Streptococcus pneumoniae in Saudi Arabia: antibiotic resistance and serotypes of recent clinical isolates

International Journal of Antimicrobial Agents 23 (2004) 32–38

Streptococcus pneumoniae in Saudi Arabia: antibiotic resistance and serotypes of recent clinical isolates Ziad A. Memish a,b,∗ , Hanan H. Balkhy b,c,1 , Atef M. Shibl d,2 , Christopher P. Barrozo e , Gregory C. Gray f,3 a

f

Department of Internal Medicine, b Department of Infection Prevention and Control, King Fahad National Guard Hospital, P.O. Box 22490, Riyadh 11426, Saudi Arabia c Department of Pediatrics, King Fahad National Guard Hospital, P.O. Box 22490, Riyadh 11426, Saudi Arabia d Department of Microbiology, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia e DoD Center for Deployment Health Research, Naval Health Research Center, San Diego, CA, USA Department of Epidemiology, College of Public Health, University of Iowa, 200 Hawkins Dr, C21K GH, Iowa City, IA, USA Received 7 February 2003; accepted 8 May 2003

Abstract During 2000, 154 clinical Streptococcus pneumoniae isolates were collected from or through three major hospitals serving the Western, Central, and Eastern regions of the Kingdom of Saudi Arabia. Ninety-one (59.1%) of the 154 isolates were resistant to penicillin with 23 strains (14.9%) highly resistant. Resistance was more prevalent among isolates obtained from patients <10 years of age and from isolates cultured from blood and cerebral spinal fluid. Decreased sensitivity to ceftriaxone, erythromycin, trimethoprim-sulphamethoxazole, and levofloxacin was found in 14.9, 15.6, 9.7, and 1.3% of isolates, respectively. There were no significant differences in the resistance pattern between isolates obtained from patients in the three different geographical areas. Serotypes of 116 isolates revealed the most prevalent types to be (descending order) 4, 3, 19F, 9V, 6A, 19A, 14 and 23F, comprising 75% of all strains typed; 9.5% of isolates were nontypable. S. pneumoniae isolates from Saudi Arabia have become more resistant to penicillin and other antimicrobials over the past 20 years. © 2003 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Streptococcus pneumoniae; Saudi Arabia; Drug resistance; Drug sensitivity; Serotypes

1. Introduction Streptococcus pneumoniae is a major health hazard among children and adults [1,2] and rates of antimicrobial resistance of clinical isolates have soared over the past three decades, notably in developing countries [3–5] and has been reported to be as high as 50–70% in some parts of the world [6,7]. It is estimated that an improved and appropriate vaccine would protect up to 30% of individuals at risk of disease [5]. The selection of S. pneumoniae serotypes for inclusion in the

23-valent pneumococcal vaccine and the conjugate vaccines was based on epidemiological studies [5,8]. Because the serotype predominance varies with time, specimen type and geographical location, a vaccine may be appropriate for one population but not for another [9]. Accordingly, we sought to evaluate the current prevalence of antibiotic resistance and distribution of serotypes among a sample of clinical S. pneumoniae isolates in Saudi Arabia.

2. Materials and methods ∗

Corresponding author. Tel.: +966-1-252-0088x3718; fax: +966-1-252-0437. E-mail addresses: [email protected] (Z.A. Memish), [email protected] (H.H. Balkhy), [email protected] (A.M. Shibl), [email protected] (C.P. Barrozo), [email protected] (G.C. Gray). 1 Tel.: +966-1-2520088x3752; fax: 966-1-2520437. 2 Tel.: +966-1-467-8042; fax: 966-1-467-6295. 3 Tel.: +319-384-5008/5004.

2.1. Study population During the period January–December 2000, three major hospitals, representing the three major provinces of the Kingdom, participated in providing S. pneumoniae isolates from inpatients and outpatients with S. pneumoniae related illnesses. These hospitals were in Jeddah, Western Province;

0924-8579/$ – see front matter © 2003 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2003.05.008

Z.A. Memish et al. / International Journal of Antimicrobial Agents 23 (2004) 32–38

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Riyadh, Central Province; and Damman, Eastern Province. Henceforth, these sites are referred to as Western, Central, and Eastern Provinces. Patient data included age, sex, site of infection, inpatient versus outpatient, and location (province).

serotypes included in the 23-valent pneumococcal vaccine. The pooled, type, and group antisera results were confirmed using the classic Quellung reaction.

2.2. Isolate identity and susceptability testing

The two-tailed Fishers exact test and the χ2 square test were used. The Epi Info statistics program 6.0 was used for statistical comparisons and ranking [11].

Isolate identity and susceptibility testing were performed as described previously [10]. Briefly S. pneumoniae specimens were preserved in tryptic soy broth with 15% glycerol at −70 ◦ C until dry-ice packaged for transport to the Naval Health Research Center in San Diego, CA. Identification was confirmed using colonial morphology, susceptibility to optochin and bile solubility (Hardy Diagnostics, Santa Maria, CA). All susceptibility testing used the E-test strip method. NCCLS guidelines for broth microdilution were used to determine quality control ranges and interpretation criteria for resistance as suggested by manufacturer of E-test strips (AB Biodisk). Control strain testing of S. pneumoniae ATCC 49619, Staphylococcus aureus ATCC 29213, Haemophilus influenzae ATCC 49247, and Enterococcus faecalis ATCC 29212 was performed in parallel with each E-test sensitivity test and interpreted according to NCCLS guidelines. Multiple drug resistance was defined as resistance to penicillin and two additional classes of antimicrobials.

2.4. Stastical analyses

3. Results From January 2000 to December 2000, 154 S. pneumoniae isolates were collected from three hospitals in the three provinces. The majority (68, 44.2%) of the isolates were from the Central Province, followed by the Western Province, (52, 33.8%), and the Eastern Province, (34, 22.1%). The male to female ratio was 2.9:1 and the majority of patients were 10–40 years of age (64.9%). One hundred four of the patients were in hospital and the rest were outpatients. Twenty-seven of the isolates were from blood, 24 from cerebrospinal fluid, 69 from the lower respiratory tract and 33 from the upper respiratory tract. One sample only was from an unidentified source. 3.1. Susceptibility testing

2.3. Serotyping Capsular typing was performed on a sample of 116 isolates using a modified latex agglutination method as reported previously [2,10]. Pooled and pneumococcal type-specific antisera were obtained from the Statens Serum Institute, Copenhagen, Denmark. All pooled sera were tested according to the manufacturer’s instructions to determine most

All isolates were confirmed to be S. pneumoniae. Only six isolates were recorded from the age group <10 years but of these, five were penicillin intermediate or highly resistant. Multidrug resistance was highest among isolates from the age groups 20–29 and 60+ years. There was no difference in the sensitivity pattern of isolates from males and females (Table 1).

Table 1 Susceptibility of Streptococcus pneumoniae isolates by age, sex and source of specimen Demographics

Number of isolates

Resistance to penicillin

%

P-value

Multi-drug resistance∗

%

P-value

1 7 8 2 2 1 3

16.7 17.5 21.1 7.1 8.3 11.1 20.0

0.81

Age (year) <10 10–19 20–29 30–39 40–49 50–59 60+

6 40 38 22 24 9 15

5 30 18 11 13 5 9

83.3 75.0 47.4 50.0 54.2 55.6 60.0

Sex Male Female

115 39

68 23

59.1 59.0

0.99

17 7

14.8 17.9

0.64

27 24 33 69

21 16 18 35

77.8 66.7 54.5 50.7

0.08

10 9 4 1

37.0 37.5 12.1 1.4

0.0001

Site of infection∗∗ Blood Cerebral spinal fluid Upper respiratory tract Lower respiratory tract ∗ ∗∗

Resistance to penicillin and 2 additional classes of antibiotics. One sample was from an unidentified source.

0.18

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Antibiotics

Total number of cases (n = 154)

Penicillin sensitivity Sensitive (n = 63 )

S Penicillin Ceftriaxone TMP-SMZ Erythromycin Levofloxacin Vancomycin

63 131 104 130 152 154

I (40.9%) (85.1%) (67.5%) (84.4%) (98.7%) (100%)

68 17 35 – 2 –

R (44.2%) (11.0%) (22.7%) (1.3%)

S: sensitive; I: intermediate; R: resistant.

23 6 15 24 – –

S (14.9%) (3.9%) (9.7%) (15.6%)

63 54 61 63 63

(100%) (85.7%) (96.8%) (100%) (100%)

Intermediate (n = 68)

I

R

S

– 8 (12.7%) – – –

– 1 (1.6%) 2 (3.2%) – –

64 46 56 66 68

(94.1%) (67.6%) (82.4%) (97.1%) (100%)

Resistant (n = 23)

I

R

S

3 (4.4%) 16 (23.5%) – 2 (2.9%) –

1 (1.5%) 6 (8.8%) 12 (17.6%) – –

4 4 13 23 23

(17.4%) (17.4%) (56.5%) (100%) (100%)

I

R

14 (60.9%) 11 (47.8%) – – –

5 (21.7%) 8 (34.8%) 10 (43.5%) – –

Z.A. Memish et al. / International Journal of Antimicrobial Agents 23 (2004) 32–38

Table 2 Sensitivity of clinical isolates of S. pneumoniae from Saudi Arabia

Z.A. Memish et al. / International Journal of Antimicrobial Agents 23 (2004) 32–38

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Table 3 Comparative ranking of serotypes Serotype

Number of isolates tested

Invasive (CSF & blood)

Non-invasive (respiratory)

Number of isolates

Rank

Number of isolates

Rank

*19F *9V *4 *14 7A *17F *23F *22F 6A 23B *9N *3 *19A *15B NT 19C *8 *7F *5 *1 9L 17A *11A *6B *20

10 10 13 5 4 4 5 4 9 2 2 12 6 4 11 2 1 1 1 1 1 1 1 4 2

6 5 4 4 3 3 3 3 2 2 2 2 2 2 2 1 1 1 1 1 0 0 0 0 0

1 2 3 3 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 – – – – –

4 5 9 1 1 1 2 1 7 0 0 10 4 2 9 1 0 0 0 0 1 1 1 4 2

5 4 2 7 7 7 6 7 3 – – 1 5 6 2 7 – – – – 7 7 7 5 6

NT: non-typable; (*) strain included in the 23-valent vaccine.

Occurrence of multidrug resistance in blood and cerebral spinal fluid (CSF) isolates was significantly higher than upper and lower respiratory tract isolates (P = 0.0001). Penicillin resistance was also higher in CSF and blood isolates. However, these differences were not statistically significant (P = 0.08) (Table 1). Ninety-one (59%) of the total 154 isolates were either intermediately or highly resistant to penicillin and 24 (15.6%) isolates were multidrug resistant. When stratified by geographical location, multidrug resistance was highest in the Eastern Province at 26.5%, followed by the Western Province at 15.4%, and least in the Central Province at 10.3% (P = 0.21). Isolates from inpatients, had a higher incidence of a multidrug resistance than those from outpatients [21% versus 4.0% (P = 0.006)]. Penicillin resistance was similar at 59.6% for inpatients, and 58.0% for outpatients (P = 0.167). Full resistance (high resistance) to penicillin, ceftriaxone, trimethoprim-sulphamethoxazole TMP-SMZ, and erythromycin was found in 14.9, 3.9, 9.7, and 15.6% of the isolates, respectively (Table 2). None were resistant to levofloxacin or to vancomycin. Of the 23 isolates that were highly resistant to penicillin, 19 (82.6%) also had decreased sensitivity to ceftriaxone with 5 isolates being highly resistant (Table 2).

3.2. Capsular typing Twenty-four different serotypes were identified in the 116 isolates tested. The most prevalent in descending order were 4, 3, 19F, 9V, 6A, and 19A. Eleven strains were untypable (Table 3). The number of isolates were too small to identify a geographical predominance. Twenty-seven of the isolates were from blood (all but one of these were typable). Of the 24 CSF isolates, all but two were typable. When stratifying the serotypes by isolation sites, the isolates were classified as invasive from CSF and blood or non-invasive, from upper and lower respiratory tract. Serotypes 19 F, 9 V, 4, and 14 were most frequently isolated from the blood and CSF. Serotype 3, followed by 4, 6A, and 9 V were most frequent among non-invasive isolates (Table 3). The most common serotypes of the 74 penicillin resistant strains were *9V, 6A, *15B, and *19F. While among the 24 multidrug resistant strains serotypes *9V, *19F and *23F made up 20, 16.6 and 12.5% of the isolates, respectively.

4. Discussion Despite efforts to reduce the antibiotic resistance selection pressures, the prevalence of resistant strains continues to

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Z.A. Memish et al. / International Journal of Antimicrobial Agents 23 (2004) 32–38

Fig. 1. Penicillin sensitivity pattern of clinical isolates from Saudi Arabia.

Z.A. Memish et al. / International Journal of Antimicrobial Agents 23 (2004) 32–38

escalate in many countries including Saudi Arabia [7,12]. The first study of antimicrobial resistance in S. pneumonia isolates in Saudi Arabia (1982–1984) found 100% to be sensitive to penicillin [13]. Further studies covering a similar time period continued to show no fully resistant strains; however, 10% were intermediately sensitive to penicillin [14,15]. In 1983, Chowdhury reported the detection of the first penicillin resistant strain from a patient in Saudi Arabia [16]. Consistent with the experience of numerous other countries, penicillin resistance among Saudi Arabian S. pneumoniae isolates has increased in recent years [3,13–15,17–21]. Before this recent study, the highest prevalence of highly resistant isolates was just above 20% as reported by Rotimi in 1995 (Fig. 1) [12]. Levofloxacin resistance is uncommon in Saudi Arabia; we found only two strains. On the other hand, the Alexander study did not show high levels of ciprofloxacin or ofloxacin resistance among 8081 clinical isolates of S. pneumoniae [6]. Rotimi reported zero resistance to ceftriaxone in 109 isolates tested in 1993 [12] and Abdul Rahman and Dixon reported 5.3% intermediate resistance to ceftriaxone [15]. Similarly, when 400 isolates from three major provinces in Saudi Arabia were tested, 4.5% were highly resistant to ceftriaxone and almost 15% were resistant to clarithromycin and cefuroxime [20]. A 20-fold increase in erythromycin resistance was recorded by Ismaeel [21] and Bannatyne and Shibl reported a similarly high percentage of resistance to erythromycin at the turn of the century [19]. After the release of the 23-valent pneumococcal vaccine in 1983 a marked decrease in severe illness in high-risk patients such as sickle cell anaemia patients or those with asplenia has been noticed [22]. More recently the seven-valent conjugate vaccine has been licensed [5]. When considering the national employment of such a vaccine, representative data on the prevalence and serotype distribution becomes important. The collection of representative national data will assist public health officials in making pubic health decisions such as employing the new seven-valent protein conjugate vaccine among children. From our data, we have shown that 26.7% of isolated serotypes leading to blood and CSF disease were included in the seven-valent vaccine or are cross-covered by serotypes in the vaccine [23]. The 9-valent and the 11-valent vaccines included 28.4 and 30.2% of the invasive serotypes respectively. The non-conjugate 23-valent vaccine would have covered 34.5% of invasive serotypes in our study [8,24]. In a study published by the CDC over a 16-year period [9], serotypes in US children less than 6 years of age suggested that the seven-valent protein conjugate vaccine would probably not cover the most common serotypes leading to invasive disease such as bacteraemia and meningitis, and at the same time protect against serotypes causing otitis media. A conjugate vaccine including 9–11 serotypes may become more cost effective [9]. In two earlier reports from the Kingdom, when clinical isolates of S. pneumoniae were serotyped, serotype 14 was the most prevalent, followed by

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serotypes 3, 7, 1, and 2. In neither study, however, did the authors report the clinical site of the specimen [13,17]. In summary, our study points to a possible changing pattern of serotype predominance among S. pneumoniae clinical isolates. However, studies including larger number of isolates are needed to confirm these results. More importantly, we document an increase in resistance to penicillin for almost all serotypes. Finally, our data emphasize the desperate need for judicious use of antimicrobials in an effort to suppress the rapid growth of antimicrobial resistance among S. pneumoniae as well as other microorganisms.

References [1] Prevention of Pneumococcal Disease. Recommendations of the Advisory Committee on Immunization Practices (ACIP). 1997;46:1–19. [2] Facklam RR, Breiman RF. Current trends in bacterial respiratory pathogens. Am J Med 1991;91:3S–11S. [3] Qadri SM, Kroschinsky R. Prevalence of pneumococci with increased resistance to penicillin in Saudi Arabia. Ann Trop Med Parasitol 1991;85:259–62. [4] Reichler MR, Rakovsky J, Sobotova A, et al. Multiple antimicrobial resistance of pneumococci in children with otitis media, bacteremia, and meningitis in Slovakia. J Infect Dis 1995;171:1491–6. [5] Preventing pneumococcal disease among infants and young children. Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep 2000;49:1–35. [6] Felmingham D, Gruneberg RN. The Alexander Project 1996–1997: latest susceptibility data from this international study of bacterial pathogens from community-acquired lower respiratory tract infections. J Antimicrob Chemother 2000;45:191–203, and the Alexander project Group. [7] Paredes A, Taber LH, Yow MD, Clark D, Nathan W. Prolonged pneumococcal meningitis due to an organism with increased resistance to penicillin. Pediatrics 1976;58:378–81. [8] Eskola J, Kilpi T, Palmu A, Jokinen J, et al. Efficacy of pneumococcal conjugate vaccine against acute otitis media. N Eng J Med 2001;344:403–9. [9] Butler J, Breiman RF, Lipman H, Hofmann J, Facklam RR. Serotype distribution of Streptococcus pneumoniae infections among preschool children in the United States, 1978–1994: implications for development of a conjugate vaccine. J Infect Dis 1995;171:885–9. [10] Hudspeth MK, Smith TC, Barrozo CP, Hawksworth AW, Ryan MA, Gray GC. National Department of Defence surveillance for invasive Streptococcus pneumoniae: antibiotic resistance, serotype distribution, and arbitrarily primed polymerase chain reaction analyses. J Infect Dis 2001;184:591–6. [11] Dean AG, Dean JA, Burton AH, Dicker RC. Epi Info, version 6: a word-processing, database, and statistics program for epidemiology on micro computers. Atlanta: Centers for Disease Control and Prevention; 1998. [12] Rotimi VO, Feteih J, Barbor PRH. Prevalence of penicillin-resistant Streptococcus pneumoniae in a Saudi Arabian hospital. Eur J Clin Microbiol Infect Dis 1995;14:149–51. [13] Mahgoub ES, Hussein SS. Streptococcus pneumoniae serotypes and their minimal inhibitory concentration to penicillin in Riyadh. Saudi Med J 1986;7:149–54. [14] Shibl AM, Hussain SS, Bahakime HM, Sofan MM. Activity of penicillin imipenem and teicoplanin against various serotypes of Streptococcus pneumoniae isolated in Riyadh. In: Berkarda B, Kuemmerle HP, editors. Proceedings of the 15th International Congress of Chemotherapy—Progress in Antimicrobial and Anti-

38

[15]

[16] [17]

[18] [19]

Z.A. Memish et al. / International Journal of Antimicrobial Agents 23 (2004) 32–38 cancer Chemotherapy, Antimicrobial Section I. Istanbul; 1987. p. 757–9. Shibl AM, Hussain SS, Sofan MM. Surveillance of Streptococcus pneumoniae serotypes existing in Riyadh and their antimicrobial susceptibility patterns. In: Proceedings of the Second International Congress of Hospital Infections. London; 1990. p. 151 (Abstract No. 13/1). Chowdhury MNH. A penicillin-resistant Streptococcus pneumoniae; case report. Trop Geograph Med 1983;35:309–11. Shibl AM, Hussein SS. Surveillance of Streptococcus pneumoniae serotypes in Riyadh and their susceptibility to penicillin and other commonly prescribed antibiotics. J Antimicrob Chemother 1992;29:149–57. Abdel Rahman E, Dixon RA. Resistance of Streptococcus pneumoniae from Saudi Arabia. Internat J Antimicrob Agents 1999;13:61–2. Bannatyne RM, Memish ZA, Jackson MC. Correlation between serotype and in vitro antibiotic susceptibility of pneumococci

[20]

[21]

[22] [23]

[24]

isolated in Saudi Arabia. J Antimicrob Chemother 1999;43:161– 2. Shibl AM, Rasheed AM, Elbashier AM, Osoba AO. Penicillinresistant and intermediate Streptococcus pneumoniae in Saudi Arabia. J Chemother 2000;12:134–7. Ismaeel NA. Serotype distribution, antimicrobial susceptibility, and clinical character of Streptococcus pneumoniae isolates. Microbios 1993;74:233–40. Butler JC, Shapiro ED, Carlone GM. Pneumococcal vaccines: history, current status, and future directions. Am J Med 1999;107:69S–76S. Joloba ML, Windau A, Bajaksouzian S, Applebaum PC, Hausdorff WP, Jacobs MR. Pneumococcal conjugate vaccine serotypes of Streptococcus pneumoniae isolates and the antimicrobial susceptibility of such isolates in children with otitis media. Clin Infect Dis 2001;33:1489–94. Klein DL. Pneumococcal conjugate vaccines: review and update. Microbial Drug Resis 1995;1:49–58.