Commensal Pseudomonas Species Isolated from Wastewater and Freshwater Milieus in the Eastern Cape Province, South Africa, as Reservoir of Antibiotic Resistant Determinants

Pseudomonas species are opportunistic pathogens with implications in a wide range of diseases including cystic fibrosis and sickle cell anaemia. Because of their status as multidrug resistant (MDR) and extremely drug resistant (XDR) bacteria Pseudomonas species represent a threat to public health. Prevalence, antibiogram and associated antibiotic resistant genes of Pseudomonas species isolated from freshwater and mixed liquor environments in the Eastern Cape Province of South Africa were assessed. Polymerase chain reaction (PCR) based technique was used to identify the isolates and screen for antibiotic resistant genes. The result shows occurrence of Pseudomonas spp. in freshwater and mixed liquor as follows: 71.42% and 37.5% (P. putida), 14.28% and 31.25% (P. flourescens), 7.14% and 6.25% (P. aeruginosa) and 7.14% and 25% for other Pseudomonas species respectively. Disk diffusion antibiogram of the Pseudomonas isolates from the two locations showed 100% resistance to penicillin, oxacillin, clindamycin, rifampicin and 100% susceptibility to ciprofloxacin and gentamicin with varied percentage resistances to cephalothin, nalidixic acid, tetracycline, and ampicillin. The blaTEM antibiotic resistant gene was detected in 12.5% of P. putida, 57.14% of P. fluorescens, 100% P. aeruginosa and 40% in other Pseudomonas species. Similarly, Integrons conserved segment were detected in 12.5% of P. putida, 57.14% of P. fluorescens, 100% of P. aeruginosa and 40% of other Pseudomonas species. The presence of blaTEM gene and integrons conserved segment in some of the isolates is worrisome and suggest Pseudomonas species as important reservoirs of multidrug resistance genes in the Eastern Cape Province environment.


Introduction
Antibiotic resistance by bacteria has been recognized as a major medical problem facing humankind and to prevent this scourge, the knowledge of their antibiotic susceptibilities, antibiotic resistance genes and their dissemination is required [1]. Most studies on antibiotic resistance in the environment have focused on enteric pathogens including Escherichia coli [2], Enterococci [3], Aeromonas spp. [4] and Campylobacter [5]. However, antibiotic resistant bacteria in the environments are autochthonous, and as reservoirs of antibiotic resistant determinants, they could perpetuate the spread of antibiotic-resistance genes to human and animal pathogens by horizontal gene transfer through such mobile genetic elements as plasmids, transposons and integrons [2], especially in wastewater treatment facilities (WWTP) [6,7]. Integrons are genetic elements that aid the acquisition and expression of gene cassettes in bacteria, most of them are involved in antibiotic resistance. WWTP have been reported as important reservoirs of antibiotic resistant organisms/determinants which could persist in the treated effluent and subsequently released into the natural environment [8][9][10] and thus impact on the ecology of antimicrobial resistance in bacterial populations [11][12][13]. However, reports of commensal bacteria including the pseudomonads as sources of antibiotic resistance determinants in the environment are rare.
Pseudomonas species are Gram negative motile rods belonging to the family Pseudomonaceae and found in various environments. Their ability to utilize different organic compounds as carbon and energy source as well as survival in the apparent absence of nutrients has been attributed to their genetic versatility which translates into enhanced metabolic activity with exceptional ability to adapt and colonize a wide variety of ecological niches including water, soil and rhizosphere [14].
Pseudomonas spp. are so well adapted in their environment that they survive extremes which includes temperatures ranging from 4 °C to 43 °C, and weak ion concentrations, among others. In this study, we assessed the incidence of Pseudomonas spp. in some freshwater environment and wastewater in the Eastern Cape Province of South Africa as well as the prevalence of antibiotic resistance genes in the isolates.

Sample Collection
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).

Isolation Processing of Samples
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.

Identification of Isolates by Polymerase Chain Reaction (PCR)
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).

Specie Specificity Screening of Pseudomonas isolates
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

Antibiotic Susceptibility Testing
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

PCR Detection of Antibiotic Resistant Genes
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 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 Isolates
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.

Identification of Antibiotic Resistance Genes
The screening for antibiotic resistance genes revealed the absence of bla OXA , bla amp C 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.

Discussion
The incidences of Pseudomonas species in the studied sampling sites appeared to be season dependent as variation in seasonal distribution reflected different recovery rates of the bacteria. Nevertheless, it must be appreciated that this recovery rates may not represent the total population of viable Pseudomonas species present in the samples, but selective for some species based on the incubation temperature used, especially considering that some Pseudomonas species such as P. syringae do not grow at temperatures above 30 °C. The absence of the Pseudomonas during winter suggests that the recovered isolates could not strive at low temperature in line with their mesophilic nature. Higher prevalence of Pseudomonas isolates were recovered during spring followed by summer especially in the mixed liquor samples, suggesting that warmer temperature favoured the recovery of these isolates. Freshwater samples from Alice consistently showed higher incidences of Pseudomonas species when compared to Fort Beaufort as evident from the incidence of 71.42% compared to that of Fort Beaufort (28.57%). Conversely, a relatively higher number of isolates was recovered in mixed liquor from Fort Beaufort (56.25%) as against Alice (43.25%). These variations may be attributed to human activities at various sites of the rivers, however; this explanation will not suffice for mixed liquor, although the limitations on overreliance on one primer pair/species for speciation of the Pseudomonas species must be appreciated. Similarly, the variation of incidence with season needs to be further investigated as it is not clear why season play a role in the occurrence of Pseudomonas species.
Resistance to different classes of antibiotics shown by the Pseudomonas species isolated from both freshwater and mixed liquor is an indication of the potential of the environment as a reservoir for antibiotic resistant organisms. Wastewater treatment process has been put forward as a potential vehicle for the selective enhancement and increase of multidrug resistant bacteria in the aquatic environment [8]. Although the findings of Gilliver et al. [27] in England reported on the occurrence of acquire antibiotic resistance characters in faecal bacteria from wild rodents in woodland sites and Pallecchi et al. [28] who studied a secluded population of the Peruvian Amazonas observed the presence of qnrB gene in commensal enterobacteria, both findings were with no prior exposure to antibiotics, as the areas were remote and devoid of human activities. Nevertheless the report by Osterblad et al. [29] and Thaller et al. [30] potentiate the presence of anthropic activities as a selective enrichment of multidrug resistant bacteria in the environment which is in accordance with the results of our findings, and suggests that the restriction of the misuse and overuse of antibiotics is a vital instrument in antibiotic resistance control.
The antibiotics, ciprofloxacin and gentamicin are the only broad spectrum antibiotics that showed high activities against all the isolates, while other antibiotics showed little or no activity against them, thus suggesting these isolates to be multi-drug resistant. However, an intriguing situation arose where all isolates were resistant to oxacillin but bla OXA gene was not detected in any of the isolates, despite that bla OXA codes for oxacillin resistance. Hence, it becomes obvious that resistance to antibiotics may be a function of more than one gene, or better still a combination of both genetic and environmental factors.
Li et al. [7] reported the presence of bla TEM in 17.3% of the bacteria isolated from penicillin production wastewater treatment plant effluent and 11% from the river downstream the plant, however, in these organisms, bla OXA gene was not detected. Similar trend was observed in our current study, however, bla TEM gene was detected only in isolates from mixed liquor, portending mixed liquor as a reservoir for antibiotic resistant genes. Also, noting that Pseudomonas is the most competent bacteria with regards to DNA uptake [31] and ability to produce transformants [32] in different environmental conditions, the chances that it picks up genes from the environment is high and this could very well be the source of these resistance genes in the isolates. The Pseudomonas isolates from freshwater and mixed liquor showed the presence of integrons, however those from mixed liquor similarly harbour bla TEM gene. The survival of bla TEM gene containing Pseudomonas species in wastewater treatment processes [33] could result in the dissemination of the β-lactamase genes into the environment and consequently increase the risk of the environment as reservoirs of antibiotic resistance determinants.

Conclusions
Pseudomonas species pose a threat to public health as it has been shown to harbour some antibiotic resistance genes. These resistance genes could be transferred to pathogenic organisms, and result in difficulty in treatment and limitation in treatment options. Although resistance is shown to various antibiotics; they have high susceptibilities to ciprofloxacin and gentamicin. Similarly, the presence of antibiotic resistance genes in these environmental isolates suggests Pseudomonas species as carriers and sources of antibiotics resistant genes with the potential to disseminate these genes into the environment for other organisms to pick up or transfer horizontally to other competent bacteria. A detailed assessment of the role of season on the incidence of Pseudomonas species, and how their antibiotic resistant genes contribute individually and collectively to antibiotic resistance is a subject of ongoing investigation in our group.