Prevalence and Antibiotic Susceptibility among Gram Negative Bacteria Isolated from Intensive Care Units at a Tertiary Care Hospital in Riyadh, Saudi Arabia

Antibiotic resistance is an essential issue mostly in the intensive care units (ICUs). The Goal of this study was to investigate the widespread of multidrug resistance (MDR) gram-negative bacterial pathogens isolated from ICUs at King Khalid University Hospital (KKUH), Riyadh, KSA, and their ability to produce ESBL and MBL enzymes. All organisms were isolated from different ICUs at (KKUH) between June to December 2016. Identification and antimicrobial susceptibility were committed according to the laboratory policy. The bacterial Isolates flagged as ESBL or MBL by Vitek 2 were confirmed using E-test method recommended by CLSI. 70 isolates from different body sites comprising 25 (35.7 %) were P. aeruginosa, 23 (32.9 % ) were K. pneumoniae, 16 (22.9%) were E. coli, and 6 (8.6 %) were A. baumannii. Among the 23 isolates K. pneumonia and 16 of E. coli, 19 (82.6%) and 16 (100%) were detected as ESBL (+) by double-disk diffusion method according to guidelines of CLSI. On the contrary, ESBL was not detected in any isolates of P. aeruginosa or in A. baumannii. All P. aeruginosa and A. baumannii isolates were carbapenem resistant. MBL was found in all P. aeruginosa, A. baumannii and 4 (17.4 %) of K. pneumonia where E. coli strains did not appear any MBL action. The essential resistance mechanisms in the evaluated strains were ESBL and MBL. Molecular testing is recommended to confirm the phenotypic results and to detect the resistant genes.


INTRODUCTION
The growth of MDR gram negative bacteria is an important healthcare challenge worldwide 1 . The infections by MDR gram negative pathogens often lead to enhanced mortality, prolonged hospital stays and unreasonable healthcare costs. In total, this evidence emphasize the significance for hospitals to start reacting in a proactive way rather than in a reactive one to withstand the increasing resistance rates 2 .
The increase at intervals percentages of antibiotic resistance can be a serious interest in every non-fermenting bacilli and isolates of the Enterobacteriaceae family. β-lactams are the base of medication for genuine contaminations. The Most effective of those are the carbapenems, which are recommended to be used for the treatment of infections brought about by strains of the emerging Enterobacteriaceae, principally Escherichia coli and Klebsiella pneumonia, and non-fermenters, Pseudomonas spp., and Acinetobacter spp 3 , which possess extendedspectrum-β-lactamase (ESBL).
β-lactamases are proteins made by specific bacteria that break down the β-lactam ring of β-lactam anti-infection agents (monobactams, penicillin's, cephalosporins, carbapenems, and monobactams), is one amongst the foremost necessary mechanisms of microbial resistance to β-lactam antibiotics 4 . The decompounded β-lactam drugs result in a dormant product once the ring is broken 5 .
One of the foremost crucial resistant mechanisms in Gram-negative bacteria against β-lactam anti-infection agents is iatrogenic by the creation of β-lactamase catalysts. β-lactamase proteins could be characterized into four gatherings which are: A, B, C and D. Predictable with this order, expansive range β-lactamases are sorted among gathering A. The Gram-negative microscopic organisms have brisk enlarged protection from expansive range β-lactam antimicrobial agents. In excess of 200 kinds of expanded range β-lactamases (ESBLs) are discovered around the world, most having a place with the Enterobacteriaceae family 6 .
Resistance in Gram-negative microscopic organisms is principally mediated through the yield of (ESBL), AmpC β -lactamases and Carbapenemases 7 .
MBLs producing Gram-negative bacteria is an emerging threat worldwide ascribed to their resistance to any or all β-lactams except aztreonam 10 . The genes of MBL are either transferable plasmids or a piece of the bacterial chromosome 11 .

Aim of the work
To detect the antimicrobial resistance pattern, ESBL and MBLase producers among strains of gram-negative bacteria were secluded from King Khalid University Hospital ICUs.

MATERIAL AND METHOD Bacterial isolates
This retrospective study was done at King Khalid University Hospital, Riyadh, Saudi Arabia. The study included 70 successive, noncopied clinical isolates of ESBL K. pneumoniae, E. coli, MDR P. aeruginosa and A. baumannii that were identified from June to December 2016. These isolates were from various specimen types including, blood, wound, sputum, tracheal aspirate, eye, and groin, which were routinely cultured in the bacteriology laboratory.

Phenotypic detection of ESBls and MBLs
All strains were examined for ESBL production by CLSI phenotypic corroborative test by double-disk diffusion method 12 . ESBL generation was affirmed by E-test ESBL strips (AB Biodisk, Solana, Sweden).
All strains were investigated for Metallobetalactamase production by utilizing the Etest MBL strip (AB Biodisk, Solana, Sweden), containing two-sided seven-dilution scope of imipenem(IP) and imipenem+ EDTA (IPI). The test was done with accordance to the manufacturer's directions MIC ratio of ≥8 for the two reagent sides, a phantom zone between IP/IP inhibitor and distortion of each ellipse was considered as a positive result 13 .

Statistical analysis
All data were stored in Microsoft Excel, Version 2016. Data management and statistical analyses were also performed in Excel. Descriptive statistics of the data and variables are shown in the form of frequencies and percentages.

ETHICAL STATEMENT
The Institutional Review Board, college of medicine, King Khalid University Hospital, Riyadh, Saudi Arabia, has granted permission to conduct this study.

Phenotypic detection of ESBLS and MBLS
Among the 23 isolates K. pneumonia and 16 of E. coli ,19 (82.6%) and 16 ( 100%) were ESBL producers, sequentially. As opposed to this outcome, ESBL was not detected in any strains of P. aeruginosa or in A. baumannii.
All P. aeruginosa and A. baumannii strains were carbapenem-resistant. MBL was detected in all the specimens of P. aeruginosa, A. baumannii and 4 (17.4%) of K. pneumonia where E coli specimens did not demonstrate any MBL action ( Table 2).

DISCUSSION
There is a remarkable increase of multidrug-resistant (MDR) gram-negative bacteria in hospitals and significantly in the intensive care unit (ICU) setting. The clinical outcomes of infections brought about by MDR pathogens as yet dubious 14 . The resistance pattern is most likely noted in ICUs due to the unlimited use of antibiotics in these units compared to the other hospital departments 15 and 70% of these infections were caused by GNB 16 .
In this study, most of bacterial pathogens were recovered from female patients. This may be due to higher antibiotic intake prior to hospitalization in women 17 . In contrast with Ibrahim's findings (2018), who stated that the majority of bacterial pathogens were recovered from male patients 18 . Moreover, most of the isolates were recovered from the respiratory system and this finding agrees with a previous surveillance studies in Riyadh indicated that the most of bacterial strains were from the respiratory tract 19,20 . Our findings indicated that respiratory diseases were the most widely recognized in our ICUs.
In the current study, the most commonly isolated organism was P. aeruginosa followed by K. pneumoniae, E. coli, and A. baumannii.
In a study of Altun et al., (2013), who studied 78 specimens isolated from ICUs, Altun et al., (2013) found that A. baumannii was the most normally isolated organism pursued by Klebsiella spp., E. coli, and P. aeruginosa 21 .
Here in our study, the isolates of K. pneumoniae demonstrated a high percentage of resistance to Ceftazidime (96%), followed by cefotaxime and cefuroxime (87%), ciprofloxacin (39%), amoxicillin/clavulanic and gentamicin (35 %). Ibrahim (2018) reported that more than 60% of K. pneumoniae strains were resistant to cephalosporins, trimethoprim/sulfamethoxazole, aztreonam, piperacillin, and piperacillin/ Results of the present study reported that E. coli specimens demonstrated a high percentage of resistance to Ceftazidime, cefotaxime, and cefuroxime (56%). Amer et al., (2017), stated that most of E. coli isolates exhibited ESBL activity, and resistance is significantly increased in all betalactams antibiotics especially ceftazidime (38% to 100%); while the previous surveillance study showed E. coli resistance to ceftazidime (24% to 54%) 23 .
In the present investigation, A . baumannii isolates showed a high percentage of resistance(83%) for imipenem and meropenem which is similar to the finding of Ibrahim (2018), who reported that high resistance rates for carbapenems such as imipenem (97.5%) and meropenem (96.2%) 18 . The high resistant rates pose a major concern in our ICU as carbapenems are the medication of decision for Acinetobacter infections. Our findings suggest the existence of carbapenemase-producing strains, which has also been reported in other studies in Saudi Arabia and Gulf Cooperation Council States 25,26 . Therefore, determining the carbapenem-resistance mechanisms could lead to improvements in the outcomes of infections brought about by these microscopic organisms 25 .
In the present investigation, ESBL was detected in 35 of 39 (90%) isolates (19 of K. pneumonia and 16 of E. coli). ESBL was distinguished by Vitek 2 compact system.
In the investigation of Altun et al. 21  In the current investigation, MBL was affirmed in all the bacterial specimens of P. aeruginosa and A. baumannii 31 of 35 (86%). P. aeruginosa displayed resistance to anti-infection agents as a consequence of a permeability barrier of the cell surface, multidrug efflux pumps, and yielding of β-lactamases (AmpC βlactamase, ESBLs, and MBLS) 28 . MBL generation in P. aeruginosa in the current investigation (71%) is comparable with Altun et al. 21 (57% ) and Tankhiwale (16.87%) 29 .
C a r b a p e n e m a s e -g e n e r a t i o n Acinetobacter strains are rising worldwide due to frequent carbapene mutilization and the choice of resistant bacteria under antibiotic pressure [30][31][32] .
In our study, it was found that 6 of 6 (100%) A. baumannii strains were MBL producers by E test. Where Altun et al. (2013) stated that 31 of 44 (70%) A. baumannii strains were detected as MBL producers 21 .

CONCLUSION
To sum up, Gram-negative bacterial resistance is still a major issue in KKUH ICUs, Riyadh. ESBL and MBL were the fundamental resistance patterns of the strains assessed in this investigation. The most commonly secluded organisms were P. aeruginosa pursued by K. pneumonia, E. coli, and A. baumannii. ESBL was confirmed in 35 of 39 (90%) isolates (19 of K. pneumonia and 16 of E. coli. And MBL was affirmed in all the specimens of P. aeruginosa and A .baumannii 31 of 35 (86%). Imipenem, Meropenem, and Amikacin were the most effective agents for K. pneumonia and E. coli. Colistin was the drug of choice for most cases of A. baumannii. P. aeruginosa, Amikacin and Colistin were the most effective agents.