P. mirabilis is recognized as an emerging healthcare concern due to its status as an opportunistic human pathogen, commonly found in the gastrointestinal tract, skin, and oral mucosa. Immunocompromised patients or those undergoing antibiotic therapy are particularly susceptible to its proliferation (Kushwaha et al. 2014).

Additionally, P. mirabilis exhibits intrinsic resistance to several antibiotics, including nitrofurans, tigecycline, tetracycline, and polymyxins (colistin) (Girlich et al. 2020; Stock 2003). Concern has escalated with the emergence of extended-spectrum β-lactamases (ESBLs), which confer resistance to cephalosporins (Stürenburg and Mack 2003), and the increasing prevalence of carbapenemase-encoding genes, limiting the effectiveness of existing antimicrobials (Adeolu et al. 2016; Firmo et al. 2020).

As human health depends greatly on the management of infectious diseases, particularly in light of the ongoing rise of MDR, XDR, or even PDR bacteria. Therefore, effective stewardship program implementation in any nation or region depends critically on an assessment of the local antimicrobial resistance trends and underlying resistance causes. That is why genomic profiling is highly beneficial in managing infections caused by pan-drug resistant P. mirabilis.

Herein, “Bacteria_11” and “Bacteria_27” were identified as pan drug resistant (PDR) P. mirabilis as they are non-susceptible to all tested agents in all antimicrobial categories (Magiorakos et al. 2012). They demonstrated substantial levels of resistance to various antibiotic classes in addition to their intrinsic resistance profiles. In a previous study, one of the first instance of PDR P. mirabilis in Egypt to be reported, 22.8% of isolates were MDR, 31.4% were XDR, and 8.5% were PDR (Algammal et al. 2021).

The phylogenetic analysis of the two isolates revealed that the closest strain to bacteria_27 was (Proteus mirabilis strain L90-1) (genome sequence ID: CP045257.1) with two plasmids. Sample was obtained from human stool specimen in China. Also, the closest strain to bacteria_11 was (Proteus mirabilis strain DY.F1.2) (genome sequence ID: CP046049.1) with a plasmid. It was taken from a swine in China as well. Many antimicrobial resistance genes were carried one the chromosome of both related strains while some were carried on the plasmids. This could be an indication that bacteria are borderless, and the global trade contribute to the spread of resistant bacteria worldwide. This strengthens the argument that antibiotic resistance is a global issue and adds to its complexity. Where a resistant bacterium emerges is irrelevant. In a globalized society, if it has a good chance of succeeding and becoming more and more popular, it might spread quickly to other regions of the world. The carriers of our two strains both were ICU patients. One was diabetic patient admitted to the hospital for above knee amputation as a result of infected diabetic foot. Unfortunately, patient died after 3 months of ICU- stay. The other was admitted as a post-operative patient, smoker, and hypertensive. He acquired chest infection and ventilated. He also passed away after 5 months of ICU- stay.

Our investigation revealed that the “Bacteria_11” and “Bacteria_27” resistance genes are all chromosomally situated. The four resistance mechanisms that caused this acquired resistance were antibiotic inactivation, antibiotic efflux, antibiotic target substitution, and antibiotic target modification.

We found that genes resistant to aminoglycosides were highly represented in both samples. The isolates exhibited resistance to gentamycin, amikacin, and tobramycin both phenotypically and genotypically. In both genomes, aph (3'')-Ib, aph(6)-Id, aadA1, aadA7, aadA5, aac(3)-Id, ant(2")-Ia, and aac(6)-Ib, were identified, which play a significant role in aminoglycoside resistance (Hua et al. 2020). These two isolates have high expression levels of genes like aph (3′)Ib and aadA1, which result in resistance to aminoglycoside drugs like streptomycin, kanamycin, and spectinomycin, which are rarely utilized in clinical settings and are not phenotypically assessed. In agreement with a previous study by (Yu et al. 2024) , these genes were highly represented in all tested P.s mirabilis isolates. In Egypt, this is not surprising, given that fosfomycin and aminoglycosides are often used to treat a variety of respiratory diseases, therefore the presence of this resistance pattern is expected (Mohamed et al. 2018). Moreover, this is in accordance with a recent study conducted in the same region (Zagazig) in Egypt except for isolates source (Tartor et al. 2021). They detected similar antimicrobial resistance (AMR) genes in three P. mirabilis strains isolated from both clinical and subclinical mastitis milk samples. These results underscore the principle of One Health and draw attention to the critical issue of the transfer of AMR genes between humans, animals, and the environment (Tartor et al. 2021).

P. mirabilis tested isolates were resistance to different beta-lactam antibiotic classes either penicillins (Ticracillin, Ticarcillin/ Clavulanic, Piperacillin, Piperacillin/Tazobactam), monobactams (Azetreonam) or broad spectrum cephalosporins (ceftazidime, cefepime). In fact, P. mirabilis producing (ESBL) genes is increasing aggressively in Egypt. A previous study reported that (57.6%) of the examined isolates produced (ESBLs) (ElTaweel et al. 2024). In our study the resistance may be assigned to the presence of extended spectrum beta-lactamase (ESBL) genes whether blaCMY gene in bacteria_11, a beta-lactamase that mainly occurs in Klebsiella pneumoniae and provides resistance to carbapenems, cephalosporins, penam, and cephamycins. Also, it is commonly found in P. mirabilis. As it was reported in another study that blaCMY-2 was present in 22% of the isolates (Chalmers et al. 2023). Furthermore, blaVEB-6 found in bacteria_27 which is a beta-lactamase commonly found in P. mirabilis (Zong et al. 2008) which has a role in resistance of cephalosporins and monobactams. In addition to the genes kpnH, kpnF, rsmA and crp that were found in both isolates, related to multidrug efflux pumps. Although Proteus by nature has reduced sensitivity to imipenem (Girlich et al. 2020), but these genes also may be the core reason for carbapenems (meropenem, imipenem) resistance as there were no detection for carbapenemases in both isolates. Although carbapenemase resistant P. mirabilis is definitely increasing worldwide (Girlich et al. 2020), the detected genes in previous studies were relatively low. In Egypt, a recent study found carbapenem resistant isolates were about (10.6%) (ElTaweel et al. 2024). Also, in Korean study, detection of carbapenemase genes were only in (15.6%) of isolated proteus samples (Yu et al. 2024). The World Health Organization (WHO) believes this to be a major public health problem since these antibiotics are used to treat diseases that are difficult to treat and because the number of bacterial strains that are resistant to them is rising and there are fewer therapeutic options available (Langford et al. 2023).

It was noticed the significant increase in infection caused by P. mirabilis in Egypt over the years especially after COVID era and the tremendous usage of antibiotic that contributed to this major range of resistant antibiotics(Negm et al. 2023).

We also figured out that there was no surprise for trimethoprim/ sulfamethoxazole resistance as this combination always used worldwide for urinary tract infection treatment, Pneumocystis carinii pneumonia, otitis media in infants and prostatic infections (Tuomisto et al. 2009; Kester et al. 2012). As sul1 and sul2 responsible for sulphonamide resistance were found repeated several times in both chromosomes and dfrA1, dfrA14, dfrA15, dfrA17, the reason for trimethoprim resistance also were detected. Also, a study conducted in Iran, reported the prevalence of sul1 and sul2 by (51.7%) and (56.7%); respectively (Human and Genom 2022). Similarly, this was found in the previously mentioned recent study (Tartor et al. 2021) which in turn emphasizes the concept of inter-species transfer between animals, the environment, and humans and again further underscores the One Health paradigm.

As β-lactams become resistant, usually quinolones offer a potential and comparatively safe alternative. In this study, quinolones (ciprofloxacin) resistance was detected phenotypically and genotypically by the presence of gyrB in both isolates, CRP, and multidrug efflux pump genes such as (kpnH, kpnF, rsmA). Unfortunately, P. mirabilis quinolones resistance is highly reported in Egypt. A significant (75.8%) quinolone resistance was found in the isolates that were examined (ElTaweel et al. 2024).

In addition to intrinsic resistance of P. mirabilis (Girlich et al. 2020), the presence of all those previously listed antimicrobial resistance genes (Table 2) in a single bacterial strain in addition to genes that provide resistance to antiseptics, diaminopyrimidines and chloramphenicol (qacJ, KpnF, RsmA, catA, and floR) is a huge concern and more worrying than expected. The isolates assessed in this study were obtained from nosocomial infections that were resistant to a wide variety of antibiotic groups in addition to having a high number of resistance genes. This implies that managing P. mirabilis infection with antibiotics poses significant challenges and entails a highly intricate process.

We may contribute the intrinsic resistant to tigecycline and some other antibiotics of P. mirabilis to the presence of the AcrAB transport system. The AcrAB efflux system transports hydrophobic substrates, such as detergents, dyes, and antibiotics, outside the cell directly with no accumulation in the periplasm (efflux) (Visalli et al. 2003).

Although our study is limited to two isolates only, the identified antimicrobial resistant genes may be present in many other Proteus isolates from the same region. This might help in choosing alternative therapeutic plans for treating hospital acquired infections from the beginning. As starting with initial loading dose followed with treatment period for 7 to 14 days. Also, combination therapy may be considered in clinical practice.

Alternatively, many virulence factors, such as the genes responsible for adhesion and swarming ability, are encoded by bacteria_11 and bacteria_27.

It is known that P. mirabilis, like other bacteria, uses flagella to swim through liquids and towards chemical ingredients. No surprise that both isolates have the main virulence genes encoding the flagellar components including the two flagellins flic1 and flic2 which comprise the wipe structure of flagellum (Schaffer and Pearson 2015a). As motility and swarming ability on solid surfaces is a main feature in P. mirabilis a lot of genes involved in flagella formation were identified coding 5 different diverse of flagellum (Armbruster and Mobley 2012).

Pathogenicity of P. mirabilis is highly detected in our isolates. Genes contribute to adherence were highly detected in both samples. It is known that the bacterial adherence of P. mirabilis is a crucial stage in the colonization and development of infections (Hasan 2020) Also, fliA, fliM, cheA and a lot of other genes contributing to bacterial swarming and motility were highly detected in both samples. According to Kuan et al. the primary virulence factor of P. mirabilis that affects the invasion and dissemination of infection in urinary tract segments is its motility (Kuan et al. 2014). This bacterium swarming motility increases the expression of its pathogenicity (Kotian et al. 2020) Furthermore, one of the key factors for P. mirabilis pathogenicity is biofilm formation. P. mirabilis expresses several virulence factors necessary for the formation of biofilms. Such as adhesion proteins, quorum sensing molecules, lipopolysaccharides, efflux pumps, and urease enzymes are a few examples of these variables (Schaffer and Pearson 2015b) Unfortunately, Crystalline biofilm-embedded bacteria develop a strong resistance to both the immune system and traditional antimicrobial drugs.

Very few studies were concerned with P. mirabilis acquired infection, especially in Egypt. Our study highlights P. mirabilis ability to acquire resistance for different antimicrobial classes used commonly in clinical aspects. In addition to its large virulence profile, to defeat host defensive mechanisms. In this study we were allowed to know the behaviour, adaptation, and pathogenicity of these bacteria. Also, we were able to pinpoint acquired resistance genes to make better informed antibiotic treatment choices.

Antibiotic resistance genes, virulence factors, and the all-other isolates traits that may be inferred from genomic sequences provide helpful information on the range of P. mirabilis pathogenicity and antimicrobial resistance acquired by them. Unfortunately, this study is limited to two isolates only, but it demonstrates the huge range of acquired antimicrobial resistance of P. mirabilis species. Further research is needed in the future to potentially uncover more of these transfer events, particularly in hospital-acquired illnesses. Our analysis of the pathogenicity and antibiotic resistance of these isolates could contribute to the future studies of multidrug-resistant P. mirabilis emergence and, hopefully, could lead to effective treatment strategies.

The availability of more whole genomes sequenced especially those sequenced with long-read technologies such as Nanopore will make it easier to comprehend how P. mirabilis acquires antimicrobial resistance.