Prevalence and characterization of Morganella morganii in beef cattle from Sichuan Province, China

Background Morganella morganii (M. morganii) is a member of the genera Morganella in the Enterobacteriaceae family. Methods To better understand the prevalence and characterization of M. morganii strains in cattle, 191 nasal swabs samples from beef cattle in Guang'an, Yibin, and Ziyang, Sichuan Province, China were collected. The presence of M. morganii in swabs was determined by PCR. Then positive swabs were processed by using bacterial isolation and identication. The M. morganii isolates were assayed for antimicrobial susceptibility and pathogenicity using a mouse model. Results The prevalence of Morgenella morganii in three cities was 10.5% (20/191). Three M. morganii strains (16GA7, 17GA61.2, and 17YB9) were isolated and identied from positive samples. Phylogenetic analysis demonstrated that the 16S rRNA gene of M. morganii isolates could be clustered with known Genbank M. morganii strains. All three M. morganii strains were sensitive to penicillin-type and quinolone antibiotics, and were resistant to some cephalosporins, carbapenems, macrolide antibiotics and polymyxin B. Lesions in mice inoculated with M. morganii included pulmonary hemorrhage, thickened alveolar walls, and pulmonary inammatory cell inltration, although lesions varied by strain.


Background
Morganella morganii (M. morganii) is a Gram-negative bacterium in the family Enterobacteriaceae that was rst isolated in 1906 from a pediatric fecal culture [1]. This bacterium is part of the normal bacterial ora of humans and other animals, and is widely distributed in the environment. M. morganii is an opportunistic pathogen that rarely causes infections, but can be associated with a diverse number of conditions, including sepsis, abscessation, urinary tract infection, cellulitis, diarrhea, and bacteremia [2][3][4][5][6]. Furthermore, it is capable of causing nosocomial infections and high mortality rates are possible. M. morganii infections have been reported in a variety of domestic animals that are associated with humans, including broiler chickens, piglets, rabbits, and dairy calves [7][8][9][10][11][12][13][14]. It is noteworthy that M. morganii can live in the oral cavity of animals and thereby cause infections in humans via bites [3,[15][16][17]. Although the M. morganii can rarely cause the occurrence of zoonotic disease, it is noteworthy that M. morganii can spread in weak persons [18,19].
Since the 1970s, M. morganii has been considered an important cause of nosocomial infections in adults, such as urinary tract and wound infections [2]. Some of these infections have had high mortality rates due to M. morganii's virulence and increasing drug resistance [20]. M. morganii has also caused disease in a variety of animals. M. morganii was isolated from chickens suspected with fowl typhoid or pullorum disease in Nigeria, and important pathologic lesions, including enlarged and congested spleens, enlarged livers congested and friable areas with necrosis, congested lungs, congested, mis-shapened and atrophic ovaries [21]. Large numbers of M. morganii were isolated from the pneumonic lesion of a piglet with sero brinous pleuropneumonia [11]. M. morganii was isolated from the lungs, blood, liver, and blowhole mucosa of a dead dolphin with brino-hemorrhagic bronchopneumonia. This death was attributed to septicemia, based on the ecchymoses and petechiae of the spleen, pancreas, forestomach, lungs, visceral peritoneum, and small intestine [7]. In 2018, Li et al. reported a case of M. morganii infection in Holstein calves in Tai'an, Shandong province that originated from lactating cattle with latent infections. In this instance, calves were infected from the drinking milk and the presented clinical signs including depression, poor appetite, paralysis, and feces with white ocs [12]. Little is known about the prevalence of M. morganii in Chinese cattle, and this agent was not previously reported in beef cattle.
In the present study, prevalence of M. morganii in beef cattle was analyzed, three strains of M. morganii were isolated from cow nasal swabs in Guang'an, Yibin and Ziyang, Sichuan Province, China. Furthermore, the biochemical characteristics, drug sensitivity and pathogenicity of these M. morganii isolates were investigated.

Mouse virulence
Mice inoculated with M. morganii were depressed, huddled together, had abdominal breathing, and sticky ocular secretions, and moribund mice had open mouth breathing. No clinical abnormalities were evident in control mice. All mice inoculated with strain 16GA7 dead, 3 mice inoculated with strain 17GA61.2 dead, and no mice inoculated with strain 17YB9 dead. Pulmonary hemorrhage was evident in inoculated mice, with no other gross post-mortem pathology evident in other organs. Control mice had no gross pathological abnormalities ( Figure 4). Each M. morganii strain was re-isolated from the tissue samples of mice inoculated with the corresponding strain.
Each M. morganii strain presented with similar histopathological ndings, although differences were evident ( Figure 5). Strain 16GA7 was associated with pulmonary hemorrhage, alveolar wall thickening, pulmonary in ammatory cell in ltration, spleen congestion, hepatic cord disorder, hepatocyte swelling and necrosis, and narrow renal cystic spaces. Strain 17GA61.2 was associated with pulmonary hemorrhage, necrosis, collapsed alveolar structures, pulmonary in ammatory cell in ltration, increased splenic multinuclear macrophages, indistinct splenic white pulp structure, hepatic cords derangement, hepatocyte swelling and necrosis, and narrowing of the renal cystic cavity ( Figure 5). Strain 17YB9 was associated with pulmonary hemorrhage, alveolar wall thickening, pulmonary in ammatory cell in ltration, splenic congestion, vacuolar degeneration, hepatic cord derangement, hepatocyte swelling and necrosis, vacuolar degeneration; narrowing of the renal cystic space, and vacuolar degeneration. There were no abnormalities in the cardiomyocytes of the mice infected any of the M. morganii strains. There were no histopathological abnormalities in control mice.

Discussion
Morganella morganii can be an opportunistic pathogen when the host's immune system is compromised and the agent spreads systemically [22]. Morganella morganii is widely distributed in nature and infections have been reported in sh, amphibians, reptiles, birds, and mammals including humans [3,23]. This agent has also been associated with food poisoning in humans [14,24,25]. Cattle farming has become a fast-growing industry in China. However, intensive beef cattle farming is facing challenges from infectious diseases. Our ndings suggest that bovine M. morganii infections are a potential infectious disease concern for the cattle industry.
In our current study, the overall prevalence of Morgenella morganii in Guang'an, Yibin and Ziyang was 10.5% (20/191). This result suggests that the risk of exposure to M. morganii in cattle is high in Sichuan. We isolated three M. morganii strains 16GA7, 17GA61.2 and 17YB9 from M. morganii positive nasal swabs collected from beef cattle farms in Sichuan. According to the medical history investigation, these cattle had a history of long-distance transportation shortly prior to disease onset. Transportation-related stressors and may have compromised immunity in these animals, and thereby favoring development of opportunistic M. morganii infections. A dolphin died from septicemia caused by M. morganii infection, and transport stress and introduction to a new facility were also considered to be the most likely risk factors [7].
All three M. morganii isolates were sensitive to quinolone and some penicillins-type antibiotics, and resistant to some cephalosporins, carbapenems, macrolide antibiotics and polymyxin B. The spectrum of antibiotics for which M. morganii has apparent resistance raises concerns for this agent becoming a multidrug-resistant or extensively drug-resistant zoonosis. A pediatric patient with sepsis was reported to have a M. morganii isolate that had NDM-1 and cephalosporinases, although this patient was managed successfully with a combination of aztreonam and ceftazidime-avibactam [26]. A Chinese cobra (Naja naja atra) isolate of M. morganii was reportedly susceptible to piperacillin [23]. Morganella morganii isolated from cattle experiencing high mortality was sensitive to streptomycin, imipenem, aztreonam, and cefoperazone [12]. Taiwanese patients had M. morganii isolates with resistance to rst-generation cephalosporins and ampicillin-clavulanate [2]. Based on our results, we recommended using enro oxacin or amikacin for treatment. Differences between our results and previous M. morganii studies drug resistance studies [2,3,12,26,27] may be caused by differences between individual strains and environment, including the application history of antibiotic use.
The virulence of M. morganii strains 16GA7, 17GA61.2 and 17YB9 differed, as did the pathology associated with each strain. Among these three M. morganii strains, 16GA7 had the strongest virulence, followed by 17GA61.2, and with 17YB9 having the least virulence. Pathology documented in infected mice included pulmonary hemorrhage, thickened alveolar walls, and pulmonary in ammatory cell in ltration, as well as varying degrees of splenic, hepatic, and renal lesions. These results differ from previous results for sdta1-sdta5 M. morganii isolates that were associated with hepatic and renal necrosis, hepatic nodules, and renal pelvis hemorrhage with the absence of pulmonary hemorrhage [12].
Comparative genomic analyses demonstrate that M. morganii pathogenesis varies with evolution of the agent's virulence [28]. Hosts are also likely more susceptible to M. morganii infection when they are immunocompromised. In short, differences among M. morganii isolates need to be further characterized for an improved understanding of pathogenicity, appropriate diagnostic screening, vaccine development, and human public health control measures.
To the best of our knowledge, this study is the rst to report the prevalence of M. morganii in cattle, and only one case report about dairy calves previously addressed M. morganii risks [12]. Nevertheless, M. morganii infections may have existed in cattle for a long term without being diagnosed or misdiagnosed. Therefore, M. morganii infections are of concern for the potential to become an emerging disease threat to the Chinese commercial cattle industry. Thus, this study is important for understanding the prevalence and pathogenic characteristics of M. morganii, and potential therapeutic antibiotics that may be effective for treating infections.

Conclusion
In our study, prevalence of M. morganii was surveyed and three M. morganii strains were isolated from beef cattle nasal swabs. In addition, our drug sensitivity assay results demonstrated that M. morganii was resistant to multiple antibiotics including cephalosporins, carbapenems, macrolide antibiotics and polymyxin B. Therefore, the development of appropriate management programs for M. morganii is vitally important and urgent for the Chinese commercial cattle industry. It is worth pondering that the risk of M. morganii infection in humans through contact with cattle or beef foods cannot be ignored.

Clinical sample collection
From April 2016 to October 2019, nasal swabs samples were collected from Sichuan Province beef cattle farms located in Guang'an (n = 77), Yibin (n = 64) and Ziyang (n = 50) ( Table 1). Samples were collected from cattle with recently developed clinical signs following transport from other provinces. Clinical signs included nasal discharge, cough, and dyspnea.
Prevalence of M. morganii determined by PCR DNA was extracted from nasal swab sample us ing a One-tube General Sample DNAup for PCR kit (Sangon Biotech (Shanghai) Co., Ltd.). The speci c operation was carried out according to the instructions provided by the manufacturer, and the extracted DNA samples were stored at -20℃ until analysis by PCR. The speci c PCR primers Mm208F (5'-CTC GCA CCA TCA GAT GAA CCC ATA T-3') and Mm1017R (5'-CAA AGC ATC TCT GCT AAG TTC TCT GGA TG-3') were previously developed by Kim and others for detection of M. morganii based on the 16S rRNA sequence [29]. Primers were synthesized by Sangon Biotech (Shanghai). 2×Master Mix (Chengdu Qingke Zixi biotechnology Co., Ltd.) was used for ampli cation. M. morganii preserved in our lab was used as positive control in PCR. Pooled PCR products were con rmed by comparing DNA size with a DNA mass ladder (Chengdu Qingke Zixi biotechnology Co., Ltd.) on 1% agarose (Biowest, Spain) gel stained with ethidium bromide (Chengdu Haoboyou biotechnology Co., Ltd.). Gels were placed on Gel Documentation system (Bio-Rad Laboratories) for band visualization and photography.

Isolation and identi cation of M. morganii
Each M. morganii positive nasal swab sample was placed in Tryptic Soy Broth (TSB) medium (Qingdao Hai Bo Biotechnology Co., Ltd.) for pre-enrichment. After incubation at 37℃for 24 h, one loop of each preenriched culture was streak-inoculated on to blood agar and MacConkey plates (Qingdao Hai Bo Biotechnology Co., Ltd.) and incubated at 37℃ for an additional 24 h. Suspected single colonies were selected for microscopic examination with gram staining. Next, the suspected M. morganii puri cation cultures were identi ed using bacterial trace biochemical identi cation tubes (Hangzhou microbial reagent Co., LTD) and PCR ampli cation. Primer synthesis and PCR products sequencing were conducted by Sangon Biotech (Shanghai) using a 16S rRNA gene primer (Forward primer 5'-AGAGTTTGATCCTGGCTCAG-3', Reverse primer 5'-TACGGCTACCTTGTTACGACTT-3'). Phylogenetic analysis was performed based on the 16S rRNA gene sequences available in the GenBank database. Phylogenetic trees were reconstructed using MEGA7.0 software [30], with sequences aligned to M. morganii reference strains retrieved from GenBank. Bootstrap values were tested with 1000 replicates using a neighbor-joining algorithm, and evolutionary distances were determined using the Kimura twoparameter model.
Mouse virulence 4-week-old SPF-Kunming mice (n = 24, male, weight range = 18-22 g) were purchased from Chengdu Da Shuo Laboratory Animal Company (China). Mice were randomly assigned to four treatment groups with six mice in each group. Three treatment groups were inoculated with a unique M. morganii strain, and one group was set as control. Inoculations were performed using intraperitoneal injections of a suspension made from M. morganii cultures containing 4×10 8 CFU/mL (0.2 mL/per mouse) in a sterile common broth medium. Control group mice were intraperitoneally injected with equivalent volumes of sterile common broth medium. All mice were monitored for mortality and clinical signs such as ru ed fur, lethargy and dyspnea for 7 d post-inoculation. Mouse hearts, livers, spleens, lungs and kidneys were collected aseptically post-mortem and xed in 4% paraformaldehyde (Solarbio Life Science) for routine processing and embedded in para n. Para n-embedded tissue samples were sectioned (3 μm) and stained with hematoxylin and eosin (H&E) (Beyotime, Beijing). Organ histopathology of all mice was documented by two pathologists who were blinded to treatments. to food and water for seven days. Then, they were randomly divided into different group. Mice were euthanized using sodium phenobarbital according to American Veterinary Medical Association guidelines (2013, AVMA) [32], and every effort was made to minimize animal suffering.

Consent for publication
Not applicable.

Availability of data and materials
The 16S rDNA gene sequences of 3 M. morganii isolates (16GA7, 17GA61.2, 17YB9) have been deposited in the GenBank database under accession numbers MN807692, MN807693, and MN807694, were free downloaded for scienti c research. The other data information during the current study are available from the corresponding author (Zhicai Zuo, zzcjl@126.com) on reasonable request.

Competing interests
All authors declared no competing con ict of interest.     Note: Sensitive (S); dose-dependent sensitivity (S-DD); drug resistance (R). Figure 1 The partial ampli ed positive PCR products analyzed by agarose gel electrophoresis. M: DNA Marker, P: positive control, 1-4: samples PCR products, 5: negative control.

Figure 3
Phylogenetic tree analysis based on 16SrRNA gene sequences.  Representative photomicrographs of H&E-stained heart, liver, spleen, lung, and kidney sections (200x). Arrows for 16GA7, 17GA61.2, and17YB9 photomicrographs identify areas of hepatocyte necrosis. In addition to hepatocyte necrosis, livers from 17YB9 group have vacuolar degeneration. The arrow for the 16GA7 group's spleen indicates splenic congestion, whereas the arrows for the 17GA61.2 group's spleen indicates multinuclear macrophage proliferation, and the arrow for the 17YB9 group's spleen indicates splenic congestion and vacuolar degeneration. The arrows for the 16GA7 and 17YB9 groups in lung tissue indicate areas of pulmonary hemorrhage, alveolar wall thickening and pulmonary in ammatory cell in ltration, and the arrow for the 17GA61.2 group indicates pulmonary hemorrhage. The arrows for the 16GA7, 17GA61.2, and 17YB9 group's kidney photomicrographs indicate narrowing of the renal