Antibiotic Resistance Profiling of Pathogenic Staphylococcus Species from Urinary Tract Infection Patients in Benin

Staphylococci can cause urinary tract infections (UTIs). These UTIs are among the significant causes of antibiotic resistance and the spread of antibiotic-resistant diseases. The current study is aimed at establishing a resistance profile and determining the pathogenicity of Staphylococcus strains isolated from UTI samples collected in Benin. For this purpose, urine samples (one hundred and seventy) that were collected from clinics and hospitals showed UTI in patients admitted/visited in Benin. The biochemical assay method was used to identify Staphylococcus spp., and the disk diffusion method tested the antimicrobial susceptibility. The biofilm formation ability of the isolates of Staphylococcus spp. was investigated by the colorimetric method. The presence of mecA, edinB, edinC, cna, bbp, and ebp genes was examined by multiplex polymerase chain reaction (PCR). The results showed that Staphylococcus species were identified in 15.29% of all infected individuals and that 58% of these strains formed biofilms. Most Staphylococcus strains (80.76%) were isolated in female samples, and the age group below 30 years appeared to be the most affected, with a rate of 50%. All Staphylococcus strains isolated were 100% resistant to penicillin and oxacillin. The lowest resistance rates were seen with ciprofloxacin (30.8%), gentamicin, and amikacin (26.90%). Amikacin was the best antibiotic against Staphylococcus strains isolated from UTIs. The isolates carried mecA (42.31%), bbp (19.23%), and ebp (26.92%) genes in varying proportions. This study provides new information on the risks posed to the population by the overuse of antibiotics. In addition, it will play an essential role in restoring people's public health and controlling the spread of antibiotic resistance in urinary tract infections in Benin.


Introduction
In clinical settings, the most common infections are urinary tract infections (UTIs) that are the second most common after respiratory tract infections [1]. UTIs affect, worldwide, approximately 150 million people annually [2]. The prevalence of UTIs was 67.96% in Benin, with a predominance of women (62.50%) [3]. However, the etiology of these infections has been extensively studied in recent decades, and only a few bacterial species are considered authentic uropathogens [4]. Identification of bacteria and preparation of antibiograms are routine laboratory procedures [5]. Enterobacteriaceae and Gram-positive bacteria are the most isolated infectious agents in UTIs. Bacteria typically responsible for 5-10% of urinary tract infections are Enterococcus faecalis, Streptococcus agalactiae, and Staphylococcus spp. [6][7][8][9][10][11]. In sexually active women, the second most common cause of uncomplicated UTIs is Staphylococcus spp. [12]. The mainly isolated Staphylococcus species are S. saprophyticus, S. aureus, S. warneri, S. epidermidis, S. hominis, S. lentus, and S. haemolyticus [13]. After identifying pathogens, UTIs are treated with broad-spectrum antibiotics [14]. However, antibiotics' inappropriate use has increased antimicrobial resistance (AMR). Thus, the occurrence of AMR induces a negative course after antibiotic treatment among UTI patients and is responsible for severe clinical complications [15,16]. Multidrug-resistant of urinary tract infection isolates have been reported in Italy [11]. A study conducted in Afghanistan found increasing antibiotic resistance in staphylococci to ampicillin, amoxicillin, and erythromycin [17].
Strains of Staphylococcus spp. have been increasingly isolated from urine specimens, although urine is not a reservoir for staphylococci [18]. This leads to the consideration of staphylococci as urinary pathogens. Staphylococci have a simple technique to respond to the threats of a hostile host that opposes it with antibodies, phagocytosis, or cytotoxicity [19]. Staphylococcal infections rely on the production of surface proteins and toxins. Those toxins are used to (i) initiate adhesion of the bacterium to host tissues, (ii) secrete extracellular toxins and enzymes that destroy the host, (iii) inactivate the host immune system and the growth, and (iv) spread the bacteria in host cells and tissues [20]. Virulence factors associated with clinical staphylococcal infections include inhibitors of epidermal cell differentiation (edin), bacterial collagen adhesins (cna), bone-binding proteins (bbp), and activators of binding proteins (ebp). In addition, Staphylococcus spp. can form biofilms, which increase their virulence and antibiotic tolerance by a factor of 100 to 1000 compared with isolates that do not form biofilm [21]. Biofilm confers antibiotic resistance through processes including encoding antibiotic resistance genes, antibiotics, and even overcoming host immunity [22].
Due to the rapid increase in bacteria's adaptive strategies, a significant change in the etiology and antibiotic resistance profile of uropathogens bacterial has been noted [7,13]. The presence of Staphylococcus strains in urine, in conjunction with their resistance to antibiotics, threatens the prognosis of human life and poses a public health problem. New diagnostic and therapeutic strategies are needed for better treatment of urinary tract infections [23]. Against this background, the present study is aimed at drawing the antibiotic resistance profile and evaluating the virulence potential of staphylococci strains isolated from urine samples collected in Benin.

Material and Methods
2.1. Sampling. The formula from Schwartz [24]: n = t 2 × p ð1 − pÞ/m 2 was used to determine the sample size (n is the sample size, t is the 95% confidence level, p is the prevalence of urinary tract infections (11.7%) [25], and m is the 5% margin of error (typical value 0.05). Thus, 170 urine samples were collected from different health facilities ("Bon Samaritain" Clinic of Porto-Novo, Borgou/Alibori Departmental and University Hospital Center, Ouémé/Plateau Departmental and University Hospital Center, Mono/Couffo Departmental Hospital Center, Zou/Collines Departmental Hospital Center, Area Hospital of Djougou, Area Hospital of Ménontin, and Area Hospital of Natitingou) in Benin with sterile wide-mouthed urine cups. These specimens were collected from hospitalized and nonhospitalized patients complaining of urinary tract infections from 05 January 2021 to 30 October 2021. The request form completed by the physicians was used as standard protocol to document the sociodemographic data of study participants. Inclusion criteria were as follows: (i) participation agreement, (ii) presumed diagnosis of UTI, and (iii) no antibacterial therapy two weeks before participation.

Staphylococcus
Strains' Isolation and Identification. After collection, samples were subjected to standard microbiological tests, including fresh state and Gram stain [26]. The culture was then established on Chapman at 37°C for 24 hours, and colonies suspected to be Staphylococcus spp. were confirmed by DNase assay, catalase assay, and the use of API® Staph (bioMérieux, France) [26]. The isolated Staphylococcus spp. was confirmed by molecular biology using classical polymerase chain reaction (PCR) [27].

Bacterial Ability to Form Biofilms.
We used a qualitative assay to detect biofilm-producing Staphylococcus isolates [29,30]. The biofilm formation based on the appearance of a visible film was accessed in vitro on a microplate. Thus, from an 18-hour culture in Brain Heart Infusion, a microplate was inoculated with 10 μl of bacterial suspension supplemented with 150 μl of Brain Heart Infusion. The microplates were incubated at 37°C for 24 hours and then washed thrice to eliminate free bacteria. Crystal violet (0.1%) was used to stain for 10 minutes sessile organisms to the polystyrene support in each of the wells. An additional thorough washing with sterile distilled water was performed to remove excess stain, and the plates were dried at room temperature [31]. The appearance, after air-drying, of a visible film on the walls of the microplate indicated the formation of a biofilm.

Molecular Identification of Virulence Factors.
Virulence factors were identified by the molecular method of multiplex PCR (3Prime Thermal Cycler) on extracted DNA using specific primers (Table 1). DNA extraction and amplifications were carried out using the technic of Socohou et al. [30]. The obtained amplicons were separated using agarose gel electrophoresis in 1x TBE buffer with ethidium bromide. After migration at 100 V for 30 minutes, gels were visualized with a UV table. The genes sought were mecA, edinB, edinC, can, ebp, and bbp.
2.6. Data Analysis. Data were entered and analyzed using the MS Excel 2013 spreadsheet. The resistance percentage was calculated for each. R 4.2.1 software was used to analyze the resistant and biofilm-forming Staphylococcus species possessing virulence genes. The threshold for statistical significance was set at p < 0:05.

Frequency of Isolation of Staphylococcus spp.
Of the 170 urine samples included in this study, Gram stain evaluation and biochemical identification revealed that Staphylococcus species were found in 15.29% of the cases. Of the Staphylococcus spp. positive samples, 80.76% (n = 21) were from women. Analysis of Table 2 shows that the 20 to 30 years (50%) age group is the most commonly affected, followed by those between 31 and 40 years (48.15%). Of the isolated staphylococci, 73.07% were coagulase positive and 26.93% were coagulase negative ( Figure 1).

3.2.
Susceptibility to Antibiotics of Strains of Staphylococcus spp. Isolated. All isolates of Staphylococcus spp. were resistant to penicillin and oxacillin. Resistance was 96.2% to cotrimoxazole and 76.9% to Augmentin. The strains were 42.30% resistant to vancomycin. The lowest resistance was found in ciprofloxacin (30.8%), gentamicin, and amikacin (26.90%), a highly significant difference between the efficacy of antibiotics and UTI strains.

Discussion
UTIs are common in hospitals and the community and are caused by Gram-negative and Gram-positive bacteria. Our study shows that 15.2% of urine samples contained strains of Staphylococcus spp. This rate remains close to the 16.97% found in a survey conducted in a provincial hospital in northern Morocco [32]. However, lower rates (4.2% to 8.13%) were reported in studies conducted in Rabat [33], in the city of Nouakchott [34], and in Dakar [35]. These variations in the prevalence of Staphylococcus strains in urine may be due to the detection methods used in the laboratory and the geographic regions where samples were collected. The high proportion of staphylococci in our study indicates that Staphylococcus spp. are more common in the urine, may be considered uropathogenic, and need to be investigated for an excellent treatment strategy for these infections.

BioMed Research International
Among patients with urinary tract infections caused by Staphylococcus spp., we found a significant difference in gender distribution (men 19.24% and women 80.76%). These data are consistent with the epidemiological data reported in Benin [18] and elsewhere [36][37][38], which show that UTIs are more common in women than in men. There are several reasons for this preponderance of women: the anatomy of the female urinary tract, which consists of a short urethra that does not provide adequate protection against contamination from the vagina and rectum, the imbalance of the saprophyte flora in the urethra and vagina due to excessive hygiene, the prescription of estrogen-progestin treatments, the use of spermicidal gel, and the diaphragm. The effect of prostatic secretions allows for additional protection in men [39].
The age distribution in our study shows that the patients most commonly affected by urinary tract infections caused by Staphylococcus spp. are under 30 years old. In the literature, UTI is usually found in older people (>60 years) due to urinary stasis, estrogen deficiency in postmenopausal women, and decreased immune response in the elderly [40].
In our study, 73.07% of isolated staphylococcal strains were positive for coagulase (Staphylococcus aureus). This predominance of Staphylococcus aureus in urine samples was previously observed in Algeria at 74% [41]. Increased Gram-positive cocci in UTIs have also been reported [42]. In addition, coagulase-negative staphylococci play an essential role in diseases such as UTI [43]. These differences concerning our results could be explained by the timing of the  Figure 2: Coagulase-negative staphylococci (CNS) isolates' antibiotic resistance rate. 4 BioMed Research International study and sociodemographic parameters. The presence of S. aureus in the urine could be defined by its ability to adhere to the extracellular matrix's components and cells, releasing toxins such as enterotoxins, its virulence factors (synthetic bacterial amino acid), which is not usual in urine, and its ability to penetrate from the urethra into the bladder. The susceptibility of isolates has shown that resistance is present to varying degrees depending on the antibiotic family and the isolated species. Staphylococcus strains (coagulase-positive and -negative) show a relatively high resistance rate to penicillin, oxacillin, Augmentin, and cotrimoxazole. This observation is shared by other authors in Benin [3] and Morocco [44]. Coagulase-positive staphylococci (S. aureus) strains showed high resistance to vancomycin (52.6%). Compared to the vancomycin resistance rate, our results differ from those obtained in Mauritania and Morocco in S. aureus, where vancomycin was more active with a susceptibility rate of over 90% [33,34]. This  5 BioMed Research International difference could be explained by the degree of contact between the strains of S. aureus and this antibiotic in our country. Several hypotheses can explain the high antibiotic resistance profiles observed in our study, not only the excessive use of antibiotics in medical care and agriculture but also the lack of regulation of the marketing and acquisition of antibacterial agents. This is compounded by self-medication with often arbitrary dosages due to inadequate regulatory requirements and premature treatment discontinuation as soon as symptoms disappear. Trafficking of smuggled molecules is also a factor in the emergence of antibiotic resistance, with molecules often containing low doses or no active ingredients. Weak resistance of S. aureus strains has been recorded with ciprofloxacin, amikacin, and gentamicin. The Gabon study on urine samples showed low resistance rates of S. aureus isolates to amikacin and gentamicin [45]. A low resistance rate to ciprofloxacin, amikacin, and vancomycin was recorded in the CNS.
Many staphylococci can form biofilm. In our study, 58% of isolated staphylococcal strains formed biofilm. Our results are lower than the 87.87% obtained in Hungary on clinical strains [46] and the 88.70% obtained in Algeria for staphylococcal strains isolated from human infections, animals, and foods [47]. However, fewer biofilm formations than our results were obtained in Benin for S. aureus strains from surfaces and medical devices [30]. This difference could be due to the fact that the formation of biofilm is a complex phenomenon influenced by many factors, including the surrounding environment and genetic regulation factors [48]. Biofilm formation is found in S. aureus (67%) and coagulase-negative staphylococci (33%). Our  BioMed Research International biofilm-forming strains can therefore be considered pathogenic germs, as their virulence also lies in their ability to produce an extracellular matrix and form a biofilm [49]. Forming a biofilm can also hinder the action of antibiotics and lead to the persistence of infections. Staphylococci possess several factors that contribute to host cell destruction and thus increase the infection risk. Our study of some virulence factors revealed that our staphylococcal strains carried the genes encoding adhesion factors such as ebp (26.92%) and bbp (19.23%). The presence of these genes encoding adhesion factors at high levels in our isolated strains can be justified by the fact that S. aureus is an enteropathogenic bacterium [50]. Our staphylococcal strains' results on the nonproduction of the edin B, edin C, and cna genes are reassuring since the edin and cna exotoxins are essential virulence factors in host tissue invasion and promoting bacterial colonization during clinical infections.
In our study, 42.31% of Staphylococcus strains produced the mecA gene. This result is nearly 50.6% of the mecA gene detected in Brazil's S. aureus strains isolated from blood culture bottles [51]. Thus, the presence of the gene encoding for mecA gives a better idea of the methicillin-resistant strains isolated in our samples.

Conclusion
This study conducted in Benin provided information on the prevalence of Staphylococcus spp. involved in UTIs and their resistance rate to eight antibiotics. Women are most affected by urinary tract infections. Antibiotic resistance is relatively high for molecules such as penicillin, oxacillin, amoxicillin-clavulanic acid, and cotrimoxazole. However, amikacin, gentamicin, and ciprofloxacin showed good antimicrobial activity against Staphylococcus isolates in our study. The emergence of S. aureus strains with decreased vancomycin sensitivity may pose severe therapeutic problems. Indeed, these data can guide clinicians in choosing a first-line antibiotic. Still, a second antibiogram is necessary to verify the efficacy of the initial treatment and choose the best antibiotics for secondary treatment. These results also highlight the need for new therapeutic agents with new mechanisms of action. Antitoxin therapies aimed at reducing the virulence of bacteria may be a promising alternative. However, some factors determining Staphylococcus spp. pathogenicity have been identified; further exploration of virulence factors is a prerequisite for developing a comprehensive understanding of UTI Staphylococcus spp.

Data Availability
The data used to support the findings of this work are available from the corresponding author upon request.

Conflicts of Interest
The authors declare no conflicts of interest.