Interspecies Transfer of the Penicillin-Binding Protein 3-Encoding Gene ftsI between Haemophilus influenzae and Haemophilus haemolyticus Can Confer Reduced Susceptibility to β-Lactam Antimicrobial Agents

Abstract

Mutations in ftsI, encoding penicillin-binding protein 3, can cause decreased β-lactam susceptibility in Haemophilus influenzae. Sequencing of ftsI from clinical strains has indicated interspecies recombination of ftsI between H. influenzae and Haemophilus haemolyticus. This study documented apparently unrestricted homologous recombination of ftsI between H. influenzae and H. haemolyticus in vitro. Transfer of ftsI from resistant isolates conferred similar but not identical increases in the MICs of susceptible strains of H. influenzae and H. haemolyticus.

TEXT

Haemophilus influenzae is the major human pathogen of the genus Haemophilus (1, 2), and infections are usually treated with β-lactam antimicrobial agents. Amino acid substitutions in penicillin-binding protein 3 (PBP3), encoded by the ftsI gene, can confer decreased susceptibility to β-lactams in strains lacking β-lactamase genes (3, 4). Genetically defined β-lactamase-negative ampicillin-resistant (gBLNAR) isolates carry either the N526K (Ubukata group II) or the R517H substitution in PBP3, while the additional substitutions S385T and/or L389F (Ubukata group III) further reduce susceptibility (3, 5–7). A range of other amino acid substitutions has been identified, but their significance is unclear (3–5, 8, 9).

Whether the worldwide spread of gBLNAR isolates is caused by clonal dissemination or horizontal gene transfer is controversial (8, 10, 11). H. influenzae and Haemophilus haemolyticus are close relatives, (1) and putative transfer of ftsI between these two species is indicated by mosaic structures of ftsI from clinical and nasopharyngeal carriage isolates (12, 13).

(Part of these data was presented on a poster at the 2014 International Pasteurellaceae Conference, Prato, Italy, 15 May 2014.)

The present study was undertaken to examine putative species barriers and delineate transformation events after ftsI transfer under standardized in vitro conditions. ftsI genes (1,833 bp) plus flanking regions from two gBLNAR H. influenzae strains and two gBLNAR H. haemolyticus strains were amplified by PCR (primers used for amplification and sequencing are listed in Table S1 in the supplemental material) and used for electroporetic transformation of susceptible strains of H. influenzae and H. haemolyticus (one representative each) as previously described (3). Characteristics of the donor and recipient strains are listed in Table 1, and overall similarity of ftsI and PBP3 are given in Table S2 in the supplemental material. Transformants were selected on chocolate agar (Columbia agar [Oxoid] supplemented with 5% horse blood and Vitox [Oxoid]) containing 0.5 μg/ml ampicillin (AMP) and screened for the presence of the N526K substitution using real-time PCR as previously described (14). We observed intraspecies ftsI transformation frequencies between 4.2 × 10⁻⁷ and 6.7 × 10⁻⁷ with H. influenzae strain Rd as the recipient and between 7 × 10⁻⁵ and 1 × 10⁻⁴ with H. haemolyticus strain ATCC 33390^T as the recipient; we observed interspecies ftsI transformation frequencies between 3.3 × 10⁻⁷ and 1.7 × 10⁻⁶ with H. influenzae strain Rd as the recipient and between 1.6 × 10⁻⁵ and 2.4 × 10⁻⁵ with H. haemolyticus strain ATCC 33390^T as the recipient. Thus, we observed no obvious difference in transformation frequencies of ftsI between strains of the same species and strains of separate species, although transformation occurred more frequently in H. haemolyticus recipient strain ATCC 33390^T than in H. influenzae recipient strain Rd.

TABLE 1.

Bacterial strains used in this study

Strain	Species	Groupa	Amino acid at positionb:																			MIC rangec (μg/ml) of:		Reference or source
			273	274	344	350	352	355	356	357	377	385	389	449	502	526	547	554	561	562	569	AMP	CTX
Recipient
Rd (KW20)	H. influenzae		S	E	K	D	T	K	V	S	M	S	L	I	A	N	V	A	A	V	N	0.125–0.19	0.008–0.016	18
ATCC 33390T	H. haemolyticus			D	R	N	G	T	V		I						I				S	0.19–0.25	0.012–0.016	19
Donor
ATCC 49247	H. influenzae	II	A													K	I	T	E		S	3	0.19–0.25	19
UTAS252	H. influenzae	III				N				N	I	T	F			K	I			L	S	0.5–1	0.5–1	This study
L23	H. haemolyticus	II				N					I				V	K	I				S	1.5–2	0.094–0.125	20
L48	H. haemolyticus	II		D	R	N	G	T	V		I			V		K	I				S	1–1.5	0.012–0.023	20

^aftsI mutation classification according to Ubukata et al. (3).

^bAmino acid (aa) positions of substitutions in the transpeptidase region (aa 265 to 580 of PBP3) relative to H. influenzae strain Rd (3). The critical mutations for group classification are in bold.

^cMIC range from two to four independent measurements.

Recombination events were delineated by sequencing the ftsI gene in five transformants of each recipient-donor combination. Mosaic structures of ftsI (Fig. 1) with sequence identities of >99.6% to the donor DNA in these segments documented homologous recombination in all 40 transformants. Recombination sites varied between recombinants of the same experiment and between different combinations of donor and recipient cells. Recombined fragments tended to be larger in intraspecies recombinations. When H. influenzae strain Rd was transformed with ftsI from H. influenzae strain ATCC 49247, all sequenced recombinants harbored almost the entire ftsI open reading frame (ORF) (1,833 nucleotides [nt]) of strain ATCC 49247 (Fig. 1A), while the recombined fragment varied from <600 to >1,500 nt when H. haemolyticus strain ATCC 33390^T was transformed with the same DNA (Fig. 1B); however, even the smallest of these fragments encoded five (N526K, V547I, A554T, A561E, and N569S) of the six amino acid substitutions in the transpeptidase domain of PBP3 (see Fig. S1B in the supplemental material). The difference in size of the recombined fragments between intraspecies and interspecies recombinations was less prominent for other donor-recipient combinations (Fig. 1C to F). β-Lactams target the transpeptidase region of PBP3 that is encoded by nt 796 to 1,741 of ftsI (3). The entire transpeptidase region of the donor strain was present in almost all of the recombinants (Fig. 1).

FIG 1.

Schematic representation of single nucleotide polymorphisms (vertical lines) in donor H. influenzae (D_Hi) or donor H. haemolyticus (D_Hh) and recombinant strains relative to recipient strains (R_Hi or R_Hh). Left lane (A, C, E, G), intraspecies gene transfer; right lane (B, D, F, H), interspecies gene transfer. (A and B) H. influenzae strain ATCC 49247 donor; (C and D) H. influenzae strain UTAS252 donor; (E and F) H. haemolyticus strain L23 donor; (G and H) H. haemolyticus strain L48 donor. (A, C, F, and H) H. influenzae strain Rd recipient; (B, D, E, and G) H. haemolyticus strain ATCC 33390^T recipient. White region, ftsI ORF; gray region, flanking regions. Numbers are relative to the first nucleotide of the ftsI ORF.

A previous comparison of ftsI sequences from clinical and surveillance strains of H. influenzae and H. haemolyticus clustered mosaic fragments of the gene into distinct groups of recombination profiles, indicating a preference for specific recombination events (13). In that analysis, there was no indication of horizontal transfer of the entire ORF of ftsI despite H. influenzae being capable of specific uptake and homologous recombination of segments in excess of 10 kb (15). In this in vitro study, we observed a wide variation in the size of recombined fragments, and for 8 of 40 recombinants, the entire ORF of ftsI was replaced. Moreover, the size and position of inserted fragments did not cluster into distinct groups of recombination profiles. The reason for these differences is not clear. We used electroporation to introduce DNA into recipient cells; hence, the recombined fragments did not depend on the specific uptake of genomic DNA fragments carrying the DNA uptake signal sequences that facilitates transformation in vivo (16). Also, we have no indication of the fitness of our recombinants when exposed to the selective forces of a commensal lifestyle. On the other hand, the apparent high frequency of interspecies recombination of ftsI in vivo (13) indicates events that may occur on multiple occasions, thereby blurring the origin of separate regions of the gene.

Ampicillin and cefotaxime (CTX) MICs were assessed using Etest (bioMérieux) on Mueller-Hinton agar (Oxoid) with 5% horse blood and HTM supplement (Oxoid). The results showed that transfer of segments of ftsI from gBLNAR strains conferred a rise in the AMP and CTX MICs for all recombinants (Table 2; see also Table S3A and B in the supplemental material). The increase in the AMP MIC was modest (3-fold to 6-fold increase) except when H. haemolyticus strain ATCC 33390^T was transformed with the Ubukata group III ftsI gene from H. influenzae UTAS252; this transfer increased the AMP MIC of H. haemolyticus ATCC 33390^T recombinants profoundly (13-fold), even surpassing the AMP MIC of the donor strain. The largest change in CTX susceptibility was also observed after transfer of the ftsI gene from strain UTAS252, which increased the CTX MICs of H. influenzae and H. haemolyticus recombinants more than 50 times (Table 2). Transfer of the Ubukata group II ftsI gene from H. influenzae strain ATCC 49247 ftsI altered the susceptibility of H. haemolyticus recombinants to a larger extent than that of the H. influenzae recombinants (6-fold and 9-fold versus 3-fold and 5-fold, respectively). Thus, ftsI from the same donor may alter the antimicrobial susceptibilities of the two species differently. The exceptionally high AMP MIC of the H. influenzae BLNAR reference strain ATCC 49247 was only partially transferred by horizontal transfer of ftsI (Table 2). The additional, non-PBP3 and non-β-lactamase resistance mechanisms have not been clearly identified (4, 17).

TABLE 2.

MICs of AMP and CTX for ftsI recombinants

Groupa	Recombinant		AMP MIC (μg/ml)			CTX MIC (μg/ml)
	Donor	Recipient	GMb	Range	Fold change	GM	Range	Fold change
A	ATCC 49247	H. influenzae strain Rd	0.47	0.25–0.75	3	0.056	0.032–0.094	5
B	ATCC 49247	H. haemolyticus strain ATCC 33390T	1.35	1–2	6	0.118	0.064–0.19	9
C	UTAS252	H. influenzae strain Rd	0.66	0.5–0.75	4	0.596	0.38–0.75	53
D	UTAS252	H. haemolyticus strain ATCC 33390T	2.86	1.5–4	13	0.772	0.75–1	56
E	L23	H. haemolyticus strain ATCC 33390T	1.11	0.5–2	5	0.047	0.012–0.094	3
F	L23	H. influenzae strain Rd	0.53	0.25–1	3	0.034	0.012–0.064	3
G	L48	H. haemolyticus strain ATCC 33390T	0.89	0.38–1.5	4	0.023	0.012–0.032	2
H	L48	H. influenzae strain Rd	0.49	0.25–0.75	3	0.023	0.008–0.047	2

^aLetters relate to recombinant groups depicted in Fig. 1.

^bGM, geometric mean of five sequenced transformants, each determined twice in separate experiments.

The present study unambiguously documents intraspecies and interspecies recombination of ftsI in H. influenzae and H. haemolyticus in vitro, resulting in mosaic structures of the gene. Unexpectedly, the interspecies recombination of ftsI appeared relatively unaffected by the sequence divergence between the two species. However, phenotypic expression of the same ftsI gene may differ between H. haemolyticus and H. influenzae.