Gram Negative Extended Spectrum Beta-Lactamase Producing Bacteria Prevalence in Jouf Region Tertiary Care Hospital, Saudi Arabia

Extended Spectrum beta lactamase producing pathogens are reported in many clinical samples and pose an emerging threat health. To find the prevalence of ESBL producing Enterobactriacea pathogens isolated from inpatients (Medical and Surgical Wards; Hospital stay > 72 h) admitted to Prince, Mutib, Hospital Sakaka, Jouf. A total of 1043 Enterobactriacea were isolated during April 2015 to October 2016 study period. Vitek -2 compact (biomeriux Leon, France) was used for identification and antimicrobial sensitivity (AST) test. A Total of 115/1043 (11.02%) ESBL producing pathogens were isolated from urine 15.07% (n=242), sputum 13.6 % (n=220), wound 10.8% (n=287) and blood 5.4 % (n=294). Klebsiella pneumonia is most dominant followed by E coli and Proteus miribilis. Cephamycins and carbapenems were found most effective (100% sensitivity) against the pathogens isolated. The aminoglycosides, beta lactams and Fluroquinolnes class of antibiotics, microorganisms have gained the resistance of 20%, 22.7%, 40-70% respectively. The infection control measures should be taken seriously and making of effective use of antibiotics is need of hour.


iNtROduCtiON
The antimicrobial resistance (AMR) is a matter of concern worldwide 1 . Genes encoding beta-lactamase and modification of enzymes in bacteria results in drug resistance 2 .The Enterobacteriaceae carry the genes for resistance to βlactams and the β-lactamase family is divided into four groups: Extended Spectrum βLacatamases (ESBLs), Amp-C type, carbapenmases and Penicillinases. The antimicrobial resistance results in treatment failure, economic loss and a source of resistant genes in bacteria that leads to risk of human health. Among the members of Enterobacteriaceae family (Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter. Baumannii and Escherichia coli) are known to produce extended-spectrum β-lacatamse (ESBL) and are a serious threat to human health 3 . E. coli the most important pathogen in community acquired infections and have been studied in detail 4 . ESBL producing pathogens delays suitable antibiotic therapy which in turn leads to longer hospital stay and more economic burden 5 . The resistance to antibiotics causes higher mortality rates in patients. In Asia 70% of ESBL infections are due to nosocomial acquired. ESBL producing microorganisms are one of the cause of antimicrobial resistance (AMR) 6 . In Europe year, 1980 first case of ESBL was reported and subsequently in USA. Emegence of ESBL present a significant threat to human health, particulary klebsiella and E. coli pathogens for which only few antibiotics are available 7 . Countries with low and moderate income are facing problems to overcome or in managing antimicrobial resistance due to lack of surveillance and non-availability of high class microbiology laboratories for diagnosis of pathogens and their antibiograms 8 . The pathogenic microbes belongs to gram positive 9 and gram negative 10 have been isolated from wound, blood, sputum and urine samples were reported as antibiotic resistant in nature 11 . The distribution of antimicrobial resistant pathogens changes with time and among the hospitals located in different regions 12 . The prevalence of ESBLs in the middle east and Gulf Cooperation countries (GCC)have been reported 13 . The plasmid encoded genes are largely responsible for the ESBL and they belong to class A and are divided into three genotypes CTX-M, SHV and TEM 14 . This is the first study conducted in Jouf region to explore the prevalence of ESBL producing pathogens among the patients attending the Prince, Mutib, Hospital Sakaka, Jouf, Saudi Arabia.

MATERIALS ANd METHOdS
A retrospective study, was conducted during April, 2015 to October 2016 at secondary care 150 bed hospital in rural area of Northern Provence of Saudi Arabia, covering the population of 2.7 million approximately. The total number of 1043 samples ( blood, wound, Urine and Sputum) were obtained from patients ( Medical and ICUs Wards) attending to the Prince, Muteb, Hospital Sakaka, Jouf, Saudi Arabia..

Bacterial isolation and identification
Samples ( blood, wound, sputum and urine) were streaked on blood agar , Mac Conkey agar , and chocolate agar and were incubated in aerobic condition at 35°C growth was observed after 24 hrs and isolates were identified based on morphological and biochemical characteristics using standard procedures of Clinical and Laboratory Standard Institute (CLSI) 15 .

Antimicrobial Susceptibility Test
The automated Vitek 2 Compac system was used to perform antimicrobial susceptibility tests as per clinical laboratory standards institute (CSLI) protocols 15 . The ESBL isolates were confirmed by the method described by bilal et al., 2018 29 and the following American type culture collection strains Klebseilla pneumoniae ATCC 700603, Proteus mirabilis ATCC BAA-856 and E. coli ATCC 25922 were used as controls.

Statistical analysis
The study data was analyzed with the aid of SPSS V.17 (statistical package for the social sciences) software.

disCussiON
In this study 115 ESBL producing Enterobacteriaceae were found among inpatients (Medical and ICUs wards) in Prince Muteb Hospital, Jouf, Saudi Arabia. Our results revealed ESBL producing E. coli and klebseilla pneumoniae is significantly higher than previous studies reported form Abha, Riayadh, Dammam and Al Khobar, Saudi Arabia [16][17][18] . The ESBL producing proteus mirabilisin our study subjects were found 36.8% which is higher as compared to other studies 18 .
In Europe, Turkey, Russia and Poland the prevalence of ESBLs was found 2-47% 19 , from the southern America (Brazil) 60% 20,21 . In Asia, ESBL producing Enterobacteriaceae (E. coli) 5% in Japan, 57% in China and Thailand were reported. In Pakisthan the prevelance was reported as highest (73%) 22 . In Middle East, Kuwait, and UAE the ESBLs were reported 25% and 36% respectively 23,24 . In our study 115/1043were ESBL producing Enterobacteriaceae (K. pneumoniae, E. coli and P.mirabilis). To our knowledge, this is the first study conducted in the northern region of Saudi Arabia to explore the ESBLs prevalence and its susceptibility pattern. The frequency of ESBLs in E. coli K. pneumoniae and P. mirabilisis 36.5%, 34.7% and 28.6% respectively. From Al Khobar, the E. coli and K. pneumoniae was reported 10.3% and 12.3% 25 . This variation may be due to differences in the number of isolates (either Tertiary care hospital or community based).
Further, in our study we did not find any strains resistance to cephamycins and carbapenems. The efficiency of these drug classes has been attributed to the stability of beta lactamases. Hence, cephamycins and carbapenems are the choice of drugs for treating the patients with infections caused by ESBL producing Enterobacteriaceae. The ESBL producing P. mirabilis, E. coli and K. pneumoniae, isolates in our study are resistance to all the generations (I-IV) of cephalosporins in hospitalized patients. Whereas, Menyfah et al., (2018) reported that, Aminoglycosides, Nitrofurantoin and sulpha drugs were 70% resistance to clinical isolates in our study. Whereas, in Norway, E. coli and K. pneumoniae showed high resistance to aminoglycoside due to the presence of modified enzymes AAC(6)-1b 26 . Higher rate of resistance against ceprofloxin, levofloxacin, ceftriaxone, cepfepem, Tigylcyline, Azetranoam and Ampicillin in community is probably due to over use, misuse or self-medications.
Previous studies has shown that, the antibiotic resistance against the Ceftriaxone, Cefuroxime, Ciprofloxacin, Ampicillin for ESBL producing pathogens 17,27,28 . However, our study also revealed that, the antibiotic resistance against Levofloxacin, Cefepime and Aztreonam. This ineffectivity against the Gram negative bacteria occur due to the mutations and spread of ESBLs with in the Enterobacteriaceae family and extensive use of these antibiotics for prophylaxis.

ACKNOWLEdGMENTS
We would like to express our heartfelt thanks to Mr. Shameem Khan for providing assistance on methods (detection of ESBL isolates). We would like to thank the microbiology department of Muteb Hospital for providing access to data files of the test reports.