Antimicrobial resistance profile and multidrug resistance patterns of Streptococcus pneumoniae isolates from patients suspected of pneumococcal infections in Ethiopia

Background Antimicrobial-resistant strains of Streptococcus pneumoniae have become one of the greatest challenges to global public health today and inappropriate use of antibiotics and high level of antibiotic use is probably the main factor driving the emergence of resistance worldwide. The aim of this study is, therefore, to assess the antimicrobial resistance profiles and multidrug resistance patterns of S. pneumoniae isolates from patients suspected of pneumococcal infections in Ethiopia. Methods A hospital-based prospective study was conducted from January 2018 to December 2019 at Addis Ababa city and Amhara National Region State Referral Hospitals. Antimicrobial resistance tests were performed from isolates of S. pneumoniae that were collected from pediatric and adult patients. Samples (cerebrospinal fluid, blood, sputum, eye discharge, ear discharge, and pleural and peritoneal fluids) from all collection sites were initially cultured on 5% sheep blood agar plates and incubated overnight at 37 °C in a 5% CO2 atmosphere. Streptococcus pneumoniae was identified and confirmed by typical colony morphology, alpha-hemolysis, Gram staining, optochin susceptibility, and bile solubility test. Drug resistance testing was performed using the E-test method according to recommendations of the Clinical and Laboratory Standards Institute. Results Of the 57 isolates, 17.5% were fully resistant to penicillin. The corresponding value for both cefotaxime and ceftriaxone was 1.8%. Resistance rates to erythromycin, clindamycin, tetracycline, chloramphenicol and trimethoprim-sulfamethoxazole were 59.6%, 17.5%, 38.6%, 17.5 and 24.6%, respectively. Multidrug resistance (MDR) was seen in 33.3% isolates. The most common pattern was co-resistance to penicillin, erythromycin, clindamycin, and tetracycline. Conclusions Most S. pneumoniae isolates were susceptible to ceftriaxone and cefotaxime. Penicillin has been used as a drug of choice for treating S. pneumoniae infection. However, antimicrobial resistance including multidrug resistance was observed to several commonly used antibiotics including penicillin. Hence, it is important to periodically monitor the antimicrobial resistance patterns to select empirical treatments for better management of pneumococcal infection.


Introduction
Streptococcus pneumoniae is one of the leading causes of bacterial infections, ranging from self-limiting respiratory tract infections to severe invasive infections. It is a major public health concern, being responsible for an estimated 3.7 million episodes (2.7 million to 4.3 million) in children globally and approximately 50% of all pneumococcal deaths in 2015 occurred in four countries in Africa and Asia [1].
Antimicrobial resistance has been detected in all parts of the world; it is one of the greatest challenges to global public health today. The problem is increasing resistance to commonly used antimicrobial drugs which have elevated multidrug resistance (MDR). The fight against pneumococcal infections is based on curative treatment with antibiotics and preventive treatment using vaccination [2]. However, the emergence of resistant strains globally poses therapeutic problems. Nearly 40% of strains are resistant to penicillin, and penicillin resistance often correlates with resistance to other additional antibiotics such as macrolides, tetracyclines, etc. [3,4].
In Asia, an alarming prevalence of penicillin and erythromycin-resistant S. pneumoniae has been described [5]. In 1995, an increase in the prevalence of resistance to penicillins, extended-spectrum cephalosporins, trimethoprim-sulfamethoxazole, and macrolides as well as MDR began to be recognized in Taiwan [6]. In Southern Finland, the proportion of MDR pneumococci doubled from 2007 to 2008, when it reached 3.6% [7].
Data on antimicrobial resistance from the African region are limited, but high-level penicillin-resistant S. pneumoniae have been described in central Africa [8]. The increasing trend of S. pneumoniae antimicrobialresistance and the emergence of MDR isolates, which may result from inappropriate use of antibiotics and high level of antibiotic use is probably the main factor driving the emergence of resistance worldwide [9,10].
In Ethiopia, there is lack of literatures on streptococcal drug resistance. A few studies have reported nasopharyngeal carriage rates [11,12] and CSF sample sources [13,14], for the presence of resistance to commonly prescribed antibiotics. But there is still a need for adequate baseline information for clinicians and other health professionals, and policy makers about the distribution of S. pneumoniae and to produce a structured data on drug resistance profile of this isolates in a regular base.
The present study aimed to investigate the antimicrobial resistance profile and multidrug resistance patterns of S. pneumoniae isolated from patients suspected of pneumococcal infections in Ethiopia using an E-test, where minimum inhibitory concentrations (MICs) determination is not routinely available.

Study area, design, and period
A hospital-based prospective study was conducted between January 2018 to December 2019 at Addis Ababa city (Yekatite12 Hospital, Alert Hospital, and International Clinical Laboratory) and Amhara National Regional State (ANRS) (University of Gondar Comprehensive Specialized Hospital, Felege Hiwot Comprehensive Specialized Hospital, and Dessie Regional Laboratory) Hospitals, Ethiopia. During the study period, 57 isolates were further confirmed as S. pneumoniae from clinical suspected patients. Suspected S. pneumoniae case means any reported case lacking confirmation of isolation from samples but has been diagnosed with S. pneumoniae by a clinician or other medical practitioner using sign and symptoms of the disease. The clinical sources of isolates were cerebrospinal fluid (CSF), blood, eye discharge, sputum, pleural fluid, ear discharge and peritoneal fluid.

Isolation of Streptococcus pneumoniae
As part of routine service, samples (CSF, blood, sputum, eye discharge, ear discharge, and other body fluids) were initially cultured on 5% sheep blood agar plates. All growing isolates were stored at − 20 °C and shipped to the University of Gondar Microbiology Laboratory for identification through ice box. All isolates received from the collection site were frozen at − 80 °C until further testing. Isolates were recovered by plating onto 5% sheep blood and incubated overnight at 37 °C in a 5% CO 2 atmosphere. Streptococcus pneumoniae was identified and confirmed by typical colony morphology, alpha-hemolysis, Gram staining, optochin susceptibility, and bile solubility test. After confirmation, the isolates were stocked in Skimmed Milk-Trypticase Soy-Glucose-Glycerol (STGG) medium and preserved at − 80 °C. monitor the antimicrobial resistance patterns to select empirical treatments for better management of pneumococcal infection.

Streptococcus pneumoniae antimicrobial susceptibility testing
The strains were shipped in dry ice to the Norwegian Reference laboratory for pneumococci at the Norwegian Institute of Public Health where antimicrobial susceptibility testing was performed. Minimum inhibitory concentrations (MICs) were determined by E-test on Mueller-Hinton agar supplemented with 5% sheep blood by the recommendation of the manufacturer (Biomerieux ® ). The antimicrobials tested were penicillin G, cefotaxime, ceftriaxone, erythromycin, clindamycin, tetracycline, chloramphenicol, trimethoprim/ sulfamethoxazole, norfloxacin, and oxacillin. The Clinical and Laboratory Standards Institute (CLSI) 2020 clinical breakpoints for S. pneumoniae were applied to categorize isolates as susceptible, intermediate, or resistant [15]. The CLSI states that the 1-µg oxacillin disk diffusion test is an effective screening method commonly used in clinical laboratories for the detection of penicillin-resistant pneumococci. The CLSI breakpoints for penicillin, cefotaxime, and ceftriaxone, differ between meningeal and nonmeningeal infections. For penicillin, the breakpoints for meningitis is susceptible ≤ 0.06 µg/ml and resistant ≥ 0.12 µg/ml, and for non-meningitis ≤ 2 µg/ ml susceptible, 4 µg/ml intermediate and ≥ 8 µg/ ml resistance. For Cefotaxime and ceftriaxone, the non-meningitis breakpoint was used to classify isolates as susceptible (MIC ≤ 1 µg/ml), intermediate (MIC = 2 µg/ml) or resistant (MIC ≥ 4 µg/ml) and for meningitis susceptible (MIC ≤ 0.5 µg/ml), intermediate (MIC = 1 µg/ml) or resistant (MIC ≥ 2 µg/ml).
Interpretive criteria for norfloxacin are not given by the CLSI 2020. Thus, we used the breakpoints for norfloxacin obtained from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [16]. Norfloxacin disk diffusion test is an effective screening method for the detection of Moxifloxacin and Levofloxacin resistant pneumococci. MDR was defined as resistant to at least one agent in three or more antimicrobial classes. S. pneumoniae ATCC 49619 was used as the quality control strain and was included in each set of tests to ensure accurate results.

Data analysis
Data were entered and analyzed using the Statistical Package for the Social Science (version 20; SPSS Inc, Chicago, IL, USA). Discrete variables were expressed as percentages and proportions.

Discussion
Streptococcus pneumoniae is responsible for many community infections, with the main ones being pneumonia and meningitis. Pneumococcus has developed increased resistance to multiple classes of antibiotics. Developing countries face significant problems of antimicrobial resistance with poor diagnostic facilities, unauthorized sale of antimicrobials, lack of appropriate functioning drug regulatory mechanisms, and non-human use of antimicrobials such as in animal production [17,18]. In this work, we investigated the antimicrobial resistance profile and MDR pattern of S. pneumoniae from clinical isolates in two regions in Ethiopia. Currently, the antibiotic resistance patterns of S. pneumoniae isolates vary widely from one country to another, but many studies are reporting a high frequency of antimicrobial resistance among pneumococcal isolates.
These differences in antimicrobial resistance may be explained by differences in the source of the isolates, geographical variability, high prevalence of HIV infection, and antibiotics usage. Higher rates of antibiotic resistance have been linked to high antimicrobial consuming countries [10]. In Ethiopia, antibiotics can be bought without a prescription and this probably leads to overuse and misuse of antibiotics which can promote the widespread antibiotic resistance strain in the area [28].
The emergence of MDR S. pneumoniae isolates has been a worldwide public health concern for several years.
The most common MDR phenotype was resistance to penicillin, erythromycin, tetracycline, and clindamycin, detected in 33.3% of the total isolates. Such MDR rates were higher than those observed in China (21.4%) [22], Russia (22%) [23] and Portugal (26%) [29]. However, it was lower than studies reported from Tunisia (96.5%) [19], Nigeria (53.8%) [24] and China (99.4%) [30]. Moreover, the results reveal a common pattern of co-resistance to penicillin, erythromycin, clindamycin, and tetracycline. The empirical treatment of pneumococcal infections, especially invasive infections, often requires a combination of two or more antibiotics and longer durations. These conditions inevitably lead to the initiation of high or multidrug resistance among patients with infections. The limitation of this study was the small sample size, and also absence of data on the prior use of antibiotics, which could potentially bias the pneumococcal collection regarding frequency of antimicrobial resistant isolates. This could potentially restrict the scope of our findings not making them general for the rest of the country. This study has also some strength which providing a useful information that may be used to guide public health measures to contain antimicrobial resistance and pneumococcal disease.

Conclusions
In the present study, most bacterial isolates were susceptible to ceftriaxone and cefotaxime. Penicillin has been used as a drug of choice for treating S. pneumoniae infection. However, antimicrobial resistance including multidrug resistance was observed to several commonly used antibiotics including penicillin. Hence, it is important to periodically monitor the antibiotic resistance patterns to choose empirical treatments for better management of pneumococcal infection. We recommend developing an action plan for the promotion of rational use of antimicrobials, strengthening of antimicrobial resistance surveillance, and implementation of an antimicrobial stewardship program.