In Vitro Selection of Resistance in Haemophilus influenzae by Amoxicillin-Clavulanate, Cefpodoxime, Cefprozil, Azithromycin, and Clarithromycin

ABSTRACT Abilities of amoxicillin-clavulanate, cefpodoxime, cefprozil, azithromycin, and clarithromycin to select resistant mutants of Haemophilus influenzae were tested by multistep and single-step methodologies. For multistep studies, 10 random strains were tested: 5 of these were β-lactamase positive. After 50 daily subcultures in amoxicillin-clavulanate, MICs did not increase more than fourfold. However, cefprozil MICs increased eightfold for one strain. Clarithromycin and azithromycin gave a >4-fold increase in 8 and 10 strains after 14 to 46 and 20 to 50 days, respectively. Mutants selected by clarithromycin and azithromycin were associated with mutations in 23S rRNA and ribosomal proteins L4 and L22. Three mutants selected by clarithromycin or azithromycin had alterations in ribosomal protein L4, while five had alterations in ribosomal protein L22. Two mutants selected by azithromycin had mutations in the gene encoding 23S rRNA: one at position 2058 and the other at position 2059 (Escherichia coli numbering), with replacement of A by G. One clone selected by clarithromycin became hypersusceptible to macrolides. In single-step studies azithromycin and clarithromycin had the highest mutation rates, while amoxicillin-clavulanate had the lowest. All resistant clones were identical to parents as observed by pulsed-field gel electrophoresis. The MICs of azithromycin for azithromycin-resistant clones were 16 to >128 μg/ml, and those of clarithromycin for clarithromycin-resistant clones were 32 to >128 μg/ml in multistep studies. For strains selected by azithromycin, the MICs of clarithromycin were high and vice versa. After 50 daily subcultures in the presence of drugs, MICs of amoxicillin-clavulanate and cefpodoxime against H. influenzae did not rise more than fourfold, in contrast to cefprozil, azithromycin, and clarithromycin, whose MICs rose to variable degrees.

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In Vitro Selection and Characterization of Resistance to Macrolides and Related Antibiotics in Mycoplasma pneumoniae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.2.460-465.2004 ◽

2004 ◽

Vol 48 (2) ◽

pp. 460-465 ◽

Cited By ~ 90

Author(s):

S. Pereyre ◽

C. Guyot ◽

H. Renaudin ◽

A. Charron ◽

C. Bébéar ◽

...

Keyword(s):

Ribosomal Protein ◽

Mycoplasma Pneumoniae ◽

Ribosomal Proteins ◽

In Vitro Selection ◽

Point Mutations ◽

Single Amino Acid ◽

23S Rrna ◽

Single Amino Acid Change ◽

Erythromycin A

ABSTRACT Macrolide-resistant mutants of Mycoplasma pneumoniae were selected in vitro from the susceptible reference strain M129, by 23 to 50 serial passages in subinhibitory concentrations of macrolides and related antibiotics, erythromycin A, azithromycin, josamycin, clindamycin, quinupristin, quinupristin-dalfopristin, pristinamycin, and telithromycin. Mutants for which the MICs are increased could be selected with all antibiotics except the streptogramin B quinupristin. Portions of genes encoding 23S rRNA (domains II and V) and ribosomal proteins L4 and L22 of mutants were amplified by PCR, and their nucleotide sequences were compared to those of the susceptible strain M129. No mutation could be detected in domain II of 23S rRNA. Two point mutations in domain V of 23S rRNA, C2611A and A2062G, were selected in the presence of erythromycin A, azithromycin, josamycin, quinupristin-dalfopristin, and telithromycin. Mutants selected in the presence of clindamycin and telithromycin harbored a single amino acid change (H70R or H70L, respectively) in ribosomal protein L4, whereas insertions of one, two, or three adjacent glycines at position 60 (M. pneumoniae numbering) were selected in the presence of both streptogramin combinations. Telithromycin was the sole antibiotic that selected for substitutions (P112R and A114T) and deletions (111IPRA114) in ribosomal protein L22. Three sequential mutational events in 23S rRNA and in both ribosomal proteins were required to categorize the strain as resistant to the ketolide. Azithromycin and erythromycin A were the only selector antibiotics that remained active (MICs, 0.06 and 1 μg/ml, respectively) on their mutants selected after 50 passages.

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Activities of Two Novel Macrolides, GW 773546 and GW 708408, Compared with Those of Telithromycin, Erythromycin, Azithromycin, and Clarithromycin against Haemophilus influenzae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.11.4113-4119.2004 ◽

2004 ◽

Vol 48 (11) ◽

pp. 4113-4119 ◽

Cited By ~ 14

Author(s):

Klaudia Kosowska ◽

Kim Credito ◽

Glenn A. Pankuch ◽

Dianne Hoellman ◽

Gengrong Lin ◽

...

Keyword(s):

Haemophilus Influenzae ◽

Ribosomal Proteins ◽

Selection Process ◽

Single Step ◽

23S Rrna ◽

Resistance Selection ◽

Resistant Strains ◽

Step Selection ◽

Better Than

ABSTRACT The MIC at which 50% of strains are inhibited (MIC50) and the MIC90 of GW 773546, a novel macrolide, were 1.0 and 2.0 μg/ml, respectively, for 223 β-lactamase-positive, β-lactamase-negative, and β-lactamase-negative ampicillin-resistant Haemophilus influenzae strains. The MIC50s and MIC90s of GW 708408, a second novel macrolide, and telithromycin, an established ketolide, were 2.0 and 4.0 μg/ml, respectively, while the MIC50 and MIC90 of azithromycin were 1.0 and 2.0 μg/ml, respectively. The MIC50 and MIC90 of erythromycin were 4.0 and 8.0 μg/ml, respectively; and those of clarithromycin were 4.0 and 16.0 μg/ml, respectively. All compounds except telithromycin were bactericidal (99.9% killing) against nine strains at two times the MIC after 24 h. Telithromycin was bactericidal against eight of the nine strains. In addition, both novel macrolides and telithromycin at two times the MIC showed 99% killing of all nine strains after 12 h and 90% killing of all strains after 6 h. After 24 h, all drugs were bactericidal against four to seven strains when they were tested at the MIC. Ten of 11 strains tested by multistep selection analysis yielded resistant clones after 14 to 43 passages with erythromycin. Azithromycin gave resistant clones of all strains after 20 to 50 passages, and clarithromycin gave resistant clones of 9 of 11 strains after 14 to 41 passages. By comparison, GW 708408 gave resistant clones of 9 of 11 strains after 14 to 44 passages, and GW 773546 gave resistant clones of 10 of 11 strains after 14 to 45 passages. Telithromycin gave resistant clones of 7 of 11 strains after 18 to 45 passages. Mutations mostly in the L22 and L4 ribosomal proteins and 23S rRNA were detected in resistant strains selected with all compounds, with alterations in the L22 protein predominating. Single-step resistance selection studies at the MIC yielded spontaneous resistant mutants at frequencies of 1.5 × 10−9 to 2.2 × 10−6 with GW 773546, 1.5 × 10−9 to 6.0 × 10−4 with GW 708408, and 7.1 × 10−9 to 3.8 × 10−4 with telithromycin, whereas the frequencies were 1.3 × 10−9 to 6.0 × 10−4 with erythromycin and azithromycin and 2.0 × 10−9 to 2.0 × 10−3 with clarithromycin. Alterations in the L22 protein (which were predominant) and the L4 protein were present in mutants selected by the single-step selection process. The postantibiotic effects of GW 773546, GW 708408, and telithromycin for seven H. influenzae strains were 6.6 h (range, 5.2 to 8.8 h), 4.7 h (range, 2.6 to 6.9 h), and 6.4 h (range, 3.8 to 9.7 h), respectively. The results of in vitro studies obtained with both novel macrolides were similar to those obtained with telithromycin and better than those obtained with older macrolides.

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In vitro selection of aztreonam/avibactam resistance in dual-carbapenemase-producing Klebsiella pneumoniae

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkz468 ◽

2019 ◽

Vol 75 (3) ◽

pp. 559-565 ◽

Cited By ~ 6

Author(s):

Siqiang Niu ◽

Jie Wei ◽

Chunhong Zou ◽

Kalyan D Chavda ◽

Jingnan Lv ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

In Vitro Selection ◽

Fold Increase ◽

Single Step ◽

Mutant Selection ◽

Microdilution Method ◽

Broth Microdilution Method ◽

Resistant Mutants ◽

Selection Of

Abstract Objectives To examine the in vitro selection of aztreonam/avibactam resistance among MBL-producing Klebsiella pneumoniae and to understand the mechanism of increased resistance. Methods The MICs of aztreonam were determined with and without avibactam (4 mg/L) using a broth microdilution method. Single-step and multi-step mutant selection was conducted on five MBL-producing K. pneumoniae strains, including two dual carbapenemase producers. Genomic sequencing and gene cloning were performed to investigate the mechanism of increased resistance. Results We examined the MICs for 68 MBL-producing K. pneumoniae isolates, including 13 dual carbapenemase producers. Compared with aztreonam alone, the addition of avibactam (4 mg/L) reduced the MICs for all isolates by >128-fold, with MIC50 and MIC90 values of 0.25 and 1 mg/L, respectively. One NDM-1-, OXA-48-, CTX-M-15- and CMY-16-positive ST101 K. pneumoniae strain was selected to be resistant to aztreonam/avibactam, with a >16-fold increase in MIC (>128 mg/L). WGS revealed that the resistant mutants lost the blaNDM-1 gene, but acquired amino acid substitutions in CMY-16 (Tyr150Ser and Asn346His). Construction of blaCMY-16 mutants confirmed that the substitutions (Tyr150Ser and Asn346His) were primarily responsible for the decreased susceptibility to aztreonam/avibactam. In addition, transfer of blaCMY-16 mutant (Tyr150Ser and Asn346His) plasmid constructs into certain clinical carbapenemase-producing isolates demonstrated >64-fold increased MICs of aztreonam/avibactam and aztreonam/avibactam/ceftazidime. Conclusions Aztreonam in combination with avibactam showed potent in vitro activity against MBL-producing K. pneumoniae. However, our study suggested the likelihood of aztreonam/avibactam resistance among MBL- and AmpC-co-producing strains and clinical practice should beware of the possibility of the emerging resistance.

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Mutations in 23S rRNA and Ribosomal Protein L4 Account for Resistance in Pneumococcal Strains Selected In Vitro by Macrolide Passage

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.8.2118-2125.2000 ◽

2000 ◽

Vol 44 (8) ◽

pp. 2118-2125 ◽

Cited By ~ 185

Author(s):

A. Tait-Kamradt ◽

T. Davies ◽

M. Cronan ◽

M. R. Jacobs ◽

P. C. Appelbaum ◽

...

Keyword(s):

Amino Acids ◽

Ribosomal Protein ◽

Ribosomal Proteins ◽

Serial Passage ◽

Single Amino Acid ◽

Rrna Genes ◽

23S Rrna ◽

Erythromycin Resistance ◽

Single Amino Acid Change

ABSTRACT The mechanisms responsible for macrolide resistance inStreptococcus pneumoniae mutants, selected from susceptible strains by serial passage in azithromycin, were investigated. These mutants were resistant to 14- and 15-membered macrolides, but resistance could not be explained by any clinically relevant resistance determinant [mef(A),erm(A), erm(B), erm(C),erm(TR), msr(A), mph(A),mph(B), mph(C), ere(A),ere(B)]. An investigation into the sequences of 23S rRNAs in the mutant and parental strains revealed individual changes of C2611A, C2611G, A2058G, and A2059G (Escherichia colinumbering) in four mutants. Mutations at these residues in domain V of 23S rRNA have been noted to confer erythromycin resistance in other species. Not all four 23S rRNA alleles have to contain the mutation to confer resistance. Some of the mutations also confer coresistance to streptogramin B (C2611A, C2611G, and A2058G), 16-membered macrolides (all changes), and clindamycin (A2058G and A2059G). Interestingly, none of these mutations confer high-level resistance to telithromycin (HMR-3647). Further, two of the mutants which had no changes in their 23S rRNA sequences had changes in a highly conserved stretch of amino acids (63KPWRQKGTGRAR74) in ribosomal protein L4. One mutant contained a single amino acid change (G69C), while the other mutant had a 6-base insert, resulting in two amino acids (S and Q) being inserted between amino acids Q67 and K68. To our knowledge, this is the first description of mutations in 23S rRNA genes or ribosomal proteins in macrolide-resistant S. pneumoniae strains.

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In vitro reconstitution of the GTPase-associated centre of the archaebacterial ribosome: the functional features observed in a hybrid form with Escherichia coli 50S subunits

Biochemical Journal ◽

10.1042/bj20060038 ◽

2006 ◽

Vol 396 (3) ◽

pp. 565-571 ◽

Cited By ~ 28

Author(s):

Takaomi Nomura ◽

Kohji Nakano ◽

Yasushi Maki ◽

Takao Naganuma ◽

Takashi Nakashima ◽

...

Keyword(s):

Escherichia Coli ◽

Ribosomal Proteins ◽

Synthetic Activity ◽

23S Rrna ◽

Elongation Factors ◽

Hybrid Form ◽

Pyrococcus Horikoshii ◽

E Coli ◽

Functional Features

We cloned the genes encoding the ribosomal proteins Ph (Pyrococcus horikoshii)-P0, Ph-L12 and Ph-L11, which constitute the GTPase-associated centre of the archaebacterium Pyrococcus horikoshii. These proteins are homologues of the eukaryotic P0, P1/P2 and eL12 proteins, and correspond to Escherichia coli L10, L7/L12 and L11 proteins respectively. The proteins and the truncation mutants of Ph-P0 were overexpressed in E. coli cells and used for in vitro assembly on to the conserved domain around position 1070 of 23S rRNA (E. coli numbering). Ph-L12 tightly associated as a homodimer and bound to the C-terminal half of Ph-P0. The Ph-P0·Ph-L12 complex and Ph-L11 bound to the 1070 rRNA fragments from the three biological kingdoms in the same manner as the equivalent proteins of eukaryotic and eubacterial ribosomes. The Ph-P0·Ph-L12 complex and Ph-L11 could replace L10·L7/L12 and L11 respectively, on the E. coli 50S subunit in vitro. The resultant hybrid ribosome was accessible for eukaryotic, as well as archaebacterial elongation factors, but not for prokaryotic elongation factors. The GTPase and polyphenylalanine-synthetic activity that is dependent on eukaryotic elongation factors was comparable with that of the hybrid ribosomes carrying the eukaryotic ribosomal proteins. The results suggest that the archaebacterial proteins, including the Ph-L12 homodimer, are functionally accessible to eukaryotic translation factors.

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Halobacterium cutirubrum ribosomes. Properties of the ribosomal proteins and ribonucleic acid

Biochemical Journal ◽

10.1042/bj1300103 ◽

1972 ◽

Vol 130 (1) ◽

pp. 103-110 ◽

Cited By ~ 51

Author(s):

L. P. Visentin ◽

C. Chow ◽

A. T. Matheson ◽

M. Yaguchi ◽

F. Rollin

Keyword(s):

Ribosomal Protein ◽

Ribosomal Proteins ◽

Soluble Fraction ◽

Ribosomal Subunit ◽

23S Rrna ◽

Core Particle ◽

Fractionation Procedure ◽

Additional Protein ◽

70S Ribosome ◽

Low Ionic Strength

1. The 30S ribosomal subunit of the extreme halophile Halobacterium cutirubrum is unstable and loses 75% of its ribosomal protein when the 70S ribosome is dissociated into the two subunits. A stable 30S subunit is obtained if the dissociation of the 70S particle is carried out in the presence of the soluble fraction. 2. A fractionation procedure was developed for the selective removal of groups of proteins from the 30S and 50S subunits. When the ribosomes, which are stable in 4m-K+ and 0.1m-Mg2+, were extracted with low-ionic-strength buffer 75–80% of the 30S proteins and 60–65% of the 50S proteins as well as the 5S rRNA were released. The proteins in this fraction are the most acidic of the H. cutirubrum ribosomal proteins. Further extraction with Li+–EDTA releases additional protein, leaving a core particle containing either 16S rRNA or 23S rRNA and about 5% of the total ribosomal protein. The amino acid composition, mobility on polyacrylamide gels at pH4.5 and 8.7, and the molecular-weight distribution of the various protein fractions were determined. 3. The s values of the rRNA are 5S, 16S and 23S. The C+G contents of the 16S and 23S rRNA were 56.1 and 58.8% respectively and these are higher than C+G contents of the corresponding Escherichia coli rRNA (53.8 and 54.1%).

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1582. Delafloxacin Activity Against Drug-Resistant Streptococcus pneumoniae, Haemophilus influenzae, Haemophilus parainfluenzae, and Moraxella catarrhalis from US Medical Centers (2014–2018)

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz360.1446 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S577-S578

Author(s):

Dee Shortridge ◽

Jennifer M Streit ◽

Michael D Huband ◽

Robert K Flamm

Keyword(s):

Streptococcus Pneumoniae ◽

Haemophilus Influenzae ◽

Moraxella Catarrhalis ◽

Active Agent ◽

The United States ◽

Multidrug Resistant ◽

In Vitro Susceptibility ◽

Amoxicillin Clavulanate ◽

Tract Infections

Abstract Background Delafloxacin (DLX) is an anionic fluoroquinolone (FQ) antimicrobial that was approved in 2017 by the United States (US) Food and Drug Administration for the treatment of acute bacterial skin and skin structure infections. DLX recently successfully completed a clinical trial for the treatment of community-acquired bacterial pneumonia (CABP). In the present study, in vitro susceptibility (S) results for DLX and comparator agents were determined for CABP pathogens including Streptococcus pneumoniae (SPN), Haemophilus influenzae (HI), H. parainfluenzae (HP) and Moraxella catarrhalis (MC) clinical isolates from US hospitals participating in the SENTRY Program during 2014–2018. Methods A total of 1,975 SPN, 1,128 HI, 684 MC, and 43 HP isolates were collected from community-acquired respiratory tract infections (CARTI) during 2014–2018 from US hospitals. Sites included only 1 isolate/patient/infection episode. Isolate identifications were confirmed at JMI Laboratories. Susceptibility testing was performed according to CLSI broth microdilution methodology, and CLSI (2019) breakpoints were applied where applicable. Other antimicrobials tested included levofloxacin (LEV) and moxifloxacin (MOX; not tested in 2015). Multidrug-resistant (MDR) SPN isolates were categorized as being nonsusceptible (NS) to amoxicillin-clavulanate, erythromycin, and tetracycline; other SPN phenotypes were LEV-NS or penicillin (PEN)-NS. β-Lactamase (BL) presence was determined for HI, HP, and MC. Results The activities of the 3 FQs are shown in the table. The most active agent against SPN was DLX, with the lowest MIC50/90 values of 0.015/0.03 mg/L. DLX activities were similar when tested against the MDR or PEN-NS for SPN phenotypes. LEV-NS isolates had DLX MIC50/90 results of 0.12/0.25 mg/L. DLX was the most active FQ against HI, HP, and MC. BL presence did not affect FQ MIC values for HI or MC; only 2 HP isolates were BL-positive. Conclusion DLX demonstrated potent in vitro antibacterial activity against SPN, HI, HP, and MC. DLX was active against MDR SPN that were NS to the agents commonly used as treatments for CABP. DLX had excellent activity against LEV-NS SPN. These data support the continued study of DLX as a potential treatment for CABP. Disclosures All authors: No reported disclosures.

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Targeted Disruption of the Ribosomal Protein S19 Gene Is Lethal Prior to Implantation

Molecular and Cellular Biology ◽

10.1128/mcb.24.9.4032-4037.2004 ◽

2004 ◽

Vol 24 (9) ◽

pp. 4032-4037 ◽

Cited By ~ 101

Author(s):

Hans Matsson ◽

Edward J. Davey ◽

Natalia Draptchinskaia ◽

Isao Hamaguchi ◽

Andreas Ooka ◽

...

Keyword(s):

Ribosomal Protein ◽

Ribosomal Proteins ◽

Normal Growth ◽

Lethal Effect ◽

Blastocyst Stage ◽

Targeted Disruption ◽

Gene Encoding ◽

Diamond Blackfan Anemia ◽

Associated Malformations ◽

Ribosomal Protein S19

ABSTRACT The ribosomal protein S19 (RPS19) is located in the small (40S) subunit and is one of 79 ribosomal proteins. The gene encoding RPS19 is mutated in approximately 25% of patients with Diamond-Blackfan anemia, which is a rare congenital erythroblastopenia. Affected individuals present with decreased numbers or the absence of erythroid precursors in the bone marrow, and associated malformations of various organs are common. We produced C57BL/6J mice with a targeted disruption of murine Rps19 to study its role in erythropoiesis and development. Mice homozygous for the disrupted Rps19 were not identified as early as the blastocyst stage, indicating a lethal effect. In contrast, mice heterozygous for the disrupted Rps19 allele have normal growth and organ development, including that of the hematopoietic system. Our findings indicate that zygotes which are Rps19 −/− do not form blastocysts, whereas one normal Rps19 allele in C57BL/6J mice is sufficient to maintain normal ribosomal and possibly extraribosomal functions.

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Antipneumococcal Activities of Two Novel Macrolides, GW 773546 and GW 708408, Compared with Those of Erythromycin, Azithromycin, Clarithromycin, Clindamycin, and Telithromycin

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.11.4103-4112.2004 ◽

2004 ◽

Vol 48 (11) ◽

pp. 4103-4112 ◽

Cited By ~ 9

Author(s):

Vlatka Matic ◽

Klaudia Kosowska ◽

Bulent Bozdogan ◽

Linda M. Kelly ◽

Kathy Smith ◽

...

Keyword(s):

Ribosomal Protein ◽

Protein Sequence ◽

Single Step ◽

23S Rrna ◽

Susceptible Parent ◽

Rrna Sequence ◽

Resistant Strains ◽

Resistant Mutants ◽

Rrna Sequences ◽

Selection Of

ABSTRACT The MICs of GW 773546, GW 708408, and telithromycin for 164 macrolide-susceptible and 161 macrolide-resistant pneumococci were low. The MICs of GW 773546, GW 708408, and telithromycin for macrolide-resistant strains were similar, irrespective of the resistance genotypes of the strains. Clindamycin was active against all macrolide-resistant strains except those with erm(B) and one strain with a 23S rRNA mutation. GW 773546, GW 708408, and telithromycin at two times their MICs were bactericidal after 24 h for 7 to 8 of 12 strains. Serial passages of 12 strains in the presence of sub-MICs yielded 54 mutants, 29 of which had changes in the L4 or L22 protein or the 23S rRNA sequence. Among the macrolide-susceptible strains, resistant mutants developed most rapidly after passage in the presence of clindamycin, GW 773546, erythromycin, azithromycin, and clarithromycin and slowest after passage in the presence of GW 708408 and telithromycin. Selection of strains for which MICs were ≥0.5 μg/ml from susceptible parents occurred only with erythromycin, azithromycin, clarithromycin, and clindamycin; 36 resistant clones from susceptible parent strains had changes in the sequences of the L4 or L22 protein or 23S rRNA. No mef(E) strains yielded resistant clones after passage in the presence of erythromycin and azithromycin. Selection with GW 773546, GW 708408, telithromycin, and clindamycin in two mef(E) strains did not raise the erythromycin, azithromycin, and clarithromycin MICs more than twofold. There were no change in the ribosomal protein (L4 or L22) or 23S rRNA sequences for 15 of 18 mutants selected for macrolide resistance; 3 mutants had changes in the L22-protein sequence. GW 773546, GW 708408, and telithromycin selected clones for which MICs were 0.03 to >2.0 μg/ml. Single-step studies showed mutation frequencies <5.0 × 10−10 to 3.5 × 10−7 for GW 773546, GW 708408, and telithromycin for macrolide-susceptible strains and 1.1 × 10−7 to >4.3 × 10−3 for resistant strains. The postantibiotic effects of GW 773546, GW 708408, and telithromycin were 2.4 to 9.8 h.

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Effect of Efflux on Telithromycin and Macrolide Susceptibility in Haemophilus influenzae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.50.3.893-898.2006 ◽

2006 ◽

Vol 50 (3) ◽

pp. 893-898 ◽

Cited By ~ 27

Author(s):

Tatiana Bogdanovich ◽

Bülent Bozdogan ◽

Peter C. Appelbaum

Keyword(s):

Haemophilus Influenzae ◽

Ribosomal Proteins ◽

Efflux Pump ◽

Efflux Pumps ◽

23S Rrna ◽

Clinical Strains ◽

Low Levels ◽

Presence And Absence

ABSTRACT This study investigated the presence of telithromycin and azithromycin efflux in 58 clinical strains of Haemophilus influenzae with various susceptibilities to macrolides, azalides, and ketolides. Efflux pumps were studied by measuring accumulation of radioactive [3H]telithromycin and [N-methyl-3H]azithromycin in the presence and absence of carbonyl m-chlorophenylhydrazone (CCCP), a protonophore. In 17 strains for which the telithromycin MICs were 0.06 to 0.5 μg/ml (azithromycin MICs, ≤0.06 to 0.125 μg/ml; clarithromycin MICs, ≤0.06 to 2 μg/ml), telithromycin and azithromycin accumulations were high without CCCP and not affected by its addition, which indicates absence of efflux. In 22 strains for which the telithromycin MICs were 0.25 to 4 μg/ml (azithromycin MICs, 0.25 to 1 μg/ml; clarithromycin MICs, 1 to 8 μg/ml), initially low levels of telithromycin accumulation became higher after addition of CCCP, indicating a functioning efflux pump. Nineteen strains for which the telithromycin MICs were ≥2 μg/ml had efflux as well as various mutations in ribosomal proteins L4, L22, and/or 23S rRNA (domains II and V). Of these 19 strains, the telithromycin MICs (≥8 μg/ml) for 17 of them were significantly raised (azithromycin, MICs 4 to >32 μg/ml; clarithromycin MICs, 8 to >32 μg/ml). From these results we conclude that telithromycin efflux with or without additional ribosomal alterations is present in all H. influenzae strains, except for those for which the telithromycin MICs were very low.

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