Introduction

Antimicrobial discovery, development, and administration overturned the modern medical approach substantially by improving the infection management.¹ However, with the emergence of drug resistance, the worldwide clinical burden is excruciating. Antimicrobial drug resistance (AMR) among microorganisms is evoking a substantial threat to the health of the patients as well as the treating physicians.

Drug resistance (DR) acquisition in the organisms is through transposons or resistance plasmids with gene accumulation, each coding explicitly to a specific agent or by multidrug efflux pumps. Gradually, due to these multiple mechanisms, the organisms acquire resistance to multiple drugs. Based on the organism’s resistance capability to various classes of drugs, the different types of resistance are termed as multidrug resistance, extensive drug resistance, and pan-drug resistance.^{2, 3} The multidrug-resistant (MDR) isolates are creating a potential economic burden on patients due to the hospital cost. Previous studies revealed a 3-fold increase in the healthcare costs during the management of patients with resistant bacterial isolates compared to nonresistant isolates.^{4, 5} Healthcare expenditure on drug resistant organisms is around 6000–30,000 dollars higher than that of the nonresistant organisms.⁵

Development of resistance among organisms requires considerably less time as compared to antibiotic discovery and development, which require years to formulate. Unfortunately, since the 21^st century, only four antibiotics were approved by the Food and Drug Administration (FDA) for clinical intervention.³ A study from the Middle East discovered that various pharmacies around the Gulf Cooperation Council (GCC) countries are dispensing the antimicrobials without prior prescription and without patient’s request, which is not unusual.⁶ In the Arabian Peninsula, the prevalence of MDR extended-spectrum β–lactamase-producing Escherichia coli and Klebsiella pneumoniae is 29% and 65%, respectively and, as a result the mortality range has intensified between 11% and 40%.7 In Saudi Arabia, susceptibility rate of Acinetobacter baumannii in 2006, was 64–81.2% for imipenem and meropenem, whereas, in 2012 susceptibility rate was only 8.3–11%.⁸ In the Middle East, frequently encountered MDR isolates are extended-spectrum β–lactamase-producing Pseudomonas aeruginosa in Qatar,_⁹ carbapenem-resistant Enterobacteriaceae in United Arab Emirates,¹⁰ and pan drug-resistant (PDR) K. pneumoniae, Staphylococcus aureus, Methicillin-resistant S. aureus (MRSA), and Methicillin-resistant S. epidermidis (MRSE), in Saudi Arabia.¹¹ This explains the extent of MDR spread around the Middle East and Southeast Asian regions.¹² In recent studies, prevalence of extensively drug-resistant (XDR) isolates is increasing, especially with vancomycin-resistant enterococci and carbapenem-producing K. pneumoniae, which are complex to treat.¹³

The approach of researchers towards MDR and XDR organisms is focused particularly on tuberculosiscausing organisms or Pseudomonas species, neglecting other MDR and XDR pathogens. Studies focusing on MDR tuberculosis-causing organisms around the GCC countries reported approximately 58,000 cases of MDR tuberculosis in 2014; Bahrain alone accounted for 6.3% of new MDR tuberculosis cases in 2015.¹⁴ Hence, it becomes essential to pursue the changing trends in antimicrobial susceptibility of all bacteria to provide suitable antimicrobial therapy for controlling MDR. Only few studies, especially from the Middle Eastern countries, have evaluated the commonly encountered organisms in MDR and XDR. In this view, the current study considered evaluating the prevalence of MDR, XDR, and PDR bacterial isolates cultured from clinical samples.

Materials & methods

Study design and data collection

The current cross-sectional study was conducted in the department of Microbiology from January 2017 to December 2017. The bacterial isolates were procured from different clinical samples, including abscess swabs, bile, blood, body fluid, catheter tip, colonic tissue, ear swab, eye swab,fluid drains, high vaginal swab, nasal swab, peritoneal fluid, pharynx, semen, sputum, stool, throat, tissue, tracheal secretion, urethral swab, urine, and wound swabs. The organisms were identified through conventional microbiological techniques, such as morphological

Gram-staining, and biochemical tests. Automated methods, such as Vitex analyzer (bioMerieux, USA) were used as well for identification. ¹⁵ Antibiotic susceptibility profile of the organisms were assessed through the standard microbiological techniques using disk diffusion method¹⁶ as well as automated methods (Vitex analyzer, bioMerieux). Susceptibility profiles were derived using Kirby–Bauer chart as per Clinical Laboratory Standard Institute (CLSI) guidelines.¹⁷

Criteria for MDR, XDR, and PDR patterns

MDR, XDR, and PDR patterns were evaluated for the isolates as per criteria recommended by European Center for Disease Prevention and Control and Centers for Disease Control and Prevention.³The isolates were considered as MDR, possible MDR (pMDR), XDR, possible XDR (pXDR), and PDR based on the following criteria:

MDR: If nonsusceptibility was observed for at least one antimicrobial agent in minimum three antimicrobial categories;

pMDR: If the isolates were non-susceptible to at least one agent in < 2 antimicrobial categories and other antibiotic categories were not tested;

 XDR: If nonsusceptibility was observed to at least one agent in all antimicrobial categories, except for two or fewer categories (i.e., bacterial isolates remain susceptible to only one or two antimicrobial categories);

 pXDR: If nonsusceptibility was observed to > 3 antimicrobial categories and antibiotics are not tested for more than one category;

 PDR: If nonsusceptibility was observed in all agents of all antimicrobial categories.¹⁸

Various antimicrobial categories, including aminoglycosides (amikacin, gentamicin), β–lactams (penicillin, ampicillin), carbapenems (imipenem, ertapenem, meropenem), cephalosporins (cefotaxime, cefixime, cefuroxime, ceftriaxone, ceftazidime, cefepime, cefazolin), fluoroquinolones (levofloxacin, ciprofloxacin, moxifloxacin, norfloxacin), folate pathway inhibitor (trimethoprim/ sulfamethoxazole), glycopeptides (vancomycin), lincosamides (clindamycin), macrolides (erythromycin, azithromycin), monobactam (aztreonam), and β–lactam/β–lactamase inhibitor combinations (piperacillin/tazobactam, amoxicillin/ clavulanate) were assessed.

 Data analysis

The data obtained were coded and entered in Microsoft Excel spreadsheet. Data were expressed as rates, ratios, and percentages and compared with different groups which includes susceptible group, followed by MDR, XDR and PDR groups.

Results

 A total of 1862 bacterial isolates were assessed for antimicrobial pattern wherein, 61.6% and 38.4% were Gram-negative and Gram-positive isolates, respectively. Among the total isolates, 65.2%, 12.8%, 17.9%, 3.1%, and 0.8% were susceptible, pMDR, MDR, pXDR, and XDR, respectively (Figure 1). Most common isolates included E. coli (15.9%), followed by S. aureus (13.8%), K. pneumoniae (8.4%), and Streptococci (7.7%; Table 1). Among susceptible isolates, majority were E. coli (19.1%) S. aureus (18.4%), and K. pneumoniae (12.2%). Among the pMDR isolates, majority were Haemophilus influenzae (28.1%), E. coli (20.1%), streptococci group A (10.5%), and S. aureus (9.6%). Among MDR isolates, 35.2%, 16.4%, 14.3%, and 11.3% isolates were extended spectrum β–lactamase-producing E. coli, MRSA, MRSE, and K. pneumoniae, respectively. Extendedspectrum β–lactamase-producing E. coli was the most common (48.2%) isolate among pXDR group, followed by A. baumannii (25.8%) and Proteus mirabilis (12.1%). Out of the total XDR isolates, 68.7%, 18.7%, and 12.5% were MRSE, MRSA, and extended-spectrum β–lactamase-producing K. pneumoniae, respectively (Table 1).

Majority of the Gram-positive isolates were observed on throat swab (18.1%), followed by nasal (15.6%) and wound (11.3%) swabs. Among the 715 gram-positive isolates, 66.8%, 13.8%, 16.5%, 1.9%, and 0.8% isolates were susceptible, pMDR, MDR, XDR, and pXDR, respectively (Table 2).

Majority of the Gram-negative isolates were observed in samples of urine (41.7%), followed by sputum (10.9%) and wound (9.6%) swabs. Out of 1147 Gram-negative isolates, 64.2%, 12.1%, 18.9%, 4.5%, and 0.17% were susceptible, pMDR, MDR, pXDR, and XDR, respectively (Table 3).

Among the MDR, pMDR, and XDR isolates, most of the organisms were resistant to fluoroquinolones, β–lactams, cephalosporins, and aminoglycosides. Interestingly, among 23 isolates of Neisseria gonorrhoeae, seven were penicillin-resistant N. gonorrhoeae (30%; Table 4).

Discussion

Deriving the DR profile of clinical bacterial isolates is essential in reducing the intensity of the infection. Contemporary MDR research focuses majorly on tuberculosis-causing organisms and pseudomonas. However, MDR pathogens, such as MRSA, extended-spectrum β–lactamase-producing Enterobacteriaceae, and vancomycin resistant enterococci are predominantly encountered and scarcely researched upon in the GCC.^{19, 20} Currently, the development of resistance, especially the emergence of PDR isolates is gaining importance due to scarcity of novel antibiotics as well as associated mortality.^{3, 19} These facts establish the risk bound around the physicians while treating the patients infected with MDR/XDR organisms. Considering these alarming scenarios, the current study attempted to analyze the prevalence of susceptible, MDR, XDR, and PDR profiles of organisms isolated from different clinical samples in a tertiary care hospital.

Among the total bacterial isolates considered, majority were Gram-negative; of these, most of the isolates were observed in urine, sputum, and wound swabs. Among Gram-positive isolates, majority of them were collected from the throat, followed by nasal, and wound swabs. A study conducted by Ten Hove et.al.,²¹ evaluated the drug susceptibility in 1983 cultures. Majority were collected from urine, wounds, blood, sputum, throat, and nasal swabs. These findings represent that urine samples possess larger amounts of bacterial isolates than other samples. Eshetie et.al.²² revealed that greater MDR isolates belonging to Enterobacteriaceae, especially K. pneumoniae and E. coli were observed prominently in urine samples.²² In accordance to the findings of Eshetie et.al., the current study also observed majority of the isolates—E. coli, S.aureus, and K. pneumoniae—in urine swabs. In a Gulf-centered review study,²³ out of 45 published research articles in and around the Middle Eastern countries, the most common bacterial isolates encountered were E. coli, K. pneumoniae, P.aeruginosa, and MRSA.

In the current study, majority of the isolates werebelonged to susceptible group, followed by MDR isolates, pMDR, pXDR, and XDR isolates group. Similarly, in a study conducted by Basak et.al.,³37.1% and 13.7% of the isolates were MDRand XDR, respectively. Cornejo-Juárez et al.²⁰reported 39% MDR isolates in their study. The rapid elevation in DR can be attributed to various risk factors associated with bacterial proliferation and resistance development, which include indiscriminate antimicrobial usage, unfamiliarity of previous antibiotic administered, unhygienic lifestyle, and cross-transmission.²⁰ MDR and XDR bacterial isolates were encountered exclusively in nosocomial infections.¹³ Further, this suggests that unoptimized prescription and administration ofantimicrobials is a predominant factor for resistance development. Developing guiding programs for healthcare workers and public regarding hygiene compliance and promoting sanitary practices can reduce the nosocomial infections and the spread of MDR.

In the present study, 12.78% and 17.99% isolates were pMDR and MDR. E. coli, extended-spectrumβ–lactamase-producing E. coli, K. pneumoniae, streptococci, MRSA, MRSE, and S. aureus were prevalent among MDR isolates, including Citrobacter freundii and P. vulgaris.

Extended-spectrum β–lactamase-producing E.coli, A. baumannii, MRSE, MRSA, and extended spectrum β–lactamase-producing K. pneumoniae  were prevalent among XDR isolates, which is similar to the observations of Basak et.al.³ Majority of the resistant isolates were MDR and XDR with E. coli and P. aeruginosa being the most common isolates, respectively. Saderi et.al.,²⁴ observed 54.5% and29% of MDR and XDR isolates (P. aeruginosa),respectively. In a study performed by Ochoa et.al.,²⁵ among 500 E. coli uropathogens, 16.4% and 4.20%isolates were MDR and XDR. In another study by Huang et.al.,²⁶ among 1672 cases, 37% were colonized with MDR-acquired organisms. Wherein, extended-spectrum β–lactamase-producing Enterobacteriaceae followed by MRSA were the most common isolates. Ramirez-Castillo et.al.²⁷ focused specifially on urinary tract infections caused by E. coli and observed that around 63% of the isolates were MDR. Various studies around the world observed distinct drug resistant organisms; the most frequent were E. coli and K. pneumoniae.^28-32 Although, studies regarding commonly encountered isolates were not available in the Middle Eastern countries, the above findings suggest that the prevalence of MDR and XDR around the globe is aggravating and the societal need for novel effective antimicrobials is intensifying. Educating the public regarding the sensible use of antibiotics is one of the effective control measures. Strict regulations must be enforced on all pharmacies throughout the nation to barricade over-the-counter marketing of antimicrobials.

Resistance acquisition to antimicrobials is through various mechanisms. Resistance to floroquinolone occurs primarily through three distinct biochemical ways: gene alterations that encode the target site of floroquinolones, efflx pumps, and floroquinolone target site protection.¹ Conversely, resistance of β–lactams and cephalosporins is through within-the organism-resistance-development mechanisms, such as production of β–lactamase. Moreover, interaction within the organisms as well as gene transfer through carriers, such as plasmids and transposons, increase the rate of acquisition and spread of resistance. These mechanisms have overpowered organisms to withstand against the multigeneration antibiotics.¹ In the current study, most of the MDR and XDR organisms showed resistance mostly to floroquinolones, β–lactams, cephalosporins, and aminoglycosides. Similarly, Worku et.al.³³ reported that MDR/XDR isolates showed resistance predominantly to β–lactams, macrolides,  cephalosporins, and aminoglycosides. Optimized use of antibiotics can be achieved by introducing surveillance programs and developing active social media programs. Further, initiatives and campaigns should be developed throughout the nation to create awareness regarding infection control and prevention to reduce the development of resistance.

Conclusion

Among the bacterial isolates evaluated, majority were Gram-negative. Most commonly encountered isolates included E. coli, S. aureus, K. pneumoniae,and streptococci. Majority of the isolates were belonged to susceptible group, followed by MDR and XDR isolates group; PDR isolates were notidentifid. Most frequently encountered organisms among MDR include E. coli, extended-spectrumβ–lactamase-producing E. coli, K. pneumoniae, streptococci, MRSA, MRSE, and S. aureus. Among XDR, extended-spectrum β–lactamase producing E. coli, A. baumannii, MRSE, MRSA, and extended-spectrum β–lactamase-producing K. pneumoniae were prevalent. The gradual amplifiation of resistance development among the commonly encountered organisms can be controlled through optimized antibiotic usage. Furthermore, developing public awareness camps and initiatives and strict regulations on drug dispensing practices must be put in force to reduce the spread of AMR.

*Tables and figures can be found in pdf file. 

 Conflicts of interest

 Authors have no conflict of interest to declare.

 Acknowledgements

Authors are grateful to Dr. Jafar Alsaid, Mrs.Tejashree Pawar, Mr. Nadim Navde for their continued support during the study period. The study was funded by Bahrain Specialist Hospital.