Proteus faecis: a putative pathogen of the freshwater Yangtze finless porpoise

doi:10.21203/rs.3.rs-4302864/v1

Proteus faecis is a Gram-negative facultative anaerobic rod-shaped bacterium capable of swarming motility. It has been isolated from numerous sources such as humans, animals, and refuse and is considered a putative human pathogen. In this study, bacteria were isolated from the blowhole of a Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis; YFP) living in captivity in China. One bacterium, P. faecis porpoise, was isolated and whole genome sequencing done. Biofilm formation, motility and antimicrobial resistance were also investigated. To find putative virulence factors, the genome of P. faecis strain porpoise was compared to the genomic sequences of eight other P. faecis isolates using the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) (https://www.bv-brc.org/). The goal of this study was to initially characterize the pathogenicity of this bacterium isolated from a cetacean species using both pathogenomics and conventional approaches.

Proteus faecis

Virulence factors

Pathogenicity

BV-BRC

Yangtze finless porpoise

The Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis, YFP) is a type of small toothed whale. It is a freshwater porpoise living in the middle and lower reaches of the Yangtze River, as well as Poyang and Dongting Lakes of China (Committee on Taxonomy 2012; Gao and Zhou 1995). Past surveys have shown a constant sharp decline in the porpoise population (Zhao et al. 2008; Mei et al. 2012; Huang et al. 2020) which was mainly due to detrimental human activities (Wang 2009; Zhao et al. 2008; Mei et al. 2012). As a result, the YFP is listed as a critically endangered population in the International Union for Conservation of Nature Red Data Book (Wang et al. 2013), and as the First Order of Key Protected Animals in China. Fortunately, the results of a recent survey done in 2022 showed an increase in this porpoise population.

To date, the genus Proteus is comprised of only ten species which have been validly published (Dai et al. 2020) in which all members belong to the family Morganellaceae (formerly Enterobacteriaceae). They are Gram-stain-negative rods containing the appendages flagella and fimbria. Some are opportunistic human pathogens (Drzewiecka 2016) capable of causing urinary tract (Li et al. 2002), ear, nose, throat and skin infections (O'Hara et al 2000). P. faecis was first isolated from human fecal and sputum samples (Dai et al. 2019). The bacterium has been described as a putative human pathogen (Bartlett et al. 2022; Paul et al. 2024). However, the pathogenicity of this bacterium has never been characterized. In this study P. faecis porpoise was isolated from the blowhole of a YFP living in captivity in China. The bacterium was subjected to whole genome sequencing and then compared to the genome sequences of eight other isolates from a refuse dump, animal, and human sources and candidate virulence factors were determined. In addition, in vitro biofilm, motility and haemolysis assays using P. faecis porpoise were performed.

Sample collection

The Yangtze finless porpoise sampled in this study is a female with a body length of 142 cm, about four years old. She was introduced from the Hubei Tian’e-Zhou Oxbow for captive breeding purposes. She has been reared in captivity in an indoor pool together with other three porpoises, at the Wuhan Baiji Dolphinarium, China. Upon exhaling an abnormal odour was detected which has lasted several months. And thus, a medical examination was conducted on July 17, 2023. During this examination, a blood agar plate was held over the blowhole of this porpoise and the animal was allowed to exhale freely.

The experimental procedures used in this study were approved by the Regulations of the People’s Republic of China for the Implementation of Wild Aquatic Animal Protection (promulgated in 1993), adhering to all ethical guidelines and legal requirements in China.

Isolation of bacteria from the blowhole

The blood agar plate was incubated aerobically at 30℃ for 2 days. Colonies were sub-cultured onto nutrient agar plates and allowed to grow. The 16S rRNA gene was subject to PCR amplified using the primers 27F and 1492R (Lane 1991) and then sequenced according to the methods outlined in the study by Edwards et al. 2019. For identification purposes, sequences generated in this study were compared to 16S rRNA gene sequences of known bacteria deposited in the EzBioCloud 16S database (Yoon et al. 2017). The 16S rRNA gene sequence from the isolate of interest in this study has been deposited in the GenBank nucleotide database under the accession number PP420222.

Genome sequencing

Genomic DNA from P. faecis strain porpoise was assayed with 1% agarose gel electrophoresis after genomic DNA extraction and was subject to whole genome sequencing (WGS) as outlined in the study by Edwards et al. 2019. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession JBBAXO000000000. The version described in this paper is version JBBAXO010000000.

Comparison of the genomes of nine P. faecis strains

The BV-BRC database was used to obtain the sequences of nine publicly available P. faecis genomes. The Type (Strain) Genome Server (TYGS) (Meier-Kolthoff and Göker 2019) was used to verify the identity of all the isolates used in this study. Plasmid replicons were predicted using PLSDB (Schmartz et al. 2022). The Comprehensive Antibiotic Resistance Database (CARD) - Resistance Gene Identifier (RGI) (Alcock et al. 2023) was used to further identify putative antimicrobial resistance genes. Using Codon Tree (Davis et al. 2020) a phylogenetic tree was constructed (Fig. 1). For the identification of putative virulence factors the protein families for all genomes were compared using the protein family sorter tool from BV-BRC. Sequences were filtered by genus-specific families (PLfams) which uses a k-mer based approach (Davis et al. 2016). Keyword searches were then used to identify putative virulence factors. BLAST was also used to search the function of sequences (Altschul et al. 1990). Default settings were used for all databases.

Biofilm assay

P. faecis porpoise was inoculated into Luria–Bertani broth (LB) grown overnight at 37℃ under static condition before being subcultured at a 1:100 dilution into Dulbecco’s modified Eagle Medium (DMEM; Invitrogen). The bacteria were then allocated to a 24-well tissue culture plate embedded with coverslips and incubated at 25℃ at static under 5% (vol/vol) CO₂ atmosphere condition for 24 h to develop biofilm. The ∆eseB and ∆eseC strains were included as the negative control and positive control, respectively. They both are derived from Edwardsiella piscicida PPD130/91 (Ling et al. 2000; Liu et al., 2019; Shao et al. 2015). The culture supernatants were removed carefully at 24 h post-subculture, and the coverslips were gently rinsed three times with prewarmed PBS, and the bacteria attached to the coverslips were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet for 30 min. Each biofilm stained with crystal violet was photographed before being resolved with 1% SDS buffer (200 per well) to evaluate the OD₅₉₅with a microplate reader (SpectraMax, M5).

Motility assay

To measure motility, P. faecis porpoise was subcultured at a 1:10 dilution into LB broth at 37°C for 3 h, similar amounts of each strain were spotted onto fresh TSA plates containing 0.3% agar or LB plates containing 0.6% agar. Twelve hours later, the motility of the bacterium was determined by comparing the diameters of motility halos on soft agar. The ∆flhB strain is the derivative of the WT strain (Edwardsiella piscicida PPD130/91). The ∆flhB strain fails to move on soft agar, because FlhB is an essential component for assembling flagellar (Xie et al. 2014).

Antimicrobial susceptibility testing

The antimicrobial susceptibility of the P. faecis porpoise was determined using a VITEK® 2 System (bioMérieux) according to the manufacturer’s instructions. In this system, the in vitro susceptibility testing criteria and quality control parameters follow the CLSI standards (Wayne, 2008).

Genome characteristics

Pairwise DNA sequence comparisons were made to sequences deposited in the EzBioCloud database. The 16S rRNA gene sequence (1,436 bp) from our isolate, strain porpoise, showed >99% identity to six different Proteus species. To identify the bacterium more accurately, WGS was performed. Genome-based taxonomy determined strain porpoise belongs within the species faecis.

The genome of P. faecis porpoise consists of 3,802,708 bp. The number of contigs is 74. The contig L50 is 9 and the contig N50 is 135, 430. No plasmid replicons were detected. The features of all nine genomes used in this study are presented in Table 1.

Putative virulence factors

Table 2 lists the putative virulence factors found in the genomes of the nine different P. faecis strains used in this study. The production of cytotoxic haemolysins is very common in pathogens (Hacker and Hughes 1985). For example, P. mirabilis secrete haemolysins which insert themselves into the membrane of host cells resulting in pore formation, and cytotoxicity (Braun and Focareta 1991). In our study P. faecis porpoise exhibited beta-haemolysis when grown on blood agar. A putative haemolysin protein (Fig. S1) was found in all nine strains.

A type VI secretion system (T6SS) was found in all nine strains. This is not unexpected since T6SS gene clusters have been found in over 8000 genomes (Zhang et al. 2022). Twenty five percent of gram-negative pathogens have this apparatus (Bingle et al. 2008). A T6SS functions in a contact dependent manner. Neighboring cells are first punctured and then injected with multiple lethal effectors (Ho et al. 2014; Basler et al. 2015). The T6SS of Proteus mirabilis HI4320 is encoded by 17 highly conserved genes (Alteri and Mobley 2016). From our study, the amino acid sequences of a putative T6SS component TssG are shown in Fig. S2. We also found Phospholipase A1 which is an antibacterial T6SS effector. For delivery into a bacterial cell, this protein interacts with the VgrG cargo protein (Flaugnatti et al. 2016). The amino acid sequences of Phospholipase A1 are shown in Fig. S3.

The type III secretion system (T3SS) is present in numerous Gram-negative bacteria. Some are human pathogens capable of causing dysentery, plague, and typhoid fever (Hueck 1998; Abby and Rocha 2012). The T3SS machinery allows bacteria to interact with target cells (Hueck 1998; Galán et al. 2014) and disrupt cellular processes, such as signal transduction, vesicle transport and cytoskeletal dynamics (Büttner 2012). Based on sequence analysis, there were genes encoding a potential T3SS found on the genome of P. mirabilis HI4320 (Pearson et al. 2008). In our study, a putative T3SS gene cluster was found (Fig. 2). The amino acid sequences of a Type III secretion outer membrane pore forming protein are shown in Fig. S4. The T3SS machinery delivers virulence proteins called type III secreted effectors (T3SEs) into the cytoplasm of a host cell. T3SEs can cause changes which are detrimental to the host cell resulting in a disease (Galán et al. 2014; Jennings et al. 2017). However, only a few T3SEs have been found for bacteria with a known and functional T3SS (Hu et al. 2017). In this study two type III secretion proteins were identified (Fig. 2). Since these proteins are part of a T3SS gene cluster they could be T3SEs. The amino acid sequences are shown in Fig. S5a and b.

Biofilm is a highly structured microbial communities where bacterial cells attach to a biotic or abiotic surface and embedded in a matrix, blocking the killing from antimicrobial agents or host defense (Kostakioti et al., 2013; Lebeaux et al., 2014). Sometimes mineral crystals can also be found in the polysaccharide matrix (Donlan 2002). P. mirabilis is capable of biofilm formation. This can be seen in chronic wound (Rajpaul 2015) and in urinary tract infections (Armbruster et al. 2018). Interestingly, the bacterium can form a biofilm of a crystalline nature due to its ability to produce urease which can in some cases lead to encrustation (Jacoben et al. 2008). In this study a putative biofilm regulator and urease alpha subunit were found. The amino acid sequences are shown in Figs. S6 and S7, respectively.

In this study the ability of biofilm formation by P. faecis porpoise was investigated in DMEM medium, which is of low nutrition, simulating an in vivo environment. As shown in Fig. 3, P. faecis develops a biofilm. The ∆eseB strain and the ∆eseC strain were included as the negative control and positive control, respectively. The EseB filament-mediated bacterial cell-cell interaction prompts biofilm formation, however, EseC inhibits biofilm formation through sequestering EseE, a positive regulator for EseB (Gao et al. 2015; Liu et al., 2019). Since biofilms act as a primary barrier to protect bacteria from the detrimental effects of antibiotics, cytokines, and complement(Wakimoto et al. 2004), our results suggest P. faecis could evade host defenses and survive in vivo through biofilm formation.

Adherence to a biotic or abiotic surface is a critical first step in biofilm formation (Harriott et al. 2019). In Gram-negative bacteria, flagella and pili/fimbriae, often aid in the attachment (Berne et al., 2015). Fimbriae allow the adherence of P. mirabilis to medical devices and uroepithelial cells which can result in a urinary tract infection (Armbruster et al. 2018). From our study, the amino acid sequences from type IV pilus protein PilA are shown in Fig. S8.

Flagella are whip-like appendages which allow for the locomotion of bacteria (Haiko and Westerlund-Wikstrom 2013). Swarming motility is defined as the movement across a semisolid surface, which is operationally defined as multicellular, flagella-mediated surface migration of bacteria (Ha et al. 2014). It is well known that P. mirabilis is capable of swarming motility. In a mouse model it has been demonstrated that P. mirabilis swarmers were able to migration across urethral catheters and enter into the urinary tract (Li et al. 2002). It has also been shown that P. mirabilis swarmer cells are able to establish an ascending infection in a mouse kidney (Allison et al. 1994). Swarming is an important virulence factor since P. mirabilis strains that exhibit non-swarming or weakly swarming capabilities have a reduced ability to colonize the bladder and kidney of mice (Mobley et al. 1996). In this study the motility of the P. faecis porpoise was analysed on LB medium with 0.6% agar or on TSA medium with 0.3% agar. E. piscicida strains were included as the controls. It was observed that P. faecis porpoise can mobilize on the surface of soft agar. The WT strain (E. piscicida PPD130/91) can also move on a semisolid surface but failed to move with the depletion of FlhB (Fig. 4). This experiment indicates that P. faecis porpoise could have flagella, which allows for swarming. Some bacteria, such as Bacillus subtilis, swarm on a wide range of energy-rich media, whereas other bacteria, such as Salmonella enterica and Yersinia entercolitica, require the presence of particular supplements, such as glucose (Julkowska et al. 2005; Young et al. 1999; Harshey et al. 1994). Our results show that P. faecis porpoise can swarm on an agar surface without the need for special supplementation (Fig. 4). The amino acid sequences from flagellar cap protein FliD are shown in Fig. S9.

The twin-arginine translocation (Tat) is a type of protein transport pathway which can be found in both bacteria and archaea. This system is specific for precursor proteins which harbour a twin-arginine pair in the signal sequence. Unlike other translocase systems Tat translocases can translocate proteins which are completely folded across the phospholipid bilayer (Fröbel et al. 2012). Importantly, virulence factor proteins and virulence-associated proteins are Tat substrates (Pradel et al. 2003; Rossier Cianciotto 2005; Zhang et al. 2009). In P. mirabilis it was shown that the twin arginine translocation system is very important in motility, fitness and translocation of virulence factors within the bloodstream during an infection (Armbruster et al. 2019). Sequence information for the Twin-arginine translocation protein TatA, TatB and TatC in P. faecis strains are shown in Figs. S10, S11 and S12, respectively.

The caseinolytic protease (ClpP) is a serine protease which is conserved across several different kingdoms. In Mycobacterium tuberculosis a ClpP depletion resulted in a reduction in colony forming units (CFUs) as shown both in vitro and using a mouse model (Raju et al. 2012). In our study a putative ClpP was found. The amino acid sequences from the ATP-dependent Clp protease proteolytic subunit ClpP are shown in Fig. S13.

Iron is essential for all forms of life. Many proteins and enzymes require this metal in order to function. As a way of combating bacterial infections iron is sequestered by the host. In early studies it was thought that P. mirabilis lacked iron-scavenging systems (Miles and Khimji 1975; Evanylo et al. 1984). However, more recent studies have shown the bacterium is able to scavenge iron. For example, it is unable to make the siderophore enterobactin, but it can still use this molecule which was produced by other bacteria (Himpsl et al. 2010). In this study a ferric anguibactin-binding protein was found in all nine strains. The amino acid sequences are shown in Fig. S14. This siderophore has been documented as an important virulence factor in the fish pathogens Vibrio anguillarum (Wolf and Crosa 1986) and Photobacterium damselae subsp. piscicida (Osorio et al. 2015). In bacteria the ferric uptake regulator (Fur) protein tightly regulates the transport of siderophores in response to the availability of iron (Ernst et al., 1978; Köster 2001). The amino acid sequences of a putative Fur protein from P. faecis strains are shown in Fig. S15. In Escherichia coli, there are different importer systems. The FhuCDB system is used to import hydroxamate-based siderophores (Fecker and Braun 1983). In this study putative ferrichrome transport system permease protein FhuB (Fig. S16) and ferrichrome transport ATP-binding protein FhuC were found (Fig. S17). A putative iron transport gene cluster is shown in Fig. 5.

Antimicrobial resistance

P. mirabilis does not contain any beta-lactamases which are chromosomally encoded. As a result, wild-type strains are sensitive to all lactams (Girlich et al. 2020). However, since there have been several bla_OXA carbapenemases, originating in Acinetobacter spp., found also in P. mirabilis strains (Girlich et al. 2020) the authors examined the antimicrobial resistance status of P. faecis porpoise. The bacterium was not resistant to the carbapenems tested in this study. It was resistant to ampicillin, but not other β-lactam antimicrobials (Table 3). The CARD database identified genes which could confer resistance to ampicillin, through mutations in the penicillin-binding proteins (D350N). The resistome of relatively few P. faecis isolates have been examined. No antimicrobial resistance genes were found in the genome of P. faecis CR112 isolated from a healthy fish (Paul et al. 2024). However, multidrug resistant (MDR) P. faecis FZP1097 was isolated from a wound secretion from a hospital located in Sichuan Province, China. The bacterium was resistant to ampicillin, aztreonam, ciprofloxacin, ceftriaxone and trimethoprim/sulfamethoxazole. It was believed there was an unknown resistance mechanism involved in the MDR phenotype (Li et al. 2002).

In this study a putative pathogen P. faecis was firstly isolated from a captive freshwater porpoise, and the whole genome of this bacterium was sequenced. And then P. faecisgenomes from human, animal and environmental sources were compared. Using in silico methods, the results of this study showed P. faecis contain putative virulence factors, such as haemolysins, flagella, T3SS, T6SS, urease, pili, and siderophore receptors which could aid in pathogenesis. P. faecis porpoise was shown in vitro to have the ability to form a biofilm, exhibit swarming motility on agar plates without the need for special supplementation and to produce a haemolysin. Finally, the antimicrobial resistance of the bacterium was tested, and the results compared to other isolates. In conclusion, our study finds that P. faecisis a putative pathogen isolated from a cetacean species, and provides a baseline for future studies which will examine the pathogenicity of the bacterium in greater depth.

Acknowledgments

We thank the veterinarian and trainers of the Wuhan Baiji Dolphinarium, especially KouXuan Yuan, ZhengYu Deng, YuanZe Feng, Ke Ma, Tiao Chen and ZhangBing Kou, for their help in collecting samples.

Funding

This work was partly supported by grants from the National Key Research and Development Program of China (Grant No. 2022YFF1301600) and the National Natural Science Foundation of China (Grant No. 31870372) to JSZ.

Authors and Affiliations

R.W. McLaughlin, Y.L. Wang, H Liu, Y.J. Hao, C.Q. Wang, J.S. Zheng

Innovation Research Center for Aquatic Mammals; Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China.

R.W. McLaughlin

School of Liberal Arts & Sciences, Gateway Technical College, Kenosha, WI 53144, USA.

S.Y. Zhang, H.X. Xie

State Key Laboratory of Freshwater Ecology and Biotechnology; Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China.

X.L. Wan

School of Animal Science and Nutritional Engineering, Wuhan Polytechnic University, Wuhan, 430023, China.

Y.L. Wang, S.Y. Zhang, H. Liu

University of Chinese Academy of Sciences, Beijing 101408, China.

Email addresses for all authors: [email protected] (R.W. McLaughlin), [email protected] (Y.L. Wang), [email protected] (S.Y. Zhang), [email protected] (H.X. Xie), [email protected] (X.L. Wan), [email protected] (H. Liu), [email protected] (Y.J. Hao), [email protected] (C.Q. Wang), [email protected] (J.S. Zheng).

Corresponding author: Tel.: 86-27-87801331; Fax: 86-27-87491267.

Email address: [email protected] (J.S. Zheng).

Contributions

J.S.Z. and R.W.M. designed this project. Y.L.W. and R.W.M. isolated bacteria, preformed PCR and sent bacteria for DNA and genome sequencing. S.Y.Z. and H.X.X. performed the biofilm and motility assays. R.W.M., S.Y.Z., H.L., X.L.W. and J.S.Z. were responsible for analyzing data. R.W.M. wrote the first draft of the manuscript. All authors contributed to discussions, revisions, and approved the final manuscript.

Corresponding author

Correspondence should be sent to JinSong Zheng

Tel.: 86-27-87801331; Fax: 86-27-87491267.

Email address:[email protected]

Ethics declarations

Competing interests

The authors declare there are no competing interests.

Conflict of interest

The authors declare there are no conflicts of interest.

Consent for publication

Not Applicable.

Abby SS, Rocha EPC (2012) The non-flagellar type III secretion system evolved from the bacterial flagellum and diversified into host-cell adapted systems. PLoS Genet 8:e1001983
Alcock BP, Huynh W, Chalil R, Smith KW, Raphenya AR, Wlodarski MA, Edalatmand A, Petkau A, Syed SA, Tsang KK, Baker SJC, Dave M, McCarthy MC, Mukiri KM, Nasir JA, Golbon B, Imtiaz H, Jiang X, Kaur K, Kwong M, Liang ZC, Niu KC, Shan P, Yang JYJ, Gray KL, Hoad GR, Jia B, Bhando T, Carfrae LA, Farha MA, French S, Gordzevich R, Rachwalski K, Tu MM, Bordeleau E, Dooley D, Griffiths E, Zubyk HL, Brown ED, Maguire F, Beiko RG, Hsiao WWL, Brinkman FSL, Van Domselaar G, McArthur AG (2023) CARD 2023: expanded curation, support for machine learning, and resistome prediction at the Comprehensive Antibiotic Resistance Database. Nucleic Acids Res 51:D690-D699
Allison C, Emody L, Coleman N, Hughes C (1994) The role of swarm cell differentiation and multicellular migration in the uropathogenicity of Proteus mirabilis. J Infect Dis 169:1155-1158
Alteri CJ, Mobley HLT (2016) The versatile type VI secretion system. Microbiol Spectr 4:10
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403-410
Armbruster CE, Mobley HL, Pearson MM (2018) Pathogenesis of Proteus mirabilis infection. EcoSal Plus 8:100-128
Armbruster CE, Forsyth VS, Johnson AO, Smith SN, White AN, Brauer AL, Learman BS, Zhao L, Wu W, Anderson MT, Bachman MA, Mobley HLT (2019) Twin arginine translocation, ammonia incorporation, and polyamine biosynthesis are crucial for Proteus mirabilis fitness during bloodstream infection. PLoS Pathog 15:e1007653
Basler M (2015) Type VI secretion system: secretion by a contractile nanomachine. Philos Trans R Soc Lond B Biol Sci 370:20150021
Berne C, Ducret A, Hardy GG, Brun YV (2015) Adhesins involved in attachment to abiotic surfaces by Gram-negative bacteria. Microbiol Spectr 3:10.1128/microbiolspec.MB-0018-2015
Bingle LE, Bailey CM, Pallen MJ (2008) Type VI secretion: a beginner's guide. Curr Opin Microbiol 11:3-8
Braun V, Focareta T (1991) Pore-forming bacterial protein hemolysins (cytolysins). Crit Rev Microbiol 18:115-158
Büttner D (2012) Protein export according to schedule: architecture, assembly, and regulation of type III secretion systems from plant- and animal-pathogenic bacteria. Microbiol Mol Biol Rev 76:262-310
Committee on Taxonomy (2012) List of Marine Mammal Species and Subspecies. Society for Marine Mammalogy, www.marinemammalscience.org. Accessed 25.07.2012
Dai H, Chen A, Wang Y, Lu B, Wang Y, Chen J, Huang Y, Li Z, Fang Y, Xiao T, Cai H, Du Z, Wei Q, Kan B, Wang D (2019) Proteus faecis sp. nov., and Proteus cibi sp. nov., two new species isolated from food and clinical samples in China. Int J Syst Evol Microbiol 69:852-858
Dai H, Lu B, Li Z, Huang Z, Cai H, Yu K, Wang D (2020) Multilocus sequence analysis for the taxonomic updating and identification of the genus Proteus and reclassification of Proteus genospecies 5 O'Hara et al. 2000, Proteus cibarius Hyun et al. 2016 as later heterotypic synonyms of Proteus terrae Behrendt et al. 2015. BMC Microbiol 20:152
Davis JJ, Wattam AR, Aziz RK, Brettin T, Butler R, Butler RM, Chlenski P, Conrad N, Dickerman A, Dietrich EM, Gabbard JL, Gerdes S, Guard A, Kenyon RW, Machi D, Mao C, Murphy-Olson D, Nguyen M, Nordberg EK, Olsen GJ, Olson RD, Overbeek JC, Overbeek R, Parrello B, Pusch GD, Shukla M, Thomas C, VanOefelen M, Vonstein V, Warren AS, Xia F, Xie D, Yoo H, Stevens R (2020) The PATRIC Bioinformatics Resource Center: expanding data and analysis capabilities. Nucleic Acids Res 48:D606-D612
Donlan RM (2002) Biofilms: microbial life on surfaces. Emerg Infect Dis 8:881-890
Drzewiecka D (2016) Significance and roles of Proteus spp. bacteria in natural environments. Microb Ecol 72:741-758
Edwards MS, McLaughlin RW, Li J, Wan X, Liu Y, Xie H, Hao Y, Zheng J (2019) Putative virulence factors of Plesiomonas shigelloides. Antonie Van Leeuwenhoek 112:1815-1826
Ernst JF, Bennett RL, Rothfield LI (1978) Constitutive expression of the iron-enterochelin and ferrichrome uptake systems in a mutant strain of Salmonella typhimurium. J Bacteriol 135:928-934
Evanylo LP, Kadis S, Maudsley JR (1984) Siderophore production by Proteus mirabilis. Can J Microbiol 30:1046-1051
Fecker L, Braun V (1983) Cloning and expression of the fhu genes involved in iron(III)-hydroxamate uptake by Escherichia coli. J Bacteriol 156:1301-1314
Flaugnatti N, Le TT, Canaan S, Aschtgen MS, Nguyen VS, Blangy S, Kellenberger C, Roussel A, Cambillau C, Cascales E, Journet L (2016) A phospholipase A1 antibacterial Type VI secretion effector interacts directly with the C-terminal domain of the VgrG spike protein for delivery. Mol Microbiol 99:1099-1118
Fröbel J, Rose P, Müller M (2012) Twin-arginine-dependent translocation of folded proteins. Philos Trans R Soc Lond B Biol Sci 367:1029-1046
Galan JE, Lara-Tejero M, Marlovits TC, Wagner S (2014) Bacterial type III secretion systems: specialized nanomachines for protein delivery into target cells. Annu Rev Microbiol 68:415-438
Gao A, Zhou K (1995) Geographical variation of external measurements and three subspecies of Neophocaena phocaenoides in Chinese waters. Acta Theriol Sinica 15:81-92
Gao ZP, Nie P, Lu JF, Liu LY, Xiao TY, Liu W, Liu JS, Xie HX (2015) Type III secretion system translocon component EseB forms filaments on and mediates autoaggregation of and biofilm formation by Edwardsiella tarda. Appl Environ Microbiol 81:6078-6087
Girlich D, Bonnin RA, Dortet L, Naas T (2020) Genetics of acquired antibiotic resistance genes in Proteus spp. Front Microbiol 11:256
Ha DG, Kuchma SL, O'Toole GA (2014) Plate-based assay for swarming motility in Pseudomonas aeruginosa. Methods Mol Biol 1149:67-72
Hacker J, Hughes C (1985) Genetic analysis of bacterial hemolysin production. Bull Inst Pasteur 83:149-165
Haiko J, Westerlund-Wikstrom B (2013) The role of the bacterial flagellum in adhesion and virulence. Biology (Basel) 2:1242-1267
Harriott BS (2019) Biofilms and antibiotics. In: Caplan MJ (ed) Reference module in biomedical sciences. Elsevier, Amsterdam
Harshey RM, Matsuyama T (1994) Dimorphic transition in Escherichia coli and Salmonella typhimurium: surface-induced differentiation into hyperflagellate swarmer cells. Proc Natl Acad Sci 91:8631-8635
Ho BT, Dong TG, Mekalanos JJ (2014) A view to a kill: the bacterial type VI secretion system. Cell Host Microbe 15:9-21
Hu Y, Huang H, Cheng X, Shu X, White AP, Stavrinides J, Köster W, Zhu G, Zhao Z, Wang Y (2017) A global survey of bacterial type III secretion systems and their effectors. Environ Microbiol 19:3879-3895
Huang J, Mei ZG, Chen M, Han Y, Zhang XQ, Moore J, Zhao XJ, Hao YJ, Wang KX, Wang D (2020) Population survey showing hope for population recovery of the critically endangered Yangtze finless porpoise. Biol Conserv 241:108315
Hueck CJ (1998) Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev 62:379-433
Jacoben S, Stickler D, Mobley H, Shirtlif M (2008) Complicated catheter-associated urinary tract infections due to Escherichia coli and Proteus mirabilis. Clin Microbiol Rev 21:26-59
Jennings E, Thurston TLM, Holden DW (2017) Salmonella SPI-2 Type III secretion system effectors: molecular mechanisms and physiological consequences. Cell Host Microbe 22:217-231
Julkowska D, Obuchowski M, Holland BI, Séror SJ (2005) Comparative analysis of the development of swarming communities of Bacillus subtilis 168 and a natural wild type: critical effects of surfactin and the composition of the medium. J Bacteriol 187:65-76
Kostakioti M, Hadjifrangiskou M, Hultgren SJ (2013) Bacterial biofilms: development, dispersal, and therapeutic strategies in the dawn of the postantibiotic era. Cold Spring Harb Perspect Med 3:a010306
Köster W (2001) ABC transporter-mediated uptake of iron, siderophores, heme and vitamin B12. Res Microbiol 152:291-301
Lane DJ (1991) 16S/23S rRNA sequencing. Nucleic Acid Techniques in Bacterial Systematics 125-175
Lebeaux D, Ghigo JM, Beloin C (2014) Biofilm-related infections: bridging the gap between clinical management and fundamental aspects of recalcitrance toward antibiotics. Microbiol Mol Biol Rev 78:510-543
Li X, Zhao H, Lockatell CV, Drachenberg CB, Johnson DE, Mobley HL (2002) Visualization of Proteus mirabilis within the matrix of urease-induced bladder stones during experimental urinary tract infection. Infect Immun 70:389-394
Li Y, Liu Q, Qiu Y, Fang C, Zhou Y, She J, Chen H, Dai X, Zhang L (2022) Genomic characteristics of clinical multidrug-resistant Proteus isolates from a tertiary care hospital in southwest China. Front Microbiol 13:977356
Liu YL, He TT, Liu LY, Yi J, Nie P, Yu HB, Xie HX. (2019) The Edwardsiella tarda type III translocon protein EseC inhibits biofilm formation by sequestering EseE. Appl Envrion Microb 85:e02133-18
Mei Z, Huang SL, Hao Y, Turvey ST, Gong W, Wang D (2012) Accelerating population decline of Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis). Biol Conserv 153:192-200
Meier-Kolthoff JP, Göker M (2019) TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nat Commun 10:2182
Miles AA, Khimji PL (1975) Enterobacterial chelators of iron: their occurrence, detection, and relation to pathogenicity. J Med Microbiol 8:477-490
Mobley HLT, Belas R, Lockatell V, Chippendale G, Trifillis AL, Johnson DE, Warren JW (1996) Construction of a flagellum-negative mutant of Proteus mirabilis: Effect on internalization by human renal epithelial cells and virulence in a mouse model of ascending urinary tract infection. Infect Immun 64:5332-5340
O'Hara CM, Brenner FW, Miller JM (2000) Classification, identification, and clinical
significance of Proteus, Providencia, and Morganella. Clin Microbiol Rev 13:534-546
Osorio CR, Rivas AJ, Balado M, Fuentes-Monteverde JC, Rodríguez J, Jiménez C, Lemos ML, Waldor MK (2015) A transmissible plasmid-borne pathogenicity island confers piscibactin biosynthesis in the fish pathogen Photobacterium damselae subsp. piscicida. Appl Environ Microbiol 81:5867-5879
Paul SI, Khan SU, Foysal MJ, Rahman MM (2024) Whole-genome sequence of Proteus faecis CR112, isolated from the gut of a healthy Labeo rohita. Microbiol Resour Announc 13:e0095323
Pearson MM, Sebaihia M, Churcher C, Quail MA, Seshasayee AS, Luscombe NM, Abdellah Z, Arrosmith C, Atkin B, Chillingworth T, Hauser H, Jagels K, Moule S, Mungall K, Norbertczak H, Rabbinowitsch E, Walker D, Whithead S, Thomson NR, Rather PN, Parkhill J, Mobley HL (2008) Complete genome sequence of uropathogenic Proteus mirabilis, a master of both adherence and motility. J Bacteriol 190:4027-4037
Pradel N, Ye C, Livrelli V, Xu J, Joly B, Wu LF (2003) Contribution of the twin arginine translocation system to the virulence of enterohemorrhagic Escherichia coli O157:H7. Infect Immun 71:4908-4916
Rajpaul K (2015) Biofilm in wound care. Br J Community Nurs 20:S6-S11
Raju RM, Unnikrishnan M, Rubin DH, Krishnamoorthy V, Kandror O, Akopian TN, Goldberg AL, Rubin EJ (2012) Mycobacterium tuberculosis ClpP1 and ClpP2 function together in protein degradation and are required for viability in vitro and during infection. PLoS Pathog 8:e1002511
Rossier O, Cianciotto NP (2005) The Legionella pneumophila tatB gene facilitates secretion of phospholipase C, growth under iron-limiting conditions, and intracellular infection. Infect Immun 73:2020-2032
Schmartz GP, Hartung A, Hirsch P, Kern F, Fehlmann T, Müller R, Keller A (2022) PLSDB: advancing a comprehensive database of bacterial plasmids. Nucleic Acids Res 50:D273-D278
Wakimoto N, Nishi J, Sheikh J, Nataro JP, Sarantuya JAV, Iwashita M, Kawano Y. (2004) Quantitative biofilm assay using a microtiter plate to screen for enteroaggregative Escherichia coli. J Trop Med Hyg 71:687-690
Wang D (2009) Population status, threats and conservation of the Yangtze finless porpoise. Chinese Sci Bull 54:3473-3484
Wang D, Turve ST, Zhao XJ, Mei Z (2013) Neophocaena asiaeorientalis ssp. asiaeorientalis. The IUCN Red List of Threatened Species. Version 2013.1. http://www.iucnredlist.org. Accessed on 16 July 2013
Wayne PA. Development of in vitro susceptibility testing criteria and quality control parameters for veterinary antimicrobial agents; Approved guideline-third edition CLSI document VET02-A3. Clinical and Laboratory Standards Institute, 2008.
Wolf MK, Crosa JH (1986) Evidence for the role of a siderophore in promoting Vibrio anguillarum infections. J Gen Microbiol 132:2949-2952
Xie HX, Lu JF, Rolhion N, Holden DW, Nie P, Zhou Y, Yu XJ (2014) Edwardsiella tarda-Induced cytotoxicity depends on its type III secretion system and flagellin. Infect Immun 82:3436-3445
Yoon SH, Ha SM, Kwon S, Lim J, Kim Y, Seo H, Chun J (2017) Introducing EzBioCloud: A taxonomically united database of 16S rRNA and whole genome assemblies. Int J Syst Evol Microbiol 67:1613-1617
Young GM, Smith MJ, Minnich SA, Miller VL (1999) The Yersinia enterocolitica motility master regulatory operon, flhDC, is required for flagellin production, swimming motility, and swarming motility. J Bacteriol 181:2823-2833
Zhang J, Guan J, Wang M, Li G, Djordjevic M, Tai C, Wang H, Deng Z, Chen Z, Ou HY (2023) SecReT6 update: a comprehensive resource of bacterial Type VI Secretion Systems. Sci China Life Sci 66:626-634
Zhang L, Zhu Z, Jing H, Zhang J, Xiong Y, Yan M, Gao S, Wu LF, Xu J, Kan B (2009) Pleiotropic effects of the twin-arginine translocation system on biofilm formation, colonization, and virulence in Vibrio cholerae. BMC Microbiol 9:114
Zhao X, Jay B, Taylor BL, Pitman RL, Wang K, Wei Z, Stewart BS, Turvey ST, Tomonari A, Reeves R, et al. (2008) Abundance and conservation status of the Yangtze finless porpoise in the Yangtze River, China. Biol Conserv 141:3006-3018

Table 1 The genomes of the Proteus faecis strains used in this study.

Strains	Isolation source	CDS	tRNA	rRNA	GC content	Genome length	GenBank accession #
Porpoise	Porpoise blowhole	3411	74	2	37.70	3802708	JBBAXO010000000
4d_1	Environmental	3534	58	3	38.19	3711274	JAIKTZ000000000
OB21_1	Refuse dump	3576	58	3	37.89	3795388	JAIKTY000000000
FZP1097	Human	3421	77	5	37.68	3766980	JAMOJQ000000000
EPP6_2	Mouse	3718	79	7	38.23	4018015	JASVXB000000000
HY1316	Bat	3490	78	10	38.17	3739844	JAKJMP000000000
CR112	Fish	3273	76	7	38.45	3563334	JARZHJ000000000
08MAS1600	Egg	3332	83	7	37.69	3757698	QEXA00000000
TJ1636	Human	3551	91	2	37.81	3889216	PENZ00000000

Table 3 The antimicrobial susceptibility of the Proteus faecis porpoise.

Antibiotics	MIC (µg mL⁻¹)	Antibiotics	MIC (µg mL⁻¹)
Ampicillin	16 (R)	Cefepime	≤1 (S)
Amoxicillin	S	Aztreonam	≤1 (S)
Sulbactam	≤2 (S)	Ertapenem	≤0.5 (S)
Piperacillin	≤4 (S)	Meropenem	S
Ceftroline	≥64 (S)	Amikacin	≤2 (S)
Cefuroxime	R	Gentamicin	≤1 (S)
Cefotetan	≤4 (S)	Tobramycin	≤1 (S)
Cefotaxime	S	Ciprofloxacin	≤0.25 (S)
Ceftazidime	≤1 (S)	Levofloxacin	≤0.25 (S)
Ceftriaxone	≤1 (S)	Macrodantin	128 (R)
Cefoperazone	S	Trimethoprim	≤32 (S)

MIC: Minimum Inhibitory Concentration; S susceptible; R resistant.

No competing interests reported.

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Proteus faecis: a putative pathogen of the freshwater Yangtze finless porpoise

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