Cross-resistance, relatedness and allele analysis of fluoroquinolone-resistant US clinical isolates of Streptococcus pneumoniae (1998–2000)

Todd A. Davies1,*, Raul Goldschmidt1, Sharon Pfleger2, Mike Loeloff1, Karen Bush1, Daniel F. Sahm3 and Alan Evangelista2

1 Johnson & Johnson Pharmaceutical Research and Development, L.L.C., Room B201C, 1000 Route 202, Raritan, NJ 08869; 2 Ortho-McNeil Pharmaceutical, Inc., Raritan, NJ 08869; 3 Focus Technologies, Herndon, VA 20171, USA

Received 7 March 2003; returned 6 April 2003; revised 24 April 2003; accepted 29 April 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: To detect relatedness among 68 (0.5%) of 13 795 US clinical isolates of Streptococcus pneumoniae from the TRUST 3 (1998–1999) and TRUST 4 (1999–2000) surveillance studies that were resistant to levofloxacin (MIC >= 8 mg/L).

Methods: All levofloxacin-resistant isolates were analysed by broth microdilution reference method for susceptibility to four fluoroquinolones, DNA sequencing of quinolone resistance determining region (QRDR) of topoisomerase IV and DNA gyrase genes, serotyping, and pulsed-field gel electrophoresis (PFGE).

Results: All levofloxacin-resistant isolates were ciprofloxacin resistant (MIC >= 4 mg/L, FDA breakpoint) and non-susceptible to gatifloxacin (MIC >= 2 mg/L); 62 were non-susceptible to moxifloxacin (MIC >= 2 mg/L). Resistant isolates were in 48 (20%) of 238 institutions in 29 states. Three institutions had levofloxacin-resistant isolates in both surveillance studies. Among the resistant isolates were 17 serotypes and 48 different PFGE patterns. Fourteen isolates had PFGE patterns closely related to the Spain23F-1 clone; one strain had a PFGE pattern closely related to the French9V-3 clone. All levofloxacin-resistant isolates had two or more mutations within the QRDR of parC, parE, gyrA and gyrB.

Conclusions: US levofloxacin-resistant S. pneumoniae isolates were rare and most were unrelated with minimal clonal spread, and were associated with multiple QRDR mutations with extensive cross-resistance noted among fluoroquinolones.

Keywords: fluoroquinolones, QRDR mutations, PFGE


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Streptococcus pneumoniae is the leading cause of outpatient respiratory tract infections including community-acquired pneumonia, acute sinusitis and otitis media.1,2 The introduction of fluoroquinolones, such as levofloxacin, gatifloxacin and moxifloxacin, that have potent activity against S. pneumoniae, Haemophilus influenzae and atypical respiratory pathogens was an important advance in the treatment of respiratory infections. Medical practice guidelines for the treatment of community-acquired pneumonia in adults recommend monotherapy with the newer fluoroquinolones or combination therapy with an extended-spectrum cephalosporin and a macrolide.1,2

Recent surveillance studies of S. pneumoniae in the USA have demonstrated that resistance to respiratory fluoroquinolones has remained low even as resistance to most other classes, including macrolides and penicillins, has increased.311 For example, TRUST 5 (Tracking Resistance in the US Today) surveillance data from 2000–2001 showed that 0.8% of the 6362 clinical isolates of S. pneumoniae isolated from 240 institutions in the USA were resistant to levofloxacin, while resistance to azithromycin and clarithromycin was >27% and resistance to penicillin (MIC >= 2 mg/L) was 16.9%.12 In addition, susceptibilities to levofloxacin have remained relatively constant over the last several years, with data from TRUST 3 (1998–1999) and TRUST 4 (1999–2000) showing that 99.4% of isolates in both studies were susceptible to levofloxacin,7,11 and TRUST 5 (2000–2001) showing 99.1% of isolates susceptible to levofloxacin.12

Surveillance studies from various parts of Asia (excluding Hong Kong), Western Europe and Latin America have also shown that fluoroquinolone resistance in S. pneumoniae remains low, with resistance to levofloxacin, gatifloxacin and moxifloxacin being <1%.1319 Previous publications from Chen et al.20 and Linares et al.21 reported increasing ciprofloxacin resistance (MICs >= 4 mg/L) in S. pneumoniae from Canada and Spain, respectively. However, more recent reports have shown that ciprofloxacin resistance in S. pneumoniae from Canada and Spain during 1999–2000 has remained at about the same levels since 1997–1998, and resistance to respiratory fluoroquinolones is still <1%.22,23 Increased resistance to all fluoroquinolones currently used has been reported in Hong Kong.24 This particular increase in fluoroquinolone resistance has been linked to the regional dissemination of a fluoroquinolone-resistant clone closely related to the Spain23F-1 clone.25 Currently, the extent to which fluoroquinolone-resistant isolates in the USA are clonally related or related to any of the major pandemic clones is not known.

The objective of this study was to determine whether levofloxacin-resistant (levofloxacin MICs >= 8 mg/L) clinical isolates of S. pneumoniae collected in the TRUST 3 (1998–1999) and TRUST 4 (1999–2000) surveillance studies were cross-resistant to other fluoroquinolones, and whether these isolates were genetically related to each other or one of the major pandemic clones.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains and antimicrobial susceptibility testing

A total of 4296 S. pneumoniae isolates were collected in the TRUST 3 surveillance study from 1998–1999,7 and 9499 isolates were collected in TRUST 4 from 1999–2000.11 Sixty-eight levofloxacin-resistant isolates (levofloxacin MICs >= 8 mg/L; 24 from TRUST 3 and 44 from TRUST 4) were found in the 13 795 isolates collected. These levofloxacin-resistant isolates were analysed as described below.

Antimicrobial agents tested herein (range in mg/L) were azithromycin (0.015–128), ciprofloxacin (0.06–256), gatifloxacin (0.008–256), levofloxacin (0.004–256), moxifloxacin (0.008–256) and penicillin (0.015–32). MICs were determined by microbroth dilution using panels manufactured by Trek Diagnostics (Westlake, OH, USA). MICs were performed according to NCCLS recommendations using cation-adjusted Mueller–Hinton broth with 5% lysed defibrinated horse blood.26 Quality control strain S. pneumoniae ATCC 49619 was included in each run of microbroth dilution MICs. Breakpoints (in mg/L, susceptible/intermediate/resistant) for levofloxacin (<=2/4/>=8), gatifloxacin (<=1/2/>=4), moxifloxacin (<=1/2/>=4), azithromycin (<=0.5/1/>=2) and penicillin (<=0.06/0.125–1/>=2) were those approved by the NCCLS.27 Breakpoints for ciprofloxacin (<=1/2/>=4) were those approved by the Food and Drug Administration (FDA).

Pulsed-field gel electrophoresis

Agarose-embedded genomic DNA was prepared using a method based on Lefevre et al.28 in conjunction with a CHEF Bacterial Genomic DNA Plug Kit (Bio-Rad, Hercules, CA, USA). Agarose-embedded DNA was digested with SmaI (New England Biolabs, Beverly, MA, USA) and separated using a CHEF DR III apparatus (Bio-Rad) under the following conditions: 1% GTG agarose (BioWhittaker, Rockland, ME, USA); 0.5x TBE; 6 V/cm; 14°C; block 1: 17 h, 1–30 s; block 2: 6 h, 5–9 s. Strains representative of three of the international multi-resistant pneumococcal clones Spain6B-2 (ATCC 700670), French9V-3 (ATCC 700671) and Spain23F-1 (ATCC 700669) were purchased from the ATCC (Manassas, VA, USA) and their pulsed-field gel electrophoresis (PFGE) patterns were compared with the fluoroquinolone-resistant TRUST isolates. PFGE patterns were compared among isolates using the criteria described by Tenover et al.29

Serotyping

Serotyping of strains was performed by the standard Quellung method with sera from Statens Seruminstitut (Copenhagen, Denmark).

PCR of quinolone resistance determinants and DNA sequencing

The quinolone resistance determining regions (QRDRs) of topoisomerase type II genes parC, parE, gyrA and gyrB were amplified by PCR using primers and cycling conditions described by Pan et al.30 PCR products from two separate reactions were sequenced bidirectionally by ACGT, Inc. (Northbrook, IL, USA) or Genome Therapeutics Corporation (Waltham, MA, USA).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Demographics

Fluoroquinolone-resistant isolates were identified from 48 of 238 institutions in 29 states during the TRUST 3 and 4 studies. Three institutions had fluoroquinolone-resistant isolates in both TRUST studies. Seventeen states had two or more fluoroquinolone-resistant isolates. North Carolina had the highest number of fluoroquinolone-resistant isolates with six, while Michigan, New York and Ohio each had five resistant isolates.

Forty-one of the 68 fluoroquinolone-resistant isolates (60%) were recovered from males. The majority of the isolates (n = 46, 68%) were from inpatients. Fifty isolates (74%) were from respiratory sources (sputum, nasopharynx, bronchial washing, tracheal aspirate, bronchial alveolar lavage) and 17 isolates (25%) were isolated from blood. One isolate was from an unknown source. One isolate was from a 17-year-old patient. For two of the isolates, the age of the patients was unknown. The other resistant isolates were evenly distributed among patients 18–64 years old (n = 33, 49%) and >=65 years old (n = 32, 47%).

Susceptibilities

MICs of levofloxacin, ciprofloxacin, gatifloxacin and moxifloxacin are listed in Table 1. Most of the isolates (91%) were non-susceptible to all fluoroquinolones tested. All levofloxacin-resistant isolates were resistant to ciprofloxacin (MIC >= 4 mg/L) and non-susceptible to gatifloxacin (six isolates intermediate, 62 isolates resistant). Sixty-two of the isolates (91%) were also non-susceptible to moxifloxacin (36 isolates intermediate, 26 isolates resistant). Eighteen (26%) of the isolates were penicillin-intermediate (MIC 0.125–1 mg/L) and 17 (25%) were penicillin-resistant (MIC >= 2 mg/L). Thirty-five (51%) of the isolates were azithromycin-resistant (MIC >= 2 mg/L) and two (3%) isolates were azithromycin-intermediate (MIC 1 mg/L).


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Table 1.  MIC distribution of fluoroquinolone-resistant S. pneumoniae
 
Serotype distribution

Seventeen different serotypes were observed (Table 2). Serotypes 19F (15 isolates), 23F (12 isolates) and 6A (eight isolates), were the most prevalent in both TRUST 3 and 4. Six serotypes (3, 4, 9V, 15C, 28A and 29) were unique to the TRUST 4 isolates and two serotypes (11A, 15B) were unique to the TRUST 3 isolates.


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Table 2.  PFGE and QRDR patterns associated with serotypes found among fluoroquinolone-resistant TRUST 3 and 4 isolates
 
PFGE patterns

The most prevalent SmaI PFGE pattern was C, including subtypes C1–C10 (Table 2). This pattern was very similar to the Spain23F-1 clone. Both 19F and 23F serotypes were found in isolates with PFGE patterns similar to the Spain23F-1 clone (Table 2). Fourteen isolates (21%: five from TRUST 3 and nine from TRUST 4) had PFGE pattern C or a subtype of C (Table 2). Five of the 14 isolates (36%) were from three different institutions in North Carolina and three isolates (21%) were from two different institutions in New York. The remaining six isolates were from six different states (California, Georgia, Indiana, Ohio, Pennsylvania and Texas). Isolate 5478 had a PFGE pattern similar to the French9V-3 clone.

Of the remaining 54 isolates, 41 had unique PFGE patterns and were from 33 different institutions in 26 states; 13 isolates had one of six PFGE patterns (A, B, D, E, F, G) (Table 2). The isolates with the same PFGE pattern had the same serotype (Table 2). Three of these patterns (E, F, G) were found in both the TRUST 3 and TRUST 4 studies. Isolates with duplicate PFGE patterns B, D and E were from the same institutions in Michigan, Oklahoma and Kansas, respectively.

QRDR substitution profile

Isolates were characterized by the amino acid substitutions in their QRDRs of ParC, ParE, GyrA and GyrB. Among 68 isolates there were 40 different QRDR profiles; the seven most common are listed in Table 3. Replicate QRDR profiles were found in seven sets of isolates, while 33 were found in only one isolate. Four of the QRDR profiles (a, b, c, d) accounted for 29 (43%) of the isolates (Table 3).


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Table 3.  Seven most common QRDR substitution profiles among TRUST 3 and TRUST 4 fluoroquinolone-resistant isolates
 
Isolates with the same PFGE pattern in most cases did not havethe same QRDR profile. Of the seven PFGE patterns found in two or more isolates, only isolates with PFGE patterns D and subtype D1 or E had the same QRDR profiles. The two isolates with PFGE patterns D/D1 (QRDR profile a, serotype 23F) were isolated from the same institution in Oklahoma during TRUST 4. The two isolates with PFGE pattern E (QRDR profile e, serotype 16F) were isolated from one institution in Kansas; one each during TRUST 3 and 4. Only four of the 14 isolates with PFGE pattern C, similar to Spain23F-1 clone, had the same QRDR profiles (profile c).

All isolates had at least two amino acid substitutions within the QRDR region of topoisomerase IV and/or DNA gyrase. The majority of isolates (69%) had three (28 isolates) or four (19 isolates) amino acid substitutions. Three isolates had five amino acid substitutions.

Distribution of QRDR mutations

Most amino acid substitutions were found in ParC and GyrA. Sixty-one isolates had at least one QRDR substitution in both ParC and GyrA. Substitutions at Ser-79 in ParC were found in 88% and 84% of TRUST 3 and 4 isolates, respectively; corresponding substitutions at Ser-81 in GyrA were found in 79% and 81% of the isolates. The Ser-79->Phe change (n = 39) and the Ser-81->Phe change (n = 49) were the most common substitutions in ParC and GyrA, respectively, for both the TRUST 3 and TRUST 4 isolates (Table 4). At both positions the Ser->Phe change was favoured over the Ser->Tyr change by a ratio of 2:1 in ParC and 8:1 in GyrA (Table 4).


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Table 4.  Distribution of QRDR mutations
 
Substitutions were found less frequently in ParE and GyrB. Forty-eight isolates had at least one QRDR substitution in ParE, whereas four isolates had a substitution in GyrB (Table 4). The Ile-460->Val change was the most common substitution in ParE (n = 44) (Table 4). No substitutions were found in GyrB in the TRUST 3 isolates; the TRUST 4 isolates had changes of Asp-435->Asn or Glu-474->Lys in GyrB (Table 4).

In comparing the frequencies of specific QRDR mutations between the TRUST 3 and 4 isolates, in most cases the frequencies differed by <10%. However, an increase in frequency from 54% to 70% was observed for the Ile-460->Val in ParE in comparison of the TRUST 3 and 4 sets of isolates; likewise an increase in frequency from 21% to 36% was observed for the Lys-137->Asn in ParC (Table 4). Furthermore, 9% of the fluoroquinolone-resistant isolates from TRUST 4 and none from TRUST 3 contained mutations in gyrB that resulted in changes of either Asp-435->Asn or Glu-474->Lys (Table 4).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Most S. pneumoniae clinical isolates from the USA are susceptible to respiratory fluoroquinolones. For example, according to TRUST 5 surveillance data (2000–2001) 99.1% of S. pneumoniae isolates are levofloxacin-susceptible.12 When fluoroquinolone-resistant clinical isolates are found, they are often cross-resistant to all the currently used respiratory fluoroquinolones and ciprofloxacin. In this study all the levofloxacin-resistant isolates were non-susceptible to gatifloxacin and most (91%) were non-susceptible to moxifloxacin. In addition all levofloxacin-resistant isolates were cross-resistant to ciprofloxacin. Similar results were reported in a year 2000 Canadian surveillance study that showed susceptibilities to levofloxacin, gatifloxacin and moxifloxacin in S. pneumoniae to be 99.0%, 99.1% and 99.1% respectively.22

In the TRUST 5 (2000–2001) surveillance data, 1.9% of isolates were resistant to ciprofloxacin.12 This is in good agreement with another US surveillance report by Brueggemann et al.10 and a Canadian surveillance study by Low et al.,22 both of which reported 1.4% of S. pneumoniae isolates to be resistant to ciprofloxacin. However, the prevalence of isolates that were ciprofloxacin intermediate (MIC 2 mg/L, FDA breakpoint) differed between the US and Canadian studies. TRUST 5 data from the USA10 showed that 13.4% of isolates had ciprofloxacin MICs of 2 mg/L, while data from the Canadian study22 showed only 3.0% of the isolates had ciprofloxacin MICs of 2 mg/L. The reason for this difference between US and Canadian isolates is unknown.

The majority of amino acid mutations in the resistant isolates occurred at Ser-79, Asp-83 and Lys-137 in ParC, Ser-81 and Glu-85 in GyrA, and Ile-460 in ParE. Although common, mutations at Lys-137 in ParC and Ile-460 in ParE are not known to contribute to fluoroquinolone resistance.31 Four isolates were identified with mutations in gyrB. The low incidence of gyrB mutations is similar to observations in many other studies.10,3135 Of particular interest is the comparison of the ratio of Ser-79->Phe to Ser-79->Tyr substitutions in ParC in levofloxacin-resistant and levofloxacin-susceptible isolates. In this study the Ser-79->Phe mutation was found almost twice as often as Ser-79->Tyr. However, among levofloxacin-susceptible TRUST 4 isolates containing first step mutations, the Ser-79->Phe mutation was found more than eight times as often as Ser-79->Tyr.36 The reason and significance of this difference is unknown at present.

Based on the molecular analysis (PFGE and QRDR profiles) of the fluoroquinolone-resistant isolates in this study, the great majority of fluoroquinolone-resistant isolates (60/68) were not clonally related. Similarly, Jones et al.4 recently reported that most of the US fluoroquinolone-resistant S. pneumoniae isolates from 1999 had unique PFGE patterns. In our study PFGE patterns from fluoroquinolone-resistant isolates were also compared with PFGE patterns from three prevalent international multi-resistant pneumococcal clones (Spain23F-1, Spain6B-2, French9V-3). Of the 68 fluoroquinolone-resistant isolates, 14 had PFGE patterns closely related to the Spain23F-1 clone and one isolate had a PFGE pattern closely related to the French9V-2 clone. Others also have described fluoroquinolone resistance in international multi-resistant clones.25,37,38 Ho et al.25 have identified 22/24 (92%) fluoroquinolone-resistant isolates in Hong Kong related to the Spain23F-1 clone. Alou et al.37 have noted that 27 of 82 (33%) of fluoroquinolone-resistant isolates from Spain were related to the Spain23F-1, France9V-3 or Spain14-5 clones. According to McGee et al.,38 four fluoroquinolone-resistant isolates from France and Spain were related to the Spain23F-1 clone and seven fluoroquinolone-resistant isolates from France and Ireland were related to the Spain9V-3 clone.

Concern has been raised over the acquisition of fluoroquinolone resistance by prevalent international multi-resistant pneumococcal clones and hence the widespread dissemination of these clones. This appears to have happened in Hong Kong.25,39 Even though 14 of the 68 fluoroquinolone-resistant isolates in this study had PFGE patterns similar to the Spain23F-1 clone, these isolates do not appear to have been spread by clonal dissemination. Of the 14 isolates, only four have the same QRDR substitution profile; each of the remaining 10 isolates has a different QRDR substitution profile. If these isolates were clonal, all would have been expected to have similar or closely related QRDR substitution profiles as well as similar PFGE patterns. The observation of Spain23F-1-like fluoroquinolone-resistant isolates probably reflects the fact that the Spain23F-1 clone is common in the penicillin-resistant S. pneumoniae population.4042 Thus, fluoroquinolone resistance in these Spain23F-1-like isolates may have arisen by independent mutational events. Isolates having PFGE patterns related to the Spain23F-1 clone were found among both the TRUST 3 and 4 studies. However, no evidence for an increase in its prevalence was found; the incidence of the Spain23F-1-related isolates (PFGE pattern C and subtypes) was 21% (five of 24) in the TRUST 3 fluoroquinolone-resistant isolates and 20.5% (nine of 44) in the TRUST 4 fluoroquinolone-resistant isolates.

Some areas of the world that report higher than normal fluoroquinolone resistance rates have observed problems with nosocomial spread of fluoroquinolone-resistant S. pneumoniae.35,43 In these instances, having more resources funneled toward infection control measures should reduce resistance rates. Another practice that could reduce the spread of fluoroquinolone resistance is proper dosing. Ho et al.44 have shown a history of suboptimal dosing of fluoroquinolones among patients with fluoroquinolone-resistant S. pneumoniae in Hong Kong.

In summary, >99% of S. pneumoniae clinical isolates from the USA are still susceptible to levofloxacin and other respiratory fluoroquinolones. Isolates resistant to levofloxacin are usually cross-resistant to other fluoroquinolones and associated with at least two QRDR mutations in topoisomerase IV and/or DNA gyrase genes. The majority of fluoroquinolone-resistant isolates (at least 60/68) were not the result of clonal spread of a single strain. Continued monitoring is warranted to track resistance levels of this important class of agents, especially in light of a recent development in the management of pneumococcal disease in the USA. The 7-valent pneumococcal vaccine has been added to the recommended childhood immunization schedule, and whether this will have an effect on the serotype distribution of fluoroquinolone-resistant isolates in the future remains to be determined. In this study 62% of the levofloxacin-resistant isolates had serotypes covered by the 7-valent vaccine. Serotyping, together with molecular epidemiology analysis (i.e. PFGE and QRDR sequencing) of S. pneumoniae isolates, are convenient tools to monitor the spread and mechanism of fluoroquinolone resistance. It will be important to determine whether this spread is due to relatively rare and sporadic independent mutational events, which appears to be the current situation in most of the USA, or whether resistance occurs, as in Hong Kong, due to dissemination of a few clones.


    Acknowledgements
 
This work was supported by Johnson & Johnson Pharmaceutical Research & Development, L.L.C., Raritan, NJ, and Ortho-McNeil Pharmaceutical Inc., Raritan, NJ, USA.


    Footnotes
 
* Corresponding author. Tel: +1-908-707-3465; Fax: +1-908-707-3501; E-mail: tdavies{at}prdus.jnj.com Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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