Evaluating the resistance pattern of gram-negative bacteria during three years at the nephrology ward of a referral hospital in southwest of Iran

Background: Gram-negative bacteria are associated with an increase in rates of antibacterial resistance. In most low- and middle-income countries such as Iran, there is no continuous surveillance system for antibiotic resistance. Objective: The purpose of this survey was to determine the pattern of antimicrobial sensitivity of gram-negative bacteria within 3 consecutive years at a nephrology ward of Nemazee hospital in Shiraz. Materials and Methods: During a 3-year period from 2013 to 2015 at the adult nephrology ward, bacteriological data of all biological samples of hospitalized patients in favor of gram-negative microorganisms were analyzed retrospectively. Antimicrobial susceptibility was performed by the Kirby-Bauer disc diffusion method. Results: The most common gram negative bacterium isolated from biological samples was Escherichia coli (43.9%). The highest (86.3%-94.1%) antibacterial resistance rate was associated with Acinetobacter spp. The most frequent resistance was seen with cephalosporins. In contrast to ceftriaxone, ciprofloxacin, and trimethoprim/sulfamethoxazole, nitrofurantoin and aminoglycosides remained their acceptable activity against E. coli. At least three-fourths (75%) of Acinetobacter spp. isolates was resistant to either aminoglycosides or imipenem. All (100%) isolated Acinetobacter spp. and Pseudomonas aeruginosa species were susceptible to colistin. The rate of Acinetobacter spp. and P. aeruginosa resistant to three or more drugs was 81.7% and 74.6%, respectively. Conclusions: The resistant rate of gram negative pathogens to different tested antibacterial agents was considerably high and has increased during the recent three years in our center.

23 000 deaths each year in the United States (2). It has been also estimated that more than 70% of the bacteria that causes hospital acquired infections are resistant to at least one antibiotic (3). Infectious diseases are common cause of morbidity and considered as the second cause of mortality in chronic kidney disease patients (4). Sepsis-related death among hemodialysis patients is 100 times more than the general population (5). On the other hand, urinary, respiratory, and hemodialysis catheter-related bloodstream infections are common among patients with renal diseases (4). In line with gram-positive pathogens (mainly Staphylococcus aureus and Staphylococcus epidermidis), gram-negative bacteria are also associated with an increase in rates of antibacterial resistance in catheterrelated infections. Among gram negatives, extendedspectrum β-lactamase (ESBL), carbapenem-resistant, and multi-drug resistant Enterobacteriaceae specially pose a serious threat to patients in healthcare settings (6). Most low-and middle-income countries such as Iran lack national continuous surveillance systems for antibiotic resistance. Having awareness of antimicrobial resistance pattern can lead to selecting an optimized antimicrobial agent or regimen and consequently, minimizing duration of hospitalization, morbidity, mortality, and direct as well as indirect health care costs (7).

Objectives
The purpose of the current study was to determine the plausible changing pattern of antimicrobial sensitivity of gram negative bacteria within 3 consecutive years at a nephrology ward of a referral teaching hospital in Shiraz, Southwest of Iran.

Materials and Methods
A retrospective study was conducted on microbiological data of hospitalized patients between 2013 and 2015 at a 20-bed adult nephrology ward of Nemazee hospital affiliated to Shiraz University of Medical Sciences. Bacteriological data of all biological isolates including blood, urine, sputum, wound drainage, abscess, synovial, pleural, ascitic, tip of catheter, and cerebrospinal fluid taken either before as empirical therapy or within antibacterial treatment as definite therapy of both community and hospital-acquired infections sent to the laboratory of the hospital were collected. Except for hospitalization at the nephrology ward during the study period, no specific inclusionexclusion criteria were implemented for patient recruitment.

Ethical issues
1) The research followed the tenets of the Declaration of Helsinki; and 2). The study was approved by the Institutional Review Board (IRB) and the Medical Ethics Committee of the Nemazee hospital related to Shiraz University of Medical Sciences.

Statistical analysis
Continuous and categorical variables were expressed as mean ± standard deviation (SD) and percentage, respectively. Descriptive analyses were carried out by SPSS (Statistical Package for the Social Sciences) version 20 software.
The rates of imipenem-resistant Acinetobacter spp. in 2013, 2014, and 2015 were 75%, 100%, and 100%, respectively. Frequency of aminoglycosides (including amikacin and gentamicin) resistance Acinetobacter spp. increased from 93.6% in 2013 to 100% in 2015. More than one-fifth (21.1%) of Acinetobacter spp. isolates were susceptible to ciprofloxacin in 2013. However, all Acinetobacter spp. isolates became resistant to this agent in 2014 and 2015. Resistance to three or more drugs was observed in 81.7% of isolated Acinetobacter spp. during the study. In 2015, all (100%) isolates of Pseudomonas aeruginosa were sensitive to both colistin and imipenem. Frequency of P. aeruginosa resistant to aminoglycosides including amikacin and gentamicin decreased from 100% in 2013 to 40% in 2015. In contrast, the rate of ciprofloxacin-resistant P. aeruginosa increased from 25% in 2013 to 50% in 2015. The resistance of this pathogen to both trimethoprim/sulfamethoxazole and chloramphenicol was 100% within 3 years. During the study period, 74.6% of isolated P. aeruginosa were considered as MDR.
Nitrofurantoin is highly effective against E. coli with relatively low rates of resistance (<10%) in most geographic areas of the world (21). Two studies from Karaj and Ahvaz reported that the rates of E. coli susceptibility to nitrofurantoin were 90.8% (19) and 71.2% (22), respectively. Japoni et al survey on 200 E. coli isolates from 3 hospitals and 2 health centers affiliated to Shiraz University of Medical Sciences demonstrated the resistant rate of 25% to nitrofurantoin (23). E. coli strains recovered from urine samples of children with community-acquired urinary tract infections in Jahrom were susceptible to nitrofurantoin in 96.8% of cases (24). The rate of nitrofurantoin-resistant E. coli decreased considerably within 3 years in the current study. Although the rate of E. coli susceptibility to nitrofurantoin was relatively acceptable in our clinical setting, this agent seems not to be a favorable antibacterial in nephrology wards. This may be due to fact that most of admitted patients have impaired renal function and administering nitrofurantoin is not generally recommended in individuals with creatinine clearance less than 60 or 40 mL/min in spite of controversies and lack of enough clinical data in this regard (25).
The similar story appears to exist for aminoglycosides. The resistance rates of E. coli to amikacin and gentamicin in the present study were only 4%-11% and 10%-20%, respectively. In addition, the resistant rate of all our isolated gram-negative bacteria to amikacin was 27.3%-35% and to gentamicin was 42.9%-57.1%. In agreement with our data, Khalili et al implicated that only 21.3%-33.3% of gram-negative pathogens during a four-year period were resistant to amikacin at a referral infectious disease ward in Tehran (11). This ranged from 0% to 21.4% at a teaching hospital in Urmia (26). Poorabbas et al reported that with the exception of Acinetobacter spp., other gram-negative pathogens isolated from seven teaching hospitals in Iran exhibited susceptibility rate of at least 60% to amikacin (27). Investigations over a long time period in other countries such as the United States and Taiwan have also revealed that an increase in the rate of resistance to aminoglycosides has been somewhat milder compared to other antibacterial agents (28,29). Despite its relative favorable susceptibility pattern in our ward, nephrologists generally do not prefer aminoglycosides as drugs of choice for treatment of gram-negative infections (e.g., E. coli) due to the presence of an underlying kidney disease in admitted patients and also the nephrotoxic as well as ototoxic properties of these agents. Lack of routine therapeutic drug monitoring for aminoglycosides in our center can partially justify clinician concerns on this issue. Except for colistin, the resistant rate of Acinetobacter spp. to other studied antibacterials during the study period was considerably high. This is especially concerning for imipenem because carbapenems were previously considered as the most effective antibacterial agent for the treatment of Acinetobacter spp. infections. The frequency of resistance to imipenem was reported to be 13%, 8%, and 33.4% in Belgium (30), Polish (31), and Turkey (32) and P. aeruginosa isolates, respectively (37). This rate was reported to be 81.7% at 2 hospitals in Tehran (15) and 95% in Isfahan (38) within the ICU setting. These data highlight the concern over a real threat of untreatable Acinetobacter spp. infections. Inappropriate utilization pattern along with overuse of agents with activity against gram-negative bacteria especially carbapenems for the treatment of both community and hospital acquired infections, as shown in at least 2 studies from Sari (39) and Tehran (40), appear to be the main cause of high burden of Acinetobacter spp. resistant to carbapenems in our country. Interestingly, Sistanizad et al demonstrated that implementing a restriction policy through prescribing carbapenems only for infections caused by culture proven multidrug-resistant gram-negative bacteria only sensitive to these agents resulted in a significant increase in the sensitivity rate of P. aeruginosa to imipenem at ICU after 6 months (41). All isolated Acinetobacter spp. and P. aeruginosa species in our ward were susceptible to colistin. Low rates of colistin-resistant Acinetobacter spp. have been reported in our country. For example, 1% of Acinetobacter spp. isolates at Hamedan in 2011-2012 (42), 11.6% at Isfahan in 2011-2012, (38) and 12% at Tehran in 2009-2010 (43) were resistant to colistin. However, most recent studies from different parts of Iran including Tehran (44), Ahvaz (34), and Kermanshah (35) revealed that the susceptible rate of Acinetobacter spp. to colistin has been 100%. Similar findings have been reported from at least 2 surveys in Shiraz including Nemazee hospital in 2008-2009 (16) and 2013 (33). Therefore, this agent in combination with other appropriate antibiotics can be considered as the last antibacterial resort for treatment of infections caused by MDR gram-negative pathogens in our center. However, noting that routine use of colistin antibiogram for Enterobacteriaceae isolates has been initiated only in limited wards of Nemazee hospital from 2014. Thus, our data in this regard need validation with new, long enough surveys.

Conclusions
Our findings implicated that resistant rate of gramnegative pathogens to different antibacterial agents especially cephalosporins are considerably high and increased during the recent years. At least 50% of isolated gram-negative bacteria were resistant to imipenem. Among gram-negative isolates, Acinetobacter spp. is associated with the highest rate of resistance. Except for Acinetobacter spp., the resistant rate of all isolated gram-negative isolates to tested aminoglycosides appears to be relatively favorable.
Performing regular and periodic surveillance of antimicrobial resistance pattern by the comprehensive, multi-specialty infection control committee appear to have a key role in optimizing both the empirical and definite antibacterial treatment regimens and may also slow down the runaway train of antibacterial resistance at our clinical settings in Iran.

Limitations of the study
The present study suffers from 3 major limitations. First, the possible association of patients' antibiogram with their response to antimicrobial treatment in real clinical conditions could not be assessed due to the retrospective pattern of the study. Second, antimicrobial susceptibility test was performed by the classic disc diffusion method rather than more accurate methods such as microbroth dilution or E-test. Third, some antibacterial agents with defined activity against gram-negative bacteria including piperacillin/ tazobactam, cefepime, ofloxacin, levofloxacin, and gemifloxacin currently available in Iran were not tested by the hospital laboratory during the study period and their susceptibility rates remain unknown.