MULTIDRUG-RESISTANCE PATTERNS AND DETECTION OF PstS GENE IN CLINICAL ISOLATES OF PSEUDOMONAS AERUGINOSA FROM NSUKKA, SOUTHEAST NIGERIA

Objective: This study was aimed to determine the antibiotic resistance patterns of clinical Pseudomonas aeruginosa isolates and to detect the presence of PstS gene. Methods: One hundred and ninety-two clinical isolates of P. aeruginosa were characterized using polymerase chain reaction (PCR) and 16S rDNA sequencing. Antibiotic resistance patterns were determined using the disk diffusion method, while the minimum inhibitory concentrations (MICs) of selected antibiotics against resistant isolates were determined by macro broth dilution and E-test strip methods. The resistant isolates were screened for the presence of PstS gene using PCR. Results: Of 192 clinical isolates of P. aeruginosa , 136 (70.83%) were resistant to at least two antibiotics. Of these, 135 (99%) could be classified as multidrug-resistant P. aeruginosa (MDR-PA), 63 (46%) were extensively drug-resistant (XDR-PA), while 38 (28%) were pandrug-resistant (PDR-PA). The isolates exhibited high level of resistance to cefotaxime and ticarcillin, and low levels of resistance to meropenem and imipenem. The MIC values for meropenem against the resistant isolates were generally <32 mg/L, while the values for other antibiotics ranged from 32 to >128 mg/L. Multiple antibiotic resistance indexes of the MDR-PA ranged from 0.27 to 0.91 and the most prevalent pattern of resistance was Piperacillin R – Ticarcillin R – Piperacillin/Tazobactam R – Cefotaxime R – Ceftazidime R – Gentamicn R – Tobramycin R – Ciprofloxacin R . About 50% of the resistant isolates possessed the PstS gene. Conclusions: The results confirmed the presence of XDR, PDRPA, and PstS gene in P. aeruginosa strains. There is an urgent need for healthcare practitioners to address the problem of multidrug resistance, by implementing a more rational and appropriate use of antibiotics.


INTRODUCTION
Pseudomonas aeruginosa is a Gram-negative aerobic bacillus belonging to the Pseudomonadaceae family. It is highly ubiquitous in nature and may be found in most moist environments in the hospital, including sinks, cleaning buckets, drains, humidifiers, and toilet water [1]. P. aeruginosa is becoming an increasingly important cause of health care-associated infections [2][3][4][5]. It has been reported to be the sixth most common nosocomial pathogen overall and second most common pathogen in ventilator-associated pneumonia in US hospitals [4]. It has also been ranked second among Gram-negative pathogens reported to the United States National Nosocomial Infection Surveillance System [3]. P. aeruginosa constitutes a common pathogen in hospitals, particularly in intensive care units, due to its ubiquitous nature, ability to survive in moist environments, innate resistance to many antibiotics and antiseptics, and ability to acquire resistance to many drug classes [3,6,7]. It is a leading cause of septicemia, pneumonia, meningitis, wound, urinary tract, surgical wound, burn, and ear infections [8].
Resistance in Pseudomonas may be mediated through several distinct mechanisms, including the production of β-lactamases, efflux pumps, target site, and outer membrane modifications, but antimicrobial agents that have been found to be effective against P. aeruginosa include aminoglycosides (gentamicin, tobramycin, amikacin, and netilmicin); carbapenems (imipenem, meropenem, and doripenem); cephalosporins (ceftazidime and cefepime); fluoroquinolones (ciprofloxacin and levofloxacin); penicillins β-lactamase inhibitors (ticarcillin-clavulanic acid and piperacillin-tazobactam); monobactams (aztreonam); phosphonic acids (fosfomycin); and polymyxins (colistin and polymyxin B) [9,10]. Antibiotic resistant P. aeruginosa can be classified today Multidrug-resistant (MDR), extensively drug-resistant (XDR) or pandrug-resistant (PDR). MDR has been defined as a strain that develops resistance to at least one agent in three or more antimicrobial categories, XDR is a strain resistant to one agent in all categories except two or less, while PDR is a strain resistant to one agent in all categories [9,11].
Of all the anti-Pseudomonas agents, the carbapenems have been widely used for empirical or directed therapy, whenever MDR P. aeruginosa is suspected. However, recent evidence indicates that resistance to the carbapenems is emerging in P. aeruginosa and improper use of these drugs could accelerate the occurrence [4,12]. This is a global concern, not just for Pseudomonas, but for other bacterial pathogens. However, for the developing world, especially, this concern appears to be more reality than speculation. The concern arises from certain practices observed in the developing world, including unregulated use/sale of antibiotics, over-prescription of antibiotics, release of antibiotics into wastewaters by drug manufacturers, poor sanitation, poor infection control, misuse of antibiotics, and among others [13]. These problems can, however, be solved by better surveillance and monitoring of drug resistance. To this effect, the World Health Organization (WHO) has proposed a new global surveillance network, although there are still questions about how the initiative will be funded [13].
Considering the constantly evolving pattern of antibiotic resistance in P. aeruginosa and high mortality rates associated with Pseudomonas infections, it is of great public health importance to continuously track the occurrence and spread of antibiotic resistance in this rapidly emerging superbug. In a WHO global map of antibiotic resistance data from 194 member countries, Nigeria was one of the countries for which no information was obtained [13].
Studies have reported that expression of the PstS gene enhances the virulence of MDR Pseudomonas strains [14]. PstS proteins are the cell-bound phosphate binding elements of the ubiquitous bacterial ATP-binding cassette phosphate uptake mechanisms. This study was, therefore, undertaken to determine the prevalence of MDR among clinical isolates of P. aeruginosa in Nsukka, Southeast Nigeria, determine the patterns of resistance and screen the isolates for the presence of PstS gene, which has been reported to confer increased virulence on MDR P. aeruginosa (MDRPA).

The bacterial strains
A total of 192 strains of P. aeruginosa were collected from Microbiology Laboratories from various hospitals in Nsukka, from March 2016 to February 2017. Ethical approval and informed consent were not required by our Institution Ethics Committee because all bacterial isolates were collected, processed, and stored as part of routine diagnosis by the hospitals. No patient information was associated with the data. The isolates obtained from the various laboratories were subcultured onto Pseudomonas cetrimide agar (Oxoid, U.K) which was supplemented with 10 ml/l of glycerol and characterized using standard microbiological techniques such as grade like odor, pigment production, positive oxidase test, growth at 42°C, and molecular method using 16S rRNA primer targeting P. aeruginosa consensus region (Inqaba Biotechnical Company, South Africa).

Antimicrobial susceptibility test
One hundred and ninety-two P. aeruginosa isolates were used for in vitro susceptibility testing. Susceptibility to ten antibiotics, namelyticarcillin 75 µg; piperacillin 100 µg; piperacillin/tazobactam 100/10 µg; ceftazidime 30 µg; cefotaxime 30 µg; imipenem 10 µg; meropenem 10 µg; gentamicin 10 µg; tobramycin 10 µg; and ciprofloxacin 5 µg (Oxoid, UK) was evaluated on Muller-Hinton agar, using Kirby-Bauer disc diffusion method. The ten antibiotics represented six classes of antibiotic (Table 1). The isolates were considered susceptible or resistant according to the zone of inhibition recommended by the Clinical and Laboratory Standard Institute [15]. P. aeruginosa ATCC 27853 was used as the control strain. Isolates were considered MDR (MDRPA) if they showed resistance to three or more classes of the tested antibiotics.

Multiple antibiotic resistance (MAR) index
The MAR index was determined for each isolate by dividing the number of antibiotics to which the isolate is resistant by the total number of antibiotics tested [16].

MAR index =
Number of antibioticsisolateisresistant to Totalnumber rof antibioticstested

Determination of minimum inhibitory concentrations (MIC)
The MIC for the MDR P. aeruginosa was determined using macro broth dilution method and E-test method according to the CLSI standard [15]. P. aeruginosa isolates that were resistant to meropenem; ceftazidime; ciprofloxacin; and gentamicin were used for MIC study. Macro broth dilution was used to determine the MIC for ciprofloxacin and gentamicin while E-strip test was used for meropenem and ceftazidime.

Genomic DNA extraction
The genomic DNA was extracted from 20 MDR P. aeruginosa strain using Zymo research fungal/bacterial DNA Miniprep Kit (Zymo Research, USA) according to the manufacturer's protocol.

DNA amplification
The extracted DNA was amplified with 16S rRNA primer targeting P. aeruginosa consensus region and another primer targeting PstS gene that enhances the virulence of MDR Pseudomonas strains (Inqaba Biotechnical Company, South Africa),  (10 µM), each of the forward and reverse primers (Inqaba Biotechnical, South Africa), 5 µl of the extracted DNA, and 6.5 µl of sterile nuclease-free water (Norgen Biotek, Canada) to make up to 25 µl of reaction volume. This was vortexed at low speed and placed in a thermal cycler machine (BIBBY) -Scientific Ltd., UK. The parameters for amplification were as follows initial denaturation of 94°C for 5 min, followed by 35 amplification cycles of denaturation at

Data analysis
Frequency of MDRPA and percentage of resistance to antibiotics were calculated.

Analysis of MAR index
Analysis of the MAR index showed that the isolates had MAR indexes ranging from 0.27 to 0.91 (Table 4) while evaluation of the MIC of some of the antibiotics against the isolates ranged from 0.12 to 128 µg/ml ( Table 5).

Occurrence of PstS gene
The result of the PCR revealed that of 20 MDRPA isolates that were screened for PstS gene, lane 1, 3-7, 9, 10, 15, and 16 showed positive amplification of the 606 bp PstS gene (Fig. 1).

DISCUSSION
In this study, 192 clinical isolates of P. aeruginosa were screened against a panel of ten antibiotics, representing six classes of antibiotics.

Agbo et al.
The highest levels of resistance were recorded to the cephalosporin (cefotaxime and ceftazidime) and β-lactam (ticarcillin) antibiotics, while the lowest resistance levels were recorded to the carbapenems (meropenem and imipenem). P. aeruginosa is known to be intrinsically resistant to many antimicrobial agents including β-lactams, the older quinolones, chloramphenicol, tetracycline, macrolides, trimethoprimsulfamethoxazole, and rifampin [10,16]. Until recently, the most important antipseudomonal agents were some β-lactams (ticarcillin and piperacillin), cephalosporins (cefoperazone, ceftazidime, and cefotaxime), aminoglycosides (gentamicin, tobramycin, and amikacin), fluoroquinolones (notably ciprofloxacin), and β-lactam-β-lactamase inhibitor combinations [3,10]. Recently, however, there are increasing reports of emerging resistance to these antipseudomonal agents in clinical settings, worldwide [3,10,[17][18][19][20]. In this study, P. aeruginosa isolates exhibited high levels of resistance to different antibiotics used. Although fluoroquinolones are regarded as a good option in many parts of the world, in this study, the P. aeruginosa isolates exhibited high levels of resistance to this class of antibiotics.
The introduction of carbapenems into clinical practice represented a great advance for the treatment of serious bacterial infections caused by β-lactam resistant bacteria and they have been reported by many surveillance studies to be the drug of choice for MDR-P. aeruginosa [4,10,12,21]. In this study, the carbapenems (meropenem and imipenem) were found to be among the most active agents.
Although many studies have reported low resistance or outright 100% sensitivity for imipenem and meropenem [22][23][24][25][26][27][28][29], 12.5% and 24.5% resistance were recorded for both drugs, respectively, in this study, which is a cause for concern. It is also worrisome that about 46% of the clinical isolates in this study have gone beyond exhibiting mere MDR to extensive drug-resistance (XDR), while up to 28% may now be classified as PDR, as shown in Table 3. The most prevalent pattern of resistance among the isolates was PRL R -TIC R -TZP R -CTX R -CAZ R -CN R -TOB R -CIP R .
It is evident from these findings that a serious public health problem already exists in the environment under study, as a significant number of isolates is exhibiting resistance not only to the classical antipseudomonal drugs but also to the β-lactam-β-lactamase inhibitor combination and the last line carbapenem group. This can only indicate that this environment would be experiencing a high degree of treatment failure. The high degree of resistance recorded in this study is in agreement with a 2012 study in Southwest Nigeria [30]. It has been suggested that this trend may be due to selective pressure from the overuse and misuse of antibiotics, prevalent in the country.
Some authors have suggested that the MAR index may give an indirect indication of the probable source of an organism, an index of greater than 0.2, indicating that an organism must have originated from an environment where antibiotics are often used [16,31]. As can be seen in Table 4, all the MDR (XDR and PDR inclusive) isolates had MAR indexes ranging from 0.27 to 0.91. These observations tend to validate the assertion by some authors that antibiotic resistance is sweeping the developing world [13] and point to the need for tighter restrictions on the sale/use of antibiotics if the rapid spread of resistance is to be checked.
Studies have reported that expression of the PstS gene enhances the virulence of MDR Pseudomonas strains. The gene encodes a periplasmic phosphate-binding protein, which, when present, confers a highly virulent phenotype on MDR strains of P. aeruginosa. In addition, the development of MDR may be related to the overproduction of the PstS gene [14]. About 50% of the isolates in this study possessed the PstS gene, which further raises alarm about the impending public health crises due to P. aeruginosa in Nsukka, Southeast Nigeria.

CONCLUSIONS
There is the presence of XDR, PDRPA, and PstS gene in P. aeruginosa strains in Southeast Nigeria. The findings from this study show an alarming degree of resistance among P. aeruginosa isolates in Nsukka, Nigeria and an urgent need for healthcare practitioners and policymakers to address the problem of MDR by implementing a more rational and appropriate use of antibiotics. Establishment of an effective surveillance program and strict disinfection policy in hospital