The freshwater samples were collected from Kat river is situated in Fort Beaufort (geographical coordinates: S 32° 47.071′ E 026° 38.916′) and Tyume river in Alice (geographical coordinates: S 32° 46.629′ E026° 50.149′) in the Eastern Cape Province, South Africa. Similarly, the mixed liquor samples were collected from two wastewater treatment plants located in Fort Beaufort and Alice. The plants are relatively small with design capacities of 2–3 ML/day and operate using activated sludge technology. While the Alice plant empties its final effluent into the Tyume River, the Fort Beaufort plant empties its effluents into the Kat River. The latest Green Drop report on both plants suggests that they are deserving of attention towards ensuring that they produce effluents of acceptable qualities [15]. These samples were transported in cooler boxes to the laboratory of the Applied and Environmental Microbiology Research Group (AEMREG) University of Fort Hare, Alice for microbiological analyses. Sampling was conducted once during the four seasons of the year (autumn, winter spring, and summer).

All freshwater and wastewater samples were serially diluted and 100 µL of the diluted samples were plated on Glutamate Starch Phenol-red (GSP) agar and incubated overnight at 37 °C. Pseudomonas-like isolates were counted, isolated and purified on fresh GSP agar. Purified isolates were thereafter transferred unto Nutrient agar plates and incubated overnight at 37 °C and thereafter screened based on typical morphology, catalase and oxidase reactions.

The purified isolates were grown on Nutrient agar for 24 h, and afterwards cells were harvested into 100 µL nuclease free water in 1.5 mL eppendorf tubes and homogenized by vortexing. The tubes were then placed in a heating block (Dri-block DB.2A, Techne, SA) at 100 °C for 10 min. After heating, the tubes were centrifuged at 25 °C for 3 min at 11,000 rpm (revolutions per minute) and immediately placed on ice. The supernatant was transferred to a new tube and used directly as DNA template for PCR assay [16]. Specific primers for Pseudomonas genus; PA-GS-F (5′-GACGGGTGAGTAATGCCTA-3′), and PA-GS-R (5′-CACTGGTGTTCCTTCCTATA-3′) were used in a 50 µL PCR reaction [17]. PCR conditions were as follows: 95 °C for 5 min, 10 cycles of 94 °C for 15 s, 53 °C for 30 s and 72 °C for 45 s; this was repeated for another 25 cycles with the exception of the 72 °C elongation step, which was increased by 5 seconds for every cycle; a final extension phase of 72 °C for 10 min was used. Pseudomonas aeruginosa reference strain ATCC 27853 was used as positive control and a reaction mixture containing Nuclease free water as negative control. The amplified PCR products of 617 bp were analysed by gel electrophoresis in 0.8% agarose gels stained with ethidium bromide (EtBr) 0.5 mg/L, for 1 h at 100 V in 0.5 × TAE buffer (40 mM Tris-HCl, 20 mM Na-acetate, 1 mM EDTA, pH 8.5) and then visualized and photographed with a imaging system Alliance 4.7 XD-79 (UVITEC Cambridge).

All isolates confirmed to belong to the Pseudomonas genus were further screened for three specific species of interest (P. fluorescens, P. aeruginosa and P. putida) selected based on on the dominance of these species from the results obtained on the preliminary identification carried out using API 20NE kit (data not shown) using the sets of primers listed in Table 1. The PCR conditions were as follows: P. fluorescens (2 min at 94 °C; 5 cycles consisting of 94 °C for 45 s, 55 °C for 1 min, 72 °C for 2 min; 35 cycles consisting of 92 °C for 45 s, 60 °C for 45 s, 72 °C for 2 min; final extension of 72 °C for 2 min; and final cooling at 4 °C); P. aeruginosa (95 °C for 1 min; 40 cycles of denaturation at 95 °C for 15 s, annealing at 58 °C for 20 s; final extension at 68 °C for 40 s); P. putida (initial denaturation at 95 °C for 10 min, then 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 90 s and extension at 72 °C for 7 min).

Antimicrobial susceptibility testing was performed using the disk diffusion method [17] with Muller-Hinton agar as the growth medium. Antibiotics were selected to represent some major classes of antibiotic and anti-pseudomonal antibiotics used as first line drug for pseudomonal infections. Antibiotics used in the study include penicillins (10 µg), clinamycins (2 µg), ciprofloxacin (5 µg), rafamycin (5 µg), trimethoprim (5 µg), sulphamethoxazole (25 µg), gentamicin (10 µg), chloramphenicol (30 µg), tetracycline (10 µg), erythromycin (15 µg), minocycline (30 µg), vacomycin (30 µg), cefotaxime (30 µg), nalidixic acid (30 µg), nitrofurantoin (300 µg), cephalothin (30 µg), ofloxacin (5 µg), ampicillin (25 µg), ampicillin-sulbactam (20 µg), oxacillin (1 µg). Disks were purchased from Mast Diagnostics (Mast Group, Merseyside, UK). Pseudomonas isolates were identified as susceptible, intermediate or resistant according to the National Committee for Clinical Laboratory Standard Guidelines (CLSI) [26].

The DNA of the Pseudomonas isolates was extracted following the method of Sambrook and Russell [16]. The set of primers used for the detection of antibiotic resistance genes are shown in Table 1. The PCR reaction was done in a total volume of 25 µL and the following conditions: bla_TEM gene (3 min at 93 °C, 40 cycles of 1 min at 93 °C, 1 min at 55 °C and 1 min at 72 °C and finally 7 min at 72 °C); bla_OXA gene and bla_ampC gene (94 °C for 5 min, 30 cycles of 25 s of denaturation at 94 °C, 40 s of annealing at 53 °C and 50 s of extension at 72 °C and a final cycle at 7 min at 72 °C); TetC gene (3 min at 94 °C, followed by 30 cycles of 1 min at 94 °C, 1 min at 65 °C and 1 min at 72 °C followed by 10 min at 72 °C); Integrons conserved segment (initial denaturation at 94 °C for 12 min, 1 min of denaturation at 94 °C, 1 min of annealing at 55 °C and 5 min of extension at 72 °C for a total of 35 cycles; five seconds were added to the extension time at each cycle).

Molecular identification of the Pseudomonas at both genus and specie levels were carried out using the sets of primers shown in Table 1. Sixty isolates were identified to belong to the Pseudomonas genus, twenty eight (46.7%) of which were from freshwater, and 32 (53.3%) were from the wastewater mixed liquor. Freshwater and wastewater samples collected in the four seasons (winter, autumn, spring and summer) showed incidences of Pseudomonas of 50% (autumn), 21.43% (spring) and 28.57% (summer) respectively in freshwater samples with none detected in winter (Table 2). Similarly, distribution of Pseudomonas with respect to species were 85.71% for P. putida (autumn) and 14.29% for other Pseudomonas species. In spring, the distribution was 33.33% each for P. putida, P. aeruginosa and P. fluorescens (Table 2). Furthermore, the analysis of the mixed liquor showed incidences of Pseudomonas at 81.25% (spring) and 18.75% (summer) alone, but at the species level, the following were observed; 66.67% (P. putida) and 33.33% (P. fluorescens) during summer, and 30.77% each for P. putida, P. flourescens and other Pseudomonas spp. and 7.69% for P. aeruginosa during spring (Table 3), respectively.

In general, with respect to the freshwater samples collected from Alice, 70% of the isolates recovered were P. putida, while the remaining 30% were equally (10% each) made up of P. fluorescens, P. aeruginosa and other Pseudomonas spp. For the Fort Beaufort water samples, P. putida constituted 75% of the isolates, while the remaining 25% were P. fluorescens. With respect to the mixed liquor samples from Alice, P. putida, and P. fluorescens made up 42.85% each of the isolates and the other Pseudomonas spp. constituted 14.29%. For the Fort Beaufort mixed liquor samples, the isolates composition includes 33.33% (P. putida), 22.22% (P. fluorescens), 11.11% (P. aeruginosa) and 33.33% (other Pseudomonas spp.).

The antibiograms of the Pseudomonas species are as shown in Table 4 and Table 5. All isolates (100%) from the two sites were susceptible to ciprofloxacin and gentamicin. Conversely, all (100%) were resistant to penicillin, oxacillin, vacomycin, trimethoprim, clindamycin and rifampicin. Varied resistances were observed against nitrofurantion as isolates from Alice showed the following resistance regimes; 60% (P. putida), 50% (P. fluorescens), 100% (P. aeruginosa) and 50% against other Pseudomonas spp. (Table 4). Unlike isolates from Alice, those from Fort Beaufort showed 100% resistance to nitrofurantoin. Isolates from Alice showed varied susceptibilities to cefotaxime in the order 60% (P. putida), 50% (P. flourescens), 100% (P. aeruginosa) and 50% (other Pseudomonas spp.). The antibiotic resistance pattern with respect to cephalothin was 100% each for P. putida, P. aeruginosa and the other Pseudomonas spp., and 75% for P. fluorescens. Ampicillin-sulbactam showed activity against P. putida (60%) and P. fluorescens (75%) (Table 4). Similarly, ofloxacin showed activity against P. putida (90%), P. flourescens (100%), P. aeruginosa (100%) and other Pseudomonas spp. (100%). Susceptibilities of isolates from Fort Beaufort to cephalothin were 50% (P. fluorescens), while all (100%) the P. putida, P. aeruginosa and the other Pseudomonas species were resistant (Table 5).

The screening for antibiotic resistance genes revealed the absence of bla_OXA, bla_ampC and TetC genes as they were not detected in any of the Pseudomonas isolates. However, Integron conserved segment was detected in freshwater samples in 10% of P. putida and 50% of P. fluorescens isolates. On the other hand, integron was found in isolates from mixed liquor in 33.33% (P. putida), 80% (P. flourescens), 100% (P. aeruginosa) and 50% for the other Pseudomonas spp. Similarly, bla_TEM gene was detected in these same organisms in the same proportion as Integron conserved segment.