Imipenem-resistant Psedomonas aeruginosa : Epidemiology and susceptibility patterns at a Teaching Hospital in Riyadh , Saudi Arabia

Pseudomonas aeruginosa is an important nosocomial pathogen. It causes hig h morbidity and mortality. The incidence of imipenem-resistance is increasing amon g Gram negative bacilli, particularly P. aeruginosa, often associated with resistance to other antipseudomonal drugs. The aim of this study is to assess potentia l risk factors for nosocomial infections of imipenemresi tant P. aeruginosa (IRPA), and to obtain base line information on the epidemiology and susceptibility pattern of this important pathogen. In a retrospect ive study of patients with nosocomial P. aeruginosa infection from July 2008 to August 2010 in a terti ary care hospital in Riyadh, Saudi Arabia, the imipenem susceptibility o f 381 nosocomial P. aeruginosa isolates was evaluated by disk diffusion method and confirmed by determining minimum inhibitory concentration (MIC) using E test . Antimicrobial susceptibility testing for other anti biotics was performed. The features of the patients with IRPA infections were compared to those with imipenem-sen sitive P. aeruginosa (ISPA) infections. During the study period, IRPA was isolated from 60 (15.7%) of patien ts, and the ISPA was isolated from 321 (84.3%) pati ents. IRPA were most frequently isolated from urine (23.3 %) cultures (p=0.01), whereas ISPA were most freque ntly isolated from swab (57.6%) cultures (p=0.04). Risk factors that were significantly associated with IRP A included: surgery (p=0.001), diabetes mellitus (p=0.04), and trauma (p=0.01). Overall, the highest P. aeruginosa rate (33%) was obtained in the medical ward followed by ICU (2 9%), surgical ward (28.3%), oncology (5.5%), burn u nit (3%), and renal dialysis unit (2.4%). ICU patients accoun ted for 21(35%) of IRPA infected patients. All test ed antipseudomonal agents, demonstrated higher resistance rates among IRPA isolates than ISPA isolates (p=0.0 01). Pipracillin/tazobactam was the most active agent fo r both groups followed by amikacin and ceftazidime. No significant difference in the mortality rate betwee n the imipenem-resistant and susceptible groups. Is olates of P. aeruginosa that are resistant to all, or almost all, available antibiotics "panresistant" are now prevalent worldwide. Our study showed that imipenem-resistanc e in this clinical pathogen is an emerging problem. Periodical surveillance studies are therefore essen tial to determine the current susceptibility patter ns of P. aeruginosa to different antimicrobial agents, and to identify risk factors for acquisition of carbapenem-resista nt strains.

aeruginosa, due to their stability to ambler classes A, C and D ß -lactamases compared with other ß -lactams.However, over use of imipenem has been associated with the isolation of P. aeruginosa strains producing class B enzymes (IMP and VIM), called metallo-ß -lactamases that are able to hydrolyse all broad-spectrum -ß lactams except monobactams (Kokis et al., 2005), such resistance can be chromosomally encoded or plasmid mediated.
Other mechanisms of resistance to carbapenem include: decreased outer membrane permeability, increased efflux systems, alteration of penicillin-binding proteins, and the production of carbapenem hydrolyzing enzymes (carbapenemases).
The incidence of imipenem-resistance is increasing among P. aerugenosa isolates, associated with resistance to other antipseudomonal drugs (Pinar et al., 2008;Fridkin et al., 1999).Infections due to this virulent organism are difficult to treat because of limited choice of effective antimicrobial agents (Quinn, 1994;Carmeli et al., 1999).It causes high mortality among critically ill patients in the services of intensive care unit (ICU), oncology, hematology, and surgery and burn units (Fluit et al., 2000).
Of great relevance is the emergence of panresistant strains with complete or almost complete lack of treatment options.Those strains are increasingly reported from many tertiary care medical centers (David. 2006).However, resistance of P. aeruginosa to colistin or polymyxin B is worrisome, because those drugs may be the only treatment options for infection with panresistant strains (David. 2006).
There are few studies available in Saudi Arabia on the resistance of P. aeruginosa isolates (Babay, 2007;Al-Tawfiq, 2007;Al-Jasser and Elkhizzi, 2004), most of them were addressing only the susceptibility pattern with minimal data on epidemiology and risk factors.Thus, in this study, we investigated the susceptibility pattern, epidemiology and the risk factors for nosocomial infections of imipenem-resistant P. aeruginosa.

MATERIALS AND METHODS
This retrospective study was conducted in King Khalid University hospital in Riyadh, Saudi Arabia, which is a tertiary (800) bed teaching hospital, over a period of 26 months, from July 2008 to August 2010, in order to determine the epidemiology, risk factors and the susceptibility pattern of P. aeruginosa to different antibiotics.
The microbiology laboratory database was searched to identify the entire clinical cultures positive for P. aeruginosa during the study period.A total of (381) isolates from various types of inpatient specimens, such as sputum, wound, blood, urine, other specimens, and their susceptibility testing results were reviewed.Repeat isolates or isolates from out patients were excluded.The major site from which inpatient P. aeruginosa strains were isolated was swab (57%, n = 218), of which 58% were wound swabs (n = 126).The second largest source of samples was from the respiratory tract (28%, (n = 106), and these were predominantly sputum samples (84%, n = 89).Urine samples were the third major source of P. aeruginosa isolates (13%, n = 50).Blood samples only represented 1.8% (n = 7) of samples from which P. aeruginosa were isolated.

Microbiological examination
P. aeruginosa were identified by the following criteria: 1. colonial morphology; 2. a positive cytochrome oxidase reaction; 3. pyocyanin production on Muller-Hinton agar; 4. growth at 42°C on blood agar.Strains positive for all criteria were accepted as P. aeruginosa and were not identified further.API 20NE system (Biomerieux, France) was used in certain conditions.
Susceptibilities to 9 antimicrobial agents, which are primarily effective agains P. aeruginosa strains, were reviewed.The antibiotics included (amikacin, aztreonam, cefepime, ceftazidime, ciprofloxacin, gentamicin, imipenem, meropenem and piperacillin/tazobactam), and the susceptibilities to these antibiotics were determined using Microscan Neg Combo 1S panels (Microscan,Siemens,USA), which is an automated microdiluton system, that determines the minimum inhibitory concentration.Manufacturer's guidelines were followed carefully.
Kerby-Bauer disc diffusion method was used to determine the susceptibility pattern to imipenem (10 µg/disc) and meropenem (10 µg/disc) all discs were obtained from (Oxoid, England).In order to avoid false designation of resistance to imipenem, due to drug degradation during storage, isolates that tested resistant by the disk diffusion method were confirmed by determining the MIC using the E test (Biomerieux, France), for imipenem and meropenem.MIC in the E test method was defined as the lowest antibiotic concentration that inhibited the visible growth and it was read on the basis of the interception of the elliptical zone of growth inhibition with the graded E test strip.In both cases of disc diffusion method or the E test method, Muller-Hinton agar (Oxoid, USA) was used as the growth medium.It was inoculated with an inoculum of 0.5 McFarland standards and incubated aerobically at 37°C for 18 to 24 h.
All results of both methods were interpreted according to Clinical Laboratory Standards Institute (CLSI) guidelines.Intermediately susceptible isolates were considered resistant P. aeruginosa ATCC 27853 was used as a reference strain.Molecular typing was not done.

Collection of data
Data were extracted to individual forms for each patient.The form included age, sex, diagnosis, admission to ICU or other wards, comorbidity (cardiovascular disease, diabetes mellitus, renal impairment, malignancy), elective or emergency operation, pneumonia including ventilator associated pneumonia, shock, trauma, sepsis, antibiotics given to the patient, cultures and antimicrobial susceptibility test results and mortality.Information on exposure to antibiotics was not available for all patients.The patients were defined as IRSA cases if they had imipenem-resistant P. aeruginosa infections and as ISSA if they had imipenemsensitive P. aeruginosa infections.

Statistical analysis
Statistical analyses were performed using SPSS software (Statistical Package for Social Sciences version 17.0 for windows).Results are presented as frequencies (percentage) for categorical variables and mean (standard deviation) and/or median (range) for continuous variables.Chi-square test or Fisher's exact test was used to find the association between risk factors and the infection groups.
In all cases, p ≤ 0.05 was considered statistically significant.Mann-Whitney U-test was used to compare the nonnormally distributed variables between groups.

RESULTS
During the study period, a total of 381 non-repetitive P. aeruginosa isolates were detected.Of these 381 isolates, 60 (15.7%) were found to be resistant to imipenem.The

Risk factors
Table 1 lists the demographics and possible risk factors for acquiring imipenem-resistan P. aeruginosa infections (IRPA).On data analysis, prior surgery, trauma and diabetes mellitus were risk factors for acquiring imipenem-resistant P. aeruginosa (chi-square, fisher exact test p < 0.05).Prior use of cephalosporins (cefazoline, ceftazidime, ceftriaxone), carbapenems, ciprofloxacine and colistin was documented in 25% of patients infected with imipenem-resistant isolates.Analysis of isolates by ward type for both imipenem resistant and imipenem sensitive P. aeruginosa is shown in Table 2 and Figure 1.The highest P. aeruginosa rate (33%) was obtained in the medical ward followed by ICU (29%), surgical ward (28.3%), oncology (5.5%), burn unit (3%), and renal dialysis unit (2.4%).The highest proportion (35%) of IRPA isolates was found among ICU patients Figure 2.

Characteristics of infection
Overall the major site from which inpatients' P. aeruginosa strains were isolated was skin and soft tissue sites (n=211, 55.4%), of which 48% were wound swabs.The second largest source of samples was from the respiratory tract (sputum and tracheal aspirate) (n=103, 27.1%).Urine was the third major source of P. aeruginosa isolates (n=14, 3.7%).Blood samples  represented 1.8% (n = 7) of samples from which P. aeruginosa were isolated.

Susceptibility testing
When isolates from June 2008 to August 2010 that were analyzed by carbapenem susceptibility status were tested with seven other agents, all agents demonstrated higher resistance rates among carbapenem resistant isolates than among carbapenem susceptible isolates (p=0.001).The greatest observed difference was 43.2% higher rate of resistance to cefepime among carbapenem resistant isolates than among carbapenem susceptible isolates.Pipracillin/tazobactam was the most active agent for both groups followed by amikacin and ceftazidime Table 4.   Multidrug-resistant P. aeruginosa isolates, defined as being resistant to pipracillin/tazobactam, ceftazidime, carbapenem and gentamicin were found in 5 five strains.Two strains were isolated from patients admitted in surgical intensive care unit (urine, sputum).The other three strains were isolated from surgical (n=2) and medical (n=1) wards.Pan-drug-resistance, which was defined as resistance to all nine antimicrobial agents tested, was found in 3 three strains (5%).Among the 60 strains of P. aeruginosa resistant to carbapenem, 53% had MIC > 32 ug/ml Table 5.We found 100% correlation between disk diffusion and E-testing for all imipeneme resistant isolates.
In our study, the resistance rate to imipenem is relatively high and accounted for 15.7% of nosocomial P. aeruginosa isolates.A similar rate of resistance was reported from Turkey 15% (Lutfu et al., 2005).
In a recent report on antimicrobial resistance among clinical isolates from the Chinese meropenem surveillance study, the resistance rates of P. aeruginosa isolates to imipenem and meropenem were 70.5 and 76% respectively (Hui et al., 2010).
An important finding in this study is the association between diabetes mellitus and carbapenem resistance.The reason for this association is not clear, but it could be related to frequent foot infections and antibiotics exposure in such patients.Varaiya et al. (2008) has reported a high incidence (75%) of carbapeneme resistant P. aeruginosa from patients with type 2 diabetes mellitus.
Our study found that prior surgery was a risk factor for selection of multidrug resistant P. aeruginosa strains, probably due to prolonged use of prophylactic antibiotics.This finding is consistent with one study from Saudi Arabia, which showed that most multidrug resistant P. aeruginosa isolates were from surgical and diabetic patients (Babay, 2007).
ICUs are generally considered sources of antibiotic resistance and outbreaks of multi-resistant bacteria.Risk factors included excessive consumption of antibiotics exerting selective pressure on bacteria and frequent use of invasive devices (Ortega, 2004).Although not statistically significant, the proportion of imipenem resistant P. aeruginosa isolates (35%) among ICU patients is higher than sensitive strains (27.4%).This finding is comparable with studies carried out in Turkey which found that 43 to 83% of the antibiotic resistant strains of P. aeruginosa were isolated from ICU patients (Bryan et al., 2004;Inan et al., 2000;Yetkin et al., 2006).In a recent study by Sameera et al. (2010) carried out at a tertiary care hospital in Riyadh, Saudi Arabia over a 5year period, the most common bacteria isolated from the ICU were Acinetobacter spp.(886 isolates, 31.7%),followed by P. aeruginosa (855 isolates, 30.6%).Their results showed that P. aeruginosa susceptibility significantly declined for carbapenem from 66% in 2004 to 26% in 2009.
Cross resistance data among the 381 P. aeruginosa isolates revealed that, pipracillin/tazobactam, amikacin, and ceftazidime were the most active antimicrobial agents.The resistance rates for those antibiotics were lower than previous reports (Kokis et al., 2005) which demonstrated higher values (71 to 79%).In our study less than 50% of the carbapenem-resistant isolates were also resistant to amikacin (28.3%), ceftazidime (36.7%) and tazocin (18.3%).This finding indicates that these antibiotics may be good options for the treatment of infections due to carbapeneme-resistant P. aeruginosa.However, the use of piperacillin and other ß-lactams must be monitored, as these antibiotics induce selective pressure on ß -lactamase-producing strains, resulting in the development of resistance in the hospital environment (Onguru et al., 2008).
Compared with carbapenem-succeptable isolates, the carbapenem-resistant-isolates showed higher rate of resistance to ciprofloxacin.This result is comparable to other studies, which showed high rates (79%) of resistance to this antibiotic among IRPA isolates (Kokis et al., 2005;Pinar et al., 2008;Sanchez-Romero et al., 2003).The low percentages of susceptibility to fluroquinolones may reflect the high use of these antimicrobials in the hospital settings.
Our study has some limitations, first, discrimination between infections with P. aeruginosa and colonization could not be achieved for all patients.Second, we have not attempted to establish the relative contributions of other risk factors in the development of carbapenem resistance, in particular the contribution of exposure to carbapenem due to lack of information.Third, molecular typing was not done.
In conclusion, our results suggest that the IRPA infection is an emerging problem in our hospital.The nosocomial occurrence is related to prior surgery, diabetes mellitus and trauma.Periodical surveillance studies are essential to determine the current susceptibility of P. aeruginosa to different antimicrobial agents, and to identify risk factors for acquisition of carbapenem-resistant strains.

Figure 1 .
Figure 1.Location of patients with IRSA and ISSA Infection.

Figure 2 .
Figure 2. Location of patients with IRPA Infection.

Table 2 .
Location of patients with IRPA and ISPA infection.

Table 3 .
Site of infection of IRPA and ISPA.
*Some patients have more than one specimen.

Table 4 .
Antimicrobial resistance rate of imipenem-sensitive P.aeruginosa (ISPA) and imipenemresistant P.aeruginosa(IRPA) isolates to the other antibiotics.
. Isolates of P. aeruginosa that