Molecular Characteristics and Antimicrobial Susceptibility Profile of Pseudomonas aeruginosa isolated from patients attending Healthcare Facilities in Mthatha, South Africa

Background Pseudomonas aeruginosa is a common pathogen causing healthcare-associated infections most especially in critically ill and immunocompromised patients. This pathogen poses a public health threat due to its innate resistance to many antimicrobial agents and its ability to acquire new resistance mechanisms under pressure. Infections with Extended spectrum β-lactamases (ESBL)-producing isolates result into outbreaks that lead to serious antibiotic management concerns with higher mortality and morbidity and significant economic causatives. In this study, we evaluated the antimicrobial resistance patterns and characterized genetically the ESBLs and Metallo- β-lactamases (MBL) produced by this pathogen. Methods Isolates of P. aeruginosa cultured from patients who attended Nelson Mandela Academic Hospital and other clinics in the four district municipalities of the Eastern Cape between August 2017 and May 2019 were identified; and their antibiotic resistance patterns were tested against amikacin, aztreonam, cefepime, ceftazidime, ciprofloxacin, doripenem, gentamicin, imipenem, levofloxacin, meropenem, piperacillin, piperacillin/tazobactam and tobramycin using the bioMérieux VITEK® 2 and confirmed by Beckman autoSCAN-4 System. Real-time PCR was done using Roche Light Cycler 2.0 to detect the presence of ESBLs; blaSHV, blaTEM and blaCTX-M genes; and MBLs; blaIMP, blaVIM. Results High antibiotic resistance in decreasing order was observed in piperacillin (64.2%), aztreonam (57.8%), cefepime (51.5%), ceftazidime (51.0%), piperacillin/tazobactam (50.5%), and imipenem (46.6%). A total of 75 (36.8%) multidrug resistant (MDR) isolates were observed of the total pool of isolates. The blaTEM, blaSHV and blaCTX-M was detected in 79.3%, 69.5% and 31.7% isolates (n=82),

Background Pseudomonas aeruginosa is a common pathogen causing healthcare-associated infections most especially in critically ill and immunocompromised patients. This pathogen poses a public health threat due to its innate resistance to many antimicrobial agents and its ability to acquire new resistance mechanisms under pressure. Infections with Extended spectrum β-lactamases (ESBL)producing isolates result into outbreaks that lead to serious antibiotic management concerns with higher mortality and morbidity and significant economic causatives. In this study, we evaluated the antimicrobial resistance patterns and characterized genetically the ESBLs and Metallo-β-lactamases Results High antibiotic resistance in decreasing order was observed in piperacillin (64.2%), aztreonam (57.8%), cefepime (51.5%), ceftazidime (51.0%), piperacillin/tazobactam (50.5%), and imipenem (46.6%). A total of 75 (36.8%) multidrug resistant (MDR) isolates were observed of the total pool of isolates. The blaTEM, blaSHV and blaCTX-M was detected in 79.3%, 69.5% and 31.7% isolates (n=82), respectively. The blaIMP was detected in 1.25% while no blaVIM was detected in any of the isolates tested.
Conclusions The study showed a high rate of MDR P. aeruginosa in our setting. The vast majority of these resistant isolates carried blaTEM and blaSHV genes. Continuous monitoring of antimicrobial resistance and strict compliance towards infection prevention and control practices are the best defence against spread of MDR P. aeruginosa. Background 3 P. aeruginosa is an opportunistic pathogen causing infections especially in immunocompromised patients. It is the leading cause of nosocomial infections such as urinary tract infections, surgical site infections, pneumonia, bacteremia and septicaemia [1][2][3] It is one of the ESKAPE pathogens that is most medically and epidemiologically significant and has been implicated as a principal cause of chronic lung infections in cystic fibrosis (CF) patients and severe infections in burn victims [4][5][6][7]. The World Health Organization (WHO) has categorized P. aeruginosa as a critical priority pathogen which needs urgent novel antibiotics intervention and has been given a serious threat level due to multidrug resistance displayed to many antibiotics [8,9]. The growing resistance of P. aeruginosa to several antibiotics, as a result of excessive antibiotic administration, has resulted to the accumulation of antibiotic resistance and cross-resistance between antibiotics and the advent of multidrug-resistant (MDR) forms of P. aeruginosa. P. aeruginosa infections are generally linked with high mortality; this is due to its innate resistance to several antimicrobial agents and acquired resistance via mutation and horizontal transfer [10,11] Various mechanisms involved in the resistance of P. aeruginosa include over expression of efflux pump, acquisition of Extended-Spectrum β-Lactamases (ESBLs) and Metalloβ-Lactamases (MBLs) [12]. ESBLs are a cluster of β-lactamases that inactivates β-lactams especially oxymino-β-lactams and monobactams, and are repressed by β-lactamase inhibitors, such as clavulanic acid. They are encoded on plasmids and can easily be conveyed from one organism to another [13,14]. ESBLs were first discovered in 1983 and ever since over 500 diverse β-lactamases have been described to date [15,16]. ESBL enzymes according to Ambler classification are categorized into two, A and D. The most prevalent enzymes in class A include bla TEM , bla CTX−M and bla SHV , and has been described in P. aeruginosa strains [13,17,18]. The emergence of betalactamase enzymes is majorly due to chromosomal mutation and procurement of resistance genes which are moved about on various mobile genetic elements (MGEs) such as bacteriophages, genomic islands, integrons, plasmids and insertion sequences [19]. The production of these enzymes is a going concern for infection control supervision because it restricts therapeutic choices. Continuous monitoring and timely detection of ESBL and MBL producing organisms is critical to establish suitable antimicrobial therapy and to thwart their spread [16]. Polymerase chain reaction (PCR)-based methods are critical to establish the prevalence and characterization of beta lactamases due to the presence of multiple resistance genes in some microorganisms [20]. Real-time PCR detection of ESBL enhances faster diagnosis and timely management of epidemiological information for monitoring outbreak situations [21]. Several studies have documented the antimicrobial resistance in P.
aeruginosa in South Africa have also been documented [26][27][28][29] but scarce data exist in the Eastern Clinical samples were sent from various primary and secondary clinics in the afore-mentioned municipalities to Mthatha at the NHLS for analysis.

Specimen collection and analysis
Non-duplicate P. aeruginosa isolates were collected from Mthatha, other clinics and hospitals from the four district municipalities. Specimens included throat swabs, wound swabs, swabs from abscesses, sputum, urine, blood culture and catheter tips. Demographic characteristics of patients and medical histories were collected from medical records including date of specimen collection, gender, age, test ordered and hospital/clinic. All samples were routinely cultured on MacConkey and Blood agar plates.
Blood and sputum were also cultured on chocolate agar. Suspected colonies were plated on Cetrimide agar and identified by gram staining, colony characteristics, motility, pyocyanin production and characteristics grape-like odour (30,31). Strains were identified to the species level with Vitek 2 GN (bioMérieux, Inc. USA) ID cards and confirmed by Microscan NID 2 panels (Beckman Coulter, Inc. USA).

Criterion for Multidrug resistance
The classification of MDR was performed according to Magiorakos et al., [35]. (MDR was defined as non-susceptibility to at least one agent in three or more antimicrobial categories).

Molecular confirmation of strains and Real-Time PCR for identification
DNA extraction was done using Roche MagNA Pure Bacteria lysis buffer, MagNA Pure Compact Nucleic Acid Isolation kit and PCR grade water (Roche Applied Science, Indianapolis), following manufacturer's instructions.
Real time PCR was carried out in the Light Cycler 2.0 instrument (Roche Applied Science, Germany) using Fast start Light Cycler 480 Hybprobes Master kit (Roche Diagnostics, USA). Specific primers targeting the genes gyrB were amplified by singleplex rPCR using primers and probes shown in Table  Germany). rPCR assay was performed according to previously published protocol [18]. Absolute quantification was carried out using the Light Cycler software 4.05. P. aeruginosa ATCC 27853 was used as a positive control.

Molecular ESBL and MBL Detection by Singleplex rPCR
Real-time PCR for bla CTX-M , bla SHV , bla TEM , bla IMP and bla VIM . Real time PCR was carried out in the Light Cycler 2.0 instrument (Roche Applied Science, Germany) using Fast start Light Cycler 480 Hybridization probes Master kit (Roche Diagnostics, USA). Specific primers and probes (Table 1) targeting the genes bla CTX-M , bla SHV , bla TEM , bla IMP and bla VIM were amplified by singleplex rPCR.
Primers were designed by TIB-Molbiol (Berlin, Germany). rPCR assay was performed in a 32 carousels  The isolates were confirmed by Vitek 2 system (bioMérieux, Inc., USA), Microscan autoscan-4 system (Beckman Coulter, Inc. USA) and rPCR using specific primer and probes targeting gyrB [39]. The majority of the isolates were recovered from male patients (60%) while 40% belonged to female patients. The isolates were predominantly from pus and wound swabs (80.4%) while the samples originated from Surgical (33.3%), General (18.1%) and Paediatrics (11.3%) wards. Table 2 General characteristics of patients and specimen from whose isolates were recovered  (Fig. 1) The study also revealed a total of seventy-five isolates (36.8%) were multidrug resistant out of the tested isolates while non-MDR constituted 63.2% of the total.
The relationship between specimen type and ESBL production revealed that sputum was found to be associated with ESBL-producing P. aeruginosa at 95% confidence interval range of 0.03-0.92 and OR of 0.16 (Table 6).
( Fig. 1). Low resistance in reducing order were observed in gentamicin (35.3%), meropenem (24.0%), amikacin (20.1%), fluoroquinolones (levofloxacin 19.1% and ciprofloxacin 11.3%), doripenem (11.3%), and tobramycin (8.3%). Data from surveillance on isolates of P. aeruginosa in the South African private laboratories is not in agreement with our study [40]. They reported a much lower resistance rates of 28.2%, 26.3%, 31.9%, 37.8%, 35.5% and 35.5% in cefepime, ceftazidime, doripenem, imipenem, meropenem and piperacillin respectively. The isolates were recovered from blood culture only, possibly this might account for the difference in resistance rate, alternatively this might be due to regional variations in the empirical use of these antimicrobials [40]. In our study, the percentage of resistance of 11.3% to ciprofloxacin was within the same range of 13.4% described by Ramsamy et al., [28]. The data obtained were from nine public sector hospitals in Kwazulu-Natal Province.
Additionally, gentamicin resistance of 17% and imipenem resistance of 13% as reported in the study was lower in comparison with resistance reported in our study at 35.3% and 46.6% respectively. The susceptibility ranges of 75%-92% of P. aeruginosa isolates in this study to routine antibiotics considered for therapy is encouraging but the increase in resistance exhibited to cephalosporins and imipenem is concerning. This might be due to selective pressure to those antibiotics and it will be important to monitor the prescription of these antibiotics. Owing to endless alteration, resistance exhibited to range of β-lactam antibiotics is challenging, thus making β-lactamase production the commonest cause of drug resistance and antimicrobial treatment tragedy [10,41]. Our study detected an average resistance of 45.7% to cephalosporins (ceftazidime and cefepime) in ESBL P. aeruginosa.
Piperacillin and gentamicin resistance in ESBL isolates was 52.4% and 40.2% respectively (Table 4) which is similar to the findings of Farhan et al., [10]. The emerging level of resistance displayed to the cephalosporins highlight the development of cephalosporinases among resistant strains of these organisms. The cephalosporins due to their wide spectrum of activity are a significant class of antimicrobials used in controlling several infections however, the emergence of cephalosporinases can in effect hamper their clinical usefulness [42]. The reported increasing penicillinase-producing β-lactamases strains among these organisms validates the noticeably observable high rate resistance of our isolates to piperacillin [42]. Antibiotic resistance is a public health menace with an alarming proportion that is getting collective attention more so that several studies have found a correlation between level of antibiotic prescription with the prevalence of antibiotic resistance [43][44]. Patients with resistant P. aeruginosa infections have a poor prognosis hence it is imperative that P. aeruginosa strains presenting severe drug resistance is monitored [23]. The swift spread and the emergence of MBL-and ESBLproducing P. aeruginosa of clinical origin is distressing and of great threat. Furthermore, level of antibiotic usage, horizontal gene transfer (HGT) event and environmental factors may account for variations in resistance patterns among strains isolated from diverse countries and regions. Antimicrobial susceptibility testing and proper screening for ESBL and MBL production has to be embarked upon before antimicrobial therapy [10]. In the present study, 36.8% of our isolates were MDR (defined as non-susceptibility to at least one agent in three or more antimicrobial categories). Similarly, MDR rate of 45% was reported by Fazeli et al., [45] however Sahoo et al., [46] reported a higher rate of resistance at 72.69%.
The emergence of ESBL-producing P. aeruginosa is increasingly reported as a major cause of health-care associated infections. In the hospital locale, infections resulting from these resistant organisms are increasingly challenging to treat due to the intensity of resistance exhibited to the most commonly recommended antibiotics.
Antimicrobial treatment is further hampered by the production of extended spectrum beta-lactamases and metallo beta-lactamases [20,47]. As at now, ESBLs in P. aeruginosa are described globally with MBLs also being reported on a growing basis in recent years [15,22]. Various studies have reported the existence of ESBLs in clinical isolates of P. aeruginosa [16,19,48]. Class A ESBLs are typically recognized in P. aeruginosa isolates presenting resistance to extended-spectrum cephalosporin. Traditional ESBLs have developed from restrictedspectrum class A TEM and SHV β-lactamases although a variety of non-TEM and non-SHV class A ESBLs have been described including CTX-M, PER, VEB, GES, and BEL [49]. The ESBL genotype differs in various parts of the world. This study found out that the most prevalent genotype for ESBL production was bla TEM which was detected in 65 (79.3%) isolates followed by bla SHV at 57 (69.5%) and bla CTX−M at 26 (31.7%) ( Table 3) Since it has been reported that ESBL genes show variation depending on the geographical location, the findings of Erhlers et al., [50], Chen et al., [15], Miranda et al., [51], Bokaeian et al., [52] and Khurana et al., [20] in South Africa, China, Brazil, Iran and India respectively corroborated the prevalent genotype to be bla TEM while contrarily Jamali et al., [53] and Rezai et al., [54] reported the prevalent gene to be bla SHV  and 29% respectively in VIM-2. MDR P. aeruginosa encoding bla VIM−2 gene have been reported in a tertiary hospital in Cape Town, which was responsible for an outbreak, and in a public hospital in Port Elizabeth [57,58].
The study of Arunagiri et al., [59] reported low rate of bla IMP in 2(3%) isolates which is similar to our study. The phenotypic resistance displayed to the carbapenems particularly imipenem which is not validated by the genotypic MBL result may be due to other resistance mechanism such as efflux over expression or forfeiture of exterior membrane protein [60,61].
Several researchers have reported on the concurrence of different β-lactamase genes found in the same isolates [46,49,62]. The most common ESBL combination among our isolates by genotype was a combination of bla TEM + bla SHV at 49 (40.5%) but contrary to Chen et al., [15] and Sahoo et al., [46], who reported the commonest to be bla SHV + bla CTX−M . The second most common genotype combination was a combination of bla SHV + bla CTX−M which is similar to the study from Syria and Philippines [63,64]. Our study showed the most predominant ESBL gene to be TEM which was corroborated by other studies. Prior to now, TEM used to be the most prevalent but recent reports suggest that the CTX-M-type group of ESBLs may now be the most predominant type globally [46,65].

Conclusion
Our study is the first surveillance report on molecular characteristics and antimicrobial susceptibility testing of P. The authors declare that they did not have any funding source or grant to support their research work.