Antimicrobial susceptibility breakpoints and first-step parC mutations in Streptococcus pneumoniae: redefining fluoroquinolone resistance - Research

Emerging Infectious Diseases, July, 2003 by Sue Lim, Darrin Bast, Allison McGeer, Joyce de Azavedo, Donald E. Low

Clinical antimicrobial susceptibility breakpoints are used to predict the clinical outcome of antimicrobial treatment. In contrast, microbiologic breakpoints are used to identify isolates that may be categorized as susceptible when applying clinical breakpoints but harbor resistance mechanisms that result in their reduced susceptibility to the agent being tested. Currently, the National Committee for Clinical Laboratory Standards (NCCLS) guidelines utilize clinical breakpoints to characterize the activity of the fluoroquinolones against Streptococcus pneumoniae. To determine whether levofloxacin breakpoints can identify isolates that harbor recognized resistance mechanisms, we examined 115 S. pneumoniae isolates with a levofloxacin MIC of >2 [micro]g/mL for first-step parC mutations. A total of 48 (59%) of 82 isolates with a levofloxacin MIC of 2 [micro]g/mL, a level considered susceptible by NCCLS criteria, had a first-step mutation in parC. Whether surveillance programs that use levofloxacin data can effectively detect emerging resistance and whether fluoroquinolones can effectively treat infections caused by such isolates should be evaluated.

**********

The emergence of Streptococcus pneumoniae resistance to [beta]-lactam and macrolide antimicrobial agents has led to recommendations that fluoroquinolones with increased activity against S. pneumoniae, such as levofloxacin, moxifloxacin, and gatifloxacin, be used to treat patients at risk for infection caused by such multidrug-resistant strains (1-6). Fluoroquinolone resistance in S. pneumoniae is primarily due to mutations in the genes encoding the target topoisomerase enzymes, namely parC, which encodes the A subunit of DNA topoisomerase IV, and gyrA, which encodes the A subunit of DNA gyrase (7). Mutations in parE and gyrB have been reported, but to a lesser extent (8-10). Most pneumococcal isolates with reduced susceptibilities to fluoroquinolones have amino acid substitutions in either ParC alone or ParC and GyrA (11-14). Resistance can also be mediated by active efflux (15), although the role of efflux in contributing to resistance by the newer fluoroquinolones is unclear (16).

The MIC of an antimicrobial agent is a value that has been used to determine breakpoints that predict the probability of clinical success, detect resistant populations, or both (17). Clinical breakpoints are dependent on the antimicrobial activity and pharmacology of the drug; such breakpoints are ascertained with the goals of eradicating the infection and ultimately achieving clinical success with the antimicrobial agent. In contrast, microbiologic breakpoints are established to identify isolates that may be categorized as susceptible when applying clinical breakpoints but harbor resistance mechanisms that result in their reduced susceptibility to the agent being tested. These microbiologic breakpoints are therefore useful in monitoring the emergence of resistance. The current National Committee for Clinical Laboratory Standards (NCCLS) guidelines make no distinction between these two interpretations of MIC, with clinical breakpoints used to characterize most antimicrobial agents, including the fluoroquinolones.

Levofloxacin has been used as a surrogate marker to predict fluoroquinolone susceptibility in clinical laboratories and surveillance studies (18). To establish whether current levofloxacin breakpoints are also able to function as microbiologic breakpoints, we determined the percentage of S. pneumoniae isolates with first-step parC mutations that would go undetected by using the current NCCLS breakpoints for levofloxacin (19).

Materials and Methods

A total of 6,076 clinical isolates of S. pneumoniae were collected as part of a 1993-1998 surveillance program throughout Canada. All isolates were identified as S. pneumoniae by standard methods. The isolates were frozen at -70[degrees]C, thawed, subcultured onto blood agar, and incubated at 37[degrees]C in 5% C[O.sub.2] for 24 h twice before testing. In vitro susceptibility testing was performed by broth microdilution, according to NCCLS guidelines (20,21). Susceptibility interpretive criteria used were those published in the NCCLS M100-S12 document (19). The nonsusceptible category was defined as those isolates with MICs of fluoroquinolines in the intermediate and resistant category. The parC gene of 115 isolates with a levofloxacin MIC [greater than or equal to] 2 [micro]g/mL (82 = MIC 2 [micro]g/mL; 8 = MIC 4 [micro]g/mL; 10 = MIC 8 [micro]g/mL; and 15 = MIC [greater than or equal to] 16 [micro]g/mL) was amplified by polymerase chain reaction (PCR), and the nucleotide sequence determined as previously described (9). All isolates (n=33) with a levofloxacin MIC of [greater than or equal to] 4 [micro]g/mL, and a random sample of 29 isolates with a levofloxacin MIC of 2 [micro]g/mL were examined for gyrA mutations. For comparative purposes, the parC gene of 14 isolates with a ciprofloxacin MIC of 2 [micro]g/mL, regardless of their levofloxacin MIC, was amplified and sequenced. Although numerous single mutational events occur in parC, the focus of this investigation was on amino acid substitutions for Ser-79 or Asp-83, because previous studies have consistently demonstrated that mutations at either of these positions are associated with decreased susceptibility (9,14).

Crude cell lysates were used as DNA templates for PCR. After overnight growth on Columbia nutrient agar and supplemented with 5% sheep blood, a loopful of growth was suspended in 100 [micro]L of lysis buffer (100 mM NaCl, 10 mM Tris-HCl [pH 8.3], 1 mM EDTA, 1% Triton X-100) and boiled for 10 min. Ten microliters of the super-natant was used as the template in a 50-[micro]L reaction volume. The quinolone-resistance--determining regions of parC and gyrA were amplified by PCR. Primers used were based on published sequences (7,8), and amplification products were purified with either the QIAquick PCR purification kit (Qiagen Inc., Mississauga, Ontario, Canada) or the Concert Rapid PCR purification kit (Life Technologies, Burlington, Ontario, Canada).