Multi-Drug Resistance in Health Care-Associated Bacteremia in Intensive Care Units at King Fahad Specialized Hospital, Buraidah, Saudi Arabia

Health care-associated bacteremia cause important morbid ity and mortality and have a considerable impact on healthcare costs. Infections caused by extended spectrum beta-lactamase producing bacteria are of clin ical and epidemiological importance, since their mobile genetic elements facilitate cross-infection. Objectives: To analyze health care-associated bacteremia in ICUs of King Fahad Specialized Hospital and to assess ESBL production in the isolated Gram negative bacteria. Methods: This study included 519 patients. Their blood samples were co llected for blood culture. The isolates were identified and antibiotic sensitivity tests were performed. The type of B-lactamase gene was determined by polymerase chain reaction (PCR). Results: The rate of health care-associated bacteremia was 9.8%. Gram positive organisms were detected in 67.8 %; methithilin resistant Staph.aureus (MRSA) was the most prevalent (17.8 %). Gram negative bacilli were detected in 30.6 %. E.coli was the most common (12.9%). The production of extended spectrum β-lactamase (ESBL) enzyme was positive in 84.2% of the isolated Gram negative isolates. Temoniera (TEM) was the main type of β-lactamase. Conclusion: The isolation of multi-drug resistant bacteria and ESBL producing Gram negative organisms in ICU patients resulted in a greater awareness of implementation of new ru les for microbiological screening and infection control measures.


Introduction
Health care-associated infections (HAI) cause important morb idity and mortality and have a considerable impact on healthcare costs. Effect ive infection control programs, such as surveillance, can reduce the infect ion rate by up to 32% [1].
Health care-associated b lood-st ream in fect ions (BSI) create a serious health problem in hospitals all over the world. In addit ion, patients admitted to intensive care units (ICUs) carry an even higher risk of BSI than those admitted to other types of units (non -ICU) and data fro m the surveillance and contro l o f pathogens of ep idemio log ic importance in United States hospitals showed that 49.4% of all BSI o ccu rred in th e ICU [2]. HA I are frequen t ly associated with drug-resistant microorganis ms, including meth icillin resistant Staphylococcus aureus (M RSA) and extended spectrum β-lactamase (ESBL)-producing Gram negative bacteria, wh ich can pose considerable therapeutic problems [3].
Antimicrobial resistance is an increasing threat in hospitalized patients, and both mortality and morbid ity fro m infections are greater when caused by antimicrobialresistant bacteria [4]. It is also noted that there is an increase in the resistance among Gram negative bacilli to third generation cephalosporins which is caused by expression of ESBL enzy mes.
The aim of the present study is to identify health care-associated bacteremia in the ICUs and to describe the resistance patterns of the isolates especially those caused by ESBLs. The study also aimed to determine the types of β-lactamase genes responsible for resistance among K pneumoniae isolates by polymerase chain reaction because K pneumoniae bacteremia is a very important cause of morb idity and mo rtality in Gram negative bacteremia in most medically well-developed countries.

Study Design and Subjects
The present study was conducted over a period of 12 months fro m May 2011 to April 2012 on patients admitted to different ICUs at KFSH. The total number of patients included in the study was 519 patients. They were monitored daily by the attending physicians for subsequent development of health care-associated bacteremia.

Sample Collection and Processing
Blood samples for culture were collected before the start of antibiotic therapy. The samples were inoculated immed iately under co mp lete aseptic conditions into bottles containing 50ml brain heart infusion broth. The bottles were incubated aerobically at 37 ℃ fo r 7 days and were examined daily for evidence of bacterial gro wth. Subcultures were done on blood agar.

Identification of B acterial Isol ates
Isolated colonies were evaluated according to their morphology, motility, Gram staining characteristics, catalase oxidase, coagulase tests, PYR test and latex agglutination test for Streptococci identificat ion (Omega Diagnostica, Scotland, UK). Gram negative bacilli were further identified by API 20E system (Bo meriu x SA, Monlien Vercica and France). Wet mount preparations were used to determine Candida species which was identified by germ tube test.

Detection of Oxacillin Resistance
Detection of oxacillin resistance was made by using Oxacillin Resistant Screening Agar Base [8].

Anti microbi al Suscepti bility
Susceptibility was tested by modified Kirby-Bauer disc diffusion method using 0.5 McFarland turbidity of each isolate [9].

Detection of β-Lactamase Producti on
Beta-lactamase enzy me is detected among the Gram negative bacilli by the chro mogenic method (Nitrocefin test-Oxoid, UK).

Genotypic Detection and Determi nation of the Type of β-Lactamase
Isolated strains of Klebsiella pneumoniae were investigated to determine the probable type of β-lactamase enzy me. The isolates were tested for TEM, SHV, CTX-M-1 and TOHO-1 genes by using PCR. DNA was extracted fro m clinical isolates and controls according to the method described by [11]. PCR react ion was performed in a final volume o f 50µl containing 0.2 µM of each primer, 200µM of each dNTPs, 1x reaction buffer, 2.5 units of Taq polymerase and 5 µM of the temp late DNA. Klebsiella strains with known ESBL types: TEM , SHV, CTX-M-1 and TOHO-1were included as positive controls. A reagent blank containing all co mponents of the reaction mixture except template DNA (substituted with sterile distilled water) was included in PCR reaction as a negative control. The amp lification conditions were as follows: 94℃ for 5 min; then 35 cycles at 94℃ for 30 s, 58℃ for 30 s, and 72℃ for 2 min; and, finally, one cycle at 72 ℃ fo r 15 min. [12][13][14]. Primers sequences for are shown in table (1).

Gene
Primers sequences

Results
A total of 51 patients developed health care-associated BSI (9.8 %). The age of the patients ranged between 5 and 75 years with a mean age of 42.4 ± 16.7 years. Nu mber of males was 36 out of 51 (70.6%) The rate of bacteremia among different intensive care units (ICUs) is illustrated in table (2) which shows that the highest rate was in the chest ICU (19.5%) followed by the trauma care unit (12.5%) then the general ICU (9.9%).

Determination of the type of β-lactamase by PCR in K pneumoniae isolates
Types of β-lactamase results are shown in table (6). It can be noticed that TEM was the main type of β -lactamase, SHV was the second, follo wed by CTX-M 1, no TOHO-1 genes were detected in the tested isolates.

ESB L Pr oduc tion by Phenotypic Methods and Correlation wi th PCR
Out of 6 isolates of K pneumoniae strains, 5 strains (83.3%) could be identified as ESBL producers by PCR, 4 of them (66.7%) could be identified as ESBL producers by Co mbined Disk method and ESBL E-Test, the strain which was negative by phenotypic methods showed TEM enzy me by PCR.

Discussion
NBSI create a serious health problem in hospitals all over the world. They contribute to greater morbid ity and mortality rates, as well as to increasing length of hospital stay and health care cost [3]. In the p resent study, the rate of health care-associated BSI was 9.8%. This result was higher than those reported by [15] in Canada (0.69%).
The high rate of health care associated BSI in this study may be due to the high risk patients enrolled in the study and the presence of several risk factors associated with those patients.
In this study we tried to assess the risk factors associated with BSI. There was a h igher significant d ifference in patients with previous prescription of antib iotics who developed BSI (34/ 51, 66.7%) co mpared to 216/ 468 of patients without BSI (46.2%) (P = 0.02), there was also a higher significant difference in patients on mechanical ventilation who developed BSI (18/51, 35.3%) co mpared to 102/468 of patients without BSI (21.8%) (P = 0.03). There was also higher percentage of patients with urinary catheterization who developed BSI 20/51 (39.2%) compared to 129/468 of patients who had no BSI (27.6%), but with non-significant difference (P = 0.081), This is supported by the results of another study [2] who reported that risk factors for acquisition of BSI in the ICU included previous prescription of antibiotics, mechanical ventilation and the use of nasogastric tube.
Our results were in concordance with surveillance for nosocomial bloodstream infect ions at 49 hospitals over a 3-year period detected >10,000 infect ions. Gram positive organisms accounted for 64% of cases, Gram negative organisms accounted for 27%. The most co mmon organisms were coagulase negative staphylococci (32%), Staphylococcus aureus (16%) [16].
In our study, few isolates (9.1%) were found to be vancomycin resistant. This report paralleled what is reported by different studies which indicated that vancomycin intermed iate S. aureus strains are beginning to eme rge [4].
Kanamori et al [18] showed that 71.4% of the isolates were resistant to gentamycin, wh ile all of the ESBL-producing isolates were susceptible to imepenem. It has been reported that the prevalence of coresistance to CIP, GEN, and SXT was high among phenotypically identified ESBL-producing E. co li and K. pneumoniae in the Philippines [19]. Imipenem remains fully effective against ESBL producers and carbapenems may be the most appropriate agents for severe infections due to ESBL-producing Enterobacteriaceae [14], [17][18].
In the present study, a total of 63.2% (12/ 19) o f isolates were confirmed to be ESBL producers. These results are in concordance to [14] who found 73.2% ESBL phenotypic expression among Enterobacteria. Akujobi and Ewuru Chika et al [9] reported ESBL production in 11.4% of Klebsiella isolates. A study confirmed ESBL production in 17/ 91 (18.7%) of strains of Enterobacteriacae and among all of the 3 klebsiella species tested [18]. Another study reported ESBL production in 65/207 (31.4%) of Klebsiella, Enterobacter and Serratia strains with a predo minance of Klebsiella pneu moniae (29/65) [19]. The resistance phenomenon is on the increase, this increasing resistance is due to inappropriate usage of antimicrobial drugs such as overuse, misuse, suboptimal dosage and non-compliance with treat ment duration [9].
In the present study, the type of β-lactamase gene was determined among K pneumoniae strains by PCR in which TEM was the main type of β-lactamase, followed by SHV, then CTX-M 1. So me K pneumoniae strains produced more than one type of β-lactamase. Vasques et al [14] reported TEM in 13/24, CTX-M 1 in 7/ 24 and SHV in 6/24 of ESBL producing Enterobacteria and they also reported that Klebsiella and E co li predominated in the ESBL p roducing species identified. Predo minance of SHV in 13/ 14 of K pneumoniae strains follo wed by TEM and CTX-M (9/ 14 for each of them) was reported [17]. Several studies showed expression of more than one ESBL genes which is in concordance to the present study [14], [17], [20].

Conclusions
The isolation of mu lti-drug resistant bacteria and ESBL producing Gram negative organisms in ICU patients during the study period resulted in a greater awareness to the presence of mult i-drug resistance among bacterial isolates in our hospital ICU units. New rules for microb iological screening and infection control measures are recommended i.e. testing for beta-lactamase production and isolation precautions for patients in who m these microorganis ms are identified. In addition, restriction of the use of in Intensive Care Units at King Fahad Specialized Hospital, Buraidah, Saudi Arabia third-generation cephalosporins, along with care in the use and abuse of antimicrobials to min imize the emergence of resistant strains, are the most effective strategies for controlling and decreasing b lood stream in fections and the spread of ESBL-producing and multi-drug resistant organisms.