Bacterial profile and their antibiotic resistance patterns from blood culture in Intensive Care Unit patients at the University Of Gondar Comprehensive Specialized Hospital, Northwest Ethiopia. CURRENT STATUS: POSTED

Abstract Background: The burden of bloodstream infection and antibiotic resistance in intensive care unit (ICU) is high when compared with other settings. The data on the changing antibiotic resistance trends are important for infection control activities. There is no sufficient data in the intensive care units of this hospital. Objective: The aims of this study were to assess the magnitude and risk factors of bacterial pathogens and their antibiotic resistance patterns of blood culture isolates from ICU patients at the University of Gondar Comprehensive Specialized Hospital, Gondar, Ethiopia. Materials and Methods: A cross-sectional study was conducted from February to May 2018.Blood specimen were collected from 384 patients and inoculated on appropriate culture media. Identification of bacterial pathogens and antibiotic susceptibility tests were performed using bacteriological standard methods. Presence of Extended Spectrum Beta Lactamase (ESBL) enzymes was confirmed by combined disk diffusion method. Data were analyzed using SPSS version 20. Result: Of the total 384 study participants, 96(25%) were culture positive. Majority of bacteria isolates 67(69.8%) were Gram negative. The commonly isolated bacterial species were K.pneumoniae 18(18.8%), Coagulase negative Staphylococcus 13(13.5%), K.ozanae 10(10.4%), K.rhinose 8(8.3%) and E.coli, E.cloacae, Citrobacter species, S.aureus each accounts 7(7.3%). Gram negative isolates were found to be resistant to ampicillin 66(98%), amoxicillin/clavulanate 64(95%) and ceftriaxone 52(77.6%). However, amikacin 64(95.5%), meropenem 58(87%) and Ciprofloxacin 56(83.6%) were relatively effective drugs. Gram positive isolates were found to be resistant to Penicillin 28(96.5%), amoxicillin/clavulanate27 (93.1%) and erythromycin 26(90%). They were sensitive to ciprofloxacin 22(79%) and clindamycin 24(83%). Methicillin resistant S.aureus accounted 4(57.1%) and ESBL enzyme producing Gram negative isolates accounted 41(78.8%). Conclusion: Klebsiella species were the predominant bacterial isolates in ICU settings. Antibiotic resistance due to ESBL enzyme production is alarmingly high. This result might be a reflection of inappropriate use of antibiotics and poor infection prevention control practice in these settings.


Introduction
Intensive care units (ICUs) are the most lifesaving units in a hospital in looking after critically ill patients by highly specialized clinicians and care giving nurses( 1). Most condition which are treated at ICU are life threatening, severe injuries and illnesses, which require constant follow up and support from specialized equipment's and medications in order to ensure normal body functions ( 2).
The rate of infections in the ICUs is rising, mainly because of increasing use of invasive procedures and specialized devices used ( 3).Some contribute to the wide spread of drug resistance among the hospital acquired organisms includes; antibiotic overuse and misuse partly due to incorrect diagnosis, irrational antibiotic market combinations; and irregular consumption due to either wrong prescription or poor compliance are among the reasons( 4,

5).
Other risk factors, such as prolonged length of hospital stay, also seem to predispose patients to infection with antibiotic-resistant bacteria ( 6). Occurrence of antibiotic-resistant bacterial infection due to inadequate infection control practices, failure to identify the presence of antibiotic resistance are also important factors promoting the spread of antibiotic resistance ( 7,8).
Antibiotic resistance can be reduced by using antibiotics carefully based on guidelines of antimicrobial stewardship programs ( 9).The most frequently reported pathogens in ICU settings are Staphylococcus aureus, enterobacteriaceae, Pseudomonas species. Enterococci species, Candida species, coagulase-negative staphylococci (CoNS) and Acinetobacter species ( 10,11).
ICU-acquired infections have been associated with significant morbidity, mortality and rising healthcare costs in developing countries with limited resources ( 12,13).Globally, patients in the ICU have encountered an increasing emergence and spread of antibiotic-resistant pathogens. The overall incidence rate is 23.7 infections per 1000 ICU admission days. Nosocomial infections in ICU account for about 20% to 25% of all nosocomial infection ( [13][14][15]. Infection is a leading cause of death in the ICU as high as 60% and twice as much in those patients The mortality and morbidity rates of bloodstream infections (BSIs) in ICU are higher due to limited resources available associated with the quality of health care system in ICU. However, available data are lacking in developing countries like Ethiopia. Therefore, the overall aim of this study was to assess the magnitude of bacterial isolates, predominant associated risk factors and their resistance patterns from blood specimens in the ICU at the University of Gondar Comprehensive Specialized Hospital. Socio demographic data and sample collection Socio demographic data such as age, sex, educational background, occupation, residence, patient setting, reason for ICU admission, antibiotic taken, and devices used during admission were collected using structured questioner. Two bottles of blood samples were collected by trained nurse working in the ICU under the supervision of the investigator by taking all aseptic techniques. Ten ml, 5ml and 1ml of blood sample was collected from adults, pediatrics and neonates respectively which were then inoculated in to tryptic soya broth. Blood samples were collected twice from different sites ( 19).

Laboratory processing
After inoculation the blood samples were incubated immediately at 35-37 0 C aerobically. After 18 hour of incubation, Gram stain was done and subcultured on to 5% sheep blood agar, MacConkey agar and Chocolate agar. MacConkey agar was incubated at 35-37 o Cfor 18 to 24hours. Blood agar and Chocolate agar plates were incubated at 35-37 o C in 5% CO 2 for 18 to 24hours.If there is no growth on the Blood agar and chocolate agar; they were further incubated for 24 hours. Still if no growth Chocolate agar only was incubated for further 24 hours. The bottles were incubated for up to seven days under observation. If there is sign of growth sub cultured it: if not, sub cultured again at 3 rd and 6 th days of incubation.
Presence of ESBL for Enterobacteriaceae was suggested by screening resistance to a third generation cephalosporins such as cefotaxime (30μg), ceftriaxone (30μg) or ceftazidime (30μg) according to the CLSI screening criteria. The breakpoints indicative of suspicion for ESBL production was ≤22 mm for ceftazidime, ≤25 mm for ceftriaxone or ≤27 mm for cefotaxime. A combined disk method was used as a confirmatory phenotypic method for ESBL detection. ceftazidime (30 μg) and cefotaxime (30 μg) disks alone and their combinations with clavulanate (30 μg/10 μg) were used for phenotypic confirmations of ESBL presence. A ≥5 mm increase in zone diameters for either of the cephalosporin disks or their respective cephalosporin/clavulanate disks was interpreted as ESBL producer (   20).

Data analysis and interpretation
Data were entered, coded and cleaned using Epi-info version 7.0 statistical software and then transferred to SPSS software version 20 for further data processing and analysis. Text descriptions, tables, and figures were used to describe the relevant findings of the study. The crude odds ratios with a 95% confidence interval were estimated in the binary logistic regression analysis to assess the association between each independent variable and the outcome variable, and to select candidate variables for the multivariate logistic regression analysis. Thus, only those independent variables with a p -values of 0.20 or less in the bivariate logistic regression were included in the multivariate logistic regression to get the adjusted effect of each covariate. Adjusted Odds Ratio with a 95% confidence interval was estimated to assess the strength of the association. P-value ≤ 0.05 were considered as statistically significant.

Socio-demographic characteristics of study participants
From the total of 384 septicemia suspected ICU patients 205 (53.4%) of the study participants were males and 179 (46.6%) were females. The age of the study participants ranged from 1day-80 years with a median age of 21 days. Majority of the study participants were Neonates 207(53.9%) and rural dwellers, 267(69.5) ( Table 1).
Extended spectrum beta lactamase detection Among the 67 Gram negative isolates of blood culture, 52 (77.6%) were resistant to third generation cephalosporins (ceftriaxone, ceftazidime and cefotaxime). Based on the CLSI guideline, 43 isolates were checked for the confirmation of ESBL production by combined disk diffusion method. Providencia species, Serratia species, Citrobacter species and E.cloacae were excluded from ESBL testing.
Whereas residence, place of admission, prior antibiotic therapy and device used were not significantly associated with bacterial infection in ICU settings. Those neonates were 2.65 times more likely to develop bacterial infection than adult age group. Patients who are male were 2 times more likely to develop bacterial infection than females (Table 7).

Discussion
In the current study, the overall prevalence of bacterial isolates was 25% which is similar with other studies conducted in India (23% and 24.7%)( 21, 22). However it is higher than another study in India (11.8%), Pakistan (16%), Jimma  . However, our study is different from study reported from North India in which Gram negative to Gram positive rate was 39.3% and 53% respectively ( 22). This discrepancy may be due to the study participants were in the ICU admitted patients and quality of the specimen collection and processing methods.
According to the present study, the predominant and the commonest bacterial isolates was K.
pneunoniae (18.8% Majority (67.7%) of the bacterial isolates were identified from NICU which is in line with the finding in India (66.3%) but higher than the study conducted in Gondar(46.6%).The most prevalent isolate in this ICU was K.pneumoniae (15.4%) similar to a previous study conducted in Gondar ( 28,30). This may be due to the same study area after five years.
Among common isolates of Gram positive bacteria, S.aureus was found to be 7.3%.Of these 57.1% was MRSA which was higher than other research conducted in Saudi Arabia(43%) ( 31).Cefoxitin resistance CoNS has become the predominant pathogen. Cefoxitin resistance CoNS was also isolated in our findings (61.5%).
In this investigation ciprofloxacin were relatively sensitive (78%) to both Gram positive and Gram negative bacteria. In the current finding amikacin (95.5%) was a drug of choice for gram negative bacteria similar to in North India(90.8%)( 32 ).
In the current findings, the overall prevalence of confirmed ESBL producing bacteria was 39(90.6%).
This result is higher than the finding in Mali (61.8%), Addis Ababa (78.57%) and Jimma (38.4%) ( 33-35). The prevalence of ESBL production in E.coliand K.pneumonia was 7 1.4% and 89% respectively. The prevalence of ESBL producing K.pneumonia was higher from a report in Mali (71.4%) and E. coli lower than a report in Ethiopia (100%). Our study also was inconsistence with a report in Dhaka which was 81.8% of E.coli and 11.7% of Klebsiella species had ESBL activity(
Furthermore, the logistic regression result indicated that independent risk factor for bacterial isolation in this finding was age (AOR=2.65, 95%CI; 1.29-5.42, p=0.008), which is consistence with result from United States and Sex (AOR=2.0, 95%CI; 1.23-3.27, p=0.005), similarly results shown in Italy (6.0% vs. 8.9%, P = 0.001). Sex is significantly associated with severe sepsis in men than women( 36, 37). This may be due to a combination of differences in chronic disease burden, environmental and social factors, and genetic disposition causing differences in the host immune response to infection likely contribute to the observed differences( 38).

Limitations of the study
This study did not include other important pathogens responsible for sepsis such as anaerobic bacteria, fungus and viruses due to lack of laboratory facility. The study was also limited to conduct carbapenemase production among Gram negative isolates due to shortage of materials.

Conclusion
Gram-negative pathogens were the predominate isolates in bloodstream infections. Klebsiella species were the predominant bacterial isolates followed by CoNS identified in this study. The distribution of bacterial isolates was high in NICU followed by PICU and AICU. Ciprofloxacin and clindamycin were effective for Gram positive isolates. For Gram negative isolates, ciprofloxacin, amikacin and meropenem were effective. Extended spectrum beta lactamase production in gram negative isolates as well as methicillin resistant Staphylococcus species is alarmingly high. This leads to an increased patient hospital stay, cost and mortality. The current finding may be useful to formulate treatment guideline to avoid unnecessary use of broad spectrum antibiotics for the ICU patients and to promote researchers to conduct advanced studies on current challenges of antibiotic resistance. The data on the changing antibiotic resistance trends is also important for disease control activities in ICU settings.  N/A* = Patients who are <6-years-old, others** = Carpenter, Beiger.       ESBL*-Extended spectrum beta lactamase.