Antibiogram of Pseudomonas isolates and potential public health impact of an abattoir effluent in Benin City, Nigeria

Background: Bacteria from abattoir wastes are often linked to livestock carcasses previously exposed to continuous antimicrobial use and misuse; thereby creating opportunity for community spread of multidrug resistant (MDR) strains such as Pseudomonas spp. The aim of this study was to investigate the antibiogram of Pseudomonas isolates and bacteriological quality of an abattoir effluent in lieu of its potential public health impact. Methodology: Water samples were collected weekly for six weeks from discharge point (DP) of the abattoir effluent, effluent receiving canal confluence point (CP), and 500 m upstream (US) and 500 m downstream (DS) from points where CP made contact with the Ikpoba River, Benin City, Nigeria. Bacteria spp. were isolated, enumerated (heterotrophic bacterial plate, coliform, Escherichia coli and Pseudomonas counts) and identified using standard microbiological techniques. Identity of Pseudomonas isolates was confirmed by PCR while antibiogram of selected isolates was evaluated and interpreted according to the disk diffusion method of the Clinical and Laboratory Standards Institute (CLSI). Results: Heterotrophic bacteria plate counts (HPC) varied from 1.1×103 ± 0.28 CFU/ml to 1.95×106 ± 0.48 CFU/ml; total coliform counts ranged between 0.0 and 1.2×106 ± 0.28 CFU/ml while mean E. coli count varied from 0.0 to 4.9×105 ± 0.49 CFU/ml, and Pseudomonas counts were between 0.0 to 1.4×103 CFU/ml. The selected strains of Pseudomonas spp (n=50) showed resistance to oxacillin (100%), vancomycin (52%), tetracycline (50%), gentamycin (26%) and ceftriaxone (20%), while they were sensitive to ceftazidime (82%), ofloxacin (80%) and amikacin (74%). MDR phenotype was observed in 9 (18%) of the test isolates. Conclusion: The study revealed that untreated abattoir effluent was a considerable source of MDR Pseudomonas spp. among other bacteriological pollutants (e.g. HPC, coliform and E. coli) that could compromise the quality of the receiving river in lieu of public health concerns of riverside communities that depend on this vital water resource for their subsistence. Keywords: Pseudomonas; MDR; antibiogram; abattoir effluent; public healt

Wastewater treatment plants as a source of microbial pathogens in receiving watersheds

Wastewater treatment facilities have become sin quo non in ensuring the discharges of high quality wastewater effluents into receiving water bodies and consequence, a healthier environment. Due to massive worldwide increases in human population, water has been predicted to become one of the scarcest resources in the 21st century, and despite large advances in water and wastewater treatments, waterborne diseases still pose a major threat to public health worldwide. Several questions have been raised on the capacity of current wastewater treatment regimes to remove pathogens from wastewaterwith many waterborne diseases linked to supposedly treated water supplies. One of the major gaps in the knowledge of pathogenic microorganisms in wastewater is the lack of a thorough understanding ofthe survival and persistence of the different microbial types in different conditions and environments. This therefore brings to the fore the need for a thorough research into the movement and behavior of these microorganisms in wastewaters. In this review paper we give an overview of wastewater treatment practices with particular emphasis on the removal of microbial pathogens

Isolation and evaluation of autochthonous microalgae strains for biodiesel production and wastewater treatment.

Doctoral Degree. University of KwaZulu-Natal, Durban.The current depletion of global fossil fuel reserves and increasing CO2 emission are generating global climate change concerns hence, the increasing calls for the development of renewable biofuels with low CO2 emission. Microalgae has been touted as the most suitable feedstock of renewable fuels due to a number of reasons including high biomass productivity, fast growth rates, cost-effective cultivation systems and alleviation of food security concerns associated with use of other feedstock for biofuel production. Bioprospecting could yet reveal microalgae with high lipid contents for biodiesel production and novel characteristics such as production of unique bioactive compounds. With an attractive climate and plenty of sunlight all year round, South Africa is in a unique position to take advantage of these organisms to guarantee her future energy needs. This research thus aims to bioprospect for indigenous strains of microalgae in aquatic habitats and wastewater treatment plants in KwaZulu-Natal province of South Africa and evaluate their potential for biofuel production and wastewater remediation. DGGE and T-RFLP fingerprinting techniques were adapted to give an overview of the diversity of microalgae in various aquatic habitats including wastewater treatment plants (WWTP), rivers, estuaries and marine ecosystems in order to identify areas for potential bioprospecting. Ten (10) microalgae species from these aquatic environments were isolated, identified and evaluated for lipid, total carbohydrate and protein accumulation using well defined procedures. The lipid profile, biodiesel characteristics as well as effect of starvation on lipid accumulation were also determined. The biomass production and simultaneous phycoremediation potential of two autochthonous isolates Asterarcys quadricellulare and Neochloris aquatica were also investigated. Microalgae classes; Trebouxiophyceae and Chlorophyceae were observed to be dominant in fresh water habitat, while those belonging to the class Ulvophycea (Oltmannseillopsis spp.) dominated the salt water environment. At the brackish water habitat, microalgae belonging to the classes Trebouxiophyceae (Chlorella minutissima) and Chlorophyceae (M. sturmi) were dominant. A shift in community structure was observed at the maturation ponds of WWTPs and in rivers over time. The growth rates of 10 isolated microalgae ranged from 0.219 ± 0.003 to 0.175 ± 0.023 gL-1day-1 while dry weight ranged from 0.433 ± 0.208 to 1.167 ± 0.153 gL-1. Chlorococcum LM1 showed high accumulation of lipid (11.93 ± 0.76 mg/L). The microalgae isolated in this study accumulated high carbohydrate content ranging from 25 to 61 % of their dry weight while protein content ranged from 1.06 ± 0.1 mg/L to 1.39 ± 0.1 mg/L accounting for 21 to 28% dry weight. Lipid accumulation also varied under nutrient limitation condition. Lipid accumulation was enhanced in some of the isolates such as Chlorococcum sp. LM1 (17.2%) and C. sorokiniana NWS5 (24%) while a decrease was observed in others such as C. minutissima TS9 (16%), N. aquatica Toti4 (3.5%) and Chlorococcum sp. LM2 (6.5%). Response to enhanced lipid accumulation via starvation seem to be unique to each algal strain irrespective of species. The lipid profile consisted mainly of saturated fatty acid such as oleic acid (C18:1), palmitic acid (C16:0) and stearic acid (C18:0) with low amounts of polyunsaturated fatty acids such as linoleic acid (C18:2 n-6). Characteristic of the biodiesel based on the lipid profile reveal a low viscosity and density. The biodiesel was determined to be of good quality with high oxidation stability, low viscosity and conformed to the ASTM guidelines. Low total phosphorus concentration in the wastewater resulted in an unbalanced N:P ratio of 44 at the Northern wastewater treatment works (NWWTW) and 4 at the Umbilo wastewater treatment works (UWWTW). Asterarcys quadricellulare utilized the wastewater for growth and reduced the COD of the wastewater effluent from the UWWTW by 12.4% in contrast to Neochloris aquatica which did not show any growth. Asterarcys quadricellulare was able to utilise the wastewater achieving a growth rate up to 0.18 day−1 in sterilized wastewater from the NWWTW and 0.17 day−1 in the unsterilized wastewater from UWWTW. Asterarcys quadricellulare accumulated high biomass of 460 mg/L compared to 180 mg/L in Neochloris aquatica. Total nitrogen (TN) and Phosphorus (TP) were reduced by 48% and 50% respectively by Asterarcys quadricellulare cultivated in sterile wastewater from NWWTW while, Neochloris reduced the TP by 37% and TN by 29%. At the UWWTP, TP and TN were reduced by 32% and 44% respectively by Asterarcys quadricellulare cultivated in sterile wastewater while 29% and 19% reduction were recorded in Neochloris aquatica. The study showed the diversity and community structure of microalgae in aquatic ecosystems in the study area. Autochthonous microalgae were rich in lipid, carbohydrate and protein and could be applied for biofuel production. Wastewater effluent can be used to generate biomass for biodiesel production while treating wastewater. However, optimization of the N:P ratio and carbon source are necessary to improve remediation and biomass productivity for future commercial scale production

Phenotypic and genotypic characterization of Salmonella species isolated from treated wastewater effluents and receiving rivers in Durban.

M. Sc. University of KwaZulu-Natal, Durban 2013.Salmonella and Shigella spp. are major pathogens of humans and they cause diseases ranging from mild food poisoning to chronic diarrhoea, especially in children under the age of 5. They are commonly found in the gastrointestinal tract of animals and humans and contaminate water surfaces through faecal pollution. Discharge of inadequately treated wastewater has been known to be conduits of these pathogens to surface waters. Emergence of antibiotic resistant bacteria is a public health concern worldwide especially in developing countries where disease burden is high. This study investigated the efficiency of two Wastewater Treatment Plants (WWTPs) in Durban for wastewater treatment, and assessed the impact of treated effluent discharge on the receiving surface water. The genotypic characteristics and antibiogram profile of Salmonella spp. recovered from the treated effluent samples of the WWTPs and the receiving river was also determined. Water samples were collected from the WWTPs over a 12 month period and analyzed for physico-chemical parameters including temperature, pH, turbidity, BOD and COD using standard methods; while presumptive Salmonella and Shigella spp. were enumerated on Salmonella-Shigella and xylose-lysine-desoxycholate agar, respectively, via membrane filtration technique. Isolation of Salmonella spp. was done by enrichment of samples in Rappaort Vassiliadis soy broth followed by spread plating on Salmonella chromogenic agar and aerobic incubation at 37°C for 18 to 24 h. Presumptive isolates were biochemically characterized and confirmed via PCR amplification of the invA gene. Isolates were tested against 20 selected antibiotics to determine their antibiotic resistance profile. Presence of virulence markers; spiC, misL, orfL and pipD genes were also determined using PCR. Unacceptably high levels of turbidity (5.52-37.58 NTU), BOD (2.19-9.1 mg/l) and COD (67.67-294 mg/l) were observed in the water samples, while temperature (14°C-25°C) and pH (6.72-7.3) fell within the recommended maximum of 25°C and 10 7.5, respectively, for treated wastewater effluent. Significant positive correlation (p < 0.05) was observed between pH and BOD, temperature and COD, and between turbidity and presumptive Salmonella count. Presumptive Salmonella and Shigella spp. were prevalent at all sampling points, with population ranging from 8.5×102 to 1.59×105 CFU/ml and 0.1×102 to 7.5×103 CFU/ml, respectively. The isolates were highly susceptible to β-lactams, Chloramphenicol, Tetracycline, Quinolones and Trimethoprim-Sulfamethoxazole (99% to 100%). Complete antibiotics resistance was observed against Sulfamethoxazole (100%), Nalidixic acid (27%) and Streptomycin (14%). Intermediate resistance was observed against Streptomycin (74%), Nalidixic acid (44%) and Fosfomycin (8.5%). Of the 200 isolates tested, 93% harboured the spiC gene, 84% harbored the misL gene, while 87.5% and 87 % of the isolates harboured the orfL and pipD gene, respectively. Results from this study indicate the inefficiency of the WWTPs investigated to totally eradicate Salmonella spp. from the final effluent and discharge of such effluent. Discharge of these effluent to surface water resources could pose health threat to the end-users of the surface water for daily domestic and recreational activities . Thus, appropriate intervention by the regulatory agencies is required to ensure compliance of WWTPs to the stipulated guidelines for safe disposal of treated effluent

In vitro assessment of antioxidant, phytochemical and nutritional properties of extracts from the leaves of Ocimum Gratissimum (linn).

The antioxidant, phytochemical and nutritional properties of acetone, methanol and aqueous extracts of the leaves of Ocimum gratissimum (Linn) were investigated to evaluate the therapeutic and nutritional potential of the leaves of this plant. The antioxidant of the plant extracts were assessed against 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and 2,2-azinobis-(3- ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and ferric reducing agent. Total phenolics, flavonoids, flavonols and proanthocyanidins were determined to assess their corresponding effect on the antioxidant activity of this plant. The extracts exhibited DPPH and ABTS.+ radical scavenging activities, which was comparable to butylated hydroxytoluene (BHT). The phytochemical screening revealed the presence of alkaloids, tannins, saponin, steroids, cardiacglycoside, flavonoid, terpenoids and phenol. The proximate analysis confirms that the leaves contain appreciable amount of ash, crude protein, lipids, fibre and carbohydrates. The macro and micro elements and constituents revealed that the leaves contain significant amount of sodium, potassium, calcium, magnesium, iron, zinc, phosphorus, copper, nitrogen, and manganese. This study shows that the leaf can be used as a therapeutic agent and justifies its application in folkloric medicine.Keywords: Ocimum gratissimum, oxidative stress, polyphenolic, proximate composition, therapeutic activit

Prevalence of listeria pathogens in effluents of some wastewater treatment facilities in the Eastern Cape province of South Africa

Wastewater discharges may contain health compromising pathogens and carcinogenic and/or chemical substances that could compromise the public health and impact negatively on the environment. The present study was conducted between August 2007 and July 2008 to evaluate the Listeria abundance (as free-living and plankton associated species) and physicochemical qualities of the final effluents of three wastewater treatment facilities in the Eastern Cape Province of South Africa selected to represent typical urban, peri-urban and rural communities and the impact of the discharged final effluents on their respective receiving watershed, as well as to elucidated the in vitro antibiotic susceptibilities and resistance genes profile of Listeria species isolated from the final effluents. The suitability of the secondary effluent of the urban treatment facility (as a case study) for use in agriculture and aquaculture with reference to recommended standards was also determined. Wastewater samples were collected from the raw sewage, secondary effluent, final treated effluent, discharge point, 500 m upstream discharge point, and 500 m downstream discharge point from all three locations on a monthly basis throughout the study period. Listeria abundance in the final effluents and the receiving watersheds varied between 2.9× 100 and 3.52 × 105cfu/ml across the sampled locations. Free-living listerial density across the sampled locations ranged between 0 and 3.2 × 103cfu/ml while counts of Listeria species attached to large (180 μm) planktons varied from 0 to 1.58 × 105 cfu/ml and those of the 60 and 20 μm categories were in the range of 0 to 1.32 × 103 cfu/ml and 0 to 2.82 × 105 cfu/ml respectively. Listeria abundance did not vary significantly with location and season; there was however, significant (P < 0.05; P < 0.01) variance in Listeria abundance with plankton sizes across the locations. Free-living Listeria species were more abundant in the rural and urban xii communities than plankton attached Listeria species; whereas the reverse was the case in the peri-urban community. Prevalence of Listeria in terms of total counts was 100 percent across all sampled locations. Free-living Listeria species showed prevalence ranging from 84-96 percent across the sampling locations; while Listeria species attached to large (180 μm) planktons exhibited prevalence ranging from 75 percent to 90 percent. The prevalence of medium-sized (60 μm) plankton associated Listeria species varied between 58 percent and 92.5 percent; whereas those of Listeria species attached to small (20 μm) planktons ranged from 65-100 percent across all three communities. Listeria prevalence was generally a reflection of the turbidity of the water system, with free-living Listeria species being more prevalent than plankton associated cells in the relatively less turbid rural and urban waters compared to the more turbid peri-urban waters where plankton attached cells were more prevalent in comparison with their free living counterparts The final treated effluent quality fell short of recommended standards for turbidity, chemical oxygen demand and phosphate across all three communities. In addition, the final effluent of the rural treatment plant also fell short of recommended standard for NO3, while that of the urban treatment plant did not comply with acceptable limits for dissolved oxygen and nitrite. Other physicochemical parameters were compliant with set standards after treatment. An inverse relationship was observed between chlorine residual and listerial density across the sampled facilities; the effect of chlorine was however not enough to eliminate the pathogen from the water systems. At the urban treatment plant and its receiving watershed, pH, temperature, EC, turbidity, TDS, DO, and nitrate varied significantly with season and sampling point (P < 0.05; P < 0.01). Salinity also varied significantly with sampling point (P < 0.01), while COD and nitrite varied significantly with season (P < 0.05). Although, the treated effluent fell within recommended water quality standard for pH, TDS, nitrate and nitrite, it fell short of stipulated standards for other parameters. Whereas the microbial quality of the secondary treated effluent at this (urban) facility fell short of recommended standard after secondary treatment, its physicochemical quality were generally compliant with recommended standards for reuse wastewater in agriculture and aquaculture. Listeria pathogens isolated from effluents of the rural wastewater facility were sensitive to 11 (55 percent) of the 20 test antibiotics, and showed varying (7-71 percent) levels of resistance to 8 antibiotics; whereas those isolated from the peri-urban community showed sensitivity to 6 (30 percent) of the 20 test antibiotics, and varying (6-94 percent) levels of resistance to 12 antibiotics; while the urban effluent isolates were sensitive to 3 (15 percent) of the 20 test antibiotics, and showed varying (4.5-91 percent) levels of resistance to 17 antibiotics. Multiple antibiotic resistances involving 78.5-100 percent of isolates and antibiotics combination ranging from 2-10 antibiotics was observed across the sampled locations. Penicillin G and ampicillin showed remarkably high (64-91 percent) phenotypic resistance across the three sampled facilities. Other antibiotics, to which isolates showed significant resistance, were linezolid (22-88 percent); erythromycin (43-94 percent) and sulphamethoxazole (7-94 percent). Two of the 14 Listeria strains isolated from the rural effluents were positive for ereA and sul1 antibiotic resistance genes; while sulII genes were detected in five of the 23 Listeria isolates from the urban effluent and none was detected in isolates from the peri-urban community. The presence of antimicrobial resistance genes in the isolates did not correlate with phenotypic antibiotic resistance. The current study demonstrated that Listeria pathogens easily survived the activated sludge treatment process as free-living and plankton attached entities and suggests that municipal wastewater treatment plants are a significant source of multiple resistant Listeria pathogens in the South African aquatic milieu. While the physicochemical quality of the urban final effluent suggests that it is a major source of pollution to the receiving watershed, the secondary effluent quality demonstrated a great potential for use in agriculture and aquaculture

Assessment of physicochemical qualities, heavy metal concentrations and bacterial pathogens in Shanomi Creek in the Niger Delta, Nigeria

The physicochemical and microbial qualities of Shanomi creeks in the Niger Delta of Nigeria were assessed between January and October 2011. The temperature across sampling stations ranged between 26 and 27.7‹C, while pH varied from 7.49 to 8.74. Turbidity ranged from 176.62-189.96 NTU and conductivity varied between 360.45 and 454.88 ƒÊS/cm. The concentrations of other physicochemical parameters were as follows: BOD (6.39-7.64 mg/L) COD (84.25-97.27 mg/L); ammonia (26.83-33.98 mg/L); nitrate (37.25-43.89 mg/L); nitrite (37.35-41.75 mg/L); and phosphate (28.83-37.85 mg/L). The relative dominance of metals in the water followed the sequence: Al &gt; Zn &gt; Cu &gt; Fe &gt; Mn &gt; Cd &gt; Pb &gt; Hg &gt; As. Feacal and total coliform densities ranged from 1.05 ~ 102 to 4.25 ~ 103 (cfu/mL) and 1.56 ~ 102 to 6.40 ~ 104 (cfu/mL) respectively. The study reveals that the water under study was heavily polluted and of serious threat to the aquatic biota and public health.Key words: Aquatic biota, contamination, pollution, public health, microbial indicators, toxic effects

Antibiotic Synergy Interaction against Multidrug-Resistant Pseudomonas aeruginosa Isolated from an Abattoir Effluent Environment

Pseudomonas aeruginosa is an opportunistic pathogen in environmental waters with a high prevalence of multidrug resistance. In this study the synergistic efficacy of synergy antibiotic combinations in multidrug-resistant P. aeruginosa strains isolated from an abattoir effluent was investigated. Water samples were processed using membrane filtration; Pseudomonas was isolated with Pseudomonas Isolation Agar and confirmed using polymerase chain reaction with specie-specific primer. Susceptibility studies and in vitro synergy interaction testing were carried out, employing agar dilution and Etest procedure, respectively. Resistance was noted for clinically relevant antipseudomonal agents tested. Finding from antibiotic synergy interaction studies revealed that cefepime, imipenem, and meropenem combined with amikacin resulted in statistically significant (P < 0.0001) in vitro antibiotics synergy interaction, indicating the possible use of this regimen in treatment of pseudomonal infections

Municipal Wastewater Effluents as a Source of Listerial Pathogens in the Aquatic Milieu of the Eastern Cape Province of South Africa: A Concern of Public Health Importance

We evaluated the effluent quality of an urban wastewater treatment facility in South Africa and its impact on the receiving watershed for a period of 12 months. The prevalence and antimicrobial susceptibility of potential Listeria pathogens (L. ivanovii and L. innocua) and the physicochemical quality of the treated wastewater effluent was assessed, with a view to ascertain the potential health and environmental hazards of the discharged effluent. Total listerial density varied between 2.9 × 100 and 1.2 × 105 cfu/mL; free living Listeria species were more prevalent (84%), compared to Listeria species attached to planktons (59–75%). The treated effluent quality fell short of recommended standards for turbidity, dissolved oxygen, chemical oxygen demand, nitrite, phosphate and Listeria density; while pH, temperature, total dissolved solids and nitrate contents were compliant with target quality limits after treatment. The Listeria isolates (23) were sensitive to three (15%) of the 20 test antibiotics, and showed varying (4.5–91%) levels of resistance to 17 antibiotics. Of seven resistance gene markers assayed, only sulII genes were detected in five (22%) Listeria strains. The study demonstrates a potential negative impact of the wastewater effluent on the receiving environment and suggests a serious public health implication for those who depend on the receiving watershed for drinking and other purposes

IN VITRO ASSESSMENT OF ANTIOXIDANT, PHYTOCHEMICAL AND NUTRITIONAL PROPERTIES OF EXTRACTS FROM THE LEAVES OF OCIMUM GRATISSIMUM (LINN)

The antioxidant, phytochemical and nutritional properties of acetone, methanol and aqueous extracts of the leaves of Ocimum gratissimum (Linn) were investigated to evaluate the therapeutic and nutritional potential of the leaves of this plant. The antioxidant of the plant extracts were assessed against 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and ferric reducing agent. Total phenolics, flavonoids, flavonols and proanthocyanidins were determined to assess their corresponding effect on the antioxidant activity of this plant. The extracts exhibited DPPH and ABTS.+ radical scavenging activities, which was comparable to butylated hydroxytoluene (BHT). The phytochemical screening revealed the presence of alkaloids, tannins, saponin, steroids, cardiacglycoside, flavonoid, terpenoids and phenol. The proximate analysis confirms that the leaves contain appreciable amount of ash, crude protein, lipids, fibre and carbohydrates. The macro and micro elements and constituents revealed that the leaves contain significant amount of sodium, potassium, calcium, magnesium, iron, zinc, phosphorus, copper, nitrogen, and manganese. This study shows that the leaf can be used as a therapeutic agent and justifies its application in folkloric medicine