Sparfloxacin Resistance in Clinical Isolates ofStreptococcus pneumoniae: Involvement of Multiple Mutations in gyrA and parC Genes

ABSTRACT Antimicrobial susceptibility testing revealed among 150 clinical isolates of Streptococcus pneumoniae 4 pneumococcal isolates with resistance to fluoroquinolones (MIC of ciprofloxacin, ≥32 μg/ml; MIC of sparfloxacin, ≥16 μg/ml). Gene amplification and sequencing analysis of gyrA andparC revealed nucleotide changes leading to amino acid substitutions in both GyrA and ParC of all four fluoroquinolone-resistant isolates. In the case of strains 182 and 674 for which sparfloxacin MICs were 16 and 64 μg/ml, respectively, nucleotide changes were detected at codon 81 in gyrA and codon 79 in parC; these changes led to an Ser→Phe substitution in GyrA and an Ser→Phe substitution in ParC. Strains 354 and 252, for which sparfloxacin MICs were 128 μg/ml, revealed multiple mutations in both gyrA and parC. These strains exhibited nucleotide changes at codon 85 leading to a Glu→Lys substitution in GyrA, in addition to Ser-79→Tyr and Lys-137→Asn substitutions in ParC. Moreover, strain 252 showed additional nucleotide changes at codon 93, which led to a Trp→Arg substitution in GyrA. These results suggest that sparfloxacin resistance could be due to the multiple mutations in GyrA and ParC. However, it is possible that other yet unidentified mutations may also be involved in the high-level resistance to fluoroquinolones in S. pneumoniae.

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Diversity of Substitutions within or Adjacent to Conserved Amino Acid Motifs of Penicillin-Binding Protein 2X in Cephalosporin-Resistant Streptococcus pneumoniaeIsolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.5.1252 ◽

1999 ◽

Vol 43 (5) ◽

pp. 1252-1255 ◽

Cited By ~ 60

Author(s):

Yasuko Asahi ◽

Yasuo Takeuchi ◽

Kimiko Ubukata

Keyword(s):

Streptococcus Pneumoniae ◽

Amino Acid ◽

Three Dimensional ◽

Crystallographic Structure ◽

Amino Acid Substitutions ◽

Penicillin Binding Protein ◽

Amino Acid Motif ◽

Dna Fragment ◽

High Level ◽

Level Resistance

ABSTRACT The sequence of an approximately 1.1-kb DNA fragment of thepbp2x gene, which encodes the transpeptidase domain, was determined for 35 clinical isolates of Streptococcus pneumoniae for which the cefotaxime (CTX) MICs varied. Strains with substitutions within a conserved amino acid motif changing STMK to SAFK and a Leu-to-Val change just before the KSG motif were highly resistant to CTX (MIC, ≧2 μg/ml). Strains with substitutions adjacent to SSN or KSG motifs had low-level resistance. The amino acid substitutions were plotted on the three-dimensional crystallographic structure of the transpeptidase domain of PBP2X. Transformants containing pbp2x from strains with high-level CTX resistance increased the CTX MIC from 0.016 μg/ml to 0.5 to 1.0 μg/ml.

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Fluoroquinolone Resistance in Clinical Isolates ofStreptococcus pneumoniae: Contributions of Type II Topoisomerase Mutations and Efflux to Levels of Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.11.3049-3054.2000 ◽

2000 ◽

Vol 44 (11) ◽

pp. 3049-3054 ◽

Cited By ~ 91

Author(s):

Darrin J. Bast ◽

Donald E. Low ◽

Carla L. Duncan ◽

Laurie Kilburn ◽

Lionel A. Mandell ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Substitution ◽

Clinical Isolates ◽

Fluoroquinolone Resistance ◽

Amino Acid Substitutions ◽

Type Ii ◽

Low Level ◽

High Level ◽

The Impact ◽

Level Resistance

ABSTRACT We report on amino acid substitutions in the quinolone resistance-determining region of type II topisomerases and the prevalence of reserpine-inhibited efflux for 70 clinical isolates ofS. pneumoniae for which the ciprofloxacin MIC is ≥4 μg/ml and 28 isolates for which the ciprofloxacin MIC is ≤2 μg/ml. The amino acid substitutions in ParC conferring low-level resistance (MICs, 4 to 8 μg/ml) included Phe, Tyr, and Ala for Ser-79; Asn, Ala, Gly, Tyr, and Val for Asp-83; Asn for Asp-78; and Pro for Ala-115. Isolates with intermediate-level (MICs, 16 to 32 μg/ml) and high-level (MICs, 64 μg/ml) resistance harbored substitutions of Phe and Tyr for Ser-79 or Asn and Ala for Asp-83 in ParC and an additional substitution in GyrA which included either Glu-85-Lys (Gly) or Ser-81-Phe (Tyr). Glu-85-Lys was found exclusively in isolates with high-level resistance. Efflux contributed primarily to low-level resistance in isolates with or without an amino acid substitution in ParC. The impact of amino acid substitutions in ParE was minimal, and no substitutions in GyrB were identified.

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Impact of Drug Resistance-Associated Amino Acid Changes in HIV-1 Subtype C on Susceptibility to Newer Nonnucleoside Reverse Transcriptase Inhibitors

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04215-14 ◽

2014 ◽

Vol 59 (2) ◽

pp. 960-971 ◽

Cited By ~ 34

Author(s):

Adriaan E. Basson ◽

Soo-Yon Rhee ◽

Chris M. Parry ◽

Ziad El-Khatib ◽

Salome Charalambous ◽

...

Keyword(s):

Amino Acid ◽

Reverse Transcriptase ◽

Resistance Testing ◽

Amino Acid Substitutions ◽

Subtype C ◽

Nnrti Resistance ◽

High Level ◽

Hiv 1 ◽

Phenotypic Susceptibility ◽

Level Resistance

ABSTRACTThe objective of this study was to assess the phenotypic susceptibility of HIV-1 subtype C isolates, with nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance-associated amino acid changes, to newer NNRTIs. A panel of 52 site-directed mutants and 38 clinically derived HIV-1 subtype C clones was created, and the isolates were assessed for phenotypic susceptibility to etravirine (ETR), rilpivirine (RPV), efavirenz (EFV), and nevirapine (NVP) in anin vitrosingle-cycle phenotypic assay. The amino acid substitutions E138Q/R, Y181I/V, and M230L conferred high-level resistance to ETR, while K101P and Y181I/V conferred high-level resistance to RPV. Y181C, a major NNRTI resistance-associated amino acid substitution, caused decreased susceptibility to ETR and, to a lesser extent, RPV when combined with other mutations. These included N348I and T369I, amino acid changes in the connection domain that are not generally assessed during resistance testing. However, the prevalence of these genotypes among subtype C sequences was, in most cases, <1%. The more common EFV/NVP resistance-associated substitutions, such as K103N, V106M, and G190A, had no major impact on ETR or RPV susceptibility. The low-level resistance to RPV and ETR conferred by E138K was not significantly enhanced in the presence of M184V/I, unlike for EFV and NVP. Among patient samples, 97% were resistant to EFV and/or NVP, while only 24% and 16% were resistant to ETR and RPV, respectively. Overall, only a few, relatively rare NNRTI resistance-associated amino acid substitutions caused resistance to ETR and/or RPV in an HIV-1 subtype C background, suggesting that these newer NNRTIs would be effective in NVP/EFV-experienced HIV-1 subtype C-infected patients.

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Canadian national survey of prevalence of antimicrobial resistance among clinical isolates of Streptococcus pneumoniae. Canadian Bacterial Surveillance Network.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.9.2190 ◽

1996 ◽

Vol 40 (9) ◽

pp. 2190-2193 ◽

Cited By ~ 49

Author(s):

A E Simor ◽

M Louie ◽

D E Low

Keyword(s):

Streptococcus Pneumoniae ◽

Antimicrobial Agents ◽

Clinical Isolates ◽

Beta Lactam ◽

Surveillance Network ◽

Antibiotic Resistant ◽

Intermediate Resistance ◽

Resistant Strains ◽

High Level ◽

Level Resistance

The antimicrobial susceptibilities of 1,089 clinical isolates of Streptococcus pneumoniae obtained from 39 laboratories across Canada between October 1994 and August 1995 were determined. A total of 91 isolates (8.4%) demonstrated intermediate resistance (MIC, 0.1 to 1.0 microgram/ml) and 36 (3.3%) had high-level resistance (MIC, > or = 2.0 micrograms/ml) to penicillin. Penicillin-resistant strains were more likely to have been recovered from normally sterile sites (P = 0.005) and to be cross-resistant to several beta-lactam and non-beta-lactam antimicrobial agents (P < 0.05). These results indicate that there has been a recent significant increase in the prevalence of antibiotic-resistant S. pneumoniae in Canada.

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Genetic Analyses of Mutations Contributing to Fluoroquinolone Resistance in Clinical Isolates ofStreptococcus pneumoniae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.12.3517-3523.2001 ◽

2001 ◽

Vol 45 (12) ◽

pp. 3517-3523 ◽

Cited By ~ 56

Author(s):

L. M. Weigel ◽

G. J. Anderson ◽

R. R. Facklam ◽

F. C. Tenover

Keyword(s):

Amino Acid ◽

Antimicrobial Agents ◽

Clinical Isolates ◽

Fluoroquinolone Resistance ◽

Cross Resistance ◽

Genetic Analyses ◽

High Level ◽

Susceptibility Profiles ◽

Level Resistance

ABSTRACT Twenty-one clinical isolates of Streptococcus pneumoniae showing reduced susceptibility or resistance to fluoroquinolones were characterized by serotype, antimicrobial susceptibility, and genetic analyses of the quinolone resistance-determining regions (QRDRs) of gyrA,gyrB, parC, and parE. Five strains were resistant to three or more classes of antimicrobial agents. In susceptibility profiles for gatifloxacin, gemifloxacin, levofloxacin, moxifloxacin, ofloxacin, sparfloxacin, and trovafloxacin, 14 isolates had intermediate- or high-level resistance to all fluoroquinolones tested except gemifloxacin (no breakpoints assigned). Fluoroquinolone resistance was not associated with serotype or with resistance to other antimicrobial agents. Mutations in the QRDRs of these isolates were more heterogeneous than those previously reported for mutants selected in vitro. Eight isolates had amino acid changes at sites other than ParC/S79 and GyrA/S81; several strains contained mutations in gyrB, parE, or both loci. Contributions to fluoroquinolone resistance by individual amino acid changes, including GyrB/E474K, ParE/E474K, and ParC/A63T, were confirmed by genetic transformation of S. pneumoniae R6. Mutations in gyrB were important for resistance to gatifloxacin but not moxifloxacin, and mutation of gyrAwas associated with resistance to moxifloxacin but not gatifloxacin, suggesting differences in the drug-target interactions of the two 8-methoxyquinolones. The positions of amino acid changes within the four genes affected resistance more than did the total number of QRDR mutations. However, the effect of a specific mutation varied significantly depending on the agent tested. These data suggest that the heterogeneity of mutations will likely increase as pneumococci are exposed to novel fluoroquinolone structures, complicating the prediction of cross-resistance within this class of antimicrobial agents.

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Novel Ser79Leu and Ser81Ile Substitutions in the Quinolone Resistance-Determining Regions of ParC Topoisomerase IV and GyrA DNA Gyrase Subunits from Recent Fluoroquinolone-Resistant Streptococcus pneumoniae Clinical Isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.6.2479-2486.2005 ◽

2005 ◽

Vol 49 (6) ◽

pp. 2479-2486 ◽

Cited By ~ 3

Author(s):

Nataliya Korzheva ◽

Todd A. Davies ◽

Raul Goldschmidt

Keyword(s):

Streptococcus Pneumoniae ◽

Amino Acid ◽

Dna Gyrase ◽

Quinolone Resistance ◽

Clinical Isolates ◽

Fluoroquinolone Resistance ◽

Amino Acid Substitutions ◽

Topoisomerase Iv ◽

Isogenic Strains ◽

New Mutations

ABSTRACT Resistance of Streptococcus pneumoniae to fluoroquinolones is caused predominantly by amino acid substitutions at positions Ser79 of ParC and Ser81 of GyrA to either Phe or Tyr encoded in the quinolone resistance-determining regions of the parC topoisomerase IV and gyrA DNA gyrase genes. Analysis of highly resistant clinical isolates identified novel second-step substitutions, Ser79Leu (ParC) and Ser81Ile (GyrA). To determine contributions of these new mutations to fluoroquinolone resistance either alone or in combination with other Ser79/81 alleles, the substitutions Ser79Leu/Phe/Tyr in ParC and Ser81Ile/Phe/Tyr in GyrA were introduced into the R6 background, resulting in 15 isogenic strains. Their level of fluoroquinolone resistance was determined by susceptibility testing for ciprofloxacin, levofloxacin, moxifloxacin, gatifloxacin, gemifloxacin, garenoxacin, and norfloxacin. Leu79 and Ile81 alone as well as 79/81Phe/Tyr substitutions did not contribute significantly to resistance, with fluoroquinolone MICs increasing two- to fourfold compared to wild type for all agents tested. Fluoroquinolone MICs for double transformants ParC Ser79Phe/Tyr/Leu-GyrA Ser81Phe/Tyr were uniformly increased by 8- to 64-fold regardless of pairs of amino acid substitutions. However, combinations including Ile81 conferred two- to fourfold-higher levels of resistance than did combinations including any other Ser81 GyrA substitution, thus demonstrating the differential effects of diverse amino acid substitutions at particular hotspots on fluoroquinolone MICs.

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Antimicrobial Susceptibilities of Group B Streptococci Isolated from Patients with Invasive Disease: 10-Year Perspective

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.12.3623-3624.2001 ◽

2001 ◽

Vol 45 (12) ◽

pp. 3623-3624 ◽

Cited By ~ 45

Author(s):

David R. Murdoch ◽

L. Barth Reller

Keyword(s):

Antimicrobial Susceptibility ◽

Susceptibility Testing ◽

Invasive Disease ◽

Antimicrobial Susceptibility Testing ◽

Streptococcal Infections ◽

Group B Streptococci ◽

Treatment And Prevention ◽

Group B ◽

High Level ◽

Level Resistance

ABSTRACT Antimicrobial susceptibility testing of 192 group B streptococcal isolates from patients with invasive disease demonstrated that 31 (16%) were resistant to erythromycin and 17 (9%) were resistant to clindamycin. One isolate demonstrated high-level resistance to streptomycin, but none was highly resistant to gentamicin. Erythromycin and clindamycin are no longer reliable empirical alternatives to penicillin for the treatment and prevention of group B streptococcal infections.

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Activities of Newer Fluoroquinolones against Streptococcus pneumoniae Clinical Isolates Including Those with Mutations in the gyrA, parC, and parELoci

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.2.329 ◽

1999 ◽

Vol 43 (2) ◽

pp. 329-334 ◽

Cited By ~ 79

Author(s):

J. H. Jorgensen ◽

L. M. Weigel ◽

M. J. Ferraro ◽

J. M. Swenson ◽

F. C. Tenover

Keyword(s):

Streptococcus Pneumoniae ◽

Amino Acid ◽

Amino Acid Sequences ◽

Clinical Isolates ◽

Single Amino Acid ◽

Single Mutation ◽

Topoisomerase Iv ◽

In Vitro Susceptibility ◽

Resistant Strains ◽

Level Resistance

ABSTRACT Resistance to fluoroquinolone (FQ) antibiotics inStreptococcus pneumoniae has been attributed primarily to specific mutations in the genes for DNA gyrase (gyrA andgyrB) and topoisomerase IV (parC andparE). Resistance to some FQs can result from a single mutation in one or more of the genes encoding these essential enzymes. A group of 160 clinical isolates of pneumococci was examined in this study, including 36 ofloxacin-resistant isolates (MICs, ≥8 μg/ml) recovered from patients in North America, France, and Belgium. The susceptibilities of all isolates to clinafloxacin, grepafloxacin, levofloxacin, sparfloxacin, and trovafloxacin were examined by the National Committee for Clinical Laboratory Standards reference broth microdilution and disk diffusion susceptibility testing methods. Among the ofloxacin-resistant strains, 32 of 36 were also categorized as resistant to levofloxacin, 35 were resistant to sparfloxacin, 29 were resistant to grepafloxacin, and 19 were resistant to trovafloxacin. In vitro susceptibility to clinafloxacin appeared to be least affected by resistance to the other FQs. Eight isolates with high- and low-level resistance to the newer FQs were selected for DNA sequence analysis of the quinolone resistance-determining regions (QRDRs) ofgyrA, gyrB, parC, andparE. The DNA and the inferred amino acid sequences of the resistant strains were compared with the analogous sequences of reference strain S. pneumoniae ATCC 49619 and FQ-susceptible laboratory strain R6. Reduced susceptibilities to grepafloxacin and sparfloxacin (MICs, 1 to 2 μg/ml) and trovafloxacin (MICs, 0.5 to 1 μg/ml) were associated with either a mutation inparC that led to a single amino acid substitution (Ser-79 to Phe or Tyr) or double mutations that involved the genes for both GyrA (Ser-81 to Phe) and ParE (Asp-435 to Asn). High-level resistance to all of the compounds except clinafloxacin was associated with two or more amino acid substitutions involving both GyrA (Ser-81 to Phe) and ParC (Ser-79 to Phe or Ser-80 to Pro and Asp-83 to Tyr). No mutations were observed in the gyrB sequences of resistant strains. These data indicate that mutations in pneumococcal gyrA,parC, and parE genes all contribute to decreased susceptibility to the newer FQs, and genetic analysis of the QRDR of a single gene, either gyrA or parC, is not predictive of pneumococcal resistance to these agents.

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