TolC mediated resistance
13. Discussion and perspectives
13.2. Rapid antibiotic susceptibility testing method 1. Disk diffusion susceptibility method
Traditional antimicrobial susceptibility (AST) testing for H. influenzae can take 48-72 hours before yielding a result which can be explained by the lack of automation of the test. Unfortunately, the long time needed to perform the AST may lead in infected patients to detrimental outcomes.
13.2.2. Procedure for H. influenzae AST by disk diffusion susceptibility method (traditional procedure)
The inoculum suspension is prepared by selecting several colonies from overnight growth (16-24 hours of incubation) on chocolate agar plates and suspending the colonies in sterile saline to the density of a 0.5 McFarland standard. The inoculum is spread over the entire surface of the Müller-Hinton agar plate supplemented with 5%
defibrinated horse blood and 20 mg/mL β-NAD (MH-F) and the plates are incubated in a humid atmosphere containing 5% CO2 at 35 ± 1°C for 18 ± 2 hours. Thereby, the AST results are only available after 18 hours.
13.2.3. Rapid AST directly from blood culture bottles
Last year, EUCAST came up with a list of recommendations for short incubation (4, 6 and 8 hours) AST directly from positive blood culture bottles for Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis and Enterococcus faecium.
However, no recommendations were provided for H. influenzae despite the steady increase of invasive infections caused by NTHi among frail patients.
13.2.4. The EUCAST rapid AST procedure directly from positive blood culture bottles (source: http://www.eucast.org/rapid_ast_in_blood_cultures/)
o Direct inoculation of disk diffusion Müller-Hinton agar plates using 100 - 150 µL directly from a positive blood culture bottle.
o Reduced incubation time from 18 ± 2 hours to 4, 6 and 8 hours. The disk diffusion Müller-Hinton agar plates should be interpreted by using the breakpoints adapted to each incubation time.
o The interpretation of rapid AST results will be done after the identification of
bacteria species.
o The zone diameters will be read only when an obvious zone edge can be
determined. Or else, Müller-Hinton agar plates should be re-incubated and read after 6 or 8 hours.
13.3. H. influenzae rapid AST project 13.3.1. Objectives
This project will be divided into two parts:
1. Development of H. influenzae rapid AST directly from bacterial colonies.
2. Development of H. influenzae rapid AST directly from positive blood culture bottles.
13.3.2. Study design for H. influenzae rapid AST directly from bacterial colonies
I plan to adapt for the H. influenzae rapid AST project the procedures described previously (115).
In order to identify the shortest incubation times for disk diffusion MH-F plates with optimal analytical performances, I will perform time-series image acquisitions on the WASPLab several hours before and up to the traditional incubation duration specific for H. influenzae AST. I plan to compare the results obtained by WASPLab automation (automated inoculation, and automated incubation combined with timely-defined high-resolution digital imaging) against conventional incubation and manual diagnostic, which represents the routine method used in our laboratory. AST analysis for each workflow will be performed by trained clinical microbiologists, blinded of the results obtained by the other method. Results will be compared and optimal imaging times will be defined in the derivation cohort. The assessment between the two methods will be performed on an independent validation cohort using routine clinical H. influenzae strains collected in the bacteriology laboratory at Geneva University Hospitals. In the derivation set, the incubation period of disk diffusion MH-F plates on the WASPLab will be assessed at eight incubation time points (4, 6, 8, 10, 12, 14, 16, and 18h). For each incubation time assessed on the WASPLab, several high-resolution digital images will be taken under different light and exposure conditions according to the manufacturer’s instructions. For the independent validation set, I will perform the same analysis as for the derivation set but only on the incubation time points and imaging conditions that were selected for their optimal analytical performances. The zone diameters will be read only when an obvious zone edge can be determined. I expect to analyse approximatively 300 H. influenzae clinical strains collected in bacteriology laboratory at Geneva University Hospitals.
Figure-1: WASPLab automation (automated inoculation, and automated incubation combined with timely-defined high-resolution digital imaging) and traditional AST testing
13.3.3. Study design for H. influenzae rapid AST directly from positive blood culture The evaluation of the H. influenzae rapid AST directly from positive blood culture will be performed by spiking 200 H. influenzae strains associated with bloodstream infections in 200 negative BD BACTEC™ Plus Aerobic blood culture bottles, in order to mimic positive blood cultures. The inoculum suspensions will be prepared by selecting several colonies from overnight growth on chocolate agar and suspending the colonies in sterile saline to the density of a 0.5 McFarland standard, corresponding to approximately 3x108 CFU/mL. Five hundred microliters of different inoculum suspensions will be injected in negative BD BACTEC™ Plus Aerobic blood culture bottles. Inoculated bottles will be immediately placed in the BD BACTEC™ FX system.
Individual blood culture bottles will be removed from the automated blood culture system when growth was detected. One 4mL aliquot were aspirated from each positive bottle with sterile airway needle. The aliquot will be used in WASPLab to perform the
AST according to EUCAST rapid AST procedure directly from positive blood culture bottle. The incubation period of disk diffusion MH-F plates on the WASPLab will be assessed at three incubation time points (4, 6, and 8h). For each incubation time evaluated on the WASPLab, several high-resolution digital images will be taken under different light and exposure conditions according to manufacturer’s instructions. The zone diameters will be read only when an obvious zone edge can be defined.
Figure-2: The workflow of H. influenzae rapid AST directly from positive blood culture 13.3.4. Importance of the H. influenzae rapid AST project
As mentioned previously, in the post-vaccine era, NTHi was recognized as the main cause of increased invasive H. influenzae infection like sepsis and meningitis in elderly persons. This increase is coupled to steadily increase of antibiotic resistance in NTHi.
This project will enable the antimicrobial susceptibility results in hours instead of days and will allow effective antibiotic stewardship interventions. In addition, this project will apply knowledge acquired during my thesis work to techniques and tools that address critical medical needs linked to H. influenzae (translational research).
14. Conclusions
In spite of the many studies showing interesting results, the mechanisms responsible for the heterogeneous expression of resistance have not been fully established. In S. aureus for example the mecA gene, which encodes a low-affinity PBP2a, is involved in methicillin resistance; nonetheless, as reported previously the autolysins and the presence of multiple unrelated regulatory mechanisms influence the resistance level and the heterogeneous resistance phenotype (116). Likewise, mutations in the ftsI gene may be the initial condition for imipenem resistance in H. influenzae, but as documented in this thesis work, additional levels of regulation in the efflux system and the drug influx contribute to the heterogeneous expression of resistance. Finally, NTHi small colony variants (SCVs) were shown to arise under imipenem stress. Thus, the mechanisms of SCVs persistence and resistance, as well as the population dynamics of NTHi, will also need to be thoroughly studied, by adding tools that are currently lacking in routine clinical microbiology laboratories.
15. References
1. Anonymous. 2011. Manual of Clinical Microbiology, 10 ed, vol 1.
2. World Health Organization. September 2013. Haemophilus influenzae type b (Hib) Vaccination Weekly epidemiological record, Position Paper – No 39, 2013, 88, 413–
428
3. Office fédéral de la santé publique. 2013. Maladies invasives à H. influenzae 1988–
2011. Bull OFSP 2013; no 37: 635-639.
4. Office fédéral de la santé publique. November 1991. Recommandations pour la prévention des infections pédiatriques à Haemophilus influenzae de type b. Directives et recommandations (précédemment Supplément VII).
5. Whittaker R, Economopoulou A, Dias JG, Bancroft E, Ramliden M, Celentano LP.
2017. Epidemiology of Invasive Haemophilus influenzae Disease, Europe, 2007-2014.
Emerg Infect Dis 23:396-404.
6. van Wessel K, Rodenburg GD, Veenhoven RH, Spanjaard L, van der Ende A, Sanders EA. 2011. Nontypeable Haemophilus influenzae invasive disease in The Netherlands: a retrospective surveillance study 2001-2008. Clin Infect Dis 53:e1-7.
7. MacNeil JR, Cohn AC, Farley M, Mair R, Baumbach J, Bennett N, Gershman K, Harrison LH, Lynfield R, Petit S, Reingold A, Schaffner W, Thomas A, Coronado F, Zell ER, Mayer LW, Clark TA, Messonnier NE. 2011. Current epidemiology and trends in invasive Haemophilus influenzae disease--United States, 1989-2008. Clin Infect Dis 53:1230-6.
8. Rubin LG, Moxon ER. 1983. Pathogenesis of bloodstream invasion with Haemophilus influenzae type b. Infect Immun 41:280-4.
9. Agrawal A, Murphy TF. 2011. Haemophilus influenzae infections in the H. influenzae type b conjugate vaccine era. J Clin Microbiol 49:3728-32.
10. Cole FS, Daum RS, Teller L, Goldmann DA, Smith AL. 1979. Effect of ampicillin and chloramphenicol alone and in combination on ampicillin-susceptible and -resistant Haemophilus influenzae type B. Antimicrob Agents Chemother 15:415-9.
11. Hoban D, Felmingham D. 2002. The PROTEKT surveillance study: antimicrobial susceptibility of Haemophilus influenzae and Moraxella catarrhalis from community-acquired respiratory tract infections. J Antimicrob Chemother 50 Suppl S1:49-59.
12. 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 51:3155-61.
13. Osaki Y, Sanbongi Y, Ishikawa M, Kataoka H, Suzuki T, Maeda K, Ida T. 2005. Genetic approach to study the relationship between penicillin-binding protein 3 mutations and Haemophilus influenzae beta-lactam resistance by using site-directed mutagenesis and gene recombinants. Antimicrob Agents Chemother 49:2834-9.
14. Schuchat A, Messonnier NR. 2007. From pandemic suspect to the postvaccine era: the Haemophilus influenzae story. Clin Infect Dis 44:817-9.
15. Adams WG, Deaver KA, Cochi SL, Plikaytis BD, Zell ER, Broome CV, Wenger JD.
1993. Decline of childhood Haemophilus influenzae type b (Hib) disease in the Hib vaccine era. JAMA 269:221-6.
16. Peltola H, Kayhty H, Sivonen A, Makela H. 1977. Haemophilus influenzae type b capsular polysaccharide vaccine in children: a double-blind field study of 100,000 vaccinees 3 months to 5 years of age in Finland. Pediatrics 60:730-7.
17. Schuchat A, Robinson K, Wenger JD, Harrison LH, Farley M, Reingold AL, Lefkowitz L, Perkins BA. 1997. Bacterial meningitis in the United States in 1995. Active Surveillance Team. N Engl J Med 337:970-6.
Greenwood B. 1997. Randomised trial of Haemophilus influenzae type-b tetanus protein conjugate vaccine [corrected] for prevention of pneumonia and meningitis in Gambian infants. Lancet 349:1191-7.
19. Levine OS, Lagos R, Munoz A, Villaroel J, Alvarez AM, Abrego P, Levine MM. 1999.
Defining the burden of pneumonia in children preventable by vaccination against Haemophilus influenzae type b. Pediatr Infect Dis J 18:1060-4.
20. Peltola H. 2000. Worldwide Haemophilus influenzae type b disease at the beginning of the 21st century: global analysis of the disease burden 25 years after the use of the polysaccharide vaccine and a decade after the advent of conjugates. Clin Microbiol Rev 13:302-17.
21. Rubach MP, Bender JM, Mottice S, Hanson K, Weng HY, Korgenski K, Daly JA, Pavia AT. 2011. Increasing incidence of invasive Haemophilus influenzae disease in adults, Utah, USA. Emerg Infect Dis 17:1645-50.
22. Georges S, Lepoutre A, Dabernat H, Levy-Bruhl D. 2013. Impact of Haemophilus influenzae type b vaccination on the incidence of invasive Haemophilus influenzae disease in France, 15 years after its introduction. Epidemiol Infect 141:1787-96.
23. Cherkaoui A, Diene SM, Emonet S, Renzi G, Francois P, Schrenzel J. 2015. Ampicillin-resistant Haemophilus influenzae isolates in Geneva: serotype, antimicrobial susceptibility, and beta-lactam resistance mechanisms. Eur J Clin Microbiol Infect Dis 34:1937-45.
24. Hoiseth SK, Gilsdorf JR. 1988. The relationship between type b and nontypable Haemophilus influenzae isolated from the same patient. J Infect Dis 158:643-5.
25. CDC. MMWR September 05, 1986 / 35(35);553-4. Preliminary report: epidemic fatal purpuric fever among children--Brazil.
26. Harrison LH, Simonsen V, Waldman EA. 2008. Emergence and disappearance of a virulent clone of Haemophilus influenzae biogroup aegyptius, cause of Brazilian purpuric fever. Clin Microbiol Rev 21:594-605.
27. Warren S, Tristram S, Bradbury RS. 2010. Maternal and neonatal sepsis caused by Haemophilus influenzae type d. J Med Microbiol 59:370-2.
28. Schumacher SK, Marchant CD, Loughlin AM, Bouchet V, Stevenson A, Pelton SI.
2012. Prevalence and genetic diversity of nontypeable haemophilus influenzae in the respiratory tract of infants and primary caregivers. Pediatr Infect Dis J 31:145-9.
29. Ladhani S, Slack MP, Heath PT, von Gottberg A, Chandra M, Ramsay ME. 2010.
Invasive Haemophilus influenzae Disease, Europe, 1996-2006. Emerg Infect Dis 16:455-63.
30. Faden H, Duffy L, Williams A, Krystofik DA, Wolf J. 1995. Epidemiology of nasopharyngeal colonization with nontypeable Haemophilus influenzae in the first 2 years of life. J Infect Dis 172:132-5.
31. Goetz MB, O'Brien H, Musser JM, Ward JI. 1994. Nosocomial transmission of disease caused by nontypeable strains of Haemophilus influenzae. Am J Med 96:342-7.
32. Loos BG, Bernstein JM, Dryja DM, Murphy TF, Dickinson DP. 1989. Determination of the epidemiology and transmission of nontypable Haemophilus influenzae in children with otitis media by comparison of total genomic DNA restriction fingerprints. Infect Immun 57:2751-7.
33. Watanabe H, Hoshino K, Sugita R, Asoh N, Watanabe K, Oishi K, Nagatake T. 2004.
Possible high rate of transmission of nontypeable Haemophilus influenzae, including beta-lactamase-negative ampicillin-resistant strains, between children and their parents.
J Clin Microbiol 42:362-5.
34. Erwin AL, Nelson KL, Mhlanga-Mutangadura T, Bonthuis PJ, Geelhood JL, Morlin G, Unrath WC, Campos J, Crook DW, Farley MM, Henderson FW, Jacobs RF, Muhlemann K, Satola SW, van Alphen L, Golomb M, Smith AL. 2005. Characterization
of genetic and phenotypic diversity of invasive nontypeable Haemophilus influenzae.
Infect Immun 73:5853-63.
35. Cherkaoui A, Gaia N, Baud D, Leo S, Fischer A, Ruppe E, Francois P, Schrenzel J.
2018. Molecular characterization of fluoroquinolones, macrolides, and imipenem resistance in Haemophilus influenzae: analysis of the mutations in QRDRs and assessment of the extent of the AcrAB-TolC-mediated resistance. Eur J Clin Microbiol Infect Dis 37:2201-2210.
36. Van Eldere J, Slack MP, Ladhani S, Cripps AW. 2014. Non-typeable Haemophilus influenzae, an under-recognised pathogen. Lancet Infect Dis 14:1281-92.
37. Wan Sai Cheong J, Smith H, Heney C, Robson J, Schlebusch S, Fu J, Nourse C. 2015.
Trends in the epidemiology of invasive Haemophilus influenzae disease in Queensland, Australia from 2000 to 2013: what is the impact of an increase in invasive non-typable H. influenzae (NTHi)? Epidemiol Infect 143:2993-3000.
38. Murphy TF, Faden H, Bakaletz LO, Kyd JM, Forsgren A, Campos J, Virji M, Pelton SI. 2009. Nontypeable Haemophilus influenzae as a pathogen in children. Pediatr Infect Dis J 28:43-8.
39. Swords WE, Moore ML, Godzicki L, Bukofzer G, Mitten MJ, VonCannon J. 2004.
Sialylation of lipooligosaccharides promotes biofilm formation by nontypeable Haemophilus influenzae. Infect Immun 72:106-13.
40. Starner TD, Zhang N, Kim G, Apicella MA, McCray PB, Jr. 2006. Haemophilus influenzae forms biofilms on airway epithelia: implications in cystic fibrosis. Am J Respir Crit Care Med 174:213-20.
41. Ehrlich GD, Veeh R, Wang X, Costerton JW, Hayes JD, Hu FZ, Daigle BJ, Ehrlich MD, Post JC. 2002. Mucosal biofilm formation on middle-ear mucosa in the chinchilla model of otitis media. JAMA 287:1710-5.
42. Foxwell AR, Kyd JM, Cripps AW. 1998. Nontypeable Haemophilus influenzae:
pathogenesis and prevention. Microbiol Mol Biol Rev 62:294-308.
43. Collins S, Ramsay M, Slack MP, Campbell H, Flynn S, Litt D, Ladhani SN. 2014. Risk of invasive Haemophilus influenzae infection during pregnancy and association with adverse fetal outcomes. JAMA 311:1125-32.
44. Sriram KB, Cox AJ, Clancy RL, Slack MPE, Cripps AW. 2017. Nontypeable Haemophilus influenzae and chronic obstructive pulmonary disease: a review for clinicians. Crit Rev Microbiol doi:10.1080/1040841X.2017.1329274:1-18.
45. Craig JE, Cliffe A, Garnett K, High NJ. 2001. Survival of nontypeable Haemophilus influenzae in macrophages. FEMS Microbiol Lett 203:55-61.
46. Ahren IL, Williams DL, Rice PJ, Forsgren A, Riesbeck K. 2001. The importance of a beta-glucan receptor in the nonopsonic entry of nontypeable Haemophilus influenzae into human monocytic and epithelial cells. J Infect Dis 184:150-8.
47. Rosadini CV, Ram S, Akerley BJ. 2014. Outer membrane protein P5 is required for resistance of nontypeable Haemophilus influenzae to both the classical and alternative complement pathways. Infect Immun 82:640-9.
48. Langereis JD, de Jonge MI. 2015. Invasive Disease Caused by Nontypeable Haemophilus influenzae. Emerg Infect Dis 21:1711-8.
49. Andersen C, Maier E, Kemmer G, Blass J, Hilpert AK, Benz R, Reidl J. 2003. Porin OmpP2 of Haemophilus influenzae shows specificity for nicotinamide-derived nucleotide substrates. J Biol Chem 278:24269-76.
50. Tristram S, Jacobs MR, Appelbaum PC. 2007. Antimicrobial resistance in Haemophilus influenzae. Clin Microbiol Rev 20:368-89.
51. Williamson R, Collatz E, Gutmann L. 1986. [Mechanisms of action of beta-lactam antibiotics and mechanisms of non-enzymatic resistance]. Presse Med 15:2282-9.
53. Fluit AC, Florijn A, Verhoef J, Milatovic D. 2005. Susceptibility of European beta-lactamase-positive and -negative Haemophilus influenzae isolates from the periods 1997/1998 and 2002/2003. J Antimicrob Chemother 56:133-8.
54. Jansen WT, Verel A, Beitsma M, Verhoef J, Milatovic D. 2006. Longitudinal European surveillance study of antibiotic resistance of Haemophilus influenzae. J Antimicrob Chemother 58:873-7.
55. Skaare D, Lia A, Hannisdal A, Tveten Y, Matuschek E, Kahlmeter G, Kristiansen BE.
2015. Haemophilus influenzae with Non-Beta-Lactamase-Mediated Beta-Lactam Resistance: Easy To Find but Hard To Categorize. J Clin Microbiol 53:3589-95.
56. Medeiros AA, O'Brien TF. 1975. Ampicillin-resistant Haemophilus influenzae type B possessing a TEM-type beta-lactamase but little permeability barrier to ampicillin.
Lancet 1:716-9.
57. Rubin LG, Medeiros AA, Yolken RH, Moxon ER. 1981. Ampicillin treatment failure of apparently beta-lactamase-negative Haemophilus influenzae type b meningitis due to novel beta-lactamase. Lancet 2:1008-10.
58. Farrell DJ, Morrissey I, Bakker S, Buckridge S, Felmingham D. 2005. Global distribution of TEM-1 and ROB-1 beta-lactamases in Haemophilus influenzae. J Antimicrob Chemother 56:773-6.
59. Bradford PA. 2001. Extended-spectrum beta-lactamases in the 21st century:
characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev 14:933-51, table of contents.
60. Goussard S, Courvalin P. 1991. Sequence of the genes blaT-1B and blaT-2. Gene 102:71-3.
61. Paterson DL, Bonomo RA. 2005. Extended-spectrum beta-lactamases: a clinical update.
Clin Microbiol Rev 18:657-86.
62. Mohd-Zain Z, Turner SL, Cerdeno-Tarraga AM, Lilley AK, Inzana TJ, Duncan AJ, Harding RM, Hood DW, Peto TE, Crook DW. 2004. Transferable antibiotic resistance elements in Haemophilus influenzae share a common evolutionary origin with a diverse family of syntenic genomic islands. J Bacteriol 186:8114-22.
63. Lartigue MF, Leflon-Guibout V, Poirel L, Nordmann P, Nicolas-Chanoine MH. 2002.
Promoters P3, Pa/Pb, P4, and P5 upstream from bla(TEM) genes and their relationship to beta-lactam resistance. Antimicrob Agents Chemother 46:4035-7.
64. Tristram SG, Nichols S. 2006. A multiplex PCR for beta-lactamase genes of Haemophilus influenzae and description of a new blaTEM promoter variant. J Antimicrob Chemother 58:183-5.
65. Saunders JR, Elwell LP, Falkow S, Sykes RB, Richmond MH. 1978. beta-lactamases and R-plasmids of Haemophilus influenzae. Scand J Infect Dis Suppl:16-22.
66. Leaves NI, Dimopoulou I, Hayes I, Kerridge S, Falla T, Secka O, Adegbola RA, Slack MP, Peto TE, Crook DW. 2000. Epidemiological studies of large resistance plasmids in Haemophilus. J Antimicrob Chemother 45:599-604.
67. Tristram SG. 2003. Effect of extended-spectrum beta-lactamases on the susceptibility of Haemophilus influenzae to cephalosporins. J Antimicrob Chemother 51:39-43.
68. Sondergaard A, Norskov-Lauritsen N. 2016. Contribution of PBP3 Substitutions and TEM-1, TEM-15, and ROB-1 Beta-Lactamases to Cefotaxime Resistance in Haemophilus influenzae and Haemophilus parainfluenzae. Microb Drug Resist 22:247-52.
69. Tristram SG, Pitout MJ, Forward K, Campbell S, Nichols S, Davidson RJ. 2008.
Characterization of extended-spectrum beta-lactamase-producing isolates of Haemophilus parainfluenzae. J Antimicrob Chemother 61:509-14.
70. Scheifele DW, Fussell SJ, Roberts MC. 1982. Characterization of ampicillin-resistant Haemophilus parainfluenzae. Antimicrob Agents Chemother 21:734-9.
71. Livrelli VO, Darfeuille-Richaud A, Rich CD, Joly BH, Martel JL. 1988. Genetic determinant of the ROB-1 beta-lactamase in bovine and porcine Pasteurella strains.
Antimicrob Agents Chemother 32:1282-4.
72. Maclean IW, Slaney L, Juteau JM, Levesque RC, Albritton WL, Ronald AR. 1992.
Identification of a ROB-1 beta-lactamase in Haemophilus ducreyi. Antimicrob Agents Chemother 36:467-9.
73. Medeiros AA, Levesque R, Jacoby GA. 1986. An animal source for the ROB-1 beta-lactamase of Haemophilus influenzae type b. Antimicrob Agents Chemother 29:212-5.
74. Thornsberry C, Kirven LA. 1974. Ampicillin resistance in Haemophilus influenzae as determined by a rapid test for beta-lactamase production. Antimicrob Agents Chemother 6:653-4.
75. Markowitz SM. 1980. Isolation of an ampicillin-resistant, non-beta-lactamase-producing strain of Haemophilus influenzae. Antimicrob Agents Chemother 17:80-3.
76. Parr TR, Jr., Bryan LE. 1984. Mechanism of resistance of an ampicillin-resistant, beta-lactamase-negative clinical isolate of Haemophilus influenzae type b to beta-lactam antibiotics. Antimicrob Agents Chemother 25:747-53.
77. Ubukata K, Shibasaki Y, Yamamoto K, Chiba N, Hasegawa K, Takeuchi Y, Sunakawa K, Inoue M, Konno M. 2001. Association of amino acid substitutions in penicillin-binding protein 3 with beta-lactam resistance in beta-lactamase-negative ampicillin-resistant Haemophilus influenzae. Antimicrob Agents Chemother 45:1693-9.
78. Clairoux N, Picard M, Brochu A, Rousseau N, Gourde P, Beauchamp D, Parr TR, Jr., Bergeron MG, Malouin F. 1992. Molecular basis of the non-beta-lactamase-mediated resistance to beta-lactam antibiotics in strains of Haemophilus influenzae isolated in Canada. Antimicrob Agents Chemother 36:1504-13.
79. Mendelman PM, Chaffin DO, Kalaitzoglou G. 1990. Penicillin-binding proteins and ampicillin resistance in Haemophilus influenzae. J Antimicrob Chemother 25:525-34.
80. 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 46:2208-18.
81. Hasegawa K, Chiba N, Kobayashi R, Murayama SY, Iwata S, Sunakawa K, Ubukata K.
2004. Rapidly increasing prevalence of beta-lactamase-nonproducing, ampicillin-resistant Haemophilus influenzae type b in patients with meningitis. Antimicrob Agents Chemother 48:1509-14.
82. 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 48:1630-9.
83. El-Halfawy OM, Valvano MA. 2015. Antimicrobial heteroresistance: an emerging field
83. El-Halfawy OM, Valvano MA. 2015. Antimicrobial heteroresistance: an emerging field