PATTERNS OF ANTIMICROBIAL RESISTANCE AMONG MAJOR BACTERIAL PATHOGENS ISOLATED FROM CLINICAL SAMPLES IN TWO TERTIARY’S HOSPITALS, IN SANA'A, YEMEN

Huda Zaid Al-Shami¹, Muhamed Ahmed Al-Haimi², Omar Ahmed Esma’il Al-dossary¹, 

Abeer Abdulmahmood Mohamed Nasher¹, Mohammed Mohammed Ali Al-Najhi³, 

Hassan Abdulwahab Al-Shamahy^1,4, Azhar Azher Mohammed Al-Ankoshy⁵

¹Medical Microbiology and Clinical Immunology Department, Faculty of Medicine and Health Sciences, Sana’a University. 

²Depertement of Medicine, Faculty of Medicine and Health Sciences, Sana’a University, Republic of Yemen.

³Orthodontics, Pedodontics and Prevention Department Faculty of Dentistry, Genius University for Sciences & Technology, Dhamar city, Republic of Yemen. 

⁴Medical Microbiology department, Faculty of Medicine, Genius University for Sciences & Technology, Dhamar city.

 ⁵Jabir Ibn Hayyan Medical University, Faculty of medicine, Department of Physiology, An-Najaf, Iraq.

ABSTRACT 

Background and objectives: At the present time, antimicrobial resistance (AMR) is a major public health hazard, with antimicrobial resistance bacteria increasing exponentially. This study estimates the epidemiological profiles and antimicrobial resistance of Gram-positive bacteria (GPB) and Gram-negative bacteria (GNB) isolated from clinical samples among patients admitted to two University hospitals in Sana'a city for one year (2019). 

Methods: This was a retrospective study of clinical samples of patients collected from January 1, 2019 to December 30, 2019. All samples were appraised to determine presence of infectious agents using standard methods for isolation and identification of bacteria and yeasts from clinical samples of patients admitted to Al-Gumhouri University Hospital and Al-Kuwait University Hospital in Sana'a city. Antibiotic resistance was done using Kirby-Bauer disc diffusion methods. 

Results:  2,931 different pathogenic bacteria were detected from 24,690 different clinical specimens. The samples had an overall detection rate of 11.9% (2931/24,690). Among the bacterial pathogens isolated from clinical samples, 52.4% (n=1536) had GPB and 41.2% (n=1207) had GNB. The predominant GNB isolates were E.coli (22.04%), Klebsiella spp (6.03%), Pseudomonas aeruginosa (7.1%), Acinetobacter baumannii (1.46%), Enterobacter spp. (1.09%), Citrobacter spp. (1.16%), respectively. Among the GPB, S.aureus was the most common (26.3%), Coagulase-negative Staphylococcus (8.1%), Non-hemolytic Streptococcus (9.1%), Other alpha-hemolytic Streptococcus (3.9%), Streptococcus pyogenes (1.9%), and Streptococcus pneumoniae (0.5% ). A high rate of antibiotic resistance was recorded for sulfamethoxazole/trimethoprim (85.5%), ceftazidime (81.07%), ampicillin (70.4%), cefuroxime (66.4%). 

Conclusions:  The current study results revealed that the rate of resistance between GNB and GPB is associated with the incidence of different infections in patients attending two major tertiary hospitals in Sana'a city is very high. These results indicate ongoing screening and follow-up programs to detect antibiotic resistance, and also suggest the development of antimicrobial stewardship programs in Sana'a, Yemen.

Keywords: Antimicrobial resistance, bacterial, infection, Gram-negative bacteria, Gram-positive bacteria, Yemen.

INTRODUCTION

Global resistance to antimicrobials is increasing for many reasons, the most important of which is the increase in prescriptions, dispensation in developing countries, and indiscriminate use. It is estimated that 700,000 to several million deaths occur annually and remain a major public health threat worldwide¹. Millions of patients contract antibiotic-resistant bacteria, many more die, and billions of dollars are lost in health care costs and lost productivity^2,3. According to estimates by the World Health Organization (WHO) and United Nations report, deaths due to antimicrobial resistance could increase with the time^4,5. Nowadays, antimicrobial resistance (AMR) is a major public health threat,^6,7 and antimicrobial resistance bacteria in different hospital departments are increasing dramatically all over the world and in Yemen this problem is more extensive and complex^8-20. As for some details of the previous work in Yemen, these studies mostly focused on studying the sensitivity to antibiotics for each bacteria separately^8-20, while the current study studied the resistance to all bacterial isolates, and the current study also identified the temporal correlation of the rate of increase in the prevalence of resistance of bacterial isolates to the antibiotics used in the study area. It has been predicted that if appropriate control and prevention measures are not taken, antimicrobial resistance will become one of the leading causes of death among hospitalized or non-hospitalized patients in developing and developed countries. Proper use and administration of antibiotics are essential to treat bacterial infections²¹. Consequently, inappropriate prescription and abuse of antibiotics can be a factor in the emergence of pathogenic bacteria that are resistant to antibiotics, restriction of treatment options, increased hospitalization time, higher treatment costs and, finally, higher mortality²².

According to the WHO Global Action Plan on Antimicrobial Resistance²³, it is essential to increase awareness of antimicrobial resistance throughout research and monitoring programs in all parts of the world. Monitoring antimicrobial resistance is critical and has many benefits including: 1). providing data on the rate of bacterial resistance, 2). helping to select appropriate antibiotics and thus reducing the rate of antimicrobial resistance, 3). lowering hospitalization rate and treatment costs, and 4). Low mortality rate²¹. Hence, the present study assesses the epidemiological profiles and antimicrobial resistance of GNB and GPB isolated from clinical samples among patients admitted to two tertiary hospitals in 2019 in Sana'a city. 

MATERIALS AND METHODS 

Study design and identification of microorganisms: This was a retrospective study of clinical samples of patients collected from January 1, 2019 to December 30, 2019 at the Microbiology Department of the National Center for Public Health Laboratories (NCPHL) Sana'a, Yemen. NCPHL is the reference laboratory for the whole country, in the microbiology department there are 6 benches with 12 bacteriologists working in the department at a rate of 6 samples per day per worker. Samples were provided by two major hospitals in Sana'a: Al-Gumhouri University Hospital and Al-Kuwait University Hospital. This research used microbiological laboratory data for 24690 different clinical samples (Table 1) collected from different inpatient hospital wards and different clinics of the same hospitals. Clinical samples were cultured in an appropriate medium according to standard methods for bacterial isolation and identification for different samples²³. Using conventional biochemical assays including IMVIC assay  (Indole, Methyl red, Voges Proskauer and Citrate), catalase and oxidase assay, growth on triglyceride Agar and Kligler Iron Agar, and Bile Esculin agar, H₂S production, motility test, growth on 6% NaCl and DNase assay;  the isolation and identification of different bacterial strains from positive cultures was performed²³.

Antibiotic susceptibility testing: Antibiotic resistance was done using Kirby-Bauer disc diffusion methods and interpretation of antibiotic sensitivity results was done according to CLSI²⁴. Antibiotic disks and media powders used in NCPHL are usually Sigma-Aldrich sources. GPB and GNB isolates consisting of Pseudomonas aeruginosa (ATCC 27853) Escherichia coli (ATCC 25922), and Staphylococcus aureus subsp Aureus ATCC 25923 was used as quality control for a routine DDM test recommended in the NCPHL Department of Microbiology.  Antimicrobial susceptibility to Gram-positive bacteria and GNB was determined using the antibiotic disks mentioned in Table 3. The research results were documented as either sensitive (S), intermediate (I) or resistant (R).

RESULTS

Number and distribution of specimens and positive cultures: During this year, a total of 24690 different clinical cultures were collected from January 2019 until the end of December 2019. Among them, 2931 (11.9%) positive cultures were isolated from different types of bacteria. Among the GPB, about 52.4% and 41.2% of the total GNB cultures were positive and the remaining positive was Candida albicans (6.4%). The frequency of different clinical samples from which bacterial strains were isolated is shown in Table 1. The most common positive samples were as follows: urine (n=1043; 35.6%), pus (n=680; 23.2%), semen (337, 11.5%), sputum (n=203; 6.9%) and ear swab (n=163; 5.6%) (Table 1).

Table 1 : Characterization of collected samples.
 

Pathogen distribution: GNB and GPB comprised 41.2% (n=1207) and 52.4% (n=1536) of the total bacteria, respectively. The most prevalent isolated GPB were Staphylococcus aureus (n=772; 26.3%), non-hemolytic streptococcus (n=266; 9.1%), coagulase negative staphylococcus (n=238; 8.1%) and alpha-hemolytic streptococcus (n=115 ; 3.9%) (Table 2). The most prevalent isolated GNB were Escherichia coli (n=646; 22.04%), Pseudomonas aeruginosa (n=209; 7.1%), Klebsiella spp (n=177; 6.03%) Acinetobacter spp (n=43; 1.46%) and Citrobacter. spp (n=34; 1.16%) (Table 2).

Antimicrobial susceptibility: The resistance rates of isolated bacteria to commonly used antimicrobials are shown in Table 3. In bacteria isolated from different samples, the highest rates of resistance belonged to sulphamethoxazole/trimethoprime (n=900; 85.5%), ceftazidime (n=1114; 81.07%), ampicillin ( n=1055; 70.4%), ceftoroxime (n=886; 66.4%), and cefotaxime (n=597; 32.6%).

Table 2: Frequency rate of isolated pathogens from inpatients and outpatients.

DISCUSSION

In the current study, the highest rates of resistance occurred to sulphamethoxazole/trimethoprime (85.5%), ceftazidime (81.07%), ampicillin (70.4%), cefturixime (66.4%), cefotaxime (32.6%) (Table 3). This generally high rate of resistance and  can be explained by the fact that the rise in drug resistance is mainly attributable to the use of antimicrobials in humans and other animals, and the prevalence of resistant strains between the two. Increased resistance has too been associated to the release of insufficiently treated effluents from the industry pharmaceutical, particularly in countries everywhere bulk pharmaceuticals are manufactured. Antibiotics increase the selective pressure in bacterial populations, causing the susceptible bacteria to die; this increases the rate of resistant bacteria that remain to grow. 

Table 3: Antibiotics susceptibility profile of isolated bacteria.

Even at very low levels of antibiotics, resistant bacteria can have the advantage of growing and growing faster than weak bacteria. As antibiotic resistance becomes more frequent, so does the need for alternative treatments. There have been calls for new antibiotic treatments, but new drug development is becoming scarce^25,26. The current study inspected the rate of antibiotic resistance among major pathogenic bacteria isolated from inpatient and outpatient settings in two tertiary hospitals, in Sana'a city, Yemen. Certain that these antibiotic resistance to GNB and GPB be able to cause serious infections in hospitalized patients, particularly in immunocompromised patients, the elderly, neonates and children, the occurrence and spreading of these agents is one of the most important concerns of clinicians^19,20,27. The application of several classes of antibiotics is not permitted in neonates and children and because there are different patterns of antimicrobial resistance in different areas, selection and prescribing of appropriate antibiotics to treat different infections in immunocompromised, elderly, neonates and children is difficult. Moreover, knowing the patterns of antimicrobial resistance can help clinicians and policy makers to find solutions to resistance problems in their countries²⁸. The lack of public surveillance programs for antimicrobial resistance in development such as Yemen and many developed countries will lead to inappropriate use among patients and health care personnel^29-31. Therefore, investigation of antimicrobial resistance patterns is critical and important, especially in developing countries such as Yemen, where there are no systematic guidelines for antibiotic use. On the other hand, it is necessary to investigate the antibiotic resistance patterns of GPB and GNB in hospitals and clinics in Sana’a city, during 2019, which could be a precious model for both policy makers and clinicians in applying experimental treatment. 

The results of the current study showed that among 24690 diverse clinical samples of patients, 2931 (11.9%) were positive cultures from which different bacteria were isolated. The minimal rate of positive culture in the current study could be as a result of: 1) The current study used different types of clinical specimens such as cerebrospinal fluid, pleural fluid, dialysis fluid and luminal fluid as the prevalence of pathogens varies in these samples, 2) efficient guidance for correct administration of antibiotics, 3) improved managing and control of infection, and 4) pre-hospitalization antibiotic use.

In the current study, the most prevalent isolated GPB were Staphylococcus aureus (26.3%), non-hemolytic streptococci (9.1%), coagulase-negative staphylococci (8.1%) and alpha-hemolytic streptococcus (3.9%). In addition, the most common GNB isolated were Escherichia coli (22.04%), Pseudomonas aeruginosa (7.1%), Klebsiella spp (6.03%) Acinetobacter spp (1.46%) and Citrobacter spp (1.16%) (Table 2), which is in agreement with two different studies conducted in Tehran, Iran^22,32. Though, in investigations previously conducted in Yemen^19,20,33-35, Saudi Arabia³⁶ and Iran by Ibrahim Saray et al.,³⁷ and Alam et al.,³⁵, Acinetobacter spp. GNB was most common in positive culture samples. The result of  published studies^18,38 revealed that E. coli was the most frequent Gram-negative pathogen in positive cultures of the specimens as in current study (22.04%) (Table 2). The detected differences in proportions of GNB and GPB could be due to the diversity of specimen type, specimen size and applied detecting methods. The results also showed that coagulase-negative staphylococci isolated from clinical samples may have been considered a common contaminant. Therefore, more effective measures such as hygiene of the hands of health care workers, regular disinfection of medical devices, and disinfection of the sampling site during sampling should be taken. However, although rare, CoNS can cause many infections including infections of the skin and soft tissues, and therefore should not be considered as contaminants at all times^20,39. Persistent CoNS infection is likely to be associated with various serious complications such as embolic complications, metastatic seeding and septic thrombophlebitis⁴⁰. For that reason, the evaluation the medical association of CoNS is a challenging problem. In medical diagnostic laboratories, as in the present findings, the main diagnostic challenge is to assess whether the expected CoNS sequestration represents: 1) common coloniza-tion of the skin, soft tissues, or mucous membranes, 2) sample contamination during sample collection, handling, and handling, or 3) clinically significant infection^16,19,20,40. In the situation of co-infection of CoNS with further bacterial infections (multimicrobial infections by CoNS), different bacteria isolates showed different patterns of sensitivity and resistance, this difficult diagnostic situation becomes more complex^40,41. Close cooperation between physicians and diagnostic laboratory specialists can resolve this medical and diagnostic dilemma. In the false positive CoNS situation, patients are treated with several antibiotics, and it is expected that in addition to the extra costs, extreme antibiotic selection pressures happen that can lead to the emergence of antibiotic resistance⁴². Consequently, it is essential to answer the question that CoNS isolated from a clinical sample is a true infection or just a common cutaneous colonization. Some of the key factors useful in predicting true infection are: 1) isolating similar strains repeatedly during infection after isolating a strain in pure culture from the infected site, 2) in bloodstream infection, patients must have clinical evidence of infection with a single positive blood culture or Only two positive blood cultures were in CoNS within 5 days, and 3) if CoNS was isolated from the skin or soft tissue bacterial culture of a suspected infectious lesion, the isolated organism should be suggested as the pathogen and appropriate treatment should be started^43-45.

Among the antibiotics tested differently, the results of current study showed that the rate of resistance to linezolid was very low (0.42%) (Table 3)  making it highly efficient antibiotics against Enterococcus spp and S. aureus which it was in accord with the rates previously described by Al-Safani et al.,²⁰, by Azimi et al., in Iran³², Dharmapalan et al., from India⁴⁶, He et al., from China⁴⁷, Li Tian et al., from China⁴⁸ and Al-Naqshbandi and others from Iraq⁴⁹. Nevertheless, the findings of several studies were inconsistent with the current research and it has been reported that the resistance to linezolid is high^50,51. In current vancomycin resistance in Enterococcus spp. was much higher (7/32); 21.8% of Enterococcus spp. were resistant to vancomycin. Even though the classification of Enterococcus spp. not completed at the species level, therefore, most vancomycin-resistant isolates are likely to be Enterococcus faecium. According to several published studies and reports, effective measures have been taken to reduce the risk of VRSA in many countries such as the USA, and some guidelines have been developed to control infections caused by these pathogenic microorganisms⁵². Thus, we suggest similar guidelines and programs designed for patients in Sana'a, Yemen. Current study also revealed that colistin (3.45% resistant rate), in comparison with ciprofloxacin (15.8% resistant rate).  These finding were similar to the results of Mahmoudi et al., from Iran²² and Dharmapalan et al., from India ⁴⁶, but different from that reported by Azimi et al., in which colistin has a higher rate of resistance than ciprofloxacin³².

Overall, the results of the current study showed that sulfamethoxazole/trimethoprim, ceftazidime, ampici-llin, ceftorexime and cefotaxime are ineffective antibiotics against GPB or GNB. It is worth mentioning that these antibiotics in different hospitals in Sana'a are often used to control various infections especially sepsis and septicemia. It is well understood that resistance to these antibiotics is increasing daily, which is the result of the selective pressure that is secreted by bystander selection and the misuse or overuse of antibiotics⁵³. Consistent with the high antibiotic resistance among bacteria, in an attempt to stop the unwanted consequence of sepsis and septicemia, as well as with the purpose of reduce the mortality rate because of these infections, accurate recognition and employ of efficient antibiotics for effective treatment is critical^54-57. Thus, awareness of antibiotic resistance patterns among common pathogens, holding work-shops to correct prescribing for empirical therapy, and changes in antimicrobial use are necessary and highly recommended. Finally, the results of the DDM are of great importance, and individuals' free access to access to antibiotics should be prevented. In this study, we revealed that GNB and GPB are resistant to different groups of antibiotics. However, it should be noted that these bacteria have two types of antibiotic resistance: acquired resistance and endogenous resistance. For example, according to EUCAST guidelines, most GNB (Enterobacteriaceae, Pseudomonas spp.) are self-resistant to various antibiotics including penicillin G, oxacillin, macrolides (eg, azithromycin, erythromycin, tylosin), lincosamides (eg lincomycin), streptogramins (eg, Virginiamycin), glycopeptides (eg, vancomycin) and bacitracin. Moreover, based on these guidelines, most GPB are intrinsically resistant to polymyxins and quinolones/fluoroquinolones (eg, enrofloxacin, ciprofloxacin, difloxacin, marboflo-xacin)⁵⁸. Therefore, these resistances should be known by clinicians in order to avoid unsuitable and ineffective therapy.

CONCLUSION 

To counter these threats there have been increasing public calls for global collective action, including a proposal for an international treaty on antimicrobial resistance. It must be recognized that more details and attention are still needed in order to identify and measure resistance trends at the international level; the idea of a global tracking system has been proposed but implementation has yet to take place. A system of this nature will provide insight into areas of high resistance as well as information needed to evaluate programs and other changes that have been made to combat or reverse antibiotic resistance. Moreover, based on the fact that we did not have full access to patients’ information such as treatment outcomes, mortality rate, etc., no specific analysis was carried out, so this information should be provided and an additional study should be carried out to clarify the picture of this problem. According to this data, choosing the right antibiotic is vital in treating bacterial infections. Therefore, awareness of antibiotic resistance patterns in pathogenic bacteria can be helpful in making the right therapeutic choice.

ACKNOWLEDGMENTS 

The authors also would like to acknowledge the Microbiology Department of the National Center of Public Health Laboratories (NCPHL) Sana'a, Yemen for support.

CONFLICT OF INTEREST 

No conflict of interest associated with this work. 

AUTHOR’S CONTRIBUTIONS

All authors contributed to data analysis, drafting and revising the paper, gave final approval of the version to be published and agree to be accountable for all aspects of the work. Huda Al-Shami has first authorship.

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