Type III Secretion System (Exoenzymes) as a Virulence Determinant in Pseudomonas aeruginosa Isolated from Burn Patients in Mansoura University Hospitals, Egypt

Elnagar, Rasha Mokhtar; Elshaer, Mohammed; Osama Shouman, Omar; Sabry El-Kazzaz, Samah

doi:10.30699/ijmm.16.6.520

year 16, Issue 6 (November - December 2022) Iran J Med Microbiol 2022, 16(6): 520-527 | Back to browse issues page

‎ 10.30699/ijmm.16.6.520

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Elnagar R M, Elshaer M, Osama Shouman O, Sabry El-Kazzaz S. Type III Secretion System (Exoenzymes) as a Virulence Determinant in Pseudomonas aeruginosa Isolated from Burn Patients in Mansoura University Hospitals, Egypt. Iran J Med Microbiol 2022; 16 (6) :520-527
URL: http://ijmm.ir/article-1-1748-en.html

Type III Secretion System (Exoenzymes) as a Virulence Determinant in Pseudomonas aeruginosa Isolated from Burn Patients in Mansoura University Hospitals, Egypt

Rasha Mokhtar Elnagar

¹, Mohammed Elshaer²

, Omar Osama Shouman³

, Samah Sabry El-Kazzaz⁴

1- Department of Medical Microbiology and Immunology, Faculty of Medicine, Mansoura University, Mansoura, Egypt , drrasha_m@mans.edu.eg
2- Department of Clinical Pathology, Faculty of Medicine, Mansoura University, Mansoura, Egypt
3- Department of Plastic and Reconstructive Surgery, Faculty of Medicine, Mansoura University, Mansoura, Egypt
4- Department of Medical Microbiology and Immunology, Faculty of Medicine, Mansoura University, Mansoura, Egypt

Keywords: Type III secretion system, Pseudomonas aeruginosa, Antimicrobial resistance, Burn infection

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Introduction

Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium implicated in serious Healthcare-Associated Infections (HAIs), especially in burn and chronic pulmonary infections (1). Many sources within healthcare facilities are considered reservoirs of P. aeruginosa isolates, including taps, showers, drains and sink traps. Also, Cross infections of P. aeruginosa may occur from other patients, hands of healthcare workers and contaminated medical devices (2).
Several virulence factors are implicated in P. aeruginosa colonization, invasion, and dissemination. These factors include lipopolysaccharides, adhesion factor (pili type IV), flagella, phospholipase C, exotoxins "Exo A", exoenzymes (exo), sialidase adherence factor, as well as exo-proteases that been involved in tissue destruction as (alkaline protease "Apr A", proteases like elastase, staphylolysin and protease IV). In addition to the ability of P. aeruginosa to produce biofilms (3). With the presence of these virulence factors, morbidity and mortality due to P. aeruginosa infection markedly increased (4).
The virulence determinant involved in P. aeruginosa pathogenicity and antimicrobial resistance that results in the poor clinical outcome of P. aeruginosa infection is type III secretion system (T3SS) effector proteins (5).
So far, four effectors' proteins belonging to the T3SS have been reported, including two exoenzymes with ADP-ribosyltransferase (ADPRT) and GTPase activities as exoenzyme S (ExoS) and exoenzyme T (ExoT), and other two exoenzymes with cytolytic and adenylate cyclase activity as exoenzyme U (ExoU) and exoenzyme Y (ExoY) respectively. In addition, ExoU has a phospholipase A2-like activity (6). These exoenzymes help P. aeruginosa evade the host immune system and cell apoptosis by modulating the host inflammatory response; also, it may damage physical barriers such as actin cytoskeleton and endothelial barriers. This results in major tissue destruction, worsens P. aeruginosa burn infection, and hinders wound healing (7).
Previous studies reported that all clinical P. aeruginosa isolates possess ExoY and ExoT effector proteins, while few isolates can express either ExoS or ExoU proteins (8).
Due to the lack of studies that evaluate the frequency of T3SS proteins among resistant P. aeruginosa isolates in Egypt, particularly those isolated from burn infection, this work aims to assess the proportion of genes encoding T3SS effector proteins among burn patients with P. aeruginosa wound infection.

Materials and Methods

Study Design

This descriptive cross-sectional study was conducted at the Bacteriology Lab, Microbiology Diagnostics, and Infection Control Unit (MDICU), Department of Medical Microbiology and Immunology, Faculty of Medicine, Mansoura University, Dakahliya, Egypt, during the period from May 2020 to October 2021.

Sample Collection and Bacterial Isolation

Burn wound specimens were collected from patients hospitalized in the Burn unit of Plastic and Reconstructive Surgery Center, Mansoura University, Egypt. The wound swabs specimens were taken from all patients showing signs and symptoms of burn infection, using a sterile cotton swab moistened with sterile physiological saline. They were placed into Stuart's transport medium tubes (Oxoid, UK) and transported to the laboratory within 2h after collection; if a delay in transporting the samples to the laboratory is expected, they were kept in the refrigerator at 4°C (9).
Swabs were streaked on suitable culture media as nutrient agar, blood agar, and MacConkey's agar plates (Oxoid, UK) and incubated for 24-48 h at 37˚C. Colonies of P. aeruginosa were identified by Gram staining, growth at 42°C, exopigment production on a nutrient agar plate, and different biochemical reactions as oxidase test using oxidase detection strips (Oxoid, UK), Kligler iron agar (KIA) test, Lysine iron agar (LIA) test, Motility Indole Ornithine (MIO) test, and Citrate utilization test (Oxoid, UK) (10). Isolates of P. aeruginosa were kept at -80°C in tryptic soy broth (TSB) (Oxoid, UK) containing 30% glycerol for further study; reference strain P. aeruginosa (ATCC 27853) obtained from NAMRU-3 Institute (Naval Medical Research Unit Three), Cairo, Egypt was used as control.

Antimicrobial Susceptibility Testing

Modified Kirby Bauer's disc diffusion technique was used to detect the susceptibility of a group of antibiotics on Mueller-Hinton agar plates (Oxoid, UK) according to the guidelines adopted by the clinical and laboratory standards institute (CLSI) (11). The following antibiotics were used: Ampicillin/sulbactam (SAM) (10/10 µg), Piperacillin/tazobactam (TPZ) (100/10 μg), Cefotaxime (CTX) (30 μg), Ceftazidime (CAZ) (30 μg), Ceftriaxone (CRO) (30 μg), Cefepime (FEP) (30 μg), Azteronam (ATM) (30 μg), Gentamicin (CN) (10 μg), Amikacin (AK) (30 μg), Tobramycin (10 μg), Ciprofloxacin (CIP) (5 μg), and Imipenem (IMP) (10 μg) (Oxoid, UK). Multi-drug resistant (MDR) was defined as non-susceptible (including intermediate or resistant) to at least one agent in ≥ three antimicrobial categories based on previous definitions (12).

Molecular Detection of T3SS Effector Proteins Genes

The DNA was extracted from the overnight P. aeruginosa cultures using (QIA amp® DNA mini kit, Qiagen Inc.) according to the instructions by the manufacturer. Using the multiplex PCR technique, detection of the genes encoding for T3SS effector proteins (exoS, exoT, exoU and exoY) was done with a set of specific oligonucleotide primers obtained from Sigma, Aldrich, Germany (Table 1) (13).

Table 1. List of primers used in the present study and length of the PCR products

Target genes	Sequences	Amplicon length (bp)	Reference
*exoS*	F5'-GCG AGG TCA GCA GAG TAT CG-3' R5'-TTC GGC GTC ACT GTG GAT GC-3	118	(13)
*exoT*	F5'-AAT CGC CGT CCA ACT GCA TGC G-3' R5'-TGT TCG CCG AGG TAC TGC TC-3'	152
*exoU*	F5'-CCGTTG TGG TGCCGT TGA AG-3' R5'-CCA GAT GTT CAC CGA CTC GC-3'	134
*exoY*	F5'-CGG ATT CTA TGG CAG GGA GG-3' R5'-GCC CTT GAT GCA CTC GAC CA-3'	289

DNA amplification was performed in a 50 μL reaction mix containing 25 μL of 2X GoTaq Green Master Mix (Willowfort, UK), 5 μL of genomic DNA, 2 μL from each of the forward and reverse primer of four genes (40 nmol), and DNase free water. The following PCR cycling conditions were used: initial denaturation at 95ºC for 3min; followed by 36 cycles of denaturation at 95ºC for 1min, annealing at 58ºC for 40 s, and extension at 72ºC for 1 min; finally, a single extension step at 72ºC for 10 min. The amplified PCR products were visualized by 2% agarose gel electrophoresis stained with ethidium bromide based on fragment size as compared with the 100bp DNA marker (Lonza Rockland. Inc, USA) using UV transilluminator (FBTIV-88, Fisher, USA) (14).

Data Analysis

Data were tabulated and statistically analyzed using Microsoft Excel 2010 and Statistical Package of Social Science (SPSS) version 23 (SPSS Inc., Chicago, IL, USA) software. Categorical data were presented as numbers and percentages. Quantitative variables were expressed as the mean ± standard deviation (SD). Categorical data were analyzed using the Chi-square and Fisher's test. The P value of 0.05 or less was considered statistically significant.

Ethical approval statements

This study obtained approval from the Mansoura University Institutional Review Board (R.21.09.1459). Informed written consent was obtained from each patient. All methods were done according to the Helsinki declarations.

Results

Forty-five P. aeruginosa isolates were obtained from non-repetitive one hundred and one patients' wound specimens included in this study. Of these 45 P. aeruginosa isolates, 27 (60.00%) were recovered from males and 18 (40.00%) from females with a mean age of 15.78±2.65 years old, ranging from 1 to 65 years old. About 31 (68.89%) of P. aeruginosa infected patients had a white blood cell count of more than 11000/mL. Meanwhile, 6 (13.33%) patients had a superficial second-degree burn, 25 (55.56%) patients had a deep second-degree burn, and 14 (31.11%) patients had a third-degree burn (Table 2).
Results of the antimicrobial susceptibility of tested P. aeruginosa isolates revealed that piperacillin/tazobactam 33 (73.33%) and imipenem 28 (62.22%) were the most susceptible agents; in comparison with the lowest susceptibility rates in ceftazidime 2 (4.44%), tobramycin 2 (4.44%), and ceftriaxone 3 (6.67%). Meanwhile, the sensitivity of ampicillin/sulbactam, ciprofloxacin, and amikacin was recorded in 10 (22.22%), 9 (20.00%), and 9 (20.00%) isolates, respectively. Of the 45 tested P. aeruginosa isolates, 23 (51.11%) isolates were MDR. The antibiotic susceptibility profiles for P. aeruginosa strains are illustrated in Table 3.

Table 2. Demographic and clinical characteristics of Pseudomonas aeruginosa infected patients

Demographic and clinical characteristics	*Total no. of P. aeruginosa* infected patients =45**
Demographic and clinical characteristics	No	%
Male Female	27 18	60.00 40.00
Age	15.78 ± 2.65 years, range (1 – 65)
Site of burn: Upper limb Lower limb Head Face Chest Abdomen	28 5 3 3 4 2	62.22 11.11 6.67 6.67 8.89 4.44
Burn degree: Superficial second Deep second Third	6 25 14	13.33 55.56 31.11
White blood cell count >11000 /mL	31	68.89
Cause of burn: Hot water Flame Chemicals	33 9 3	73.33 20.00 6.67

Table 3. Antibiotic susceptibility profile for Pseudomonas aeruginosa isolates

Class	Antimicrobial agent	*Total P. aeruginosa* isolates (no = 45)**
		Sensitive		Intermediate		Resistant
		No.	%	No.	%	No.	%
β-lactam+inhibitors	Pipracillin/tazobctam	33	73.33	5	11.11	7	15.56
β-lactam+inhibitors	Ampicillin/sulbactam	10	22.22	10	22.22	25	55.56
Carbapenems	Imipenem	28	62.22	3	6.67	14	31.11
Monobactam	Azteronam	11	24.44	7	15.56	27	60.00
Aminoglycosides	Amikacin	9	20.00	13	28.89	23	51.11
	Gentamicin	5	11.11	8	17.78	32	71.11
	Tobramycin	2	4.44	9	20.00	34	75.56
Fluoroquinolones	Ciprofloxacin	9	20.00	6	13.33	30	66.67
Cephalosporins	Cefepime	7	15.56	5	11.11	33	73.33
	Ceftazidime	2	4.44	4	8.89	39	86.67
	Cefotaxime	5	11.11	0	0.00	40	88.89
	Ceftriaxone	3	6.67	0	0.00	42	93.33

All tested P. aeruginosa isolates expressed the exoY and exoT genes (100%), while 28 (62.22%) and 19 (42.22%) of the clinical isolates harbored exoS and exoU genes, respectively. The displaying of exoS and exoU genes was significantly associated with higher level of antibiotic resistance to fluoroquinolones, aminoglycosides, cephalosporins and MDR rate; meanwhile, there was no significant correlation between resistance to β-lactam+inhibitors, carbapenems, and monobactam and presence of exoS and exoU genes. On analyzing the resistance pattern to specific antimicrobial agents, the isolates harboring exoS and exoU genes were associated with increased levels of resistance to ciprofloxacin (P=0.001, 0.002); cefepime (P=0.001, 0.002); ceftazidime (P=0.063, 0.014); ceftriaxone (P=0.021, 0.008); cefotaxime (P=0.002, 0.043); as well as amikacin (P=0.006); gentamicin (P=0.002, 0.043) and tobramycin (P=0.021, 0.043). Harboring exoS and exoU genotypes was also associated with MDR (P= 0.001, 0.024) strains (Table 4).

Table 4. Correlation of exoS and exoU virulence genes with antibiotic resistance pattern

Class	Antimicrobial agent	*Total P. aeruginosa* isolates (no = 45)**
Class	Antimicrobial agent	exoS + (n= 28)	exoS – (n= 17)	P value	exoU + (n= 19)	exoU – (n= 26)	P value
β-lactam+inhibitors	Pipracillin/tazobctam (n=7)	6	1	0.195	5	2	0.670
β-lactam+inhibitors	Ampicillin/sulbactam (n=25)	23	2	0.911	16	9	0.248
Carbapenems	Imipenem (n=14)	13	1	0.616	11	3	0.787
Monobactam	Azteronam (n=27)	25	2	0.187	20	7	0.572
Aminoglycosides	Amikacin (n=23)	23	0	0.006*	21	2	0.006*
	Gentamicin (n=32)	31	1	0.002*	26	6	0.043*
	Tobramycin (n=34)	32	2	0.021*	29	5	0.043*
Fluoroquinolones	Ciprofloxacin (n=30)	29	1	0.001**	27	3	0.002*
Cephalosporins	Cefepime (n=33)	32	1	0.001**	28	5	0.002*
	Ceftazidime (n=39)	37	2	0.063	29	10	0.014*
	Cefotaxime (n=40)	36	4	0.002*	29	11	0.043*
	Ceftriaxone (n=42)	38	4	0.021*	30	12	0.008*
MDR (n=23)		21	2	0.001**	19	4	0.024*

MDR: Multi drug-resistant; *significant at the P ≤ 0.05; **highly significant at the P ≤ 0.01.

Discussion

Pseudomonas aeruginosa is a significant organism with a complex structure that enhances the excretion of virulence factors in the cytoplasm of target cells by a type III secretion system mediated by cell contact. These virulence determinants are usually associated with higher mortality outcomes in patients infected with those isolates, particularly burn patients (15).
In the current research, P. aeruginosa was detected in 45 patients among the studied 101 patients with burn infection with an infection rate of 44.6%. This means that P. aeruginosa represents nearly fifty percent of the infectious bacteria that could cause wound infection in our locality. The isolation rate of P. aeruginosa was previously found to be higher among samples that were taken from cases with respiratory system and urinary tract infections than those with burn infections. This was recorded in an Egyptian study conducted in 2012 (16). On the other hand, the present results were parallel to that observed in another study by Saleh et al., as 55% of their isolates were obtained from burn specimens (17).
The increasing rate of burn infection by P. aeruginosa was recorded in previous studies (18, 19). This encourages the need to test those resistant isolates for the presence of different virulence factors, mostly the type III secretion system examined in the present research.
Most of the examined isolates observed an elevated pattern of antibiotic resistance. 51.11% of them were recorded as MDR. A higher frequency of antimicrobial resistance was observed in P. aeruginosa isolated from burn in previous research performed on patients in Iran (93.1%) (19). This prototype of resistance to antimicrobials was a common observation, particularly for burn-recovered strains of P. aeruginosa in several studies (7, 20, 21). Contrary to the current results, a lower percentage of MDR P. aeruginosa was observed in a late study conducted in 2020 (40% only) (13). The higher antibiotic resistance in the studied strains, particularly in our locality and the previously mentioned areas, is usually supported by the heavy use of antibiotics which is usually prescribed without correct antibiotic sensitivity testing, together with the natural barriers of pseudomonas bacteria itself.
Fortunately, piperacillin/tazobactam was the most effective antibiotic against the examined P. aeruginosa isolates (73.33% sensitivity) followed by imipenem (62.22%), contrary to the present results, Jarees et al. (13) observed that 100% of the examined P. aeruginosa were found to be resistant to piperacillin/tazobactam. However, only 6% of them were found to be resistant to amikacin and ciprofloxacin, which also matches the previously documented results of Khodayary et al. (6). This is found to be against the results observed in the present research as only 20% of the examined isolates recorded sensitivity for amikacin and ciprofloxacin.
The apparent disparity in the pattern of sensitivity of P. aeruginosa isolates among different studies is usually supported by the variation in the antibiotic policy among different localities that allows the gaining of variable determinants of antimicrobial resistance in P. aeruginosa species.
The major target of the present research was to examine the burn P. aeruginosa isolates for presence of protein encoding T3SS, one hundred percent of the tested P. aeruginosa were found to harbor exoY and exoT genes, this seems to be expected in our locality as P. aeruginosa isolated from different infection sites particularly burn is usually exhibited high level of resistance, both genes were more frequently detected than exoU and exoS. Lower frequency of exoY and exoT genes among P. aeruginosa isolates was documented in previous study (60 and 85% respectively) (17).
The present results run in parallel with that of Adwan et al. and also, they confirmed the presence of exoT genes in 100% of their isolates; however, exoY gene was detected only in 72.2% of P. aeruginosa isolates, and neither exoS nor exoU was detected in P. aeruginosa isolated in that research (7).
Also, another study agreed with the present study's results and reported the high frequency of the exoT and exoY genes (100%) of the isolates (22).
ExoY and exoT genes seems to be the most frequently existing genes of T3SS in P. aeruginosa as observed in the present study and other studies that confirm the same results (6, 23).
The current data showed that exoS was found to be more prevalent than exoU, this agreed to other results which documented that exoS is more prevalent compared to exoU (24-27) and differed with another study that concluded that exoU genotype was more prevalent than exoS genotypes in clinical P. aeruginosa isolates (28). On the other hand, the co-occurrence of exoU and exoS genes was significantly associated with higher antibiotic resistance to fluoroquinolones, aminoglycosides, and cephalosporins. On the other hand, although it is not significant, the P. aeruginosa isolates displaying exoU and exoS genes were more non-susceptible to β-lactam+inhibitors, carbapenems and monobactam. These results were aligned with a study by Horna et al., as they also report the significant association of resistance to fluoroquinolones and aminoglycosides with the expression of exoU and exoS genotypes (22). However, they differed that there no significant correlation between exoU and exoS genotypes and resistance to cephalosporins and carbapenems. Other studies established the significant interrelation between decreased susceptibility to fluoroquinolones and aminoglycosides in P. aeruginosa isolates displaying exoU genotype (6, 29), contrasting with another study that concluded exoS genotype was mostly associated with a higher proportion of MDR among P. aeruginosa strains from burn patients (30).
The present research faced some limitations like the small sample size; the current data should be confirmed by a higher number of P. aeruginosa isolated from infection sites other than burn.

Conclusion

This work established the high proportion of T3SS gene expression among non-susceptible P. aeruginosa isolates recovered from patients with burn wound infection. Also, the presence of exoS and exoU genotypes may indicate increased morbidity and mortality due to P. aeruginosa infection in those patients.

Acknowledgment

We would like to thank all the patients who participated in this study.

Authors Contributions

Rasha Mokhtar Elnagarand Samah Sabry El-Kazzaz designed and carried out the study. Rasha Mokhtar Elnagar performed all microbiological and molecular laboratory work, analyzed, and interpreted all data, and wrote the manuscript. Samah Sabry El-Kazzaz contributed to a critical review of the manuscript. Mohammed Elshaer and Omar Osama Shouman collected clinical samples and patients' data. All authors revised and approved the final version of the manuscript.

Funding

The authors received no financial support for this article.

Conflicts of Interest

The authors declare no conflict of interest.

Type of Study: Original Research Article | Subject: Antibiotic Resistance
Received: 2022/05/16 | Accepted: 2022/08/8 | ePublished: 2022/09/9

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