The Frequency of Exotoxin A and Exoenzymes S and U Genes Among Clinical Isolates of Pseudomonas aeruginosa in Shiraz, Iran.

Pseudomonas aeruginosa as an opportunistic pathogen produces several virulence factors. The most important of these factors are exotoxin A and type III secretion system (T3SS). The aim of this study was to determine the frequency of toxA, exoU and exoS genes among clinical isolates of P. aeruginosa. In this cross-sectional study from September 2011 to February 2012, 156 P. aeruginosa isolates were recovered from different clinical samples. Susceptibility testing against 10 antibiotics was performed on individual isolates by the disc diffusion method according to CLSI guidelines. Extracted DNA was subjected to PCR assay for determining the presence of toxA, exoU and exoS genes. Overall, the frequency of toxA, exoU and exoS genes were 90.4%, 66.7% and 65.4%, respectively. All of the abdominal and eye isolates were exoS (+). The frequency of exoS (+)/exoU (-) and exoS (-)/exoU (+) genotypes was estimated 19.2% and 16.2%, respectively. Indeed, genotypes exoS (+)/exoU (+) and exoS (-)/exoU (-) were found with frequencies of 48.7% and 15.3%, respectively. The highest and lowest antibiotic resistance rate was seen against azteroenam (94.2%) and amikacin (44.9%), respectively. Fluoroqinolone-resistant isolates were isolated with frequency of 45.8%. Multi-drug resistant (MDR) isolates were detected in 62.8% of isolates. The resistance rate in exoU (+) isolates was 86% compared to 66% in exoS (+) isolates. The high frequencies of virulence genes detected in our clinical isolates with notable antibiotic resistance rates indicate the potential risk of these isolates in nosocomial infections.

Pseudomonas aeruginosa is a Gram-negative and opportunistic pathogen which is widesp-read throughout the environment (1). In immune competent hosts, the bacterium seldom causes disease (1, 2). P. aeruginosa causes a wide range of infections including septicemia, pneumonia, endo-carditis, burn wounds, otitis and keratitis. It is also frequently encountered in cystic fibrosis, immuno-compromised and/or hospitalized individ-uals (3-5). The pathogenesis of P. aeruginosa is multifactorial and comprises several cell-associated and extra-cellular virulence determinants. These include exo-toxin A (ExoA), phospholipase, elastase, pyocya-nin, pili, flagella and lipopolysac-charide (3,6,7). Proliferation of P. aeruginosa in host cells and overcoming defense mechanisms is due to the transfer of many proteins via specialized secretion apparatuses including the types I,II,III,V and VI secretary systems (TSS) (2, 8).

ExoA is the major member of the type II secretion system (T2SS) which inhibits protein synthesis by ADP-ribosylation of eukaryotic elongation factor 2 (1, 8, 9). Another important virulence factor recently recognized is the type III secretion system (T3SS) (3, 6). T3SS is a contact-dependent protein secretion pathway that plays a major role in the pathogenesis of serious P. aeruginosa infections (10). The four well known T3SS effectory molecules are exoenzymes (Exo) S, U, T and Y (2). It is known that these effectors are delivered to the host cells via a translocation complex consisting of products encoded by the pcrGVHpopBD operon that cause cell necrosis and modulation of actin cytoskeleton, allowing the bacteria to invade the eukaryotic cells and escape phagocytosis (3, 6, 10).

ExoS is a major cytotoxin involved in stages of colonization, invasion and dissemination of infection (11). ExoU is a potent cytotoxin with phospholipase activity, capable of killing a variety of eukaryotic cells in vitro (12, 13). Additionally, ExoU has a greater effect than other T3SS effectors on the virulence of the bacteria (10).

A key determinant of P. aeruginosa is its remarkable resistance to antibiotics and notably many of isolates are multidrug-resistant (MDR) (14). In several studies, the relationship between MDR isolates and presence of genes encoding T3SS, especially ExoU has been demonstrated (10, 14).

As the presence of T3SS encoding genes in clinical isolates of P. aeruginosa is a variable trait, distribution of these genes in different populations should be explored. Moreover, to the best of the authors' knowledge there is no previous report from Shiraz investigating the prevalence of P. aeru-ginosa virulence genes. Therefore, the present study aimed to evaluate the frequencies of toxA, exoS and exoU genes among the different clinical isolates of P. aeruginosa. We also sought to determine whether there is any correlation between the presence of these genes and antibiotic resistance profile of the isolates.

Materials and methods

Study design and clinical specimens

In this cross-sectional study, a total 156 P. aeruginosa clinical isolates were obtained from September 2011 to February 2012 in Shiraz (a major city in the south of Iran) teaching hospitals. The samples included: urine, cerebrospinal fluid (CSF), sputum, abdominal discharge (AD), endotr-acheal tube aspirates (ETT), eye, blood, and wound.

Bacterial identification

All the isolates were identified as P. aeruginosa using both the conventional microbio-logic (e.g., Gram staining, capacity for growth at 42 C, oxidase, and IMViC tests) methods and Microgen TM GnA+B-ID System (Microgen Bioproducts Ltd, U.K) diagnostic kit. Confirmed P. aeruginosa isolates were stored in tryptic soy broth (TSB) (Merck Co., Germany) containing 20% glycerol at -70 C until further study.

Antibiotic susceptibility testing

The antimicrobial susceptibility test was done by disk diffusion method on Muller-Hinton agar (Merck Co., Germany). The following antibiotics were tested: Ceftazidime (30 g), Aztreonam (30 g), Gentamicin (10 g), Piperacillin+Tazobactam (110 g), Amikacin (30 g), Ticarcillin (75 g), Ciprofloxacin (5 g), Imipenem (10 g), Ofloxacin (30 g) and Meropenem (10 g) (MAST Co., U.K), in accordance with clinical and laboratory standards institute (CLSI) recommendations (15). MDR isolates were defined if they showed simultaneous resistance to 3 antibiotics. The P. aeruginosa ATCC 27853 was used as a reference strain for the quality control of susceptibility test. The intermediate isolates were accounted as resistant in our results.

Genomic DNA purification and molecular assay

Genomic DNA was extracted from overnight TSB cultures of P. aeruginosa isolates using the small-scale phenol-chloroform extraction method (16). The evaluation of toxA, exoS and exoU genes was accomplished by previously described primers (9,17). PCR amplification was performed in 50 l reaction volume consisting of 5 l 1x PCR buffer, 2 µl of each primer (10 pmol/µl), 1 l MgCl2 (1.5 mM), 0.8 l each of the dNTPs (200 µM), 0.6 l Taq DNA polymerase (1 U), and 2 l DNA (10-1,000 ng) from each isolate. The PCR cycling conditions were: initial denaturation at 95 °C for 10 min, followed by 30 cycles (for exoA gene) and 35 cycles (for exoU and exoS genes) of denaturation at 95 °C for 30 seconds, annealing at 55 °C for 45 seconds, extension at 72 °C for 40 seconds, with final extension at 72 °C for 10 min. All reagents were obtained from Cinnagene Co., Iran. In each run of amplicons, P. aeruginosa ATCC 27853 which is positive for both toxA and exoS, and one clinical isolate which became positive for the presence of exo U gene (428 bp amplicon) and was further confirmed by DNA sequencing, and negative (E. coli ATCC 35218) controls were included in agarose gels. The amplicons were resolved in a 1% horizontal agarose gel, stained with ethidium bromide and photographed under 300 nm UV light (Figure 1).

Fig. 1

Amplification of toxA, exoS and exoU genes from clinical isolates of P. aeruginosa by PCR. M: 100 bp ladder; lane 1: toxA gene positive control; lanes 2, 8 and 15: negative control (E. coli ATCC 35218); lanes 3-6 investigated clinical isolates for toxA gene; lane 7: exoU gene positive control; lanes 9-12: investigated clinical isolates for exoU gene; lane 14: exoS gene positive control; lanes 16-18: investigated clinical isolates for exoS gene

Statistical analyzes

Statistical analyses were performed using SPSSTM software, version 21.0. The results for antimicrobial susceptibility and the current genes were presented as descriptive statistics in terms of relative frequencies. Chi–square test or Fisher's exact test was used to analyze the results wherever needed and a p-value< 0.05 was considered as significant clinical relevance.

Results

Of the total 156 P. aeruginosa isolates, majority of isolates were recovered from urine (n= 57) and sputum (n= 35) specimens (Table 1). The results of antibiotic susceptibility revealed that tested P. aeruginosa isolates were mostly sensitive to amikacin (55.1%), imipenem (53.8%) and meropenem (51.3%). On the other hand, the most resistance rates were seen against aztreonam (94.2%), ticarcillin (78.8%) and piperacillin /tazobactam (76.9%). The results of antibiotic susceptibility profile for P. aeruginosa isolates were summarized in Table 1. Moreover, among the 156 isolates, 98 (62.8%) were MDR.

Table 1

Antibiotic susceptibility profile of P. aeruginosa isolates based on source of isolation

Antibiotic Specimen	CAZ No. (%)	IMI No. (%)	OFX No. (%)	GM No. (%)	PTZ No. (%)	TC No. (%)	MER No. (%)	CIP No. (%)	AZT No. (%)	AK No. (%)
Urine (57)	21 (37)	37 (65)	28 (49)	30 (53)	16 (28)	15 (26)	32 (56)	30 (53)	3 (5.3)	37 (65)
CSF (4)	3 (75)	4 (100)	3 (75)	3 (75)	2 (50)	2 (50)	4 (100)	3 (75)	1 (25)	4 (100)
Sputum (35)	10 (29)	19 (54)	14 (40)	16 (46)	5 (14)	7 (20)	18 (51)	19 (54)	2 (5.7)	16 (46)
Abdominal (4)	1 (25)	3 (75)	1 (25)	3 (75)	0	1 (25)	3 (75)	3 (75)	0	3 (75)
ETT (19)	5 (26)	11 (58)	9 (47)	11 (58)	6 (32)	5 (26)	10 (53)	10 (53)	2 (11)	12 (63)
Eye (5)	2 (40)	1 (20)	1 (20)	2 (40)	1 (20)	0	3 (60)	3 (60)	0	3 (60)
Blood (3)	3 (100)	2 (67)	2 (67)	2 (67)	2 (67)	2 (67)	2 (67)	2 (67)	0	2 (67)
Wound (29)	7 (24)	7 (24)	6 (21)	5 (17)	4 (14)	1 (3.4)	8 (28)	6 (21)	1 (3.4)	9 (67)
Total (156)	52 (33.3)	84 (53.8)	64 (41)	72 (46.2)	36 (23.1)	33 (21.2)	80 (51.3)	76 (42.9)	9 (5.8)	86 (55.1)

CAZ: Ceftazidime; IMI: Imipenem; OFX: Ofloxacin, GM: Gentamycine; PTZ: Piperacillin/ tazobactam; TC: Ticarcillin; MER: Meropenem; CIP: Ciprofloxacin; AZT: Aztreonam; AK: Amikacin

The presence of toxA, exoS and exoU genes was detected in 90.4%, 66.7% and 65.4% of all the isolates, respectively. The distribution of virulence genes among different clinical samples is shown in Table 2. The toxA and exoS were detected among all the isolates from AD and eye samples. However, the relative frequency of exoU was higher in the eye (80%) and wound (75.9%) compared to that seen in other clinical samples. Although, relative frequencies of virulence genes were different between clinical specimens, statistical analysis showed no differences for the presence of individual genes and source of isolation. The only exception was toxA which was significantly (P <0.05) higher among wound isolates (29/29) compared to urine ones (49/57). Moreover, isolates recovered from urine, wound and ETT samples contained significantly (P<0.001) a higher frequ-ency of toxA gene than exoS and exoU. The fre-quency of exoS+/exoU-, exoS-/exoU+, exoS+/exoU+ and exoS-/exoU- genotypes were 19.2%, 16.2%, 48.7% and 15.3%, respectively.

Table 2

Distribution of P. aeruginosa isolates virulence genes based on source of isolation

Genes Specimen	toxA No. (%)	exoS b No. (%)	exoU b No. (%)
Urine (57) c,d	49 (86.0)	32 (56.1)	33 (57.9)
CSF (4)	2 (50.0)	3(75.0)	2 (50.0)
Sputum (35)	30 (85.7)	24 (68.6)	23 (65.7)
Abdominal (4)	4 (100.0)	4 (100.0)	2 (50.0)
ETT (19) c,d	19 (100.0)	14 (73.7)	14 (73.7)
Eye (5)	5 (100.0)	5 (100.0)	4 (80.0)
Blood (3)	3 (100.0)	1 (33.3)	2 (66.7)
Wound (29) a,c,d	29 (100.0)	21 (72.4)	22 (75.9)
Total	141 (90.4)	104 (66.7)	102 (65.4)

Frequency of toxA among wound samples was significantly higher than urine isolates (P< 0.05) and distribution of toxA among other sources was not significantly different

Distribution of exoS and exoU among clinical samples showed no significant differences.

Significant differences of toxA frequency compared to exoS among individuals clinical samples (P< 0.05).

Significant differences of toxA frequency compared to exoU among individuals clinical samples (P< 0.05).

The exoA+, exoS+ and exoU+ genotypes demonstrated a higher spread (96.9%, 67.3% and 72.4%, respectively) among MDR than non-MDR isolates (P <0.05). Indeed, resistance to aztreonam (74.4%) was mainly associated to the isolates harboring exoA in comparison to other genes (P <0.05).

Discussion

As detection of virulence genes in clinical isolates of P. aeruginosa is important (3,18), thus, in the present study, the frequency of some P. aeruginosa virulence genes among different clinical isolates was characterized. As among T3SS, exoT and pcrV (part of the injection apparatus of the T3SS) genes exist in nearly all P. aeruginosa isolates from both clinical and environmental origins (3,12,19), the presence of these genes was not evaluated in this study.

In our investigation, 100% of P. aeruginosa isolates from the AD, ET, eye, blood and wound samples were toxA+. The exoS gene was also detected in 100% of isolates from the AD and eye samples. According to our results, the relative frequencies of virulence genes were higher in some special clinical specimens. It has been suggested that the infection site and duration of disease influences the virulence of P. aeruginosa clinical isolates by altering the production of some virulence determinants. For example, some anatomical sites enhance the production of ExoA and ExoS (10).

In a research in Poland by Wolska et al., the prevalence of toxA among the 62 clinical isolates of P. aeruginosa was 88.7%, which is comparable with our study. However, the frequency of exoS was more than that of the present study (75.8% vs. 66.7%) (17). It seems that the exoU had been acquired through a mobile element (plasmid) integrated into the chromosome of P. aeruginosa. Therefore, the lower prevalence of the gene than the other virulence genes could be due to this phenomenon (11). However, in our analysis, this rate was nearly the same as exoS prevalence (65.4% vs. 66.7%). In the study conducted by Mitove et al. (20) on 202 cystic fibrosis (CF) and non- CF patients, the frequency of exoS was 62.4%, whereas the prevalence of exoU was found to be 30.2%, which is not in agreement with our study. Interestingly, in a survey in France, the prevalence of exoS was markedly higher than other studies (94% in CF isolates vs. 80% in non-CF isolates) (21).

Wareham et al. showed a significant correlation between the distribution of exoS+/exoU- and exoS-/exoU+ genotypes with CF (sputum sample) and blood isolates, respectively, which is in concordance with our results (13). Likewise, these results are reported by Feltman et al., who had established an association between exoS+/exoU+ genotype with CF isolates (6). In another work by Wong–Beringer et al. exoS+/exoU– (9%) and exoS-/exoU- (2%) genotypes were found among 45 clinical isolates of P. aeruginosa (14). Moreover, in a study conducted on 55 keratitis isolates, 64% of isolates contained exoS+/exoU– genotype, whereas 33% and 4% were exoS-/exoU+, and exoS+/exoU+, respectively (18).

It is generally suggested that isolates from clinical setting contain either exoS or exoU gene but not both (22). However, interestingly, in our analysis, 48.7% and 15.3% of the isolates had exoS+/exoU+ and exoS-/exoU-genotypes, respective-ly. Similar to our survey, in the study of Finnan et al., 75% of isolates were exoS+/exoU+ (23).

Our results showed that the overall resistance to azteronam (94.2%) and ticarcillin (78.8%) was remarkably high. Among clinical specimens, AD and eye isolates were 100% resistant to aztreonam. Furthermore, isolates from CSF samples were not resistant to 6 antibiotics of 10 tested antimicrobial agents. But the isolates obtained from other clinical samples showed resistance (with different rates) to all antibiotics. The lowest resistance was seen against amikacin (44.9%), which seems to be the in vitro drug of choice in our investigation.

Fluroquinolones such as ciprofloxacin and levofloxacin have a great potency in vitro on P. aeruginosa; however, because of the widespread usage of these agents, resistance to them has developed (10). In the present study, the overall resistance to ciprofloxacin and ofloxacin were 43.6% and 48.1%, respectively. In contrast to our results, in the study of Choy et al., resistance to fluoroquinolones was found to be 11%. However, this resistance was only related to ofloxacin, whereas all isolates of P. aeruginosa were sensitive to ciprofloxacin (18). A relatively high resistance was cited by Agnello et al. with 54% of pneumonic isolates of P. aeruginosa being resistant to levofloxacin (10).

On the other hand, in our investigation 62.8% of the resistant isolates exhibited MDR phenotype. The MDR results, is nearly consistent with the finding of a study in Bulgaria with 57.5% prevalence (20). It has been suggested that the clinical isolates comprising exoU gene were significantly associated with MDR phenotype (18). This finding is supported in our results, since MDR resistance rate in exoU+ isolates with 80% frequency was higher compared to 66% in exoS+ isolates. The prevalence of such high resistant P. aeruginosa in our region is not uncommon, since, previously Anvarinejad et al. and Sarhangi et al. showed high rate of MDR among isolates from the burn patients and clinical isolates from Shiraz City, respectively (24, 25).

One of the limitations of this study was the small sample size of specimens such as blood, CSF, which could affect the results. However, beside the limitations, the current study has some outcomes. First we showed a relatively high frequency of toxA, exoS and exoU genes among P. aeruginosa clinical isolates obtained in our area and this frequency was associated to high antibiotic resistance among the isolates. Second, it seems that the source of bacterial isolation is associated with the trend of acquisition of specific virulence genes and these genes may serve to cause specific infections. These results indicate the potential risk of these isolates in nosocomial infections which merit more attention. Of course, further studies are required with larger sample size and from other regions of country to reach a comprehensive conclusion.