Fluoroquinolone Resistance In Streptococcus Research Articles

Allele-specific collateral and fitness effects determine the dynamics of fluoroquinolone resistance evolution

Collateral sensitivity (CS), which arises when resistance to one antibiotic increases sensitivity toward other antibiotics, offers treatment opportunities to constrain or reverse the evolution of antibiotic resistance. The applicability of CS-informed treatments remains uncertain, in part because we lack an understanding of the generality of CS effects for different resistance mutations, singly or in combination. Here, we address this issue in the gram-positive pathogen Streptococcus pneumoniae by measuring collateral and fitness effects of clinically relevant gyrA and parC alleles and their combinations that confer resistance to fluoroquinolones. We integrated these results in a mathematical model that allowed us to evaluate how different in silico combination treatments impact the dynamics of resistance evolution. We identified common and conserved CS effects of different gyrA and parC alleles; however, the spectrum of collateral effects was unique for each allele or allelic pair. This indicated that allelic identity can impact the evolutionary dynamics of resistance evolution during monotreatment and combination treatment. Our model simulations, which included the experimentally derived antibiotic susceptibilities and fitness effects, and antibiotic-specific pharmacodynamics revealed that both collateral and fitness effects impact the population dynamics of resistance evolution. Overall, we provide evidence that allelic identity and interactions can have a pronounced impact on collateral effects to different antibiotics and suggest that these need to be considered in models examining CS-based therapies.

Fluoroquinolone resistance in Streptococcus pneumoniae isolates in Germany from 2004–2005 to 2014–2015

Streptococcus pneumoniae is a major cause of bacterial pneumonia, sepsis and meningitis worldwide. Prevalence of levofloxacin-resistant S. pneumoniae isolates in Germany and associated mutations in the quinolone resistance determining regions (QRDRs), as well as serotype distribution and multi locus sequence types (MLST) are shown. 21,764 invasive S. pneumoniae isolates from Germany, isolated in the epidemiological seasons from 2004/05 to 2014/15 were analyzed at the German National Reference Centre for Streptococci (GNRCS) for their levofloxacin resistance by micro broth dilution method. All resistant (minimal inhibitory concentration (MIC) ≥8μg/ml) and intermediate (MIC >2μg/ml and <8μg/ml) isolates were selected for the present study. Additionally, 29 susceptible isolates were randomly selected. A total of ninety isolates were tested for their levofloxacin-MIC by Etest, their serotype and sequence type, as well as for point-mutations at the QRDRs in the genes parC, parE, gyrA and gyrB. Twenty-five isolates exhibited levofloxacin MICs <2μg/ml (Etest) and no mutations in the QRDRs. Four isolates with MICs=2μg/ml had one mutation in parC; isolates with MICs >2μg/ml all had one or more mutations in the QRDRs. Four of nine intermediate isolates had a mutation in either parC or gyrA, and four isolates had mutations in both parC and gyrB. One isolate had mutations in both parC and gyrA. All isolates with MICs ≥8μg/ml (52) had mutations in both topoisomerase IV and gyrase. Serotypes associated with levofloxacin resistance shifted from a majority of PCV13 serotypes before the introduction of the PCV13 vaccine towards non-PCV serotypes. Resistant isolates were almost exclusively found among adults (98.1%).

Insights into the evolutionary trajectories of fluoroquinolone resistance in Streptococcus pneumoniae.

Fluoroquinolone resistance in Streptococcus pneumoniae typically arises through specific site mutations, but dynamic variation of mutations in the resistance evolution and interaction among these mutations have not been clearly demonstrated. The objectives of this study were to investigate the dynamics of allele frequency in populations evolved under fluoroquinolone pressure and pervasive interactions among mutations present in the evolutionary trajectories. Thirty-three evolved populations were obtained by serial passages in the presence of antibiotic pressure and these populations were sequenced by using the Paired-End Illumina method. Mutants that occurred in the evolutionary trajectories were constructed by transforming the parental strain with PCR fragments containing corresponding mutations. The number of target mutations increased progressively, consistent with phenotypic adaptation to moxifloxacin and levofloxacin. However, more mutations are required for high-level resistance to moxifloxacin than levofloxacin. Pervasive interactions, including positive epistasis between mutations, play a role in the evolutionary trajectories of resistance to the two drugs. Two mutations (R447C and P454S) in gyrB were identified to confer 2-fold increases in resistance to moxifloxacin and levofloxacin based on the background of the double mutant S81F/S79F in parC. Moreover, the dynamics of allele frequency in evolved populations was revealed and found to be directly correlated with the resistance levels of evolved populations. Clonal interference among alleles of mutations contributed to the molecular dynamics of resistance evolution. Our results provide novel insights into the evolutionary trajectories of resistance to fluoroquinolones and may serve as a theoretical basis for predicting resistance development and provide references for the clinical use of these drugs.

A novel gene amplification causes upregulation of the PatAB ABC transporter and fluoroquinolone resistance in Streptococcus pneumoniae.

Overexpression of the ABC transporter genes patA and patB confers efflux-mediated fluoroquinolone resistance in Streptococcus pneumoniae and is also linked to pneumococcal stress responses. Although upregulation of patAB has been observed in many laboratory mutants and clinical isolates, the regulatory mechanisms controlling expression of these genes are unknown. In this study, we aimed to identify the cause of high-level constitutive overexpression of patAB in M184, a multidrug-resistant mutant of S. pneumoniae R6. Using a whole-genome transformation and sequencing approach, we identified a novel duplication of a 9.2-kb region of the M184 genome which included the patAB genes. This duplication did not affect growth and was semistable with a low segregation rate. The expression levels of patAB in M184 were much higher than those that could be fully explained by doubling of the gene dosage alone, and inactivation of the first copy of patA had no effect on multidrug resistance. Using a green fluorescent protein reporter system, increased patAB expression was ascribed to transcriptional read-through from a tRNA gene upstream of the second copy of patAB. This is the first report of a large genomic duplication causing antibiotic resistance in S. pneumoniae and also of a genomic duplication causing antibiotic resistance by a promoter switching mechanism.

Advances in antibiotic therapy for community-acquired pneumonia

Community-acquired pneumonia (CAP) is a major public health problem all over the world. The increasing number of antibiotic-resistant bacteria is a matter of concern for physicians when choosing antibiotic treatment in patients with CAP. This review focuses on the current recommendations of antibiotic treatment, recent information concerning antibiotic resistance of pathogens, and the advances in antibiotic therapy in the field of CAP. A significant increase in the frequency of resistance to the antibiotics commonly used against causative pathogens of CAP, such as β-lactams or macrolides, has been reported in recent years. At present, the prevalence of fluoroquinolone resistance in Streptococcus pneumoniae remains low. Community-acquired methicillin-resistant Staphylococcus aureus and influenza A(H1N1)pdm09 have been reported as causes of severe CAP. Several newly-developed antibiotics, including cepholosporins, ketolides and quinolones, present marked activity in vitro against the main causative pathogens of CAP. Many randomized controlled trials have demonstrated equivalent efficacy of the newer antibiotics compared with conventional antimicrobial therapy in mild-to-moderate CAP. An increase of resistance to the antibiotics used in CAP has been documented over the years. Several new antibiotics have been developed for treating CAP, with promising results. However, data regarding their efficacy and safety in patients with severe CAP are lacking.

Molecular characterization of quinolone-insensitive Streptococcus pneumoniae isolates from Japanese patients

Fluoroquinolone resistance in Streptococcus pneumoniae has become a growing concern. Using S. pneumoniae isolates (n=61) for which the minimum inhibitory concentration (MIC) of levofloxacin was not less than 1μg/ml, we investigated the susceptibility of S. pneumoniae isolates to other fluoroquinolones (ciprofloxacin, pazufloxacin, moxifloxacin, garenoxacin, and sitafloxacin) and sequenced the quinolone resistance-determining regions of two topoisomerase genes, parC and gyrA, in these isolates to identify mutations. As the number of missense mutations increased, the MIC values for each drug increased. However, moxifloxacin, garenoxacin, and sitafloxacin showed potent activities against the isolates, while the MICs of ciprofloxacin and pazufloxacin were higher than the MICs of levofloxacin.

Evaluation of Melting Curve Analysis for Screening the Most Prevalent Mutations in Topoisomerase Genes from Streptococcus pneumoniae

In a recently published article, Fukushima et al. ([3][1]) described a novel PCR-melting curve analysis (PCR-MCA) method for rapid screening of the most prevalent point mutations related to fluoroquinolone resistance in Streptococcus pneumoniae . In their study, they included 22 levofloxacin (LVX)-

The Lys137Asn Mutation as Surrogate Marker for Developing Fluoroquinolone Resistance in Streptococcus pneumoniae?

(2007). The Lys137Asn Mutation as Surrogate Marker for Developing Fluoroquinolone Resistance in Streptococcus pneumoniae? Journal of Chemotherapy: Vol. 19, No. 6, pp. 750-752.

The contribution of efflux pumps to quinolone resistance in Streptococcus pneumoniae clinical isolates

Active efflux has been involved in fluoroquinolone resistance in Streptococcus pneumoniae. While only one efflux machinery has been well described (PmrA), the eventual existence of other pumps than PmrA has been suggested. The actual role of active quinolone efflux in producing ciprofloxacin resistance was examined by means of several methods. Strains HUB 2375 and HUB 3073 are clinical isolates resistant to fluoroquinolones. Since no mutations in the quinolone resistance-determining regions of these strains were found, resistance to fluoroquinolones should be attributed only to efflux. Strain R6 was transformed by DNA from both HUB 2375 and HUB 3073. Selected transformants were chosen for knockout of pmrA genes by polymerase chain reaction ligation mutagenesis in order to study the actual role and the degree of involvement of PmrA in determining ciprofloxacin resistance. Results strongly supported the hypothesis that efflux pumps other than PmrA are involved in fluoroquinolone resistance in clinical strains of pneumococci.

Call for the international adoption of microbiological breakpoints for fluoroquinolones and Streptococcus pneumoniae

The use of current Clinical and Laboratory Standards Institute levofloxacin breakpoints for assessing fluoroquinolone resistance in Streptococcus pneumoniae is inadequate for detecting isolates possessing first-step parC mutations. Consequently, the risk for development of fluoroquinolone resistance is greatly underestimated. Adopting microbiological breakpoints for fluoroquinolones and S. pneumoniae, where parC mutations are rare in susceptible isolates, more accurately describes the emergence of resistance and may help to prevent a number of future fluoroquinolone treatment failures. Additionally, we propose that the use of a second fluoroquinolone marker, such as ciprofloxacin, offers the best prediction for detecting an isolate possessing a first-step parC mutation.

In Vivo Development of High-Level Fluoroquinolone Resistance in Streptococcus pneumoniae in Chronic Obstructive Pulmonary Disease

Patients with chronic obstructive pulmonary disease are generally subjected to multiple regimens of antimicrobial treatment. The development of high-level levofloxacin resistance (i.e., a minimum inhibitory concentration >8 mu g/mL) in 8 patients whose previous pneumococcal isolates showed susceptibility is described. Molecular methods were used to characterize the strains and to study the sequential changes in fluoroquinolone targets.

Relative Fitness of Fluoroquinolone-resistantStreptococcus pneumoniae

Fluoroquinolone resistance in Streptococcus pneumoniae is primarily mediated by point mutations in the quinolone resistance–determining regions of gyrA and parC. Antimicrobial resistance mutations in housekeeping genes often decrease fitness of microorganisms. To investigate the fitness of quinolone-resistant S. pneumoniae (QRSP), the relative growth efficiencies of 2 isogenic QRSP double mutants were compared with that of their fluoroquinolone-susceptible parent, EF3030, by using murine nasopharyngeal colonization and pneumonia models. Strains containing the GyrA: Ser81Phe, ParC: Ser79Phe double mutations, which are frequently seen in clinical QRSP, competed poorly with EF3030 in competitive colonization or competitive lung infections. However, they efficiently produced lung infection even in the absence of EF3030. The strain containing the GyrA: Ser81Phe, ParC: Ser79Tyr double mutations, which is seen more frequently in laboratory-derived QRSP than in clinical QRSP, demonstrated reduced nasal colonization in competitive or noncompetitive lung infections. However, the strain was equally able to cause competitive or noncompetitive lung infections as well as EF3030.

Interspecies Recombination in Type II Topoisomerase Genes Is Not a Major Cause of Fluoroquinolone Resistance in Invasive Streptococcus pneumoniae Isolates in the United States

Mutations in the topoisomerase type II enzymes account for fluoroquinolone resistance in Streptococcus pneumoniae. These mutations can arise spontaneously or be transferred by intraspecies or interspecies recombination, primarily with viridans streptococci. We analyzed the nucleotide sequences of the quinolone resistance-determining regions of 49 invasive levofloxacin-resistant pneumococcal isolates and did not find any evidence for interspecies recombination.

Stability of Fluoroquinolone Resistance in Streptococcus pneumoniae Clinical Isolates and Laboratory-Derived Mutants

The stability of fluoroquinolone resistance in Streptococcus pneumoniae was evaluated in laboratory-derived mutants and clinical isolates. Isolates with various genotypes and phenotypes were subcultured for 20 days on antibiotic-free media and were monitored by E-tests to identify any alterations in resistance. Fluoroquinolone resistance mechanisms, whether efflux or chromosomally mediated, remained stable in both clinical and laboratory-derived mutants.

Molecular characterization and antimicrobial susceptibility of fluoroquinolone-resistant or -susceptible Streptococcus pneumoniae from Hong Kong.

Fluoroquinolone resistance in Streptococcus pneumoniae isolated from Hong Kong as part of Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin 1999/2000 was found to be due to the spread of the Spain(23F)-1 clone (mainly a Spain(23F)-1-14 variant). All the isolates were multidrug resistant but were susceptible to quinupristin-dalfopristin, linezolid, and telithromycin. The Spain(23F)-1 clone also occurred among antimicrobial-susceptible isolates, which suggests that the primary source of this clone may have been Asia rather than Iberia.

Evidence for efflux pumps, other than PmrA, associated with fluoroquinolone resistance in Streptococcus pneumoniae

Fluoroquinolone resistance in pneumococci is known to be associated with the efflux pump, PmrA. However, there may be other efflux systems that also cause drug resistance. Two types of mutants were studied. The efflux phenotype from mutants selected by sub-MIC levofloxacin or gemifloxacin was transformed into R6. These transformants did not show increased pmrA transcripts in Northern blots; insertional inactivation of pmrA in the transformants did not abolish the efflux phenotype. A second set of efflux phenotype mutants was selected in R6:cat by ethidium bromide but not by norfloxacin; accumulation of ethidium bromide in the one among these mutants studied was reduced in comparison to its parent. This evidence suggests that systems other than PmrA can contribute to efflux-mediated resistance in pneumococci.

Screening of large numbers of Streptococcus pneumoniae isolates for mutations associated with fluoroquinolone resistance using an oligonucleotide probe assay.

Mutations at a relatively small number of sites in parC, parE and gyrA account for most of the fluoroquinolone resistance in Streptococcus pneumoniae clinical isolates. A high throughput oligonucleotide probe assay was developed to screen for mutations in the quinolone-resistance determining region (QRDR) of parC (Ser79), gyrA (Ser81) and parE (Asp435) of Streptococcus pneumoniae. Eight oligonucleotide probes (17mers) were used in the presence of tetramethyl ammonium chloride so that the melting temperature was dependent on length and not on base composition. Using this assay it was possible to accurately detect QRDR mutations from several hundred S. pneumoniae clinical isolates that were grown on nylon membranes.

Screening of large numbers of Streptococcus pneumoniae isolates for mutations associated with fluoroquinolone resistance using an oligonucleotide probe assay

Mutations at a relatively small number of sites in parC, parE and gyrA account for most of the fluoroquinolone resistance in Streptococcus pneumoniae clinical isolates. A high throughput oligonucleotide probe assay was developed to screen for mutations in the quinolone-resistance determining region (QRDR) of parC (Ser79), gyrA (Ser81) and parE (Asp435) of Streptococcus pneumoniae. Eight oligonucleotide probes (17mers) were used in the presence of tetramethyl ammonium chloride so that the melting temperature was dependent on length and not on base composition. Using this assay it was possible to accurately detect QRDR mutations from several hundred S. pneumoniae clinical isolates that were grown on nylon membranes.

Fluoroquinolone resistance in Streptococcus pneumoniae in United States since 1994-1995.

The in vitro activities of ciprofloxacin, levofloxacin, gatifloxacin, and moxifloxacin against a large collection of clinical isolates of Streptococcus pneumoniae (n = 4,650) obtained over a 5-year period, 1994-1995 through 1999-2000, were assessed as part of a longitudinal multicenter U.S. surveillance study of antimicrobial resistance. Three sampling periods were used during this investigation, the winter seasons of 1994-1995, 1997-1998, and 1999-2000; and 1,523, 1,596 and 1,531 isolates were collected during these three periods, respectively. The overall rank order of activity of the four fluoroquinolones examined in this study was moxifloxacin > gatifloxacin > levofloxacin = ciprofloxacin, in which moxifloxacin (MIC at which 90% of isolates are inhibited [MIC(90)], 0.25 microg/ml; modal MIC, 0.12 microg/ml) was twofold more active than gatifloxacin (MIC(90), 0.5 microg/ml; modal MIC, 0.25 microg/ml), which in turn was fourfold more active than either levofloxacin (MIC(90), 1 microg/ml; modal MIC, 1 microg/ml) or ciprofloxacin (MIC(90), 2 microg/ml; modal MIC, 1 microg/ml). Changes in the in vitro activities of fluoroquinolones against S. pneumoniae strains in the United States over the 5-year period of the survey were assessed by comparing the MIC frequency distributions of the study drugs against the isolates obtained during the three sampling periods encompassing this investigation. These comparisons revealed no evidence of changes in the in vitro activities of the fluoroquinolones. In addition, the percentages of isolates in the three sampling periods for which MICs were above the resistance breakpoints were compared. Low percentages of resistant strains were detected, and there was no evidence of resistance rate changes over time. For example, by use of a ciprofloxacin MIC of > or = 4 microg/ml to define resistance, the proportions of isolates from the three sampling periods for which MICs were at or above this breakpoint were 1.2, 1.6, and 1.4%, respectively. A total of 164 unique isolates (n = 58 from 1994-1995, 65 from 1997-1998, and 42 from 1999-2000) were examined for evidence of mutations in the quinolone resistance-determining regions (QRDRs) of the parC and the gyrA genes. Forty-nine isolates harbored at least one mutation in the QRDRs of one or both genes (1994-1995, n = 15; 1997-1998, n = 19; 1999-2000, n = 15). Among the 4,650 isolates of S. pneumoniae examined in the study, we estimated that 0.3% had mutations in both the parC and gyrA loci. The majority of mutations (67.3% of the mutations in 49 isolates with mutations) were amino acid substitutions in the parC locus only. Four isolates had a mutation in the gyrA locus only, and 12 isolates had mutations in both genes (8.2 and 24.5% of isolates with mutations, respectively). There was no significant difference in the number of isolates with parC and/or gyrA mutations detected during each study period. Finally, because of the magnitude of the study, we had reasonably large numbers of pneumococcal isolates with genotypically defined mechanisms of fluoroquinolone resistance and were thus able to determine the effects of specific resistance mutations on the activities of different fluoroquinolones. In general, isolates with mutations in parC only were resistant to ciprofloxacin but remained susceptible to levofloxacin, gatifloxacin, and moxifloxacin, whereas isolates with mutations in gyrA only and isolates with mutations in both parC and gyrA were resistant to all four fluoroquinolones tested.

Activity of telithromycin against 26 quinolone-resistant pneumococci with known quinolone-resistance mechanisms

NCCLS agar dilution was used to test activity of telithromycin compared to clarithromycin, penicillin G, ciprofloxacin, levofloxacin, sparfloxacin and moxifloxacin against 26 pneumococci with defined quinolone resistance (type II topoisomerase and efflux) mechanisms. Thirteen strains were penicillin susceptible, six intermediate and seven resistant. Clarithromycin resistance (mef and/or erm) was seen in eight strains. Ciprofloxacin MICs (mg/L) were 8-64 compared to 1-32 (levofloxacin), 0.5 . or = 32 (sparfloxacin) and 0.125-4 (moxifloxacin). Telithromycin MIC50 and MIC90 values (mg/L) were 0.016 and 0.25, with only one strain having an MIC of 2 mg/L.