Prevalence of some virulence genes and antibiotic susceptibility pattern of Pseudomonas aeruginosa isolated from different clinical specimens

Background: Pseudomonas aeruginosa is an opportunistic human pathogen and are reported to cause acute and chronic infectious diseases. Due to its high ability to acquire resistance to many antibiotics, it has become a global public health threat. It consists of some virulence genes that may lead to its pathogenicity. The main objective of this cross-sectional study was to detect the virulence genes and antibiotic susceptibility pattern of P. aeruginosa isolated from clinical specimens collected from governmental hospital of Nepal. Methods: A total of 7898 clinical specimens were analyzed for the period of six months from November 2018 to April 2019. The specimens were cultured on Nutrient agar, Blood agar, MacConkey agar, Chocolate agar, Cysteine-Lactose, Electrolyte Decient agar plates and were incubated at 37°C for 24 hours. All the isolates were identied by standard biochemical tests and further conrmed by growth on Cetrimide agar plate. The antibiotic susceptibility testing was performed by modied Kirby-Bauer disc diffusion method following CLSI guideline. Multiplex-PCR was done to detect the virulence genes oprL and toxA. Statistical analysis was carried out using IBM SPSS Statistic ver. 25 and the p-value was calculated at signicance level (0.05%) by using Chi square. Results: Out of these specimens investigated, 87 isolates were tentatively identied to be P. aeruginosa in which 20 (22.98 %) were found to be multidrug resistant. Comparatively, most of the P. aeruginosa were isolated from outpatients 63 (72.41 %) than inpatients 24 (27.58 %), from male 56 (64.36 %) than female 31 (35.63 %) and in age group 60-79 years (41.37 %). AST result showed the highest resistance of 100% with cexime whereas susceptibilities of 83.9% and 81.6% with polymixin B and tobramycin were noticed respectively. The PCR results showed that all P. aeruginosa isolates carried oprL 87 (100%) and 83 (95.4 %) This revealed multiplex PCR may be rapid diagnostic tool for the identication of P. aeruginosa infections.


Background
Pseudomonas aeruginosa is known as one of the most-widely spread opportunistic human pathogen causing 18 to 63% infection worldwide (1,2). It has the ability to grow at temperature 42 °C, this unique character helps to differentiate from many other Pseudomonas species (3). Most strains produce water soluble pigments, such as pyocyanin, pyoverdin, pyorubin and pyomelanin (4)(5). The ability of P. aeruginosa to grow in minimum nutritional requirements and to withstand various physical condition has assigned these organism to persist in both hospital and community settings (6-7). P. aeruginosa having great diversity and capable of causing life threatening contagion infections in a multifariousness of patients population (8). There are several extracellular and cell associated virulence factors that may lead to its pathogenicity. The colonization of these factors can cause blood stream invasive, extensive tissue damage and dissemination. Some genes, normally encode and participate in the virulence factors are, toxA, exoS, exoY, exoU, oprL, oprI, lasA, lasB, oprD, plcH, plcN and nan1 etc. (9).
Virulence genes such as oprL, oprI and oprD are the major outer membrane lipoproteins, speci c to P. aeruginosa only and true factors for identi cation of P. aeruginosa infections (10). Similarly, toxA gene is one of the virulence genes that encode exotoxin A produced by P. aeruginosa and inhibits protein biosynthesis by stop elongation of polypeptide chains (11)(12)(13).
Among all the investigating tools, DNA typing techniques have been often used to investigate the divergence of P. aeruginosa (14). Proteins can also be identify by matrix-assisted leser desorption/ionization time-of-ight mass spectrometry (MALDI-TOF MS) (15).
This cross-sectional study is designed to assist the current resistant against different classes of antibiotics and to characterize P. aeruginosa by identifying virulence genes oprL and toxA using polymerase chain reaction (PCR).
In Nepal, the increasing trend of antimicrobial resistant property of P. aeruginosa is becoming an alarming health issues. There are no exact observable scheme for marking resistant pattern and its use in Nepal. Additionally, few researches and some available secondary data are not su cient to study current scenario and it is really hard to describe the true positive trends of antibiotic resistant of P. aeruginosa in Nepal. Therefore this cross-sectional study is designed to assist the current resistant pattern of P. aeruginosa against different classes of antibiotics and to identify the involvement of several virulence genes in resistant mechanisms by using Polymerase Chain Reaction (PCR) which ultimately helps to select appropriate antibiotics useful for the treatment of infectious disease caused by P. aeruginosa.

Methods
This cross-sectional study was conducted between November 2018 to April 2019 at Bir Hospital (a tertiary care hospital), National Academy of Medical Sciences (NAMS), Kathmandu, Nepal and Department of Microbiology at National College (NIST), Kathmandu Nepal. In this study, clinical and socio-demographic study of patients was also performed. In total, 7898 different clinical specimens were collected and cultured semi-quantitatively on Nutrient agar, blood agar, MacConkey agar. It was con rmed by using cetrimide agar medium and standard biochemical tests. The antibiotic susceptibility testing was performed on Muller Hinton agar by modi ed Kirby-Bauer disc diffusion method. Strains were considered as multi drug resistant (MDR) if they were resistant to at least 4 of following antimicrobial agents belonging to different classes of antibiotics: Imipenem, Ceftazidime, Tobramycin or Gentamicin and Cipro oxacin (16). Diameter of the zone of inhibition for each antibiotic was measured and interpreted as recommended by Clinical and Laboratory Standard Institute (CLSI) guideline (2018) (17)(18).

DNA Extraction
For the identi cation of virulence genes (oprL and toxA), chromosomal DNA was extracted from each P. aeruginosa isolate by phenol-chloroform assay (19).

Detection of Virulence Genes by using PCR
The PCR ampli cation was carried out by using temperature gradient thermal cycler (PCR tube 96 wells, Takara/Japan) with speci c forward and reverse primer for the detection of oprL, and toxA genes respectively. The primer was then diluted to working concentration of 10 Pm by using nuclease free water (Table 1).
The PCR was carried out in total 20 µl volume of reaction mixtures containing 2 µl of template DNA, 1 µl of each primer, 4 µl of master mixtures, 12 µl of nuclease free water and Taq-polymease enzyme with 35 cycles. The annealing temperature was 61.8 °C for oprL, and 58.2 °C for toxA. The PCR condition is depicted in Table 1. The PCR products were separated by gel electrophoresis on 1% agarose gel containing 5 µl of ethidium bromide. The band of size about 500 bp and 352 bp of oprL and toxA were produced respectively along with 100 bp DNA marker and positive control (20).
Clinical and socio-demographic study The highest number of P. aeruginosa was isolated from male patients 56 (64.36%) and community acquired infections was found to be greater which accounts 63 (72.41%). In addition, the substantial number 36 (41.37%) of P. aeruginosa were found in age group 60-75 (Table 2).  As shown in Table 4, out of 87 P. aeruginosa isolates, the maximum number of MDR P. aeruginosa (MDRPA) was isolated from pus (30.76%) which followed by urine (30%) and sputum (16.66%). Out of 87 total isolates, 83 (95.4%) P. aeruginosa isolates showed the presence of toxA virulence genes whereas oprL gene was detected in all of the collected P. aeruginosa isolates 87 (100%) ( Table 5).  (21)(22).
In this study, the prevalence of P. aeruginosa isolates was 4.29%, in which the distribution in male patients 56 (64.36%) was higher than in female patients 31 (35.63%). The possible reasons might be types of studied populations, different geographical locations, type of hospitals. In addition, other reasons may be the male have a routine outdoor work and they are frequently in the risk of infection from the infected environments (16). The prevalence rate of infections was higher in outpatients 63 (72.41%) compared to the hospital admitted patients 24 (27.58%) which may be due to frequent exposure to infected environment. In addition, the occurrence of P. aeruginosa isolates to be greater in the age group 60-79 (41.37%), infection caused by P. aeruginosa is more common in patients of old age group. This could be described as because of decrease immune system, prolonged duration of hospitalization (23)(24)(25).
In our study, the difference between gender of patients and P. aeruginosa isolation from different clinical specimens was found to be statistically signi cant (p = 0.016). Likewise, the difference between type of patients and P. aeruginosa isolated from different clinical specimens was found to be statistically signi cant (p = 0.009). There was no statistical signi cance between the different age groups and occurrence of P. aeruginosa in male and female patients (p = 0.071) at 5% level of signi cance.
In this study, the most effective drug for P. aeruginosa isolates was found to be polymyxin B 73 (83.90%), also called last resort antibiotic for Pseudomonadecae family in the hospitals and less effective antibiotic was ce xime 87 (100%). Out of 87 P. aeruginosa isolates, 20 (22.98%) were identi ed to be MDRPA. The development of antibiotic resistant towards P. aeruginosa might be due random use of antibiotics, production of different types of enzyme like carbapenamase, AmpC-lactamases, quorum sensing modi cation of different target side etc (26,13). Furthermore, one of the major cause of the emergence of P. aeruginosa is prescribing the antibiotics without performing susceptibility test due to lack of laboratory facilities in most of the healthcare centers in Nepal (16,24,(27)(28).
The PCR results showed that 87 (100%) of 87 P. aerugiosa isolates were positive for oprL genes. Similarly in this study 83 (95.40%) of 87 P. aeruginosa were positive for toxA gene. Almost comparable study was carried out by Ibraheem at 2018 which showed 100% of toxA and 98.8% oprL genes. According to the Khattab et al in (2015), 100% of oprL gene, and 63.33% toxA gene were reported among the P. aeruginosa. The divergences in the distribution of virulence factor genes in the different populations might be due to the probability that some P. aeruginosa strains are better adopted to the particular conditions found in infectious sites that may returned to the diverse geographical and environmental sources. The prevalence of P. aeruginosa and its virulence genes depends on various causes consisting nature of places, degree of contamination and type, immune status of individual patients and virulence of strains (29).
Exotoxins A are either actively secreted through the type 1 secretion system (T1SS), the type 2 secretion system (T2SS), and the type 3 secretion system (T3SS) or passively secreted via the cell (27). The exotoxin A is encoded by gene called exoA which is involved in the tissue necrosis and resistant to antibiotics (28). The L, and I are two outer membrane lipoproteins of P. aeuginosa are found only in this organism, so that they could be a suitable factors for rapid identi cation of P. aeruginosa in clinical specimens. This bacterium is also answerable for inherent resistance to antiseptics and antibiotics (29).
In this study, detection of P. aeruginosa by multiplex PCR for ampli cation of oprL gene, present study showed that of 87 tested P. aruginosa isolates 87 (100%) contained the oprL gene (sensitivity = 100%), where as other species of bacteria did not produce any positive result (speci city = 100%). While ampli cation of the toxA gene showed that of 87 tested P. aeruginosa isolates 83 (95.40%) contained the toxA gene (sensitivity = 95%), where as other species of bacteria did not yield any positive result (speci city = 100%). This study revealed that the multiplex PCR may be one of the rapid diagnostic tool for the identi cation of P. aeruginosa infections.

Conclusion
In this research, polymyxin and amynogycocides were found to be functional antibiotics for treatment and the studies revealed that almost all P. aeruginosa harbors both oprL and toxA genes. Additional studies might be required to con rm the pathogenicity and increasing trends of antibiotic resistance pattern of P. aeruginosa.

Limitations of study
Study has limitations as minimum inhibitory concentration and 16 s rRNA sequencing for P. aeruginosa were not performed because of cost and unavailability of laboratories respectively. Despite of all, only tox A and opr L genes were taken as an identi cation keys because of expensiveness.  Agarose gel (1%) electrophoresis of PCR assay of toxA gene (352 bp). From left to right bands U7, U8, U18, U19, B1, BF1, P6, P10, S12 and S24 indicates P. aerogenosa isolates, bands P4, S4, S7 and S17 indicates negative isolates with 100 bp marker and band P5 represents positive control.