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Antibiotic susceptibility and characteristics of Neisseria meningitidis isolates from the African meningitis belt, 2000 to 2006 : phenotypic and genotypic perspectives


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⫹0 doi:10.1128/AAC.00994-08

Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Antibiotic Susceptibility and Characteristics of Neisseria meningitidis

Isolates from the African Meningitis Belt, 2000 to 2006: Phenotypic

and Genotypic Perspectives

Sara Thulin Hedberg,


* Hans Fredlund,


Pierre Nicolas,


Dominique A. Caugant,


Per Olce



and Magnus Unemo


National Reference Laboratory for Pathogenic Neisseria, Department of Laboratory Medicine, Microbiology, O

¨ rebro University Hospital,


¨ rebro, Sweden


; French Forces Institute of Tropical Medicine (IMTSSA), WHO Collaborating Centre for Reference and

Research on Meningococci, Marseille, France


; and WHO Collaborating Centre for Reference and

Research on Meningococci, Norwegian Institute of Public Health, Oslo, Norway


Received 25 July 2008/Returned for modification 12 October 2008/Accepted 18 January 2009

Up-to-date information regarding the antibiotic susceptibility of Neisseria meningitidis strains from African

countries is highly limited. Our aim was to comprehensively describe the antibiotic susceptibilities of a

selection of N. meningitidis isolates recovered between 2000 and 2006 from 18 African countries, mainly those

within the meningitis belt. Susceptibilities to 11 antibiotics were determined using Etest for 137 N. meningitidis

isolates (stringently selected from 693 available isolates). The isolates were also characterized by serogrouping,

multilocus sequence typing, genosubtyping, and penA allele identification. All N. meningitidis isolates were

susceptible to ceftriaxone, chloramphenicol, and ciprofloxacin. No isolate produced

␤-lactamase. Only three

isolates (2%) displayed reduced susceptibility to penicillin G. The two isolates with the highest penicillin G

MICs were the only isolates showing reduced susceptibility to ampicillin and cefuroxime. One of these isolates

was also resistant to penicillin V. One percent of isolates displayed reduced susceptibility to rifampin, while

52% of the isolates were resistant to tetracycline, 74% were resistant to erythromycin, and 94% were resistant

to sulfadiazine. The MICs of rifampin and tetracycline seemed to be associated with the serogroup of the

isolates. In total, 18 sequence types (STs), 10 genosubtypes, and 8 different penA alleles were identified; the

most common were ST-7, P1.20,9,35-1, and penA4, respectively. A high level of correlation was found between

ST, genosubtype, and penA allele. In conclusion, N. meningitidis isolates from the African meningitis belt

remain highly susceptible to the antibiotics used. Regarding

␤-lactam antibiotics, rare isolates showed a

reduced susceptibility to penicillins, but the expanded-spectrum cephalosporins are not affected at present.

Invasive meningococcal disease is a significant health

problem worldwide. In Africa, particularly in the sub-Saharan,

so-called meningitis belt, epidemics of acute meningitis can

reach incidence rates of 1,000 cases per 100,000 inhabitants,

and in individual communities, attack rates as high as 1:10

for the population have been reported. During these

epi-demics, a mortality rate of about 10% is usually reported,

which most probably is an underestimation (14). These

epi-demics in the African meningitis belt have historically been

caused mostly by a limited number of Neisseria meningitidis

serogroup A clones. During recent years, strains of other

sero-groups, such as serogroups C, W-135, and X, have also been

involved (13).

Early antibiotic treatment of meningococcal disease is

cru-cial for keeping the case fatality rate and risk of sequelae as low

as possible. In Africa, the general recommendation for

treat-ment during endemic periods is (i) ceftriaxone in multiple

doses to also provide effective treatment of other presumptive

etiological agents of bacterial meningitis, such as Streptococcus

pneumoniae and Haemophilus influenzae; or (ii) multiple doses

of penicillins. During meningococcal epidemics, the

recom-mended treatment is (i) a single dose of chloramphenicol in oil

or (ii) a single dose of ceftriaxone (13, 30, 43). However,

increased levels of reduced susceptibility to penicillins have

been reported worldwide (22, 28, 32, 39, 42). This reduced

susceptibility has been due mainly to alterations in

penicillin-binding protein 2, encoded by the penA gene (3, 27, 32, 35).

Furthermore, though still rare, resistance to chloramphenicol

has been reported from Australia, France, and Vietnam (12,

25). This resistance is considered to be due mainly to the

presence of the catP gene, encoding the enzyme

chloramphen-icol acetyltransferase. Resistance to ceftriaxone has been

re-ported from India (18). However, these strains certainly need

to be examined further, comprehensively characterized

pheno-typically and genetically, and confirmed/disconfirmed by an

independent laboratory (20).

In the African meningitis belt, chemoprophylaxis of close

contacts of patients is rarely used and is not recommended by

the WHO (43). In the Western world, on the other hand,

chemoprophylaxis is often used, and ciprofloxacin and

ri-fampin are recommended antibiotics (30). However, in recent

years, there have been reports of reduced susceptibility to

ciprofloxacin (1, 6–8, 26, 31) as well as resistance to rifampin

from several countries worldwide (23, 29, 33).

Comprehensive data regarding the antibiotic susceptibility

of N. meningitidis in many African countries are limited, and no

* Corresponding author. Mailing address: Department of

Labora-tory Medicine, Microbiology, O

¨ rebro University Hospital, SE-701 85


¨ rebro, Sweden. Phone: 46 19 602 15 20. Fax: 46 19 12 74 16. E-mail:


Published ahead of print on 2 February 2009.


by on March 19, 2009



up-to-date extensive study of the antibiotic susceptibility of N.

meningitidis is at hand. Susceptibility testing, performed mainly

using the disc diffusion method and the most commonly used

antibiotics for treatment, is carried out mainly in hospital

lab-oratories in the meningitis belt. Disc diffusion methodology,

however, is hard to optimize, standardize, and quality assure,

which makes the results from different laboratories and

coun-tries difficult to compare.

The aims of the present study were to comprehensively

de-scribe the antibiotic susceptibilities (to 11 different antibiotics)

of invasive N. meningitidis isolates, strictly selected from 18

different African countries, mainly those within the meningitis

belt, during the period 2000–2006 and to genetically

charac-terize them.


Bacterial isolates.The N. meningitidis isolates used for this study were from collections available at the WHO Collaborating Centers in Marseille, France, and Oslo, Norway, and from the National Reference Laboratory for Pathogenic Neisseria in O¨ rebro, Sweden. In total, between 2000 and 2006, 693 invasive isolates were recovered from patients, using routine bacteriological procedures, in 18 different African countries, mainly within the meningitis belt (Table 1). From this collection, all isolates with suspected reduced susceptibility according to initial testing, as well as isolates representing the prevalent phenotypes (se-rogroup and antibiogram) from each country, were included. When several isolates were related to the same epidemic outbreak, only one representative isolate was selected. One hundred thirty-seven invasive isolates were subse-quently examined, comprising serogroups A (n⫽ 82), W-135 (n ⫽ 38), X (n ⫽ 8), Y (n⫽ 7), and C (n ⫽ 1), with one nongroupable isolate (Table 1). For quality control, one serogroup A reference strain, OR173/87 (15, 19), was included. The reference strain was susceptible to all antibiotics tested, except for erythromycin (MIC⫽ 0.75 ␮g/ml) and sulfadiazine (MIC ⬎ 256 ␮g/ml).

Phenotypic antibiotic susceptibility testing.The MICs of penicillin G, ampi-cillin, penicillin V, ceftriaxone, cefuroxime, chloramphenicol, ciprofloxacin, ri-fampin, tetracycline, erythromycin, and sulfadiazine were determined for all isolates, using the Etest method (AB Biodisk, Solna, Sweden) on Mueller-Hinton agar (Becton Dickinson and Company, Sparks, MD) supplemented with 5% sheep blood at 37°C in 5% CO2for 16 to 18 h (40). The present Etest

method-ology was previously evaluated in comparison to a reference method, i.e., agar dilution (41). The breakpoints used are shown in Table 2. The breakpoints used were from several different organizations, since no organization describes break-points for all examined antibiotics. All isolates were tested for␤-lactamase production by use of nitrocefin discs (AB Biodisk, Solna, Sweden).

Isolation of genomic DNA.Isolation of genomic DNA from the N. meningitidis isolates was performed using the MagNA Pure system (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions. The DNA preparations were stored at 4°C prior to PCR.

MLST.Multilocus sequence typing (MLST) was performed mainly as previ-ously described (15, 17). The isolates were assigned a sequence type (ST) ac-cording to the Neisseria MLST website (http://pubmlst.org/neisseria/).

Genosubtyping.The porA gene was amplified by real-time PCR as previously described (19). The porA amplicons were purified using a Multiscreen PCR␮96

plate (Millipore, Bedford, MA) and vacuum filtration according to the manu-facturer’s instructions. The purified products were resolved in 50␮l of 10 mM Tris-HCl, and cycle sequencing PCR was performed as previously described (19). Subsequently, the products were purified using ethanol-sodium acetate precipi-tation and resuspended in 10␮l formamide (Applied Biosystems, Warrington, United Kingdom) according to the manufacturer’s instructions. The nucleotide sequences were determined using an ABI PRISM 3100 genetic analyzer (Ap-plied Biosystems, Foster City, CA). The deduced amino acid sequences of vari-able region 1 (VR1) and VR2 were assigned genosubtype numbers according to the Neisseria meningitidis PorA variable regions database (http://neisseria.org /perl/agdbnet/agdbnet.pl?file⫽poravr.xml), and the sequences of VR3 were as-signed numbers according to the PorA VR3 database (http://www.smprl.scot.nhs .uk/PorA_VR3.htm).

penA gene sequencing.The penA gene was amplified and sequenced as previ-ously described (4, 35). The isolates were assigned a penA allele number by using the N. meningitidis penA database (http://neisseria.org/perl/agdbnet/agdbnet .pl?file⫽penA.xml). Based on the different penA alleles, a phylogenetic tree was

constructed with TREECON (version 1.3b) software by using the Jin and Nei substitution model, the Kimura evolutionary model, an␣ value of 0.5, and the neighbor-joining method (38), as previously described (37).


Antibiotic susceptibility.

The results of antibiotic

suscepti-bility testing are summarized in Table 2. Briefly, all isolates


␤-lactamase negative and susceptible to ceftriaxone


ⱕ 0.002 ␮g/ml), chloramphenicol (MIC, 0.38 to 1.5 ␮g/

ml), and ciprofloxacin (MIC, 0.002 to 0.012

␮g/ml). Three of

the isolates (2%) displayed reduced susceptibility to penicillin

G; two of these isolates were in serogroup A (isolated in

Ethiopia and Somalia), and one was in serogroup Y (isolated

in Senegal). The two isolates with the highest penicillin G


⫽ 0.25 ␮g/ml and 0.38 ␮g/ml for isolates in

sero-groups A and Y, respectively) were the sole isolates displaying

TABLE 1. Numbers and serogroups of N. meningitidis isolates

recovered in 18 different African countries, mainly those in

the meningitis belt, during the period 2000–2006, according

to year and country (n

⫽ 137)


Country Yr of


No. of isolates (serogroup distribution)a



2 (A)


1 (A)



3 (2 A, 1 W-135)


3 (1 A, 1 Y, 1 W-135)


3 (2 W-135, 1 Y)

Burkina Faso


5 (3 A, 2 W-135)


2 (W-135)


6 (3 A, 2 W-135, 1 Y)


12 (7 A, 3 W-135, 1 Y, 1 NG)



2 (A)



4 (A)


5 (4 A, 1 W-135)

Central African



2 (W-135)



7 (6 A, 1 W-135)


1 (W-135)


1 (W-135)


2 (W-135)



1 (A)



1 (A)


1 (A)


2 (A)


2 (A)



3 (2 A, 1 W-135)



2 (W-135)



8 (A)


5 (3 A, 1 X, 1 W-135)


11 (4 A, 3 Y, 4 W-135)


4 (3 A, 1 X)


5 (3 X, 2 W-135)



2 (1 A, 1 W-135)


2 (1 A, 1 W-135)

Republic of the Congo


4 (2 A, 1 C, 1 W-135)



2 (1 A, 1 W-135)


3 (2 A, 1 W-135)


2 (1 Y, 1 W-135)



2 (A)



6 (5 A, 1 W-135)


6 (A)



2 (X)

a NG, nongroupable. b

All isolates were from cerebrospinal fluid.

by on March 19, 2009



reduced susceptibility to ampicillin and cefuroxime. The

sero-group Y isolate was also resistant to penicillin V (MIC

⫽ 1.5

␮g/ml). Overall, 1 of 82 serogroup A isolates (⬍1%) displayed

reduced susceptibility to rifampin (MIC

⫽ 0.38 ␮g/ml).

Fifty-two percent of the isolates were resistant to tetracycline, 74%

were resistant to erythromycin, and 94% were resistant to


For rifampin, two susceptible populations could be

ob-served, one comprising mainly serogroup W-135 and Y

iso-lates, displaying very low MICs, and one with higher MICs,

comprising mainly serogroup A and X isolates (Fig. 1). For

tetracycline, a similar pattern was observed, with a resistant

population (MIC, 2 to 6

␮g/ml) of mainly serogroup A isolates,

belonging to ST-7 or ST-2859, and a susceptible population

(MIC, 0.064 to 0.38

␮g/ml) consisting of isolates representing

other serogroups and STs.

MLST, genosubtyping, and penA gene sequencing.


different STs (comprising five clonal complexes and six STs not

belonging to any clonal complex) were identified. ST-7 (47%),

ST-11 (20%), and ST-2881 (9%) were the most prevalent STs,

and clonal complex ST-5 (cc5) was the most common clonal

complex (58% of the isolates).

A total of 10 genosubtypes were identified, with P1.20,9,35-1

(58%), P1.5,2,36-2 (20%), and P1.5-1,2-36,36-2 (9%) being the

most prevalent.

Eight penA alleles were identified, with penA4 being the

most common (65%) (Fig. 2). penA4 was also found to be

associated with serogroup A and cc5 (Fig. 2). Accordingly,

TABLE 2. Susceptibilities of N. meningitidis isolates (n

⫽ 137) recovered during the period 2000–2006 in 18 different African countries,

mainly those within the meningitis belt, to 11 different antibiotics

Antibiotic Breakpoints


MIC range

(␮g/ml) (MIC␮g/ml)50 (MIC␮g/ml)90

% of isolates

Susceptible Intermediate Resistant

Penicillin G


















Penicillin V




















































































Breakpoints previously described by Taha et al., based on a combination of identified penicillin G MICs and the presence/absence of a penA mosaic allele (32).


Breakpoints proposed by the European Meningococcal Disease Society (40).


Breakpoints in accordance with the Swedish Reference Group for Antibiotics (www.srga.org).


Breakpoints in accordance with the British Society for Antimicrobial Chemotherapy (4a).


Breakpoints for sulfisoxazole were used, which displayed a similar MIC distribution to that of sulfadiazine by Etest (data not shown).

FIG. 1. Serogroup distribution and MICs of rifampin for N. meningitidis isolates (n

⫽ 137) collected in 18 different African countries, mainly

those from the meningitis belt, from 2000 to 2006. The broken line indicates the breakpoint for susceptible isolates (MIC

ⱕ 0.25 ␮g/ml).

by on March 19, 2009



93% of all serogroup A and 97% of all cc5 isolates had the

penA4 allele.

The two isolates with the highest MICs of penicillin G


⫽ 0.25 ␮g/ml and 0.38 ␮g/ml) comprised mosaic alleles

(penA52 and penA20, respectively), while all other isolates

displayed wild-type penA alleles. Consequently, there was an

association between reduced susceptibility to penicillins and

the presence of penA mosaic alleles in African meningitis belt

N. meningitidis isolates.

There was also an association between penA alleles and

genosubtypes (Fig. 2). Ninety-six percent of the isolates with

genosubtype P1.20,9,35-1 carried penA4, and 100% of the

ge-nosubtype P1.5,2,36-2 isolates contained penA1. Gege-nosubtypes

and STs were also associated, especially in studying the clonal

complexes. The most common genosubtype, P1.20,9,35-1 (n

79 isolates), consisted of only isolates belonging to cc5, and the

second most common genosubtype, P1.5,2,36-2 (n

⫽ 27),

con-sisted of only cc11 isolates.


In the present study, N. meningitidis isolates recovered in

2000 to 2006, mainly from countries constituting the African

meningitis belt, were found to remain highly susceptible to the

antibiotics used for treatment and prophylaxis. However,

iso-lates from northern and southern Africa were not included and

may differ substantially (10, 44).

Only a few isolates included in the study displayed reduced

susceptibility to penicillin G (2%) and other


antibi-otics (1% had reduced susceptibility to ampicillin, penicillin V,

and cefuroxime). Since the study was based partly on isolates

selected for reduced antibiotic susceptibility, this proportion is

most likely lower among all clinical N. meningitidis isolates in

the African meningitis belt, where numerous isolates

repre-senting identical strains are recovered in outbreak situations.

All isolates were susceptible to ceftriaxone. In spite of the

fact that there have been reports of rare

chloramphenicol-resistant N. meningitidis isolates in Australia, France, and

Viet-nam (12, 25), all of the presently examined African

meningo-cocci were fully susceptible to chloramphenicol. Previous

studies of African N. meningitidis isolates have displayed

iden-tical results, i.e., all isolates were fully susceptible to

chloram-phenicol (11, 36, 44). Furthermore, all previously described

chloramphenicol-resistant isolates have been serogroup B

iso-lates (12, 25), which is extremely rare in Africa. Serogroup B N.

meningitidis strains are highly transformable, unlike serogroup

A strains (25), and hence the lower transformation rate for

serogroup A could be a possible explanation for the lack of

chloramphenicol resistance in the examined African N.

men-ingitidis isolates. However, serogroup A accounted for only

60% of the isolates in the present study, so this is not the only


For continents other than Africa, there have been reports of

reduced susceptibility or resistance to ciprofloxacin (1, 6–8, 26,

31) and rifampin (23, 29, 33), which are commonly used

anti-biotics for prophylaxis. In the present study, however, the

ex-amined N. meningitidis isolates, mainly from the African

men-ingitis belt, were highly susceptible to both antibiotics, with

only one serogroup A isolate from Ethiopia displaying reduced

FIG. 2. Phylogenetic tree based on a 402-bp alignment of penA gene sequences in N. meningitidis isolates (n

⫽ 137) collected in 18 different

African countries, mainly those in the meningitis belt, from 2000 to 2006. The genosubtypes and clonal complexes (cc) associated with the different

penA alleles are included in the boxes.

by on March 19, 2009



susceptibility to rifampin (MIC

⫽ 0.38 ␮g/ml). This finding

may be due to the infrequent use of prophylaxis in Africa.

Tetracycline and erythromycin are not used for treatment of

meningococcal disease. Since these antibiotics are used for

treatment of many other infections, their susceptibility patterns

may reflect the overall antibiotic pressure on African N.

men-ingitidis isolates. A large number of the presently examined

African N. meningitidis isolates were resistant to tetracycline

(52%) and erythromycin (74%). A high level of resistance to

tetracycline (85%) has also been described in a previous study

including a limited number of African N. meningitidis isolates


⫽ 20) (24). According to other previous reports (9, 16),

among 441 examined N. meningitidis isolates, including 22

se-rogroup A isolates, resistance to tetracycline was found only in

the serogroup A isolates, all belonging to cc5. This resistance

was associated with the drug efflux mechanism encoded by the

gene tet(B) (9). The close association between serogroup A

isolates belonging to ST-7 or ST-2859 and tetracycline

resis-tance observed both in the present study and in the previous

one (9) may explain the low resistance to tetracycline (0.3%)

observed in a previous French study including 2,167 N.

men-ingitidis isolates, with only nine serogroup A isolates (2). In

that study, 9.1% of all invasive isolates in France between

1999 and 2002 also displayed reduced susceptibility to

eryth-romycin (2).

In the present study, almost all examined African isolates

displayed wild-type penA alleles. The most common allele was

penA4, which was present in 65% of the isolates. This can be

compared to a recent extensive study where the penA genes of

N. meningitidis isolates (n

⫽ 1,670) from 22 mainly European

countries were sequenced (32). In this study, penA4 was found

in only 10% of the isolates, while penA1 and penA3 together

accounted for 46% of the isolates (32). Accordingly, during

recent years, i.e., 2000 to 2006, penA4 may seem strongly

as-sociated with cc5, and hence serogroup A (Fig. 2). This may be

due to the clonal expansion of cc5 in the meningitis belt since

the 1980s (5, 34). The two African isolates with the highest

MICs for the different penicillins displayed the penA mosaic

alleles 52 and 20. penA52 was found in a serogroup A, ST-7

isolate from Somalia. In the previous European study (32),

penA52 was found in 19 serogroup B isolates from six different

European countries and in 1 serogroup B isolate from the

United States. In the present study, penA20 was found in a

serogroup Y, ST-23 isolate cultured in Senegal. For

compari-son, in the previous European study (32), penA20 was found in

six serogroup Y isolates and one serogroup C isolate,

recov-ered in France and Italy.

In the present study, the most common ST was ST-7 (n

⫽ 64

isolates) (in cc5 [n

⫽ 79]), followed by ST-11 (n ⫽ 28) (in cc11


⫽ 29]). These findings are in congruence with earlier studies

regarding N. meningitidis isolates from the meningitis belt (5,

15, 21, 34). In Africa, cc5 and cc11 have been predominant,

and only a few other sequence types, such as ST-2881 and

ST-181, have been detected sporadically (5). In the present

study, cc5 consisted of two subgroups of serogroup A isolates,

all of which were resistant to sulfadiazine, but 5 and

ST-2207 isolates were susceptible to tetracycline and ST-7 and

ST-2859 isolates were resistant. Both ST-7 and ST-2859 are

relatively new variants of cc5 and have recently expanded in

the meningitis belt to replace ST-5 (5). cc11 and ST-2881

consisted mainly of sulfadiazine-resistant,

tetracycline-suscep-tible serogroup W-135 isolates. In ST-181, only

sulfadiazine-and tetracycline-susceptible serogroup X isolates were found.

All of these isolates were cultured in Niger during 2002 to 2005

and probably represented a single clone, since all phenotypic

and genotypic characteristics for the five individual isolates

were identical, with the exception of slightly different MICs.

In conclusion, N. meningitidis isolates from the African

men-ingitis belt remain highly susceptible to the antibiotics used for

treatment and prophylaxis, and hence, the present results

sup-port the WHO recommendations (43). Regarding


antibiotics, a few isolates displayed reduced susceptibility to

penicillins, but at present the expanded-spectrum

cephalospo-rins, such as ceftriaxone, are not affected. However, it is crucial

to continuously monitor the antibiotic susceptibility of N.

men-ingitidis to avoid future treatment failures in Africa during both

endemic and epidemic periods.


This study was supported by grants from the O

¨ rebro County Council

Research Committee and the Foundation for Medical Research at


¨ rebro University Hospital, O

¨ rebro, Sweden.


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