*3.5. Prevalence of Antibiotic Resistance Genes*

Generally, a total of five of 10 ARGs tested were detected in one or more resistant isolates, with higher frequencies recorded in isolates recovered from seawater. Of the ten ARGs tested (*bla*Z, *mec*A, *rpo*B, *erm*B, *erm*A, *erm*C, *tet*K, *tet*M, *van*A and *van*B), the *blaZ* gene, coding for resistance to beta-lactam antibiotics (penicillin & ampicillin), was detected in 16 (55.2%, *n* = 29) of the isolates, the *mec*A gene, coding for methicillin resistance was detected in 5 (22.7%, *n* = 22), the *rpo*B gene, coding for rifampicin resistance, was detected in 11 (45.8%, *n* = 24), the *erm*B gene, coding for erythromycin resistance, in 15 (71.4%, *n* = 21) and the *tet*M gene, coding for tetracycline resistance, was detected in 8 (72.7%, *n* = 11) of the isolates. However, other ARGs such as *erm*A, *erm*C, *tet*K, *van*A and *van*B investigated were absent in the isolates. Table 2 shows the various ARGs detected in beach sand and water while Figure 3 shows a representative gel of the PCR amplified products for these genes.

**Figure 3.** Representative gel showing PCR amplified products of antibiotic resistance genes of *mec*A, *rpo*B, *bla*Z, *erm*B and *tet*M separated on 1.5% agarose. Lane 1: 100 bp DNA ladder (Fermentas Life Sciences, Vilnius, Lithuania), Lane 2: negative control, Lane 3, 4: *mec*A (499 bp) positive isolates, Lane 5, 6: *rpo*B (460 bp) positive isolates, Lane 7, 8: *bla*Z (173 bp) positive isolates, Lane 9, 10: *erm*B (142 bp) positive isolates and Lane 11, 12: *tet*M (142 bp) positive isolates.

The *fem*A gene, a factor also responsible for methicillin resistance [40], was identified in 53.3% (16/30) of the isolates. Figure 4 shows the gel electrophoresis of PCR amplified products for the *fem*A gene.

**Figure 4.** Representative gel showing PCR amplified products of *fem*A gene separated on 1.5% agarose. Lane 1: 100 bp DNA ladder (Fermentas Life Sciences, Vilnius, Lithuania), Lane 2: negative control, Lane 3–10: *fem*A (132 bp) positive isolate.


**Table 2.** Antibiotic resistance genes detected in *S. aureus* isolates from beach sand and seawater.

#### **4. Discussion**

Humans and animals have been reported as sources of antibiotic-resistant organisms in water environments and can transfer antibiotic resistance genes to other pathogens and naturally occurring water microbes through transposons, plasmids and integrons [47,48]. Bacteria isolated from beach sand, seawater and sediments have recorded resistance to various antimicrobials [48–52].

The occurrence of *S. aureus* and MRSA is on the rise, resulting in increased incidences of hospital-acquired and community-acquired infections worldwide, posing a major public health concern [53–55]. Moreover, microbial ecosystems can also be potentially altered by the presence of varying antibiotics of industrial origin, circulating in water environs [47]. *S. aureus* is one of the most successful and adaptable human pathogens due to its proficiency in acquiring antibiotic-resistant mechanisms and pathogenic determinants, leading to its emergence in both nosocomial and community settings [54]. Nosocomial colonisation of *S. aureus* and MRSA can go undetected, and signs of infection may only appear months after a patient is exposed to the infection. Infected patients may then serve as reservoirs for further transmission, especially as most of these strains carry SCC*mec* types coding for resistance to methicillin and other beta lactams [56].

To the best of our knowledge, this is the first study which has used a mixture of phenotypic and genotypic approaches simultaneously to determine the occurrence and antibiotic resistance profiles of *S. aureus* strains from beach water and sand in the study area. In this study, *S. aureus* was isolated from beach water and sand samples. Other studies have also reported this organism in marine water and/or sand [18,20–22,24,57–59], however, the frequency (12.2%) of isolation was lower in our study than observed in other studies [20,21,24]. This study only analyzed a single isolate for every sample, which could account for the lower detection frequency.

Of the isolates evaluated in this study, individual resistances of *S. aureus* to penicillin G and ampicillin was high (96.7%; 29/30). High resistance to these β-lactam antibiotics was not surprising, as ampicillin is one of the most commonly used antibiotics for treatment of infections in humans and animals [60], with penicillin developing resistance to *S. aureus* since the 1960s [61]. In addition, ampicillin-resistant isolates may cross-select for resistance to other beta-lactams [62]. Resistance to ampicillin may therefore indicate resistance of the isolates to other β-lactam antibiotics. This was observed in our study, as resistance to both ampicillin and penicillin occurred in equal proportion. Resistances observed to erythromycin, chloramphenicol, sulfamethoxazole-trimethoprim, and tetracycline were similar to that previously reported [21].

Given the relatively small number of isolates evaluated, a 50% vancomycin resistance was of concern, as this antibiotic is historically regarded as the antibiotic of final resort and the highest quality level antimicrobial for the treatment of genuine MRSA diseases [30]. The first case of a fully vancomycin-resistant *S. aureus* was described in Michigan, USA, in a renal dialysis patient [63]. The utilization of growth promoters such as tylosin, macrolide and avoparcin has been related to the occurrence of erythromycin and vancomycin resistance in *S. aureus* [64] in the environment, which might have then leached to marine waters. Based on the phenotypic identification of MRSA, 50% and 73.3% of the isolates were potentially MRSA by both methods. The poor specificity of the phenotypic methods in this study was not surprising, as higher specificity and sensitivity of these phenotypic methods have mostly been recorded in clinical isolates [38].

In this study, all *S. aureus* isolates were multidrug resistant. This point is worth noting, as it potentially could lead to failure in treatment therapy, prolonged illnesses, increased expenses for health care, and in serious cases, risk of death if humans are infected with such strains [65]. The transmission of resistance (R-factor), a plasmid-mediated genetic determinant, may be credited with the development of MAR among these isolates [62]. Studies have shown an upward pattern in the incidences of *S. aureus* isolates with multiple antibiotic resistance [66–69]. It has also been reported that *S. aureus* isolates with multiple antibiotic resistance attributes have a negative impact on the treatment of staphylococcal infections, especially in elderly, children, and immune-compromised individuals [70].

Generally, a total of five out of 10 ARGs tested were detected, with a higher frequency of detection in beach water compared to sand isolates. The higher frequency of detection in seawater could be because water is exposed to a greater variety of potential contaminants than sand. These may include runoffs from pharmaceutical, hospital, and industrial waste as well as farmlands [71,72]. Sources may also include antibiotic-resistant bacteria from poorly treated or untreated sewage, as final effluents of waste water treatment plants that may leach into seawater [52].

The *bla*Z gene is responsible for the production of β- lactamase enzyme, which confers resistance to β- lactam antibiotics such as penicillin and ampicillin [73]. This gene was only detected in small proportions compared to its phenotypic detection. Molecular confirmation identified the *mec*A gene only in five (22.7%) of the MRSA isolates detected by at least one of the phenotypic methods. The presence of this gene encodes a penicillin-binding protein 2a (PBP2a), responsible for methicillin resistance in staphylococci, with this protein, rendering a reduced affinity for β-lactam antibiotics [74]. Various studies have reported the occurrence of methicillin-resistant *S. aureus* from water sources, animal-derived food and humans [55,75,76]. MRSA has also been previously reported from marine waters [21,24] and waste water treatment plants [77]. Oxacillin has been proposed as a proxy antibiotic for testing susceptibility not only to methicillin and to all β-lactams [46], which could explain why all oxacillin-resistant isolates were not carrying the *mec*A gene. Phenotypic resistance observed to oxacillin in this study was probably achieved through other mechanisms [78], which may include alteration of the penicillin binding proteins, which brings about hyper-production of methicillinase or beta-lactamase [37,79,80].

The mode of resistance of rifampin is inhibition of the process of RNA polymerase [81]. Mutations on the gene encoding the β-subunit of RNA polymerase (*rpo*B gene) account for rifampin resistance (Rifr ) [82–84]. In our study, this gene was detected in 45.8% (11/24) of the rifampicin-resistant *S. aureus* isolates. Erythromycin resistance in staphylococci is mainly facilitated by the *erm* genes, coding for erythromycin resistant methylase [85], with *erm*A and *erm*C reported as the most frequently detected *erm* gene associated with staphylococci in human infections [86]. Results from this study however, detected *erm*B as the only gene coding for erythromycin resistance. The high incidence (72.7%) of

*tet*M in our study is similar to that (74.2%) earlier reported [87]. Another study has also reported the presence of both *tet*M and *tet*K gene from *S. aureus* isolates from public beaches [21].

The *fem*A gene was detected in 53.3% (16/30) of the confirmed *S. aureus* isolates. This gene is a chromosomally encoded factor in *Staphylococcus aureus*, which is crucial for the expression of advanced methicillin resistance, encoding proteins which influence the level of methicillin resistance [88]. Finding *fem*A gene in all *mec*A positive isolates is evidence that these isolates had a functional methicillin resistance. The detection of *fem*A together with *mec*A by PCR has long been considered a reliable indicator in the identification of MRSA [89].
