Previous studies with beta-lactamase-negative, ampicillin-resistant (BLNAR) Haemophilus influenzae from Japan, France, and North America indicate that mutations in ftsI encoding PBP3 confer ampicillin MICs of 1 to 4 μg/ml. Several BLNAR strains with ampicillin MICs of 4 to 16 μg/ml recently isolated from North America were studied. Pulsed-field gel electrophoresis identified 12 unique BLNAR strains; sequencing of their ftsI transpeptidase domains identified 1 group I and 11 group II mutants, as designated previously (K. Ubukata, Y. Shibasaki, K. Yamamoto, N. Chiba, K. Hasegawa, Y. Takeuchi, K. Sunakawa, M. Inoue, and M. Konno, Antimicrob. Agents Chemother. 45:1693-1699, 2001). Geometric mean ampicillin MICs for several clinical isolates were 8 to 10.56 μg/ml. Replacement of the ftsI gene in H. influenzae Rd with the intact ftsI from several clinical isolates resulted in integrants with typical BLNAR geometric mean ampicillin MICs of 1.7 to 2.2 μg/ml. Cloning and purification of His-tagged PBP3 from three clinical BLNAR strains showed significantly reduced Bocillin binding compared to that of PBP3 from strain Rd. Based on these data, changes in PBP3 alone could not account for the high ampicillin MICs observed for these BLNAR isolates. In an effort to determine the presence of additional mechanism(s) of ampicillin resistance, sequencing of the transpeptidase regions of pbp1a, -1b, and -2 was performed. While numerous changes were observed compared to the sequences from Rd, no consistent pattern correlating with high-level ampicillin resistance was apparent. Additional analysis of the resistant BLNAR strains revealed frame shift insertions in acrR for all four high-level, ampicillin-resistant isolates. acrR was intact for all eight low-level ampicillin-resistant and four ampicillin-susceptible strains tested. A knockout of acrB made in one clinical isolate (initial mean ampicillin MIC of 10.3 μg/ml) lowered the ampicillin MIC to 3.67 μg/ml, typical for BLNAR strains. These studies illustrate that BLNAR strains with high ampicillin MICs exist that have combined resistance mechanisms in PBP3 and in the AcrAB efflux pump.
A total of 395 Haemophilus influenzae strains from 226 Japanese institutions participating in the Nationwide Surveillance Study Group for Bacterial Meningitis were received from 1999 to 2002. All strains were analyzed by PCR to identify the resistance genes, and their susceptibilities to β-lactam agents were determined. Of these strains, 29.1% were β-lactamase nonproducing and ampicillin (AMP) susceptible (BLNAS) and lacked all resistance genes; 15.4% were β-lactamase producing and AMP resistant and had the blaTEM-1 gene; 30.6% were β-lactamase nonproducing and AMP resistant (low-BLNAR) and had a Lys-526 or His-517 amino acid substitution in ftsI encoding PBP 3; 13.9% were β-lactamase nonproducing and AMP resistant (BLNAR) and had an additional substitution of Thr-385 in ftsI; 9.1% were amoxicillin-clavulanic acid resistant (BLPACR I) and had the blaTEM-1 gene and a Lys-526 or His-517 amino acid substitution in ftsI; and 1.8% showed resistance similar to that of the BLPACR I group (BLPACR II) but had blaTEM-1 gene and ftsI substitutions, as was the case for the BLNAR strains. All but three strains were serotype b. The prevalence of BLNAR strains has increased rapidly: 0% in 1999, 5.8% in 2000, 14.1% in 2001, and 21.3% in 2002. The MICs at which 90% of BLNAR isolates were inhibited were as follows: AMP, 16 μg/ml; cefotaxime, 1 μg/ml; ceftriaxone, 0.25 μg/ml; and meropenem, 0.5 μg/ml. All of these values were higher than those for the BLNAS counterpart strains. The relatively wide distributions of the β-lactam MICs for BLNAR strains presumably reflect variations in ftsI gene mutations. Pulsed-field gel electrophoresis suggested the rapid spread of specific H. influenzae type b strains throughout Japan. Expedited vaccination, rapid identification, and judicious antibiotic use could slow their spread.
We evaluated the recent prevalence of antimicrobial-resistant Haemophilus influenzae isolated from the upper respiratory tracts (URT) of patients in Japan. Mutations in the ftsI gene, which encodes penicillin binding protein 3 (PBP3), and the clonal dissemination of the resistant strains were also investigated. A total of 264 H. influenzae isolates were collected from patients with URT infections. According to the criteria of the Clinical and Laboratory Standards Institute for the susceptibility of H. influenzae to ampicillin (AMP), the isolates were distributed as follows: 161 (61.0%) susceptible strains (MIC ≤ 1 μg/ml), 37 (14.0%) intermediately resistant strains (MIC = 2 μg/ml), and 66 (25.0%) resistant strains (MIC ≥ 4 μg/ml). According to PCR-based genotyping, 172 (65.1%) of the isolates had mutations in the ftsI gene and were negative for the β-lactamase (bla) gene. These 172 isolates were thus defined as genetically β-lactamase-negative ampicillin-resistant (gBLNAR) strains. The ftsI mutant group included 98 (37.1%) strains with group I/II mutations in the variable mutated region (group I/II gBLNAR) and 74 (28.0%) strains with group III mutations in the highly mutated region (group III gBLNAR). Eighty-seven (33.0%) of the isolates were genetically β-lactamase-negative ampicillin-susceptible (gBLNAS) strains. The group III gBLNAR strains showed resistance to β-lactams. Only five strains (1.9%) were positive for a bla gene encoding TEM-type β-lactamase. The three clusters consisting of 16 strains found among the 61 BLNAR strains (MIC ≥ 4 μg/ml and without the bla gene) showed identical or closely related DNA restriction fragment patterns. Those isolates were frequently identified among strains with a MIC to AMP of 16 μg/ml. The current study demonstrates the apparent dissemination and spread of a resistant clone of H. influenzae among medical centers in Japan. The gBLNAR strains show a remarkable prevalence among H. influenzae isolates, with the prevalence increasing with time. This fact should be taken into account when treating URT infections.
Pneumococcal and Haemophilus influenzae type b (Hib) vaccines were introduced in our national immunisation program in April 2010. The aims of this retrospective, laboratory-based study were to determine the serotypes and antibiotic resistance of Streptococcus pneumoniae and H. influenzae isolates from middle ear fluid (MEF) collected before the introduction of immunization.
S. pneumoniae (n = 128) and H. influenzae (n = 40) strains isolated from MEF of children with AOM between 1994 and 2011 were studied. MICs were determined by a microdilution assay. Serotyping of S. pneumoniae was done by Quellung method and PCR capsular typing was used for H. influenzae. Macrolide resistance genes were detected by PCR for erythromycin resistant S. pneumoniae (ERSP). DNA sequencing of ftsI gene was performed for ampicillin nonsusceptible H. influenzae.
The most common serotypes found among children with pneumococcal AOM were 19 F (20.3%), 6B (15.6%), and 19A (10.9%). The potential coverage rates by the PCV7, PCV10 and PCV13 of children aged < 5 years were 63.6%, 66.4% and 85.5%, respectively. Reduced susceptibility to oral penicillin was seen in 68.1%; resistance to erythromycin was 46.9%. We found erm(B) gene in 56.7% of the ERSP, mef(E) gene in 25%; 15% harbored both genes erm(B) + mef(E) and 3.3% had mutations of L4 ribosomal protein. Of the 40 H. influenzae isolates 97.5% were nontypeable. Nonsusceptibility to ampicillin occurred in 25%. Ampicillin resistance groups were: β-lactamase-positive ampicillin resistant (BLPAR) strains (10%), β-lactamase-negative ampicillin resistant (BLNAR) strains (12.5%) and β-lactamase-positive amoxicillin-clavulanate resistant (BLPACR) strains (2.5%). Among BLNAR and BLPACR most of the isolates (5/6) belonged to group II, defined by the Asn526Lys substitution.
The levels of antibiotic resistance among S. pneumoniae and H. influenzae causing severe AOM in children are high in our settings. The existence of multidrug-resistant S. pneumoniae serotype 19A is of particular concern. The rate of BLNAR and BLPACR strains among H. influenzae isolates was 15%.
AOM; S. pneumoniae; H. influenzae; Serotypes; Antibiotic resistance
Haemophilus influenzae is one of the main aetiological agents of community-acquired respiratory tract infections. The primary aim of this study was to evaluate the antibacterial activity of telithromycin against H. influenzae clinical isolates showing different pattern of resistance in comparison with azithromycin and clarithromycin at 1/4 ×, 1/2 ×, 1 ×, 2 ×, 4 × minimum inhibitory concentration (MIC) and to peak concentrations in epithelial lining fluid (ELF). The secondary aim was to determine the influence of CO2 enriched atmosphere on bacterial susceptibility.
Telithromycin showed high activity against H. influenzae, including strains susceptible to β-lactams (n = 200), β-lactamase producer (n = 50) and β-lactamase negative ampicillin resistant (BLNAR) (n = 10), with MIC from ≤0.03 to 4 mg/L, and MIC50/MIC90 of 1/2 mg/L with susceptibility rate of 100%, and minimum bactericidal concentrations (MBC) from 2 to 4-fold higher than the MIC. Azithromycin was the most active tested macrolide (range: 0.25 – 4 mg/L; MIC50/MIC90: 1/2 mg/L), comparable to telithromycin, while clarithromycin showed the highest MICs and MBCs (range: 0.25 – 8 mg/L; MIC50/MIC90: 2/8 mg/L). In time-kill studies, telithromycin showed a bactericidal activity at the higher concentrations (4 – 2 × MIC and ELF) against all the strains, being complete after 12 – 24 hours from drug exposition. At MIC concentrations, at ambient air, bactericidal activity of telithromycin and azithromycin was quite similar at 12 hours, and better than that of clarithromycin. Besides, telithromycin and clarithromycin at ELF concentrations were bactericidal after 12 hours of incubation for most strains, while 24 hours were needed to azithromycin to be bactericidal. Incubation in CO2 significantly influenced the MICs and MBCs, and only slightly the in vitro killing curves.
Telithromycin showed an in-vitro potency against H. influenzae comparable to azithromycin, with an in-vitro killing rate more rapid and superior to clarithromycin at 2X-MIC against β-lactamase producers and BLNAR strains, and to azithromycin at ELF concentrations against β-lactamase negative strains. Against all strains, MICs and MBCs were lower in the absence of CO2 for the tested antibiotics, showing an adverse effect of incubation in a CO2 environment. The in-vitro potency together with the tissue concentrations of the antimicrobial, should be considered in predicting efficacy.
The in vitro antibacterial activities of oral cephem antibiotics and ketolide telithromycin against major respiratory pathogens possessing β-lactam-resistant mutations (within the pbp gene) and/or macrolide-resistant genes (erm and mef) were examined in clinical isolates collected at 66 institutes in all over the Japan between 2002 and 2003. Telithromycin showed the strongest antibacterial activity against methicillin-susceptible Staphylococcus aureus strains with and without macrolide-resistant genes, such as ermA or ermC gene. All the cephem antibiotics showed potent antibacterial activity against Streptococcus pyogenes, with minimum inhibitory concentrations (MICs) of 0.015 mg/L or lower. Cefdinir had a much higher MIC90 against genotypic penicillin-resistant Streptococcus pneumoniae (gPRSP) than cefditoren and cefcapene (8 mg/L cefdinir vs. 1 mg/L cefditoren and cefcapene). The majority of gPRSP harbored either ermB or mefA, and the antibacterial activity of telithromycin against these strains was decreased however some susceptibility was still sustained. Cefditoren exerted the strongest antibacterial activity against β-lactamase-negative ampicillin-resistant Haemophilus influenzae, with an MIC90 of 0.5 mg/L. These results underline the importance of checking the susceptibility and selecting an appropriate antibiotic against target pathogens.
cefditoren; telithromycin; Microbial Sensitivity Tests; Minimum Inhibitory Concentration; beta-Lactams
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.
The affinity of [3H]benzylpenicillin for penicillin-binding protein (PBP) 3A was reduced in 25 clinical isolates of β-lactamase-negative ampicillin (AMP)-resistant (BLNAR) Haemophilus influenzae for which the AMP MIC was ≥1.0 μg/ml. The affinities of PBP 3B and PBP 4 were also reduced in some strains. The sequences of the ftsI gene encoding the transpeptidase domain of PBP 3A and/or PBP 3B and of the dacB gene encoding PBP 4 were determined for these strains and compared to those of AMP-susceptible Rd strains. The BLNAR strains were classified into three groups on the basis of deduced amino acid substitutions in the ftsI gene, which is thought to be involved in septal peptidoglycan synthesis. His-517, near the conserved Lys-Thr-Gly (KTG) motif, was substituted for Arg-517 in group I strains (n = 9), and Lys-526 was substituted for Asn-526 in group II strains (n = 12). In group III strains (n = 4), three residues (Met-377, Ser-385, and Leu-389), positioned near the conserved Ser-Ser-Asn (SSN) motif, were replaced with Ile, Thr, and Phe, respectively, in addition to the replacement with Lys-526. The MICs of cephem antibiotics with relatively high affinities for PBP 3A and PBP 3B were higher than those of AMP and meropenem for group III strains. The MICs of β-lactams for H. influenzae transformants into which the ftsI gene from BLNAR strains was introduced were as high as those for the donors, and PBP 3A and PBP 3B showed decreased affinities for β-lactams. There was no clear relationship between 7-bp deletions in the dacB gene and AMP susceptibility. Even though mutations in another gene(s) may be involved in β-lactam resistance, these data indicate that mutations in the ftsI gene are the most important for development of resistance to β-lactams in BLNAR strains.
β-Lactamase-negative ampicillin-resistant (BLNAR) isolates of Haemophilus influenzae have been emerging in some countries, including Japan. The Clinical and Laboratory Standards Institute has only a susceptible MIC breakpoint (≤1 μg/ml) for piperacillin-tazobactam and a disclaimer comment that BLNAR H. influenzae should be considered resistant, which was adapted without presentation of data. In addition, fluoroquinolone-resistant H. influenzae isolates have recently been occasionally reported worldwide. To address these problems, we examined susceptibilities to β-lactams, including piperacillin-tazobactam, and ciprofloxacin by microdilution and disk diffusion (only for piperacillin-tazobactam) methods, against a total of 400 recent H. influenzae clinical isolates, including 100 β-lactamase-negative ampicillin-susceptible, β-lactamase-positive ampicillin-resistant, BLNAR, and β-lactamase-positive amoxicillin-clavulanate-resistant (BLPACR) isolates each. BLNAR and BLPACR isolates were tested by PCR using primers that amplify specific regions of the ftsI gene. We also detected mutations in quinolone resistance-determining regions (QRDRs) by direct sequencing of the PCR products of DNA fragments. Among β-lactams, piperacillin-tazobactam exhibited potent activity against all isolates of H. influenzae, with all MICs at ≤0.5 μg/ml (susceptible). A disk diffusion breakpoint for piperacillin-tazobactam of ≥21 mm is proposed. We confirmed that all BLNAR and BLPACR isolates had amino acid substitutions in the ftsI gene and that the major pattern was group III-like (87.5%). One ciprofloxacin-resistant isolate (MIC, 16 μg/ml) and 31 ciprofloxacin-susceptible isolates (MICs, 0.06 to 0.5 μg/ml) had amino acid changes in their QRDRs. Piperacillin-tazobactam was the most potent β-lactam tested against all classes of H. influenzae isolates. It is possible that fluoroquinolone-resistant H. influenzae will emerge since several clinical isolates carried mutations in their QRDRs.
Ampicillin resistance in Haemophilus influenzae due to alterations in penicillin-binding proteins (β-lactamase negative ampicillin resistant [BLNAR]) is acquiring increasing clinical and epidemiological importance. BLNAR strains with low ampicillin MICs (0.5 to 4 μg/ml) represent the majority of this population in Europe and the United States, but separating them from susceptible isolates is challenging. To investigate the best method to identify low-BLNAR strains, we studied the antibiotic susceptibilities of 94 clinical isolates of H. influenzae by microdilution, Etest, and disk diffusion: 25 had no resistance mechanisms (gBLNAS), 34 had mutations in the ftsI gene only (gBLNAR), 20 were β-lactamase producers only (gBLPAR), and 15 showed β-lactamase production and mutations in the ftsI gene (gBLPACR). By current CLSI breakpoints, most gBLNAR isolates were ampicillin susceptible by microdilution (76.5%) or by Etest (88.2%). Most gBLNAR strains (79.4%) were nonsusceptible to amoxicillin (the most widely used community antibiotic in the United States and Europe) when tested by microdilution. By Etest, 15% of β-lactamase-positive isolates were nonresistant to ampicillin or amoxicillin. The poorest agreement between Etest and microdilution results was for the gBLPAR isolates (25% for ampicillin, 15% for amoxicillin, and 10% for cefaclor). Low-strength disks of ampicillin and amoxicillin-clavulanic acid poorly identified low-BLNAR isolates and are not recommended as a screening method. We suggest new amoxicillin breakpoints for BLNAR isolates as follows: susceptible, MIC ≤ 0.5 μg/ml (no resistance mechanisms; pharmacokinetic/pharmacodynamic [PK/PD] data favorable); intermediate, MICs = 1 to 2 μg/ml (resistance mechanisms present but PK/PD data favorable), and resistant, MICs ≥ 4 μg/ml (resistance mechanisms present and PK/PD data unfavorable).
The aim of this study was to characterize ampicillin resistance mechanisms in clinical isolates of Haemophilus influenzae from Portugal. Association between specific patterns of amino acid substitutions in penicillin-binding protein 3 (PBP3) (with or without β-lactamase production) and β-lactam susceptibility as well as genetic relatedness among isolates were investigated.
Two-hundred and forty non-consecutive H. influenzae isolates chosen according to their different ampicillin MICs [101 β-lactamase-non-producing ampicillin-resistant (BLNAR) isolates, 80 β-lactamase-producing ampicillin-resistant (BLPAR) isolates and 59 β-lactamase-non-producing ampicillin-susceptible (BLNAS) isolates] were analysed. The β-lactamase-encoding blaTEM-1 gene was detected by PCR. The ftsI gene encoding PBP3 was sequenced. Genetic relatedness among isolates was examined by PFGE.
Of the 240 H. influenzae isolates, 141 had mutations in the transpeptidase domain of the ftsI gene, including most BLNAR strains (94/101, 93.1%) and a high percentage of BLPAR strains (47/80, 58.8%). As previously reported, the latter have been described as β-lactamase-positive amoxicillin/clavulanic acid resistant (BLPACR). The most common amino acid substitutions were identified near the KTG motif: N526K (136/141, 96.5%), V547I (124/141, 87.9%) and N569S (121/141, 85.8%). The 141 strains were divided into 31 ftsI mutation patterns and included six groups (I, IIa, IIb, IIc, IId and III-like). BLNAR strains were genetically diverse but close genetic relationships were demonstrated among BLPACR strains.
This study shows that the non-enzymatic mechanism of resistance to β-lactams is widespread among H. influenzae isolates in Portugal. Clonal dissemination of BLPACR strains showing high resistance to ampicillin and reduced susceptibility to amoxicillin/clavulanic acid was documented.
penicillin-binding proteins; ampicillin resistance; genetic relatedness; β-lactamases
Increasing antimicrobial resistance among the key pathogens responsible for community-acquired respiratory tract infections has the potential to limit the effectiveness of antibiotics available to treat these infections. Since there are regional differences in the susceptibility patterns observed and treatment is frequently empirical, the selection of antibiotic therapy may be challenging. PROTEKT, a global, longitudinal multicentre surveillance study, tracks the activity of telithromycin and comparator antibacterial agents against key respiratory tract pathogens.
In this analysis, we examine the prevalence of antibacterial resistance in 1,336 bacterial pathogens, isolated from adult and paediatric patients clinically diagnosed with acute bacterial sinusitis (ABS).
Results and discussion
In total, 58.0%, 66.1%, and 55.8% of S. pneumoniae isolates were susceptible to penicillin, cefuroxime, and clarithromycin respectively. Combined macrolide resistance and reduced susceptibility to penicillin was present in 200/640 (31.3 %) of S. pneumoniae isolates (128 isolates were resistant to penicillin [MIC >= 2 mg/L], 72 intermediate [MIC 0.12–1 mg/L]) while 99.5% and 95.5% of isolates were susceptible to telithromycin and amoxicillin-clavulanate, respectively. In total, 88.2%, 87.5%, 99.4%, 100%, and 100% of H. influenzae isolates were susceptible to ampicillin, clarithromycin, cefuroxime, telithromycin, and amoxicillin-clavulanate, respectively. In vitro, telithromycin demonstrated the highest activity against M. catarrhalis (MIC50 = 0.06 mg/L, MIC90 = 0.12 mg/L).
The high in vitro activity of against pathogens commonly isolated in ABS, together with a once daily dosing regimen and clinical efficacy with 5-day course of therapy, suggest that telithromycin may play a role in the empiric treatment of ABS.
Horizontal gene transfer has been identified in only a small number of genes in Haemophilus influenzae, an organism which is naturally competent for transformation. This report provides evidence for the genetic transfer of the ftsI gene, which encodes penicillin-binding protein 3, in H. influenzae. Mosaic structures of the ftsI gene were found in several clinical isolates of H. influenzae. To identify the origin of the mosaic sequence, complete sequences of the corresponding gene from seven type strains of Haemophilus species were determined. Comparison of these sequences with mosaic regions identified a homologous recombination of the ftsI gene between H. influenzae and Haemophilus haemolyticus. Subsequently, ampicillin-resistant H. influenzae strains harboring identical ftsI sequences were genotyped by pulsed-field gel electrophoresis (PFGE). Divergent PFGE patterns among β-lactamase-nonproducing ampicillin-resistant (BLNAR) strains from different hospitals indicated the potential for the genetic transfer of the mutated ftsI gene between these isolates. Moreover, transfer of the ftsI gene from BLNAR strains to β-lactamase-nonproducing ampicillin-susceptible (BLNAS) H. influenzae strains was evaluated in vitro. Coincubation of a BLNAS strain (a rifampin-resistant mutant of strain Rd) and BLNAR strains resulted in the emergence of rifampin- and cefdinir-resistant clones at frequencies of 5.1 × 10−7 to 1.5 × 10−6. Characterization of these doubly resistant mutants by DNA sequencing of the ftsI gene, susceptibility testing, and genotyping by PFGE revealed that the ftsI genes of BLNAR strains had transferred to BLNAS strains during coincubation. In conclusion, horizontal transfer of the ftsI gene in H. influenzae can occur in an intraspecies and an interspecies manner.
To clarify the relationship between mutations commonly found for penicillin-binding protein 3 (PBP 3) of β-lactamase-nonproducing ampicillin-resistant (BLNAR) Haemophilus influenzae isolates and β-lactam resistance, single and multiple amino acid mutations at positions 377, 385, 389, 517, and 526 were introduced into PBP 3 of a β-lactam-susceptible Rd strain by site-directed mutagenesis. Twelve isogenic recombinant strains were challenged with nine β-lactam antibiotics. Replacement of the asparagine at position 526 with lysine (N526K) increased the resistance to imipenem eightfold and increased the resistance to various cephalosporins two- to eightfold. Substitution of threonine for serine at position 385 (S385T) and/or substitution of phenylalanine for leucine at position 389 (L389F), in addition to the N526K mutation, led to two- to fourfold additional increases in cephalosporin resistance. An isoleucine-to-methionine substitution at position 377 did not change the antibiotic sensitivity of any of the recombinant strains also carrying other PBP 3 mutations tested. Thirty-six clinical isolates carrying a PBP 3 gene (ftsI) with the S385T, L389F, R517H, and/or N526K mutation were chosen from among 279 clinical isolates collected in Japan, and the isolates were grouped into six classes on the basis of the patterns of the four mutations in PBP 3. Rd recombinants were made with each of the ftsI genes. The levels of resistance to β-lactams varied between recombinants of different classes but were comparable for those of the same class. The levels of resistance to cephalosporins of these recombinants were similar to those of the parent clinical isolates, while those to ampicillin and carbapenems were lower. These results indicate that resistance to β-lactams, at least to cephalosporins, depends in large part on the PBP 3 mutations R517H, N526K, S385T, and L389F.
The Drug-Resistant Pathogen Surveillance Group in Pediatric Infectious Disease conducted national surveillance for Haemophilus influenzae in 2007 (phase 3) and 2010 (phase 4), following the previous surveillance conducted from 2000 to 2001 (phase 1) and in 2004 (phase 2). We examined the antimicrobial susceptibility for H. influenzae derived from clinical specimens of pediatric patients collected nationwide from 27 institutions during phases 3 (386 strains) and 4 (484 strains). The frequency of β-lactamase-nonproducing ampicillin (ABPC)-resistant (BLNAR) strains, which rapidly increased from 11.4 % in phase 1 to 43.4 % in phase 2, has gradually decreased from 38.3 % in phase 3 to 37.8 % in phase 4. In contrast, On the other hand, the frequency of β-lactamase-producing strains, which continuously decreased from 8.3 % in phase 1 to 4.4 % in phase 3, has increased to 8.7 % in phase 4. Prevalence of β-lactamase-producing clavulanic acid/amoxicillin-resistant (BLPACR) strains, especially, has increased from 1.6 % in phase 3 to 4.8 % in phase 4. The oral antimicrobial agents with the lowest MIC90 were levofloxacin in both phases, and tosufloxacin in phase 4 (≤0.063 μg/ml), whereas for intravenous use the corresponding agent was tazobactam/piperacillin in both phases (0.125 μg/ml). There was no increase in the MIC90 of most β-lactams between phase 3 and phase 4. In relationship to sex, age, presence of siblings, attendance at a daycare center, siblings’ attendance at a daycare center, and prior administration of antimicrobial agents within 1 month, the frequency of β-lactamase-nonproducing ABPC-intermediately resistant (BLNAI) strains + BLNAR strains was high (P = 0.005) in cases with prior administration of antimicrobial agents in phase 3.
Pediatric infectious disease; Surveillance; Haemophilus influenzae sensitivity; Drug resistance
The increasing prevalence of resistance to established antibiotics among key bacterial respiratory tract pathogens, such as Streptococcus pneumoniae, is a major healthcare problem in the USA. The PROTEKT US study is a longitudinal surveillance study designed to monitor the susceptibility of key respiratory tract pathogens in the USA to a range of commonly used antimicrobials. Here, we assess the geographic and temporal trends in antibacterial resistance of S. pneumoniae isolates from patients with community-acquired respiratory tract infections collected between Year 1 (2000–2001) and Year 4 (2003–2004) of PROTEKT US.
Antibacterial minimum inhibitory concentrations were determined centrally using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method; susceptibility was defined according to CLSI interpretive criteria. Macrolide resistance genotypes were determined by polymerase chain reaction.
A total of 39,495 S. pneumoniae isolates were collected during 2000–2004. The percentage of isolates resistant to erythromycin, penicillin, levofloxacin, and telithromycin were 29.3%, 21.2%, 0.9%, and 0.02%, respectively, over the 4 years, with marked regional variability. The proportion of isolates exhibiting multidrug resistance (includes isolates known as penicillin-resistant S. pneumoniae and isolates resistant to ≥ 2 of the following antibiotics: penicillin; second-generation cephalosporins, e.g. cefuroxime; macrolides; tetracyclines; and trimethoprim-sulfamethoxazole) remained stable at ~30% over the study period. Overall mef(A) was the most common macrolide resistance mechanism. The proportion of mef(A) isolates decreased from 68.8% to 62.3% between Year 1 and Year 4, while the percentage of isolates carrying both erm(B) and mef(A) increased from 9.7% to 18.4%. Over 99% of the erm(B)+mef(A)-positive isolates collected over Years 1–4 exhibited multidrug resistance. Higher than previously reported levels of macrolide resistance were found for mef(A)-positive isolates.
Over the first 4 years of PROTEKT US, penicillin and erythromycin resistance among pneumococcal isolates has remained high. Although macrolide resistance rates have stabilized, the prevalence of clonal isolates, with a combined erm(B) and mef(A) genotype together with high-level macrolide and multidrug resistance, is increasing, and their spread may have serious health implications. Telithromycin and levofloxacin both showed potent in vitro activity against S. pneumoniae isolates irrespective of macrolide resistance genotype.
The in vitro activities of modithromycin against Gram-positive and -negative respiratory pathogens, including macrolide-resistant cocci with different resistance mechanisms, were compared with those of other macrolide and ketolide agents. MICs were determined by the broth microdilution method. All 595 test strains used in this study were isolated from Japanese medical facilities. The erm (ribosome methylase) and/or mef (efflux pump) gene, which correlated with resistance to erythromycin as well as clarithromycin and azithromycin, was found in 81.8%, 21.3%, and 23.2% of Streptococcus pneumoniae, Streptococcus pyogenes, and methicillin-susceptible Staphylococcus aureus (MSSA) strains, respectively. Modithromycin showed MIC90s of 0.125 μg/ml against these three cocci, including macrolide-resistant strains. In particular, the MIC of modithromycin against ermB-carrying S. pyogenes was ≥32-fold lower than that of telithromycin. The activities of modithromycin as well as telithromycin were little affected by the presence of mefA or mefE in both streptococci. Against Gram-negative pathogens, modithromycin showed MIC90s of 0.5, 8, and 0.031 μg/ml against Moraxella catarrhalis, Haemophilus influenzae, and Legionella spp., respectively. The MICs of modithromycin against M. catarrhalis and H. influenzae were higher than those of telithromycin and azithromycin. However, modithromycin showed the most potent anti-Legionella activity among the macrolide and ketolide agents tested. These results suggested that the bicyclolide agent modithromycin is a novel class of macrolides with improved antibacterial activity against Gram-positive cocci, including telithromycin-resistant streptococci and intracellular Gram-negative bacteria of the Legionella species.
By Etest determination of the susceptibilities of 229 Haemophilus influenzae strains isolated in Korea to 10 antibiotics, the isolates were found to be antibiotic nonsusceptible in the following order: ampicillin (58.1%), trimethoprim-sulfamethoxazole (52%), cefaclor (41.1%), clarithromycin (25.8%), chloramphenicol (14.0%), amoxicillin-clavulanic acid (13.5%), meropenem (11.7%), cefixime (10.9%), cefuroxime (9.2%), and levofloxacin (1.3%). The prevalences of each resistance class were 23.6% for β-lactamase-negative ampicillin-susceptible (BLNAS) strains; 37.6% for strains with the TEM-1 type β-lactamase gene; 1.3% for strains with the ROB-1 type β-lactamase gene; 29.3% for the β-lactamase-negative ampicillin-resistant (BLNAR) strains with a mutation in the ftsI gene, which encodes PBP 3; and 8.3% for β-lactamase-positive amoxicillin-clavulanate-resistant (BLPACR) strains, which showed both resistance mechanisms (i.e., a β-lactamase gene and a mutation in the ftsI gene). The MIC50s of all β-lactams, including cephem and meropenem agents, for the BLNAR strains were two to three times higher than those for the BLNAS strains. This study confirms that the prevalence of BLNAR and BLPACR strains is relatively high and for the first time confirms the presence of H. influenzae strains carrying blaROB-1 in Korea. Even though mutations in another gene(s) might be involved in β-lactam resistance, these results suggest that mutations in the ftsI gene are important for the development of resistance to β-lactams in H. influenzae strains in Korea.
A nationwide multicenter susceptibility surveillance study (Susceptibility to the Antimicrobials Used in the Community in España [SAUCE] project), SAUCE-4, including 2,559 Streptococcus pneumoniae, 2,287 Streptococcus pyogenes, and 2,736 Haemophilus influenzae isolates was carried out from May 2006 to June 2007 in 34 Spanish hospitals. Then, the results from SAUCE-4 were compared to those from all three previous SAUCE studies carried out in 1996-1997, 1998-1999, and 2001-2002 to assess the temporal trends in resistance and the phenotypes of resistance over the 11-year period. In SAUCE-4, on the basis of the CLSI breakpoints, penicillin (parenteral, nonmeningitis breakpoint) and cefotaxime were the antimicrobials that were the most active against S. pneumoniae (99.8% and 99.6%, respectively). Only 0.9% of isolates had a penicillin MIC of ≥2 μg/ml. In S. pyogenes, nonsusceptibility to erythromycin was observed in 19.4% of isolates. Among the H. influenzae isolates, a β-lactamase-positive prevalence of 15.7% was found. A statistically significant temporal decreasing trend over the 11-year period was observed for nonsusceptibility (from 60.0% to 22.9%) and resistance (from 36.5% to 0.9%) to penicillin and for the proportion of erythromycin-resistant isolates of S. pneumoniae of the macrolide-lincosamide-streptogramin B (MLSB) phenotype (from 98.4% to 81.3%). A similar trend was observed for the prevalence of ampicillin resistance (from 37.6% to 16.1%), β-lactamase production (from 25.7% to 15.7%), and β-lactamase-negative ampicillin resistance (BLNAR) in H. influenzae (from 13.5% to 0.7%). Among erythromycin-resistant isolates of S. pyogenes, a significant increasing trend in the prevalence of MLSB was observed (from 7.0% to 35.5%). SAUCE-4 confirms a generalized decline in the resistance of the main respiratory pathogens to the antimicrobials as well as a shift in their resistance phenotypes.
Beta-lactam resistance in Haemophilus influenzae due to ftsI mutations causing altered penicillin-binding protein 3 (PBP3) is increasing worldwide. Low-level resistant isolates with the N526K substitution (group II low-rPBP3) predominate in most geographical regions, while high-level resistant isolates with the additional S385T substitution (group III high-rPBP3) are common in Japan and South Korea.
Knowledge about the molecular epidemiology of rPBP3 strains is limited. We combined multilocus sequence typing (MLST) and ftsI/PBP3 typing to study the emergence and spread of rPBP3 in nontypeable H. influenzae (NTHi) in Norway.
The prevalence of rPBP3 in a population of 795 eye, ear and respiratory isolates (99% NTHi) from 2007 was 15%. The prevalence of clinical PBP3-mediated resistance to ampicillin was 9%, compared to 2.5% three years earlier. Group II low-rPBP3 predominated (96%), with significant proportions of isolates non-susceptible to cefotaxime (6%) and meropenem (20%). Group III high-rPBP3 was identified for the first time in Northern Europe.
Four MLST sequence types (ST) with characteristic, highly diverging ftsI alleles accounted for 61% of the rPBP3 isolates. The most prevalent substitution pattern (PBP3 type A) was present in 41% of rPBP3 isolates, mainly carried by ST367 and ST14. Several unrelated STs possessed identical copies of the ftsI allele encoding PBP3 type A.
Infection sites, age groups, hospitalization rates and rPBP3 frequencies differed between STs and phylogenetic groups.
This study is the first to link ftsI alleles to STs in H. influenzae. The results indicate that horizontal gene transfer contributes to the emergence of rPBP3 by phylogeny restricted transformation.
Clonally related virulent rPBP3 strains are widely disseminated and high-level resistant isolates emerge in new geographical regions, threatening current empiric antibiotic treatment. The need of continuous monitoring of beta-lactam susceptibility and a global system for molecular surveillance of rPBP3 strains is underlined. Combining MLST and ftsI/PBP3 typing is a powerful tool for this purpose.
Haemophilus influenzae; Beta-lactam resistance; ftsI; PBP3; BLNAR; MLST; PFGE; Horizontal gene transfer; Recombination; Surveillance
The in vitro activities of piperacillin (PIP) against β-lactamase-negative ampicillin (AMP)-resistant (BLNAR) Haemophilus influenzae were compared with those of cefotaxime (CTX) and ceftriaxone (CRO), and the potency of PIP as therapy for meningitis caused by BLNAR is also discussed. PIP showed good activity (MIC at which 90% of strains are inhibited, 0.25 μg/ml) against 69 BLNAR strains, and its activity was comparable to that of CRO and superior to that of CTX. No significant correlation was observed between the MICs of PIP and CTX or CRO or AMP, whereas a high correlation was observed between the MICs of CTX and CRO. In the killing study, PIP showed potent bactericidal activity compared with those of CTX and CRO. By microscopic examination, PIP caused the formation of a spindle and short filamentous cells with bulges and induced cell lysis in BLNAR strains, while treatment with CTX and CRO resulted in the formation of large, spherical cells without any obvious lysis. The affinity of Bocillin FL, a fluorescent penicillin used for determination of the 50% inhibitory concentration (IC50s) for penicillin-binding proteins (PBPs), to PBPs 3a and 3b of BLNAR strains was drastically decreased compared with that to an AMP-susceptible strain (ATCC 33391). In the case of the BLNAR strains, the IC50s for PBPs 1a, 1b, and 2 were similar to those for the PBPs of ATCC 33391. Since the affinity of binding to PBPs 3a and 3b of the BLNAR strains decreased drastically, the second targets among the PBPs were PBP 2 for PIP, PBP1 (1a and 1b) for CTX and CRO. In conclusion, PIP showed excellent activities against BLNAR strains in a manner different from those of cephem antibiotics, suggesting that it could be a candidate therapeutic agent for the treatment of meningitis caused by BLNAR strains.
Antimicrobial susceptibility patterns and β-lactam resistance mechanisms of 544 Haemophilus influenzae isolates through the nationwide Acute Respiratory Infections Surveillance (ARIS) network in Korea during 2005 and 2006 were determined. Resistance to ampicillin was 58.5%, followed by resistance to cefuroxime (23.3%), clarithromycin (18.7%), cefaclor (17.0%), amoxicillin-clavulanate (10.4%), and chloramphenicol (8.1%). Levofloxacin and cefotaxime were the most active agents tested in this study. β-Lactamase production (52.4%) was the main mechanism of ampicillin resistance, affecting 96.1% of TEM-1-type β-lactamase. According to their β-lactam resistance mechanisms, all isolates were classified into the following groups: β-lactamase-negative, ampicillin-sensitive (BLNAS) strains (n = 224; 41.5%); β-lactamase-positive, ampicillin-resistant (BLPAR) strains (n = 255; 47.2%); β-lactamase-negative, ampicillin-resistant (BLNAR) strains (n = 33; 6.1%); and β-lactamase-positive, amoxicillin-clavulanate-resistant (BLPACR) strains (n = 28; 5.2%). Among the BLNAR and BLPACR strains, there were various patterns of multiple-amino-acid substitutions in penicillin-binding protein 3. Particularly, among BLNAR, group III isolates, which had three simultaneous substitutions (Met377Ile, Ser385Thr, and Leu389Phe), were identified for the first time in Korea. Three group III strains displayed the highest MIC of cefotaxime (1 to 2 μg/ml). The results indicate the importance of monitoring a changing situation pertaining to the increase and spread of BLNAR and BLPACR strains of H. influenzae for appropriate antibiotic therapy for patients with respiratory tract infections in Korea.
In vitro activities of erythromycin A, telithromycin, and two investigational ketolides, JNJ-17155437 and JNJ-17155528, were evaluated against clinical bacterial strains, including selected common respiratory tract pathogens. Against 46 macrolide-susceptible and -resistant Streptococcus pneumoniae strains, the MIC90 (MIC at which 90% of the isolates tested were inhibited) of the investigational ketolides was 0.25 μg/ml, twofold lower than that of telithromycin and at least 64-fold lower than that of erythromycin A. Against erm(B)-containing pneumococci, the MIC90 of all the ketolides was 0.06 μg/ml. The MIC90 of the investigational ketolides against mef(A)-containing pneumococci or pneumococci with both mef(A) and erm(B) was 0.25 μg/ml, two-and fourfold lower, respectively, than that of telithromycin. In contrast, the MICs of the investigational ketolides against macrolide-resistant S. pneumoniae strains with ribosomal mutations were similar to or, in some cases, as much as eightfold higher than those of telithromycin. Against Haemophilus influenzae, MICs of all the ketolides were ≤2 μg/ml. Against three Moraxella catarrhalis isolates, the MIC of the ketolides was 0.25 μg/ml. The ketolides inhibited in vitro protein synthesis, with 50% inhibitory concentrations ranging from 0.23 to 0.27 μM. In time-kill studies against macrolide-susceptible and erm- or mef-containing pneumococci, the ketolides were bacteriostatic to slowly bactericidal, with 24-h log10 decreases ranging from 2.0 to 4.1 CFU. Intervals of postantibiotic effects for the ketolides against macrolide-susceptible and -resistant S. pneumoniae were 3.0 to 8.1 h.
A total of 1,537 clinical isolates of Haemophilus influenzae were recovered in 30 U.S. medical center laboratories between 1 November 1994 and 30 April 1995 and were characterized in a central laboratory with respect to serotype and beta-lactamase production and the in vitro activities of 15 oral antimicrobial agents. Overall, 36.4% of the isolates were found to produce beta-lactamase. The rank order of activity of six cephalosporins on the basis of MICs was cefixime > cefpodoxime > cefuroxime > loracarbef > or = cefaclor > cefprozil. On the basis of current National Committee for Clinical Laboratory Standards (NCCLS) breakpoints ages of isolates found to be resistant or intermediate to these agents were as follows: 0.1, 0.3, 6.4, 16.3, 18.3, and 29.8, respectively (National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 4th ed. M7-A4, 1995). Azithromycin was, on a weight basis, the most potent of the macrolides tested in this study, followed by erythromycin and then clarithromycin. Azithromycin was typically fourfold more active than erythromycin, which was, in turn, slightly more active than clarithromycin. However, when compared on the basis of the frequency of resistance determined by using current NCCLS breakpoints, there was essentially no difference between azithromycin and clarithromycin, i.e., 0.5 and 1.9%, respectively (P = 0.086). Interpretive breakpoints for erythromycin MIC tests versus H. influenzae have not been developed. Resistance to other non- beta-lactam agents was variable, as follows: trimethoprim-sulfamethoxazole, 9.0%; chloramphenicol, 0.2%; tetracycline, 1.3%; and rifampin, 0.3%. Two conspicuous findings in this study were the identification of 39 strains H. influenzae that were beta-lactamase negative but ampicillin intermediate or resistant (BLNAR) and, even more surprisingly, 17 beta-lactamase-positive isolates that were resistant to amoxicillin-clavulanate (BLPACR). Strains of H. influenzae in the first group have heretofore been very uncommon; organisms in the second group have not previously been described in the literature. The percentages of all study isolates comprised of BLNAR and BLPACR organisms were 2.5 and 1.1, respectively. Overall resistance to ampicillin was thus 38.9%, and that to amoxicillin-clavulanate was 4.5%.
Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen which causes a variety of respiratory infections. The objectives of the study were to determine its antimicrobial susceptibility, to characterize the β-lactam resistance, and to establish a genetic characterization of NTHi isolates. Ninety-five NTHi isolates were analyzed by pulsed field gel electrophoresis (PFGE) and multi locus sequence typing (MLST). Antimicrobial susceptibility was determined by microdilution, and the ftsI gene (encoding penicillin-binding protein 3, PBP3) was PCR amplified and sequenced. Thirty (31.6%) isolates were non-susceptible to ampicillin (MIC≥2 mg/L), with 10 of them producing β-lactamase type TEM-1 as a resistance mechanism. After ftsI sequencing, 39 (41.1%) isolates showed amino acid substitutions in PBP3, with Asn526→ Lys being the most common (69.2%). Eighty-four patients were successfully treated with amoxicillin/clavulanic acid, ceftriaxone and levofloxacin. Eight patients died due either to aspiration or complication of their comorbidities. In conclusion, NTHi causing CAP in adults shows high genetic diversity and is associated with a high rate of reduced susceptibility to ampicillin due to alterations in PBP3. The analysis of treatment and outcomes demonstrated that NTHi strains with mutations in the ftsI gene could be successfully treated with ceftriaxone or fluoroquinolones.