Interlaboratory Comparison of Agar Dilution and Etest Methods for ...

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 2003, p. 3430–3434 0066-4804/03/$08.00⫹0 DOI: 10.1128/AAC.47.11.3430–3434.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Vol. 47, No. 11

Interlaboratory Comparison of Agar Dilution and Etest Methods for Determining the MICs of Antibiotics Used in Management of Neisseria meningitidis Infections Julio A. Va´zquez,1* Luisa Arreaza,1 Colin Block,2 Ingrid Ehrhard,3 Stephen J. Gray,4 Sigrid Heuberger,5 Steen Hoffmann,6 Paula Kriz,7 Pierre Nicolas,8 Per Olcen,9 Anna Skoczynska,10 Lodewijk Spanjaard,11 Paola Stefanelli,12 Muhamed-Kheir Taha,13 and Georgina Tzanakaki14 National Institute of Health Carlos III, Majadahonda (Madrid), Spain1; Hadassah University Hospital, Jerusalem, Israel2; National Reference Center for Meningococci, Heidelberg, Germany3; Meningococcal Reference Unit, Public Health Laboratory, Manchester, United Kingdom4; Bundesstaatliche Bakterilogisch-Serologischen, Untersuchungsanstalt, Graz, Austria5; Statens Serum Institute, Copenhagen S, Denmark6; National Reference Laboratory for Meningococcal Infections, Prague, Czech Republic7; Unite ¨ rebro, Sweden9; National Meningocoque, WHO Collaborating Center, Marseille, France8; University Hospital, O Reference Center for Bacterial Meningitis, National Institute of Public Health, Warsaw, Poland10; Netherlands Reference Laboratory of Bacterial Meningitis, The Netherlands11; Istituto Superiore di Sanita ´, Rome, Italy12; 13 Institute Pasteur, Paris, France ; and National School of Public Health, Athens, Greece14 Received 7 February 2003/Returned for modification 9 June 2003/Accepted 15 July 2003

Previous studies have shown that there is considerable variation in the methods and media used to determine the susceptibility of Neisseria meningitidis to antimicrobial agents in different countries. In this study, national and regional reference laboratories used a standardized methodology to determine the MICs of antibiotics used in the management of meningococcal infection. Fourteen laboratories participated in the study, determining the susceptibility to penicillin G, rifampin, cefotaxime, ceftriaxone, ciprofloxacin, and ofloxacin of a collection of 17 meningococci, of which 11 strains were previously defined as having intermediate resistance to penicillin (PenI) by sequencing and restriction fragment length polymorphism analysis of the penA gene. The MIC was determined by agar dilution and Etest with Mueller-Hinton agar (MH), MH supplemented with sheep blood (MHⴙB), and MH supplemented with heated (chocolated) blood. Several laboratories encountered problems obtaining confluent growth with unsupplemented MH. MHⴙB was considered to give the most congruent and reproducible results among the study laboratories. The modal MIC for MHⴙB for each antibiotic and method was calculated to define the MIC consensus, allowing assessment of each individual laboratory’s data in relation to the others. The agreement in each antibiotic/method/medium combination was defined as the percentage of laboratories with a result within one dilution of the modal result. For the whole study, an agreement of 90.6% was observed between agar dilution and Etest methods. The agreement in each laboratory/antibiotic/method combination ranged from 98.2% to 69.7%, with six laboratories demonstrating agreement higher than 90% and 11 more than 80%. The ability of the laboratories to detect the PenI isolates ranged from 18.2% to 100%. The apparent difficulty in interpreting susceptibility to rifampin, particularly with the Etest method, is very interesting. Neisseria meningitidis, the etiological agent of meningococcal disease, remains susceptible to many antimicrobial agents. Apart from resistance to sulfonamides, meningococci remain susceptible to the antibiotics classically used for treatment and chemoprophylaxis (15). But, since 1985, decreased susceptibility to penicillin caused by alterations in the penicillin-binding proteins (12) has been noted in some countries (11, 14, 15). In addition, several meningococcal strains with a high level of penicillin resistance due to beta-lactamase production have also been described (4). Resistance to rifampin is only occasionally observed, and this may be identified following chemoprophylaxis (8). Because meningococcal disease is such a serious and rapidly progressing illness, it is very important to monitor trends in the

resistance to antimicrobial agents in meningococci in each country. In order to compare data between laboratories and monitor resistance trends, it is critical to use standardized protocols for the determination of susceptibility to antibiotics. The National Committee for Clinical Laboratory Standards (NCCLS) recommends a specific methodology to determine the MIC of antibiotics against meningococci (10), but breakpoints to define different categories of susceptibility have not been established. The NCCLS recommends the use of broth microdilution in cation-adjusted Mueller-Hinton (MH) broth supplemented with lysed horse blood or agar dilution with MH supplemented with sheep blood. But, the Etest method, commonly used in Europe, is a practical alternative for clinical laboratories when a quick result is clinically or epidemiologically important (9). Alternatively, the use of a disk diffusion test with 2 U of penicillin or 1 ␮g of oxacillin has been proposed for that purpose (7), but there is some concern about the reliability of this methodology (6), and further multicenter studies are required to clarify the use of this method. In order to identify the methodologies used for MIC deter-

* Corresponding author. Mailing address: National Reference Laboratory for Meningococci—Centro Nacional de Microbiología, National Institute of Health Carlos III, 28220 Majadahonda (Madrid), Spain. Phone: 34 915097901. Fax: 34 915097966. E-mail: jvazquez @isciii.es. 3430

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TABLE 1. Characteristics of the strains used in the EMGM standardization study EMGM strain no.

Antigen expressiona

Yr of isolation

Originb

1 2 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

C:2a:P1.5,2 NG:NT:NST B:4:P1.15 B:1:NST B:NT:P1.3,6 B:4:P1.15 B:4:P1.15 B:4:NST C:2b:P1.5,2 B:NT:P1.9 B:1:NST C:2b:NST C:2a:P1.5 C:2a:P1.5 C:2b:NST B:1:NST C:2b:NST

1999 1992 1999 1999 1999 1999 1999 1998 1999 1999 1999 1992 1999 1994 1992 1999 1992

Blood Nasopharynx Nasopharynx Blood Nasopharynx Nasopharynx Blood CSF Blood CSF Nasopharynx Blood CSF Blood Blood Blood Blood

a

Serogroup/serotype/serosubtype combination. CSF, cerebrospinal fluid. c NG, nongroupable; NT, nonserotypeable; NST, nonserosubtypeable. b

mination in different countries, a questionnaire compiled by the European Monitoring Group of Meningococci (EMGM) was sent to 26 national and regional reference laboratories, including not only European centers but also laboratories in Australia, Israel, and the United States (5). The results of the questionnaire highlighted the heterogeneity of methods, particularly with regard to the media used. A similar degree of variability was also observed with respect to the breakpoints used. While susceptibility data can monitor trends within a given country or region, the lack of standardization makes it very difficult to compare susceptibility data between countries. The EMGM, during its fifth meeting in Greece in 1999, concluded that it would be useful and instructive to apply a standard methodology for determining the MICs of a panel of meningococcal strains, particularly focusing on the agar dilution and Etest methods, which, according to the results of the questionnaire, were the most frequently used across Europe. The results of the project, coordinated by the Spanish Reference Laboratory for Neisseria (SRLN), are reported in this study. MATERIALS AND METHODS The SRLN prepared and distributed a collection of meningococcal strains together with 500 g of MH dehydrated medium, the antibiotics, and a protocol for performing dilutions and preparing media to determine the MICs of the strains by the agar dilution and Etest methods. The strains were tested on MH, MH supplemented with sheep blood (MH⫹B), and MH supplemented with heated (chocolated) sheep blood (MH⫹CH). Strains. A collection of 17 freeze-dried meningococcal strains isolated in Spain between 1992 and 1999 was prepared. The isolates, according to the data of the SRLN, represented a range of levels of susceptibility to antimicrobial agents, particularly to penicillin G and rifampin. The characteristics of these strains are shown in Table 1. Media and antibiotics. The same designated batch of MH (Oxoid, Basingstoke, Hampshire, England) was distributed to all the participating laboratories. The antibiotics to be used were chosen by consensus and included penicillin G, rifampin, ciprofloxacin, ofloxacin, cefotaxime, and ceftriaxone. Batches of antibiotics were sourced from Cepa Schwarz Pharma S.L. for ciprofloxacin and Sigma-Aldrich, Inc., for the other antibiotics. The Etest strips were obtained

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from the sole manufacturer (AB Biodisk, Sweden) via local suppliers by each laboratory. Protocols. Protocols for producing the dilutions and media to determine the MICs of the strains by agar dilution and Etest methods were distributed to all participating laboratories. Antimicrobial susceptibility was determined by two methods (agar dilution and Etest) with three culture media (MH, MH⫹B, and MH⫹CB) as previously described (5, 9). For agar dilution, the antibiotics were used in serial twofold dilutions, with concentrations ranging from 0.007 to 2 ␮g/ml for penicillin G, 0.0003 to 0.03 ␮g/ml for cefotaxime, 0.00007 to 0.06 ␮g/ml for ceftriaxone, 0.007 to 64 ␮g/ml for rifampin, and 0.0007 to 0.12 ␮g/ml for ciprofloxacin and ofloxacin. Although the accuracy of concentrations below 0.004 ␮g/ml is not very good, a wide range of concentrations was included to be able to compare results at these low concentrations. The organisms were applied to the plates at a final concentration of 105 CFU per spot. After allowing the surface to absorb the inoculum, all the plates were incubated for 24 h at 35°C in 5% CO2. The MIC was defined as the lowest concentration of antibiotic that prevented visible growth. For the Etest method, 0.5 McFarland standard suspensions of the test organisms were prepared and inoculated onto the plate with a nontoxic swab to produce confluent growth. Etest strips containing the antimicrobial agent were placed on the inoculated plates with sterile forceps. Some laboratories used 150-mm plates, placing all six strips on it; others used 90-mm plates, placing two strips in each; but most of the laboratories used 90-mm plates, placing only one Etest strip in each. The plates were incubated for 24 h at 35°C in 5% CO2 and then the MICs were determined according to the manufacturer’s instructions. Each laboratory used its own method to determine the inoculum concentration for the 105 CFU per spot and 0.5 McFarland. Sequence analysis and RFLP of the penA gene. Because intermediate resistance to penicillin is produced by alterations in PBP2, encoded by the penA gene, this gene was sequenced, by the SRLN, in all 17 strains as previously described (3). The Institute Pasteur also analyzed the penA gene by restriction fragment length polymorphism (RFLP) according to a published method (1). Analysis of the data. All the data were included in a Microsoft Excel file, and the analysis was done with this software. The congruence of the data was analyzed to identify the optimal medium. For this purpose, the agreement in each antibiotic/method/medium combination was defined as the percentage of laboratories with a result within one dilution of the modal result. The modal result was calculated for each strain against each antibiotic with the

TABLE 2. Agreement obtained in each antibiotic/method/medium combinationa Drug

Method

Agreement (%) MH

MH⫹B

Penicillin

AD Etest

81.2 75

91.6 87.6

MH⫹CH

90.8 82.9

Rifampin

AD Etest

77 70.7

78.8 75.6

82 74.2

Ciprofloxacin

AD Etest

87.6 77.4

85.3 91.6

88.6 87.1

Ofloxacin

AD Etest

98.5 85.9

94.1 98.8

91.5 100

Cefotaxime

AD Etest

77.3 74.7

85.6 90.9

81 86.1

Ceftriaxone

AD Etest

76.6 ND

86 ND

87.7 ND

Total

AD Etest

83.0 76.7

86.9 88.9

86.9 86.1

a The highest percentage of coincident MICs among all participating laboratories. The best agreement in each case is in italics. Two or three different media in bold for the same antibiotic and method indicates that differences were not statistically significant. ND, not determined; AD, agar dilution.

´ ZQUEZ ET AL. VA

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TABLE 3. Modal MICs obtained in MH⫹B for the both, agar dilution, and Etest methodsa Modal MIC (␮g/ml) EMGM strain no.

Method

Penicillin G

Rifampin

Cefotaxime

Ceftriaxone

Ciprofloxacin

Ofloxacin

1

AD Etest

0.03 0.032

0.015 0.016

0.003 0.003

0.0015

0.007 0.003

0.03 0.016

2

AD Etest

0.03 0.023

0.003 ⬍0.002

0.0007

0.007 0.003

0.03 0.016

4

AD Etest

0.03 0.016

0.03 0.023

0.003 ⬍0.002

0.0007

0.003 0.003

0.015 0.016

5*

AD Etest

0.06 0.064

0.007 0.008

0.0015 0.002

0.0007

0.003 0.003

0.015 0.012

6*

AD Etest

0.12 0.125

0.007 0.004

0.007 0.004

0.0015

0.003 0.003

0.03 0.016

7*

AD Etest

0.5 0.38

0.015 0.016

0.015 0.008

0.0015

0.003 0.003

0.015 0.012

8*

AD Etest

0.25 0.125

0.03 0.023

0.007 0.004

0.0015

0.007 0.002

0.03 0.012

9*

AD Etest

0.25 0.125

0.03 0.016

0.007 0.003

0.0015

0.003 0.004

0.03 0.012

10*

AD Etest

0.25 0.25

0.007 0.004

0.015 0.008

0.003

0.007 0.003

0.015 0.016

11

AD Etest

0.015 0.012

0.007 0.0023

0.0007 ⬍0.002

0.007

0.007 0.003

0.015 0.016

12*

AD Etest

0.12 0.125

0.007 0.0023

0.003 0.003

0.0015

0.003 0.003

0.015 0.012

13*

AD Etest

0.5 0.38

8 6

0.007 0.003

0.0015

0.003 0.007

0.03 0.016

14

AD Etest

0.03 0.032

0.015 0.023

0.003 0.002

0.0015

0.003 0.003

0.015 0.016

15*

AD Etest

0.5 0.5

0.03 0.032

0.015 0.004

0.003

0.007 0.004

0.015 0.016

16*

AD Etest

0.5 0.38

0.007 0.004

0.0015

0.007 0.003

0.03 0.012

17

AD Etest

0.03 0.032

0.015 0.008

0.003 ⬍0.002

0.0007

0.007 0.003

0.03 0.012

18*

AD Etest

0.5 0.38

8 6

0.007 0.004

0.0015

0.007 0.003

0.015 0.012

⬎64 ⬎32

8 ⬎32

a Results differing by more than ⫾1 dilution comparing agar dilution with Etest are in italics. *, strains identified as being intermediate to penicillin by sequencing of the penA gene and RFLP.

TABLE 4. Agreement obtained between the agar dilution and Etest methods for the determination of the consensus MIC of each antibiotica

most optimal medium. These modal results defined the MIC for each strain/ antibiotic combination. Two results were considered coincident (the same) if they were within ⫾1 dilution.

Antibiotic

RESULTS The agreement for each antibiotic/method/medium combination is shown in Table 2. The agreement was very similar in MH⫹B and MH⫹CH media, but overall, MH⫹B offered the best agreement, being the medium with the most homogeneous results among laboratories, and was therefore adopted

Agreement (%)

Penicillin G ..................................................................................100 Rifampin ...................................................................................... 82.4 Ciprofloxacin ............................................................................... 94.1 Ofloxacin ...................................................................................... 76.5 Cefotaxime................................................................................... 94.1 Total ............................................................................................. 90.6 a

Ceftriaxone is not included because the MIC was not determined by Etest.

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TABLE 5. Agreement for each laboratory/antibiotic/method combinationa Agreement (%) Laboratory no.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 a

Penicillin G

Rifampicin

Ciprofloxacin

Ofloxacin

Cefotaxime

AD

Etest

AD

Etest

AD

Etest

AD

Etest

AD

Etest

100 47.05 94.1 100 88.2 88.2 94.1 100 94.1 100 82.3 — 100 100

100 82.3 100 70.6 100 94.1 94.1 100 100 100 100 23.5 62.5 100

88.2 70.6 94.1 82.3 76.5 88.2 76.5 100 64.7 58.8 76.5 — 75 93.3

86.7 87.5 68.7 81.2 — 50 87.5 80 73.3 50 81.2 81.2 66.7 92.8

100 100 64.7 100 64.7 88.2 94.1 100 41.2 88.2 64.7 — 100 100

100 47 100 100 — 100 100 100 88.2 94.1 100 100 100 100

100 — 100 100 64.7 88.2 94.1 100 100 100 100 — 87.5 93.3

100 100 — 100 100 100 100 100 100 100 100 100 100 100

88.2 41.2 94.1 100 94.1 94.1 93.7 100 100 47 82.3 — 86.7 100

— 82.3 — 100 — 100 82.3 100 100 100 94.1 94.1 87.5 100

Ceftriaxone (AD)

100 100 100 100 0 100 100 100 100 100 100 100 100 6.7

AD, agar dilution; —, MICs not determined..

for most of the analyses. Three laboratories experienced difficulty obtaining confluent growth with MH. No laboratories reported difficulties in the interpretation of the Etest method. We were unable to analyze the agreement for ceftriaxone with the Etest because all the strains had MICs lower than the minimum (⬍0.002 ␮g/ml) present on the Etest strips. The MICs obtained by calculating the modal result in MH⫹B are shown in Table 3. The MICs were called the MIC consensus. The MIC was not calculated for ceftriaxone with Etest for the reason stated previously. An agreement of 90.6% was observed between the agar dilution and Etest methods (Table 4). The best agreement was observed with penicillin G, ciprofloxacin, and cefotaxime. The agreement of each laboratory/antibiotic/method combination is shown in Table 5. The results for each antibiotic were heterogeneous, with some laboratories showing large variations in agreement; laboratory number 12 ranged from 23.5% to 100%, and laboratories 2 and 9 ranged from 41.2% to 100%. The most homogeneous results were obtained by laboratory 8 (Table 5). Of note was the apparent difficulty in determining susceptibility to rifampin, particularly with the Etest method. In fact, no laboratory obtained a 100% agreement in this antibiotic/method combination (Table 5). The worst results (obtained by laboratories 5 and 14) were in determining MICs of ceftriaxone in agar dilution (Table 5); but, this antibiotic/ method combination offered very good and homogeneous results in the other laboratories, even at very low concentrations. Comparing the penicillin MICs with sequencing and RFLP analysis of the penA gene, 11 meningococci of the collection were defined as having intermediate resistance to penicillin (PenI). This information was used to analyze the ability of the laboratories to detect PenI strains. The meningococci identified as intermediate were those numbered EMGM 5, 6, 7, 8, 9, 10, 12, 13, 15, 16, and 18. If we accept that the PenI strains are those defined by sequence/RFLP of the penA gene, only three laboratories were able to detect all of these meningococci by the agar dilution and Etest methods. One laboratory was unable to detect five (45.4%) intermediate strains by agar dilution, while another detected only two (18.2%). The MICs of

penicillin were found to be higher than 0.06 ␮g/ml (the breakpoint used to define intermediate resistance) for all strains previously defined as PenI meningococci except strain EMGM5 (Table 3). Strain EMGM5, assigned a MIC consensus corresponding to a susceptible isolate, was the most difficult to determine as PenI. DISCUSSION Three laboratories reported problems obtaining confluent growth in MH without supplement. MH⫹B and MH⫹CH gave very similar results of general agreement (Table 2), but MH⫹B is easier to prepare and additionally gave the results closest to the consensus overall in each antibiotic/method combination. We therefore recommend the use of MH (or some other proper basal medium) supplemented with 5% blood for the routine determination of MICs for N. meningitidis. Several laboratories used their own basal medium, and the results were comparable (data not showed). The agreement between the agar dilution and Etest methods was good, being 100% for penicillin G (Table 4). This is a very important finding because many laboratories use the Etest method as the routine method to determine the MIC for meningococcal strains (5). This would allow accurate comparison of the incidence of PenI isolates between laboratories using different methods. One group detected unusual patterns of growth along the Etest strips, and that finding might have made it more difficult to determine the MIC for the strains showing that phenomena. The agreement between agar dilution and Etest was worse with ofloxacin and rifampin, but the discrepancies were very close to the range of ⫾1 dilution difference. This finding is very important for rifampin, because the discrepancies were found in strains with very low MICs (EMGM 11, EMGM 12) and also those showing resistance (EMGM 16) (Table 3). However, both methods defined the same strains as resistant or susceptible to rifampin, so the discrepancies may affect the epidemiological surveillance trends but not the clinical information. All the strains showed a high level of susceptibility to ofloxacin, so

3434

´ ZQUEZ ET AL. VA

we could not compare the sensitivity of each method in detectong different levels of susceptibility. The comparison between the agar dilution and Etest methods will be interesting to study further if strains showing decreased susceptibility to that antimicrobial agent appear (13). There was one strain (EMGM 5) possessing a sequence typical of a PenI isolate that demonstrated a MIC consensus inside the susceptibility range with both methods (Table 3). The study shows a limitation at this level: no strains with a MIC of 0.094 ␮g/ml by Etest were included. The NCCLS does not specify the breakpoint to define the susceptibility levels of penicillin with microdilution or macrodilution, but those described for Neisseria gonorrhoeae have been used for meningococci (2). These breakpoints are ⱕ0.06 ␮g/ml for susceptible, 0.12 to 1 ␮g/ml for intermediate, and ⱖ2 ␮g/ml for resistant strains (10). Both methods (microdilution and agar dilution) are based on a range of doubling dilutions. With those breakpoint definitions, there will be several gaps by Etest, and strains with MICs of 0.094 ␮g/ml or between 1 and 2 ␮g/ml might be misclassified. The definition of the breakpoints to be used to define different levels of susceptibility should take into account the possibility that Etest will be used. In the same way, it is very important that all laboratories work with the same range of doubling dilutions in order to apply definite categories. To this end, the range of doubling dilutions that allow the classification of the strains according to breakpoints defined for gonococci by the NCCLS should be used (10). Because the main area of concern with meningococci is intermediate resistance to penicillin, most of the study was focused on the ability of each laboratory to detect this level of resistance. Ten strains were defined as PenI according to the common definition. But the only mechanism well known to increase the level of resistance is genetic modification in the penA gene. The sequence of this gene and the RFLP patterns of the gene allowed us to identify 11 PenI meningococci. No isolate with a sequence characteristic of susceptible strains presented a MIC consensus higher than 0.06 ␮g/ml, so we do not think that mechanisms other than the modification of the penA gene contribute to this intermediate resistance level. The EMGM 5 strain, well defined as intermediate by the sequence or RFLP pattern, had a MIC consensus of 0.06 ␮g/ml by the agar dilution and 0.064 ␮g/ml by the Etest method (Table 3). The real MIC of this strain should be close to 0.1 ␮g/ml; in fact, five laboratories found MICs higher than 0.06 ␮g/ml by Etest, ranging between 0.094 and 0.125 ␮g/ml. The development of a molecular approach to define and determine a PenI strain may be warranted. This panel of serologically well defined meningococci with known penicillin resistance determinants could be used in further studies to define quality control limits and MIC breakpoints for meningococci. We propose the use of four isolates with a high agreement between laboratories (EMGM 1, 2, 10, and 13) as the basis of a strain collection to make available for

ANTIMICROB. AGENTS CHEMOTHER.

use by others working in the field of meningococcal antibiotic resistance. These isolates belong to the EMGM and are kept at the Spanish Reference Laboratory for Meningococci (National Institute of Health Carlos III), where they can be obtained by request. ACKNOWLEDGMENTS This work was supported by grant QLK2-CT-2001-01436 from the European Commission. The large number of laboratories working on this project makes it difficult to include everybody who might sign this paper. We thank Laura de la Fuente (Instituto de Salud Carlos III, Spain), Gillian Smollan (Hadassah University Hospital, Israel), Lene Berthelsen (Statens Serum Institute, Denmark), Paula Urbaskova (National Reference Laboratory for Antibiotics, Czech Republic), and Cecilia Fazio (Istituto Superiore di Sanita´, Italy) for their collaboration. REFERENCES 1. Antignac, A., J. M. Alonso, and M. K. Taha. 2001. Nonculture prediction of Neisseria meningitidis susceptibility to penicillin. Antimicrob. Agents Chemother. 45:3625–3628. 2. Arreaza, L., L. De La Fuente, and J. A. Va ´zquez. 2000. Antibiotic susceptibility patterns of Neisseria meningitidis isolated from patients and asymptomatic carriers. Antimicrob. Agents Chemother. 44:1705–1707. 3. Arreaza, L., and J. A. Va ´zquez. 2001. Molecular approach for the study of penicillin resistance in Neisseria meningitidis, p. 107–119. In A. J. Pollard and M. C. J. Maiden (ed.), Meningococcal disease. Methods and protocols. Humana Press, Totowa, N.J. 4. Ba ¨ckman, A., P. Orverlid, J. A. Va ´zquez, O. Sko ¨ld, and P. Olce´n. 2000. Complete sequence of a beta-lactamase encoding plasmid in Neisseria meningitidis. Antimicrob. Agents Chemother. 44:210–212. 5. Block, C. 2001. Antibiotics susceptibility testing, p. 89–106. In A. J. Pollard and M. C. J. Maiden (ed.), Meningococcal disease. Methods and protocols. Humana Press, Totowa, N.J. 6. Block, C., Y. Davidson, and N. Keller. 1998. Unreliability of disc diffusion test for screening for reduced penicillin susceptibility in Neisseria meningitidis. J. Clin. Microbiol. 36:3103–3104. 7. Campos, J., P. M. Mendelman, M. U. Sako, D. O. Chaffin, A. L. Smith, and J. A. Sa ´ez Nieto. 1987. Detection of relatively penicillin G resistant Neisseria meningitidis by disc susceptibility testing. Antimicrob. Agents Chemother. 31:1478–1482. 8. Cuevas, L. E., and C. A. Hart. 1993. Chemoprophylaxis of bacterial meningitis. J. Antimicrob. Chemother. 31(Suppl. B):79–91. 9. Hughes, J. H., D. J. Biedenbach, M. E. Erwin, and R. N. Jones. 1993. E test as susceptibility test and epidemiologic tool for evaluation of Neisseria meningitidis isolates. J. Clin. Microbiol. 31:3255–3259. 10. National Committee for Clinical Laboratory Standards. 2003. Performance standards for antimicrobial susceptibility testing; eleventh informational supplement. NCCLS document M100-S13. National Committee for Clinical Laboratory Standards, Wayne, Pa. 11. Richter, S. S., K. A. Gordon, P. R. Rhomberg, M. A. Pfaller, and R. N. Jones. 2001. Neisseria meningitidis with decreased susceptibility to penicillin: report from the SENTRY antimicrobial surveillance program, North America, 1989–99. Diagn. Microbiol. Infect. Dis. 41:83–88. 12. Sa ´ez-Nieto, J. A., R. Lujan, S. Berro ´n, J. Campos, M. Vin ˜ as, C. Fuste´, J. A. Va ´zquez, Q. Y. Zhang, L. D. Bowler, J. V. Martinez Sua ´rez, and B. G. Spratt. 1992. Epidemiology and molecular basis of penicillin resistant Neisseria meningitidis in Spain: a five year history (1985–1989). Clin. Infect. Dis. 14:394– 402. 13. Shultz, T. R., J. W. Tapsall, P. A. White, and P. J. Newton. 2000. An invasive isolate of Neisseria meningitidis showing decreased susceptibility to quinolones. Antimicrob. Agents Chemother. 44:1116. 14. Tapsall, J. W., T. Shultz, E. Limnios, R. Munro, J. Mercer, R. Porritt, J. Griffith, G. Hogg, G. Lum, A. Lawrence, D. Hansman, P. Collignon, P. Southwell, K. Ott, M. Gardam, C. J. Richardson, J. Bates, D. Murphy, and H. Smith. 2001. Surveillance of antibiotic resistance in invasive isolates of Neisseria meningitidis in Australia 1994–1999. Pathology 33:359–361. 15. Va ´zquez, J. A. 2001. The resistance of Neisseria meningitidis to the antimicrobial agents: an issue still in evolution. Rev. Med. Microbiol. 12:39–45.