Introduction

The human body harbors distinct bacterial forms as normal flora, without harming the host. Bacterial count across the human body reaches up to 38 trillion,¹ which interestingly exceeds the total human cell count in the body accounting for approximately 30 trillion.¹ Based on the requirement, bacterial communities restructure themselves to reside in different tissues or organs of the host such as the skin, gastrointestinal tract, rectum, mouth, and stomach. However, breach of the tissue integrity in any manner by either wound, cut, injury, or burn, furnishes an opportunity for the bacteria to colonize and cause disease.² Common opportunistic bacterial species include Staphylococcus aureus among the Gram-positive forms and Escherichia coli, Proteus mirabilis, and Klebsiella pneumoniae, among the Gram-negative forms.^{3, 4}

The bacterial population has the potentiality to interact within and between the species corresponding to their physiological and metabolic requirements.⁵ To sustain themselves, they can also adapt themselves to the changing ecological conditions by genetic alterations.^3,5 These characteristics along with alteration of outer membrane proteins, efflux mechanisms, and production of hydrolyzing enzymes, such as extended-spectrum β–lactamases enable the bacteria to withstand against the antimicrobials used.³ Although, antimicrobials are competent in eradicating bacteria, with the emergence of resistance, eradicating bacteria has become a substantial concern.³ Bacteria acquire antimicrobial resistance (AMR) via gene level mutations, which are transferred via interaction with other bacterial species through plasmids.⁶ Eventually, it aids in the development of multiple drug resistance in the bacteria.

Extended-spectrum β–lactamases are hydrolyzing enzymes synthesized within the organism that mediate the development of resistance to broadspectrum antimicrobials, such as monobactams.⁷ Over the past two decades, the extended-spectrum β–lactamases represented the onset of significant health constraints. The resistance is, predominantly, acquired through chromosomal DNA alteration and the acquired resistance mechanism might be encoded within the transferable genes of the bacterium, which is passed on to the neighboring bacterial species.³

The prevalence of AMR in the Arabian Gulf region is fairly large, especially, with the producers of extended-spectrum β–lactamases .⁸ There is scarcity of studies regarding the evaluation of AMR in the Kingdom of Bahrain.^{3, 9-11} Further, higher predominance of extended-spectrum β–lactamasesproducing bacterial isolates were observed in the region of Bahrain¹¹; however, researchers are emphasizing specifically on AMR without differentiating extended-spectrum β–lactamasesproducing isolates. Therefore, the present study was performed to determine the frequently isolated bacterial species from various clinical samples. The antimicrobial susceptibility profile of the isolates was also derived, to determine the impact of exposure of common antibiotics on the isolates.

Materials & Methods

Study design and data collection

The present retrospective study was conducted from January-December 2017 and was approved by the Institutional Ethical Committee. During the study period, all the clinical samples received at a tertiary care hospital, for microbiological examination, were considered for the study.

The sociodemographic data such as age, gender, nationality, and presence of any comorbidities were recorded for evaluation on predesigned forms, by accessing the medical records of the patients. Clinical samples collected from multiple sites for the diagnosis included nasal swabs, ear swabs, sputum, stool, throat, abscess, eye swab, pharynx swab, urine, blood, wound, colonic tissue culture, bronchoalveolar lavage, pleural fluid, amnicator swabs, oral swabs, bile, peritoneal fluid, tissue/ surgical culture, drain fluid, semen, catheter tip, tracheal secretion, and vaginal and urethral swabs.

Identification

The standard microbial maintenance norms and procedures were followed; organisms isolated from the clinical samples were cultured appropriately using the following media formulations, which includes MacConkey agar, Sheep blood agar, Campylobacter charcoal agar, Selenite broth, Gardnerella vaginalis agar, Sabouraud dextrose agar, Thayer Martin agar, Neomycin anaerobic agar, Fluid thioglycollate broth, Salmonella Shigella agar, and Chocolate bacitracin agar (Saudi Prepared Media Laboratory Company Ltd, Saudi Arabia). Incubation temperature was maintained at 35 °C for MacConkey agar, Sabouraud dextrose agar, Salmonella Shigella agar, and Mueller Hinton agar whereas, 43 °C with 5 % CO₂ for Campylobacter charcoal agar, 35 °C with 5 % CO2 for Sheep blood agar, Chocolate agar, Chocolate bacitracin agar, Gardnerella vaginalis agar, and Thayer Martin agar, and 35 °C with 5 % CO₂ in anaerobic agar with anaerobic gas pack for Neomycin anaerobic agar and Sheep blood agar for throat swabs.

Subsequently, majority of the cultured isolates were identified using Vitex automated analyzer (Vitex® bioMerieux, USA), whereas, few isolates were identified manually based on the routine microbiological techniques, such as morphological characteristics on culture plate, Gram staining, motility test, and biochemical characteristics analysis. ^{4, 12}

Antimicrobial susceptibility analysis

Antimicrobial susceptibility profile of each isolate was assessed using disk diffusion method (6-mm disc, OXOID, UK) by Kirby-Bauer procedure and Vitex automated analyzer (Vitex® bioMerieux, USA).13 The various antibiotics specific for Grampositive and Gram-negative organisms were tested, which included amikacin (30 μg), gentamicin (10 μg), amoxicillin/clavulanate (30 μg), piperacillin-tazobactam (110 μg), imipenem (10 μg), meropenem (10 μg), cefixime (5 μg), cefuroxime (30 μg), cefazolin (30 μg), linezolid (30 μg), ceftriaxone (30 μg), ceftazidime (30 μg), cefepime (30 μg), levofloxacin (5 μg), ciprofloxacin (5 μg), clindamycin (2 μg), erythromycin (15 μg), vancomycin (30 μg), and trimethoprimsulfamethoxazole (25 μg). The inoculated plates (0.5 Mcfarland) were incubated under standard conditions for 24 h at 37 °C. Each plate was evaluated for the zone of clearance and the diameter (mm) of the zone, if present, was measured and compared with the standard Kirby-Bauer chart for characterizing the susceptibility of the isolates.^{12, 14}

Statistical analysis

Data were recorded in Microsoft excel 2013 and analyzed using R-3.4.1. Chi-square test was used to determine the association between the commonly isolated groups of bacteria, absence or presence of common comorbidities, and association between the Bahraini and non-Bahraini culture-positive patients.

Results

Demographic characteristics

A total of 16,500 cultures were isolated from all the clinical samples received for microbiological analysis. Organisms cultured from the same site with similar sensitivity patterns of one month were excluded. After applying exclusion criteria, 1931 isolates were obtained from 1931 clinical samples and were considered for analysis.

Out of 1931 patients, four patients were excluded due to lack of demographic information. Among the 1927 patients considered for demographical analysis, 53.5 % were females and the majority (38 %) of the patients were aged between 21 and 40 years (Table 1).

Based on the patients’ geographical region, 37.8 % were from Bahrain, whereas, 62 % were non-Bahraini patients, including the Asian continent. Most (68.1 %) of the study population were inhabitants of Asia, while the remaining non-Bahraini patients include Americans (19.5 %), Europeans (7.3 %), Africans (4.6 %), and Australians (0.4 %; Table 1).

Culture characteristics of isolates

Among the clinical samples cultured, majority of them were urine samples (29.7 %) followed by wound (10.5 %), and nasal swabs (10.2 %; Table 2). A wide range of bacterial flora were isolated, accounting to 26 different bacterial species. Among the total isolates, 61.6 % belonged to Gram-negative group dominated by E. coli and K. pneumoniae and 38.4 % were Gram-positive organisms with S. aureus being the predominant organism.

*Table 2 can be referred to in the downloadable PDF

Interestingly, Campylobacter (21 %) was observed to be the second predominant organism isolated in the stool samples and Aspergillus as the third most common isolate in the ear cultures. Out of 64 patients with methicillin-resistant Staphylococcus aureus (MRSA), 32 % were from Bahrain followed by 26.5 % from America. Majority (50 %) of the Campylobacter-isolated-patients belonged to America. Most common organisms isolated from the clinical samples are represented in Table 3.

Antibiotic susceptibility profile of the isolates

Majority of the isolates showed high resistance pattern against the antimicrobial agents. Gram-negative isolates, such as extended-spectrum β– lactamases E. coli, extended-spectrum β–lactamases K. pneumoniae, Acinetobacter baumannii, and Citrobacter freundii exhibited comparatively high resistance, especially to β–lactams and cephalosporins.

Extended-spectrum β–lactamases E. coli showed greater resistance pattern to cefuroxime (98.6 %) whereas, extended-spectrum β–lactamases K. pneumoniae showed prominent resistance pattern to ceftriaxone and amoxicillin/clavulanate (89.5 %, each), followed by Enterobacter aerogenes (96.3 %; Table 4). Extended spectrum β–lactamases E. coli and K. pneumoniae isolates were relatively resistant to amoxicillin/clavulanate and all six classes of cephalosporins were tested. However, greater susceptibility to all classes of carbapenems was observed in both extended-spectrum β–lactamases isolates—E. coli and K. pneumoniae. Among non-extended spectrum β–lactamases E. coli, only 1.2 % of resistance pattern was observed towards cephalosporin antibiotics.

Among Gram-positive isolates, Enterococcus faecalis exhibited relatively greater resistance against most of the antimicrobial agents (Table 5). Ciprofloxacin resistance was prevalent among MRSA (53.1 %), methicillin-resistant Staphylococcus epidermidis (MRSE) (82.5 %), S. aureus (19.9 %), and Enterococcus faecalis (19.9 %).

Association of bacterial isolates with co-morbidities present among culture-positive patients

Among the study population, data regarding co-morbidities were available only for 657 culture positive patients. Association between isolated groups of bacteria and presence of comorbidities, such as diabetes and hypertension showed that A. baumannii, non-extended and extendedspectrum β–lactamases E. coli, and Haemophilus influenzae in the Gram-negative group and S.aureus, Streptococcus Group A (β–hemolytic), and Streptococcus pneumoniae in the Gram-positive group were significantly present in patients with diabetes (P < 0.001). Predominant isolates observed in hypertensive patients included A. baumannii, non-extended-spectrum β–lactamases E. coli, H. influenzae, MRSE, S. aureus, Stenotrophomonas maltophilia, Streptococcus Group A (β–hemolytic), and S. pneumoniae (P < 0.001; Table 6).

Association of bacterial isolates with Bahraini and non-Bahraini culture-positive patients

Organisms such as E. coli, A. baumannii, Enterococcus faecium, extended spectrum β– lactamases K. pneumoniae, and S. maltophilia were significant and predominantly isolated from the samples of the Bahraini patients (P = 0.05). Significantly observed isolates in non-Bahraini patients included non-extended and extendedspectrum β–lactamases E. coli, H. influenzae, Klebsiella oxytoca, K. pneumoniae, and MRSA (P < 0.001; Table 7).

Discussion

The present retrospective study encountered female preponderance, with majority of the patients belonging to the younger age group. Clinical samples collected from Bahraini patients were significantly more compared to non-Bahraini patients belonging to the Asian continent as well as to other foreign continents.

Among the total isolates in the present study, majority belonged to the Gram-negative group with the prevalence of E. coli (31.3 %) and K. pneumoniae (14.4 %) and among Gram-positive group S. aureus (70.6 %) was prevalent. Similarly, a study by Ali et al.³ on AMR prevalence in and around the Gulf countries reported prevalence of E. coli (14 %), K. pneumoniae (13.9 %), Enterococcus spp. (76.5 %), and MRSA (8.5 %).

In the present study, majority of the clinical samples were urine and wound swab samples. The prevalent organisms isolated from the urine samples included E. coli and K. pneumoniae, whereas, E. coli, S. aureus, and MRSA were observed to be predominant in the wound samples. Similar observations were reported in the urine samples, globally.^15-20 Urinary tract infections are believed to be the second most predominant communitybased and nosocomial infections, worldwide.^{21, 22} These findings represent that urine and urinary tract environment demonstrate a higher prevalence of uropathogens due to the longer hospital stay, sexual activity, diabetes, hormonal imbalance, and genetic susceptibility.²³

The breakthrough of infection management was the discovery of antimicrobials, which led the treating physicians to significantly manage the patients with infections. Alarming increase in antimicrobial commercialization, drug resistance development among organisms, and indiscriminate use without knowledge jeopardized the antimicrobial era into an economic burden era.²⁴ Although, many antibiotic discoveries are in place, the sensible use of antibiotics is the only means for controlling the development of AMR. In the current study, higher resistance pattern was observed to most of the antibiotics used, especially to β–lactams and cephalosporins. Probable reason might be the ease availability of antibiotics without a prescription in pharmacies and wrong psychological perception among people about antibiotics as the first-aid response to any health complications.

In the current study, amoxicillin/clavulanate and ciprofloxacin resistance were relatively high among the Gram-negative and Gram-positive groups, respectively Susceptibility profile of carbapenems was common among both Gram-positive and Gramnegative organisms. In a Gulf-centered study by Al et al.,²⁵ oral amoxicillin/clavulanate was reported to be the most unsuccessful antibiotic among the 54 patients. In another study conducted in the Kingdom of Bahrain,¹⁰ carbapenems were the most susceptible class of antibiotics. In the present study, among the Gram-positive group, Enterococcus spp. represented relatively higher resistance pattern to antimicrobial agents. This is in accordance to a study conducted by Aly et al.,³ wherein, resistance pattern was observed with Enterococcus spp. in the Bahrain region. Current study observed Enterococcus spp. and S. aureus isolates with 1.1 % and 3.1 % of resistance towards linezolid antibiotic, respectively which was uncommon in most of the institutions. Alteration of the antimicrobial’s target sites, such as efflux pumps and porin channels on the bacterium prevent the binding of antibiotic molecules resulting in the development of resistance.²⁶

Extended-spectrum β–lactamases enzymes are the class of β–lactams with a unique ability of resistance to penicillin, cephalosporins of first-, second-, and third-generation, and aztreonam with higher susceptibility profile to carbapenems. In most of the cases, extended-spectrum β–lactamases production was observed prevalent in Gram-negative organisms, especially in E. coli and K. pneumoniae isolates.¹⁰ Presence of resistance profile towards extendedspectrum antibiotics, such as cephalosporins was first reported in Bahrain during 1980’s.¹⁰ In the late 1990’s, the resistance was observed towards cephalosporins and aminoglycosides in majority of the Klebsiella isolates.⁹ The extended spectrum β–lactamases-producing K. pneumoniae was first reported in 1995 in Bahrain.^{9, 10} A study conducted by Bindayna¹¹ reported that among 2695 isolates analyzed, the majority of the isolates were E. coli (52.4 %) followed by K. pneumoniae (24.2 %) and both were extended spectrum β–lactamases producers. Taking into consideration the higher prevalence of extended spectrum β–lactamasesproducing E. coli and K. pneumoniae organisms, the current study considered distinguishing the E. coli and K. pneumoniae isolates for extended spectrum β–lactamases production. In present study, 32.9 % and 18.27 % of the E. coli and K. pneumoniae isolates, respectively, were extended spectrum β– lactamases producers; majority were susceptible to carbapenems and relatively resistant to all classes of cephalosporins. In 2011, another study by Bindayna et al.²⁷ reported that among the extended-spectrum β–lactamases producing isolates, such as E. coli and K. pneumoniae, greater AMR patterns was observed towards cephalosporins and higher susceptibility patterns towards carbapenems. Indiscriminate use, overuse, and reuse of antibiotics for broad spectrum infections have led to the development of extended spectrum β–lactamases production.²⁸

The present study reported the association between isolates and the presence of comorbid conditions— diabetes and hypertension in the patients. Organisms such as non-extended spectrum β–lactamases E. coli, H. influenzae, and A. baumannii among Gram-negative group and S. aureus, Streptococcus Group A, and S. pneumoniae among the Gram-positive group were significantly associated with the presence of co-morbidities. The probable reason behind the relationship between the prevalence of isolates and comorbidity is difficult to derive due to the complexity and heterogeneous nature of the co-morbidities. Although, studies have attempted to derive the association, it still remains unclear.^{29, 30}

E. coli was prevalent among both the Bahraini and non-Bahraini patients. Among the extended spectrum β–lactamases producers, E. coli was predominant among the Bahraini patients, whereas, the non-extended spectrum β–lactamases E. coli was prevalent among the non-Bahraini patients. Similar findings were not found in the literature. These data represent that higher prevalence of β–lactamproducing isolates are emerging significantly in the Bahrain region. Studies considering similar parameters necessary in Bahrain region.

Conclusion

The urine and wound samples are the highest source of bacterial isolates among the various clinical samples predominantly with S. aureus, E. coli, and K. pneumoniae organisms. Isolates were highly resistant to β–lactams and cephalosporins and were susceptible to carbapenems. The prevalence of extended-spectrum β–lactamases was more among the Bahraini patients compared to the non-Bahraini patients. Further, studies are required to substantiate these findings with longer follow-ups and wider range of antibiotics.

*All tables and figures can be found in the downloadable pdf.