atpD gene sequencing, multidrug resistance traits, virulence-determinants, and antimicrobial resistance genes of emerging XDR and MDR-Proteus mirabilis

Proteus mirabilis is a common opportunistic pathogen causing severe illness in humans and animals. To determine the prevalence, antibiogram, biofilm-formation, screening of virulence, and antimicrobial resistance genes in P. mirabilis isolates from ducks; 240 samples were obtained from apparently healthy and diseased ducks from private farms in Port-Said Province, Egypt. The collected samples were examined bacteriologically, and then the recovered isolates were tested for atpD gene sequencing, antimicrobial susceptibility, biofilm-formation, PCR detection of virulence, and antimicrobial resistance genes. The prevalence of P. mirabilis in the examined samples was 14.6% (35/240). The identification of the recovered isolates was confirmed by the atpD gene sequencing, where the tested isolates shared a common ancestor. Besides, 94.3% of P. mirabilis isolates were biofilm producers. The recovered isolates were resistant to penicillins, sulfonamides, β-Lactam-β-lactamase-inhibitor-combinations, tetracyclines, cephalosporins, macrolides, and quinolones. Using PCR, the retrieved strains harbored atpD, ureC, rsbA, and zapA virulence genes with a prevalence of 100%, 100%, 94.3%, and 91.4%, respectively. Moreover, 31.4% (11/35) of the recovered strains were XDR to 8 antimicrobial classes that harbored blaTEM, blaOXA-1, blaCTX-M, tetA, and sul1 genes. Besides, 22.8% (8/35) of the tested strains were MDR to 3 antimicrobial classes and possessed blaTEM, tetA, and sul1genes. Furthermore, 17.1% (6/35) of the tested strains were MDR to 7 antimicrobial classes and harbored blaTEM, blaOXA-1, blaCTX-M, tetA, and sul1 genes. Alarmingly, three strains were carbapenem-resistant that exhibited PDR to all the tested 10 antimicrobial classes and shared blaTEM, blaOXA-1, blaCTX-M, tetA, and sul1 genes. Of them, two strains harbored the blaNDM-1 gene, and one strain carried the blaKPC gene. In brief, to the best of our knowledge, this is the first study demonstrating the emergence of XDR and MDR-P.mirabilis in ducks. Norfloxacin exhibited promising antibacterial activity against the recovered XDR and MDR-P. mirabilis. The emergence of PDR, XDR, and MDR-strains constitutes a threat alarm that indicates the complicated treatment of the infections caused by these superbugs.

Sampling. Approximately, 240 specimens were obtained from apparently healthy (n = 40) and diseased ducks (n = 40) from private duck commercial farms (Muscovy duck with average age 70 days) at Port-Said Province, Egypt (From May 2020 to August 2020). Tracheal and cloacal swabs were collected from live birds, while the internal organs were collected separately under complete aseptic conditions from freshly dead and slaughtered ducks as illustrated in Table1. Diseased ducks exhibited diarrhea and respiratory manifestations. Specimens were collected in peptone water (Oxoid, UK) and rapidly transmitted to the lab as soon as possible for bacteriological examination.  The identification of suspected colonies was performed according to their culture characters, swarming activity, hemolytic activity, morphological characteristics using Gram's-staining, and biochemical characters as described by Quinn 24 . Moreover, the identification of P. mirabilis was confirmed by the PCR detection of the atpD gene as described by Bi 25 (Table 2), followed by gene sequencing of the PCR products.
The atpD gene sequencing and phylogenetic analyses. Since the retrieved isolates exhibited harmony in their phenotypic and biochemical characteristics: the PCR products of 5 randomly selected isolates were purified with a QIAquick PCR-Product extraction kit (QIAGEN Sciences Inc., Germantown, MD, USA) and sent for direct sequencing using the same set of primers. The sequencing was carried out using the Bigdye Terminator V3.1 cycle sequencing kit (Thermo Fisher Scientific, Waltham, MA, USA). The sequencing was performed using the Applied Biosystems 3130 genetic analyzer (HITACHI, Japan), and the retrieved sequences were deposited in the GenBank with accession numbers: MW357650, MW357651, MW357652, MW357653, and MW357654. To detect the sequence identity to GenBank accessions, the BLAST analysis (Basic Local Alignment Search Tool) was done. The phylogenetic tree was generated by the MegAlign module of LasergeneD-NAStar version 12.1 using maximum likelihood, neighbor-joining, and maximum parsimony in MEGA6 26 .
Antimicrobial susceptibility testing of P. mirabilis. The  Estimation of the biofilm formation in the isolated P. mirabilis. Estimation of biofilm formation was carried out in glass test tubes as previously described by Kadam 29 . Briefly, each P. mirabilis strain was inoculated in tryptic soy broth (Oxoid, Hampshire, UK), and left incubated overnight at 28 °C without shaking. Negative control experiments were carried out with sterile broth. After discarding the broth, the incubated tubes were stained with 1% crystal violet (to observe cells attached to the test tube) and were incubated for 15 min. Then, the tubes were washed with sterile distilled water. The test was repeated three times for each strain. Positive results indicated by the formation of purple biofilms.  GTA TCA TGA ACG TTC TGG GTAC  TGA AGT GAT ACG CTC TTG CAG  595  94 °C  30 s   58 °C  40 s   72 °C  45 s   25   ureC  GTT ATT CGT GAT GGT ATG GG  ATA AAG GTG GTT ACG CCA GA  317  94 °C  30 s   56 °C  40 s   72 °C  40 s   30   rsbA  TTG AAG GAC GCG ATC AGA CC ACT  CTG CTG TCC TGT GGG TA  467  94 °C  30 s   58 Table 2. The agar gel electrophoresis was carried out for the separation of the obtained PCR-products using 1.5% agarose stained with ethidium bromide 0.5 μg/ml and followed by photographing the gel.
Statistical analyses. The obtained findings were analysed using the Chi-square test (SAS software, version 9.4, SAS Institute, Cary, NC, USA) (Significance-level; P < 0.05). Besides, the correlation coefficient and the nonparametric Wilcox signed-rank test were performed using R-software (version 4.0.2; https//www.r-proje ct. org/).

Results
Phenotypic characteristics and prevalence of P. mirabilis in the examined samples. The recovered colonies are red with black center on XLD, pale colonies (non-lactose fermenter) on MacConkey agar, black colonies on TSI (H 2 S producer), hemolytic on blood agar, and undergo the characteristic swarming activity. Biochemically, the retrieved isolates were positive for catalase, H 2 S production, urease, methyl red, and citrate utilization tests, while are negative for oxidase, lactose fermentation, indole, and Voges-Proskauer tests. The prevalence of P. mirabilis among the examined birds was 25% (20/80); the prevalence was 15% (6/40) in the examined apparently healthy ducks, while it was 35% (14/40) in the examined diseased ducks (Table 3). Concerning the distribution of P. mirabilis in the examined samples, the total prevalence of P. mirabilis was 14.6% (35/240); the prevalence of P. mirabilis was 10.8% (13/120) in the examined samples of apparently healthy ducks, while the prevalence was 18.3% (22/120) in the examined samples of diseased ducks. The most predominant infected organ was the liver, followed by the heart and lung. Statistically, there is a significant difference in the prevalence of P. mirabilis between the examined apparently healthy and diseased ducks (P < 0.05), whereas there is no significant difference (P > 0.05) among different examined samples (Table 4 and Fig. 1).
Sequence analysis of the atpD gene. The atpD gene sequencing and the phylogenetic analysis proved that the tested P. mirabilis isolates (n = 5) shared a common ancestor. Moreover, the tested isolates showed high genetic identity to other strains of P. mirabilis such as P. mirabilis strain HI4320 of United Kingdom (Accession No. AM942759), P. mirabilis strain BB2000 of China (Accession No. MF576130), P. mirabilis strain BB2000 (Accession No. CP045538) and strain AOUC-001 (Accession No. CP015347) of Italy, and P. mirabilis strain BB2000 of USA (Accession No. CP004022) as illustrated in Fig. 2.

Discussion
Proteus mirabilis is frequently incriminated in food-borne infections and urinary tract infections in humans. Few studies are concerning the emergence of P. mirabilis in birds. The current study was directed to investigate the prevalence, atpD gene sequencing, antimicrobial-resistance profiles, PCR-based detection of virulence genes (ureC, zapA, and rsbA), and antimicrobial resistance genes (bla TEM , bla CTX , bla KPC , bla NDM-1 , bla OXA-1 , sul1, and tetA) of emerging P. mirabilis in ducks.
The bacteriological examination evidenced that the prevalence of P. mirabilis in the examined samples was 14.6% (35/240). Besides, there is no ambivalence in the phenotypic characteristics of the retrieved P. mirabilis strains that revealed a significant harmony between the isolates: red colonies with black center on XLD, pale colonies (non-lactose fermenter) on MacConkey agar, black colonies on TSI, and undergo a characteristic swarming activity. Biochemically: the retrieved isolates are positive for catalase, H 2 S, urease, methyl-red, and citrate utilization tests, whereas they are negative for oxidase, lactose fermentation, indole, and Voges-Proskauer tests. These results are in agreement with those obtained by Lei 6 and Reich 7 . In the present study, P. mirabilis was isolated from the internal organs of the examined birds in a pure form suggesting that the retrieved isolates were the primary bacterial cause of these infections in ducks. These results were supported by the previous findings that were reported by Barbour 36 and Yeh 37 . P. mirabilis is a ubiquitous pathogen widely distributed in the environment 38 . P. mirabilis is an opportunistic pathogen that is incriminated in various infections in humans, animals, and poultry. Recently, several studies reported the emergence of P. mirabilis in food-producing animals, especially poultry 36,38,39 .
In the present study, P. mirabilis could be isolated from the internal organs of both apparently healthy and diseased birds. P. mirabilis is an opportunistic microorganism that normally inhabits the alimentary tract of birds, animals, and humans. The microorganism could escape from the intestinal tract and reach other internal organs. Thus, it could be responsible for other illnesses associated with the spread of P. mirabilis to other internal organs, and in severe cases, it could cause sepsis. In the meantime, the development of the clinical signs depends mainly on the onset of the disease as well as the immune status of the bird 40 .
The atpD gene phylogenetic analysis revealed that the tested P. mirabilis isolates (n = 5) are shared a common ancestor. Besides, they exhibited high genetic identity with other P. mirabilis strains of human origin that were previously isolated in Italy 41,42 , China 43 , USA 44 , and United Kingdom 45 . Our findings conceived the epidemiological map and emphasized the zoonotic impact of P. mirabilis that is considered a public health threat.
Concerning the in-vitro antimicrobial-resistance profiles; the recovered P. mirabilis strains showed remarkable resistance-patterns to penicillins, β-Lactam β-lactamase-inhibitor combinations, cephalosporins, sulfonamides, tetracyclines, macrolides, and quinolones. The development of such resistant strains reflected as a public health alarm. Moreover, the retrieved strains were sensitive to norfloxacin (85.7%), meropenem (77.1%), and imipenem (74.3%). Our findings are consistent with those reported by Wong 46 and Nahar 47 . The improper application of antimicrobial agents in the poultry industry and the ability of P. mirabilis to acquire the antimicrobial-resistant genes from other resistant pathogens are the major causes of the emergence of these MDR-strains. Unfortunately, P. mirabilis could resist various antimicrobial classes due to the presence of chromosomal antibiotic-resistant genes as well as the resistant-plasmids 37 .
The biofilm assay revealed that 94.3% (33/35) of the isolated P. mirabilis strains are biofilm producers. Our findings are consistent with those reported by Kwiecinska-Piróg 48 . The biofilm is one of the most important virulence determinants of bacteria. It preserves bacteria during adverse environmental conditions. Moreover, biofilm protects bacteria from phagocytosis, antibodies, and antibiotics. Besides, it plays a vital role in antimicrobial resistance. P. mirabilis produces biofilm in various environments includes: biological and non-biotic surfaces such as glass, silicone, and polystyrene. The formation of biofilm on the non-biotic surface is considered the main source of nosocomial infections 49,50 .
The PCR proved that the recovered P. mirabilis strains are virulent and harbored atpD, ureC, rsbA, and zapA virulence genes with a prevalence of 100%, 100%, 94.3%, and 91.4%, respectively. Our findings are nearly agreed with those reported by Pathirana 30 and Sun 51 . The atpD gene is encoded for ATP synthase β-subunit for the production of ATP from ADP. The atpD gene is more conservative in Proteus species when compared with 16SrRNA 25 . Infections caused by P. mirabilis are controlled by several virulence-determinants that are regulated by specific virulence genes. IgA-degrading proteases are commonly accompanied by the pathogenic strains of P. mirabilis. ZapA-protease could degrade IgG, IgA1, and IgA2. It is regulated by the zapA gene. P. mirabilis is frequently incriminated in urinary tract infections that are mediated by stone-formation due to the release of urease enzyme. Urease is a metalloenzyme that acts by increasing the pH of urine that induces crystal formation. The urease production is controlled by the ureC gene. Besides, the characteristic swarming activity of P. mirabilis is encoded by the rsbA gene. The rsbA gene expresses a membrane sensor that induces the production of extracellular polysaccharides. Also, it regulates the swarming phenomena and enhances the biofilm formation by P. mirabilis 23 54 . ESBLs are responsible for the hydrolysis of Broad-spectrum β-lactam antibiotics including penicillins, piperacillin, and cephalosporins. EBSLs are frequently produced by Enterobacteriales. Recently, P. mirabilis strains reported harboring various acquired antimicrobial resistance genes. The high prevalence of the bla TEM gene among the recovered P. mirabilis strains enabling them to resist penicillins (amoxicillin and ampicillin). Moreover, the resistance to cephalosporins (cefotaxime, and ceftazidime) is mediated by the presence of the bla CTX-M gene. The resistance to piperacillin is mainly attributed to the bla OXA-1 gene which also promoting the resistance to cephalosporins. Besides, both bla OXA-1 and In addition, P. mirabilis is frequently resistant to tetracyclines and sulfonamides due to the presence of tetA and sul1genes, respectively. On the other hand, P. mirabilis is usually susceptible to fluoroquinolones such as norfloxacin 57 . The polymyxins exert their effect by increasing the permeability of the Gram-negative bacterial cell membrane through displacing Mg2 + and Ca2 + from the lipid A content of LPS that results in leakage of the cell contents. The resistance to polymyxins is common in the mutant P. mirabilis due to the alteration of LPS that is controlled by the expression of the eptC gene and the modification of the L-Ara4N. However, several previous studies reported the sensitivity of some P. mirabilis isolates to polymyxins, especially those of animal origin as reported by Sun 51 . The eptC gene may be present but not expressed. Besides, the alterations of the LPS in the cell envelop occurs only in the mutant strains and varies among different strains of P. mirabilis as previously reported by McCoy 58 .
In the present study, three strains are carbapenem-resistant as well as PDR to all the tested ten antimicrobial classes and are sharing bla TEM , bla OXA-1 , bla CTX-M , tetA, and sul1 genes. Of them, two strains harbored the bla NDM-1 gene, and one strain carried the bla KPC gene. Globally, the emergence of carbapenem-resistance in P. mirabilis is relatively low; however, it inclines to increase over time. The carbapenem-resistance is attributed to the presence of bla NDM-1 and bla KPC genes. The existence of the bla KPC gene in P. mirabilis was recorded for the first time in a diabetic Patient in the USA in 2008 59 , followed by China in 2010 60 , and Brazil in 2015 61 . Moreover, the bla NDM-1 is recognized for the first time in P. mirabilis strain retrieved from urinary infection in France in 2012 62 and followed by China in 2015 63 .
Concerning the correlation between the antimicrobial resistance genes and the virulence determinants, a previous study that was reported by Filipiak 64 revealed an inversed correlation between the virulence factors and the presence of the resistance genes in the retrieved P. mirabilis strains. However, in the present study, the majority of the screened virulence genes were found in the recovered isolates. Besides, there is no significant difference in the distribution of the virulence genes among the retrieved isolates either the susceptible or the antimicrobial-resistant strains. These findings suggest that the P. mirabilis pathogenicity is not affected by the presence of antimicrobial resistance genes.

Study limitations.
Multilocus sequence typing (MLST) should be carried out to illustrate the genetic relatedness among the recovered P. mirabilis strains.
In conclusion, to the best of our knowledge, this is the first report regarding the emergence of XDR and MDR-P. mirabilis in ducks. P. mirabilis is more prevalent in diseased birds than the apparently healthy ones, and the liver is the most prominent infected organ. P. mirabilis is a common biofilm-producing pathogen. The recovered P. mirabilis isolates commonly harbor the atpD, ureC, zapA, and rsbA virulence genes. The retrieved P. mirabilis strains are extensively drug-resistant (XDR) or multidrug-resistant (MDR) to several antimicrobial classes (penicillins, β-Lactam-β-lactamase-inhibitor-combinations, cephalosporins, sulfonamides, tetracyclines, quinolones, macrolides, and polymyxins), and commonly harbored bla TEM , bla OXA-1, bla CTX-M, tetA , and sul1 antimicrobial www.nature.com/scientificreports/ resistance genes. In-vitro, norfloxacin exhibited promising antibacterial activity against the recovered XDR and MDR-P. mirabilis. Furthermore, the emergence of carbapenem-resistant (harbored either bla KPC or bla NDM-1 genes) and PDR-strains constitutes a threat alarm that indicates a complicated treatment of the diseases caused by such superbugs. Accordingly, it endorses the incessant surveillance of antimicrobial susceptibility testing as well as the limited and appropriate use of antibiotics in health and veterinary practices.