OXA-48-like carbapenemases in Proteus mirabilis - novel genetic environments and a challenge for detection.

OXA-48-like enzymes represent the most frequently detected carbapenemases in Enterobacterales in Western Europe, North Africa and the Middle East. In contrast to other species, the presence of OXA-48-like in Proteus mirabilis leads to an unusually susceptible phenotype with low MICs for carbapenems and piperacillin-tazobactam, which is easily missed in the diagnostic laboratory. So far, there is little data available on the genetic environments of the corresponding genes, blaOXA-48-like, in P. mirabilis. In this study susceptibility phenotypes and genomic data of 13 OXA-48-like-producing P. mirabilis were investigated (OXA-48, n = 9; OXA-181, n = 3; OXA-162, n = 1). Ten isolates were susceptible to meropenem and ertapenem and three isolates were susceptible to piperacillin-tazobactam. The gene blaOXA-48 was chromosomally located in 7/9 isolates. Thereof, in three isolates blaOXA-48 was inserted into a P. mirabilis genomic island. Of the three isolates harbouring blaOXA-181 one was located on an IncX3 plasmid and two were located on a novel MOBF plasmid, pOXA-P12, within the new transposon Tn7713. In 5/6 isolates with plasmidic location of blaOXA-48-like, the plasmids could conjugate to E. coli recipients in vitro. Vice versa, blaOXA-48-carrying plasmids could conjugate from other Enterobacterales into a P. mirabilis recipient. These data show a high diversity of blaOXA-48-like genetic environments compared to other Enterobacterales, where genetic environments are quite homogenous. Given the difficult-to-detect phenotype of OXA-48-like-producing P. mirabilis and the location of blaOXA-48-like on mobile genetic elements, it is likely that OXA-48-like-producing P. mirabilis can disseminate, escape most surveillance systems, and contribute to a hidden spread of OXA-48-like.

A Novel SXT/R391 Integrative and Conjugative Element Carries Two Copies of the blaNDM-1 Gene in Proteus mirabilis.

The rapid spread of the blaNDM-1 gene is a major public health concern. Here, we describe the multidrug-resistant Proteus mirabilis strain XH1653, which contains a novel SXT/R391 integrative and conjugative element (ICE), harboring two tandem copies of blaNDM-1 and 21 other resistance genes. XH1653 was resistant to all antibiotics tested, apart from aztreonam. Whole-genome data revealed that two copies of blaNDM-1 embedded in the ISCR1 element are located in HS4 of the novel ICE, which we named ICEPmiChnXH1653. A circular intermediate of ICEPmiChnXH1653 was detected by PCR, and conjugation experiments showed that the ICE can be transferred to the Escherichia coli strain EC600 with frequencies of 1.5 × 10-7. In the recipient strain, the ICE exhibited a higher excision frequency and extrachromosomal copy number than the ICE in the donor strain. We also observed that the presence of ICEPmiChnXH1653 has a negative impact on bacterial fitness and leads to changes in the transcriptome of the host. In vitro evolution experiments under nonselective conditions showed that the two tandem copies of the ISCR1 element and the ISVsa3 element can be lost during repeated laboratory passage. This is the first report of a novel SXT/R391 ICE carrying two tandem copies of blaNDM-1, which also illustrates the role that ICEs may play as platforms for the accumulation and transmission of antibiotic resistance genes. IMPORTANCE The occurrence of carbapenemase-producing Proteus mirabilis, especially those strains producing NDM-1 and its variants, is a major public health concern worldwide. The integrative conjugative element (ICE) plays an important role in horizontal acquisition of resistance genes. In this study, we characterized a novel SXT/R391 ICE from a clinical P. mirabilis isolate that we named ICEPmiChnXH1653, which contains two tandem copies of the carbapenemase gene blaNDM-1. We performed an integrative approach to gain insights into different aspects of ICEPmiChnXH1653 evolution and biology and observed that ICEPmiChnXH1653 obtained the carbapenemase gene blaNDM-1 by ISCR1-mediated homologous recombination. Our study reveals that the transmission of blaNDM-1 by ISCR1 elements or ICEs may be an important contributor to the carbapenem resistance development across species, which could improve our understanding of horizontal gene transfer in clinical environments.

Semi-rational design of L-amino acid deaminase for production of pyruvate and D-alanine by Escherichia coli whole-cell biocatalyst.

In our previous study, one-step pyruvate and D-alanine production from D,L-alanine by a whole-cell biocatalyst Escherichia coli expressing L-amino acid deaminase (Pm1) derived from Proteus mirabilis was investigated. However, due to the low catalytic efficiency of Pm1, the pyruvate titer was relatively low. Here, semi-rational design based on site-directed saturation mutagenesis was carried out to improve the catalytic efficiency of Pm1. A novel high-throughput screening (HTS) method for pyruvate based on 2,4-dinitrophenylhydrazine indicator was then established. The catalytic efficiency (kcat/Km) of the mutant V437I screened out by this method was 1.88 times higher than wild type. Next, to improve the growth of the engineered strain BLK07, the genes encoding for Xpk and Fbp were integrated into its genome to construct non-oxidative glycolysis (NOG) pathway. Finally, the CRISPR/Cas9 system was used to integrate the N6-pm1-V437I gene into the genome of BLK07. Pyruvic acid titer of the plasmid-free strain reached 42.20 g/L with an L-alanine conversion rate of 77.62% and a D-alanine resolution of 82.4%. This work would accelerate the industrial production of pyruvate and D-alanine by biocatalysis, and the HTS method established here could be used to screen other Pm1 mutants with high pyruvate titers.

Catalase Activity is Critical for Proteus mirabilis Biofilm Development, Extracellular Polymeric Substance Composition, and Dissemination during Catheter-Associated Urinary Tract Infection.

Proteus mirabilis is a leading uropathogen of catheter-associated urinary tract infections (CAUTIs), which are among the most common health care-associated infections worldwide. A key factor that contributes to P. mirabilis pathogenesis and persistence during CAUTI is the formation of catheter biofilms, which provide increased resistance to antibiotic treatment and host defense mechanisms. Another factor that is important for bacterial persistence during CAUTI is the ability to resist reactive oxygen species (ROS), such as through the action of the catalase enzyme. Potent catalase activity is one of the defining biochemical characteristics of P. mirabilis, and the single catalase (katA) gene in strain HI4320 was recently identified as a candidate fitness factor for UTI, CAUTI, and bacteremia. Here, we show that disruption of katA results in increased ROS levels, increased sensitivity to peroxide, and decreased biofilm biomass. The biomass defect was due to a decrease in the production of extracellular polymeric substances (EPS) by the ΔkatA mutant and specifically due to reduced carbohydrate content. Importantly, the biofilm defect resulted in decreased antibiotic resistance in vitro and a colonization defect during experimental CAUTI. The ΔkatA mutant also exhibited decreased fitness in a bacteremia model, supporting a dual role for catalase in P. mirabilis biofilm development and immune evasion.

Proteus mirabilis Urease: Unsuspected Non-Enzymatic Properties Relevant to Pathogenicity.

Infection by Proteus mirabilis causes urinary stones and catheter incrustation due to ammonia formed by urease (PMU), one of its virulence factors. Non-enzymatic properties, such as pro-inflammatory and neurotoxic activities, were previously reported for distinct ureases, including that of the gastric pathogen Helicobacter pylori. Here, PMU was assayed on isolated cells to evaluate its non-enzymatic properties. Purified PMU (nanomolar range) was tested in human (platelets, HEK293 and SH-SY5Y) cells, and in murine microglia (BV-2). PMU promoted platelet aggregation. It did not affect cellular viability and no ammonia was detected in the cultures' supernatants. PMU-treated HEK293 cells acquired a pro-inflammatory phenotype, producing reactive oxygen species (ROS) and cytokines IL-1ß and TNF-α. SH-SY5Y cells stimulated with PMU showed high levels of intracellular Ca2+ and ROS production, but unlike BV-2 cells, SH-SY5Y did not synthesize TNF-α and IL-1ß. Texas Red-labeled PMU was found in the cytoplasm and in the nucleus of all cell types. Bioinformatic analysis revealed two bipartite nuclear localization sequences in PMU. We have shown that PMU, besides urinary stone formation, can potentially contribute in other ways to pathogenesis. Our data suggest that PMU triggers pro-inflammatory effects and may affect cells beyond the renal system, indicating a possible role in extra-urinary diseases.

The 'Shape-Shifter' Peptide from the Disulphide Isomerase PmScsC Shows Context-Dependent Conformational Preferences.

Multiple crystal structures of the homo-trimeric protein disulphide isomerase PmScsC reveal that the peptide which links the trimerization stalk and catalytic domain can adopt helical, ß-strand and loop conformations. This region has been called a 'shape-shifter' peptide. Characterisation of this peptide using NMR experiments and MD simulations has shown that it is essentially disordered in solution. Analysis of the PmScsC crystal structures identifies the role of intermolecular contacts, within an assembly of protein molecules, in stabilising the different linker peptide conformations. These context-dependent conformational properties may be important functionally, allowing for the binding and disulphide shuffling of a variety of protein substrates to PmScsC. They also have a relevance for our understanding of protein aggregation and misfolding showing how intermolecular quaternary interactions can lead to ß-sheet formation by a sequence that in other contexts adopts a helical structure. This 'shape-shifting' peptide region within PmScsC is reminiscent of one-to-many molecular recognition features (MoRFs) found in intrinsically disordered proteins which are able to adopt different conformations when they fold upon binding to their protein partners.

A small-molecular inhibitor against Proteus mirabilis urease to treat catheter-associated urinary tract infections.

Infection and blockage of indwelling urinary catheters is significant owing to its high incidence rate and severe medical consequences. Bacterial enzymes are employed as targets for small molecular intervention in human bacterial infections. Urease is a metalloenzyme known to play a crucial role in the pathogenesis and virulence of catheter-associated Proteus mirabilis infection. Targeting urease as a therapeutic candidate facilitates the disarming of bacterial virulence without affecting bacterial fitness, thereby limiting the selective pressure placed on the invading population and lowering the rate at which it will acquire resistance. We describe the design, synthesis, and in vitro evaluation of the small molecular enzyme inhibitor 2-mercaptoacetamide (2-MA), which can prevent encrustation and blockage of urinary catheters in a physiologically representative in vitro model of the catheterized urinary tract. 2-MA is a structural analogue of urea, showing promising competitive activity against urease. In silico docking experiments demonstrated 2-MA's competitive inhibition, whilst further quantum level modelling suggests two possible binding mechanisms.

Highly selective synthesis of D-amino acids via stereoinversion of corresponding counterpart by an in vivo cascade cell factory.

BACKGROUND: D-Amino acids are increasingly used as building blocks to produce pharmaceuticals and fine chemicals. However, establishing a universal biocatalyst for the general synthesis of D-amino acids from cheap and readily available precursors with few by-products is challenging. In this study, we developed an efficient in vivo biocatalysis system for the synthesis of D-amino acids from L-amino acids by the co-expression of membrane-associated L-amino acid deaminase obtained from Proteus mirabilis (LAAD), meso-diaminopimelate dehydrogenases obtained from Symbiobacterium thermophilum (DAPDH), and formate dehydrogenase obtained from Burkholderia stabilis (FDH), in recombinant Escherichia coli. RESULTS: To generate the in vivo cascade system, three strategies were evaluated to regulate enzyme expression levels, including single-plasmid co-expression, double-plasmid co-expression, and double-plasmid MBP-fused co-expression. The double-plasmid MBP-fused co-expression strain Escherichia coli pET-21b-MBP-laad/pET-28a-dapdh-fdh, exhibiting high catalytic efficiency, was selected. Under optimal conditions, 75 mg/mL of E. coli pET-21b-MBP-laad/pET-28a-dapdh-fdh whole-cell biocatalyst asymmetrically catalyzed the stereoinversion of 150 mM L-Phe to D-Phe, with quantitative yields of over 99% ee in 24 h, by the addition of 15 mM NADP+ and 300 mM ammonium formate. In addition, the whole-cell biocatalyst was used to successfully stereoinvert a variety of aromatic and aliphatic L-amino acids to their corresponding D-amino acids. CONCLUSIONS: The newly constructed in vivo cascade biocatalysis system was effective for the highly selective synthesis of D-amino acids via stereoinversion.

Bioinformatics and experimental studies on the structural roles of a surface-exposed α-helix at the C-terminal domain of Chondroitinase ABC I.

A series of single and double mutants generated on residues of a surfaced-exposed helix at the C-terminal domain of chondroitinase ABC I (cABC I) from proteus vulgaris. M886A, G887E, and their respective double mutant, MA/GE were inspired by the sequence of a similar helix segment in 30S ribosomal protein S1. Additionally, M889I, Q891K, and the corresponding double mutant, MI/QK, were made regarding the sequence of a similar helix in chondroitin lyase from Proteus mirabilis. Circular dichroism spectra in the far-UV region, demonstrate that the ordered structure of wild-type (WT), and double mutants are the same; however, the helicity of the ordered structures in MI/QK is higher than that of the WT enzyme. When compared with the single mutants, the double mutants showed higher activity, and that the activity of MI/QK is higher than that of the WT enzyme. Heat-induced denaturation experiments showed that the stability of the tertiary structure of double mutants at moderate temperatures is higher compared with the WT, and single mutants. It concluded that this helix can be considered as one of the hot spots region that can be more manipulated to obtain improved variants of cABC I.

Biotransformation and chiral resolution of d,l-alanine into pyruvate and d-alanine with a whole-cell biocatalyst expressing l-amino acid deaminase.

Pyruvate is an important pharmaceutical intermediate and is widely used in food, nutraceuticals, and pharmaceuticals. However, high environmental pollution caused by chemical synthesis or complex separation process of microbial fermentation methods constrain the supply of pyruvate. Here, one-step pyruvate and d-alanine production from d,l-alanine by whole-cell biocatalysis was investigated. First, l-amino acid deaminase (Pm1) from Proteus mirabilis was expressed in Escherichia coli, resulting in pyruvate titer of 12.01 g/L. Then, N-terminal coding sequences were introduced to the 5'-end of the pm1 gene to enhance the expression of Pm1 and the pyruvate titer increased to 15.13 g/L. Next, product utilization by the biocatalyst was prevented by knocking out the pyruvate uptake transporters (cstA, btsT) and the pyruvate metabolic pathway genes pps, poxB, pflB, ldhA, and aceEF using CRISPR/Cas9, yielding 30.88 g/L pyruvate titer. Finally, by optimizing the reaction conditions, the pyruvate titer was further enhanced to 43.50 g/L in 8 H with a 79.99% l-alanine conversion rate; meanwhile, the resolution of d-alanine reached 84.0%. This work developed a whole-cell biocatalyst E. coli strain for high-yield, high-efficiency, and low-pollution pyruvate and d-alanine production, which has great potential for the commercial application in the future.

Unveiling the Multipath Biosynthesis Mechanism of 2-Phenylethanol in Proteus mirabilis.

Proteus mirabilis could convert l-phenylalanine into 2-phenylethanol (2-PE) via the Ehrlich pathway, the amino acid deaminase pathway, and the aromatic amino acid decarboxylase pathway. The aromatic amino acid decarboxylase pathway was proved for the first time in P. mirabilis. In this pathway, l-aromatic amino acid transferase demonstrated a unique catalytic property, transforming 2-penylethylamine into phenylacetaldehyde. Eleven enzymes were supposed to involve in 2-phenylethanol synthesis. The mRNA expression levels of 11 genes were assessed over time by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in vivo. As a result, the expression of 11 genes was significantly increased, suggesting that P. mirabilis could transform l-phenylalanine into 2-phenylethanol via three pathways under aerobic conditions; nine genes were significantly overexpressed, suggesting that P. mirabilis could synthesize 2-phenylethanol via the Ehrlich pathway under anaerobic conditions. This study reveals the multipath synthetic metabolism for 2-phenylethanol in P. mirabilis and will enrich the new ideas for natural (2-PE) synthesis.

A single Proteus mirabilis lineage from human and animal sources: a hidden reservoir of OXA-23 or OXA-58 carbapenemases in Enterobacterales.

In Enterobacterales, the most common carbapenemases are Ambler's class A (KPC-like), class B (NDM-, VIM- or IMP-like) or class D (OXA-48-like) enzymes. This study describes the characterization of twenty-four OXA-23 or OXA-58 producing-Proteus mirabilis isolates recovered from human and veterinary samples from France and Belgium. Twenty-two P. mirabilis isolates producing either OXA-23 (n = 21) or OXA-58 (n = 1), collected between 2013 and 2018, as well as 2 reference strains isolated in 1996 and 2015 were fully sequenced. Phylogenetic analysis revealed that 22 of the 24 isolates, including the isolate from 1996, belonged to a single lineage that has disseminated in humans and animals over a long period of time. The blaOXA-23 gene was located on the chromosome and was part of a composite transposon, Tn6703, bracketed by two copies of IS15∆II. Sequencing using Pacbio long read technology of OXA-23-producing P. mirabilis VAC allowed the assembly of a 55.5-kb structure encompassing the blaOXA-23 gene in that isolate. By contrast to the blaOXA-23 genes, the blaOXA-58 gene of P. mirabilis CNR20130297 was identified on a 6-kb plasmid. The acquisition of the blaOXA-58 gene on this plasmid involved XerC-XerD recombinases. Our results suggest that a major clone of OXA-23-producing P. mirabilis is circulating in France and Belgium since 1996.

Distribution of blaTEM, blaSHV, blaCTX-M, blaOXA, and blaDHA in Proteus mirabilis Isolated from Diabetic Foot Infections in Erbil, Iraq.

Diabetic foot infection is considered to be one of the most important medical, economic, and social problems and a major cause of morbidity and mortality. Proteus mirabilis is a common etiologic agent of diabetic foot infections. This study aimed to determine the prevalence of beta-lactamase genes in P. mirabilis recovered from patients with diabetic foot wounds in Erbil, Iraq. Eighteen P. mirabilis isolated from 84 patients with diabetic foot ulcers were first phenotypically examined for the existence of extended-spectrum beta-lactamases by combined disc method and double-disc synergy method that all isolates showed positive results by both methods. The results were confirmed genetically by PCR to detect beta-lactamase-encoding genes (blaTEM, blaSHV, blaCTX-M, blaOXA, and blaDHA). The results revealed that all isolates contained extended-spectrum beta-lactamase and that 80% of the P. mirabilis isolates contained blaDHA, 60% had blaTEM, 53.3% had blaOXA, and 26.7% had blaCTX-M, whereas no isolates harbored blaSHV. The coexistence of two or more beta-lactamase genes in one isolate was observed. The existence of four genes (blaTEM + blaCTX-M + blaOXA + blaDHA) in the same isolate was documented in two isolates. In conclusion, this is the first study that reports a high prevalence of blaDHA and the coexistence of four resistance genes in the same organism in P. mirabilis isolated from diabetic foot patients in Iraq.

Efficient enzymatic synthesis of α-keto acids by redesigned substrate-binding pocket of the l-amino acid deaminase (PmiLAAD).

In our previous study, we produced α-keto acids by using an L-amino acid deaminase PmiLAAD (wide-type) from Proteus mirabilis, however, the catalytic efficiency was low due to its low substrate affinity. In this study, protein engineering of PmiLAAD was performed to improve the α-keto acid production. PmiLAAD was engineered by iterative CASTing to improve its catalytic performance. The four mutant PmiLAAD-SAVS (PmiLAAD-Phe93Ser-Pro186Ala- Met394Val-Phe184Ser) with 6.6 -fold higher specific activity compared with that of wild-type PmiLAAD has been obtained by high-throughput screening. Comparative kinetics analysis showed that the four mutant PmiLAAD-SAVS had a higher substrate-binding affinity and catalytic efficiency than that of PmiLAAD wild-type. The Km, kcat, and kcat/Km values of the PmiLAAD(SAVS) variant was better (-42.7%, 75.11%, and 85.79%, respectively) than the corresponding values of PmiLAAD wild type. Finally, the whole cell biocatalyst E. coli-pETDuet-1-PmiLAAD(SAVS) has been applied to α-keto acids production. The conversion rate of L-phenylalanine reached 99% by whole-cell biocatalyst E. coli-pETDuet-1-PmiLAAD(SAVS). The conversion of (D/L)-4-phenylalanine was reached 49.5% after 7 h by whole-cell biocatalyst E. coli-pETDuet-1-PmiLAAD(SAVS), while the conversion of E. coli-pETDuet-1-PmiLAAD (wild type) was only 18% after an extension of the reaction time (24 h). This study has developed a robust whole-cell E. coli biocatalyst for α-keto acids production by protein engineering, and this strategy may be useful for the construction of other biotransformation biocatalysts.

Functionally critical residues in the aminoglycoside resistance-associated methyltransferase RmtC play distinct roles in 30S substrate recognition.

Methylation of the small ribosome subunit rRNA in the ribosomal decoding center results in exceptionally high-level aminoglycoside resistance in bacteria. Enzymes that methylate 16S rRNA on N7 of nucleotide G1405 (m7G1405) have been identified in both aminoglycoside-producing and clinically drug-resistant pathogenic bacteria. Using a fluorescence polarization 30S-binding assay and a new crystal structure of the methyltransferase RmtC at 3.14 Å resolution, here we report a structure-guided functional study of 30S substrate recognition by the aminoglycoside resistance-associated 16S rRNA (m7G1405) methyltransferases. We found that the binding site for these enzymes in the 30S subunit directly overlaps with that of a second family of aminoglycoside resistance-associated 16S rRNA (m1A1408) methyltransferases, suggesting that both groups of enzymes may exploit the same conserved rRNA tertiary surface for docking to the 30S. Within RmtC, we defined an N-terminal domain surface, comprising basic residues from both the N1 and N2 subdomains, that directly contributes to 30S-binding affinity. In contrast, additional residues lining a contiguous adjacent surface on the C-terminal domain were critical for 16S rRNA modification but did not directly contribute to the binding affinity. The results from our experiments define the critical features of m7G1405 methyltransferase-substrate recognition and distinguish at least two distinct, functionally critical contributions of the tested enzyme residues: 30S-binding affinity and stabilizing a binding-induced 16S rRNA conformation necessary for G1405 modification. Our study sets the scene for future high-resolution structural studies of the 30S-methyltransferase complex and for potential exploitation of unique aspects of substrate recognition in future therapeutic strategies.

A Rare Opportunist, Morganella morganii, Decreases Severity of Polymicrobial Catheter-Associated Urinary Tract Infection.

Catheter-associated urinary tract infections (CAUTIs) are common hospital-acquired infections and frequently polymicrobial, which complicates effective treatment. However, few studies experimentally address the consequences of polymicrobial interactions within the urinary tract, and the clinical significance of polymicrobial bacteriuria is not fully understood. Proteus mirabilis is one of the most common causes of monomicrobial and polymicrobial CAUTI and frequently cocolonizes with Enterococcus faecalis, Escherichia coli, Providencia stuartii, and Morganella morganiiP. mirabilis infections are particularly challenging due to its potent urease enzyme, which facilitates formation of struvite crystals, catheter encrustation, blockage, and formation of urinary stones. We previously determined that interactions between P. mirabilis and other uropathogens can enhance P. mirabilis urease activity, resulting in greater disease severity during experimental polymicrobial infection. Our present work reveals that M. morganii acts on P. mirabilis in a contact-independent manner to decrease urease activity. Furthermore, M. morganii actively prevents urease enhancement by E. faecalis, P. stuartii, and E. coli Importantly, these interactions translate to modulation of disease severity during experimental CAUTI, predominantly through a urease-dependent mechanism. Thus, products secreted by multiple bacterial species in the milieu of the catheterized urinary tract can directly impact prognosis.

Extended-spectrum ß-lactamase-producing Proteus mirabilis with multidrug resistance isolated from raw chicken in Singapore: Genotypic and phenotypic analysis.

OBJECTIVES: Proteus mirabilis is ubiquitous in soil and water. It is an important catheter-associated urinary tract pathogen and has reportedly been associated with antimicrobial-resistant infections. This study reports the draft genome of a multidrug-resistant P. mirabilis isolated from raw retail chicken meat in Singapore. METHODS: The P. mirabilis strain was isolated on BrillianceTM ESBL Agar and was screened for antimicrobial susceptibility against 29 antimicrobial agents using a MicroScan® Neg MIC Panel Type 44. The double-disk synergy test (DDST) was used for confirmation of extended-spectrum ß-lactamase (ESBL) production. Genomic DNA from the pure culture isolate was extracted and was sent for sequencing based on Illumina HiSeq 2500 technology. Further bioinformatics analysis was performed using online tools available at the Center for Genomic Epidemiology. RESULTS: Species identification of the isolate was performed by KmerFinder. Antimicrobial susceptibility testing of the isolate showed multidrug resistance to broad-spectrum ß-lactams, fluoroquinolones and aminoglycosides, among others. ESBL production was confirmed by the DDST. A total of 29 antimicrobial resistance genes were detected by ResFinder. CONCLUSION: To the best of our knowledge, this is the first report of the whole-genome sequence of a multidrug-resistant P. mirabilis producing an ESBL from raw chicken meat in Singapore. This indicates that raw meat in Singapore can be a reservoir for drug-resistant pathogens.

Quantification of Urease Activity.

Urease is one of the most distinctive virulence factors of Proteus mirabilis pathogenesis. Urease activity correlates with many landmark side effects of P. mirabilis catheter-associated urinary tract infections, such as urolithiasis and bacteremia. Here we describe two simple and inexpensive colorimetric methods for quantifying urease activity in single species cultures as well as cocultures.

Disinfection of water and wastewater by biosynthesized magnetite and zerovalent iron nanoparticles via NAP-NAR enzymes of Proteus mirabilis 10B.

Disinfection of water and wastewater strongly contributes to solving the problem of water shortage in arid/semi-arid areas; cheap and ecofriendly approaches have to be used to meet water quality standards. In the present study, a green synthesis of iron nanoparticles (INPs) under aerobic and anaerobic conditions via nitrate reductases (NAP/NAR) enzymes produced by Proteus mirabilis strain 10B were employed for this target. The biosynthesized INPs were characterized; UV-Vis spectroscopy revealed surface plasmon resonance at 410 (aerobic) and 265 nm (anaerobic). XRD indicated crystalline magnetite ((MNPs) aerobically synthesized) and zerovalent INPs (ZVINPs anaerobically synthesized). EDX demonstrated strong iron signal with atomic percentages 73.3% (MNPs) and 61.7% (ZVINPs). TEM micrographs illustrated tiny, spherical, periplasmic MNPs (1.44-1.92 nm) and cytoplasmic ZVINPs with 11.7-60.8 nm. Zeta potential recorded - 31.8 mV (ZVINPs) and - 66.4 mV (MNPs) affirming colloidal stability. Moreover, the disinfection power of INPs was evaluated for standards organisms and real water (fresh, sea and salt mine) and wastewater (municipal, agricultural and industrial) samples. The results reported that INPs displayed higher antagonistic effect than iron precursor, 700 and 850 µg/mL of MNPs and ZVINPs, respectively, was sufficient to show a drastic algicidal effect on algal growth. Both types of INPs demonstrated obvious dose-dependent antibiofilm efficiency. Due to their smaller size, MNPs were more efficient than ZVINPs at the suppression of microbial growth in all examined water samples. Overall, MNPs showed superior antagonistic activity, which promotes their exploitation in enhancing water/wastewater quality.

Virulence targeted inhibitory effect of linalool against the exclusive uropathogen Proteus mirabilis.

Proteus mirabilis is one of the leading causes of catheter-associated UTIs (CAUTI) in individuals with prolonged urinary catheterization. Since, biofilm assisted antibiotic resistance is reported to complicate the treatment strategies of P. mirabilis infections, the present study was aimed to attenuate biofilm and virulence factor production in P. mirabilis. Linalool is a naturally occurring monoterpene alcohol found in a wide range of flowers and spice plants and has many biological applications. In this study, linalool exhibited concentration dependent anti-biofilm activity against crystalline biofilm of P. mirabilis through reduced production of the virulence enzyme urease that raises the urinary pH and drives the formation of crystals (struvite) in the biofilm. The results of q-PCR analysis unveiled the down regulation of biofilm/virulence associated genes upon linalool treatment, which was in correspondence with the in vitro bioassays. Thus, this study reports the feasibility of linalool acting as a promising anti-biofilm agent against P. mirabilis mediated CAUTI.