Molecular characterization of Pseudomonas aeruginosa isolates from various clinical specimens in Khartoum/Sudan: Antimicrobial resistance and virulence genes

Background: Pseudomonas aeruginosa is a pathogenic organism responsible for frequent wound and nosocomial infections worldwide. Its infections are difficult to control since the organism is known to rapidly develop antibiotic resistance and becomes multidrug-resistant (MDR) during treatment of patients. Aim of the study: This study was intended to investigate the occurrence of certain important types of (ESBL) and (MBL) enzymes in association with important specific virulence factors associated with P. aeruginosa clinical isolates from Khartoum, Sudan. Methods: This study investigated 70 P. aeruginosa isolates which were collected from patients admitted to four major hospitals in Khartoum (Fedail, Ribat, Ibn Sina and Soba hospitals). These isolates were recovered from 40 wound swabs (57.1%), 27 urine samples (38.6%), and 3 pleural fluid samples (4.3%) of patients. Higher numbers of isolates were recovered from males 42 (60%) than in females 28 (40%). All P. aeruginosa isolates were first confirmed by conventional biochemical and second using molecular PCR tests. PCR methods were also used for detecting the presence of the virulence genes ToxA, AlgD, LasB, exoS, exoU, CTX, GES-1, and genes of VIM, IMP, KPC, CTX, VEB-1 and SHV-1. Results: Antimicrobial susceptibility testing of P. aeruginosa isolates showed a high resistance to azetronam 49 (70%), followed by ceftazidime 32 (45.7%), 16 ciprofloxacin (22.9%), gentamicin 13 (18.6%), Molecular characterization of Pseudomonas aeruginosa isolates from various clinical specimens in Khartoum/Sudan: Antimicrobial resistance and virulence genes Islam A. Babour1, Maha B. Mohamed2, Asem A. Shehabi1 1 Department of Pathology-Microbiology and Forensic Medicine, The University of Jordan, School of Medicine, Amman, Jordan. 2 Department of Microbiology, The University of Medical Sciences and Technology, Faculty of Medical Laboratory Sciences, Khartoum. Contact information:

P. aeruginosa needs very limited nutritional requirements. It can survive in distilled water [6]. The organism survives at wide range of temperature (4-42 °C), and it is also tolerant to disinfectants and high concentrations of salts [7]. P. aeruginosa is capable of producing several different pigments, which are pyocyanin (blue), pyoverdin (yellow and fluorescent), pyorubin (red), and pyomelanin (brown) [8].
Multidrug-resistant P. aeruginosa is considered to be a serious problem in all types of infections because the organism has intrinsic resistance to several drug classes and it is able to acquire resistance to most normally effective anti-Pseudomonal drugs within a short period [9].
There are many virulence factors that may contribute to pathogenic potential of P. aeruginosa, such as elastase, alkaline protease hemolysins (phospholipase and lecithinase) and pyocyanin diffusible pigment. In addition, most P. aeruginosa strains produce a variety of toxins especially exotoxin encoded by the toxA gene, which inhibits protein biosynthesis and is responsible for tissue necrosis. Exoenzyme S is encoded by the exoS gene, which is secreted by a type-III secretion system directly into the cytosol of host epithelial cells and subsequently causes their destruction [10]. Other important virulence factor found in P. aeruginosa include algD, which codes for GDP mannose dehydrogenase, which is transcriptionally activated in mucoid strains, and elastase B, a zinc metallopro-piperacillin-tazobactam 11 (15.7%), amikacin 9 (12.9%), and imipenem 6 (8.6%) showed the least resistance. All isolates were positive for algD and lasB (100%), followed by toxA (90%), exoS (34.3), exoU (24.3%), respectively. The rates of detected ESBL genes blaTEM, blaCTX-m, blaSHV-1,GES-1, were 3.3%, 6.6%, 10%, 3.3%, 10%, respectively, but all isolates were negative for bla-KPC and bla-VIM and IMP . The percentages of pigment production were 61.4% for pyocyanin, 37.1% for pyoverdin and 1.4% for pyorubin. tease encoded by the (lasB) gene, which attacks eukaryotic cells [11][12]. P. aeruginosa may also possess flagellae and pili, which helps in motility and adherence to host cells [13].
In recent years, a new variety of β-lactamases have been detected in P. aeruginosa clinical isolates. Broad spectrum β-lactam resistance attributed to β-lactamases is generally due to the metallo-βlactamases (MBLs) such as IMP and VIM. The over expression of chromosomally encoded cephalosporinase, AmpC, is prevalent in P. aeruginosa. These MBLs hydrolyze anti-Pseudomonal cephalosporins and carbapenems effectively and their activity is not suppressed by the β-lactamase inhibitors that are currently available for treatment [14][15].
This study aimed to investigate P. aeruginosa isolates obtained from various clinical specimens of patients in Khartoum, Sudan, for their antimicrobial resistance profile, the occurrence of most important MBL genes and the common specific virulence factors using PCR molecular and culture tests.

Materials and Methods
This study included a total of 70 different clinical P. aeruginosa isolates collected from patients admitted to four hospitals in Khartoum, Sudan, during the period between September to November 2017. All patients were on treatment with various antibiotics.
The data about each patient was obtained and registered as the following: name, gender, age, and type of specimen, and all patients are agreed for any necessary medical process once they are admitted to each hospital.

Culture and isolation
All P. aeruginosa isolates were preserved in cryo tubes containing brain heart infusion with 15% glycerol samples and transported from Sudan to Jordan. All isolates were then subcultured on Pseudomonas Agar plates and incubated for 24 to 48 hours at 37 °C, and were examined for the presence of pigmentation and grape-like odor of P. aeruginosa at the research laboratories of the School of Medicine, The University of Jordan, Amman, Jordan.

Identification and storage
All growth of P. aeruginosa isolates was first identified using conventional biochemical tests including oxidase test, develop pigmention and growth on 42 °C and produce no chemical reaction during culture in Kligler's iron agar tube were stored in cryo tubes at -70°C.

Antimicrobial susceptibility using disc diffusion method
Antimicrobial susceptibility using disc diffusion test was performed according to the guidelines of the Clinical Laboratory and Standards Institute (CLSI, 2016) [16].

Minimum inhibitory concentration (MIC) using E-test
This test was only performed on the P. aeruginosa isolates, which were MDR to three or more antibiotic classes. Four classes of antibiotics were tested (ceftazidime, ciprofloxacin, imipenem and amikacin). Results were interpreted according to the CLSI 2016 guidelines [16].

DNA extraction and PCR
The DNA extraction was performed according to the Promega kit (USA). Two PCR assays were performed to confirm the isolates; one is specific for the genus Pseudomonas, while the other is specific for P. aeruginosa. Two pairs of primers were used for each assay based on 16S ribosomal DNA (rDNA) sequence as shown in Table 1.
The bacterial plasmid of freshly cultured P. aeruginosa isolates was extracted according to manufacturer instructions using the Zyppy TM Plasmid Miniprep Kit (QIAGEN, Germantown, MD, USA)for detection of ESBLs, MBLs and virulence genes. All

Discussion
This study included P. aeruginosa isolated from different clinical specimens of patients whom were admitted for medical care at four major hospitals in Khartoum, Sudan. The majority of the isolates were recovered from wound swabs (57.1%), followed from urine (38.6%) and the least percentage was (4.3%) from pleural fluid. The study shows that all isolates have β-hemolytic activity and are mostly    producing pigmentation, either pyocyanin (61.4%) or pyoverdin (37.1%) and more rarely the pigment pyorubin (1.4%).
Many studies have reported that most important virulence factors of P. aeruginosa are production of pigments, including mostly both pyoverdin and pyocyanin and blood hemolysis, which contribute to its pathogenicity during the infection process [27,28]. However, there are few studies comparing the incidence of pyocyanin and pyoverdin production of P. aeruginosa isolates according to their clinical sources. It has been demonstrated that pyocyanin has numerous antagonistic effects and damages host tissues in vitro and in vivo, including pro-inflammatory and free radical effects [29].
Treatment of infections caused by P. aeruginosa has become challenging in recent years due to the ability of this pathogen to rapidly develop resistance to multiple classes of antibiotics. Therefore, the investigation of the genetics of antimicrobial resistance of this organism may help to control and treat its infections by selection of useful antibiotics [30].
This study also showed high to low rates of resistance markers to certain commonly used drugs in treatment of Pseudomonas infections including azotreonam (70%), ceftazidime (47%), ciprofloxacin (23%), gentamicin (19%), piperacillin-tazobactam (16%), amikacin (13%) and imipenem (9%) ( Table  3). A previous study conducted also in Sudan [31], reported that P. aeruginosa isolates from clinical samples of patients was 49.3%. The study showed that 74 of the isolates were resistant to carbenicillin [31]. While a new study conducted in Sudan found a multidrugresistant (MDR) P. aeruginosa isolate from urine of patient with a urinary tract infection was highly resistant to many antibiotics including ciprofloxacin, gentamicin, ceftazidime, piperacillin, and meropenem [32].
The most important mechanisms of resistance to carbapenems are production of carbapenemase enzymes, which have the ability to hydrolyze penicillins, cephalosporins, monobactams and carbapenems. Carbapenem-resistant Klebsiella (KPC β-lactamases) hydrolyze most β-lactams, including carbapenems. [33,34]. Therefore, early detection of this gene will help to avoid incorrect treatment, controlling hospital infections, and preventing the increase spread of resistant strains in hospital and community [35].
The study didn't find any bla-VIM/IMP and KPC genes in P. aeruginosa isolates. Other mechanisms might be responsible for the resistance other than the KPC genes. It is interesting to note here that similar results to ours have been recently reported by a Jordanian study [2].
The study has demonstrated that all P. aeruginosa isolates carried potential AlgD and lasB genes (100%), followed by other toxic genes ( Table  6). All MDR isolates were highly associated with these virulence genes compared to less resistant isolates. However, no significant associations were found between antibiotic resistance markers and putative virulence genes neither among MDR nor all P. aeruginosa isolates from urine or wound samples P. aeruginosa excreted many virulence factors and forms biofilms that contribute to its infection process. Virulence of P. aeruginosa is multifactorial, involving both secreted and cell-associated bacterial products such as elastase, alkaline protease, protease IV, exotoxin A and exoenzymeS. However, most studies consider that exotoxin A, exoenzymeS and exoenzymeU are major virulence factors produced by most clinical P. aeruginosa strains, and the most toxic effects of these factors are causing host tissue damage [2,36].

Conclusion
This study demonstrated high rates of antimicrobial resistance markers to most commonly used antibiotics in the treatment of P. aeruginosa infections. There was no significant relationship discovered between antimicrobial resistance markers and