Abstract
The purpose of this study was to analyze the molecular mechanisms of ampicillin-resistant Haemophilus influenzae isolated in Geneva, Switzerland. We investigated the association between specific patterns of amino acid substitutions in penicillin-binding protein 3 (with or without β-lactamase production) and β-lactam susceptibility. Another main focus for this study was to compare the accuracy of disk diffusion and Etest methods to detect resistance to ampicillin and amoxicillin/clavulanic acid. The antibiotic susceptibility to β-lactam antibiotics of 124 H. influenzae isolates was determined by disk diffusion and Etest methods, and interpreted by European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) breakpoints. Alterations in PBP3 were investigated by sequencing the ftsI gene. Of the 124 clinical isolates analyzed, ampicillin resistance was found in 36 % (45 out of 124). The rate of resistance to amoxicillin/clavulanic acid was 9 % and 0.8 %, using EUCAST and CLSI breakpoints respectively. For the 78 β-lactamase negative ampicillin-susceptible (BLNAS) isolates for which the Etest method indicated a high degree of susceptibility (MIC ≤ 1 mg/L), the disk diffusion method revealed resistance to ampicillin and amoxicillin/clavulanic acid in 33 cases (42 %). Most common amino acid substitutions were Asn526Lys and Val547Ile, followed by Asp569Ser, Ala502Val, Asp350Asn, Met377Ile, Ile449Val, and Arg517His. The patterns observed were classified into six groups (IIa, IIb, IIc, IId, III-like, and miscellaneous). Continued characterization of both invasive and respiratory H. influenzae isolates is necessary in order to observe changes in the microbiology and epidemiology of this pathogen that could lead to clinical failure when treated by empirical antibiotic therapy.
Similar content being viewed by others
References
Skaare D, Anthonisen IL, Kahlmeter G, Matuschek E, Natas OB, Steinbakk M, Sundsfjord A, Kristiansen BE (2014) Emergence of clonally related multidrug resistant Haemophilus influenzae with penicillin-binding protein 3-mediated resistance to extended-spectrum cephalosporins, Norway, 2006 to 2013. Euro Surveill 19(49)
Barbosa AR, Giufre M, Cerquetti M, Bajanca-Lavado MP (2011) Polymorphism in ftsI gene and {beta}-lactam susceptibility in Portuguese Haemophilus influenzae strains: clonal dissemination of beta-lactamase-positive isolates with decreased susceptibility to amoxicillin/clavulanic acid. J Antimicrob Chemother 66(4):788–796
Setchanova LP, Kostyanev T, Markovska R, Miloshev G, Mitov IG (2013) Serotypes, antimicrobial susceptibility, and beta-lactam resistance mechanisms of clinical Haemophilus influenzae isolates from Bulgaria in a pre-vaccination period. Scand J Infect Dis 45(2):81–87
Bajanca-Lavado MP, Simoes AS, Betencourt CR, Sa-Leao R (2014) Characteristics of Haemophilus influenzae invasive isolates from Portugal following routine childhood vaccination against H. influenzae serotype b (2002–2010). Eur J Clin Microbiol Infect Dis 33(4):603–610
Park C, Kim KH, Shin NY, Byun JH, Kwon EY, Lee JW, Kwon HJ, Choi EY, Lee DG, Sohn WY, Kang JH (2013) Genetic diversity of the ftsI gene in beta-lactamase-nonproducing ampicillin-resistant and beta-lactamase-producing amoxicillin-/clavulanic acid-resistant nasopharyngeal Haemophilus influenzae strains isolated from children in South Korea. Microb Drug Resist 19(3):224–230
Skaare D, Anthonisen IL, Caugant DA, Jenkins A, Steinbakk M, Strand L, Sundsfjord A, Tveten Y, Kristiansen BE (2014) Multilocus sequence typing and ftsI sequencing: a powerful tool for surveillance of penicillin-binding protein 3-mediated beta-lactam resistance in nontypeable Haemophilus influenzae. BMC Microbiol 14:131
Hasegawa K, Yamamoto K, Chiba N, Kobayashi R, Nagai K, Jacobs MR, Appelbaum PC, Sunakawa K, Ubukata K (2003) Diversity of ampicillin-resistance genes in Haemophilus influenzae in Japan and the United States. Microb Drug Resist 9(1):39–46
Resman F, Ristovski M, Forsgren A, Kaijser B, Kronvall G, Medstrand P, Melander E, Odenholt I, Riesbeck K (2012) Increase of beta-lactam-resistant invasive Haemophilus influenzae in Sweden, 1997 to 2010. Antimicrob Agents Chemother 56(8):4408–4415
Puig C, Calatayud L, Marti S, Tubau F, Garcia-Vidal C, Carratala J, Linares J, Ardanuy C (2013) Molecular epidemiology of nontypeable Haemophilus influenzae causing community-acquired pneumonia in adults. PLoS One 8(12), e82515
Kishii K, Chiba N, Morozumi M, Hamano-Hasegawa K, Kurokawa I, Masaki J, Ubukata K (2010) Diverse mutations in the ftsI gene in ampicillin-resistant Haemophilus influenzae isolates from pediatric patients with acute otitis media. J Infect Chemother : Off J Jpn Soc Chemother 16(2):87–93
Hoshino T, Sato Y, Toyonaga Y, Hanaki H, Sunakawa K (2013) Nationwide survey of the development of drug resistance in the pediatric field in 2007 and 2010: drug sensitivity of Haemophilus influenzae in Japan (second report). J Infect Chemother : Off J Jpn Soc Chemother 19(3):495–503
Dabernat H, Delmas C (2012) Epidemiology and evolution of antibiotic resistance of Haemophilus influenzae in children 5 years of age or less in France, 2001–2008: a retrospective database analysis. Eur J Clin Microbiol Infect Dis 31(10):2745–2753
Ladhani S, Slack MP, Heath PT, von Gottberg A, Chandra M, Ramsay ME (2010) Invasive Haemophilus influenzae disease, Europe, 1996–2006. Emerg Infect Dis 16(3):455–463
Bruin JP, Kostrzewa M, van der Ende A, Badoux P, Jansen R, Boers SA, Diederen BM (2014) Identification of Haemophilus influenzae and Haemophilus haemolyticus by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Eur J Clin Microbiol Infect Dis 33(2):279–284
Cherkaoui A, Hibbs J, Emonet S, Tangomo M, Girard M, Francois P, Schrenzel J (2010) Comparison of two matrix-assisted laser desorption ionization-time of flight mass spectrometry methods with conventional phenotypic identification for routine identification of bacteria to the species level. J Clin Microbiol 48(4):1169–1175
Frickmann H, Christner M, Donat M, Berger A, Essig A, Podbielski A, Hagen RM, Poppert S (2013) Rapid discrimination of Haemophilus influenzae, H. parainfluenzae, and H. haemolyticus by fluorescence in situ hybridization (FISH) and two matrix-assisted laser-desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS) platforms. PLoS One 8(4), e63222
Cerquetti M, Giufre M, Cardines R, Mastrantonio P (2007) First characterization of heterogeneous resistance to imipenem in invasive nontypeable Haemophilus influenzae isolates. Antimicrob Agents Chemother 51(9):3155–3161
Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23(21):2947–2948
Dabernat H, Delmas C, Seguy M, Pelissier R, Faucon G, Bennamani S, Pasquier C (2002) Diversity of beta-lactam resistance-conferring amino acid substitutions in penicillin-binding protein 3 of Haemophilus influenzae. Antimicrob Agents Chemother 46(7):2208–2218
Garcia-Cobos S, Campos J, Lazaro E, Roman F, Cercenado E, Garcia-Rey C, Perez-Vazquez M, Oteo J, de Abajo F (2007) Ampicillin-resistant non-beta-lactamase-producing Haemophilus influenzae in Spain: recent emergence of clonal isolates with increased resistance to cefotaxime and cefixime. Antimicrob Agents Chemother 51(7):2564–2573
Tristram S, Jacobs MR, Appelbaum PC (2007) Antimicrobial resistance in Haemophilus influenzae. Clin Microbiol Rev 20(2):368–389
Witherden EA, Bajanca-Lavado MP, Tristram SG, Nunes A (2014) Role of inter-species recombination of the ftsI gene in the dissemination of altered penicillin-binding-protein-3-mediated resistance in Haemophilus influenzae and Haemophilus haemolyticus. J Antimicrob Chemother 69(6):1501–1509
Kaczmarek FS, Gootz TD, Dib-Hajj F, Shang W, Hallowell S, Cronan M (2004) Genetic and molecular characterization of beta-lactamase-negative ampicillin-resistant Haemophilus influenzae with unusually high resistance to ampicillin. Antimicrob Agents Chemother 48(5):1630–1639
Hirakata Y, Ohmori K, Mikuriya M, Saika T, Matsuzaki K, Hasegawa M, Hatta M, Yamamoto N, Kunishima H, Yano H, Kitagawa M, Arai K, Kawakami K, Kobayashi I, Jones RN, Kohno S, Yamaguchi K, Kaku M (2009) Antimicrobial activities of piperacillin-tazobactam against Haemophilus influenzae isolates, including beta-lactamase-negative ampicillin-resistant and beta-lactamase-positive amoxicillin-clavulanate-resistant isolates, and mutations in their quinolone resistance-determining regions. Antimicrob Agents Chemother 53(10):4225–4230
Norskov-Lauritsen N, Ridderberg W, Erikstrup LT, Fuursted K (2011) Evaluation of disk diffusion methods to detect low-level beta-lactamase-negative ampicillin-resistant Haemophilus influenzae. APMIS 119(6):385–392
Transparency declarations
Nothing to declare.
Conflict of Interest
The authors declare that they have no conflict of interest.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Cherkaoui, A., Diene, S.M., Emonet, S. et al. Ampicillin-resistant Haemophilus influenzae isolates in Geneva: serotype, antimicrobial susceptibility, and β-lactam resistance mechanisms. Eur J Clin Microbiol Infect Dis 34, 1937–1945 (2015). https://doi.org/10.1007/s10096-015-2435-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10096-015-2435-5