuw N, Vanden-broucke-Grauls CMJE, Sanz M (2000) Antimicrobial resistance in the subgingival microflora in patients with adult periodontitis: a comparison between the Netherlands and Spain. J Clin Periodontol 27:79–86. https://doi.org/10.1034/j.1600-051x.2000.027002079.x

    Article  PubMed  Google Scholar 

  37. 37.

    Ready D, Bedi R, Mullany P, Wilson M (2012) Penicillin and amoxicillin resistance in oral Veillonella spp. Int J Antimicrob Agents 40:188–189. https://doi.org/10.1016/j.ijantimicag.2012.04.007

    Article  PubMed  Google Scholar 

  38. 38.

    Nyfors S, Könönen E, Bryk A, Syrjänen R, Jousimies-Somer H (2003) Age-related frequency of penicillin resistance of oral Veillonella. Diagn Microbiol Infect Dis 46:279–283. https://doi.org/10.1016/S0732-8893(03)00082-8

    Article  PubMed  Google Scholar 

  39. 39.

    Reig M, Mir N, Baquero F (1997) Penicillin resistance in Veillonella. Antimicrob Agents Chemother 41:1210. https://doi.org/10.1128/aac.41.5.1210

    Article  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Theron MM (2003) Penicillin-binding proteins involved in high-level piperacillin resistance in Veillonella spp. J Antimicrob Chemother 52:120–122. https://doi.org/10.1093/jac/dkg297

    Article  PubMed  Google Scholar 

  41. 41.

    Kuriyama T, Williams DW, Yanagisawa M, Iwahara K, Shimizu C, Nakagawa K, Yamamoto E, Karasawa T (2007) Antimicrobial susceptibility of 800 anaerobic isolates from patients with dentoalveolar infection to 13 oral antibiotics. Oral Microbiol Immunol 22:285–288. https://doi.org/10.1111/j.1399-302X.2007.00365.x

    Article  PubMed  Google Scholar 

  42. 42.

    van Winkelhoff AJ, Herrera D, Oteo A, Sanz M (2005) Antimicrobial profiles of periodontal pathogens isolated from periodontitis patients in the Netherlands and Spain. J Clin Periodontol 32:893–898. https://doi.org/10.1111/j.1600-051X.2005.00782.x

    Article  PubMed  Google Scholar 

  43. 43.

    Fernández-Canigia L, Cejas D, Gutkind G, Radice M (2015) Detection and genetic characterization of β-lactamases in Prevotella intermedia and Prevotella nigrescens isolated from oral cavity infections and peritonsillar abscesses. Anaerobe 33:8–13. https://doi.org/10.1016/j.anaerobe.2015.01.007

    Article  PubMed  Google Scholar 

  44. 44.

    Rams TE, Degener JE, van Winkelhoff AJ (2013) Prevalence of β-lactamase-producing bacteria in human periodontitis. J Periodontal Res 48:493–499. https://doi.org/10.1111/jre.12031

    Article  PubMed  Google Scholar 

  45. 45.

    Pancoast SJ (1988) Aminoglycoside antibiotics in clinical use. Med Clin North Am 72:581–612

    Article  Google Scholar 

  46. 46.

    Holbrook WP, Ogston SA, Ross PW (1978) A method for the isolation of Bacteroides melaninogenicus from the human mouth. J Med Microbiol 11:203–207. https://doi.org/10.1099/00222615-11-2-203

    Article  PubMed  Google Scholar 

  47. 47.

    McCarthy K (2015) Pseudomonas aeruginosa: evolution of antimicrobial resistance and implications for therapy. Semin Respir Crit Care Med 36:044–055. https://doi.org/10.1055/s-0034-1396907

    Article  Google Scholar 

  48. 48.

    Puzari M, Chetia P (2017) RND efflux pump mediated antibiotic resistance in gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa: a major issue worldwide. World J Microbiol Biotechnol 33:24. https://doi.org/10.1007/s11274-016-2190-5

    Article  PubMed  Google Scholar 

  49. 49.

    Dougherty TJ, Pucci MJ (2012) Antibiotic discovery and development. Springer US, Boston

    Google Scholar 

  50. 50.

    Roberts AP, Mullany P (2011) Tn916-like genetic elements: a diverse group of modular mobile elements conferring antibiotic resistance. FEMS Microbiol Rev 35:856–871. https://doi.org/10.1111/j.1574-6976.2011.00283.x

    Article  PubMed  Google Scholar 

  51. 51.

    Santoro F, Vianna ME, Roberts AP (2014) Variation on a theme; an overview of the Tn916/Tn1545 family of mobile genetic elements in the oral and nasopharyngeal streptococci. Front Microbiol 5:1. https://doi.org/10.3389/fmicb.2014.00535

    Article  Google Scholar 

  52. 52.

    Ready D, Pratten J, Roberts AP, Bedi R, Mullany P, Wilson M (2006) Potential role of Veillonella spp. as a reservoir of transferable tetracycline resistance in the oral cavity. Antimicrob Agents Chemother 50:2866–2868. https://doi.org/10.1128/AAC.00217-06

    Article  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Clewell DB, Flannagan SE, Jaworski DD, Clewell DB (1995) Unconstrained bacterial promiscuity: the Tn916-Tn1545 family of conjugative transposons. Trends Microbiol 3:229–236. https://doi.org/10.1016/S0966-842X(00)88930-1

    Article  PubMed  Google Scholar 

  54. 54.

    Edwards R, Beringer R, Greenwood D (1996) Characterization of β-lactamases of Prevotella species. Anaerobe 2:217–221. https://doi.org/10.1006/anae.1996.0030

    Article  Google Scholar 

  55. 55.

    Ehrmann E, Handal T, Tamanai-Shacoori Z, Bonnaure-Mallet M, Fosse T (2014) High prevalence of β-lactam and macrolide resistance genes in human oral Capnocytophaga species. J Antimicrob Chemother 69:381–384. https://doi.org/10.1093/jac/dkt350

    Article  PubMed  Google Scholar 

  56. 56.

    Kolenbrander PE (2000) Oral microbial communities: biofilms, interactions, and genetic systems. Annu Rev Microbiol 54:413–437. https://doi.org/10.1146/annurev.micro.54.1.413

    Article  PubMed  Google Scholar 

  57. 57.

    Roberts AP, Kreth J (2014) The impact of horizontal gene transfer on the adaptive ability of the human oral microbiome. Front Cell Infect Microbiol 4:1. https://doi.org/10.3389/fcimb.2014.00124

    Article  Google Scholar 

  58. 58.

    Ioannidis I, Sakellari D, Spala A, Arsenakis M, Konstantinidis A (2009) Prevalence of tetM, tetQ, nim and blaTEM genes in the oral cavities of Greek subjects: a pilot study. J Clin Periodontol 36:569–574. https://doi.org/10.1111/j.1600-051X.2009.01425.x

    Article  PubMed  Google Scholar 

  59. 59.

    European Centre for Disease Prevention and Control (ECDC) (2014) Surveillance of antimicrobial consumption in Europe 2012. https://www.ecdc.europa.eu/en/publications-data/surveillance-antimicrobial-consumption-europe-2012

  60. 60.

    Haenni M, Lupo A, Madec J-Y (2018) Antimicrobial resistance in Streptococcus spp. Microbiol Spectr 6:1. https://doi.org/10.1128/microbiolspec.ARBA-0008-2017

    Article  Google Scholar 

  61. 61.

    Ding Y, Zhang J, Mi Z, Qin L, Tao YZ, Qi X (2004) Study on the molecular epidemiology of beta-lactamase TEM gene in isolated Streptococcus pneumoniae. Zhonghua Liu Xing Bing Xue Za Zhi 25:970–972

    PubMed  Google Scholar 

  62. 62.

    Chang C-Y, Lin H-J, Li B-R, Li Y-K (2016) A novel metallo-β-lactamase involved in the ampicillin resistance of Streptococcus pneumoniae ATCC 49136 strain. PLoS One 11:e0155905. https://doi.org/10.1371/journal.pone.0155905

    Article  PubMed  PubMed Central  Google Scholar 

  63. 63.

    Koncan R, Valverde A, Morosini M-I, García-Castillo M, Cantón R, Cornaglia G, Baquero F, del Campo R (2007) Learning from mistakes: Taq polymerase contaminated with β-lactamase sequences results in false emergence of Streptococcus pneumoniae containing TEM. J Antimicrob Chemother 60:702–703. https://doi.org/10.1093/jac/dkm239

    Article  PubMed  Google Scholar 

  64. 64.

    Naito M, Sato K, Shoji M, Yukitake H, Ogura Y, Hayashi T, Nakayama K (2011) Characterization of the Porphyromonas gingivalis conjugative transposon CTnPg1: determination of the integration site and the genes essential for conjugal transfer. Microbiology 157:2022–2032. https://doi.org/10.1099/mic.0.047803-0

    Article  PubMed  Google Scholar 

Download references

Funding

No external funding, apart from the support of the authors’ institution, was available for this study.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Rubén León.

Ethics declarations

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the Universitat Internacional de Catalunya research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(DOCX 69.7 kb).

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Arredondo, A., Blanc, V., Mor, C. et al. Resistance to β-lactams and distribution of β-lactam resistance genes in subgingival microbiota from Spanish patients with periodontitis. Clin Oral Invest (2020). https://doi.org/10.1007/s00784-020-03333-1

Download citation

Keywords

  • Periodontitis
  • Antibiotic resistance
  • β-Lactams
  • Multidrug resistance