Prevalence of Multidrug-Resistant Pseudomonas aeruginosa in Patients with Nosocomial Infections at a University Hospital in Egypt , with Special Reference to Typing Methods

Multidrug resistant (MDR) P.aeruginosa had emerged in Egypt and seen mainly in nosocomial infections due to the selective pressure by overusage of antibiotics. This study was planned to delineate prevalence of MDR P.aeruginosa in nosocomial infection patients, and to screen for ESβLs producing P.aeruginosa with typing of P.aeruginosa isolates in Menofia University Hospitals (MUH), Egypt. Our study included 287 inpatients admitted to Menoufia University Hospital and having different nosocomial infections. Samples from medical staff and from hospital environment were collected. Antibiotyping of P. aeruginosa isolates were determined. MDR and ESBLs P. aeruginosa were detected. Plasmid DNA analysis and pyocin typing were done. In our study, a total of 57 P. aeruginosa strains were isolated. P. aeruginosa and its MDR phenoype accounted for 19 % and 9.5% respectively of nosocomial infections. MDR and ESβLs producer P. aeruginosa infections were commonly isolated from burn wound specimens followed by urinary tract, respiratory tract and wound infections. We detected high rates of MDR P. aeruginosa (52%) and of ESβLs producer strains (45.6%) and those ESBLs strains were all MDR. Amikacin and imipenem were the most effective drugs against P. aeruginosa. 23 different resistance patterns were identified, profiles from (1 8) were prevalent. The most prevalent antibiotype (2) included 12 MDR isolates, 9 clinical and 3 environmental isolates having same patterns. 61.5% of ESβLs isolates harbor plasmids. Five groups could be demonstrated among our P. aeruginosa isolates. Each had the same antibiotype and plasmid profile. In conclusions, our results clarified that threats of MDR and ESBLs P. aeruginosa become of major concern in our hospital and implementation of infection control strategies are major concerns to avoid the spread of this threat. Eenvironmental sources may have a significant role in spread of MDR P. aeruginosa among hospitalized patients. Combination of Journal of Virology & Microbiology 2 simple typing methods as antibiotyping with plasmid profiling may be the cheapest and effective method for tracing source of infection.


Introduction
P. aeruginosa is a non-fermentative Gramnegative bacteria widely distributed in nature and can survive on a wide variety of surfaces and in hospital environment, as the wards encourage bacterial growth (Arora et al., 2011).P.aeruginosa is responsible for about 10% -20% of nosocomial infections as bacteraemia and sepsis in ICU, cystic fibrosis, pneumonia, urinary tract infections, burn infection and wound infection (Carmeli et al., 1999).Multidrug-resistant (MDR) P. aeruginosa phenotype is defined as resistant to one anti-microbial agent in three or more anti-pseudomonal anti-microbial classes (carbapenems, fluoroquinolones, penicillins /cephalosporins and aminoglycosides) ( Magiorakos et al., 2011 andMaria et al., 2011).MDR P. aeruginosa are particularly problematic because the organism is inherently resistant to many drug classes and is able to acquire resistance to all effective antimicrobial drugs (Gad et al., 2007).MDR P. aeruginosa develops resistance by various mechanisms like multi-drug resistance efflux pumps, biofilm formation, production of βlactamases and aminoglycoside modifying enzymes.The risk for acquiring MDR organisms may be related to the number of carriers in the same ward as well as to individual risk factors, such as patient characteristics and in-hospital events (invasive devices and antibiotic treatment) (Carmeli et al., 2002).Extended spectrum beta lactamases (ESβLs) have been described in P. aeruginosa only recently.β-lactamases described in P. aeruginosa belong to various families as TEM and SHV types which are common among Enterobacteriaceae, PER type, VEB type which have been reported from various parts of the world (Amutha et al., 2009).Typing techniques are essential for epidemiological purposes to establish a clonal relationship between different strains isolated from patients and those isolated from surrounding environment and medical team.Phenotypic methods such as biotyping, antibiotyping, pyocin typing and serotyping and molecular methods such as plasmid profile analysis and PCR were used for this purpose.
This study was planned to delineate prevalence and resistance patterns of P. aeruginosa and MDR phenotype in nosocomial infection patients, and to screen for ESβLs producing P. aeruginosa in Menofia University Hospitals (MUH).Also to characterize clinical and environmental isolates of P. aeruginosa by plasmid profile analysis, antibioyping and pyocin typing, aiming for a better management, reducing both mortality and costs.

Materials and Methods
This study was conducted during the period from May 2009 to July 2011 and included 287 inpatients (group I) (165 males and 122 females) admitted to different wards and units of MUH and having different nosocomial infections, their ages ranged from 0 to 90 years.A total of 287clinical specimens were examined; 78 urine specimens, 46 ear swabs, 25 wound swabs and 65 burn swabs.51 hand, throat and nasal swabs were collected and processed from 17 medical staff (3 swabs each) (group II) working at MUH. 40 environmental samples were taken from surgical instruments, dressings, bath, suction devices, floors, walls, beds, commodes, sinks and antiseptic solutions.Samples were inoculated onto blood agar, nutrient agar, CLED agar and MacConkey agar (Oxoid, England).Colonies grown on different media were subjected to further morphological and biochemical identification.Suspected P. aeruginosa colonies were identified according to standard microbiological methods ( Koneman et al., 2006) 1984).Each isolated strain was streaked diametrically across the surface of tryptone soya agar to give an inoculum width of approximately 1 cm.The growth was exposed to chloroform for 15 minute.The plate was then opened exposing the medium to air for few minutes.Five standard indicator strains were grown in nutrient broth were then streaked on to the medium at right angles to the line of the original inoculum.The plate was then incubated at 37°C for 18hrs.Any pyocin produced by the original tested strains diffused into the medium during the first period of incubation and then exerted their inhibitory action on the indicator strains during the second incubation.Plates were observed for growth inhibition of indicator strains.

Results
The present study included 287 inpatients, out of them 283 clinical isolates were recovered.P. eruginosa accounted for 19 % (54 of 283) of nosocomial infections isolates.Only 3 P. aeruginosa strains were isolated from environmental sites.In our work, P. aeruginosa wasn't detected from medical staff swabs.P. aeruginosa infections were common in males (66.7 %) than female (33.3%) cases and their mean age was (44.6±25.8).In our study, P. aeruginosa mostly isolated from burn unit (32.3%),ICU (16.7%) then urology department (18.75%) (Table I) and (Figure I).

Figure (I): Distribution of P. aeruginosa Isolates among Different Departments
Table (II) summarizes the resistance patterns of 57 P. aeruginosa strains (54 clinical and 3 environmental isolates).Amikacin was the most effective drug against all P. aeruginosa isolates showed maximum sensitivity (80.5%) followed by imipenem (66.7%) and gentamicin (56.1%).On the other hand, P.

Table (V): Relation between Resistance Patterns and Plasmid
In the current study P. aeruginosa isolates (57) were typed by pyocine typing method.The results of pyocin typing classified P.aeruginosa isolates into 8 groups (I-VIII) (Table VI).Pyocine typing method couldn't discriminate MDR or ESBLs P.aeruginosa isolates in unique groups.Five clusters could be demonstrated among our MDR P. aeruginosa isolates.Each cluster had the same antibiotic sensitivity pattern and plasmid profile (Table VII), while cluster one isolates had different pyocin type.Two environmental isolates were identical by antibiotyping and plasmid profile to 3 clinical isolates and present in cluster 1 (Table VII).

Discussion
P. aeruginosa is a well-recognized nosocomial pathogen that can cause severe infections in hospitalized patients.A total of 57 P. aeruginosa strains were isolated (54 clinical and 3 environment isolates).P. aeruginosa and its MDR phenoype accounted for 19 % and 9.5% respectively of nosocomial infections in MUH.This is nearer to 18% rate of nosocomial infection reported by Gad et al., (2007) from three hospitals in Minia, Egypt.Lower incidence reported by Khan et al., (2008) in Pakistan 6.67%.In our wok P. aeruginosa and MDR-ESβLs producer P. aeruginosa infections were most common in burn wound infection followed by urinary tract, respiratory tract and wound infections.The highest incidence of P. aeruginosa was found in burn unit and ICU.In concordance with our result Aly, (2005) who founded that P.aeruginosa accounted for (44.4%) of infections in burn unit at MUH.Also Pathmanathan et al., (2009) concluded that P. aeruginosa is a nosocomial pathogen often isolated from burn infections.ICUs are generally considered epicenters of (MDR) organisms (Ramprasad et al., 2010).MDR P.aeruginosa had emerged in Egypt in recent years and seen mainly in nosocomial infections (Ali et al., 2009).In this study the isolated P. aeruginosa were investigated for MDR and ESβLs production.We found high prevalence of MDR P. aeruginosa (52%) and ESβLs producer strains (45.6%) were found in MUH.This high rate of MDR, has been reported elsewhere in previous studies.In Turkey, Unan et al., (2000) 2009) in Pakistan who found that out of 100 isolates, 33 (33%) were found to be ESβLs producers.In our work, 10 strains of the ESβLs producers were plasmidless and β -lactamase production in these strains could be chromosomally mediated.A finding which was explained by Bradford, ( 2001).In our study, pyocin typing didn't differentiate P.aeruginosa isolates belonging to different sources.There were 8 pyocin groups in our work but there were no difference between clinical and environmental isolates.Contrary to our result, Ramprasad et al., (2010) found pyocine typing is a very sensitive method of typing.A possible common source of infection in our study may be hospital environment, as two environmental isolates were identical by two typing method to 3 clinical isolates.Environmental sources may have a significant role in the transmission of P. aeruginosa (Bradford, 2001).This study highlighted the need for additional attention to the disinfection of inanimate objects in the hospital environment to limit the transfer of P. aeruginosa.A close relationship was found between the presence of certain plasmids and characteristic patterns of antibiotic resistance as five clusters could be demonstrated among our P. aeruginosa isolates, each had the same antibiotic sensitivity pattern, plasmid profile and βlactamase production.These results are nearer to Bradford, (2001) who reported that combination of multiple typing methods may lead to more precise results and more efficient tracing of the source of infection.In conclusions, our results clarified that threats of MDR and ESBLs P. aeruginosa become of major concern in our hospital and implementation of infection control strategies are major concerns to avoid the spread of this threat.Environmental sources may have a significant role in spread of MDR P. aeruginosa among hospitalized patients.Combination of simple typing methods as antibiotyping with plasmid profiling may be the cheapest and effective method for tracing source of infection.

Table ( VI): Pyocin Typing of P. aeruginosa Isolates
A, B, C, D, E are indicator strains.+ve means inhibition of growth of indicator strains.-ve means no inhibition of growth of indicator strains.

Gad et al., (2007). Earlier
reported that 60% of his P. aeruginosa isolates were MDR.studies reported that imipenem was the most effective antibiotic against P.