Serotypes , Antibiogram and Genetic Relatedness of Pseudomonas aeruginosa Isolates from Urinary Tract Infections at Urology and Nephrology Center , Mansoura , Egypt

Background: Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen that represents a major problem in many hospitals because of its increased resistance to antibiotics and the ability to cause nosocomial infections. The present study aimed to phenotype and genotype isolates of P. aeruginosa from inpatients with UTIs at Urology and Nephrology center, Mansoura, Egypt to study their relatedness. Methods: Thirty nine isolates of P. aeruginosa were phenotypically typed by determination of O-serotypes by slide agglutination technique and antimicrobial resistance patterns by disk-diffusion method. The genetic diversity of isolates was illustrated by performing RAPD-PCR using M13 primer. Results: Serotypes O11, O6 and O10 were the most prevalent. Isolates showed high resistance rates to antipseudmonal antibiotics with high incidence (51.3%) of multidrug resistance (MDR). Amikacin was the most effective. A significant correlation was found between O6, O10 and MDR. A relatively high polymorphism was demonstrated among P. aeruginosa isolates by using RAPD-M13 fingerprinting. Cross transmission was suggested by phenotypically and clonally identical isolates. Conclusion: The study demonstrates the role of combining both classical and molecular typing as a valuable mean to study the origin and cross transmission of P. aeruginosa in UTIs for better assessment of treatment and infection control.


Introduction
P. aeruginosa is a cosmopolitan Gram-negative bacterium that is considered as a major frequent cause of nosocomial infections [1].It is a leading cause of human opportunistic infections particularly in immunocompromized patients [2] [3] [4].Urinary tract infections are among the most predominant nosocomial infections.P. aeruginosa is responsible for 7% -10% of such infections [5].
For epidemiological purpose, typing techniques are required to recognize nosocomial transmission by establishing clonal relationships between isolates.P. aeruginosa is the third most common pathogen associated with hospit- al-acquired catheter-associated UTIs isolates [6].Typing of P. aeruginosa relied on phenotypic characters such as lipopolysaccharide (LPS) serotypes, susceptibility to antimicrobials, phage susceptibility typing and bacteriocin production [7].
LPS contains O antigen, a repeating polysaccharide portion that has been used for the classification of P. aeruginosa isolates.The International Antigenic Typing Scheme (IATS) reported 20 different serotypes based on the expression of the O-antigen moiety [8] [9].
Infections caused by P. aeruginosa are difficult to treat because of the limited susceptibility to antibiotics which is due to its inherent resistance to many drug classes and the ability to develop further resistance mechanisms to available antibiotics [10].MDR P. aeruginosa has increased worldwide in the last century [3].They are usually isolated from nosocomial infections [11].It is of a great importance to study the susceptibility of P. aeruginosa isolates to commonly used antibiotics.Antibiogram can be used as an epidemiological indicator that may guide the best choice of antimicrobial agents in infections' management [12].Molecular typing methods have been used to study the genetic diversity of P. aeruginosa.DNA typing methods include ribotyping, pulsed field gel electro- phoresis (PFGE), repetitive element based PCR (rep-PCR) and random amplification of polymorphic DNA (RAPD) [13] [14].RAPD-PCR is one of the molecular techniques used for P. aeruginosa typing.It is based on the use of single primers of arbitrary nucleotide sequence for amplification of random DNA segments [15].RAPD-PCR is a simple, low cost genotyping method capable of generating a large number of genetic markers using small amount of DNA without the need for molecular characterization of the genome of the species under investigation [16].
The aim of the present study was to investigate the prevalence of O-serotypes, resistance phenotypes of clinical urine P. aeruginosa isolates obtained from Urology and Nephrology center, Mansoura, Egypt.Also, RAPD genotyping was conducted to characterize their genetic diversity.Assessment of obtained data was done to verify any association of serotypes with resistance pattern or RAPD genotypes.

Serotyping
Serotyping of P. aeruginosa was performed by slide agglutination technique using specific 4 polyvalent and 16 monovalent antisera according to recommendation of the manufacturer's protocol (Bio-Rad®, France) according to Glupczynski et al. [19].However, the determinations of P. aeruginosa serogroups were based on the International Antigen Typing Scheme (IATS) according to Legakis et al. [9].

Statistical Analysis
Data analysis was done by chi square probability test using GraphPad Prism5 software to find association between studied characteristics.P value of ≤ 0.05 was considered significant.
Combined datasets of serotype, antibiogram, RAPD-PCR profile of M13 were created to construct a dendrogram based on the unweighted pair group method with an arithmetic average (UPGMA) using online software.

Results
In the current study, 39 clinical isolates of P. aeruginosa were isolated from patient with UTIs.They were identified morphologically and by conventional biochemical tests.
Amikacin was the most effective as only 25.6% of isolates was resistant.Isolates showed the same level of resistance (48.7%) to both ciprofloxacin and levofloxacin.Imipenem resistance was demonstrated by 14 (35%) isolates.While 20 (51.3%) isolates were MDR, only 10 (25.6%) isolates were sensitive to all tested antibiotics.Regarding the resistance pattern, 13 patterns were found among the studied isolates.A1 pattern that represents resistance to all tested antibiotics was demonstrated by 7 (17.9%)isolates (Table 2).

Relationship between Serotypes and Antibiotic Resistance
Regarding resistance to each antibiotic, the distribution of serotypes among resistant isolates to levofloxacin, ciprofloxacin and piperacillin differs significantly (P value = 0.0085, 0.0085 and 0.0165, respectively).There was a high association between resistance to these antibiotics and certain serotypes (O6, O10 and O11).

Discussion
P. aeruginosa is a major nosocomial pathogen that frequently causes urinary tract infections [25].Studying its serotypes, antibiotic susceptibility and genotypic characterization will help in control of infection, and to improve outcome of treatment [26].
Of the 14 serogroups (20 serotyps) identified by IATS, only six serogroups (9 srotypes) were found (B, C, E, G, H and I).The present study revealed that serotypes O11, O6 and O10 were the dominant representing 66.6% of P. aeruginosa isolates.The frequency of incidence of different O-serotypes differs considerably among publications.Similar to our results, Lu et al., [27] found that O6 followed O11 and O10 were the commonest serotypes.Our results are in partial accordance with previous studies prevalence of other serotypes that reported O6 and LEV: levofloxacin, CIP: ciprofloxacin, AK: amikacin, CN: gentamicin, IPM: imipenem, PRL: piperacillin and CFS: cefoperazone/sulbactam.MDR: multi-drug resistant.
O11 among the most common serotypes [26] [28] [29] [30].In Egypt, El-Bialy et al. reported the prevalence of O4 and O6 serotypes in their study [25].Hafez et al. [31] and Mohammed [32] reported O12 among the commonest serotypes de- tected.A study conducted by Elogne et al. [33] in Abidjan and Cattoen et al. [34] in Tunisia reported O4 as the most prevalent serotype.This may be attributed to the difference in specimen type and geographical location.Nosocomial P. aeruginosa is associated with high resistance rates to antibiotics and frequent multidrug resistance [11].This was demonstrated in the present study as more than 50% of isolates were MDR.A nearby percent of MDR P. aeruginosa (43.8%) was reported by El-Domany et al. [35] and a higher percent (64%) by Hashem et al. [36].Our isolates were associated with high resistance rate to piperacillin, cefoperazone/sulbactam, gentamicin and quinolones (48.7% -61.5%).Amikacin and imipenem were associated with lower resistance rates (25.6% and 35%, respectively).El-Bialy et al. reported similar results concerning the effectiveness of amikacin and imipenem [25].These results disagreed with Abaza et al. who reported higher resistance to imipenem (78.3%) [37].Improper use of antibiotics explains the high resistance found among isolates.Levofloxacin, ciprofloxacin and piperacillin resistant isolates were significantly observed among the most prevalent serogroups, the same result was reported by Vizujė et al. [38].
Analysis of antibiogram revealed the association of MDR isolates with serotypes O6, O10 and O11.Previous studies reported prevalence of MDR P. aeruginosa among serotype O11 [33] [38] [39] [40].In contrast to our results, Aydoğan et al. found association of susceptibility to all drugs and serotypes (O6, O11) and MDR was common among serotype O12 which was not detected in  the present study [41].High polymorphisms (25.6%) with 10 different profiles were obtained by RAPD-M13 fingerprinting.However, 6 isolates could not be genotyped, referred to as untypable, a similar result was reported by previous studies [42]  previous study [45].However, Raafat et al. did not found such association in their study [43].
In dendrogram, combining the phenotypic and genotyping methods gave a high level of discrimination between isolates.The 39 isolates were classified into 21 clusters (>70% similarity).The largest cluster comprised most of P1 genotype isolates that were MDR but of different serotypes.This confirms that P. aeruginosa of the same genotype could be discriminated by phenotypic methods such as serotyping and susceptibility to antimicrobials [25] and vice versa [46].Interestingly, two pairs of isolates (number 1, 5 and 13, 23) and three isolates (number 3, 9 and 34) were placed in the same subgroup in this clusters showing 100% similarity.This strongly suggests the common source of infection in the hospital [43] [45].In the present work, genotypic clusters show that the RAPD clonal lineage was not congruent with the serotypes of isolates.This leads to poor significant clusterization of isolates on using RAPD-M 13 alone [41].
For studying P. aeruginosa population, the use of phenotypic characteristics such as serotypes and antimicrobial susceptibility patterns together with genotypic characteristics confirms that more integrated information from a group of organisms reflects the biological reality of such population [47].

Conclusion
In the present study, serotypes O11, O6 and O10 were the most prevalent.Isolates showed high resistant rates to antipseudmonal antibiotics with high incidence of MDR isolates; that suggested the urgent need for revision of management and treatment policy to decrease the burden of resistant strains.Amikacin was the most effective antibiotic and piperacillin was the least effective one.
A significant correlation was found between O6, O10 and MDR.A relatively high polymorphism was demonstrated among P. aeruginosa isolates by using RAPD-M13 fingerprinting.No clear correlation between serotypes and genotypes was found.On the other hand, antibiotic resistance was highly associated with genotype P1.Common source of infection is clear from isolates presenting uniform phenotypic and genotypic traits.The study illustrates the role of combining both phenotypic and genotypic characterization as a valuable way to study the epidemiology of P. aeruginosa infections for better assessment of treatment and infection control.

Table 1 .
Distribution of serotypes among P. aeruginosa isolates.

Table 2 .
Antibiograms showing resistance pattern of P. aeruginosa isolates.

Table 3 .
Distribution of Antibiotic resistance among different RAPD pattern.