Evolution of antimicrobial resistance and serotype distribution of Streptococcus pneumoniae isolated from children with invasive and noninvasive pneumococcal diseases in Algeria from 2005 to 2012

Accepted Manuscript Evolution of antimicrobial resistance and serotype distribution of Streptococcus pneumoniae isolated from children with invasive and non-invasive pneumococcal diseases in Algeria from 2005 to 2012 Nadjia Ramdani-Bouguessa, Hanifa Ziane, Souad Bekhoucha, Zehor Guechi, Amina Azzam, Djamila Touati, Malek Naim, Sihame Azrou, Moufida Hamidi, Abdelouahab Mertani, Abdennour Laraba, Tahar Annane, Salah Kermani, Mohamed Tazir PII:

S2052-2975(15)00030-X

DOI:

10.1016/j.nmni.2015.02.008

Reference:

NMNI 29

To appear in:

New Microbes and New Infections

Received Date: 19 September 2014 Revised Date:

19 December 2014

Accepted Date: 24 February 2015

Please cite this article as: Ramdani-Bouguessa N, Ziane H, Bekhoucha S, Guechi Z, Azzam A, Touati D, Naim M, Azrou S, Hamidi M, Mertani A, Laraba A, Annane T, Kermani S, Tazir M, Evolution of antimicrobial resistance and serotype distribution of Streptococcus pneumoniae isolated from children with invasive and non-invasive pneumococcal diseases in Algeria from 2005 to 2012, New Microbes and New Infections (2015), doi: 10.1016/j.nmni.2015.02.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

Evolution of antimicrobial resistance and serotype distribution of

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Streptococcus pneumoniae isolated from children with invasive and non-

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invasive pneumococcal diseases in Algeria from 2005 to 2012

4 Nadjia Ramdani-Bouguessaa, Hanifa Zianea, Souad Bekhouchab, Zehor Guechic,

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Amina Azzamd, Djamila Touatie, Malek Naimf, Sihame Azroug, Moufida Hamidih,

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Abdelouahab Mertania, Abdennour Larabai, Tahar Annanei, Salah Kermanij,

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Mohamed Tazira

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a Service de Microbiologie, Centre Hospitalier Universitaire Mustapha Bacha, Place

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du 1er Mai, Sidi M'Hamed, 16000 Algiers, Algeria. Emails: NRB, AM:

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[email protected]; HZ: [email protected]; MT: [email protected]

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b Centre Hospitalier Universitaire d’Oran, Boulevard du Dr Benzerdjeb, 31000, Oran

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Algiers, Algeria. Emails: SB: [email protected]

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c Centre Hospitalier Universitaire Nafissa Hamoud, Rue Boudjemaa Moghni, 16040

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Hussen Dey, Algiers, Algeria. Emails: ZG: [email protected]

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d Centre Hospitalier Universitaire Nedir Mohamed, Rue Lamali Ahmed, 15000, Tizi

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Ouzou, Algeria. Emails: AA: [email protected]

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e Centre Hospitalier Universitaire Issad Hassani, Rue Ibrahim Hadjrass, Béni-

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Messous, 16206, Algeria. Emails: DT: [email protected].

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f Hôpital Central de l'Armée Mohamed Seghir Nekkache, BP 244, 16205, Ain Naadja,

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Algiers, Algeria. Emails: MN: [email protected]

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g Hôpital de Boufarik, Rue des frères Hamadouche, 09400, Boufarik, Blida, Algeria.

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Email: SA: [email protected]

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ACCEPTED MANUSCRIPT h Hôpital Salim Zmirli, Route de Baraki, BP 71, 16200, El Harrach, Algiers, Algeria.

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Email: MH: [email protected]

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i Centre Hospitalier Universitaire Lamine Debaghine, Boulevard Said Touati, 16009,

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Bab El Oued, Algiers, Algeria. Email: AL, TA: [email protected].

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j Hopital Ain Taya, Hai si El Houas, 16611, Ain Taya, Algiers, Algeria. Email: SK:

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[email protected].

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Corresponding author: Nadjia Ramdani-Bouguessa

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Phone: +213 21 23 57 87

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ACCEPTED MANUSCRIPT Abstract

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Pneumococcal infections are a major cause of morbidity and mortality in developing

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countries. The introduction of pneumococcal conjugate vaccines (PCVs) has

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dramatically reduced the incidence of pneumococcal diseases. PVCs are not

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currently being used in Algeria. We conduct a prospective study from 2005 to 2012 in

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Algeria to determine antimicrobial drug resistance and serotype distribution of

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Streptococcus pneumoniae from children with pneumococcal disease.

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Among 270 isolated strains from children, 97 (36%) were invasive disease; of which

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48% were not susceptible to penicillin, and 53% not susceptible to erythromycin. A

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high rate of antimicrobial non-susceptibility was observed in strains isolated from

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children with meningitis. The serotype distribution from pneumococci isolated from

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children with invasive infections was (by order of prevalence): 14, 1, 19F, 19A, 6B, 5,

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3, 6A, and 23F. Multidrug resistance was observed in serotypes 14, 19F, 19A and

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6B. The vaccine coverage of serotypes isolated from children<5 years was 55.3% for

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PCV7, 71.1% for PCV10 and 86.8% for PCV13.

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Our results highlight the burden of pneumococcal disease in Algeria and the

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increasing S. pneumoniae antibiotic resistance. The current pneumococcal vaccines

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cover a high percentage of the circulating strains, therefore vaccination would reduce

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the incidence of pneumococcal disease in Algeria.

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Keywords: Pneumococcal diseases; Streptococcus pneumoniae; Burden of disease;

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Children; Serotype distribution; Algeria; Pneumococcal conjugate vaccines; ntibio

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ACCEPTED MANUSCRIPT Introduction

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Streptococcus pneumoniae is a leading respiratory pathogen that is responsible for

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infections such as pneumonia, meningitis, bacteremia and otitis media. In 2003, the

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World Health Organization (WHO) estimated that pneumococcal disease is

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responsible for 1 million deaths annually, most of which occur in children <5 years of

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age in the developing world [1]. Although pneumococcal meningitis is relatively rare,

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it is strongly associated with mortality or subsequent neurological damage [2].

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Pneumococcal resistance to penicillin was first described in 1967, and since the

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1990s an increasing rate of resistance has been reported worldwide [3, 4]. This

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resistance makes the treatment of serious pneumococcal infections difficult and

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many antibiotic treatment failures have been reported [5].

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In Algeria, several reports have shown an increase in antibiotic resistance from 1996

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to 2010, especially among children [6-9]. Although epidemiological surveillance data

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for invasive pneumococcal infections are available, clinical data are lacking.

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Moreover, since the introduction of Hib vaccination in 2008 in Algeria, S. pneumoniae

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has become the predominant pathogen in bacterial meningitis (unpublished data).

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The S. pneumoniae polysaccharide capsule is a major virulence factor; more than 90

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serotypes have been identified, and their distribution differs in different regions and

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between developing and developed countries [10]. This highlights the importance of

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having national data before implementation of a pneumococcal vaccine. The aim of

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the study was to investigate the evolution of antibiotic resistance and serotype

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distribution of S. pneumoniae in infections in children in Algeria.

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ACCEPTED MANUSCRIPT Material and methods

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From January 2005 to June 2012, 270 unique S. pneumoniae isolates were collected

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from children aged from 0 to 16 years old with invasive and non-invasive infections;

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about 89% were from Algiers and 11% from Oran, a town located in the western part

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of the country. The hospital clinical laboratories were asked to send all their viable

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isolates. Every year, the centers isolate between 15 and 50 S.pneumoniae strains

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from children who are diagnosed by physicians with invasive pneumococcal disease

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(IPD) and non-IPD (NIPD). S. pneumoniae isolates were identified by colony

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morphology, Gram staining, catalase reaction, optochin susceptibility and bile lysis.

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Antibiotic susceptibilities for oxacillin, erythromycin, clindamycin, tetracyclin,

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chloramphenicol and cotrimoxazole were determined following the Clinical and

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Laboratory Standards Institute (CLSI) recommendations [11, 12]. Minimum inhibitory

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concentrations (MICs) for penicillin, amoxicillin and cefotaxim were determined using

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the E-test following the manufacturer’s instructions (Solna, Sweden) for all strains.

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The S. pneumoniae ATCC 49619 strain was used for quality control.

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Serotyping was performed by latex agglutination for determining pools, and serotypes

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were identified using the Neufeld test (Pneumo test latex, Statens Serum Institute,

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Copenhagen). A total of 127 isolates were serotyped, 85 from invasive samples and

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42 from non-invasive samples. The isolates that were serotyped were selected on the

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basis of the clinical data, with priority given to IPD and the number of viable isolates.

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Isolates were stored in 10% glycerol broth at –80°C after primary isolation.

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Results

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From the 270 isolates collected (IPD n=97, NIPD n=173), 197 (73.0%) were from

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children <5 years, of whom 151 (76.7%) were <2 years (Table 1). Among the isolates

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ACCEPTED MANUSCRIPT from children with IPD, 78.4% were collected from children <5 years of age, of whom

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76.3% were <2 years old (Table 1). The IPD isolates were collected from children

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with meningitis (n=53), pneumonia and pleuropneumonia (n=25), bacteremia (n=11),

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arthritis or peritonitis infections (n=8). The non-IPD isolates were from ENT infections

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(n=91), bronchopulmonary infections (n=77) and other suppurative infections (n=5).

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Among the isolates from children with NIPD, 69.9% were from children <5 years of

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age; 53.8% were <2 years old (Table 1).

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Non-susceptibility to penicillin was detected in 48% of the S. pneumoniae isolates

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(MICs ranged from 0.016 µg/ml to 4 µg/ml); 2.6% of isolates had intermediate

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resistance to amoxicillin; 7% and 1.7% of isolates had intermediate and full

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resistance to cefotaxime, respectively (Table 2). For the IPD isolates, the MIC90s

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were 2 µg/ml for penicillin and amoxicillin and 1.5 µg/ml for cefotaxime (Table 2). The

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highest rate of cefotaxim resistance was observed in isolates from meningitis: 20.8%

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intermediate and 3.8% resistant (Table 2).

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The percentages of isolates that were resistant to non-beta-lactam antibiotics were:

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53.0% for erythromycin and cotrimoxazole; 43.7% for clindamycin; 42.0% for

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tetracycline; and 5.3% for chloramphenicol. According to the new breakpoints

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suggested by CLSI, whereby meningitis and non-meningitis isolates have different

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breakpoints, 49.0% of the meningitis isolates were resistant to penicillin

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(MIC≥0.12 µg/ml), among which 26.9% had a MIC≥2 µg/ml.

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After meningitis isolates, the next highest rate of penicillin non-susceptible S.

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pneumoniae (PNSP) was observed in the isolates from non-invasive PD. From all the

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invasive isolates (n=97), 40.3% were pneumococcus non-susceptible to penicillin

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(PNSP) with 81.5% of these from children <5 years and 72.7% from children <2

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years.

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ACCEPTED MANUSCRIPT Among the 85 IPD samples that were serotyped, meningitis was the most common

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diagnosis n=50 (58.8%), followed by pneumonia and pleuropneumonia 21 (24.7%)

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and bacteremia 9 (10.6%) (Table 3). The prevalence of IPD in children <5 years and

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< 2 years was 78.3% (n=76) and 59.8% (n=58), respectively. The most frequent

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serotypes for invasive isolates were 14 (29.4%), 1 (10.6%), 19F (10.6%), 19A (7%),

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6B (7%), 5 (4.7%), 3 (4.7%), 6A (3.5%) and 23F (3.5%). Serotype 14 was the most

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prevalent in meningitis (34%) and pleuropneumonia (28.6%), while serotype 19F

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(38%) and 14 (23.8%) were the most frequent in non-invasive samples (Table 3). In

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order of prevalence, serotypes 14,19F, 6B, 19A, 1, 5, 23F, 6A, 3, 7F and 18C

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accounted for 91.0% of IPD in children <5 years old. Serotypes 5, 7F, 6B, 18C and

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19A were found exclusively in children <2 years of age.

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The serotypes that were most often antibiotic resistant were 14, 19F, 19A and 6B;

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they were mainly highly resistant to penicillin and only serotypes 14 and 19A were

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resistance to cefotaxime (Figure 2, Table 4).

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144 Discussion

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The prevalence of penicillin resistance among pneumococcal disease isolates in

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Algeria, which was measured by the oxacillin disc diffusion method, appears to have

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increased over the years from 34.6% in 1995-2000 to 48.1% in 2005-2012 (current

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study) [6-9]. The percentage of PNSP among IPD isolates in children was reported to

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be approximately 11% in Western European countries, with the highest percentage

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(49%) in Spain [1].

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Our results are more consistent with the increasing antibiotic resistance rates

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reported for North African countries. For instance, in Tunisia and Morocco, the rates

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of PNSP were respectively 52.8% and 43.3% [6-9, 13-15]. The dramatic increase in

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ACCEPTED MANUSCRIPT the antibiotic resistance rate since our previous study (PNSP of 48.1% in this study

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compared with 34.6% in 2001) could be explained by overuse of antibiotics for acute

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respiratory tract infections in Algeria [8].

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We also observed an evolution in the serotype distribution in Algeria. While the

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distribution is similar to that reported for Morocco and Tunisia, as well as other

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developing countries, it differs from that reported in our previous study where we

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found serotypes/serogroups 1, 5, 14 and 6 were the most frequent [8, 9, 15, 16].

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Some pneumococcal serotypes, such as serotypes 1, 5 and 7F, have more invasive

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potential than others; serotype 7F has been reported to be associated with a higher

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risk of severe and fatal outcomes [17]. In Africa, serotypes 1 and 5 are commonly

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associated with invasive diseases. In Algeria, the frequency of serotype 1 was low

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during the 2002-2004 and 2005-2007 period, but increased in 2008-2009

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[unpublished data]. Similar observations of cyclical peaks in serotype 1 incidence

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have been reported in many countries. Although serotypes 1 and 5 are designated as

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‘developing country serotypes’, they have also been reported to be frequently

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responsible for pediatric IPD in industrialized countries such as Germany, Sweden

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and England [18, 19]. Serotype 1 has remained one of the most prevalent invasive

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serotypes and is usually associated with meningitis outbreaks in Africa and in

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crowded communities [16, 20]. It is ranked as one of the four serotypes with the

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highest IPD burden in Africa, Asia and Latin American, while serotype 5 is ranked

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third in Africa and Latin America and fourth in Asia [10]. In Thailand, the seven most

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frequent serotypes associated with IPD in patients <5 years old were: 6B, 23F, 14,

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19F, 19A, 6A, and 4 or 9V [21].

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Two studies in Oxford and Stockholm that characterized the genotypes and

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serotypes of nasopharyngeal and invasive pneumococcal isolates from children and

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ACCEPTED MANUSCRIPT adults identified serotypes 1, 4 and 7F as having a high level of invasiveness, and

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serotypes 3 and 7F as having a higher case fatality rate compared with other

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serotypes [22].

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The predominant serotypes of PNSP in our study are similar to those reported in

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other studies and previous studies in Algeria, with the emergence of serotype 19A in

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the absence of routine pneumococcal vaccination in our country [2, 8, 9, 14, 15]. The

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most commonly multi-resistant serotypes among the PNSP were 19A, 19F, 14 and

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6B, which were resistant to erythromycin, co-trimoxazole, clindamycin and

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tetracycline.

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Serotype 14 is one of the most invasive serotypes that can cause life-threatening

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IPDs. Moreover, it can harbor multiple resistance determinants, conferring resistance

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to penicillin, erythromycin and cefotaxime [23]. In many countries the multi-resistant

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serotype 19A has become the most predominant non-vaccine serotype isolated after

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PCV7 vaccination [24].

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Based on the serotype distribution observed in our study, the PCV7 coverage in

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children < 5 years of age with IPD in Algeria is 55.3%, the PCV10 coverage is 71.1%

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and the PCV13 coverage is 86.8%. This vaccine coverage among children <2 years

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is 51.7% for PCV7, 69% for PCV10 and 87.9% for PCV13. These data may not

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reflect the situation in all cases of IPD in the studied regions because many children

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with IPD are treated empirically. In addition, the limitations of existing diagnostic tests

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impact the ability to obtain accurate IPD burden data, since only 10% of blood culture

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are positive so most patients with pneumonia are not bacteriemic [25]. In Algeria, the

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lack of laboratory facilities including no facilities for storing frozen samples at -80°C in

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different regions of the country is another limiting factor for conducting a multicenter

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study, in order to obtain accurate IPD burden data. Despite limited financial

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ACCEPTED MANUSCRIPT resources dedicated to IPD surveillance in Algeria, we have conducted a

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nasopharyngeal carriage in healthy children and the most prevalent serogroups were

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6, 14 and 19 (23rd European Congress of Clinical Microbiology and Infectious

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Diseases, abstract R2717).

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The pneumococcal conjugate vaccines currently available have been shown to

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protect children. The first licensed 7-valent vaccine, which was widely used, resulted

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in dramatic reductions in pneumococcal disease mortality and morbidity. The

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emergence of the multidrug-resistant serotype 19A and the absence of the so-called

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‘developing country serotypes’, 1 and 5, has led manufacturers to develop higher

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valency pneumococcal conjugate vaccines such as PCV9, PCV10, PCV11, and

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PCV13 [26]. The WHO recommends the inclusion of PCVs in childhood immunization

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programs worldwide, especially in countries with childhood mortality that exceeds 50

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deaths/1000 births [27]. Although there are some limitations with our study, the

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results show that the burden of IPD is high in our country. The good vaccine serotype

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coverage suggests that the introduction of childhood vaccination with PCVs could be

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expected to have a dramatic effect on the burden of IPD in our country.

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Continuous national monitoring of IPD and nasopharyngeal carriage, as well as

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judicious use of antibiotics are crucial before and after PCV implementation in order

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to evaluate the impact of vaccination and to prevent the selection of multidrug

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resistant clones.

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ACCEPTED MANUSCRIPT Acknowledgements: The authors would like to thank the following people for

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sending strains and for technical support: Mohamed Bachtarzi, Fazia Djennane,

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(Centre Hospitalier Universitaire Mustapha Bacha, Algiers, Algeria); Souad Zouagui,

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(Centre Hospitalier Universitaire d'Oran, Oran, Algeria); Sadjia Mahrane, Radia

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Boushaki, (Centre Hospitalier Universitaire Nafissa Hamoud, Algiers, Algeria); Dalila

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Haouchine, (Centre Hospitalier Universitaire Nedir Mohamed, Tizi Ouzou, Algeria);

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Azzoug Saloua, (Centre Hospitalier Universitaire Ibrahim Hadjrass, Algiers, Algeria)

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Nadjet Aggoune, Fatma Zohra Henniche, (Hôpital Central de l'Armée Mohamed,

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Algiers, Algeria); Karima Lassas, (Hôpital de Boufarik, Blida, Algeria); Farida Sahli,

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(Centre Hospitalier Universitaire Saadna Abdennour, Sétif, Algeria); Farah Lalaoui,

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(Hôpital Fares Yahia, Tipaza, Algeria); Radia Abiayad, Ilhem Boubekri,

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(Etalissement hospitalier Universitaire 1er novembre, Oran, Algeria).

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The authors would like to thank Pfizer for funding the sera used for serotyping, and

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the French National Reference Center for Pneumococci (CNR pneumo) for

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performing confirmatory serotyping. The authors acknowledge editorial assistance

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from Margaret Haugh, MediCom Consult, funded by Pfizer.

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[27] World Health Organisation. Pneumococcal vaccines WHO position paper - 2012

321

- recommendations. Vaccine. 2012;30:4717-8.

322

AC C

EP

TE D

M AN U

SC

323

RI PT

316

15

ACCEPTED MANUSCRIPT 324

Table 1: Number of S. pneumoniae isolates by sample origin and age

Invasive samples

327

CSF

30

10

13

53

328

Blood culture1

15

4

2

21

329

Puncture fluids2

13

4

6

23

330

Total

58

18

21

97

331

Non-invasive samples

332

Lower respiratory tract samples

57

8

12

77

333

Auricular swabs

31

7

9

47

334

Sinus and nasal aspirates

5

10

29

44

335

Other samples3

0

3

2

5

336

Total

93

28

52

173

337

Overall total

151

46

73

270

338

1

339

2

340

3

M AN U

TE D

Pneumonia (n=10), bacteremia (n=11)

Pleural (n=15), joint (n=5) and peritoneal (n=3).

EP

Conjunctive (n=4), genital sample (n=1)

AC C

341

<2 years 2-5 years > 5 - 16 years Total

RI PT

326

SC

325

16

ACCEPTED MANUSCRIPT 342

Table 2: Minimum inhibitory concentrations (MICs) for penicillin, amoxicillin and

343

cefotaxime in isolates from invasive pneumococcal disease (IPD) and non-invasive

344

pneumococcal disease. The global MICs were: Penicillin I=20.6%, R=27.5%;

345

Amoxicillin I=2.56%, R=0.42%; Cefotaxime I=7%, R=1.68% Penicillin

Amoxicillin

347

I

R

MIC50

MIC90

I

348

(%)

(%)

(µg/mL)

(µg/mL)

(%)

349

Meningitis (n=53)

350

R

Cefotaxime

MIC50

MIC90

I

R

MIC50

MIC90

(%) (µg/mL)

(µg/mL)

(%)

(%)

(µg/mL)

(µg/mL)

0.3

2

Other IPD (n=44)

20.4 27.2

0.5

2

6.8

2.3

0.5

2

351

Total IPD (n=97)

10

39.2

0.38

2

3.1

1

0.5

2

352

Otitis (n=47)

25.5 10.6

0.8

2

0

0

1

353

LRTI (n=77)

34.6 32.7

1

2

4.1

0

1.5

354

Other NIPD (n=49) 34.2

0.2

1.5

355

Total NIPD (n=173) 31.4 16

0.5

2

3.8

0.4

1.5

4.5

0

0.5

1.5

13.4

2

0.5

1.5

2

0

0

0.5

1

4

0

2

0.5

2

M AN U

356

20.8

SC

49

2.5

RI PT

346

2.5

0

0.3

2

2.5

0

0.2

1.5

2.2

0

0.8

4

0.8

0.8

0.5

1.5

I = intermediate resistance; R = resistant; LRTI = lower respiratory tract infections;

358

NIPD = non-invasive pneumococcal disease

EP AC C

359

TE D

357

17

ACCEPTED MANUSCRIPT

Table 3: Serotype distribution among isolates from children with invasive (IPD) and non invasive (NIPD) pneumococcal diseases

6 2 2 1 1

1

1

4 2 1 2 1 2 1 1

1 2 1

2

2

23 7 6 2 1 6 4 2 2 2 6 1 1

5 10 2 2 1

3 6

1 4

1 1 1

1 1 9

37

17

4

1 1 67

26

1 1

9

2

2

1 1

1 1 2 1

1

1

1 1

13

EP

1

1

TE D

1

31 23 8 4 2 0 6 4 2 3 2 10 1 1 0 1 1 0 1 0 0 0 1 1 102

RI PT

15 3 3 0

SC

2 2 1 1

5 - 16 years IPD (n=18) NIPD (n=7) Meningitis PleuroBone Otitis LRTI pneumonia and joint infection 2 1 1 2

AC C

14 19F 6B 23F 18C 4 1 5 7F 3 6A 19A 35B 9A 16F 23B 6C 8 12 17F 23A 24A 28F 35F Total

Total IPD<5

NIPD (n=35) Otitis LRTI Total <5

M AN U

<5 years IPD (n=67) Bacteremia Meningitis PleuroOther IPD Serotypes pneumonia

18

1

4

1

5

2

Total 5 - 16 4 2 0 1 0 1 3 0 0 3 1 0 2 1 2 1 0 1 0 1 1 1 0 0 25

Overall total 35 25 8 5 2 1 9 4 2 6 3 10 3 2 2 2 1 1 1 1 1 1 1 1 127

ACCEPTED MANUSCRIPT

Table 4: Resistance profiles by isolated S. pneumoniae serotypes 14 19F

oxa

2

oxa, sxt

10

oxa, sxt, tet

1

oxa, ery, clin

4

oxa, ery, clin, sxt

4

6

oxa, ery, clin, tet

1

7

oxa, ery, sxt, tet

1

oxa, ery, clin, sxt, tet

5

5 7F 6A 19A

16F 24A

28F Total

2

4

1

11

SC

1

1

1 2

1

1 1

3

1

1 28 20

5

2

1 1

6

1

1

TE D

5

M AN U

4

oxa, ery, clin, chl, sxt, tet Total

6B 23F

RI PT

Antimicrobial

1

8

1

13 11 3 21 1

1

1

69

AC C

EP

oxa = oxacillin; sxt = cotrimoxazole; tet = tetracyclin; ery = erythromycin; clin = clindamycin; chl = chloramphenicol

19

ACCEPTED MANUSCRIPT Figure legends Figure 1: Serotype prevalence and pneumococcal vaccine coverage of serotypes isolated from children ≤5 years with IPD Figure 2: Resistance to penicillin and cefotaxime of S. pneumoniae serotypes isolated from

AC C

EP

TE D

M AN U

SC

RI PT

children ≤5 years. I = intermediate resistance; R = resistant

20

ACCEPTED MANUSCRIPT Figure 1

Total = 67

20 15 10

7

6

6

6 4

5

2

1

2 0

0

0

PCV7 55.3%

4

1

5

7F

2

Serotypes

PCV10 71.1%

AC C

EP

TE D

PCV13 86.8%

21

1

1

1

1

1

1

3 6 A' 19 A35 B 9 A' 6 C 12 28 F 35 F

M AN U

14 19 F 6 B 23 F 18 C 9 V

2

RI PT

23

SC

Number of isolates

25

ACCEPTED MANUSCRIPT Figure 2

30 Cefotaxime R Cefotaxime I

RI PT

20

Penicillin R Penicillin I

10

0 5

6A

6B

7F

14

16 F 19 A 19 F

23 F 24 A 28 F

EP

TE D

M AN U

SC

Serotypes

AC C

Number of isolates

40

22