Clinical Specimens are the Pool of Multidrug- resistant Pseudomonas aeruginosa Harbouring oprL and toxA Virulence Genes: Findings from a Tertiary Hospital of Nepal

The multidrug- or extensively drug-resistant (MDR/XDR) Pseudomonas aeruginosa carrying some virulence genes has become a global public health threat. However, in Nepal, there is no existing report showing the prevalence of oprL and toxA virulence genes among the clinical isolates of P. aeruginosa. Therefore, this study was conducted for the first time in the country to detect the virulence genes (oprL and toxA) and antibiotic susceptibility pattern of P. aeruginosa. A total of 7,898 clinical specimens were investigated following the standard microbiological procedures. The antibiotic susceptibility testing was examined by the modified disc diffusion method, and virulence genes oprL and toxA of P. aeruginosa were assessed using multiplex PCR. Among the analyzed specimens, 87 isolates were identified to be P. aeruginosa of which 38 (43.68%) isolates were reported as MDR. A higher ratio of P. aeruginosa was detected from urine samples 40 (45.98%), outpatients' specimens 63 (72.4%), and in patients of the age group of 60–79 years 36 (41.37%). P. aeruginosa was more prevalent in males 56 (64.36%) than in female patients 31 (35.63%). Polymyxin (83.90%) was the most effective antibiotic. P. aeruginosa (100%) isolates harboured the oprL gene, while 95.4% of isolates were positive for the toxA gene. Identification of virulence genes such as oprL and toxA carrying isolates along with the multidrug resistance warrants the need for strategic interventions to prevent the emergence and spread of antimicrobial resistance (AMR). The findings could assist in increasing awareness about antibiotic resistance and suggest the judicious prescription of antibiotics to treat the patients in clinical settings of Nepal.


Introduction
Pseudomonas aeruginosa is known as one of the most widely spread opportunistic human pathogens causing 18 to 63% of infections worldwide [1,2]. It can grow at a temperature of 42°C, this unique character helps to differentiate it from the rest of the Pseudomonas species [3]. Most strains produce water-soluble pigments, such as pyocyanin, pyoverdin, pyorubin, and pyomelanin [4,5]. e ability of P. aeruginosa to grow in minimum nutritional requirements and to withstand various physical conditions such as disinfectants has assigned this organism to persist both in hospital and community settings [6][7][8]. P. aeruginosa commonly caused a high rate of infection in immunocompromised and cystic fibrosis (CF) patients [9].
P. aeruginosa has great diversity and is capable of causing life-threatening contagious infections in a multifariousness patients' population [10], causing several diseases such as urinary tract infections (UTIs) [11], respiratory tract infections (RTIs) [12], burn wounds, skin and soft tissue infections [13], bacterial keratitis [14], and swimmer ear infections [15]. e pathogenic effect of P. aeruginosa is mainly mediated by secreted virulence factors [10]. ere are several extracellular and cell-associated virulence factors that may lead to its pathogenicity. e colonization of these factors can cause bloodstream invasive, extensive tissue damage, and dissemination [10]. e pathogenesis of P. aeruginosa is also linked with the extracellular and cellmediated virulence factors such as toxA, exoS, exoY, exoU, oprL, oprI, lasA, lasB, oprD, plcH, plcN, and nan1 which can cause host tissue destruction, invasion, and spread in the host body [16]. Virulence genes such as oprL, oprI, and oprD are the major constituents of outer membrane lipoproteins of P. aeruginosa which are also used as markers for the identification of P. aeruginosa-associated infections [17]. Similarly, the toxA gene is one of the virulence genes, which encodes exotoxin A produced by P. aeruginosa and inhibits protein biosynthesis by stopping the elongation of polypeptide chains [17][18][19].
Increasing antibiotic resistance is a global problematic issue in recent days, and drug resistance in P. aeruginosa has become a huge concern due to its ability to cause frequent nosocomial as well as chronic infections [6]. Resistance to the different classes of antibiotics by P. aeruginosa is due to the loss or reduced copy numbers of oprD and overproduction of active efflux pumps, AmpC β-lactamase and extended-spectrum β-lactamases and their resistance mechanism are classified as intrinsic, acquired, and adaptive [6]. In Nepal, the increasing trend of antimicrobial-resistance bacteria in both clincal and nonclinical settings [11,12,[20][21][22][23][24][25][26] is becoming an alarming health issue. ere is no exact observable scheme for marking antibiotic resistance patterns and their use in Nepal. Additionally, few studies and some available secondary data are not sufficient to study the current scenario and it is hard to describe the true positive trends of antibiotic resistance of P. aeruginosa in Nepal. erefore, 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.

Study Period, Design, and Sample Size.
is cross-sectional study was conducted between November 2018 and April 2019 at a public hospital in Nepal. In total, 7898 different clinical specimens comprising urine (n � 4555), sputum (n � 975), pus (n � 595), blood (n � 1241), and body fluids (n � 532) were collected. Sample collection was carried in the sterile, leak-proof, clearly labeled container and processed immediately. Only sufficiently collected and properly labeled samples were included in this study to avoid the error in results. Clinical samples with missing demographic information such as name, age, and gender were excluded from this study.

Sample Processing and Identification of Isolates.
All collected clinical specimens were cultured semiquantitively on several culture media such as nutrient agar, nutrient broth, blood agar, MacConkey agar, cysteine-lactose electrolyte deficient (CLED) medium, brain heart infusion (BHI) medium, and chocolate agar (CA). e bacterial count more than >10 5 CFU/mL is referred to as significant bacteriuria [27]. e cultural media were bought from HiMedia Laboratories, India. Further confirmation of Pseudomonas sp. was carried out by using cetrimide agar medium and following the standard microbiological procedures that included colonial morphology, Gram staining, and a series of standard biochemical tests (indole, methyl red, Voges-Proskauer, citrate utilization, urease, oxidative/fermentative, catalase, oxidase, triple sugar iron, mannitol fermentation, and motility) [28,29].
Following CLSI guidelines, the quality of MHA plate and antibiotic discs was checked from their lot number, manufacturer, expiry date, and proper storage condition. e aseptic condition was maintained during the collection and processing of the specimens to avoid any contamination from an outlying area. Likewise, the quality of each batch of culture and biochemical media was assured by incubating a randomly sampled medium at 37°C for 24 h. A reference strain Pseudomonas aeruginosa ATCC 27853 was used to maintain the quality control of AST. In the same way, the thickness and pH of MHA were kept at 4 mm and 7.2-7.4, respectively.

DNA Extraction and Detection of Virulence Genes Using
PCR. For the identification of virulence genes (oprL and toxA), bacterial DNA was extracted from each P. aeruginosa isolate by phenol-chloroform assay [33]. e PCR amplification was carried out by using a temperature gradient thermal cycler (PCR tube 96 wells, Takara/Japan) with a specific forward and reverse primer for the detection of oprL and toxA genes, respectively. e primer was then diluted to a working concentration of 10 Pm by using nuclease-free water (Table 1).
e PCR was carried out in total 20 µL volume of reaction mixture containing 2 µL of template DNA, 1 µL of each primer, 4 µL of the master mixture, and 12 µL of nuclease-free water and Taq-polymerase enzyme with 35 cycles. e annealing temperature was 61.8°C for oprL and 58.2°C for toxA. e PCR condition is depicted in Table 1. e PCR products were separated by gel electrophoresis on 1% agarose gel containing 5 µL of ethidium bromide. e band of size about 504 bp and 352 bp of oprL and toxA was produced, respectively, along with 100 bp DNA marker and positive control [18,34]. Pseudomonas aeruginosa ATCC 27853 harbouring both toxA and oprL genes was considered as the positive control, while the negative control was maintained by using nuclease-free water.

Statistical
Analysis. Data analysis was performed using the R-programming statistical analysis tool (version 1.2.5033) and Statistical Package for Social Sciences (SPSS) software (Version 16.0). e chi-square test (χ 2 ) was estimated between the clinical, sociodemographics of patients with the distribution of MDR P. aeruginosa, and the statistically significant associations were represented by P < 0.05 * (significant), while P > 0.05 refers insignificance. e graphical presentation was performed using the ggplot package (grammar of graphics) (version 3.3.2) of R-programming language.

Growth Pattern.
Of the 7898 specimens examined, 2026 (26%) showed significant growth (i.e., >10 5 CFU/mL); among them, 87 (4.29%) were positive for P. aeruginosa. e highest significant growth (1294) was observed in urine samples, while the lowest significant growth (110) was noted from the blood samples. Likewise, the highest numbers of P. aeruginosa (40) were also recovered from the urine samples, whereas the lowest numbers of them (2) were isolated from the blood samples ( Figure 1).
e antibiotic susceptibility patterns of P. aeruginosa isolates are depicted in Table 3. Out of 87 P. aeruginosa isolates, 38 (43.68%) were identified to be MDR.

Virulence
Genes Associated with P. aeruginosa. Out of 87 total isolates, 83 (95.4%) P. aeruginosa isolates showed the presence of toxA virulence genes whereas the oprL gene was detected in all the collected P. aeruginosa isolates, 87 (100%) (Figure 2). e detection of virulence genes of P. aeruginosa was performed by multiplex PCR. e present study showed that, of 87 tested P. aeruginosa isolates, 87 (100%) contained the oprL gene (sensitivity � 100%) whereas other species of bacteria did not produce any positive result (specificity � 100%), while the amplification of the toxA gene showed that, of 87 tested P. aeruginosa isolates, 83 (95.40%) contained the toxA gene (sensitivity � 95%) whereas other species of bacteria did not yield any positive result (specificity � 100%). PCR amplification of the toxA and oprL genes is shown in Figures 3 and 4.

Discussion
P. aeruginosa is associated as a versatile opportunistic human pathogen, and its ultimate infection is reported to be accomplished by attachment, colonization, local invasion, and dissemination as a systemic disease [35,36]. In this study, the prevalence of P. aeruginosa isolates was 4.29% which is in line with the study conducted in Nepal where the prevalence rate was 5.10% [37]; however, the prevalence rate was lower (2.75%) in the study performed in Pakistan [38]. is might be due to the types of studied populations, different geographical locations, and types of hospitals. e distribution of P. aeruginosa was higher in male patients, 56 (64.36%), than in female patients, 31 (35.63%). e possible reasons may be males have routine outdoor work and they are frequently at risk of infection from the infected environments [39]. e Emergency Medicine International 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 of the outpatients to the infected environment. e occurrence of P. aeruginosa isolates was greater in the age group of 60-79 years with a prevalence rate of 41.37% which illustrates that the    Emergency Medicine International infection caused by P. aeruginosa is more common in patients of the old-age group. is could be associated with the decrease in the function of the immune system and prolonged duration of hospitalization [37,40,41]. Moreover, the occurrence of MDR P. aeruginosa was higher in females, inpatients, and 40-59 years age group patients (P > 0.05). e higher percentage of MDR P. aeruginosa was isolated from urine samples (P > 0.05). e lack of national antibiotic management policies and easy access to antibiotics without the prescription of the medical personnel might have contributed to the emergence of MDR isolates [42]. e most effective drug for P. aeruginosa isolates was found to be polymyxin 73 (83.90%), also called the last resort antibiotic for the Pseudomonadaceae family in the hospitals, and the less effective antibiotic was cefixime 87 (100%). Out of 87 P. aeruginosa isolates, 38 (43.68%) were identified to be MDR.
is was in line with the study conducted in India where the prevalence of the MDRPA was 50.00% [43]. e development of antibiotic resistance towards P. aeruginosa might be due to random use of antibiotics, production of different types of enzyme-like carbapenemases, AmpC-lactamases, quorum sensing modification of different target sides, etc. [17,44]. Furthermore, one of the major causes of the emergence of P. aeruginosa is prescribing antibiotics without performing susceptibility tests due to the lack of laboratory facilities in most of the healthcare centers in Nepal [37,39,45,46]. e PCR results showed that 87 (100%) of 87 P. aeruginosa isolates were positive for oprL genes. Similarly, in this study, 83 (95.40%) of 87 P. aeruginosa were positive for the toxA gene. Almost a comparable study was carried out in Brazil reporting 81.82% P. aeruginosa isolates were positive for toxA and 100% for oprL genes [47]. Similarly, a study conducted in Iran reported 69.4% of P. aeruginosa isolates were positive for the toxA gene [48]. e 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 adapted to the conditions found in infectious sites that may be returned to the diverse geographical and environmental sources. e prevalence of P. aeruginosa and its virulence genes depends on various causes consisting the nature of places, degree of contamination and type, immune status of individual patients, and virulence of strains [49].
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 [45]. e exotoxin A is encoded by a gene called exoA which is involved in tissue necrosis and resistant to antibiotics [46]. e L and I are two outer membrane lipoproteins of P. aeruginosa found only in this  Emergency Medicine International 5 organism, so they could be a suitable factor for the rapid identification of P. aeruginosa in clinical specimens. is bacterium is also answerable for inherent resistance to antiseptics and antibiotics [49]. Moreover, the detection of virulence genes of P. aeruginosa using multiplex PCR showed high sensitivity and specificity which revealed that multiplex PCR may be one of the rapid diagnostic tools for the identification of P. aeruginosa infections.

Conclusions
In this research, polymyxin and aminoglycosides were found to be effective antibiotics for treatment, and the studies revealed that almost all P. aeruginosa harbor both oprL and toxA genes. e higher prevalence of MDR P. aeruginosa in clinical specimens is worrisome, and special attention is required in regular surveillance of antibiotic susceptibility patterns along with their careful and judicious use. Likewise, the presence of intrinsic virulence and pathogenicity of P. aeruginosa is indicated by the existence of virulence genes such as oprL and toxA, so detection of these genes by PCR is highly recommended.

Data Availability
All data needed to support the results of this study are incorporated in the manuscript.

Additional Points
Strengths and Limitations of the Study. To the best of our knowledge, this is the first study determining the prevalence of oprL and toxA virulence genes harboring multidrug-resistant P. aeruginosa from clinical specimens in Nepal. Being unique of its kind, this study could assist the policymakers of the country to formulate the holistic approach for the proper implementation of the antimicrobial policies at all kinds of medical sectors, management of the treatment procedures, prescription of antibiotics, and improving the diagnostic procedures. ese efforts might contribute to control the emergence and transmission of MDR bacteria in clinical settings. Furthermore, this research might be a significant reference for future studies on the prevalence of P. aeruginosa harboring oprL and toxA virulence genes in several other clinical settings of the country. Despite having numerous advantages, this study possesses some limitations such as the minimum inhibitory concentration test and 16S rRNA sequencing for P. aeruginosa were not performed because of cost and unavailability of laboratories, respectively. Likewise, only toxA and oprL genes were taken as identification keys because of expensiveness. Additional studies might be required to confirm the pathogenicity and increasing trends of antibiotic resistance patterns of P. aeruginosa.
Ethical Approval e ethical clearance to conduct this research work was obtained from the Institutional Review Board (IRB), National Academy of Medical Sciences (NAMS), Bir Hospital, Kathmandu (Reference No. 728). e research was conducted in accordance with the Helsinki Declaration without any potential bias.

Consent
Prior to the sampling, written informed consent was obtained from each participant. Permission was sought from the parents for sampling in the case of children.

Disclosure
Yamuna Chand, Sujan Khadka, and Sanjeep Sapkota share the first authorship. A preprint of the current study has previously been published [50].   Emergency Medicine International