Multi-drug resistance and nosocomial infections of Acinetobacter baumannii and Pseudomonas aeruginosa among patients hospitalized at Felegehiwot Referral Hospital, Northwest Ethiopia: A cross-sectional study CURRENT STATUS: UNDER REVISION

Background:Multi-drug aeruginosa are major causes of nosocomial infections globally. They are the current World Health Organization critical priority pathogens for resistance and discovery of new antibiotics. However, there is paucity of data on nosocomial infections caused by such superbugs in Ethiopia.Therfore, this study determined the magnitude and profile of nosocomial MDR Acinetobacter baumannii and Pseudomonas aeruginosa infections among patients hospitalized at Felegehiwot referral hospital, Northwest Ethiopia. Methods:A cross-sectional study was conducted at Felegehiwot referral hospital from April 1 to July 31, 2018.A total of 238 patients presumptive for blood stream, urinary tract and surgical site nosocomial infections were enrolled using convenient sampling technique.Either blood, urine and wound swab specimens were collected and processed using standard bacteriological procedures.A.baumannii and P.aeruginosa isolates were identified using standard bacteriological techniques and confirmed by automated Vitek2 Compact.Antimicrobial susceptibility testing on isolates was performed using the disk diffusion technique.The results interpreted as per the standard zone sizes of Clinical and Laboratory Standards Institute.Chi-square test was done to determine associations among variables.P.value< 0.05 was considered statistical significant. Results:The median age of participants was 29 years.Overall, 20(8.4%) of patients had nosocomial MDR A.baumannii and P.aeruginosa infections.The proportion of nosocomial MDR blood stream, urinary tract and surgical site infections were 13(8.9%),5(8.3%) and 2(6.3%), respectively. The mean age of patients with nosocomial infection was significantly lower (24.9 years) than their counter parts (29.6years) (P=0.035). All isolates of nosocomial infections were from patients with intravenous catheterization.The frequency of nosocomial MDR A.baumannii infection was 9(3.8%) and

Conclusions:Health care associated MDR A.baumannii and P.aeruginosa infections are critical problems in the study area.Therefore,urgent focused interventions required to contain the spreading of MDR NIs.Treatment of NIs for patients on health care should be guided by antimicrobial susceptibility testing.

Background
Nosocomial infection (NI) is an infection occurring in a patient at the time of care in a hospital that was not manifest or incubating during admission but developed after 48 hours of hospitalization [1]. The hospital environment contains a large number of immunocompromised individual's and patients with diverse bacterial pathogens and normal flora [2,3]. NI accounted 7% -10% prevalence in the world [3]. According to 2014 World Health Organization (WHO) report, 15% of all the hospitalized patients suffered from NIs [4].
Surgical-site, blood stream and urinary tract infections are the most frequently reported types of NIs [2,5].
Nosocomial infections due to multi-drug resistant (MDR) bacteria are the major issue to global health. It is reported as the leading causes of NIs in the world [2,6,7]. NIs with MDR organisms are very difficult for treatment and are main causes of morbidity, mortality, prolonged hospitalization and high health care costs [8,9]. The situation is true and urgent in Ethiopia.
Multi-drug resistant A. baumannii and P. aeruginosa survive in the hospital environment, transmitted easily between patients through the hands of health care workers in the health care setting [6,11]. Earlier findings elsewhere in the world reported that A. baumannii and P. aeruginosa commonly possess inherent resistance to antimicrobial agents through reduced permeability of the outer membrane, efflux pump systems, enzymatic inactivation and biofilm formation [12,13]. Thus, A. baumannii and P.
Nosocomial isolates of MDR A. baumannii and P. aeruginosa complicated the treatment of infections and had adverse effect on clinical outcomes and increases patient treatment costs [15]. Factors such as antimicrobial drug overuse, prescription of drugs without susceptibility testing, self-medication and long duration of hospitalization are reported to the occurrence of MDR [8,16]. However, there is a scarcity of data on the burden of nosocomial MDR and A. baumannii and P. aeruginosa infections in Ethiopia in general and study area in particular. Unavailability of local antibiogram data linked with self-drug prescription by patients and poor awareness on antimicrobial resistance are also big issues. Thus, the study aimed at determining the proportion of nosocomial MDR

Sample size and sampling
The sample size for NIs was determined using Epi info version 3.5.1 (public domain software, www.cdc.gov) by considering 95% confidence level and marginal error (5%). A proportion of 0.83 MDR NIs taken from previous study in other parts of South East Ethiopia [16]. Thus, the total sample size was 238. All patients who were clinically presumptive for nosocomial blood stream, urinary tract and post-operative surgical site nosocomial infections were included conveniently until the required sample size was achieved.

Variables
Nosocomial MDR A.baumannii and P. aeruginosa infections were the dependent variables while demographic variables (age, sex, residence, educational status, occupation) and clinical data on co-morbidity, urinary catheterization, intravenous catheterization, duration of catheterization, previous history of antibiotics, previous history of surgery, duration of hospitalization, wards of patients hospital and duration of operation were the independent variables.

Data collection
Information on demographic variables was collected from each participant by face-to-face interview using a structured questionnaire. Clinical data related to co-morbidities, hospitalization, surgery and use of antibiotics were collected by reviewing patient's medical record and in consultation with the respective physician. Clinical specimens (blood, urine and wound swab) were collected as soon as nosocomial infection was reported following the bacteriological standard procedures (Rods, 2014).

Wound swab collection and processing
Two wound samples from each participant were collected aseptically by sterile cotton swabs dipped in normal saline using Levine method [17]. After, the wound was cleaned with normal saline moistened sterile gauze, the swab taken by rotating the sterile cotton tipped applicator in a 1cm² area of cleaned wound for five seconds without contaminating with skin commensals. Specimens were transported to FHRH microbiology laboratory within 20 minutes of collection. The wound swabs were inoculated on MacConkey agar (MAC) and Blood agar (BA) (Oxoid, UK) at a time. Both plates were incubated at 37 °C and examined for visible bacterial growth after 48 hours of incubation.

Blood sample collection and processing
As per the standard protocol, 10, 2 and 1ml of venous blood, respectively were collected from adult, children and neonates presumptive for blood stream infection [18]. Following cleaning of the site of blood collection with 70% alcohol and 2% tincture iodine, two blood samples were collected from two different sites of peripheral vein of each febrile patient using two bottles of blood sample within 30 minutes difference. The collected blood samples were inoculated directly to 5 -10ml Tryptic Soya broth blood culture medium bottle (Oxoid, UK) and transported to FHRH microbiology laboratory. All blood culture broths were incubated aerobically at 37 °C and regular subcultures were done after 1, 2 and then 3 days later daily up to 7 days of incubation. Subcultures were made on BA and MAC (Oxoid, UK). All of the inoculated agar plates were incubated at 37ºc. Finally, plates were examined for bacterial growth after 24 hours.

Urine sample collection and processing
Clean-catch mid-stream urine was collected from catheterized and non-catheterized patients presumptive for urinary tract infection. From catheterized patients, 5 milliliters (ml) of catheterized urine transferred to a sterile container after cleansing the out let of catheter. For non-catheterized patients, the same amount of urine sample was collected by the patient using sterile screw-capped and wide-mouth container after careful instruction. Then, the urine was delivered to FHRH Microbiology Laboratory within 20 minutes. Urine samples were inoculated on MAC and BA (Oxoid, UK) using calibrated wire loop that can deliver 0.001 ml of urine. All agar plates were incubated aerobically at 37 °C for 24 hours and observed for bacterial growth. Blood agar colonies were counted using colony counter and checked for significant bacteriuria. Culture from catheterized and noncatheterized patients that grew ≥ 10² CFU/ml and 10 5 CFU/ ml, respectively was taken as a significant bacteriuria, respectively. For heterogeneous colonies, sub-culturing of individual distinct colonies was performed to ensure pure cultures.

Identification of bacterial isolates
Preliminary identification of bacteria was done based on gram reaction, colony morphology and pigment formation. A series of enzymatic tests (catalase and oxidase), carbohydrate fermentation test (triple sugar iron (TSI), protein tests (Lysine decarboxylase (LDC) and indole), and motility tests were performed on colonies of pure culture for identification of A. baumannii and P.aeruginosa isolates [18]. Non-fermentative grape-like colonies with blue-green pigment production on culture media, motile, oxidase and catalase positive, LDC and indole negative isolates were considered as P.aeruginosa.
On the other hand, non-motile, gram negative short rods, oxidase, indole and LDC negative, catalase positive and non-glucose fermenter isolates were considered as A.baumanii.All A.baumannii and P.aeruginosa suspectedisolates were also further confirmed by an automated Vitek2 Compact (BioMérieux, France).

Quality control
The prepared questionnaire was checked for its completeness and validity prior to the data collection. All the standard operating procedures (SOPs) were strictly followed at all stages of microbiological analysis. Reference strains of P.aeruginosa ATCC27853 and E.
coli ATCC 25922 were used for quality control of antimicrobial susceptibility testing. MDR A.baumanii and P.aeruginosa isolates were confirmed by using Vitek2 compact. A standardized bacteriological procedure was followed to maintain correct laboratory results. At regular intervals and whenever a new batch of strain or reagent is prepared, standard strains of P.aeruginosa ATCC27853 and E. coli ATCC 25922 were used as positive controls. The sterility of the media was checked by incubating the media overnight before its use. The data were checked for completeness and representativeness prior to entry.

Data analysis
Data were checked, entered and analyzed using Statistical Package for Social Science 23 (IBM Corp Released 2011.IBM SPSS statistics. Armonk, NY: IBM Corp). Descriptive statistics were used to describe relevant variables. Chi-square test, Fishers exact test and Independent samples T Test was obtained to determine association between dependent and independent variables. P-value of < 0.05 was considered statistical significant.

Demographic characteristics
A total of 238 patients with clinical evidence of nosocomial infection (BSI, UTI and SSI) were enrolled in the study. Of them, 129 (54.2%) were males. The majority (21.4%) of participants were found in the age group of > 51 years with median age of 29 years. One hundred twenty six (52.9%) of the study participants were from urban settings. Data on occupation showed that majority (39.1%) of participants were government employee.
Table1 depicts the demographic characteristics of the study participants.
Participants with NIs had lower mean of age (24.9 years) than those without NIs (29.6 years) and the difference was statistical significant (P = 0.035). Highest (15.4%) NIs rate was found in age groups < 10years. The proportion of NI was 13 (11.4%) in those participant with co-morbidity. All isolates of NIs were from patients with intravenous catheterization ( Table 3). Duration of operation was significantly higher in those patients with confirmed NIs (180 minutes) than their counterparts (155 minutes) (P = 0.04).
Moreover, the duration of catheterization was higher in those with confirmed NIs (13.6 days) than their counter parts (11.3 days). However, the difference was not statistical significant (P = 0.25) ( Table 3).
Multi-drug resistance profiles of A. baumannii and P. aeruginosaisolates A. baumannii and P. aeruginosa isolates were resistant to three to six antibiotics from different classes. All isolates of A. baumannii (9) and P.aeruginosa (11) were MDR (100%). Among this, 3 (33.3%) isolates of A. baumannii and 4 (36.4%) isolates of P. aeruginosa showed resistance to antibiotics from six different classes, respectively. Three (33.3%) isolates of A.baumanii and 1 (9.1%) isolates of P.aeruginosa were resistant against antibiotics from 4 different classes, respectively. Moreover, 3 (33.3%) of isolates of A.baumanii and 6 (54.5%) of P.aeruginosa isolates were resistant against antibiotics from three different classes (Table 4).

Discussion
Antibiotic resistant nosocomial infections are becoming serious health care problem in ICU and other areas of hospital care, leading to high rate of morbidity and mortality [6,21].
The epidemiological and antimicrobial resistance profiles of NIs showed variations among hospitals around the globe. Many of the infections are caused by bacteria that are resistant to multiple antibiotics [21,22]. This study showed the proportion of NIs due to two MDR non-fermentative gram negative bacilli among patients hospitalized in different wards of a referral hospital.
In the present study, 8.4% of patients were infected with nosocomial MDR A.baumanii and P.aeruginosa. This indicated that MDR A.baumanii and P.aeruginosa infections are the major health problem in the clinical area in Ethiopia. High patient load, overcrowding, poor infrastructure, poor infection control practices of the hospital and differences in trained medical staff for aseptic procedures might be the possible explanations. This finding was coherent with reports in Tikur Anbessa Hospital, Ethiopia (8.12%) [22], Uganda (7.39%) [23], Morocco (7 -8%) [24], Italy (9.3%) [25] and Gaza city (6.9%) [26]. However, it was higher compared to reports from Hiwot Fana Hospital, Ethiopia (0.5%) [27], Gabon (5.7%) [28], China (0.78%) [29] and Indonesia (3.5%) [30]. In contrast, the overall nosocomial MDR A. baumannii and P.aeruginosa infections in the present study was lower than studies done in Nigeria (12.5%) [31] and Ghana (23.5%) [32]. This might be due to variation in sample size, clinical site of infection, age of patients, hospital setting, duration of hospitalization, patients exposure to high risk devices or surgical procedures, microbiological methods employed for detection and screening of MDR resistant strains.
In this study all isolates of MDR P.aeruginosa were resistant for ampicillin and amoxacillinclavulanic acid. This was consistent with reports from Tikur Anbessa hospital, Ethiopia (22) and Southeast China [33] where 87.5% and 100% resistance levels against ampecillin and amoxacillin-clavulanic acid, respectively were noticed. In this study, all isolates of P. aeruginosa revealed 100% resistance against piperacillin. This was significantly higher than studies from Italy (25%) [25], Vietnam (17.7%) [43], Southeast China (12%) [33], Turkey (28.7%) [44] and Taiwan (66.8%) [45].This might be associated with differences in the number of MDR strains of P.aeruginosa and patient type. The frustrating level of resistance against piperacillin antibiotic is an alarm for treatment to be guided with antimicrobial susceptibility testing hence so far, piperacillin was not prescribed in the study area (FHRH).
The highest level of resistance against third generation cephalosporins might be linked with excessive use, mis and inappropriate use of these antibiotics in the study hospital that drives selective pressure and emergence of MDR.
In the present study, P.aeruginosa isolates showed 45.5% levels of resistance against meropenem. This was coherent with studies in Sodo, Ethiopia (41.7%) [15], Southeast China (36.6%) [33], India (54%) [42] and Vietnam (40%) [43]. However, higher level of resistance against meropenem was documented in Taiwan (73.2%) [45], Pakistan (86.4%) [35] and Saudi (81.8%) [46].The relatively lower proportion resistance against meropenem in the present study might be due to the absence of meropenem prescription practice for patients in the study hospital. In contrast, lower level of resistance against meropenem was reported in Uganda (14%) [23] and Turkey (20.4%) [44].This could be due to variation in the availability of meropenem in each localities, prescription difference, misuse and inappropriate use of antibiotics.
In this study, all isolates of MDR A. baumannii were resistance against ampicillin. This was parallel with studies conducted in Tikur Anbessa Hospital (88.2%), Ethiopia [22] and Southeast China (100%) [33]. Moreover, high level (88.9%) of MDR A. baumannii isolates resistance to amoxacillin clavulanic acid in the present study was comparable with earlier studies in Nigeria [47] and Southeast China [33], where all isolates of A. baumannii were resistant against amoxacillin-clavulanic acid.
In the present study, 33.3% of A.baumanii isolates showed resistance against meropenem.
The high MDR proportion observed in two non-fermenter gram negative bacilli in this study is probably related to the contaminations and cross transmission of this bacteria from hospital environment [15], hands of healthcare workers, frequent use of broad spectrum antibiotics, inherent resistance nature to many antimicrobial agents and the ability of pathogens persist in the environment, and on medical devices for a long period of time [6]. Moreover, intrinsic nature of the bacteria, high prescription practice of common antibiotics and third generation cephalosporins and use of drugs outside the hospital might contribute for the high resistance rate of A. baumannii and P. aeruginosa different classes of antibiotics.
The highest proportion of nosocomial infection due to MDR non-fermentative gram negative bacilli infection among the lower age groups in the present study is consistent with earlier studies elsewhere [28,50,51]. On the other hand, in the present study all NIs observed among patients with intravenous catheterization. The rate of nosocomial infections was also significantly higher among patients who had prolonged time of operation than their counter parts. This was consistent with previous study in Tikur Anbessa Hospital, Ethiopia (22) and India [42]. This might be due to the high rate of exposure of patients to the two MDR pathogens from the hospital environment, health care professionals, multiple invasive device and cross-contamination among patient's procedures.
This study was limited to participants admitted in hospital but nosocomial infection that arose after discharge was not detected.

Conclusions
Alarming proportion of nosocomial MDR A. baumannii and P. aeruginosa infection obtained in the study area. All isolates of the non-fermentative gram negative bacilli were MDR for atleast three antibiotics from different classes. Therefore, urgent intervention towards nosocomial infection prevention practices required. Moreover, treatment of patients on care should be guided with antimicrobial susceptibility testing.

Declarations
Ethics approval and consent to participate Ethical clearance was secured from Institutional Review Board (IRB) of College of Medicine and Health Sciences, Bahir Dar University. Permission letter was obtained from the Amhara Public Health Institute and FHRH prior to data collection. All the study participants were informed about the purpose of the study. Written informed consent was obtained from each study participants and guardian for participants under 16 years old before clinical examination and sample collection. Information obtained in the course of the study was kept confidential. Participants who were positive for the pathogen reported to physicians for treatment and any other care.

Consent for Publication
Not applicable

Availability of data and materials
The finding of this study is generated from the data collected and analyzed based on the stated methods and materials. All the data are already found in the manuscript and there are no supplementary flies. The original data supporting this finding will be available at any time upon request

Competing interests
The authors declared that no competing interest exists.

Funding
The research project was financed by College of

Medicine and Health Sciences of Bahir Dar
University. The fund was utilized for purchasing reagents, data collection, analysis and interpretation Authors' Contributions HM: Conceptualized and designed the study, performed the laboratory investigation.
Collected the data, analyzed the data and drafted the manuscript. WM: Conceptualized and designed the study, supervised the laboratory investigation and data collection, analyzed the data, wrote, revised and critically edited the manuscript. FB: Conceptualized the study, supervised the data collection and revised the manuscript. All authors have read and approved the manuscript, and ensure that this is the case. Key: Unemployed: Below school age + students; Other work: daily laborer and merchant