Susceptibility of Neisseria meningitidis to 16 Antimicrobial Agents and Characterization of Resistance Mechanisms Affecting Some Agents
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微生物临床杂志 2005年第7期
Department of Pathology, The University of Texas Health Science Center, San Antonio, Texas 78229
ABSTRACT
Neisseria meningitidis represents a pathogen of great public health importance in both developed and developing countries. Resistance to some antimicrobial agents used either for therapy of invasive infections or for prophylaxis of case contacts has long been recognized, although specific guidelines for susceptibility testing have not been fully developed. We have examined the susceptibilities of a collection of 442 meningococcal clinical isolates from 15 countries to 16 antimicrobial agents. These included isolates recovered between 1917 and 2004, with representatives of all major serogroups. All isolates were tested by the Clinical and Laboratory Standards Institute (formerly NCCLS) broth microdilution method using Mueller-Hinton lysed horse blood broth, while a subset of 102 isolates was tested by agar dilution using Mueller-Hinton sheep blood agar. Most isolates provided adequate growth for MIC determinations by both broth and agar methods. Growth in broth was enhanced by CO2 incubation and was required for two strains (1.7%). MICs of the study drugs compared favorably between the broth and agar methods (79 to 100% essential agreement), and MICs also generally agreed closely (92 to 100% essential agreement, excluding azithromycin) between broth tests incubated in the two different atmospheres. Elevated penicillin and ampicillin MICs (0.12 μg/ml and 0.25 μg/ml, respectively) occurred in 14.3% and 8.6% of strains and were associated with polymorphisms of the penA gene encoding a modified penicillin-binding protein 2. None of the 442 isolates produced beta-lactamase. Elevated tetracycline and doxycycline (but not minocycline) MICs were associated with efflux-mediated resistance encoded by tet(B) in 13 strains. Resistance to sulfisoxazole in 21.7% of strains and to trimethoprim-sulfamethoxazole in 21.0% resulted from polymorphisms of folP encoding a modified dihydropteroate synthetase. Seven strains were resistant to rifampin due to mutations in the rpoB gene, and two strains were resistant to chloramphenicol due to production of chloramphenicol acetyltransferase mediated by catP. Two strains had reduced quinolone susceptibility due to mutations of gyrA. The determination of the susceptibilities of a large group of meningococcal strains (including strains with characterized resistance mechanisms) to 16 antimicrobial agents has served as the essential first step in defining susceptibility testing breakpoints specific for this organism.
INTRODUCTION
Neisseria meningitidis is a leading cause of bacterial meningitis and severe sepsis in the United States, other industrialized countries of the Americas and Europe, and in the developing world (31, 37). It is also a major cause of periodic epidemics in sub-Saharan Africa and areas of the Middle East (31, 37). Invasive meningococcal disease (meningococcemia or meningitis) occurs in approximately 0.9 to 1.5 cases per 100,000 population per year in the United States, or 2,500 to 3,000 cases per year (31). The incidence of invasive disease in the United States has not changed significantly since the 1960s, and the disease continues to affect particularly infants, adolescents, and young adults. In recent years, the proportion of cases in the age group of 12 to 29 years has increased to 28% of all cases (31), but invasive disease can occur in adults of all ages. Respiratory infections may also be caused by meningococci, including pneumonia, otitis media, and epiglottitis (31, 37). While the number of sporadic cases of meningococcal diseases remained relatively stable, there has been a recent increase in local outbreaks in the United States, perhaps due to introduction of new clones to which immunity has not yet developed (31). Colonization of the human nasopharynx is a necessary precedent to invasive meningococcal disease and serves to transmit meningococci to others by expelled droplets or aerosols (31, 37).
In the United States, most cases of invasive meningococcal disease are caused by isolates belonging to serogroup B or C and only very rarely to group A (31, 37). Recently, disease due to serogroup Y, which is commonly associated with pneumonia and was previously associated with outbreaks in military populations, has increased to approximately one-third of all cases (31). Serogroup W135 has been associated with military population outbreaks and was responsible for a large outbreak during the Muslim pilgrimage in the Middle East, hajj, in 2000 (9, 31). While most cases in Europe and the Americas are due to serogroups B and C, serogroups A and C are most common in Africa and Asia (37). Outbreaks that occur in the "meningitis belt" of Africa in some years have had an attack rate of 500 to 1,000 cases per 100,000 population and a mortality rate of approximately 10% (31).
Close contacts of primary cases of meningococcal disease are at a significantly greater risk of acquisition of infection themselves. Family members residing in the same household of an index case are at a 400- to 800-fold increased risk of infection (31). Persons with impaired humoral immunity, particularly those with deficiencies of antibody-dependent, complement-mediated bacterial killing, are most susceptible to infection (31, 37). Individuals undergoing physical stress and living in crowded conditions with individuals from different geographic locales have a higher risk of sporadic or outbreak-associated meningococcal disease (31, 37). This includes young military recruits and perhaps also college freshman living in a dormitory setting (8, 31). Exposure to cigarette smoke in social settings frequented by adolescents and young adults also increases the risk of infection (8, 12).
Patients with invasive meningococcal infection must be treated with effective antibiotics due to the severity of meningococcemia and meningitis. Penicillin has historically been an effective antibiotic, but strains with reduced susceptibility to penicillin (often referred to as relatively resistant or penicillin-intermediate strains) have been reported in Europe, South America, Asia, Australia, and, less frequently, in the United States (3, 7, 17, 21, 23, 24, 30, 32, 36, 38). Such strains have been shown to have alterations in penicillin-binding protein 2 (PBP2; encoded by the penA gene), thought to be the result of formation of mosaic genes derived through transformation with DNA from commensal Neisseria species in the nasopharynx of colonized individuals (2, 33). While high-level penicillin resistance due to production of beta-lactamase has been reported (6, 19), such strains appear to now be extremely rare (29, 31). Resistance to other antimicrobial agents that may be used for therapy of meningococcal infections or for prophylaxis of case contacts has been reported in several countries. This includes resistance to chloramphenicol (due to production of chloramphenicol acetyltransferase [CAT] mediated by the gene catP) (20, 34), sulfonamides (3, 4, 16, 18, 36), tetracycline (14, 36), and rifampin (3, 21, 29, 35, 39). Thus far resistance has not been described to the extended-spectrum cephalosporins (e.g., cefotaxime or ceftriaxone) that may be used for treatment of meningococcal meningitis in developed countries, and in only a few instances has diminished fluoroquinolone (e.g., ciprofloxacin) susceptibility been described due to mutations in the gene (gyrA) encoding the gyrase A fluoroquinolone target (B. Alcala, C. Salcedo, L. de la Fuente, L. Arreaza, M. J. Urfa, R. Abad, R. Enriquez, J. A. Velazquez, M. Motge, and J. de Batlle, Letter, J. Antimicrob. Chemother. 53:409, 2004; T. R. Schultz, J. W. Tapsall, and P. A. White, Letter, Antimicrob. Agents Chemother. 44:1116, 2000). Fluoroquinolones are often used for prophylaxis of case contacts in developed countries (31).
The lack of consensus-derived breakpoints specific for N. meningitidis has hampered accurate estimates of resistance rates. The Clinical and Laboratory Standards Institute (CLSI; previously the National Committee for Clinical Laboratory Standards, or NCCLS) has recommended broth and agar dilution MIC susceptibility methods for N. meningitidis that are the same as the CLSI methods for Streptococcus spp. (27), although interpretive breakpoints have not previously been provided. This study has validated the recommended CLSI susceptibility testing methods and has determined MICs of 16 antimicrobial agents on a large multinational collection of meningococcal strains. These data have been augmented with studies to define the molecular mechanisms of resistance to several of the agents as a step toward development of interpretive breakpoints for this organism.
MATERIALS AND METHODS
Test isolates:. A collection of 442 meningococcal isolates was assembled, relying heavily upon U.S. surveillance isolates from the Active Bacterial Core Surveillance (ABCs) network of the Emerging Infections Program of the Centers for Disease Control and Prevention (CDC), from several state health department laboratories, and from outbreaks investigated by the Epidemiologic Investigations Laboratory of CDC. In addition, representative isolates from 14 other countries were obtained, many with diminished susceptibility or resistance to relevant antimicrobial agents. Dates of isolation ranged from 1917 to 2004. All relevant serogroups were represented in the strain collection in the following numbers: serogroup A, 22; B, 162; C, 125; W135, 28; X, 1; Y, 83; and Z, 2. Nineteen isolates were not serogrouped. The specific sources of the isolate collection are listed in Table 1. The collection included a number of strains with previously characterized resistance mechanisms. These included 13 isolates with tetracycline resistance due to the tetracycline efflux mechanism encoded by tet(B) (14), 96 isolates with resistance to trimethoprim-sulfamethoxazole or sulfisoxazole due to folP gene polymorphisms (18), 2 strains resistant to chloramphenicol due to production of CAT encoded by catP (34), and 2 strains with fluoroquinolone resistance resulting from gyrA mutations (B. Alcala, C. Salcedo, L. de la Fuente, L. Arreaza, M. J. Urfa, R. Abad, R. Enriquez, J. A. Velazquez, M. Motge, and J. de Batlle, Letter, J. Antimicrob. Chemother. 53:409, 2004; T. R. Schultz, J. W. Tapsall, and P. A. White, Letter, Antimicrob. Agents Chemother. 44:1116, 2000).
Antimicrobial agents for testing. The therapeutic agents for testing included penicillin G, ampicillin, cefotaxime, ceftriaxone, meropenem, and chloramphenicol. Agents for prophylaxis or therapy of non-central nervous system infections included rifampin, trimethprim-sulfamethoxazole, sulfisoxazole, tetracycline, doxycycline, minocyline, ciprofloxacin, levofloxacin, and azithromycin. Nalidixic acid was tested as a possible indicator of mutations in gyrA that might not be recognized by testing a potent fluoroquinolone.
Broth microdilution MIC susceptibility tests. All susceptibility testing procedures were performed in a class II laminar flow biological safety cabinet in order to prevent possible laboratory-acquired meningococcal infection (10). MICs of each agent were determined using the broth microdilution procedure described in NCCLS document M7-A6 (27). It included use of cation-adjusted Mueller-Hinton broth supplemented with 3% lysed horse blood as the test medium. Microdilution panels for testing the first 102 isolates were prepared with each antibiotic diluted in media prepared from dehydrated Mueller-Hinton medium from two different manufacturers (Becton Dickinson, Cockeysville, MD, and Oxoid, Ltd., Basingstoke, Hampshire, United Kingdom). All of the isolates were tested using Becton Dickinson (Difco formulation) Mueller-Hinton broth base. Test inocula were prepared from meningococcal colonies grown on chocolate agar plates that had been incubated for 18 to 24 h in 5% CO2. Colonies were suspended in 0.9% saline to obtain a suspension equivalent to the turbidity of a 0.5 McFarland standard and further diluted to provide a final inoculum density of 5 x 105 CFU/ml in the wells of the microdilution panels. With 68 meningococcal isolates, parallel sets of microdilution panels were inoculated, with one set incubated at 35°C in ambient air and the second set incubated in 5% CO2 for 20 to 24 h prior to visual determination of MICs in order to assess whether CO2 incubation was beneficial or necessary.
Agar dilution MIC susceptibility tests. MICs of each agent were also determined on the first 102 meningococcal isolates using the NCCLS agar dilution procedure described in NCCLS document M7-A6 (27). This included use of Mueller-Hinton agar supplemented with 5% sheep blood as the test medium. Plates containing the same dilution series of drugs contained in the initial microdilution MIC panels were prepared in molten agar and dispensed in 100-mm round plastic plates. Dehydrated Mueller-Hinton agar base produced by two different manufacturers (Becton Dickinson and Oxoid) was used for preparation of two sets of agar dilution plates for parallel testing. Test inocula were prepared as described above. The 0.5 McFarland inoculum suspension was further diluted to provide a density of 1 x 107 CFU/ml and then delivered to the surface of the agar plates using a Cathra replicator (MCT Medical, St. Paul, MN) that dispensed 1 to 2 μl to the agar surface. This resulted in a final inoculum of 1 x 104 CFU/spot on the agar. Plates were incubated at 35°C in 5% CO2 for 20 to 24 h prior to visual determination of MICs.
Quality control strains. Because the methods and media used for testing meningococci were essentially the same as the CLSI (NCCLS) test methods for streptococci, S. pneumoniae ATCC 49619 was employed for quality control of both the broth microdilution and agar dilution tests for all drugs for which there are approved CLSI control ranges (11). With ciprofloxacin, nalidixic acid, minocycline, and sulfisoxazole, which lack approved MIC control limits for the pneumococcal control strain, Escherichia coli ATCC 25922 was employed (11).
Beta-lactamase testing. Each isolate was tested for beta-lactamase production using a disk nitrocefin hydrolysis test (Cefinase; Becton-Dickinson).
Molecular genetic studies. (i) penA gene polymorphisms. A total of 85 isolates were selected for PCR restriction fragment length polymorphism (RFLP) analysis of the penA gene encoding the PBP2 of N. meningitidis using primer sets and probes previously described for this resistance determinant (1, 2). Briefly, primers AA-1 and 99-2 were used to amplify a 511-bp fragment of the penA gene that was then subjected to Taq1 restriction enzyme digestion. The bands obtained by agarose gel electrophoresis were used to identify strains as wild type or mutant, according to the scheme described by Antignac et al. (1). The strains selected were all of those with a penicillin MIC of 0.12 μg/ml and a representative sample of strains with penicillin MICs of 0.03 and 0.06 μg/ml. A previously sequenced wild-type strain, N. meningitidis 7926, was used as a control.
(ii) Detection of rpoB gene mutations. Seven isolates with notably high rifampin MICs were selected for PCR amplification and product sequencing for mutations in the rpoB gene. Two sets of primers were used to amplify a 913-base-pair sequence and a 469-base-pair sequence of the rpoB gene clusters I and II (28, 35). The amplified product was purified (QIAGEN QIAquick PCR Purification kit) and sequenced at the University of Texas Health Science Center at San Antonio Nucleic Acids core facility, using Big Dye Terminator v3.1 chemistry (ABI, Foster City, CA) and 3100 capillary sequencers (ABI, Foster City, CA). The resulting rpoB sequences were compared with wild type N. meningitidis sequences available in GenBank (5).
RESULTS
Initial studies demonstrated that better growth of test isolates was achieved in broth microdilution panels incubated in CO2 rather than in ambient air. While only 1.7% of strains failed to grow in air, the quality of growth (e.g., 4+ versus 3+ or 3+ versus 2+) was judged to be superior with 14.7% of 115 isolates (data not further depicted). MICs determined with a selected subset of 68 strains determined in parallel by incubation of microdilution panels in ambient air and CO2 did not indicate significant shifts in MICs, with the notable exception of azithromycin. The essential agreement (EA) of MICs (±1 dilution) ranged from 92.65 to 100% with all drugs except azithromycin (Table 2). Azithromycin MICs were 2- to 16-fold higher when incubated in CO2 as compared to incubation in air (Table 2 and Fig. 1). Furthermore, incubation of microdilution panels in 5% CO2 did not result in any out-of-range MICs with the quality control strain S. pneumoniae ATCC 49619, again with the exception of azithromycin (data not depicted further). Azithromycin MICs with the pneumococcal control strain were generally fourfold higher when incubated in CO2 and were thus uniformly outside the acceptable range for that agent. There was no significant effect of the lysed horse blood supplementation of the Mueller-Hinton broth or with CO2 incubation of the panels with the control strain E. coli ATCC 25922 with sulfisoxazole, minocycline, nalidixic acid, or ciprofloxacin (data not depicted further).
No significant differences in MICs of the 14 antimicrobial agents based upon the brand of broth used for microdilution tests were observed in tests with 102 meningococcal strains (data not depicted). There was generally very good agreement between MICs of the 14 drugs when determined by the broth microdilution or agar dilution methods (Table 3). The EA of MICs (±1 dilution) ranged from 79.2% to 100%, with only three drugs associated with an EA of less than 90%, and in those instances it was related to one brand of Mueller-Hinton agar that resulted in poorer growth of some strains. In fact, a number of growth failures were noted with one brand of Mueller-Hinton agar base used for the agar dilution comparison (Table 3).
Following the initial studies described above, the broth microdilution method with incubation of panels in 5% CO2 for 20 to 24 h was chosen as the most reproducible and reliable approach for further studies. Subsequently, the entire collection of 442 isolates was tested against all 14 antimicrobial agents, and the resulting MICs were compared to the resistance mechanisms detected when applicable. A total of 75 meningococcal isolates were found to have penicillin MICs of 0.12 to 1 μg/ml, and 48 isolates had ampicillin MICs of 0.25 to 1 μg/ml. Isolates with these elevated MICs and selected isolates representing the modal MIC of 0.06 μg/ml (or lower) of the two drugs were evaluated for possible PBP2 structural gene alterations. penA gene polymorphisms were detected by Taq1 RFLP digest patterns (Fig. 2) in the majority of isolates with a penicillin or ampicillin MIC of 0.25 μg or greater (Fig. 3 and Table 4). However, there was a sharper delineation of strains with penA gene polymorphisms based upon an ampicillin MIC of 0.25 μg/ml or greater (Fig. 3B). Eight different Taq1 digest patterns were noted among the isolates examined by RFLP (Table 4). None of the 442 isolates produced beta-lactamase, as evidenced by negative nitrocefin tests. MICs of cefotaxime, ceftriaxone, and meropenem were exceedingly low with this collection of isolates (Table 5). The MICs of these potent agents were not affected by the presence of penA polymorphisms (data not depicted further).
Two isolates were kindly provided (J. W. Tapsall) for this study with previously documented chloramphenicol resistance due to production of chloramphenicol acetyltransferase encoded by the catP gene. Both isolates had a chloramphenicol MIC of 16 μg/ml in this study, as compared to MICs of 2 μg/ml or less with the remaining strain collection (Fig. 4).
Thirteen isolates had elevated tetracycline MICs of 8 or 16 μg/ml and were associated with the presence of the tet(B)-mediated efflux mechanism of resistance (Fig. 5A). MICs of minocycline were not elevated in the strains that possessed tet(B), as that agent showed almost uniform inhibitory activity against this strain collection (Fig. 5B). A subset of 125 strains, including those resistant to tetracycline, was tested with doxycycline. Doxycycline MICs appeared to be only minimally affected by the presence of tet(B)-mediated resistance (Fig. 5C).
By far, the most frequent resistance in this strain collection was found to sulfisoxazole and trimethoprim-sulfamethoxazole. A total of 232 isolates had sulfisoxazole MICs of 8 μg/ml or greater, and 240 strains had trimethoprim-sulfamethoxazole MICs of 0.5 (trimethoprim)/9.5 (sulfamethoxazole) μg/ml or greater. These elevated MICs were associated with mutations in the folP gene encoding a modified dihydropteroate synthetase. Isolates with sulfisoxazole MICs of 2 μg/ml or less and trimethoprim-sulfamethoxazole MICs of 0.25/4.75 μg/ml or less lacked mutations in the folP gene (18) (Fig. 6A and B). A few isolates with a sulfisoxazole MIC of 4 μg/ml also possessed mutations in folP (Fig. 6A).
Only seven isolates in this collection demonstrated rifampin resistance with a MIC of 128 μg/ml or greater (Fig. 7). All other isolates were inhibited by 0.25 μg/ml or less of rifampin. The rpoB gene of each of the seven resistant isolates underwent sequencing following PCR amplification of chromosomal DNA. All isolates showed mutations in the rpoB gene as expected. Interestingly, there were two resistant isolates paired with pre-rifampin-exposure isolates available for study from siblings of invasive meningococcal disease cases. The sibling strain pairs differed by only one amino acid substitution: one with a histidine-to-tyrosine substitution at position 552 and the other with a serine-to-phenylalanine substitution at position 548. Pulsed-field gel electrophoresis showed the sibling pairs to be nearly identical (data not depicted).
Two isolates with previously reported fluoroquinolone resistance were kindly provided for this study by investigators in Australia and Spain. The Spanish strain had an elevated ciprofloxacin MIC of 0.25 μg/ml, as compared to MICs of 0.007 μg/ml or less for susceptible isolates, and the Australian strain had a MIC of 0.06 μg/ml (Fig. 8A). The levofloxacin MICs were also 0.25 and 0.06 μg/ml, respectively, while all other isolates were inhibited by 0.015 μg/ml or less (data not further depicted). Nalidixic acid was tested with the isolate collection as a potential surrogate reagent to detect strains with diminished fluoroquinolone susceptibility. Indeed, the nalidixic acid MIC of the strains described above was 64 μg/ml or greater, as compared with MICs of 2 μg/ml or less with all remaining isolates (Fig. 8B).
Lastly, azithromycin demonstrated relatively uniform activity against this strain collection. All isolates were inhibited by 1 μg/ml or less based upon the standard broth microdilution method with CO2 incubation used for all of the other drugs in this study (Table 5). Testing a subset of 100 of the strains in parallel with incubation in ambient air as well as 5% CO2 demonstrated much lower MICs of 0.25 μg/ml or less when incubation was carried out in air (Table 5).
DISCUSSION
This study has validated the media and test conditions recommended by the CLSI for susceptibility testing of N. meningitidis (11, 27). In particular, the broth microdilution MIC method using lysed horse blood supplemented Mueller-Hinton broth with incubation at 35°C for 20 to 24 h in a 5% CO2 atmosphere provided very reproducible results. While CO2 incubation was only required for growth of a few strains (1.7%), the quality of growth in the microdilution panels was superior when CO2 incubation was employed. There were not significant differences in MICs determined in ambient air versus CO2 with the broth microdilution method, with the notable exception of azithromycin, which is known to be adversely affected by incubation in CO2 (27). MICs determined by the agar dilution method (which necessarily employs CO2 incubation with meningococci) were quite comparable to MICs determined by broth microdilution with CO2 incubation. Likewise, there were not significant differences in MICs determined using different brands of Mueller-Hinton broth or agar. The standard CLSI pneumococcal and E. coli control strains functioned well for quality control purposes of tests performed on meningococci, despite the fact that the incubation of microdilution panels in a CO2 atmosphere in this study is in contrast to incubation in ambient air for S. pneumoniae tests and in unsupplemented Mueller-Hinton medium in ambient air with the E. coli control strain (11).
With some of the agents examined in this study, there was essentially a unimodal population of MICs, suggesting the lack of acquired resistance mechanisms significantly affecting those drugs (e.g., cefotaxime, ceftriaxone, meropenem, doxycycline, minocycline, and azithromycin). However, there were distinct populations of very susceptible strains as compared to those with diminished susceptibility (or resistance) to penicillin, ampicillin, chloramphenicol, tetracycline, sufisoxazole, trimethoprim-sulfamethoxazole, rifampin, ciprofloxacin, levofloxacin, and nalidixic acid. Elevated penicillin and ampicillin MICs were associated with the presence of penA gene polymorphisms, as previously described (1, 2, 33), and not due to beta-lactamase production. The two chloramphenicol-resistant strains produced the inactivating enzyme CAT (34), and all rifampin-resistant strains showed previously described mutations in the rpoB gene (28, 35). Recent studies conducted by our group have demonstrated that tetracycline resistance in an international clone of serogroup A meningococci is due to the active efflux mechanism encoded by tet(B) (14), while sulfisoxazole and trimethoprim-sulfamethoxazole resistance in this strain collection can be attributed to a modified dihydropteroate synthetase enzyme encoded by mutations in folP (18). For unclear reasons, the tet(B) mechanism did not significantly affect minocycline MICs and only modestly elevated doxycycline MICs in this collection of strains.
Elevated ciprofloxacin, levofloxacin, and nalidixic acid MICs were observed in two meningococcal strains with previously reported gyrA mutations. The slightly different MICs may be attributable to different sites of amino acid substitution in the gyrA QRDR, (i.e., Thr-to-Ile substitution at position 91 for the Spanish strain, and Asp-to-Asn substitution at position 95 of the Australian strain) (B. Alcala, C. Salcedo, L. de la Fuente, L. Arreaza, M. J. Urfa, R. Abad, R. Enriquez, J. A. Velazquez, M. Motge, and J. de Batlle, Letter, J. Antimicrob. Chemother. 53:409, 2004; T. R. Schultz, J. W. Tapsall, and P. A. White, Letter, Antimicrob. Agents Chemother. 44:1116, 2000). While not available for examination in this study, an additional strain from France with an elevated ciprofloxacin MIC and gyrA mutation of Asp to Gly at position 95 of GyrA has also been reported (I. Casin, B. Gandry, F. Lassau, M. Janier, P. LaGrange, and E. Collatz, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother, abstr. 2101, p. 172, 1999). More recently, an isolate with efflux-mediated ciprofloxacin resistance was reported from Argentina (M. Corso, M. Miranda, D. Faccone, L. Jorda, M. Regueira, C. Carranza, N. Castro, and M. Galas, Abstr. 44th Intersci. Conf. Antimicrob. Agents Chemother., abstr. C1-1107, p. 77, 2004). The results of this study suggest that testing nalidixic acid may be a more sensitive means of detection of gyrA QRDR mutations that affect the more potent fluoroquinolones to a less conspicuous degree.
Resistance to agents of choice for therapy (e.g., cefotaxime, ceftriaxone) and prophylaxis (fluoroquinolones, ceftriaxone, minocycline) of invasive meningococcal disease has fortunately not yet developed in the United States (31, 32). However, resistance to inexpensive agents that might be used for treatment (penicillin, ampicillin, chloramphenicol) or prophylaxis (sulfonamides, rifampin) in other developed or developing countries clearly does exist (3, 6, 7, 16, 17, 20, 24, 34, 35, 36). Indeed, sulfonamides were exceptionally effective for therapy and prophylaxis of invasive meningococcal disease in the United States from the 1940s until 1963 (15, 22, 25). However, when sulfonamide resistance initially emerged in a military population, it led to failures of prophylaxis resulting in a number of fatalities and rapid spread of the resistant strain into the civilian population (25). In order to accurately recognize resistance in sporadic case isolates of N. meningitidis and for monitoring of possible emerging resistance to currently effective agents by public health laboratories, it is important that there be standardized susceptibility testing methods for meningococci and relevant breakpoints for interpretation of MICs. This study has demonstrated that testing meningococcal clinical isolates is relatively simple and analogous to methods that are widely used for testing streptococci. Examination of the international strain collection included in this study demonstrated that there were clear separations of MICs with some drugs that corresponded to the presence of demonstrable resistance mechanisms (i.e., with penicillin, ampicillin, chloramphenicol, rifampin, tetracycline, sulfonamides). It would thus be possible to readily assign susceptibility breakpoints based strictly upon microbiological criteria. However, in establishing breakpoints, it is important to take into consideration pharmacokinetic and pharmacodynamic criteria as well as existing published clinical response data with the various antimicrobial agents (13, 26). Indeed, the MIC data obtained with this strain collection have been employed in pharmacodynamic simulations to derive rational interpretive breakpoints specific for N. meningitidis (manuscript in preparation). Those breakpoints have recently been established and published for the first time by the CLSI for all of the drugs in this study, with the exception of tetracycline and doxycycline (11).
ACKNOWLEDGMENTS
This study was supported by grant RS1/CCR622402 from the Centers for Disease Control and Prevention.
We thank the Epidemiologic Investigations Laboratory of the CDC, Tanja Popovic, Deborah Talkington, Nancy Rosenstein, and Fred Tenover for providing many of the isolates used in this study. Additional U.S. isolates were kindly provided by the Minnesota Department of Health, The New York State Department of Health, and by The Oregon Department of Health Services. Most of the U.S. strains were recovered through the Active Bacterial Core surveillance (ABCs)/Emerging Infections Program (EIP) Network of CDC. Non-U.S. isolates were generously provided by John Turnidge from Adelaide, Australia, and, notably, two chloramphenicol-resistant isolates and a strain with diminished quinolone susceptibility were kindly provided by John Tapsall from Randwick, NSW, Australia. Julio Vazquez (Spain) kindly provided a strain with diminished fluoroquinolone susceptibility. Robert Rennie (Canada) provided eight isolates with elevated penicillin MICs. We thank Letitia Fulcher and M. Leticia McElmeel for excellent technical support.
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ABSTRACT
Neisseria meningitidis represents a pathogen of great public health importance in both developed and developing countries. Resistance to some antimicrobial agents used either for therapy of invasive infections or for prophylaxis of case contacts has long been recognized, although specific guidelines for susceptibility testing have not been fully developed. We have examined the susceptibilities of a collection of 442 meningococcal clinical isolates from 15 countries to 16 antimicrobial agents. These included isolates recovered between 1917 and 2004, with representatives of all major serogroups. All isolates were tested by the Clinical and Laboratory Standards Institute (formerly NCCLS) broth microdilution method using Mueller-Hinton lysed horse blood broth, while a subset of 102 isolates was tested by agar dilution using Mueller-Hinton sheep blood agar. Most isolates provided adequate growth for MIC determinations by both broth and agar methods. Growth in broth was enhanced by CO2 incubation and was required for two strains (1.7%). MICs of the study drugs compared favorably between the broth and agar methods (79 to 100% essential agreement), and MICs also generally agreed closely (92 to 100% essential agreement, excluding azithromycin) between broth tests incubated in the two different atmospheres. Elevated penicillin and ampicillin MICs (0.12 μg/ml and 0.25 μg/ml, respectively) occurred in 14.3% and 8.6% of strains and were associated with polymorphisms of the penA gene encoding a modified penicillin-binding protein 2. None of the 442 isolates produced beta-lactamase. Elevated tetracycline and doxycycline (but not minocycline) MICs were associated with efflux-mediated resistance encoded by tet(B) in 13 strains. Resistance to sulfisoxazole in 21.7% of strains and to trimethoprim-sulfamethoxazole in 21.0% resulted from polymorphisms of folP encoding a modified dihydropteroate synthetase. Seven strains were resistant to rifampin due to mutations in the rpoB gene, and two strains were resistant to chloramphenicol due to production of chloramphenicol acetyltransferase mediated by catP. Two strains had reduced quinolone susceptibility due to mutations of gyrA. The determination of the susceptibilities of a large group of meningococcal strains (including strains with characterized resistance mechanisms) to 16 antimicrobial agents has served as the essential first step in defining susceptibility testing breakpoints specific for this organism.
INTRODUCTION
Neisseria meningitidis is a leading cause of bacterial meningitis and severe sepsis in the United States, other industrialized countries of the Americas and Europe, and in the developing world (31, 37). It is also a major cause of periodic epidemics in sub-Saharan Africa and areas of the Middle East (31, 37). Invasive meningococcal disease (meningococcemia or meningitis) occurs in approximately 0.9 to 1.5 cases per 100,000 population per year in the United States, or 2,500 to 3,000 cases per year (31). The incidence of invasive disease in the United States has not changed significantly since the 1960s, and the disease continues to affect particularly infants, adolescents, and young adults. In recent years, the proportion of cases in the age group of 12 to 29 years has increased to 28% of all cases (31), but invasive disease can occur in adults of all ages. Respiratory infections may also be caused by meningococci, including pneumonia, otitis media, and epiglottitis (31, 37). While the number of sporadic cases of meningococcal diseases remained relatively stable, there has been a recent increase in local outbreaks in the United States, perhaps due to introduction of new clones to which immunity has not yet developed (31). Colonization of the human nasopharynx is a necessary precedent to invasive meningococcal disease and serves to transmit meningococci to others by expelled droplets or aerosols (31, 37).
In the United States, most cases of invasive meningococcal disease are caused by isolates belonging to serogroup B or C and only very rarely to group A (31, 37). Recently, disease due to serogroup Y, which is commonly associated with pneumonia and was previously associated with outbreaks in military populations, has increased to approximately one-third of all cases (31). Serogroup W135 has been associated with military population outbreaks and was responsible for a large outbreak during the Muslim pilgrimage in the Middle East, hajj, in 2000 (9, 31). While most cases in Europe and the Americas are due to serogroups B and C, serogroups A and C are most common in Africa and Asia (37). Outbreaks that occur in the "meningitis belt" of Africa in some years have had an attack rate of 500 to 1,000 cases per 100,000 population and a mortality rate of approximately 10% (31).
Close contacts of primary cases of meningococcal disease are at a significantly greater risk of acquisition of infection themselves. Family members residing in the same household of an index case are at a 400- to 800-fold increased risk of infection (31). Persons with impaired humoral immunity, particularly those with deficiencies of antibody-dependent, complement-mediated bacterial killing, are most susceptible to infection (31, 37). Individuals undergoing physical stress and living in crowded conditions with individuals from different geographic locales have a higher risk of sporadic or outbreak-associated meningococcal disease (31, 37). This includes young military recruits and perhaps also college freshman living in a dormitory setting (8, 31). Exposure to cigarette smoke in social settings frequented by adolescents and young adults also increases the risk of infection (8, 12).
Patients with invasive meningococcal infection must be treated with effective antibiotics due to the severity of meningococcemia and meningitis. Penicillin has historically been an effective antibiotic, but strains with reduced susceptibility to penicillin (often referred to as relatively resistant or penicillin-intermediate strains) have been reported in Europe, South America, Asia, Australia, and, less frequently, in the United States (3, 7, 17, 21, 23, 24, 30, 32, 36, 38). Such strains have been shown to have alterations in penicillin-binding protein 2 (PBP2; encoded by the penA gene), thought to be the result of formation of mosaic genes derived through transformation with DNA from commensal Neisseria species in the nasopharynx of colonized individuals (2, 33). While high-level penicillin resistance due to production of beta-lactamase has been reported (6, 19), such strains appear to now be extremely rare (29, 31). Resistance to other antimicrobial agents that may be used for therapy of meningococcal infections or for prophylaxis of case contacts has been reported in several countries. This includes resistance to chloramphenicol (due to production of chloramphenicol acetyltransferase [CAT] mediated by the gene catP) (20, 34), sulfonamides (3, 4, 16, 18, 36), tetracycline (14, 36), and rifampin (3, 21, 29, 35, 39). Thus far resistance has not been described to the extended-spectrum cephalosporins (e.g., cefotaxime or ceftriaxone) that may be used for treatment of meningococcal meningitis in developed countries, and in only a few instances has diminished fluoroquinolone (e.g., ciprofloxacin) susceptibility been described due to mutations in the gene (gyrA) encoding the gyrase A fluoroquinolone target (B. Alcala, C. Salcedo, L. de la Fuente, L. Arreaza, M. J. Urfa, R. Abad, R. Enriquez, J. A. Velazquez, M. Motge, and J. de Batlle, Letter, J. Antimicrob. Chemother. 53:409, 2004; T. R. Schultz, J. W. Tapsall, and P. A. White, Letter, Antimicrob. Agents Chemother. 44:1116, 2000). Fluoroquinolones are often used for prophylaxis of case contacts in developed countries (31).
The lack of consensus-derived breakpoints specific for N. meningitidis has hampered accurate estimates of resistance rates. The Clinical and Laboratory Standards Institute (CLSI; previously the National Committee for Clinical Laboratory Standards, or NCCLS) has recommended broth and agar dilution MIC susceptibility methods for N. meningitidis that are the same as the CLSI methods for Streptococcus spp. (27), although interpretive breakpoints have not previously been provided. This study has validated the recommended CLSI susceptibility testing methods and has determined MICs of 16 antimicrobial agents on a large multinational collection of meningococcal strains. These data have been augmented with studies to define the molecular mechanisms of resistance to several of the agents as a step toward development of interpretive breakpoints for this organism.
MATERIALS AND METHODS
Test isolates:. A collection of 442 meningococcal isolates was assembled, relying heavily upon U.S. surveillance isolates from the Active Bacterial Core Surveillance (ABCs) network of the Emerging Infections Program of the Centers for Disease Control and Prevention (CDC), from several state health department laboratories, and from outbreaks investigated by the Epidemiologic Investigations Laboratory of CDC. In addition, representative isolates from 14 other countries were obtained, many with diminished susceptibility or resistance to relevant antimicrobial agents. Dates of isolation ranged from 1917 to 2004. All relevant serogroups were represented in the strain collection in the following numbers: serogroup A, 22; B, 162; C, 125; W135, 28; X, 1; Y, 83; and Z, 2. Nineteen isolates were not serogrouped. The specific sources of the isolate collection are listed in Table 1. The collection included a number of strains with previously characterized resistance mechanisms. These included 13 isolates with tetracycline resistance due to the tetracycline efflux mechanism encoded by tet(B) (14), 96 isolates with resistance to trimethoprim-sulfamethoxazole or sulfisoxazole due to folP gene polymorphisms (18), 2 strains resistant to chloramphenicol due to production of CAT encoded by catP (34), and 2 strains with fluoroquinolone resistance resulting from gyrA mutations (B. Alcala, C. Salcedo, L. de la Fuente, L. Arreaza, M. J. Urfa, R. Abad, R. Enriquez, J. A. Velazquez, M. Motge, and J. de Batlle, Letter, J. Antimicrob. Chemother. 53:409, 2004; T. R. Schultz, J. W. Tapsall, and P. A. White, Letter, Antimicrob. Agents Chemother. 44:1116, 2000).
Antimicrobial agents for testing. The therapeutic agents for testing included penicillin G, ampicillin, cefotaxime, ceftriaxone, meropenem, and chloramphenicol. Agents for prophylaxis or therapy of non-central nervous system infections included rifampin, trimethprim-sulfamethoxazole, sulfisoxazole, tetracycline, doxycycline, minocyline, ciprofloxacin, levofloxacin, and azithromycin. Nalidixic acid was tested as a possible indicator of mutations in gyrA that might not be recognized by testing a potent fluoroquinolone.
Broth microdilution MIC susceptibility tests. All susceptibility testing procedures were performed in a class II laminar flow biological safety cabinet in order to prevent possible laboratory-acquired meningococcal infection (10). MICs of each agent were determined using the broth microdilution procedure described in NCCLS document M7-A6 (27). It included use of cation-adjusted Mueller-Hinton broth supplemented with 3% lysed horse blood as the test medium. Microdilution panels for testing the first 102 isolates were prepared with each antibiotic diluted in media prepared from dehydrated Mueller-Hinton medium from two different manufacturers (Becton Dickinson, Cockeysville, MD, and Oxoid, Ltd., Basingstoke, Hampshire, United Kingdom). All of the isolates were tested using Becton Dickinson (Difco formulation) Mueller-Hinton broth base. Test inocula were prepared from meningococcal colonies grown on chocolate agar plates that had been incubated for 18 to 24 h in 5% CO2. Colonies were suspended in 0.9% saline to obtain a suspension equivalent to the turbidity of a 0.5 McFarland standard and further diluted to provide a final inoculum density of 5 x 105 CFU/ml in the wells of the microdilution panels. With 68 meningococcal isolates, parallel sets of microdilution panels were inoculated, with one set incubated at 35°C in ambient air and the second set incubated in 5% CO2 for 20 to 24 h prior to visual determination of MICs in order to assess whether CO2 incubation was beneficial or necessary.
Agar dilution MIC susceptibility tests. MICs of each agent were also determined on the first 102 meningococcal isolates using the NCCLS agar dilution procedure described in NCCLS document M7-A6 (27). This included use of Mueller-Hinton agar supplemented with 5% sheep blood as the test medium. Plates containing the same dilution series of drugs contained in the initial microdilution MIC panels were prepared in molten agar and dispensed in 100-mm round plastic plates. Dehydrated Mueller-Hinton agar base produced by two different manufacturers (Becton Dickinson and Oxoid) was used for preparation of two sets of agar dilution plates for parallel testing. Test inocula were prepared as described above. The 0.5 McFarland inoculum suspension was further diluted to provide a density of 1 x 107 CFU/ml and then delivered to the surface of the agar plates using a Cathra replicator (MCT Medical, St. Paul, MN) that dispensed 1 to 2 μl to the agar surface. This resulted in a final inoculum of 1 x 104 CFU/spot on the agar. Plates were incubated at 35°C in 5% CO2 for 20 to 24 h prior to visual determination of MICs.
Quality control strains. Because the methods and media used for testing meningococci were essentially the same as the CLSI (NCCLS) test methods for streptococci, S. pneumoniae ATCC 49619 was employed for quality control of both the broth microdilution and agar dilution tests for all drugs for which there are approved CLSI control ranges (11). With ciprofloxacin, nalidixic acid, minocycline, and sulfisoxazole, which lack approved MIC control limits for the pneumococcal control strain, Escherichia coli ATCC 25922 was employed (11).
Beta-lactamase testing. Each isolate was tested for beta-lactamase production using a disk nitrocefin hydrolysis test (Cefinase; Becton-Dickinson).
Molecular genetic studies. (i) penA gene polymorphisms. A total of 85 isolates were selected for PCR restriction fragment length polymorphism (RFLP) analysis of the penA gene encoding the PBP2 of N. meningitidis using primer sets and probes previously described for this resistance determinant (1, 2). Briefly, primers AA-1 and 99-2 were used to amplify a 511-bp fragment of the penA gene that was then subjected to Taq1 restriction enzyme digestion. The bands obtained by agarose gel electrophoresis were used to identify strains as wild type or mutant, according to the scheme described by Antignac et al. (1). The strains selected were all of those with a penicillin MIC of 0.12 μg/ml and a representative sample of strains with penicillin MICs of 0.03 and 0.06 μg/ml. A previously sequenced wild-type strain, N. meningitidis 7926, was used as a control.
(ii) Detection of rpoB gene mutations. Seven isolates with notably high rifampin MICs were selected for PCR amplification and product sequencing for mutations in the rpoB gene. Two sets of primers were used to amplify a 913-base-pair sequence and a 469-base-pair sequence of the rpoB gene clusters I and II (28, 35). The amplified product was purified (QIAGEN QIAquick PCR Purification kit) and sequenced at the University of Texas Health Science Center at San Antonio Nucleic Acids core facility, using Big Dye Terminator v3.1 chemistry (ABI, Foster City, CA) and 3100 capillary sequencers (ABI, Foster City, CA). The resulting rpoB sequences were compared with wild type N. meningitidis sequences available in GenBank (5).
RESULTS
Initial studies demonstrated that better growth of test isolates was achieved in broth microdilution panels incubated in CO2 rather than in ambient air. While only 1.7% of strains failed to grow in air, the quality of growth (e.g., 4+ versus 3+ or 3+ versus 2+) was judged to be superior with 14.7% of 115 isolates (data not further depicted). MICs determined with a selected subset of 68 strains determined in parallel by incubation of microdilution panels in ambient air and CO2 did not indicate significant shifts in MICs, with the notable exception of azithromycin. The essential agreement (EA) of MICs (±1 dilution) ranged from 92.65 to 100% with all drugs except azithromycin (Table 2). Azithromycin MICs were 2- to 16-fold higher when incubated in CO2 as compared to incubation in air (Table 2 and Fig. 1). Furthermore, incubation of microdilution panels in 5% CO2 did not result in any out-of-range MICs with the quality control strain S. pneumoniae ATCC 49619, again with the exception of azithromycin (data not depicted further). Azithromycin MICs with the pneumococcal control strain were generally fourfold higher when incubated in CO2 and were thus uniformly outside the acceptable range for that agent. There was no significant effect of the lysed horse blood supplementation of the Mueller-Hinton broth or with CO2 incubation of the panels with the control strain E. coli ATCC 25922 with sulfisoxazole, minocycline, nalidixic acid, or ciprofloxacin (data not depicted further).
No significant differences in MICs of the 14 antimicrobial agents based upon the brand of broth used for microdilution tests were observed in tests with 102 meningococcal strains (data not depicted). There was generally very good agreement between MICs of the 14 drugs when determined by the broth microdilution or agar dilution methods (Table 3). The EA of MICs (±1 dilution) ranged from 79.2% to 100%, with only three drugs associated with an EA of less than 90%, and in those instances it was related to one brand of Mueller-Hinton agar that resulted in poorer growth of some strains. In fact, a number of growth failures were noted with one brand of Mueller-Hinton agar base used for the agar dilution comparison (Table 3).
Following the initial studies described above, the broth microdilution method with incubation of panels in 5% CO2 for 20 to 24 h was chosen as the most reproducible and reliable approach for further studies. Subsequently, the entire collection of 442 isolates was tested against all 14 antimicrobial agents, and the resulting MICs were compared to the resistance mechanisms detected when applicable. A total of 75 meningococcal isolates were found to have penicillin MICs of 0.12 to 1 μg/ml, and 48 isolates had ampicillin MICs of 0.25 to 1 μg/ml. Isolates with these elevated MICs and selected isolates representing the modal MIC of 0.06 μg/ml (or lower) of the two drugs were evaluated for possible PBP2 structural gene alterations. penA gene polymorphisms were detected by Taq1 RFLP digest patterns (Fig. 2) in the majority of isolates with a penicillin or ampicillin MIC of 0.25 μg or greater (Fig. 3 and Table 4). However, there was a sharper delineation of strains with penA gene polymorphisms based upon an ampicillin MIC of 0.25 μg/ml or greater (Fig. 3B). Eight different Taq1 digest patterns were noted among the isolates examined by RFLP (Table 4). None of the 442 isolates produced beta-lactamase, as evidenced by negative nitrocefin tests. MICs of cefotaxime, ceftriaxone, and meropenem were exceedingly low with this collection of isolates (Table 5). The MICs of these potent agents were not affected by the presence of penA polymorphisms (data not depicted further).
Two isolates were kindly provided (J. W. Tapsall) for this study with previously documented chloramphenicol resistance due to production of chloramphenicol acetyltransferase encoded by the catP gene. Both isolates had a chloramphenicol MIC of 16 μg/ml in this study, as compared to MICs of 2 μg/ml or less with the remaining strain collection (Fig. 4).
Thirteen isolates had elevated tetracycline MICs of 8 or 16 μg/ml and were associated with the presence of the tet(B)-mediated efflux mechanism of resistance (Fig. 5A). MICs of minocycline were not elevated in the strains that possessed tet(B), as that agent showed almost uniform inhibitory activity against this strain collection (Fig. 5B). A subset of 125 strains, including those resistant to tetracycline, was tested with doxycycline. Doxycycline MICs appeared to be only minimally affected by the presence of tet(B)-mediated resistance (Fig. 5C).
By far, the most frequent resistance in this strain collection was found to sulfisoxazole and trimethoprim-sulfamethoxazole. A total of 232 isolates had sulfisoxazole MICs of 8 μg/ml or greater, and 240 strains had trimethoprim-sulfamethoxazole MICs of 0.5 (trimethoprim)/9.5 (sulfamethoxazole) μg/ml or greater. These elevated MICs were associated with mutations in the folP gene encoding a modified dihydropteroate synthetase. Isolates with sulfisoxazole MICs of 2 μg/ml or less and trimethoprim-sulfamethoxazole MICs of 0.25/4.75 μg/ml or less lacked mutations in the folP gene (18) (Fig. 6A and B). A few isolates with a sulfisoxazole MIC of 4 μg/ml also possessed mutations in folP (Fig. 6A).
Only seven isolates in this collection demonstrated rifampin resistance with a MIC of 128 μg/ml or greater (Fig. 7). All other isolates were inhibited by 0.25 μg/ml or less of rifampin. The rpoB gene of each of the seven resistant isolates underwent sequencing following PCR amplification of chromosomal DNA. All isolates showed mutations in the rpoB gene as expected. Interestingly, there were two resistant isolates paired with pre-rifampin-exposure isolates available for study from siblings of invasive meningococcal disease cases. The sibling strain pairs differed by only one amino acid substitution: one with a histidine-to-tyrosine substitution at position 552 and the other with a serine-to-phenylalanine substitution at position 548. Pulsed-field gel electrophoresis showed the sibling pairs to be nearly identical (data not depicted).
Two isolates with previously reported fluoroquinolone resistance were kindly provided for this study by investigators in Australia and Spain. The Spanish strain had an elevated ciprofloxacin MIC of 0.25 μg/ml, as compared to MICs of 0.007 μg/ml or less for susceptible isolates, and the Australian strain had a MIC of 0.06 μg/ml (Fig. 8A). The levofloxacin MICs were also 0.25 and 0.06 μg/ml, respectively, while all other isolates were inhibited by 0.015 μg/ml or less (data not further depicted). Nalidixic acid was tested with the isolate collection as a potential surrogate reagent to detect strains with diminished fluoroquinolone susceptibility. Indeed, the nalidixic acid MIC of the strains described above was 64 μg/ml or greater, as compared with MICs of 2 μg/ml or less with all remaining isolates (Fig. 8B).
Lastly, azithromycin demonstrated relatively uniform activity against this strain collection. All isolates were inhibited by 1 μg/ml or less based upon the standard broth microdilution method with CO2 incubation used for all of the other drugs in this study (Table 5). Testing a subset of 100 of the strains in parallel with incubation in ambient air as well as 5% CO2 demonstrated much lower MICs of 0.25 μg/ml or less when incubation was carried out in air (Table 5).
DISCUSSION
This study has validated the media and test conditions recommended by the CLSI for susceptibility testing of N. meningitidis (11, 27). In particular, the broth microdilution MIC method using lysed horse blood supplemented Mueller-Hinton broth with incubation at 35°C for 20 to 24 h in a 5% CO2 atmosphere provided very reproducible results. While CO2 incubation was only required for growth of a few strains (1.7%), the quality of growth in the microdilution panels was superior when CO2 incubation was employed. There were not significant differences in MICs determined in ambient air versus CO2 with the broth microdilution method, with the notable exception of azithromycin, which is known to be adversely affected by incubation in CO2 (27). MICs determined by the agar dilution method (which necessarily employs CO2 incubation with meningococci) were quite comparable to MICs determined by broth microdilution with CO2 incubation. Likewise, there were not significant differences in MICs determined using different brands of Mueller-Hinton broth or agar. The standard CLSI pneumococcal and E. coli control strains functioned well for quality control purposes of tests performed on meningococci, despite the fact that the incubation of microdilution panels in a CO2 atmosphere in this study is in contrast to incubation in ambient air for S. pneumoniae tests and in unsupplemented Mueller-Hinton medium in ambient air with the E. coli control strain (11).
With some of the agents examined in this study, there was essentially a unimodal population of MICs, suggesting the lack of acquired resistance mechanisms significantly affecting those drugs (e.g., cefotaxime, ceftriaxone, meropenem, doxycycline, minocycline, and azithromycin). However, there were distinct populations of very susceptible strains as compared to those with diminished susceptibility (or resistance) to penicillin, ampicillin, chloramphenicol, tetracycline, sufisoxazole, trimethoprim-sulfamethoxazole, rifampin, ciprofloxacin, levofloxacin, and nalidixic acid. Elevated penicillin and ampicillin MICs were associated with the presence of penA gene polymorphisms, as previously described (1, 2, 33), and not due to beta-lactamase production. The two chloramphenicol-resistant strains produced the inactivating enzyme CAT (34), and all rifampin-resistant strains showed previously described mutations in the rpoB gene (28, 35). Recent studies conducted by our group have demonstrated that tetracycline resistance in an international clone of serogroup A meningococci is due to the active efflux mechanism encoded by tet(B) (14), while sulfisoxazole and trimethoprim-sulfamethoxazole resistance in this strain collection can be attributed to a modified dihydropteroate synthetase enzyme encoded by mutations in folP (18). For unclear reasons, the tet(B) mechanism did not significantly affect minocycline MICs and only modestly elevated doxycycline MICs in this collection of strains.
Elevated ciprofloxacin, levofloxacin, and nalidixic acid MICs were observed in two meningococcal strains with previously reported gyrA mutations. The slightly different MICs may be attributable to different sites of amino acid substitution in the gyrA QRDR, (i.e., Thr-to-Ile substitution at position 91 for the Spanish strain, and Asp-to-Asn substitution at position 95 of the Australian strain) (B. Alcala, C. Salcedo, L. de la Fuente, L. Arreaza, M. J. Urfa, R. Abad, R. Enriquez, J. A. Velazquez, M. Motge, and J. de Batlle, Letter, J. Antimicrob. Chemother. 53:409, 2004; T. R. Schultz, J. W. Tapsall, and P. A. White, Letter, Antimicrob. Agents Chemother. 44:1116, 2000). While not available for examination in this study, an additional strain from France with an elevated ciprofloxacin MIC and gyrA mutation of Asp to Gly at position 95 of GyrA has also been reported (I. Casin, B. Gandry, F. Lassau, M. Janier, P. LaGrange, and E. Collatz, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother, abstr. 2101, p. 172, 1999). More recently, an isolate with efflux-mediated ciprofloxacin resistance was reported from Argentina (M. Corso, M. Miranda, D. Faccone, L. Jorda, M. Regueira, C. Carranza, N. Castro, and M. Galas, Abstr. 44th Intersci. Conf. Antimicrob. Agents Chemother., abstr. C1-1107, p. 77, 2004). The results of this study suggest that testing nalidixic acid may be a more sensitive means of detection of gyrA QRDR mutations that affect the more potent fluoroquinolones to a less conspicuous degree.
Resistance to agents of choice for therapy (e.g., cefotaxime, ceftriaxone) and prophylaxis (fluoroquinolones, ceftriaxone, minocycline) of invasive meningococcal disease has fortunately not yet developed in the United States (31, 32). However, resistance to inexpensive agents that might be used for treatment (penicillin, ampicillin, chloramphenicol) or prophylaxis (sulfonamides, rifampin) in other developed or developing countries clearly does exist (3, 6, 7, 16, 17, 20, 24, 34, 35, 36). Indeed, sulfonamides were exceptionally effective for therapy and prophylaxis of invasive meningococcal disease in the United States from the 1940s until 1963 (15, 22, 25). However, when sulfonamide resistance initially emerged in a military population, it led to failures of prophylaxis resulting in a number of fatalities and rapid spread of the resistant strain into the civilian population (25). In order to accurately recognize resistance in sporadic case isolates of N. meningitidis and for monitoring of possible emerging resistance to currently effective agents by public health laboratories, it is important that there be standardized susceptibility testing methods for meningococci and relevant breakpoints for interpretation of MICs. This study has demonstrated that testing meningococcal clinical isolates is relatively simple and analogous to methods that are widely used for testing streptococci. Examination of the international strain collection included in this study demonstrated that there were clear separations of MICs with some drugs that corresponded to the presence of demonstrable resistance mechanisms (i.e., with penicillin, ampicillin, chloramphenicol, rifampin, tetracycline, sulfonamides). It would thus be possible to readily assign susceptibility breakpoints based strictly upon microbiological criteria. However, in establishing breakpoints, it is important to take into consideration pharmacokinetic and pharmacodynamic criteria as well as existing published clinical response data with the various antimicrobial agents (13, 26). Indeed, the MIC data obtained with this strain collection have been employed in pharmacodynamic simulations to derive rational interpretive breakpoints specific for N. meningitidis (manuscript in preparation). Those breakpoints have recently been established and published for the first time by the CLSI for all of the drugs in this study, with the exception of tetracycline and doxycycline (11).
ACKNOWLEDGMENTS
This study was supported by grant RS1/CCR622402 from the Centers for Disease Control and Prevention.
We thank the Epidemiologic Investigations Laboratory of the CDC, Tanja Popovic, Deborah Talkington, Nancy Rosenstein, and Fred Tenover for providing many of the isolates used in this study. Additional U.S. isolates were kindly provided by the Minnesota Department of Health, The New York State Department of Health, and by The Oregon Department of Health Services. Most of the U.S. strains were recovered through the Active Bacterial Core surveillance (ABCs)/Emerging Infections Program (EIP) Network of CDC. Non-U.S. isolates were generously provided by John Turnidge from Adelaide, Australia, and, notably, two chloramphenicol-resistant isolates and a strain with diminished quinolone susceptibility were kindly provided by John Tapsall from Randwick, NSW, Australia. Julio Vazquez (Spain) kindly provided a strain with diminished fluoroquinolone susceptibility. Robert Rennie (Canada) provided eight isolates with elevated penicillin MICs. We thank Letitia Fulcher and M. Leticia McElmeel for excellent technical support.
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