Inappropriate use and selection of antimicrobial agents are risk factors for the ongoing development of genetic resistance leading to a notable increase in bacterial pathogens that are multidrug-resistant (MDR) and even extremely drug-resistant (XDR) [1]. These bacteria have become a global burden resulting in an increase in morbidity, mortality, and healthcare costs [1, 2]. In the past few years, reports have shown an unprecedented increase in the risk of antibiotic resistance. For example, the US Centers for Disease Control and Prevention has identified antibiotic resistance as one of the global public health problems, with tens of thousands of Americans dying each year. As a result, this subject has become a hot topic for researchers [3]. The World Health Organization (WHO) released a report in February 2017 on antibiotic-resistant bacteria to highlight the importance of this issue and encourage researchers to develop new and effective antimicrobial drugs [4]. Studies have revealed that the availability of documented information about antimicrobial use and bacterial resistance helps in selecting appropriate antibiotics and reducing antibiotics-associated adverse effects [1]. Little is known about antibiotic resistance in developing countries, particularly in Iraq. Therefore, this study aims to explore potential bacterial species that are resistant to antimicrobial drugs used in health centers and hospitals in Najaf city, Iraq.

A total of 165 subjects were collected, including 42 males and 123 females. The sampling period was four months, commencing in September 2022. Bacteria were evaluated semi-quantitatively to determine bacterial growth and distinguish them from infections.

The prepared solutions in this study were sterilized at 121 \(^{\circ}\)C for 15 minutes for heat-tolerant solutions, while sensitive solutions were sterilized using 0.22 \(\mu\)m Millipore filters. The acidity of the medium was adjusted using 1 mol of sodium hydroxide or 1 mol of hydrochloric acid. Gram stain reagents were prepared. The materials were prepared as follows: to complete the volume to 100 mL, 21 mL of 70% hydrogen peroxide (HO) was added to 50 mL of distilled water (DW), and 0.1 g of N, N, N, N-tetramethyl-P-phenylene dihydrochloride was dissolved in 10 mL of DW to determine the ability of bacteria to produce the lactase enzyme [1]. Blood agar, Mannitol agar, and MacConkey agar were prepared and sterilized according to supplier instructions. Samples were directly inoculated and spread on the aforementioned media plates. These plates were then incubated for 24 hours at 35 \(^{\circ}\)C and subcultured for further identification [2]. The oxidase test was prepared to detect the ability of bacteria to produce the oxidase enzyme.

In this study, Staphylococcus aureus isolates were subjected to antimicrobial drug resistance testing using the Kirby-Bauer method for disc diffusion. After culturing, the samples were incubated in Mueller-Hinton broth at 37 \(^{\circ}\)C for 24 hours, and various antimicrobial discs were placed on the agar plates within 15 minutes. The zones of inhibition and results were measured based on laboratory institute standards.

The antibiotics tested for multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan drug-resistant (PDR) Staphylococcus aureus isolates included aminoglycosides (gentamicin, rifampin/rifampicin), ceftaroline, oxacillin (or cefoxitin), fluoroquinolones (ciprofloxacin, moxifloxacin), fusidic acid, trimethoprim/sulfamethoxazole, glycopeptides (vancomycin, teicoplanin, telavancin), tigecycline, clindamycin, daptomycin, erythromycin, linezolid, chloramphenicol, fosfomycin, quinupristin-dalfopristin, tetracyclines (doxycycline, minocycline). For Enterococcus spp., antibiotics tested for MDR, XDR, and PDR included gentamicin, streptomycin, carbapenems (imipenem, meropenem, doripenem). For Enterococcus faecium, the antibiotics were ciprofloxacin, levofloxacin, moxifloxacin, vancomycin, teicoplanin, tigecycline, daptomycin, linezolid, ampicillin, quinupristin-dalfopristin. For Enterococcus faecalis, the antibiotics were tetracycline, doxycycline, minocycline. Antibiotics tested for MDR, XDR, and PDR in Enterobacteriaceae isolates included amikacin, ceftaroline, antipseudomonal penicillins with $\beta$-lactamase inhibitors, ticarcillin-clavulanic acid, piperacillin-tazobactam, ertapenem, imipenem, meropenem, doripenem, cefotaxime, ceftriaxone, ceftazidime, cefepime, cefoxitin, ciprofloxacin, trimethoprim-sulfamethoxazole, aztreonam, ampicillin, amoxicillin-clavulanic acid, ampicillin-sulbactam, chloramphenicol, fosfomycin, polymyxins (colistin). Antibiotics tested for MDR, XDR, and PDR in Pseudomonas aeruginosa isolates included tobramycin, amikacin, netilmicin, imipenem, meropenem, doripenem, ceftazidime, cefepime, ciprofloxacin, levofloxacin, ticarcillin-clavulanic acid, piperacillin-tazobactam, aztreonam, fosfomycin, polymyxins (colistin).

Data were represented and analyzed using SPSS software, version 20. Descriptive analysis techniques such as percentages, frequencies, means, and standard deviations were applied to process the data, unless otherwise stated. The threshold for the probability of significance was set at less than 0.05.

The current study strictly adheres to the criteria and recommendations of the committee at the University of Kufa.

A total of 42 males and 123 females diagnosed with bacterial infections were enrolled in the present study. The study revealed that approximately 44% of the total specimens consisted of Enterobacteriaceae species (Escherichia coli, Klebsiella, and Proteus), followed by Streptococcus saprophyticus, which accounted for approximately 26% of the total samples (Table 1). Staphylococcus aureus isolates ranked third, comprising about 20%. Conversely, Salmonella and Neisseria gonorrhoeae were uncommon and were only detected in specimens taken from blood and vaginal swabs, respectively (Table 1).

The present study revealed that 126 clinical isolates taken from patients were classified as MDR resistant (Table 2). Additionally, six isolates were categorized as XDR, and two isolates were classified as PDR (Table 2). In contrast, only 31 clinical isolates were classified as susceptible (Table 2).

In this study, men (n=42) comprised approximately 25% of the total participants. The resistant isolates among men were as follows: MDR, 14; XDR, 4; PDR, 2 (Table 3). On the other hand, there were 123 female participants, accounting for 74.5% of the total. Among females, 112 isolates were found to be resistant in the MDR category, and there were two resistant isolates classified as XDR with no PDR isolates (Table 3).

Majority of the bacterial resistance isolates among all types of specimens (n=126) were classified as MDR, followed by six samples of XDR and two samples of PDR (Table 4). Bacteria with MDR were found in high percentages in urine (38.88%) and vagina swab (23.80%). Microorganisms with XDR were present at lower levels and found in urine, vagina swab, and seminal fluid (Table 4). Additionally, one isolate from a wound and another from urine were classified as PDR (Table 4).

The current investigation highlights the frequent occurrence of bacterial strains (such as Staphylococcus aureus, Enterococcus spp., Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter spp.) exhibiting MDR, XDR, and PDR features in some hospitals and health centers in Najaf city, Iraq.

In this surveillance, significant differences were observed among the samples and strains. The study included 42.4% males and 74.5% females as participants. Hidron and colleagues reported an increased number of samples taken from women due to their greater vulnerability to bacterial infections [5]. Gender, as a biological variable, influences immune responses, with women being more prone to infections, particularly urinary tract infections caused by a variety of pathogens including fungi, viruses, bacteria, parasites, and allergens [6,7]. These results are consistent with previous studies [8,9,10].

Consistent with the literature, the present study revealed that about 44% of the total specimens were infected with E. coli (28.48%), Klebsiella (13.30%), and Proteus (3.03%), suggesting that E. coli is a dominant uropathogen [11].

Out of the 126 isolates classified as MDR, 36 isolates were E. coli, followed by Staph saprophyticus (33 isolates), and Staph aureus (26 isolates), in agreement with earlier observations [12]. However, these figures were higher than those obtained from a study conducted by Rasheed [13]. Consistent with AL-Mohana's findings, two isolates of Staph. aureus were classified as XDR and one as PDR [14,15]. This could be attributed to factors such as the presence of the mecA gene, deformation/mutation of porin proteins, membrane impermeability, and the accumulation of a lipid layer covering the cell wall [15]. The present study reported strains of Staphylococcus aureus that were more drug-resistant than other staphylococcal strains, particularly showing resistance against third-generation cephalosporins [16]. Furthermore, out of 126 isolates, 33 isolates were Staph. saprophyticus, constituting approximately 26% of the MDR-classified isolates. Additionally, only one isolate was classified as XDR with no isolates classified as PDR, which aligns with previous reports [17].

These differences can be partially explained by the ongoing evolution of MDR strains across different regions of the world and the significant geographic heterogeneity in MDR strains among various countries [18,19].

Regarding the type of specimens, out of 126 MDR isolates, 49 specimens (38%) were from urine samples, with MDR bacteria accounting for 38%. Additionally, 30 specimens (23%) were from vaginal swabs, while lower percentages were observed in the remaining specimens. These findings align with certain studies [20], but lower percentages were recorded by Aljanaby [21]. Notably, strains isolated from urine often comprised gram-negative bacteria resistant to antibiotic therapy, indicating a potential for developing resistance, as previously reported [4,7].

The current study found that the majority of isolated E. coli strains exhibited resistance to more than half of the antibiotics tested, indicating a high degree of resistance compared to other detected isolates [22]. The results also showed that Klebsiella pneumoniae exhibited higher resistance than other Enterobacteriaceae strains after E. coli. This could be due to these strains having a $\beta$-lactam ring supported by Zwitterionic bridges, providing protection against these antibiotics [4]. Some reports from the same city as the current study indicated susceptibility of most isolates to ceftazidime and ceftriaxone [21,23]. Consistent with the literature, bacterial isolates in the present study demonstrated resistance to ceftriaxone and ceftazidime [19].

Furthermore, strains of Escherichia coli and Staphylococcus aureus exhibited resistance to more than two antibiotics, particularly third-generation cephalosporins, amikacin, sulfamethoxazole-trimethoprim, piperacillin, and ciprofloxacin. This high level of resistance could be attributed to a high rate of adaptive mutations. Antibiotics in the drug discovery pipeline continue to face challenges due to increased drug-resistant strains, putting patients at risk of bacterial infections. Hence, investigating bacterial strains and their resistance to antimicrobial medications could provide valuable insights for drug discovery.

The research has identified that the majority of bacteria isolated from patients, categorized as MDR, exhibit resistance to over three antibiotics. Notably, Escherichia coli, Staphylococcus aureus, Staphylococcus saprophyticus, and Klebsiella species all demonstrated this resistance pattern. In contrast, Proteus species displayed resistance to more than two medications, whereas Streptococcus spp. revealed resistance to at least two drugs.

The authors declare that they have no conflicts of interest.

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