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Antimicrobial Agents and Chemotherapy, July 2007, p. 2564-2573, Vol. 51, No. 7
0066-4804/07/$08.00+0     doi:10.1128/AAC.00354-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Ampicillin-Resistant Non-β-Lactamase-Producing Haemophilus influenzae in Spain: Recent Emergence of Clonal Isolates with Increased Resistance to Cefotaxime and Cefixime{triangledown}

Silvia García-Cobos,1 José Campos,1,2* Edurne Lázaro,3 Federico Román,1 Emilia Cercenado,4 César García-Rey,5 María Pérez-Vázquez,1 Jesús Oteo,1 and Francisco de Abajo3

Antibiotic Laboratory, Bacteriology Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain,1 Consejo Superior de Investigaciones Científicas, Madrid, Spain,2 División de Farmacovigilancia, Agencia Española del Medicamento, Madrid, Spain,3 Microbiology Department, Hospital Gregorio Marañón, Madrid, Spain,4 GlaxoSmithKline S.A., Madrid, Spain5

Received 16 March 2007/ Returned for modification 7 April 2007/ Accepted 20 April 2007


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ABSTRACT
 
The sequence of the ftsI gene encoding the transpeptidase domain of penicillin-binding protein 3 (PBP 3) was determined for 354 nonconsecutive Haemophilus influenzae isolates from Spain; 17.8% of them were ampicillin susceptible, 56% were β-lactamase nonproducing ampicillin resistant (BLNAR), 15.8% were β-lactamase producers and ampicillin resistant, and 10.4% displayed both resistance mechanisms. The ftsI gene sequences had 28 different mutation patterns and amino acid substitutions at 23 positions. Some 93.2% of the BLNAR strains had amino acid substitutions at the Lys-Thr-Gly (KTG) motif, the two most common being Asn526 to Lys (83.9%) and Arg517 to His (9.3%). Amino acid substitutions at positions 377, 385, and 389, which conferred cefotaxime and cefixime MICs 10 to 60 times higher than those of susceptible strains, were found for the first time in Europe. In 72 isolates for which the repressor acrR gene of the AcrAB efflux pump was sequenced, numerous amino acid substitutions were found. Eight isolates with ampicillin MICs of 0.25 to 2 µg/ml showed changes that predicted the early termination of the acrR reading frame. Pulsed-field gel electrophoresis analysis demonstrated that most BLNAR strains were genetically diverse, although clonal dissemination was detected in a group of isolates presenting with increased resistance to cefotaxime and cefixime. Background antibiotic use at the community level revealed a marked trend toward increased amoxicillin-clavulanic acid consumption. BLNAR H. influenzae strains have arisen by vertical and horizontal spread and have evolved to adapt rapidly to the increased selective pressures posed by the use of oral penicillins and cephalosporins.


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INTRODUCTION
 
Haemophilus influenzae is commonly involved in community-acquired respiratory infections in both children and adults. The resistance of this microbe to β-lactam antibiotics can have important clinical implications. Two primary mechanisms are implicated in the resistance of H. influenzae to aminopenicillins: enzymatic hydrolysis of the antibiotic and alterations in penicillin-binding proteins (PBPs). Enzymatic hydrolysis is the most common mechanism and results from the production of β-lactamases, usually of the TEM-1 type and sometimes of the ROB-1 type (8).

The β-lactam resistance mechanism due to amino acid substitutions in PBPs is known as the β-lactamase-nonproducing ampicillin resistance (BLNAR) mechanism. The BLNAR phenotype was initially reported in the 1980s in type b capsulated (24, 30) and noncapsulated (2, 28) strains. Substitutions, particularly in PBP 3, which mediates septal peptidoglycan synthesis, produce a decreased affinity of PBPs for β-lactam antibiotics (36). In addition, β-lactamase production and changes in PBPs have been reported simultaneously in clinical isolates. Such strains are termed β-lactamase-producing amoxicillin-clavulanic acid-resistant (BLPACR) strains (18, 25, 35).

In the United States, a prevalence of BLNAR strains of <5% has been reported in multicenter studies (9, 21). Although the presence of BLNAR strains with high ampicillin resistance levels (MICs, >4 µg/ml) has been documented, these strains required amino acid mutations in the acrR gene, which codes for the repressor of the AcrAB efflux pump, in addition to the PBP 3 modifications (20).

A recent European study suggested that the resistance of H. influenzae to amoxicillin might be decreasing due to a reduction in the number of β-lactamase-positive ampicillin-resistant (BLPAR) strains, whereas the prevalence of the BLNAR phenotype remains relatively constant (19). In Japan, a rapid increase in the number of BLNAR isolates has been observed (32).

Spain used to report high levels of antibiotic resistance in H. influenzae (3, 4). Three multicenter studies (the Susceptibility to the Antimicrobials Used in the Community in Spain [SAUCE] studies) have been carried out to evaluate the antibiotic resistance rates among respiratory pathogens, including H. influenzae (13, 23, 31). One of the most remarkable findings was the increasing prevalence of isolates belonging to the BLNAR phenotype. However, neither the molecular basis of the Spanish BLNAR strains nor their molecular epidemiology has been characterized so far.

Accordingly, the aims of this study were (i) to describe the mutation patterns in the sequence of the ftsI gene, which encodes the transpeptidase domain of PBP 3, among Spanish isolates; (ii) to determine the molecular epidemiology of BLNAR H. influenzae strains in Spain; (iii) to determine whether or not susceptibility to oral cephalosporins in H. influenzae is affected by circulating BLNAR phenotypes; and (iv) to ascertain the patterns and evolution of community β-lactam antibiotic consumption in connection with the development of the BLNAR H. influenzae phenotype in Spain.


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MATERIALS AND METHODS
 
Clinical isolates. A total of 354 clinical isolates of H. influenzae isolated from children (26%) and adults (69.5%) were studied; for 4.5% of the isolates the age of the patient was not known. A total of 214 (8.09%) isolates were selected from the SAUCE 3 multicenter study, which was carried out in 25 Spanish hospitals from 2001 to 2002 and which included a total of 2,645 isolates (31). In the SAUCE study, the proportion of β-lactamase-negative isolates with ampicillin MICs ≥1 µg/ml was 22.2%, while 20% were β-lactamase positive (31). The study collection was completed with 140 additional isolates selected from the national Haemophilus reference laboratory, which had received the isolates from 21 Spanish clinical laboratories (2004 to 2006).

Three major criteria were followed for the initial selection of the study collection, either from the SAUCE 3 study or from the Haemophilus reference laboratory collections. First, all geographical regions of Spain (which has about 45 million inhabitants) were represented; second, almost all MIC intervals for initial susceptibility to ampicillin and/or amoxicillin and oral cephalosporins were represented, although special emphasis was given to isolates suggestive of the BLNAR phenotype in such a way that the final selection did not represent sequential isolates; and, finally, the isolates selected represented isolates recovered over a long period of time, from 2001 to 2006.

In total, 23 isolates (6.5%) in the study collection were isolated from cerebrospinal fluid, blood, and pleural fluid; and the remaining 331 were from community-acquired respiratory tract infections. All strains were identified according to standard microbiological methods (6). Antibiotic susceptibility testing was carried out according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (7). All strains were serotyped by a coagglutination test (Phadebact Haemophilus test; Boule Diagnostics AB, Huddinge, Sweden), and the serotypes were further confirmed by a PCR reference method (10).

H. influenzae ATCC 49247 was used as a control for the susceptibility testing procedures, as recommended by the CLSI (7). H. influenzae Rd KW20, a strain whose whole genome has been sequenced (11), was used as a control for the molecular procedures.

Antimicrobial susceptibility testing. Antibiotic susceptibility was determined by broth microdilution and disk diffusion susceptibility testing in Haemophilus test medium, according to the CLSI guidelines (7). Other susceptibility testing procedures used were the disk diffusion method, Etest (AB-Biodisk, Solna, Sweden), and the low-charge disk diffusion method (7). The antibiotics evaluated were amoxicillin, amoxicillin-clavulanic acid (2:1 ratio), cefaclor, cefuroxime, cefotaxime, and cefixime.

β-Lactamase production was determined by the chromogenic cephalosporin test with nitrocefin as the substrate (29).

PCR and DNA sequencing. Following extraction of the genetic material, the specific amplification of ftsI from nucleotide positions 936 to 1640 bp, corresponding to amino acid positions 327 to 540, was determined by using the primers and PCR conditions described previously (8).

To establish whether the increased efflux of ampicillin may play a role in conferring high ampicillin MICs in selected BLNAR strains, the coding sequence of the acrR gene from nucleotides 1 to 564 (amino acids 1 to 188) was determined and compared with the H. influenzae Rd KW20 coding sequence by using the primers and PCR conditions described by Kaczmarek et al. (20). AcrR sequences were amplified from isolates with ampicillin MICs of 4 µg/ml (n = 2), 2 µg/ml (n = 31), 1 µg/ml (n = 19), and ≤0.5 µg/ml (n = 20).

PCRs were performed in a final volume of 25 µl with PureTaq Ready-To-Go PCR beads (Amersham Biosciences) containing 5 µl of DNA template, 10 mM Tris-HCl (pH 9), 50 mM KCl, 1.5 mM MgCl2, 1 µM each primer, 200 µM each deoxynucleoside triphosphate, and 2.5 U of Taq polymerase. The PCR products were visualized by 1% agarose gel electrophoresis and ethidium bromide staining, and all PCR products were purified with a PCR purification kit (QIAGEN, Inc.) and were subjected to automated DNA sequencing.

The purified products were sequenced with an Applied Biosystems BigDye Terminator (version 3.1) cycle sequencing kit (Perkin-Elmer, Warrington, United Kingdom), according to the instructions of the manufacturer. The products were resolved and analyzed with an ABI PRISM 377 DNA sequencer. The nucleotide sequences were analyzed with DNAstar (Madison, WI) software. Strain Rd KW20 (11) was used as a reference in the sequence alignment procedures.

Phenotype definition. On the basis of the sequencing results and β-lactamase determinations, we classified the H. influenzae strains into four main classes: β-lactamase-nonproducing ampicillin-susceptible (BLNAS) strains that had no resistance mechanism, BLNAR strains that had mutations in the ftsI gene, BLPAR strains that had no ftsI amino acid substitutions but that were β-lactamase producers, and BLPACR strains that had both mechanisms (β-lactamase production and amino acid substitutions in the ftsI gene) (22).

Molecular epidemiology studies. The molecular epidemiology of the H. influenzae isolates was determined by pulsed-field gel electrophoresis (PFGE), as described previously (1, 5). Total bacterial DNA was digested with SmaI (MBI Fermentas, Vilnius, Lithuania), and the restriction fragments were separated by PFGE on a 1% agarose gel in 0.5x Tris-borate-EDTA buffer with a CHEF Mapper apparatus (Bio-Rad, Madrid, Spain). The initial pulse time of 5 s was increased linearly to 50 s over 23.3 h at 6 V/cm at 13°C. Bacteriophage {lambda} ladders were applied as molecular size markers (48.5 to 1,000 kb; PFGE marker I; Boehringer-Mannheim, Mannheim, Germany). The gels were then stained with ethidium bromide and photographed under UV light. A genetic similarity dendrogram was obtained according to the Dice correlation coefficient with a tolerance level of 1% (Fingerprinting II software; Bio-Rad). Well-resolved bands, corresponding to fragments exceeding 48.5 kb, were included in the computer analysis. PFGE patterns with coefficients of similarity greater than 85% were considered to define a particular clone.

The PFGE study included 71 isolates representing each one of the mutation patterns found in the ftsI gene, as follows: 44 isolates for subgroup IIc (30 isolates with the Asp350Asn, Ala502Thr, and Asn526Lys substitutions and 14 with a mutation pattern composed of Ala502Thr and Asn526Lys), 17 isolates for subgroup IIb (Asp350Asn, Met377Ile, Ala502Val, and Asn526Lys), and 10 isolates belonging to subgroup III-like (described below). All these isolates were nontypeable. Reference strain ATCC 49247 was studied in parallel.

Antibiotic consumption. The Spanish Ministry of Health and Consumer Affairs maintains a drug database with a packet-by-packet record of all retail pharmacy sales of medications acquired with National Health System prescriptions. This database was used to gather information on β-lactam antibiotic sales in Spain over the period from 2000 to 2004. The information was tabulated, and the number of units sold was converted into defined daily doses (DDDs) of active drug ingredients, according to the World Health Organization methodology (37). The number of DDDs per 1,000 inhabitants per day for each of the active drug ingredients was then calculated.


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RESULTS
 
H. influenzae susceptibility in relation to resistance phenotype. In total, 349 isolates (98.6%) were nontypeable (noncapsulated) and 5 (1.4%) were typeable (3 isolates were type b, 1 was type e, and 1 was type f). The collection was grouped into the following classes according to their resistance patterns: BLNAS strains (n = 63; 17.8%), BLNAR strains (n = 198; 56%), BLPAR strains (n = 56; 15.8%), and BLPACR strains (n = 37; 10.4%). Of the BLNAR strains, 196 (98.5%) were nontypeable and 2 were capsulated (1 was type e and 1 was type f). Of 23 invasive isolates, 14 (60.9%) were BLNAR and 3 (13.0%) were BLPACR. Table 1 shows the activities of five β-lactam antibiotics against the 354 H. influenzae isolates studied.


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  		TABLE 1. Susceptibilities to five β-lactam antibiotics of H. influenzae strains, according to resistance class

The MIC90s of ampicillin, amoxicillin-clavulanic acid, cefaclor, cefuroxime, cefotaxime, and cefixime for the BLNAR strains were about fourfold higher than those for the BLNAS strains (Table 1). Approximately half of the BLNAR strains had ampicillin MICs less than 1.0 µg/ml.

Mutation patterns in ftsI gene. Of the 354 isolates, 236 (66.7%) had amino acid substitutions in the ftsI gene, corresponding to 28 different mutation patterns (Table 2). When possible, we followed the BLNAR classifications proposed by Dabernat et al. (8) and Ubukata et al. (36). Of the 236 isolates with mutations, 93.2% had amino acid substitutions at the Lys-Thr-Gly (KTG) motif, the two most common being Asn526 to Lys (83.9%) and Arg517 to His (9.3%), analogous to previously described groups II and I, respectively (36) (Table 2). These particular changes appeared to be mutually exclusive, as they were not simultaneously present in any of the isolates studied.


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  		TABLE 2. Amino acid substitutions identified in the ftsI gene of 355 H. influenzae strains

Ten BLNAR strains (4.2% of the total BLNAR strains) could be classified as group I and displayed the Arg517His change alone (seven isolates) or in combination with other amino acid substitutions (three isolates) (Table 2). Group II (197 isolates, 83.5%) could be further subdivided, according to previous proposals (8), into four subgroups. Twenty-one isolates belonged to subgroup IIa, and 11 of these isolates (including control strain ATCC 49247) had the Asn526Lys substitution alone, while 10 displayed other mutations as well (Table 2). Subgroup IIb (53 isolates) was characterized by the substitution Ala502Val, usually combined with the substitutions Asp350Asn and/or Gly490Glu. Subgroup IIc (defined by the Ala502Thr substitution) was the most common, as it contained 112 of the 236 BLNAR strains (47.4%). Finally, subgroup IId, defined by the Ile449Val substitution, had 12 isolates.

We observed an additional mutation pattern in 12 BLNAR strains (5%) characterized by the following amino acid substitutions: Met377Ile, Ser385Thr, and/or Leu389Phe in the Ser-Ser-Asn (SSN) motif; Arg517His and Thr532Ser in the KTG motif; Asp350Asn in the Ser-Thr-Val-Lys (STVK) motif; and finally, Ser357Asn (Table 2). We named the last group the "III-like group" because it had the amino acid changes described by Ubukata et al. (36) at the SSN motif (and defined as group III by those authors) but had additional combinations at the KTG and STVK motifs. Most BLNAR strains in this group were isolated recently (2005 and 2006).

An additional miscellaneous group, consisting of six low-prevalence patterns, was characterized by the following changes: Ile348Val (one isolate), Ala368Thr (two isolates), Asp350Asn (eight isolates), Met391Ile (one isolate), Ala502Ser (one isolate), and Ala502Thr (three isolates) (Table 2).

The β-lactam susceptibilities of the isolates according to their ftsI mutation patterns are depicted in Table 3. All groups gave similar MIC90s for ampicillin and amoxicillin-clavulanic acid (about 2 to 4 µg/ml), cefaclor (8 to 16 µg/ml), and cefuroxime (2 to 4 µg/ml). However, important differences were observed with cefotaxime and cefixime, as the so-called III-like group had MIC90s of 4.0 µg/ml in both cases, while for the remaining groups, the MIC90s were 0.12 to 0.25 µg/ml.


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  		TABLE 3. β-Lactam antibiotic susceptibilities of H. influenzae isolates according to ftsI gene mutations patternsa

Sequence analysis of acrR of the AcrAB efflux pump. In relation to the sequence of the reference strain, strain Rd KW20, all 72 isolates from which we amplified the acrR regulatory gene (including 34 with ampicillin MICs ≤1 µg/ml) had amino acid substitutions that affected 21 different positions. The isolates (including control strain ATCC 49247) could be classified into 18 groups on the basis of their acrR mutation patterns (Table 4).


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  		TABLE 4. Amino acid substitutions identified in acrR gene from 72 strains of H. influenzae strains according to their ampicillin MICs

In addition to the amino acid substitutions described above, 8 of the 72 isolates studied (11.1%) had changes in the acrR sequence that predicted the early termination of the acrR reading frame (Table 5). Four of those isolates presented changes that caused a frame shift, three (with ampicillin MICs of 2, 1, and 0.5 µg/ml, respectively) had a single nucleotide insertion, and one isolate (ampicillin MIC, 1 µg/ml) had a 3-base deletion that caused the disappearance of an amino acid. Four additional isolates (one of them with an ampicillin MIC of 2 µg/ml and the other three with ampicillin MICs of 0.5 µg/ml) presented a single nucleotide change (T->G) that generated an early stop codon (Table 5).


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  		TABLE 5. Modifications found in the sequence of the acrR gene in a collection of 72 strains of Haemophilus influenzae

PFGE profiles. The 72 BLNAR strains were grouped into 57 different PFGE profiles (Fig. 1). Most profiles contained individual isolates. However, 13 patterns emerged that contained two to four genetically related isolates (>85% identity) (Fig. 1).


Figure 1
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  		FIG. 1. Dendrogram displaying the genetic relatedness of 72 BLNAR isolates of H. influenzae obtained by PFGE according to Dice's similarity index. Strains are identified on the right column, and the BLNAR subtypes are described in Table 2.

The 44 isolates of ftsI subgroup IIc were distributed into 36 PFGE profiles, the 17 isolates of subgroup IIb were distributed into 16 profiles, and the 10 isolates of the III-like group were distributed into 5 profiles.

PFGE pattern 1 consisted of four isolates (isolates 51221, 51179, 51156, and 2299). The first three isolates were collected from three children with conjunctivitis, from the same hospital, and within a 1-month period. The last isolate was isolated 3 years earlier from a bronchial aspirate and from a geographic area 300 km away from where the first three isolates were recovered (Fig. 1). All four isolates shared the III-like group amino acid substitution pattern (Asp350Asn, Ser357Asn, Met377Ile, Ser385Thr, Arg517His, Thr532Ser) and had cefixime MICs of 0.5 to 1.0 µg/ml and cefotaxime MICs of 0.5 µg/ml.

PFGE pattern 2 also included four identical isolates (isolates 50730, 51403, 51517, and 51105) (Fig. 1) that had the same III-like group mutation pattern in the ftsI gene and increased resistance to cefixime and cefotaxime. The first three isolates were isolated in the same hospital: strain 50730 was isolated from the blood of one adult patient in February 2005, isolate 51403 was from the conjunctiva of a child, and isolate 51517 was from a bronchial aspirate from an adult patient. A 1.3-year interval existed between the isolation of isolate 50730 and the isolation of isolate 51517. Isolate 51105 was isolated from the sputum of one adult in a geographical area 400 km away from where the first isolates were recovered. In addition, a difference of only three bands was observed between PFGE profiles 1 and 2, suggesting that all of these isolates could be related (33).

PFGE pattern 3 consisted of isolates 2108, 2166, 2111, and 3257. Isolates 2108, 2111, and 3257 were isolated in northern Spain from cases of otitis in children, while isolate 2166 was obtained from the bronchial aspirate of one adult in northwestern Spain. These four isolates had an identical substitution pattern in the ftsI gene (Asp350Asn, Ala502Thr, Asn526Lys) and similar MICs for ampicillin (2 µg/ml), amoxicillin-clavulanic acid (2 to 4 µg/ml), and cefixime (0.12 µg/ml).

Two isolates (isolates 1980 and 2557) collected from two adults with upper respiratory infections in the same hospital gave rise to PFGE pattern 4. PFGE pattern 5 consisted of two isolates (isolates 1876 and 2518) from the same hospital, and both were from adults with upper respiratory infections. The four isolates with patterns 4 and 5 were distantly related (Fig. 1), but they had identical ftsI mutations and similar β-lactam MICs.

Two β-lactamase-positive isolates (isolates 51027 and 51028) had pattern 6 and were isolated from two sisters with conjunctivitis. The two isolates (isolates 51165 and 51157) with pattern 7 came from the same hospital and were isolated on the same day from two children with conjunctivitis (Fig. 1). Again, the four isolates with patterns 6 and 7 were only distantly related (Fig. 1) but shared the same ftsI mutations and similar β-lactam MICs.

Isolates with two PFGE patterns, patterns 8 (isolates 133 and 1055) and 9 (isolates 3164 and 50343), presented the same PFGE profile, even though they came from separate geographical areas.

Finally, there were four PFGE patterns (Fig. 1, patterns 10 to 13) that contained two isolates each (isolates 583 and 143, pattern 10; isolates 50687 and 51337, pattern 13; isolates 503 and 50957, pattern 11; and isolates 51131 and 2671, pattern 12). The pairs of isolates with each pattern were isolated from the same geographic area but from different hospitals. In all cases, the isolates belonging to the same PFGE group presented the same ftsI mutation pattern. Isolates 50687 and 51337 had the highest cefixime and cefotaxime MICs of the collection (4 µg/ml).

Antibiotic consumption. The most commonly used Anatomical Therapeutic Chemical (ATC) classification group was JO1C, the consumption of which showed an increase of 5.8% in 5 years (Table 6). Although the rate of amoxicillin use decreased by 21.3%, the rate of use amoxicillin in combination with β-lactamase inhibitors increased by 37.8% (Table 6). The most frequently used cephalosporins were cefuroxime (6.5% reduction over 5 years), cefixime (16.2% reduction), cefaclor (73.4% reduction), and cefpodoxime (33.3% increase, with very low DDDs) (Table 6). Over the study period, the proportion of cephalosporin usage with respect to that for ATC group JO1C did not increase.


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  		TABLE 6. Antimicrobial consumption in Spain (2000 to 2004)


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DISCUSSION
 
We found that 56% of our H. influenzae isolates were BLNAR; however, this percentage refers to the 354 isolates studied and is not necessarily indicative of the entire population of Spanish H. influenzae isolates. According to pharmacokinetic/pharmacodynamic data (7, 31), only isolates with ampicillin or amoxicillin MICs ≤2 µg/ml should be considered fully susceptible to these two antibiotics. We found that all β-lactamase-negative isolates with ampicillin MICs ≥1 µg/ml had a modified PBP 3. Accordingly, precaution is advised when infections caused by clinical H. influenzae isolates with ampicillin or amoxicillin MICs of 1 to 2 µg/ml or higher are treated with ampicillin or amoxicillin, particularly in patients with invasive infections or in patients with impaired immunity.

In H. influenzae, β-lactam resistance due to changes in PBPs has been encountered in several countries (8, 19, 36). This is especially true regarding PBP 3, which is involved in septal peptidoglycan synthesis (26). A collection of French BLNAR isolates was classified into two main categories on the basis of the Arg517His and Asn526Lys mutations in the ftsI gene, which encodes the transpeptidase domain of PBP 3 (8). We have classified our BLNAR collection in the same way because the Arg517His and Asn526Lys mutations were also prevalent in Spanish isolates, with the Asn526Lys substitution by far being the most frequent (Table 2). We also found other PBP 3 changes of interest, mainly changes that affected the KTG motif.

Among Japanese BLNAR strains, the Arg517His substitution has decreased, whereas the frequency of the Asn526Lys substitution has remained constant (32). The Japanese study also found increases in the Met377Ile, Ser385Thr, and Leu389Phe substitutions in isolates collected between 2002 and 2003. Strains with these mutations exhibited ampicillin MICs >4 µg/ml and decreased susceptibility to cephalosporins (32).

To date, this unique class of BLNAR strains has not been observed in the United States, Europe, or Korea (12, 19, 22). However, 4% of our recent BLNAR strains had a ftsI mutation pattern that produced a remarkable increase in the rates of resistance to cefotaxime and cefixime. Interestingly, one group of 10 isolates displayed Asp350Asn, Ser357Asn, Met377Ile, Ser385Thr, Arg517His, and Thr532Ser substitutions and had cefotaxime MICs of 0.5 µg/ml and cefixime MICs of 0.5 to 1.0 µg/ml (about 10- to 20-fold higher than those for fully susceptible isolates). Two additional isolates with the same changes plus Leu389Phe (Table 2) had cefotaxime and cefixime MICs of 4 µg/ml (about 60-fold higher than those for susceptible isolates). It seems, then, that the initial step leading to resistance to cefotaxime and cefixime is achieved by the ftsI mutations Met377Ile and Ser385Thr. An additional Leu389Phe mutation further increases resistance. In our isolates with increased resistance to cephalosporins, ampicillin and amoxicillin MICs were between 0.5 and 2 µg/ml, which is lower than the MICs of >4 µg/ml reported for the Japanese strains (32).

It is important to note that the BLNAR group that displayed increased resistance to cephalosporins had some additional relevant features. Nearly all of them were isolated recently (in 2005 and 2006) and were more clonally related (Fig. 1) than the rest of the BLNAR collection of isolates. Additionally, most of them were restricted to specific geographical areas. These facts suggest their recent emergence and further clonal dissemination.

It has been shown (20) that BLNAR strains expressing high levels of resistance to ampicillin have insertions in the acrR regulatory region. We analyzed the sequence of the acrR repressor in strains with ampicillin MICs ranging from 0.25 to 4 µg/ml (Table 4). First, we found that the acrR gene is highly polymorphic in H. influenzae strains, as a large number of amino acid substitutions were observed independently of the ampicillin MICs. Additionally, we found these changes in both BLNAR and BLNAS strains displaying ampicillin and amoxicillin MICs ranging from 0.25 to 2.0 µg/ml (Table 5). Furthermore, none of our BLNAR strains presenting the highest ampicillin MICs (4.0 µg/ml) had changes that predicted an early termination of the acrR reading frame. These findings may indicate that in our BLNAR collection, multiple changes in or the absence of the acrR repressor may be unrelated to high levels of ampicillin resistance, in contrast to the findings presented in previous reports (20).

Similar to other studies, most Spanish BLNAR strains were genetically diverse (12, 14, 18, 27), suggesting that the BLNAR H. influenzae strains have independently appeared on many occasions in several countries, possibly due to the widespread use of antibiotics for the treatment of respiratory infections.

However, other epidemiological models of BLNAR dissemination were also possible, as we detected 13 PFGE clusters, each of which contained two to four BLNAR strains, and most of these strains were associated with otitis and conjunctivitis in children. The most remarkable clonal dissemination found was for the group of isolates in clusters 1 and 2, characterized by increased resistance to cefotaxime and cefixime.

The most common mechanism of β-lactam resistance in H. influenzae is production of the TEM-1 β-lactamase (3), which does not protect H. influenzae from amoxicillin-clavulanic acid or cefixime. Data from our study suggest that H. influenzae PBP 3 has experienced two major adaptive changes that confer enhanced antibiotic resistance. One change comprised amino acid substitutions at the KTG motif (amino acid positions Ala502, Arg517, and Asn526) (Table 2) that typically increased ampicillin and amoxicillin-clavulanic acid MICs about four times but that had little impact on resistance to the latest oral cephalosporins. The other change consisted of amino acid substitutions at the SSN motif (amino acids Met377, Ser385, and Leu389) that increased the cefixime and cefotaxime MICs from 10 to 60 times. For those isolates, ampicillin and amoxicillin-clavulanic acid MICs were similar to those for BLNAR groups I and II (Table 3). It is possible that our cephalosporin-resistant BLNAR group arose from BLNAR group I, characterized by the Arg517His substitution (Table 2).

Although there is a consensus (34) that antibiotic use and misuse are key factors in the development of antibiotic resistance, none of the previous studies on BLNAR H. influenzae has provided background data on β-lactam antibiotic consumption. This knowledge is important in several ways, as it can help to explain how BLNAR strains have appeared and evolved and how they could be controlled. In Spain, the most frequently used antibiotics in 2004 were amoxicillin-clavulanic acid, amoxicillin, cefuroxime, and cefixime (Table 6). However, the total oral cephalosporin use was about 1.80 DDDs in that year, which is between 2.2 and 3.6 times lower than the amoxicillin and amoxicillin-clavulanic acid use of 3.96 and 6.52 DDDs, respectively. Also, the use of amoxicillin-clavulanic acid greatly increased over the study period (Table 6).

Spain's antibiotic consumption pattern is similar to those reported from some European countries and the United States (15), but it might be quite different from the antibiotic use pattern in Japan, in which the high prevalence of BLNAR strains has been attributed to the widespread use of oral and intravenous cephem antibiotics (16, 22). Also, many Japanese BLNAR strains are H. influenzae type b (17), which could be easily prevented by vaccination; widespread anti-H. influenzae type b vaccination programs are in place in the majority of occidental countries, including Spain.

In summary, we have determined the genetic basis and the molecular epidemiology of a large collection of BLNAR H. influenzae strains mostly isolated from respiratory specimens. The most common BLNAR genotypes were well represented, but we observed for the first time in Europe the recent emergence of a new type of BLNAR phenotype characterized by increased levels of resistance to cefotaxime and cefixime. Although most BLNAR strains arose independently, we documented some clonal dissemination linked to conjunctivitis and otitis. It is likely that H. influenzae PBP 3 has undergone two major changes at the KTG and SSN motifs in response to the selective pressure engendered by the use of penicillins and cephalosporins.


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ACKNOWLEDGMENTS
 
This study was supported by research grants from the Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias (reference 04/0899), the REIPI Network (reference RD 06/0008/0023), the Network of Excellence GRACE (PL 518226), and by an unrestricted grant from GlaxoSmithKline S.A., Madrid, Spain. S. García-Cobos is a recipient of a fellowship from the Instituto de Salud Carlos III (reference 05/0033).


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FOOTNOTES
 
* Corresponding author. Mailing address: Centro Nacional de Microbiología, Instituto de Salud Carlos III, Carretera Pozuelo a Majadahonda, 28220 Majadahonda, Madrid, Spain. Phone: (34) 91-822-3650. Fax: (34) 91-509-7966. E-mail: jcampos{at}isciii.es Back

{triangledown} Published ahead of print on 30 April 2007. Back


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Antimicrobial Agents and Chemotherapy, July 2007, p. 2564-2573, Vol. 51, No. 7
0066-4804/07/$08.00+0     doi:10.1128/AAC.00354-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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