Molecular identification and antibiotics resistance genes profile of Pseudomonas aeruginosa isolated from Iraqi patients

Pseudomonas aeruginosa is one of the leading Gram-negative organisms associated with nosocomial infections. The increasing frequency of multidrug-resistant P. aeruginosa (MDRPA) strains affects the efficacious antimicrobial options which are severely limited. In this prospective study, forty two different types of samples collected from patients with multiple types of infections attainted to ALKadhyimia Teaching Hospital-Baghdad-Iraq, from February, 2012 to August, 2012. Morphological characteristics, biochemical testing by Vitek 2 Compact for Gram-negative Identification, card 2GN and amplification of species-specific 16SrDNA gene were used to identify P. aeruginosa. The antibiotic sensitivity profiles of the strains were determined against ten antibiotics belonging to different classes using the BioMérieux Vitek2 compact system AST card. In order to identify the genes implicated in antimicrobial resistance mechanisms, DNA was extracted from collected samples and resistance genes PstS, blaOXA-50, blaOXA-2 and bla IMP-13 were amplified using polymerase chain reaction (PCR). The results showed that there were difference in number of P. aeruginosa isolates that were identified using Vitek 2 Compact for Gram-negative Identification, card 2GN (25/42) and 16SrDNA gene(29/42), but it was not statistically significant (p value= 1.000). Also, it showed that the highest resistance percentages of strains to tested antibiotics was for Ceftriaxone (96.55%) and the lowest resistant percentages was (17.24%) for Meropenem. Seven strains out of twenty nine (24%) were resistant to all tested antibiotics and were identified as a multidrug resistance strain (MDR). The study detects the presence of PstS gene and blaOXA-50 in (65.5% and 93% of strains, respectively), but it did not identify the presence of blaOXA-2 or blaIMP 13 at any of the tested strains of P. aeruginosa. There was no significant relationship between the presence of blaOXA-50 and resistance to Meropenem, Imipenem, Ceftazidime and Cefepim (p value 1.000, 1.000, 0.474, and 0308, respectively). We concluded that Meropenem is the most effective antibiotic and can be considered as the drug of choice against P. aeruginosa. This study is the first report presence of PstS and blaOXA50 in P. aeruginosa in Iraq. The presence of blaOXA-50 is important in order to identify and track the spread of multidrug-resistant P. aeruginosa clones since blaOXA-50 may be potential clonality marker.


INTRODUCTION
Pseudomonas aeruginosa is an opportunistic pathogen.Its infections in hospitals mainly affect the patients in intensive care units and those having catheterization, burn, and/or chronic illnesses (Yetkin et al., 2006).The ability of P. aeruginosa to survive on minimal nutritional requirements and to tolerate a variety of physical conditions has allowed this organism to persist in both community and hospital settings (Lister et al., 2009).Thou, It infects healthy tissues rarely but when defenses are compromised, it can infect different tissues.This explains why most infections are nosocomial (Mesaros et al., 2007).P. aeruginosa characterized by inherent resistances to a wide variety of antimicrobials.Its intrinsic resistance to many antimicrobial agents and its ability to develop multidrug resistance and mutational acquired resistance to antibiotics through chromosomal mutations imposes a serious therapeutic problem (Gales et al., 2001;Gorgani, 2009;Al-Grawi, 2011).A number of antimicrobial agents, including several Beta-lactams are active against P. aeruginosa.Extended-spectrum Penicillins, often used to treat infections caused by this bacterium.
Although most Cephalosporins are not active against P. aeruginosa, Ceftazidime, a third generation agent, and Cefepime, a fourth-generation agent, have excellent and about equivalent activity.Of Carbapenems, a class of broad-spectrum β-lactam antibiotics, Meropenem has slightly greater activity against P. aeruginosa (Ayalew et al., 2003;Shah and Narang, 2005;Baldwin et al., 2008).Of the fluoroquinolones agents, Ciprofloxacin is the most active against P. aeruginosa.Finally, the Aminoglycosides have been mainstays in the treatment of these infections (Hauser and Sriram, 2005;Katzung et al., 2009).However, P. aeruginosa adaptive ability causes difficulties for the sensitivity of microbial identification methods and it has become necessary to develop genotype-based characterization systems capable of accurately identifying these bacteria despite any phenoltypic modifications.So, molecular identification eliminates the problem of variable phenotype and allows for more accurate identification of bacteria (Drancourt et al., 2000).16SrDNA genes are highly conserved among all organisms and they possess various unique speciesspecific regions that allow for bacterial identification.Polymerase chain reaction (PCR) is highly sensitive, specific and rapid method which vastly improved the detection of P. aeruginosa especially when using species-specific primer for 16SrDNA (Spilker et al., 2004).
In the present study, we used two methods to identify P. aeruginosa, Vitek2 system and molecular technique using PCR to amplified species-specific 16SrDNA.Also, we study the relationships between the presence of resistance genes PstS, blaOXA-50, blaOXA-2 and blaIMP-13 and the sensitivity to ten antibiotics.

Collection of samples
Forty two different types of samples were collected from patients with multiple types of infections attainted to AL-Kadhyimia Teaching Hospital from February 2012 to August 2012, Table 1.

Identification of P. aeruginosa
Collected samples were cultured on MacConkey agar and incubated overnight at 37°C.The selected colonies were cultured on 0.03% cetrimide agar and King B agar.Gram stain was done to examine cell shape and Gram reaction (Atlas et al., 1995).Also, Oxidase test was done (Mcfadden, 2000).The isolates were identified with Vitek 2 Compact system for Gram-Negative Identification, card 2GN (bioMérieux-France).Antibiotics susceptibility was examined using the BioMérieux VITEK2 compact system AST card (bioMérieux-France) according to manufacturer instructions, Table 2.

Molecular study
Genomic DNA was extracted from P. aeruginosa strains using DNA extraction kit (Promega, USA) following manufacture instructions.The sequences of primer sets used in PCR to amplify speciesspecific 16S rDNA gene for P. aeruginosa and resistance genes were shown in Table 3 (Spilker et al., 2004;Carciunas et al., 2010).*Corresponding author.E-mail: dr_maysaa@yahoo.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License  Briefly, five master mixes (each of 25 µl per reaction) were prepared, one for each gene as in the following: 1X of 5XPCR buffer (Promega, USA), 200 µm of dNTPs (Promega,USA), 25 pmol of each primer (Alpha, USA), 1 U/reaction of Green Go Taq DNA polymerase (Promega,USA).The above components were mixed well by pipetting several times after each addition.Two microliter (equivalent to 100ng) of DNA was added for each reaction tube, except the no template control tube.DNA extracted from Escherichia coli, Burkholderia cepacia, Enterobacter and Klebsiella pneumonia were used as P. aeruginosa negative controls in testing the specify of primer set for amplifying 16S rDNA gene specific for P. aeruginosa.PCR reaction tubes were transferred into thermal cycler (eppendroff, Germany) that was programmed as following: 94•C for 2 mints (X1), (92°C for 1 mint, 56°C (or 55°C) for 1 mint, 72°C for 1 mint) (X30) and final extension at 72°C for 10 mints.Amplified products were electrophoresed on 1.5% agarose for 90 mints at 4 V/cm.

Statistical analysis
Data were analyzed using SPSS version 16 and Microsoft Office Excel 2007.Nominal data were expressed as number and percent.Fischer Exact test was used for comparison of frequency.P-value less than 0.05 were considered significant.

Isolation and identification of P. aeruginosa
This study included 29 P. aeruginosa strains isolated from different clinical samples.The strains were identified using a variety of techniques, which are morphological characteristics; biochemical testing using automated Vitek2 GN, pigment production, and molecular technique.The identifications probabilities that was performed using the commercially available identification Vitek 2 GN card were ranged from 86 to 99%.
In this study, 16S rDNA species-specific primers for P. aeruginosa were used.Twenty nine strains identified as P. aeruginosa after amplification of 16S rDNA genes using PCR technique (Figure 1).

Antimicrobial sensitivity
The results of antibiotic sensitivity tests among P. aeruginosa strains using BioMérieux VITEK2 compact system AST card were shown in Table 4.The percentage of resistant strains to each antibiotic shown in Figure 2. Twenty eight strains (96.5%) were found to be resistant to ceftriaxone, 8 (27.59%) strains resistant to ceftazidime and gentamicin, 7 (24.14%)strains resistant to cefepim, imipenem, amikacin, tobramycin, ciprofloxacin and levofloxacin and 5 (17.24%) strains were resistant to meropenem.According to that, seven (24%) strains were considered as MDR.

Occurrences of PstS, blaoxa50, blaoxa2 and imp13, and their relation with sensitivity to meropenem, imipenem, ceftazidime and cefepime
The results of amplification of common sequence of psts, blaoxa50, blaoxa2 and imp13 in relation to sensitivity patterns of meropenem, imipenem, ceftazidime and cefepime were shown in Figures 3, 4, 5 and 6.
Figure 7 show the results of agarose gel electrophoresis of amplification of psts and blaoxa50.Amplification of common sequence of blaoxa2 gene and imp13 gene revealed that all isolates were negative.In this study there were no significant relationship between psts and blaOXA-50 presence and resistance to meropenem, imipenem, ceftazidime and cefepim (P value 0.281, 1.000, 0.375, 0.375 and 1.000, 1.000, 0.474, 0308, respectively).

Isolation and identification of P. aeruginosa
In disease process in which delay in the initiation of appropriate therapy has significant consequences for patient outcomes, diagnostic tests such as PCR offer more rapid and sensitive results compared with traditional phenotypic laboratory methods.It was shown that when this rapid test is used, there is significant reduction in the time to initiating effective antimicrobial therapy, decreesed mortality and decreased hospital costs (Goff et al., 2012).
In this study, from Forty Two isolates, 25 isolates were identified as P. aeruginosa using Vitek 2 system.The correct identification rates of P. aeruginosa using this automated technique were 90.7%.Results from other in-vestigators indicate that the Vitek ID-GNB cards correctly identified 85.3 to 100% of P. aeruginosa strains (Funke et al., 1998;Jossart and Courcol, 1999).Joyanes et al., tested 146 routinely isolated strains with the Vitek 2 system and ID-GNB cards and found correct identification rates of 91.6% (Joyanes et al., 2001).Using the same vitek identification card, Ines et al. (2009) found that correct identification rates of P. aeruginosa were 90.1%.
Molecular identification was done using primers targeted the variable regions in the 16S rDNA gene, which is a sequence offered a useful method for the identification of bacterial genus-or species (Spilker et al., 2004;Drancourt et al., 2000).From Forty Two isolates, 29 isolates were identified as P. aeruginosa using PCR.Spilker et al. (2004) tested 42 culture collection strains (including 14 P. aeruginosa strains and 28 strains representing 16 other closely related Pseudomonas species) and 43 strains that had been previously identified as belonging to 28 non-pseudomonal species also recovered from cystic fibrosis patient sputum.Based on these 85 strains, the specificity and sensitivity of used 16S ribosomal DNA (rDNA) sequence data to design PCR assays were 100%.
This variation that occurred may be due to the fact that the phenotypic test systems have potential inherent problems, example, (i) not all strains within a given species may exhibit a particular characteristic, (ii) the same strain may give different results upon repeated testing, and (iii) the corresponding databases are limited (Bosshard et al., 2006).The difference in the results of Vitek2 and those found by 16S rDNA for identification of P. aeruginosa isolates was not statistically significant (P value = 1.000).However, the rate of incorrect identifications showed that there was a need for a more precise method such as molecular methods of identification (Burd, 2010).

Distribution of P. aeruginosa isolates according to its sources
The incident of infection with P. aeruginosa varies according to the site of infection.It affect when introduced into areas devoid of normal defenses example, when membranes and skin are disrupted by direct tissue damage, when intravenous or urinary catheter are used (Cao et al., 2004).
In this regard, the isolates distributions in the current study (Table 1) were in agreement with that reported by local and global studies.In a study conducted by R'auf (2003), the highest percentage of P. aeruginosa were recorded among burn infections followed by wound (41.7%) and ear infection (28%) while Miteb (2006) in Najaf found highest percentages of P. aeruginosa were obtained from wound (44%) followed by burn (30%), urine (20.5%), ear (4.2%).Al-Derzi (2012) in the North of Iraq (Mosul and Duhok) revealed that the most common P. aeruginosa isolates come from purulent specimens      collected from skin wounds and burns (44.4%) followed by isolates from urine (31.8%) and ear discharge specimens (12.4%).Also, the study of Hasan et al. (2012) in Kurdistan region of Iraq found highest percentage of P. aeruginosa was obtained from burn samples (%10.9)whereas the lowest percentage were obtained from Otitis samples (%1.81).Manhal indicated that P. aeruginosa constituted 7.3 % of hospital contamination in Iraq (Hassan et al., 2012;Manhal, 2006;Rauf, 2003;Miteb, 2006;Al-Derzi, 2012).

Strain number Sample type Antibiotic MEP IMP CAZ CEF CRO GEN AK TOB CIP LEV
The discrepancy in the percentages of P. aeruginosa isolated from urine samples, septic surgical wound exudates and burn samples could be attributed to the difference in numbers of clinical specimens investigated in different studies.It is important to say that there was no burns ward in the hospital during sample collection period.

Antibiotic resistance of P. aeruginosa
Molecular methods can be used to identify antimicrobialresistant organisms directly in a variety of clinical sam- ples to optimize therapy early in the course of infectious illnesses (Tenover, 2010).Moreover, it enables the detection of resistance determinants in viable but not cultivable microorganisms (Volkmann et al., 2004).
In this study (Table 3), Meropenm MIC ranged from ≤0.25 to ≥16 μg/ml and it appeared to have the lowest resistance percent (17.24%) among tested antibiotics.This may be contributed to meropenem being a newly introduce drug in Iraq and there is certain prescription criteria in hospitals that it should not be use until it's the only antibiotic available and because it is expensive, this limits its use in private sector but the appearance of meropenem resistant strain despite previously mentioned restrictions is a great concern because it's considered as one of last-line agents and its resistance puts to threat its clinical effectiveness in treating MDR infections.
The activity of meropenem against P. aeruginosa is comparable to that of imipenem in this study.Imipenem resistance was 24.4% with MIC ≤1 to ≥16μg/ml.Lautenbach et al. (2010) referred to it as the greater intrinsic activity of meropenem over imipenem which may be explained, at least in part, by improved stability against common serine β-lactamases.In cases when porin synthesis is suppressed and Imipenem resistance emerges, Meropenem retains its activity on P. aeruginosa, suggesting the existence of another undefined route of Meropenem transport through the outer membrane.
We found that there were about 27.59% of P. aeruginosa isolates resistant to Ceftazidime MIC ≤1-≥64 μg/ml and about 24.41% of them were resistant to cefepime MIC ≤1 to ≥64μg/ml.The highest resistance percent was for ceftriaxone (96.55%)MIC 16 to ≥64 μg/ml.Cefepime is less susceptible to destruction by βlactamases than earlier generation Cephalosporins and thus remains active against bacterial mutants that produce such enzymes.In addition, cefepime may require Gram-negative bacteria to have more than one mutation to become resistant and it has a lower affinity than other Cephalosporins for binding many of the common bacterial β-lactamases (Jazani et al., 2010).The low susceptibility percentage of Ceftriaxone may be due to its extensive use in clinical practice in Iraq.Excessive use of broad-spectrum antibiotics in hospitals has led to the emergence of highly resistant strains of P. aeruginosa (Al-Grawi, 2011).
Imipenem and Ceftriaxone antibiotic sensitivity pattern were close to the antibiotic sensitivity pattern of Rabea , which found that resistance of burn isolated pseudomonas isolates in Najaf were 20%, 100% respectively but far from other antibiotics sensitivity pattern were (100%) found to be resistant to Ceftazidime, Cefepim and 85% of isolates were found to be resistant to Gentamicin and 75 of isolates were found to be resistant to Tobramycin and Ciprofloxacin; 60 of isolates were found to be resistant to Levofloxacin and Amikacin (Rabea, 2010).
In this study, MDR represent 24% of tested isolates.The definition of MDR organisms can vary from study to study; this study used the USA definition MDR P. aeruginosa as those strains that are resistant to all of the agents in two or more classes of antibiotic defined as (1) beta-lactam (including carbapenem agents), (2) aminoglycoside and (3) quinolone agents.

Occurrence of PstS
In this study, PstS was identified in 65.5% of all tested isolates.It is presents in 85.7% MDR and 59% of non-MDR isolates.Craciunas et al. (2010) in Romania found that PstS gene is present in almost all tested isolates.
PstS proteins are the cell-bound phosphate-binding elements of the ubiquitous bacterial ABC phosphate uptake mechanisms.PstS proteins are induced by phosphate deprivation in Pseudomonas non-fermenters species (Morales et al., 2007).The presence of this periplasmic phosphate binding protein (PstS) confers a highly virulent phenotype of MDR isolates of P. aeruginosa.Also the development of multi-drug resistance in P. aeruginosa clinical isolates might be related to the overproduction of PstS proteins (Zaborina et al., 2008).

Occurrences of blaOXA-50 and blaOXA-2
The blaOXA-50 gene is naturally occurring in that P. aeruginosa species since that blaOXA-50 may be another potential clonality marker for P. aeruginosa.This aspect is important in order to identify and track the spread of multidrug-resistant P.aeruginosa (Aktas et al., 2005).The prevalence of blaOXA-50 in clinical strains of P. aeruginosa raises an alarm, as the high frequency of horizontal gene transfer among bacteria may likely introduce Class D β-lactamases to other co-inhabiting bacteria species (Girlich et al., 2004;Kong et al., 2005).
blaOXA-2 was the first class D β-lactamases to be discovered (Dale et al., 1985).The first characterized class D β-lactamases was referred to as oxacillinases because they commonly hydrolyse the isoxazolyl penicillins, oxacillin and cloxacillin (Sun et al., 2003).OXA-2 has evolved variants, which confer resistance to extendedspectrum cephalosporins including ceftazidime and cefotaxime.These variants arise from single amino acid substitutions, which alter the substrate specificity of the enzyme (Paetzel et al., 2000).
A blaOXA-50 gene was identified in 93% of the P. aeruginosa strains tested and there were lack in presence of blaoxa -2.blaoxa-50 and blaoxa-2genes were found mainly in P. aeruginosa isolates from Turkey (Aktas et al., 2005;Kolayli et al., France (Bert et al., 2002) and Romania (Carciunas et al., 2010).In this study there were no significant relationship between blaOXA-50 presence and resistance to meropenem, imipenem, ceftazidime and cefepim (P value 1.000, 1.000, 0.474, and 0308, respectively).This may indicate the presence of other resistance mechanisms such isolates may express other β-lactam resistance mechanisms like other types of β-lactamases such as ESBLs or express MexAB-OprM efflux pump, or may exist simultaneously or in various combinations.

Occurrence of IMP13
In this study, the results of PCR amplification showed the lack of IMP13 in tested strains.That was similar to what was recommended by Craciunas which also did not find IMP13 among its isolates (Carciunas et al., 2010).IMPtype metallo-B-lactamases (MBLs) were the first acquired MBLs detected in Gram-negative pathogens, in the early 1990s, and is among the most relevant due to their worldwide distribution (Queenan and Bush, 2007).Since MBL production may confer resistance to virtually all clinically available β-lactams, the continued spread of MBL is a major clinical concern (Walsh, 2003).Several IMP-type variants have been described (Bebrone, 2007).IMP-13 was first detected in clinical isolates of P. aeruginosa from Italy (Toleman et al., 2003) where IMP-13 has become a widespread carbapenem resistance determinant, even involved in relatively large outbreaks (Santella et al., 2010).IMP-13 was also occasionally detected in P. aeruginosa isolates from other European countries, including Romania (Mereuta et al., 2007), France (Cuzon et al., 2008) and Austria (Duljasz et al., 2009).

Conclusion
The molecular method (16SrDNA gene amplification) offered a useful method for the identification of bacteria used for genus -species -level identification.The isolates included in this study showed the presence of Psts and blaoxa50 genes and lack the presence of blaoxa2 and IMP13.More efforts are needed to control the spread of carbapenem resistant P. aeruginosa strains.

Figure 2 .
Figure 2. Percentage of P. aeruginosa resistant isolates to ten antibiotics.

Figure 3 .
Figure 3. Presence of psts and blaoxa 50 and its relation with sensitivity to meropenem.

Figure 4 .Figure 5 .Figure 6 .
Figure 4. Presence of psts and blaoxa 50 and its relation with sensitivity to imipenem

Table 1 .
Distribution of isolates according to their sources.

Table 3 .
The sequences and molecular size of primers used in PCR reaction.

Table 4 .
Results of Vitek 2 antibiotic sensitivity test.