Antibiotic-resistant staphylococci from the wastewater treatment plant and greywater samples in Obafemi Awolowo University , Ile-Ife , Nigeria

This study examined the occurrence and molecular basis for antibiotic-resistant staphylococci from the wastewater treatment plant and grey-water samples in Obafemi Awolowo University, Nigeria. Standard microbiological techniques and molecular methods were utilized. The species identified (MALDI score >1.7) comprised S. saprophyticus (19), S. cohnii (8), S. sciuri (7), S. aureus (4), S. epidermidis (3), S. warneri (2), S. equorum (1), S. haemolyticus (1), S. nepalensis (1), S. condimenti (1), and S. pasteuri (1). Resistance to trimethoprim, tetracycline and cefoxitin were observed in 78.3% (47/60), 36.7% (22/60) and 25% (15/60) of the isolates, respectively. The rate of multidrug resistance was 53.3% (32/60) and observed in eight species from different sampling sites. Seven (S. sciuri; n1⁄4 5; S. aureus; n1⁄4 1; S. warneri; n1⁄4 1) of the 20 selected (representing the various staphylococcal species and antibiotypes) isolates were mecA-positive. Furthermore, the tetK gene was detected in nine isolates, six with dfrA, and four were positive for the dfrG gene. One S. aureus was mecA, tetK and dfrG gene positive. The study provides insights on antibiotic-resistant staphylococci from a nonclinical setting and highlights the need for active surveillance to understand the burden of antimicrobial resistance in Nigeria. This is key to improve synergy across the human, animal and environmental health sectors in Nigeria.


INTRODUCTION
Wastewater (including grey-waterhousehold waste devoid of fecal matter) is an integral part of human activities. However, its indiscriminate discharge to various environmental receptors (stream/rivers, seas) has profound consequences on human health and ecosystems (Naidoo & Olaniran ). These adverse effects could be reduced by a combi- were employed for fusidic acid and mupirocin. Multidrug resistance (MDR) was defined as resistance to at least three classes of antibiotics.

Detection of antibiotic resistance and virulence genes
The resistance genes for trimethoprim (dfrA and dfrG), methicillin (mecA), and tetracycline (tetK) were investigated by PCR based on the widespread use of these antibiotics (trimethoprim and tetracycline) and clinical importance (methicillin) in Nigeria. Twenty isolates were selected based on their antibiotypes and to represent the various staphylococcal species identified in the study. A multiplex PCR reaction was performed for the detection of staphylococcal protein A (spa), Panton-Valentine Leukocidin (PVL) and methicillin resistance (mecA) genes as previously described (Larsen et al. ). A uniplex PCR was performed for the detection of the dfrA, dfrG, and tetK genes (Ng et al. ; Argudín et al. ). Thereafter, a multiplex PCR was developed for the detection of the above-mentioned genes. The multiplex PCR conditions were as follows: initial denaturation at 94 C for 5 minutes followed by 35 cycles of denaturation at 94 C for 60 seconds, annealing at 54 C for 60 seconds, and elongation at 72 C for 60 seconds. The final extension was at 72 C for 4 minutes. The primers for the detection of virulence and resistance genes are described in Table 1
One methicillin-susceptible S. aureus (MSSA) was PVL positive (Table 3) it indicates that the non-clinical environment could play a role in its transmission to humans (Porrero et al. ).
Although data on microbial exposure on WWTP workers are not available in Nigeria, the identification of a PVL-positive MSSA from WWTP in this study is also of concern, particularly for WWTP workers and individuals who could be exposed through inhalation or dermal exposure to reclaimed wastewater (Goldstein et al. ).
Trimethoprim and tetracycline are widely used in Nigeria due to their low cost and broad availability (Shittu et al. ). However, trimethoprim resistance in staphylococci has been reported to be as high as 85% in humans (Nurjadi et al. ; Ayepola et al. ). Furthermore, the prevalence of tetracycline-resistant staphylococci has been reported to be about 55% in human samples (Shittu et al. ). The molecular basis for trimethoprim resistance is attributed to these resistance (dfrA, dfrB, dfrG and dfrK) genes. Similarly, resistance to tetracycline is conferred by various mechanisms including efflux pump, enzymatic and ribosomal protections, but mainly through the tetK and tetM genes (Schwarz et al. ). In this study, resistance to trimethoprim and tetracycline was observed across different staphylococcal species. The multiplex PCR assay also provided some insights into their gene determinants. Resistance to trimethoprim was mediated by the dfrA and dfrG genes. This resistance determinant (dfrG) has been reported as predominant in clinical S. aureus isolates in Nigeria (Nurjadi et al. ). We observed that some isolates were resistant to trimethoprim but were dfrA and dfrG gene negative, and should be further investigated. In addition, the tetK gene was detected in most of the tetracycline-resistant S. sciuri (7) S. aureus (4)

ETHICAL STATEMENT
Formal approval of the ethics committee was not required as environmental samples were analyzed.

DATA AVAILABILITY STATEMENT
All relevant data are included in the paper or its Supplementary Information.
Figure 1 | Detection of the dfrA, dfrG, and tetK genes by multiplex PCR among staphylococcal isolates obtained from WWTP and grey-water samples.