Detection of SHV type Extended-Spectrum B-lactamase and Risk Factors in Pseudomonas aeruginosa Clinical Isolates.

OBJECTIVE
Pseudomonas aeruginosa is one of the most important causes of nosocomial infections and can acquire resistant to many antimicrobials, including β-lactams. The aim of this study was to detect the prevalence of SHV type extended-spectrum beta-lactamase (ESBL), antimicrobial resistance patterns of the P. aeroginusa and risk factors in hospitalized patients in two teaching hospitals in Sanandaj, Iran.


METHODOLOGY
123 P. aeruginosa were isolated from various clinical specimens. All samples were prepared for double-disk synergy test on the isolates for detection of ESBL. SHV was confirmed by PCR method. Risk factors were evaluated for infection due to P. aeruginosa.


RESULTS
The incidence of multiple drug resistance (MDR) in P. aeroginusa isolates was 3.85%. The prevalence of ESBL-SHV gene was 10.57%. Days of hospitalization (OR=14.34 CI95% 2.87-25.8), ICU hospitalization (OR=3.4 CI95% 1.24- 9.29), presence of catheter (OR=3.63 CI 95% 1.34-9.84), use of antimicrobials within previous two weeks (OR=5.51 CI95% 1.85-16.43) and use of ventilator (OR=3.7557 CI95%1.29-9) were risk factors for Pseudomonas nosocomial infection SHV positive ESBL.


CONCLUSION
In this study Prevalence of ESBL, SHV gene and MDR in P. aeroginosa infection was lower than the prevalence reported from other studies in Iran and this indicated appropriate antimicrobial managements strategies and infection control. In addition, our research data indicate that risk factors such as use of ventilator, use of antimicrobials and ICU hospitalization can be effective in managing Pseudomonas infection.


INTRODUCTION
Pseudomonas aeruginosa is one of the most important pathogens causing nosocomial infections including pneumonia, urinary tract infections, and bacteremia; and in recent years has shown multidrug resistant to many antimicrobials, including b-lactams. 1 Nowadays antimicrobial resistance is a great problem for the treatment and management of infectious disease.Patients with drug resistance organisms are at risk of negative outcome, even mortality.Designing controlling program will require insights from a range of disciplines including epidemiology, molecular biology and evolutionary biology of resistance genes. 2 The most prevalent of antimicrobial resistance mechanism in bacterial strains are enzymatic destruction.Among these enzymes, extended spectrum beta-lactamase (ESBL) have been studied extensively.In P. aeruginosa, various classes of ESBLs (A, B and D) have been found.Five types of class A ESBLs (PER, VEB, GES and IBC, TEM and SHV) were recently reported. 3][6] Multiple drug resistance P. aeruginosa (MDR) are defined as isolates resistant to ceftazidime, cefepime, aztreonam, ciprofloxacin, piperacillin, and gentamicin. 7Evaluation of the risk factors association Multiple drug resistance P. aeruginosa (MDR) are defined as isolates ociated with the acquisition of MDR P. aeruginosa has been an area of active research for epidemiologists and physicians around the world. 8These factors may provide an im provement in treatment outcome.We survey this study to analysis the risk factors for acquisition of SHV enzyme in P. aeruginosa strains isolated from hospitalized patients at two teaching hospitals in Sanandaj, Iran.

Detection ESBL-SHV by polymerase chain reaction (PCR):
Polymerase chain reaction (PCR) was performed for all isolates identified as ESBL producers, showing resistance to b-lactams.DNA was extracted by the boiling method. 10Template DNA was prepared, a cell pellet from 1.5 ml of overnight culture was resuspended in 500 μl of TE (10 mM Tris, 1 mM EDTA, pH 8.0) after centrifugation and boiling for 10 min for PCR was used as supernatant.Primers and conditions of polymerase chain reaction used in this study were SHV-F 5´-GGGTTATTCTTATTTGTCGC-3.´, and SHV-R 5´-TTAGCGTTGCCAGTGCTC-3.´ PCR condition 94°C 5min, 35 cycles of, 94° C 1min, 58°C 1min, and 72°C 1min.Statistical analysis: Data were entered into a database using SPSS 11.5 for Windows.Differences between proportions were analyzed using the c2 test.All differences in which the probability of the null hypothesis was p < 0.05 were considered significant.

DISCUSSION
In our study 123 specimens P.aeroginosa were isolated; blood 9.7%, lung 30%, urine 46.34%, wound 7.32%, brain shunt 0.81%, swab 3.25% and the other 1.63% while the highest specimen was urine , that is similar to other findings reported at a referral center in Iran. 11n 2003 from the National Nosocomial Infections Surveillance System regarding intensive care unit (ICU) patients across the USA showed that, Pseudomonas spp.were responsible for 18.1% of pneumonias, 3.4% of bloodstream infections, 9.5% of surgical site infections and 16.3% of urinary tract infections.8 We surveyed risk factors in Pseudomonas nosocomial infection correlation with SHV positive ESBL that days of hospitalization (OR14.34CI95% 2.87-25.8),ICU hospitalization( OR 3.4CI95% 1.24-9.29),presence catheter(OR 3.63 CI 95% 1.34-9.84), use of antimicrobials within previous two weeks (OR5.51CI95% 1.85-16.43)and use of ventilator (OR3.57CI95% 1.29-9.83)were risk factor but there are no significant difference between gender, trauma, mean age and blood transfusion with pseudomonas infection. Silarly, to a research in France, there were significant differences between days of hospitalization 0.003, ICU hospitalization 0.014, and presence catheter 0.011with pseudomonas infection.12 As reported from previous studies use of invasive devices and urinary catheterization were significant 8,12 as a risk factor for resistance development.Recently, resistance to antimicrobial agents of P. aeruginosa has become a serious problem in clinic and outbreak of MDR phenotype is increasing among P. ae ruginosa in patients.13 In this study, the most of resistant in P. aeruginosa to ceftazidime and cefotaxim were (23.58 %), and (30.48%), respectively.These resistance rates are similar in some of studies 11 and lower than other reports from Iran.1,14,15 The outbreak of ESBL has previously been described in Iran but the rate of that was different from this study results.In this study frequency of ESBL-producing strains in pseudomonas was 17.89% that lower than to the rates of other studies in Iran 1,15-17 and higher than United Kingdom (3.7%).18 In the present study, reported prevalence of SHV gene 10.57%.In Turkey, prevalence SHV production by Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii and Pseudomonas aeruginosa was 21.87% has been reported 19 and in Iran was reported by shacheraghi 22% in P. aeruginosa 15 and 26% for klebsiella pneumonia by Nasehi 20 which may reflect these differences in infection control policies.The rates of these enzymes in our study are lower than previous reports.SHV in P. aeruginosa mostly are located in chromosome and can play a hidden reservoir for these enzymes.Furthermore, the isolates harboring SHV-type genes are scarcely reported.21 Multidrug Resistance (MDR) in Pseudomonas is different from throughout the world.In our study the rate of MDR isolate was 3.85% that lower than Turkey and Iran 11,22 and higher than Europe and some American countries.23

CONCLUSION
In this study prevalence of ESBL in pseudomonas infection has decreased in comparison with other studies in Iran.Blind therapy the P. aeruginosa infection without information from antimicrobial resistant may lead to increasing in risk factors, long hospitalization, persistence of infection and mortality rate.Appropriate infection control can prevent spread and outbreaks of ESBL-producing and MDR P. aeruginosa.

Table I :
Demographics of patients at both the hospitals.Table-II:Multiple drug resistance patterns of the P. aeroginusa isolates at both the hospitals for SHV gene positive and negative.

Table -
III: Risk factors for acquisition of SHV type's enzyme in P. aeruginosa strains isolated from 123 patients.