Genetic Diversity of the ftsI Gene in β-Lactamase-Nonproducing Ampicillin-Resistant and β-Lactamase-Producing Amoxicillin-/Clavulanic Acid-Resistant Nasopharyngeal Haemophilus influenzae Strains Isolated from Children in South Korea

Abstract

Haemophilus influenzae frequently colonizes the nasopharynx of children and adults, which can lead to a variety of infections. We investigated H. influenzae carriage in the nasopharynx of 360 children, in terms of (1) the prevalence of strains with decreased susceptibility, and (2) the presence of amino acid substitutions in PBP3. One hundred twenty-three strains were isolated (34.2%, 123/360), 122 of which were classified as nontypable H. influenzae (NTHi). Of these, β-lactamase-nonproducing ampicillin-susceptible strains accounted for 26.2%, β-lactamase-producing-ampicillin-resistant strains for 9.0%, β-lactamase-nonproducing ampicillin-resistant (BLNAR) strains for 40.2%, and β-lactamase-producing amoxicillin-/clavulanic acid-resistant (BLPACR) for 24.6%, respectively. Pulsed field gel electrophoresis (PFGE) patterns were so diverse that they were clustered into 41 groups. The amino acid substitutions in the transpeptidase domain (292 amino acids) of ftsI in BLNAR isolates showed that group IIb accounted for 30.6%, IIc for 8.2%, IId for 16.3%, III for 32.7%, and the others for 12.2%. Moreover, groups IIb (56.7%; 17/30) and III (23.3%; 7/30) were prevalent among BLPACR strains. They were subclassified into more diverse sequence subtypes by analysis of the entire PBP3 (610 amino acids). Groups IIb, IIc, IId, and III exhibited 13, four, six, and four sequence subtypes, respectively. Such a genetic diversity is likely indicative of significant potential for decreased antimicrobial susceptibility in nasopharyngeal-colonizing NTHi strains.

Introduction

H aemophilus influenzae is one of the major etiological agents that cause respiratory tract, ear, nose, and throat infections despite the availability of the H. influenzae type b vaccine (Tristram et al.). Nontypeable H. influenzae (NTHi) is a particularly important cause of recurrent acute otitis media (AOM) and pneumonia, with several reports emphasizing the issue of antibiotic resistance within these strains.^{7,23,24,27,34} With widespread implementation of PCV-7 vaccination, NTHi has gained greater significance as a cause of AOM, underscoring the need for epidemiological studies of this organism.²⁹

The principal mechanisms of resistance to ampicillin in H. influenzae result from enzymatic hydrolysis by β-lactamase (the TEM-1 and ROB-1 type) and a decreased affinity for β-lactams, mainly correlated with alteration of penicillin-binding protein 3 (PBP3) encoded by ftsI.^{3,17,26,30,34,35} More specifically, amino acid substitutions surrounding the conserved STVK (Ser327-Thr-Val-Lys), SSN (Ser379-Ser-Asn), and KTG (Lys512-Thr-Gly) motifs in the transpeptidase domain of PBP3 are associated with decreased affinity for β-lactams in β-lactamase-nonproducing ampicillin-resistant (BLNAR) strains.^5,11,20,35 Strains with a single substitution of Asn526Lys or Arg517His have the characteristic low ampicillin resistance, resulting in slightly lower ampicillin MICs than in BLNAR strains.^12,20

β-Lactamase-producing strains (BLPAR) are prevalent globally¹³; however, the proportion of BLNAR strains has increased since the first report in the early 1980s.^{2,9,12,15,22,25,28} Additionally, the incidence of β-lactamase-producing strains with PBP3 mutations (BLPACR strains) that exhibit decreased susceptibility to amoxicillin/clavulanic acid is increasing.^2,6

In South Korea, NTHi strains consisted of 38.9% BLPAR, 29.3% BLNAR, and 8.3% BLPACR from 2000 to 2005.¹⁸ Another study of nationwide acute respiratory infection during 2005 and 2006 reported 47.2% BLPAR, 6.1% BLNAR, and 5.2% BLPACR strains among NTHi.¹ Taken together, these findings suggest that BLPAR strains are the most prevalent strains in the clinical setting in Korea, although some differences between studies have been noted. However, there is no information regarding the proportion of nasopharyngeal strains that are BLNAR or BLPACR.

Some studies showed a high prevalence of BLNAR strains in the nasopharynx and the possibility of intrafamilial transmission of NTHi.^14,36 Nasopharyngeal-colonizing H. influenzae with decreased β-lactam susceptibility is a major concern in pediatrics and otolaryngology. Here we describe nasopharyngeal carriage of H. influenzae by children, the prevalence of strains with decreased susceptibility, and the diversity of PBP3 mutations that are associated with decreased affinity for β-lactams.

Methods

Bacterial isolates and culture

During January and February 2010, nasopharyngeal specimens were collected from 360 children under 5 years of age in three tertiary-care hospitals. Children had no history of infection or antibiotic treatment within the previous 2 weeks. Ethics approval was received from the institutional review board of each hospital. H. influenzae was identified in a clinical microbiology company (Neogene, Seoul, Korea), and then isolates were transferred to our laboratory. One hundred twenty-three strains of H. influenzae were isolated from these specimens. Isolates were cultured in a Haemophilus test medium (HTM; Oxoid, Ltd., Basingstoke, Hampshire, United Kingdom) containing supplement (X and V factors) for antibiotic susceptibility and molecular characterization.

With the exception of one isolate (type a), 122 isolates were determined to be NTHi by polymerase chain reaction (PCR) for capsular typing, as described previously.⁸

Antibiotic susceptibility

The MICs of ampicillin, amoxicillin/clavulanate, cefaclor, and cefditoren were determined by the broth microdilution method on HTM following the guidelines of the Clinical and Laboratory Standards Institute.⁴ The presence of β-lactamase was tested by nitrocefin disks (Cefinase, Becton-Dickinson Microbiology Systems, Cockeysville, MD), as instructed by the manufacturer. H. influenzae ATCC 49766 (ampicillin susceptible) and ATCC 49247 (BLNAR) were used as controls for antibiotic susceptibility and molecular assays.

PCR amplification, ftsI sequencing, and pulsed field gel electrophoresis

The presence of the blaTEM and blaROB ampicillin resistance genes was determined by PCR, as described previously.³³ PCR products were of 526 bp (bla_TEM) and 692 bp (bla_ROB). Also, PCR for capsular typing was performed as previously described.⁸ To detect alterations of PBP3 in the BLNAR and BLPACR strains, the 2719-bp PCR product was amplified by primers 5′-CTCGTTATCCGTTACAGCAG-3′ (1604–1623 in U32793) and 5′-GCCAAACCGTGTGATGAAAC-3′ (4303–4322 in U32793), newly designed for this study. Sequencing was performed with PCR primers, A601F (5′-GTAAGATTGAAAATGGAC-3′), A91R (5′-ATTTCATCTCGTTTAACG-3′), C96R (5′-CCACAATTTCTTTACCGC-3′), and C464F (5′-AACTAAAGATATTGTGGG-3′).

Amplification was carried out in a 50-μl total volume containing 50 μM of each primer, 10 mM deoxynucleoside triphosphates, 1× PCR buffer containing 25 mM MgCl₂, and 1 U ExTaq polymerase (Takara Bio, Inc., Otsu, Shiga, Japan). PCR cycling was carried out in MyCycler (Biorad, Hercules, CA) as follows: an initial denaturation step at 94°C for 5 min, 30 amplification cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 3 min, and an extension step at 72°C for 10 min.

Sequence homology searches were performed using BLASTN and BLASTP (www.ncbi.nlm.nih.gov/BLAST). Open reading frames were predicted using ORF Finder at the NCBI (www.ncbi.nlm.nih.gov/gorf/gorf.html). The sequence of strain Rd (GenBank no. L42023) was used as a reference template. Multiple sequence alignments were performed with ClustalW2 (www.ebi.ac.uk/Tools/clustalw2).²¹ Phylogenetic analysis was conducted with MEGA version 4.0³¹ using the maximum likelihood method based on the JTT matrix-based model.¹⁶ In the convenience, sequence types were determined, as the identical sequences (100% of homology) were grouped into one sequence type.

Pulsed field gel electrophoresis (PFGE) analysis was performed as described previously.³² Electrophoresis was performed in CHEF DR III (Biorad) using 1–5 sec of the switching time, 6 V/cm, at 14°C for 18 hr. The patterns were analyzed based on a similarity cutoff of 80% (analyzed by the Dice coefficient and UPGMA, with 1% tolerance and 0.5% optimization settings).

Nucleotide sequence accession numbers

Nucleotide sequences were deposited in GenBank (www.ncbi.nlm.nih.gov/GenBank) under accession numbers JN944445 to JN944483 and JX861248 to JX861257.

Results

Antibiotic susceptibility and β-lactamase assay of NTHi isolates

From a total of 360 children, 122 (33.9%) H. influenzae isolates were determined to be NTHi by the capsular PCR method.

Of these NTHi strains, 26.2% (32 isolates) were ampicillin sensitive, whereas 73.8% (90 isolates) exhibited reduced susceptibility (intermediate and resistant). A β-lactamase test by a cefinase disk showed that positive strains were 33.6% (41 isolates) of isolates. Among them, strains with bla_TEM were 90.2% (37/41), and strains with bla_ROB were 9.8% (4/41).

We classified NTHi isolates as BLNAS, BLPAR, BLNAR, or BLPACR based on their ampicillin susceptibility and possession of β-lactamase. Of 122 NTHi isolates, BLNAS strains accounted for 26.2% (32 isolates), BLPAR for 9.0% (11 isolates), BLNAR for 40.2% (49 isolates), and BLPACR for 24.6% (30 isolates). With regard to ampicillin, BLPACR isolates exhibited higher MICs (MIC₅₀=128 μg/ml) than either BLPAR (MIC₅₀=32 μg/ml) or BLNAR (MIC₅₀=2 μg/ml) isolates. The amoxicillin/clavulanate MIC₉₀ values (32 μg/ml) of BLNAR isolates were higher than those of BLPAR isolates (MIC₉₀=4 μg/ml). In terms of cefditoren susceptibility, the MIC₅₀ of BLNAS isolates was 0.125 μg/ml, BLPAR was 0.5 μg/ml, BLNAR was 0.5 μg/ml, and BLPACR was 1 μg/ml.

Alteration of transpeptidase domain of PBP3 in BLNAR and BLPACR strains

All BLNAR and BLPACR isolates contained amino acid substitutions in the transpeptidase domain (280–571) of PBP3. With the exception of one isolate, the substitution occurred in the conserved SSN (Ser379-Ser-Asn) and/or KTG (Lys512-Thr-Gly) motifs (Table 1). One isolate that exhibited a BLPACR phenotype had no mutation in these conserved motifs, but did have substitutions surrounding these motifs. No amino acid substitutions in the STVK (Ser327-Thr-Val-Lys) motif were detected.

Table 1.

Amino Acid Substitutions in the Transpeptidase Domain of the ftsI Gene of BLNAR and BLPACR Strains

	No. of isolates		Amino acid substitution
Group ^a	BLNAR	BLPACR	348 Ile	350 Asp	357 Ser	368 Ala	377 Met	385 Ser	389 Leu	437 Ala	449 Ile	502 Ala	517 Arg	526 Asn	530 Ala	532 Thr	547 Val	551 Asp	554 Ala	562 Val	569 Asn
I	1	0		Asn									His				Ile				Ser
IIa	1	0												Lys	Ser		Ile
IIb	11	15		Asn			Ile					Val		Lys			Ile				Ser
	1	0		Asn			Ile					Val		Lys			Ile
	1	2										Val		Lys			Ile				Ser
	1	0		Asn								Val		Lys			Ile				Ser
	1	0					Ile					Val		Lys			Ile				Ser
IIc	1	0				Thr								Lys
	0	1				Thr						Thr		Lys
	1	0				Thr								Lys							Ser
	1	0				Thr								Lys			Ile				Ser
	1	0		Asn	Asn							Thr		Lys
IId	8	1									Val			Lys			Ile				Ser
	0	1									Val			Lys
III	8	7		Asn	Asn		Ile	Thr	Phe					Lys			Ile			Leu	Ser
	8	0		Asn	Asn		Ile	Thr	Phe					Lys						Leu
III+IIc	2	1		Asn	Asn		Ile	Thr	Phe			Thr		Lys
III like	2	1		Asn	Asn		Ile	Thr					His			Ser	Ile
Miscellaneous^b	0	1															Ile				Ser
Total	49	30

Groups were identified as described by Dabernat et al.⁵ III+IIc was defined in this study, and III-like was as defined by García-Cobos et al.¹⁰

This group has not been defined in any previous reports.

BLNAR, β-lactamase-nonproducing ampicillin-resistant; BLPACR, β-lactamase-producing amoxicillin-/clavulanic acid-resistant.

The mutations in the ftsI transpeptidase domain were classified into nine groups, 18 subgroups, and one singleton (Table 1). Classification was performed as described by Dabernat et al.⁵ III+IIc was defined in this study, and III-like was as defined by García-Cobos et al.¹⁰ There was no group I or IIa in the BLPACR isolates. Overall, the most frequent amino acid substitutions occurred near the KTG motif. Group II, with the substitution Asn526Lys, was the most prevalent in both BLNAR (57.1%, 28/49) and BLPACR (90.0%, 27/30). In BLNAR isolates, group IIb accounted for 30.6% (15/49), IIc for 8.2% (4/49), IId for 16.3% (8/49), and III for 32.7% (16/49) (Table 1). Group IIb (56.7%; 17/30) and III (23.3%; 7/30) were most prevalent in BLPACR isolates (Table 1). Group III+IIc presented the characteristics of both III and IIc, which included the following amino acid substitutions: Asp350Asn, Ser357Asn; Met377Ile, Ser385Thr, and Leu389Phe in the SSN motif; and Ala502Thr and Asn526Lys in the KTG motif. The present study indicated that group III strains of BLNAR or BLPACR had a higher cefaclor MIC₅₀ (16 μg/ml of BLNAR and 32 μg/ml of BLPACR) than did the other groups (Table 2).

Table 2.

Susceptibilities to β-Lactam of PBP3 Mutation Groups in the BLNAR and BLPACR Strains

		Ampicillin		Amoxicillin-clavulanate		Cefaclor		Cefditoren
Group (No. of isolates)		MIC50 (μg/ml)	MIC90 (μg/ml)	MIC50 (μg/ml)	MIC90 (μg/ml)	MIC50 (μg/ml)	MIC90 (μg/ml)	MIC50 (μg/ml)	MIC90 (μg/ml)
BLNAR	I (1)	16	16	16	16	16	16	4	4
	IIa (1)	2	2	4	4	4	4	0.25	0.25
	IIb (15)	2	8	4	16	8	16	0.25	2
	IIc (4)	4	8	4	16	8	8	0.5	1
	IId (8)	2	8	4	64	4	16	0.12	4
	III (16)	4	16	8	32	16	128	0.5	8
	III+IIc (2)	2	4	4	4	8	8	0.5	2
	III like	4	4	8	8	8	8	0.25	2
BLPACR	IIb (17)	128	128	8	64	8	64	0.5	2
	IIc (1)	128	128	8	8	8	8	0.015	0.015
	IId (2)	4	128	8	16	4	8	2	2
	III (7)	128	128	16	64	32	64	1	1
	III+IIc (1)	128	128	32	32	16	16	4	4
	III like	(1)	128	128	32	32	8	8	4
	Miscellaneous (1)	128	128	8	8	16	16	1	1

Furthermore, mutations were detected in the BLNAS and BLPAR strains. Two isolates in BLPAR and 5 isolates in BLNAS belonged to group IIb, and 4 isolates in BLNAS belonged to IId.

PFGE and phylogenic analysis of the entire ftsI gene

PFGE patterns of all isolates were so diverse that they were clustered into 26 groups and 18 singletons with a similarity of 80%. There were no distinctive PFGE patterns among the BLNAS, BLPAR, BLNAR, and BLPACR isolates. Moreover, most strains within the same ftsI mutation groups had diverse PFGE patterns, resulting in wide dissemination into different groups. The isolates of Group IIb were disseminated into 9 PFGE groups and 12 singletons, group IId 7 groups and 2 singletons, and group III 5 groups and 4 singletons. There were no distinctive PFGE patterns among the ftsI mutation groups. PFGE analysis showed the clonal diversity of nasopharyngeal-colonizing NTHi isolates.

The phylogenetic relationships of the amino acid sequences of the entire ftsI gene are displayed in Fig. 1. In Fig. 1, each isolates represented a typical sequence type. In the convenience, a sequence type was determined, as the identical sequences (100% of homology) were grouped into one sequence type. Total 45 sequence types were determined in those of all isolates (Fig. 1). Among them, 12 sequence types were determined in BLNAS and BLPAR mainly due to the variation of other domain, not the transpeptidase domain (Fig. 1). These results showed that the mutation groups of the transpeptidase domain could be subclassified into more diverse types. Group IIb was subclassified into 13 sequence subtypes, group IIc into four, group IId into six, and group III into four subtypes. Group IIb strains were widely disseminated into five major clusters with related distances. No significant differences between BLNAR and BLPACR strains were found in the phylogenetic analysis.

FIG. 1.

A phylogenetic tree based on a comparison of the entire ftsI sequences of all isolates, created with MEGA version 4.0³¹ using the maximum likelihood method based on the JTT matrix-based model.¹⁶

Discussion

NTHi frequently colonizes the nasopharynx in children and causes a variety of infections. A study in healthy children reported that the prevalence of H. influenzae type b and BLPAR strains was higher in North India.¹⁴ However, H. influenzae type b strains were not isolated in this study, possibly due to the introduction of the Hib vaccine in the mid-1990s in South Korea. Previous studies in South Korea showed that BLPAR strains were the most prevalent in the clinical setting, with BLNAR or BLPACR strains accounting for a smaller proportion.^1,18,19 However, in this study, both the prevalence of colonizing NTHi (34.2%) and rates of resistance (73.8%) were very high in the nasopharynx of hospitalized children with no infection history.

BLNAR strains are usually classified into three groups (I, II, and III) based on the presence of different amino acid substitutions in the transpeptidase region of PBP3, as previously described by Ubukata et al.³⁵ Group II was further divided into subgroups IIa, IIb, IIc, and IId, as described by Dabernat et al.,⁵ and III-like was further classified according to García-Cobos et al.¹⁰ Although other classifications exist, for example, that of Hasegawa et al.,¹² the former classifications seem to be more convenient for our comparison of the amino acid substitution groups of nasopharyngeal strains to groups derived from infectious strains reported by Bae et al.¹ and Kim et al.¹⁸ More groups (19 groups) were found in these nasopharyngeal strains compared with the 14 groups noted by Kim et al.¹⁸ and 11 by Bae et al.¹ in the clinical setting. Compared with the report by Kim et al.,¹⁸ the proportion of IIa strains was markedly lower, and IIb strains were more prevalent in the nasopharyngeal BLNAR and BLPACR isolates (Table 1). However, the prevalence of IIb was similar to that noted by Bae et al.¹ The most prevalent types of both BLNAR and BLPACR strains were also reported to be the most prevalent in a recent Portuguese study by Barbosa et al.² (Table 1). The ftsI mutation groups of the BLPACR strains exhibited lower diversity than did those of the BLNAR strains, likely because the group of BLPACR strains originated from a small number of types (IIb and III) (Table 1).

Additionally, amino acid sequence analysis of the entire ftsI, including of the transpeptidase domain, showed the sequence type of ftsI to be highly diverse, as some amino acid substitution groups defined by transpeptidase mutation comprised many variable subgroups (Fig. 1). Also, PFGE analysis suggested that diverse clones were capable of acquiring the variations associated with β-lactam resistance. Taken together, these findings suggest that acquisition of resistance may originate from diverse NTHi clones.

In conclusion, the high prevalence of nasopharyngeal BLNAR and BLPACR strains in the nasopharyngeal carriage of children represents a major risk of increased respiratory infections, including recurrent AOM and pneumonia, as well as systemic infections. Therefore, it is necessary to pay close attention to the selection of antibiotics, establish a prudent treatment policy, and monitor the emergence of resistance to other antibiotics. Also, an effective anti-NTHi vaccine should be developed.

Footnotes

Acknowledgments

This study was supported by GlaxoSmithKline Biologicals, Seoul, Korea. This study was approved by the institutional review board at the Seoul St. Mary's Hospital (Approval No. K009EIMI0402; Approval Date 01/Jan/2010).

Author Disclosure Statement

No competing financial interests exist.

References

Bae

, Lee

, Kim

, Lee

, Yu

, Kang

2010. Antimicrobial resistance in Haemophilus influenzae respiratory tract soilates in Korea: results of a nationwide acute respiratory infections surveillance. Antimicrob. Agents Chemother., 54:65–71.

Barbosa

A.R.

, Giufrè

, Cerquetti

, Bajanca-Lavado

M.P.

2011. Polymorphism in ftsI gene and β-lactam susceptibility in Portuguese Haemophilus influenzae strains: clonal dissemination of β-lactamase-positive isolates with decreased susceptibility to amoxicillin/clavulanic acid. J. Antimicrob. Chemother., 66:788–796.

Clairoux

, Picard

, Brochu

, Rousseau

, Gourde

, Beauchamp

, Parr

T.R.

, Bergeron

M.G.

, Malouin

1992. Molecular basis of the non-β-lactamase-mediated resistance to β-lactam antibiotics in strains of Haemophilus influenzae isolated in Canada. Antimicrob. Agents Chemother., 36:1504–1513.

[CLSI] Clinical, Laboratory Standards Institute. 2010. Performance standards for antimicrobial susceptibility testing. Eighteenth informational supplementApproved standard M100-S18CLSI: Wayne, PA.

Dabernat

, Delmas

, Seguy

, Pelissier

, Faucon

, Bennamani

, Pasquier

2002. Diversity of beta-lactam resistance-conferring amino acid substitutions in penicillin-binding protein 3 of Haemophilus influenzae. Antimicrob. Agents Chemother., 46:2208–2218.

Doern

G.V.

, Brueggemann

A.B.

, Pierce

, Holley

H.P.

Jr. , Rauch

1997. Antibiotic resistance among clinical isolates of Haemophilus influenzae in the United States in 1994 and 1995 and detection of β-lactamase-positive strains resistant to amoxicillin–clavulanate: results of a national multicenter surveillance study. Antimicrob. Agents Chemother., 41:292–297.

Faden

, Duffy

, Wasielewski

, Wolf

, Krystofik

, Tung

1997. Relationship between nasopharyngeal colonization and the development of otitis media in children. J. Infect. Dis., 175:1440–1445.

Falla

T.J.

, Crook

D.W.

, Brophy

L.N.

, Maskell

, Kroll

J.S.

, Moxon

E.R.

1994. PCR for capsular typing of Haemophilus influenzae. J. Clin. Microbiol., 32:2382–2386.

Fluit

A.C.

, Florijn

, Verhoef

, Milatovic

2005. Susceptibility of European β-lactamase-positive and β-negative Haemophilus influenzae isolates from the periods 1997/1998 and 2002/2003. J. Antimicrob. Chemother., 56:133–138.

10.

García-Cobos

, Campos

, Lázaro

, Román

, Cercenado

, García-Rey

, Pérez-Vázquez

, Oteo

, de Abajo

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., 51:2564–2573.

11.

Hasegawa

, Chiba

, Kobayashi

, Murayama

S.Y.

, Iwata

, Sunakawa

, Ubukata

2004. Rapidly increasing prevalence of beta-lactamase nonproducing, ampicillin-resistant Haemophilus influenzae type b in patients with meningitis. Antimicrob. Agents Chemother., 48:1509–1514.

12.

Hasegawa

, Kobayashi

, Takada

, Ono

, Chiba

, Morozumi

, Iwata

, Sunakawa

, Ubukata

the Working Group of the Nationwide Surveillance for Bacterial Meningitis. 2006. High prevalence of type b β-lactamase-non-producing ampicillin-resistant Haemophilus influenzae in meningitis: the situation in Japan where Hib vaccine has not been introduced. J. Antimicrob. Chemother., 57:1077–1082.

13.

Hoban

, Felmingham

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.

14.

Jain

, Kumar

, Agarwal

S.K.

2008. High nasopharyngeal carriage of beta-lactamase-negative ampicillin-resistant Haemophilus influenzae in north Indian school-going children. J. Infect. Chemother., 14:727–724.

15.

Jansen

W.T.

, Verel

, Beitsma

, Verhoef

, Milatovic

2006. Longitudinal European surveillance study of antibiotic resistance of Haemophilus influenzae. J. Antimicrob. Chemother., 58:873–877.

16.

Jones

D.T.

, Taylor

W.R.

, Thornton

J.M.

1992. The rapid generation of mutation data matrices from protein sequences. Comput. Appl. Biosci., 8:275–282.

17.

Jorgensen

J.H.

1992. Update on mechanisms and prevalence of antimicrobial resistance in Haemophilus influenzae. Clin. Infect. Dis., 14:1119–1123.

18.

Kim

I.S.

, Ki

C.S.

, Kim

, Oh

W.S.

, Peck

K.R.

, Song

J.H.

, Lee

N.Y.

2007. Diversity of ampicillin resistance genes and antimicrobial susceptibility patterns in Haemophilus influenzae strains isolated in Korea. Antimicrob. Agents Chemother., 51:453–460.

19.

Kim

K.H.

, Lim

K.A.

, Whang

I.T.

, Hong

Y.M.

, Kang

E.S.

, Hong

K.S.

2001. Carriage and antimicrobial susceptibility of Haemophilus influenzae isolated from oropharynges of children. Kor. J. Pediatr., 44:509–516

20.

Kishii

, Chiba

, Morozumi

, Hamano-Hasegawa

, Kurokawa

, Masaki

, Ubukata

2010. Diverse mutations in the ftsI gene in ampicillin-resistant Haemophilus influenzae isolates from pediatric patients with acute otitis media. J. Infect. Chemother., 16:87–93.

21.

Larkin

M.A.

, Blackshields

, Brown

N.P.

, Chenna

, McGettigan

P.A.

, McWilliam

, Valentin

, Wallace

I.M.

, Wilm

, Lopez

, Thompson

J.D.

, Gibson

T.J.

, Higgins

D.G.

2007. ClustalW2 and ClustalX version 2. Bioinformatics, 23:2947–2948.

22.

Markowitz

S.M.

1980. Isolation of an ampicillin-resistant, non-β-lactamase producing strain of Haemophilus influenzae. Antimicrob. Agents Chemother., 17:80–83.

23.

Moxon

E.R.

1986. The carrier state: Haemophilus influenzae. J. Antimicrob. Chemother., 18,Suppl A:17–24.

24.

Murphy

T.F.

, Apicella

M.A.

1987. Nontypeable Haemophilus influenzae: a review of clinical aspects, surface antigens, and the human immune response to infection. Rev. Infect. Dis., 9:1–15.

25.

Okusu

, Nakamura

, Sawada

2000. Antibiotic resistance among recent clinical isolates of Haemophilus influenzae in Japanese children. Diagn. Microbiol. Infect. Dis., 36:249–254.

26.

Parr

T.R.

Jr. , Bryan

L.E.

1984. Mechanism of resistance of an ampicillin-resistant, β-lactamase-negative clinical isolate of Haemophilus influenzae type b to β-lactam antibiotics. Antimicrob. Agents Chemother., 25:747–753.

27.

Pettigrew

M.M.

, Foxman

, Ecevit

, Marrs

C.F.

, Gilsdorf

2002. Use of pulsed-field gel electrophoresis, enterobacterial repetitive intergenic consensus typing, and automated ribotyping to assess genomic variability among strains of nontypeable Haemophilus influenzae. J. Clin. Microbiol., 40:660–662.

28.

Sanbongi

, Suzuki

, Osaki

, Senju

, Ida

, Ubukata

2006. Molecular evolution of β-lactam-resistant Haemophilus influenzae: 9-year surveillance of penicillin-binding protein 3 mutations in isolates from Japan. Antimicrob. Agents Chemother., 50:2487–2492.

29.

De Schutter

, de Wachter

, Crokaert

, Verhaegen

, Soetens

, Piérard

, Malfroot

2011. Microbiology of bronchoalveolar lavage fluid in children with acute nonresponding or recurrent community acquired pneumonia: identification of nontypeable Haemophilus influenza as a major pathogen. Clin. Infect. Dis., 52:1437–1444.

30.

Scriver

S.R.

, Walmsley

S.L.

, Kau

C.L.

, Hoban

D.J.

, Brunton

, McGeer

, Moore

T.C.

, Witwicki

the Canadian Haemophilus Study Group. Low

D.E.

1994. Determination of antimicrobial susceptibilities of Canadian isolates of Haemophilus influenzae and characterization of their β-lactamases. Antimicrob. Agents Chemother., 38:1678–1680.

31.

Tamura

, Dudley

, Nei

, Kumar

2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol., 24:1596–1599.

32.

Tarasi

, D'Ambrosio

, Perrone

, Pantosti

1998. Susceptibility and genetic relatedness of invasive Haemophilus influenzae type b in Italy. Microb. Drug Resist., 4:301–306.

33.

Tenover

F.C.

, Huang

M.B.

, Rasheed

J.K.

, Persing

D. H.

1994. Development of PCR assays to detect ampicillin resistance genes in cerebrospinal fluid samples containing Haemophilus influenzae. J. Clin. Microbiol., 32:2729–2737.

34.

Tristram

, Jacobs

M.R.

, Appelbaum

P.C.

2007. Antimicrobial resistance in Haemophilus influenzae. Clin. Microbiol. Rev., 20:368–389.

35.

Ubukata

, Shibasaki

, Yamamoto

, Chiba

, Hasegawa

, Takeuchi

, Sunakawa

, Inoue

, Konno

2001. Association of amino acid substitutions in penicillin-binding protein 3 with β-lactam resistance in β-lactamase-negative ampicillin-resistant Haemophilus influenzae. Antimicrob. Agents. Chemother., 45:1693–1699.

36.

Watanabe

, Hoshino

, Sugita

, Asoh

, Watanabe

, Oishi

, Nagatake

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., 42:362–365.