Isolation, phenotypical and genotypical characterization of Mannheimia haemolytica serotype A5 strain associated with pneumonic Pasteurellosis

doi:10.21203/rs.3.rs-4929192/v1

Download PDF

Research Article

Isolation, phenotypical and genotypical characterization of Mannheimia haemolytica serotype A5 strain associated with pneumonic Pasteurellosis

https://doi.org/10.21203/rs.3.rs-4929192/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Background

Mannheimia haemolytica (M. haemolytica) is the primary pathogen responsible for respiratory diseases in ruminants. As an opportunistic pathogen, it often co-infects with other bacteria and viruses, leading to severe pneumonia. In this study, a suspected M. haemolytica pathogen was isolated from the lungs of sheep on a farm in Luoyang that exhibited respiratory symptoms and died acutely. The species classification, biological characteristics, and genome sequence analysis of the pathogen were determined.

Results

Morphological observations, biochemical tests, and phylogenetic analysis confirmed that the isolate was closely related to serotype A1 M. haemolytica GCA-900474405.1. Furthermore, sequence comparison of the capsular gene region revealed that the bacterium belonged to serotype A5 M. haemolytica and was named MH-1. Antibiotic sensitivity tests showed that MH-1 was resistant to tetracycline, erythromycin, spectinomycin and penicillin-G, and sensitive to other selected antibiotics. In animal experiments conducted on mice via intraperitoneal inoculation with MH-1, depression symptoms and dishevelled hair were observed in all mice in the highest-dose group, leading to death. The LD₅₀ value for mice was determined to be 1.27×10⁹ CFU. Whole-genome sequence analysis revealed that MH-1 had a total of 20 open reading frames (ORFs) encoded genes related to pathogenicity, including proteins involved in adhesion, invasion, iron uptake, and antiphagocytosis. While 8 ORFs were responsible for drug resistance genes, such as the macrolide resistance gene macB, the tetracycline resistance gene tet(35), the aminoglycoside resistance gene APH(3')-Ia, and the β-lactam resistance gene CRP. These findings were consistent with the results obtained from the antibiotic susceptibility test.

Conclusion

In conclusion, we successfully isolated and identified a strain of M. haemolytica serotype A5 from sheep. Through whole-genome sequencing and biological characterization analysis, we have enriched the understanding of the pathogenic properties of M. haemolytica in sheep. This information provides valuable insights for prevention and treatment strategies against M. haemolytica infections in sheep.

Mannheimia haemolytica

genome sequence analysis

antimicrobial resistance

pathogenicity

M. haemolytica, an important conditioned pathogen, commonly resides in the nasal cavity and tonsillar recess of the upper respiratory tract in ruminants^[1]. When the host’s defense system is compromised due to stress, respiratory virus infection or mycoplasma infection, it infiltrates the lungs and can lead to diseases such as pneumonia in goats, mastitis in sheep, and acute septicemia in newborn lambs^[2]. At the same time, it can also increase the incidence of other pathogens. Research has indicated that M. haemolytica can exacerbate the progression of respiratory diseases caused by Mycoplasma^[3]. Sheep and goats are valuable livestock worldwide, providing meat, milk, wool, and leather. However, they are highly susceptible to respiratory diseases, which contribute to approximately 50% of their mortality, regardless of the underlying cause^[4]. While respiratory diseases have multifactorial etiologies globally, specific serotypes of M. haemolytica and Pasteurella multocida (P. multocida) are recognized as major bacterial agents responsible for respiratory illnesses in sheep and goats^[5]. The looming challenge posed by respiratory diseases stems primarily from economic losses due to reduced livestock productivity and high mortality rates, significantly impacting the industry.

M. haemolytica belongs to the Pasteurella family within the Mannheimia genus. Microscopically, it appears as a short gram-negative bacillus with bluntly rounded ends and a capsular membrane. It can ferment glucose, mannitol, sorbitol, xylose, and maltose, but cannot ferment sorbose or mannose^[6]. Sheep infected with M. haemolytica typically exhibit clinical signs of pneumonia, including coughing, dyspnea, lethargy, and sticky nasal discharge^[7]. This bacterium has been identified to have 12 capsular serotypes (A1, A2, A5 ~ 9, A12 ~ 14, A16 and A17). Research data indicate that serotypes A1 and A6 are closely associated with bovine respiratory diseases^[8]. However, serotype A2 is the primary pathogen for pneumonia in sheep and goats^[9]. With the rapid development of the farming industry, the incidence of serotypes A5 and A7 is also increasing. Owing to the diversity of pathogenic serotypes and significant biological differences among them, effective prevention and control measures for this disease are lacking. In recent years, numerous studies have attempted to identify the involved serotypes of ovine/goat pneumonia caused by M. haemolytica to understand their genetic mechanisms of transmission. For example, dominant reports of multiple capsular types from both P. multocida type-A and M. haemolytica serotypes A1, A2 and A7 were reported in goats from the Tanqua Aberegelle and Kola Tembien districts, northern Ethiopia^[10]. Similar research^[11] revealed that at least four different serotypes present in cases of ovine/goat pneumonia caused by M. haemolytica within the Tanqua Aberegelle region. Another study carried out in Wollo, northeast Ethiopia, revealed that ovine cases presented with M. haemolytica serotypes A1, A5, A8, A9 and A13^[12].

In recent years, M. haemolytica has been frequently reported at home and abroad, mostly in endemic or sporadic epidemics^[13]. Virulence factors play crucial roles in its pathogenesis. Upon infecting the host, these bacteria must undergo adhesion steps followed by invasion and proliferation while interfering with the body's defense mechanisms. They produce toxins that harm the body, leading to immune damage along with evasion of clearance mechanisms, which ultimately activate inflammatory responses^[14]. Furthermore, the current methods for the clinical prevention and treatment of this disease rely primarily on antibiotic therapy. However, the widespread use of antibiotics contributes to bacterial resistance enhancement as well as drug residue accumulation within organisms, resulting in an increase in multiple antimicrobial-resistant bacterial strains. This issue has become another major concern regarding disease prevention^[15]. Whole-genome sequencing of microorganisms will not only reveal physiological and evolutionary information about microbial species, but also provide new methods for the diagnosis and treatment of infectious diseases^[16]. The high-efficiency and low-cost characteristics of high-throughput sequencing have greatly contributed to the development of genome sequencing, which has led to the widespread use of genomics in laboratory research^[17]. The aim of this study was to isolate and identify pathogens from the lungs of sheep that died of respiratory diseases in a sheep farm in Luoyang, and to characterize them biologically to provide a reference for further elucidation of the molecular mechanism of the pathogenic mechanism, and thus to provide suggestions for the prevention and treatment of the disease.

Isolation and identification of M. haemolytica

The isolate grew well on BHI solid media and formed round, milky white, translucent colonies (see Fig. 1a). Pinpoint-sized red single colonies were observed in MacConkey agar medium (see Fig. 1b), and weak haemolysis was observed on the blood agar plate (Fig. 1c). Bacterial Gram’s staining, and short red bacilli were visible under the microscope in scattered or clustered arrangements (Fig. 1d). The Swiss staining was bipolar intensive staining (Fig. 1e), which was consistent with the morphological characteristics of M. haemolytica. The scanning electron microscopy morphology of the bacterium is shown in Fig. 1f, without spores or flagella on the surface.

The results of biochemical tests (Table 1) revealed that the isolate could ferment carbohydrates such as glucose, lactose, maltose, sucrose, xylose and L-arabinose without producing indoles. These results were consistent with the biochemical characteristics of M. haemolytica. According to colony morphology, Gram staining, scanning electron microscopy and standard biochemical identification, the isolate was initially identified as M. haemolytica, and the isolate was named MH-1.

Table 1

Biochemical identification results. + represents a positive reaction; - represents a negative reaction.
Phenotypic characteristics	Expected criteria		MH-1
Phenotypic characteristics	M. haemolytica	P. multocida	MH-1
Haemolysis	+	-	+
MacConkey	+	-	+
Indole	-	+	-
Catalase	+	+	+
Oxidase	+	+	+
Glucose	+	+	+
Lactose	+	+	+
Maltose	+	+	+
Sucrose	+	+	+
Xylose	+	+	+
Arabinose	+	-	+
Motility	-	-	-
Gluconate	-	-	-

16S rRNA and sequence evolution analysis

The 16S rRNA amplification product of the isolate was sequenced and compared with the GenBank database, and the results revealed that the similarity of this sequence with other M. haemolytica sequences in the database was as high as 97%, and the evolutionary tree of the 16S rRNA sequence (Fig. 2) revealed that the isolate MH-1 isolated in this study genetically evolved in close relationship with the serotype A1 M. haemolytica GCA- 900474405.1, further indicating that this isolate was M. haemolytica. In addition, GCA-023516355.1, which belongs to the same branch, is also serotype A1, so the serotype of the isolated MH-1 is extremely important.

Growth trends of MH-1

To evaluate the bacterial growth rate and determine the optimal time point for subsequent experiments, we monitored the OD_600nm values and bacterial quantity from a single colony inoculated in BHI medium. The results revealed that with increasing culture time, the OD_600nm value of the bacterial mixture also increased, indicating logarithmic growth at 4 ~ 10 h, slow growth after 10 h, and a decrease after 12 h, as shown in Fig. 3. The average number of viable bacteria was 2×10⁹ CFU/mL after 10 h of culture.

Antibiotic susceptibility testing of MH-1

The results of the antibiotic susceptibility testing (see Table 2) revealed that the isolate MH-1 was sensitive to chloramphenicol, gentamicin, amikacin, streptomycin, neomycin, amoxicillin, cefradine, cefotaxime, cephalexin, ciprofloxacin, oxfloxacin, enrofloxacin, lomefloxacin, clindamycin and polymyxin B; it was moderately susceptible to azithromycin; and it was resistant to tetracycline, erythromycin, spectinomycin and penicillin-G.

Table 2

Antibiotic susceptibility testing of MH-1 to 20 antibiotics.
Antibiotic	Judgment criteria/mm			IZD/mm	Results
Antibiotic	R:Resistance	I:Intermediary	S:Sensitivity	IZD/mm	Results
tetracycline (30 µg)	≤ 14	15 ~ 18	≥ 19	13	R
chloramphenicol (30 µg)	≤ 12	13 ~ 17	≥ 18	19	S
erythromycin (15 µg)	≤ 15	16 ~ 20	≥ 21	15	R
azithromycin (15 µg)	≤ 13	14 ~ 18	≥ 19	17	I
gentamicin (10 µg)	≤ 12	13 ~ 14	≥ 15	25	S
amikacin (30 µg)	≤ 14	15 ~ 16	≥ 17	20	S
streptomycin (10 µg)	≤ 11	12 ~ 14	≥ 15	19	S
neomycin (30 µg)	≤ 12	13 ~ 16	≥ 17	19	S
spectinomycin (100 µg)	≤ 12	13 ~ 14	≥ 15	13	R
penicillin-G (10 IU)	≤ 19	20 ~ 28	≥ 29	9	R
amoxicillin(30 µg)	≤ 16	17 ~ 19	≥ 20	26	S
cefradine (30 µg)	≤ 14	15 ~ 17	≥ 18	25	S
cefotaxime (30 µg)	≤ 22	23 ~ 25	≥ 26	29	S
cephalexin (30 µg)	≤ 14	15 ~ 17	≥ 18	35	S
ciprofloxacin (5 µg)	≤ 15	16 ~ 20	≥ 21	24	S
ofloxacin (5 µg)	≤ 12	13 ~ 15	≥ 16	19	S
enrofloxacin (10 µg)	≤ 15	16 ~ 20	≥ 21	30	S
lomefloxacin (10 µg)	≤ 16	17 ~ 19	≥ 20	21	S
clindamycin (2 µg)	≤ 14	15 ~ 20	≥ 21	26	S
polymyxin B (300 µg)	≤ 7	8 ~ 10	≥ 11	18	S

Pathogenicity test of MH-1 inoculated mice

In the pathogenicity test, the mice in the 2×10¹⁰ CFU group appeared depressed and fried hair 1 h after the attack, and died one after another after 4 h. We continued to observe the state of the mice in the low-dose group, which also appeared similar to the phenomenon of the mice in the high-dose group, with anorexia and depressed spirit. Five mice in the 2×10⁹ CFU group died after 6 h, and the state of the mice in the remaining dose group gradually returned to normal without death. There were no obvious abnormalities in the state of the mice in the PBS group. On the basis of the deaths of the mice, the Koch method was used to calculate the LD₅₀ as the minimum amount of MH-1 required to kill half of the mice weighing 18 ~ 22 g by intraperitoneal injection within one week, which was 1.27×10⁹ CFU. Jiao et al.^[18] isolated the M. haemolytica strain at a 5×10⁹ CFU dose in a mouse model of peritoneal attack, and no death was observed in the mice. In contrast, MH-1 was a strong strain. The pathological changes in the organs of the mice after autopsy are shown in Fig. 4. In group (a), the lungs were bleeding, and the other organs were normal in shape. Group (b) had the most severe lesions, with severe congestion and necrosis of the lungs, and the liver was congested with a dark red color with the oozing of pus on the surface. The lungs of the other groups of mice also presented different degrees of congestion, and the degree of lesions was correlated with the dose. Histopathological examination was subsequently performed on the lungs of the mice in the different groups. As shown in Fig. 5, a large amount of inflammatory cell infiltration was observed in the lungs of group (a), and a large amount of red blood cell infiltration and small blood vessel congestion were observed in groups (b), (c) and (d). Subsequently, strains with the same morphological characteristics as the infected bacteria were isolated from the diseased organs of the dead mice and identified as M. haemolytica via specific PCR (Fig. 6).

Whole-genome basic information of MH-1

Whole-genome sequencing of MH-1 was performed via the Illumina platform, and 15632272 reads were obtained, with an average read length of 149.41 bp. A total of 54 contigs were obtained via reassembly via Spades, and the largest contigs were 292622 bp long. The whole genome size of MH-1 was 2457929 bp, and the N50 was 116123 bp, with 41% GC content. NCBI prokaryotic genome annotation predicted a total of 2407 protein coding regions, of which 1586 genes were predicted to be functional and 821 genes were annotated as hypothetical proteins, with 5 rRNA genes and 54 tRNA genes predicted (Fig. 7). The strain spliced sequences were uploaded to the Genome database as FASTA files to obtain the accession number JBCEYN000000000. The MLST database revealed that the sequence type of the MH-1 strain was ST-16 on the basis of the seven housekeeping genes of M. haemolytica, including adk, aroE, deoD, gapDH, gnd, mdh, and Zwf. According to the database created by Christensen et al.^[19], the sequence comparison between the capsule gene region of this strain and the database predicted that the capsular serotype of this strain was A5.

Annotation of virulence genes

Virulence factors, including bacterial toxins, cell surface proteins that regulate bacterial attachment, and carbohydrate and protein hydrolytic enzymes that protect the bacterial cell surface, are the main substances that cause bacterial colonization and pathogenesis in the host. Different combinations of virulence factors are able to cause different diseases. VFDB is a database used to identify the virulence factors of the pathogenic bacteria, chlamydia and mycoplasma. After VFDB annotation and BLAST comparison, those with ≥ 70% identity were selected, and 20 ORFs encoding MH-1 genes associated with pathogenicity, including proteins mediating adhesion, invasion, iron uptake, and antiphagocytosis, were identified. The related results are shown in Table 3. M. haemolytica produces a series of adhesins that help it adhere to epithelial cells and prevent phagocytosis^[20]. The endotoxin-secreting lipo-oligosaccharide (LOS) acts as a virulence factor that directly exerts toxicity on the host, and the capsule, like other proteins related to the regulation of biofilm formation via its antiphagocytosis effect, also regulates virulence factors to evade the host immune system and indirectly exerts its own pathogenic effects.

Table 3

Virulence factors associated with pathogenicity in the MH-1 genome.
Virulence factors	Related genes
LOS	msbA、yhxB/manB、rfaE、lpxB、rfaF、lpxD、rfaD、lpxC、galU、kdsA、kdsB、lpxA、orfM、gmhA/lpcA
KatA	katA
HxuABC	hxuB
Hsp60	htpB
Capsule	bexC、bexB
Autoinducer-2	luxS

Annotation of drug resistance genes

The gene protein sequences were aligned with the CARD database via BLAST to combine the annotation information of the genes and their corresponding drug resistance functions. A total of eight ORFs encoding multiple resistance genes in the genome of the MH-1 strain, including the macrolide resistance genes macB and CRP, the tetracycline resistance gene tet(35), the aminoglycoside resistance gene APH(3')-Ia, the fluoroquinolone resistance genes CRP and rsmA, the β-lactam resistance gene CRP and the phenicol resistance gene, were identified. Among them, 2 ORFs encoded the same resistance gene, the peptide resistance gene rosA. The annotations of related resistance genes are shown in Table 4.

Table 4

Predicted resistance genes in the MH-1 genome.
Resistant gene category	Resistant gene name
Macrolide antibiotic	macB、CRP
Tetracycline antibiotic	tet(35)
Aminoglycoside antibiotic	APH(3')-Ia
Fluoroquinolone antibiotic	CRP、rsmA
β-lactam antibiotics	CRP
Peptide antibiotic	rosA
Disinfecting agents and antiseptics	qacL
Diaminopyrimidine antibiotic	rsmA
Phenicol antibiotic	rsmA

Pan-genome analysis

The pan-genome consists of 2870 genes, including 2034 core genes, 700 accessory genes, and 136 unique genes (Fig. 8a). In the pan-genome map, the pan-gene size increases with the number of sequenced genomes, which indicates that the pan-genome of strain MH-1 is open; in contrast, the core genes become stable with the addition of certain genomes, which indicates that the core genome is stable (Fig. 8b). Heatmaps based on the homologous genes of M. haemolytica revealed that strain MH-1 was highly similar to GCA-900474405.1 and presented significant genetic differences from the other six strains of M. haemolytica (Fig. 8c). Compared with the genomes of the other 7 strains of M. haemolytica, the petal diagram shows the number of genes common to and unique to those of MH-1 and the related strains, with the flower heart indicating that all the strains were present and that the petals were unique, indicating that the genome of strain MH-1 contained 40 unique genes, mainly some enzymes and transcriptional regulators, as well as the virulence factors kpsM and lktA (Fig. 8d).

In recent years, there have been many reports on the isolation and identification of M. haemolytica from cattle around the world^{[21, 22]}, whereas relatively few reports exist on the isolation and identification of M. haemolytica from sheep^[23]. Respiratory diseases in small ruminants can lead to reduced productivity, acute fever and lung infections, which, if not diagnosed and treated in time, can lead to animal death and reduce economic efficiency. A sheep farm in Luoyang presented severe respiratory symptoms, and one of them died. To investigate the cause of the disease, dead sheep were found to have severe lung and liver lesions, from which bacterial pathogens were isolated. After purification, the isolate was identified as M. haemolytica by morphological observation, biochemical tests and 16S comparisons, and was named MH-1. Next, we sequenced and analyzed the biological characteristics of MH-1 to help us better understand its pathogenesis and transmission in the feedlot.

M. haemolytica can distinguish between different capsule serotypes on the basis of capsule antigens, and the virulence of different serotypes varies greatly. Importantly, inactivated vaccines mainly provide immune protection against homologous serotypes^[1], which is not conducive to the prevention and control of the disease. Therefore, it is highly important to identify and compare the serotypes of M. haemolytica that cause disease in sheep. First, we used existing multiplex PCR methods^[24] to determine whether MH-1 was a common A1, A2, or A6 serotype, and the serotype PCR test was negative (not shown here). However, phylogenetic analysis revealed that MH-1 is most closely related to the known serotype A1 of M. haemolytica, GCA-900474405.1, and the upper branch, GCA-023516355.1, also belongs to serotype A1. To characterize the serotype of MH-1 quickly and accurately, we conducted whole-genome sequencing and bioinformatics analysis and found that the strain was serotype A5. However, the correlation between serotypes and virulence is not absolute. Pathogenicity tests in mice also revealed that MH-1 has high virulence in mice. The study result of Dassanayake et al.^[25] reported that at least some M. haemolytica type A1 strains were nonpathogenic to domestic sheep but highly pathogenic to bighorn sheep. Therefore, we speculate that isolated MH-1 may undergo recombination or mutation, resulting in a change in its serotype. Second, we need to perform further challenge tests on sheep to verify its virulence effect on native animals.

MLST was used to study the genetic characteristics of MH-1, and the MH-1 strain isolated in this study was found to have a unique sequence. Only one strain of ST-16, which was isolated from goat lung in 2013, was included in the Pub-MLST database in China, and no epidemiological reports on this type have been published thus far. However, Garcia-Alvarez et al.^[26] found that the ST16, ST28 and ST8 isolates were the most prevalent (81%) in their study on the typing of M. haemolytica in sheep populations. These genotypes are widely distributed in small ruminants and have never been reported in bovine isolates. This may indicate the adaptability of certain genetic lineages of M. haemolytica to small ruminants.

The presence of various virulence factors and helper genes promotes the pathogenesis and colonization of M. haemolytica in different host organisms. A comparison with the VFDB database revealed 20 ORF-encoding genes related to pathogenicity in MH-1. Among the virulence factors of MH-1, the heat shock protein Hsp60 plays an adhesion role^[27], helping bacteria invade the host cell, continuing to multiply in the host and causing infection^[28]. Lipopolysaccharides (LPS) is a structural component of the cell wall of gram-negative bacteria. Like LPS, LOS also have the function of secreting endotoxin, the main difference is that LPS does not have an O antigen^[29]. Endotoxin is an effective pro-inflammatory medium, which can trigger the release of inflammatory cytokines and stimulate the influx of inflammatory cells, contributing to the occurrence of diseases. In addition, endotoxin has a synergistic effect with M. haemolytica leukocytotoxin (lkt) to increase pathogenicity^[30]. The iron uptake and binding of bacteria with heme are carried out via the HxuABC gene^[31], which can not only transfer host transferrin and lactoferrin as transferrin binding proteins, but also transfer host heme as a heme-binding enzyme to achieve haemolysis properties. Among these virulence factors, a variety of genes regulate the formation of biofilms, which together constitute the first line of defense against antibiotics. Among them, the capsule-related genes bexC and bexB protect bacteria against phagocytosis and immune escape, thus assisting bacteria in avoiding clearance by the host immune system^[32].

The presence of resistance genes in bacteria indicates that they are highly adaptable in harsh environments^[33]. In recent years, resistance to M. haemolytica has increased, and similarly, extensively drug-resistant isolates are becoming more common. New research suggests that antimicrobials routinely used on some farms may be responsible for the development of resistance patterns. Importantly, the genetic mechanism driving multidrug resistance in this bacterium is mainly a mobile genetic element called the integrated conjugated element (ICE)^{[34, 35]}, and ICE can easily spread across genus boundaries. Multidrug resistance is obtained through horizontal gene transfer^[36]. Studies have shown that the drug resistance genes aph and tet are located in ICE^[37]. The results of the drug resistance genes predicted in this study revealed that MH-1 contains tet(35), the most common gene encoding tetracycline resistance^[38], which encodes an effluent pump that can actively pump tetracycline out of bacterial cells^[39]. APH(3')-Ia carried by MH-1 is a resistance gene encoding aminoglycoside O-phosphotransferase, which can modify and inactivate aminoglycosides^[40]. In addition, MH-1 also carries the β-lactam resistance gene CRP and the macrolide resistance gene macB. To verify the drug resistance genotype of the MH-1 strain, 20 antibiotics were used to test its antibiotic sensitivity. The results revealed that MH-1 was moderately sensitive to azithromycin in macrolides, resistant to erythromycin, tetracycline, the aminoglycoside spectinomycin, and the β-lactam penicillin-G. These phenotypic results are basically consistent with the genotypic prediction results, suggesting that analyzing and studying their genetic characteristics via whole-gene technology is reliable. Timsit et al.^[41] also reported that M. haemolytica had a high level of resistance to penicillin-G, whereas parafenicol, enrofloxacin and cefotifore had no or little resistance. These antibiotics with high resistance rates should not be reused to control and treat this disease in clinical prevention and treatment.

Common genes and specific genes correspond to the commonalities and characteristics of samples, and can be used as the basis for the study of functional differences between samples. The functional annotation of the core genes of strain MH-1 revealed that the largest class contained 156 genes (9%), which are involved in the biosynthesis of amino acid transport metabolism products and their derivatives. Further analysis revealed that 13 genes (1%) associated with bacterial growth metabolism, virulence, disease, and defense mechanisms were associated with the MH-1 genome. Specifically, eight genes (dapH, rhaS-2, mdaB, frmA, tufB, cdhR, rng-2, and rng-3) are involved in various anabolic processes of MH-1, helping bacteria establish infection in the host by regulating the expression of virulence-related genes, and enhancing its ability to resist the host immune response; rep-2 and ucpA are important for maintaining the stability and tolerance of bacteria. kpsM is the gene responsible for the synthesis of capsular polysaccharide, which can destroy the host immune system and thus play a pathogenic role^[42]. Importantly, in the prediction results, MH-1 carried the virulence factor leukocytotoxin (lktA), and the presence of the lktA gene in MH-1 was confirmed by specific PCR. A low concentration of lktA can cause an inflammatory response of in sheep white blood cells, whereas a relatively high concentration can cause target cells to form transmembrane channels, thus causing cell lysis and apoptosis^[43]. In addition, some researchers have reported that isolates of M. haemolytica with the lktA gene may not have leukocyte toxicity, and the β-hemolysis is not a reliable indicator of leukocyte toxicity in M. haemolytica isolates^[44]. Although MH-1 exhibit weak β-hemolysis, the cytotoxicity of MH-1 to leukocytes is uncertain. Therefore, it is necessary to determine the leukocyte toxicity of the MH-1 culture supernatant via a target cell cytotoxicity test. In addition, neo may be an antibiotic resistance gene that affects the resistance of bacteria to specific antibiotics. In conclusion, in the face of the epidemic and transmission of M. haemolytica, we need to recognize the role of virulence factors and antimicrobial resistance in its pathogenesis, and consider these factors in the design of disease treatment and control programs focused on these factors.

In this study, pathogenic bacteria were isolated and purified from sheep infected with morbidity and showing typical clinical symptoms of respiratory diseases in the Luoyang area, and the bacteria were identified as M. haemolytica by morphology and molecular biology. Whole-genome sequencing revealed that bacterium was serotype A5, which was named MH-1. By comparing the virulence factors and resistance genes in the genome, a more comprehensive prediction of the pathogenic characteristics of MH-1 such as resistance genes, resistance profiles and related virulence genes was made, and the presence of all these factors elucidated the virulence potential of M. haemolytica strains in sheep and their ability to survive in the host environment. The results of this study may provide a reference for the prevention and control of Mannheimia haemolytica disease and the rational use of antibiotics in the Luoyang area.

Sample collection

Disease materials were obtained from morbid sheep at a large-scale sheep farm in Luoyang, with clinical manifestations of obvious respiratory symptoms such as coughing, runny nose and dyspnea, and trachea and lung tissues were collected after autopsy.

Isolation and phenotypic identification

First, the collected tissue samples were placed in brain heart infusion (BHI) broth and incubated aerobically at 37℃ for 24 h. A loop full of the broth was streaked onto blood agar supplemented with 5% defibrillated sheep blood and MacConkey agar and incubated aerobically for 24 h at 37℃. Second, the typical colonies were selected from the culture plates for Gram’s staining and Swiss staining, and the staining reaction and morphology of the bacteria were observed under a light microscope at 100× magnification. Third, single-colony pure cultures from blood and MacConkey agar were passed onto nutrient agar slants. The following primary biochemical tests were performed: catalase and oxidase. The final identification of the bacteria at the species level was aided by secondary tests, which included the metabolism of sugars such as glucose, lactose, maltose, sucrose, xylose and L-arabinose. In addition, the production of indole, the final product of metabolism, was tested. Finally, presumptive identification was performed on the basis of colony morphology, haemolysis, Gram’s staining, Swiss staining and biochemical tests. In addition, the isolated and identified bacterial samples were observed via scanning electron microscopy.

16S rRNA sequencing

The DNA was extracted according to the instructions of the Bacterial Whole Genome DNA Extraction Kit, and the DNA samples were stored at -20℃. PCR amplification was performed via generic primers for bacterial 16S rRNA: 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACCTTGTTACGACTT-3′). The amplification products were detected by 1% agarose gel electrophoresis, recovered and sent to Sangon Biotech Co., Ltd. (Shanghai, China) for sequencing. The predicted 16S rRNA sequences were subsequently compared with the 16S database of NCBI via BLAST. A phylogenetic tree based on the 16S rRNA genes was constructed via Molecular Evolutionary Genetics Analysis (MEGA) version 6^[45] via the maximum likelihood method.

Growth curve measurement

A single colony of freshly cultured MH-1 was selected, inserted into a test tube containing 5 mL of BHI medium, and cultured at 37℃ and 180 g/min for 12 h as the seed mixture. Twelve test tubes containing 5 mL of BHI medium were supplemented with 50 µL of seed mixture and cultured at 37℃ and 180 g/min. The samples were removed at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h, respectively. The OD_600nm value of the bacterial mixture was determined at different culture times. The growth curve of bacteria was drawn with BHI culture medium without inoculated bacteria as blank control. At the same time, the bacterial mixture at each time point was taken, diluted to an appropriate concentration with a normal saline gradient, and 100 µL was taken from each plate coated with BHI and cultured at 37℃ for 12 h. The colony number of each plate was observed and recorded, and the count was repeated three times.

Antibiotic susceptibility testing

Kirby Bauer’s disk diffusion method was used to determine antibiotic susceptibility. Escherichia coli ATCC 25922 was used as a quality control strain for the antimicrobial susceptibility test. These antibiotics were chloramphenicol (30 µg), penicillin-G (10 IU), streptomycin (10 µg), amoxicillin (30 µg), tetracycline (30 µg), gentamicin (10 µg), amikacin (30 µg), neomycin (30 µg), cefradine (30 µg), cefotaxime (30 µg), cephalexin (30 µg), ciprofloxacin (5 µg), ofloxacin (5 µg), enrofloxacin (10 µg), lomefloxacin (10 µg), clindamycin (2 µg), polymyxin B (300 µg), erythromycin (15 µg), azithromycin (15 µg), and spectinomycin (100 µg). The MH-1 suspension was made with sterile physiological saline and adjusted to 0.5 McFarland standards before being spread on BHI agar with a sterile cotton swab and allowed to stand for 3 ~ 5 min before the antimicrobial discs were applied. The ring of each disc containing a single concentration of each antimicrobial agent was then gently pressed with the point of the forceps to ensure complete contact with the agar surface and left for 30 min for antibiotic diffusion in the disc. The plates were inverted and incubated at 37°C for 24 h. The diameter of the clear zone formed by antibiotic inhibition of bacterial growth was the inhibitory zone diameter (IZD), which was measured in millimeters, and interpreted as sensitive, intermediate, and resistant according to the Clinical and Laboratory Standards Institute (CLSI) breakpoints^[46].

Pathogenicity to mice

Mice weighing 18 ~ 22 g SPF grade were randomly divided into five test groups and one control group, with six mice in each group, and the half lethal dose (LD₅₀) was determined. A 10-fold gradient of 1×10¹¹ CFU/mL bacteria-boosting solution was diluted to different concentrations, with 1 concentration gradient corresponding to each test group, and 0.2 mL was injected intraperitoneally into each, while the control group was injected with an equal amount of PBS. The mice in the test and control groups were subsequently observed regularly for appetite and mental status, and the LD₅₀ was subsequently calculated according to Karber’s method on the basis of the death of the mice. The dead mice were dissected immediately, pathological changes were observed, and the lungs were examined via histopathological slides. The bacteria were reisolated from each organ, and the specific primer Lkt was identified according to the method established by Alexander et al.^[47]. The forward primer sequence was 5′-GCAGGAGGTGATTATTAAAGTGG-3′, and the reverse primer sequence was 5′-CAGCAGTTATTGTCATACCTGAAC-3′.

Whole-genome sequencing

Whole-genome sequencing was performed by Sangon Biotech Co., Ltd. (Shanghai, China). The Illumina^[48] platform was used for sequencing, FastQC was used for data statistics and quality assessment of the original sequencing data, and quality cutting was performed at the same time to obtain relatively accurate and effective data. SPAdes^[49] was used to splice the second-generation sequencing data, GapFiller^[50] was used to fill the GAP between the spliced contig, and Pilon was used for sequence correction to correct the editing errors and insertion loss of small fragments during the splicing process. NCBI-PGAP/Prokka^[51] software was used to predict gene elements CDS, tRNA, rRNA, etc. Finally, CGView^[52] was used to analyze the genome of this strain and generate a circular genomic map. Using the M. haemolytica classification database (https://pubmlst.org/organisms/mannheimia-haemolytica/)^[53], Multi-Locus Sequence Typing (MLST) was performed on seven housekeeping genes (adk, aroE, deoD, gapDH, gnd, mdh and Zwf). In accordance with the database created by Christensen et al.^[19], sequence alignment of the capsule gene region of this strain was performed to predict the serotype of this strain.

Prediction of virulence genes, resistance genes

Virulence Factors of Pathogenic Bacteria (VFDB)^[54] is a database dedicated to the study of pathogenic bacteria, chlamydia, and mycoplasma pathogenic agents, using VFDB to annotate virulence genes of isolated bacteria. The Comprehensive Antibiotic Resistance Database (CARD) is a classic database of antimicrobial resistance genes^[55], that provides a research basis for studying the effects of drugs, and uses the CARD to annotate the antimicrobial resistance genes of isolated bacteria.

Pan-genome analysis

We obtained genomic information and gene sequences from the NCBI RefSeq database on the basis of the names of related strains of M. haemolytica, used Roray to obtain the common and unique genes of the sequenced strains and related strains, and performed in-depth analysis.

Ethics approval and consent to participate

All experimental and animal management procedures were approved by the Animal Welfare and Ethics Committees of Henan University of Science and Technology (Approval Code: 20240508048; Approval Date: 8 May 2024), and complied with the guidelines of the Institutional Administrative Committee and Ethics Committee of Laboratory Animals.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Funding

This research is sponsored by the National Natural Science Foundation of China (grant no. 32072771) and the Natural Science Foundation of Henan Province (grant no. 182300410052).

Author Contribution

Conceptualization, J.W.; methodology, J.W. and Z.Y.; software, J.W.; validation, R.G. and J.L.; formal analysis, J.W.; investigation, S.C.; resources, C.L. and L.H.; data curation, J.W.; writing—original draft preparation, J.W.; writing—review and editing, J.W. and R.G.; visualization, K.S. and Y.W.; supervision, L.W. and X.X.; project administration, Y.J. and K.D.; funding acquisition, Z.Y. and K.D. All authors have read and agreed to the published version of the manuscript.

Acknowledgements

Not applicable.

Availability of data and materials

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Rice J A, Carrasco-Medina L, Hodgins D C, Shewen P E. Mannheimia haemolytica and bovine respiratory disease [J]. Animal health research reviews, 2007, 8(2): 117-28.
Rainbolt S, Pillai D K, Lubbers B V, Moore M, Davis R, Amrine D, et al. Comparison of Mannheimia haemolytica isolates from an outbreak of bovine respiratory disease [J]. Veterinary Microbiology, 2016, 182: 82-6.
Prysliak T, Vulikh K, Caswell J L, Perez-Casal J. Mannheimia haemolytica increases Mycoplasma bovis disease in a bovine experimental model of BRD [J]. Vet Microbiol, 2023, 283: 109793.
Kumar A, Tikoo S K, Malik P, Kumar A T. Respiratory diseases of small ruminants [J]. Veterinary medicine international, 2014, 2014: 373642.
Arnal J L, Fernández A, Vela A I, Sanz C, Fernández-Garyzábal J F, Cid D. Capsular type diversity of Mannheimia haemolytica determined by multiplex real-time PCR and indirect hemagglutination in clinical isolates from cattle, sheep, and goats in Spain [J]. Vet Microbiol, 2021, 258: 109121.
Highlander S K. Molecular genetic analysis of virulence in Mannheimia (pasteurella) haemolytica [J]. Frontiers in bioscience : a journal and virtual library, 2001, 6: D1128-50.
Laishevtsev A I, Iop. Mannheimiosis of cattle, sheep and goats; proceedings of the 3rd International Conference on Agribusiness, Environmental Engineering and Biotechnologies (AGRITECH), Krasnoyarsk, RUSSIA, F Jun 18-20, 2020 [C]. 2020.
Kayal A, Nahar N, Barker L, Tran T, Williams M, Blackall P J, et al. Molecular identification and characterisation of Mannheimia haemolytica [J]. Vet Microbiol, 2024, 288: 109930.
Omaleki L, Browning G F, Allen J L, Markham P F, Barber S R. Molecular epidemiology of an outbreak of clinical mastitis in sheep caused by Mannheimia haemolytica [J]. Veterinary Microbiology, 2016, 191: 82-7.
Assefa G A, Kelkay M Z. Goat pasteurellosis: serological analysis of circulating Pasteurella serotypes in Tanqua Aberegelle and Kola Tembien Districts, Northern Ethiopia [J]. BMC research notes, 2018, 11(1): 485.
Berhe K, Weldeselassie G, Bettridge J, Christley R M, Abdi R D. Small ruminant pasteurellosis in Tigray region, Ethiopia: marked serotype diversity may affect vaccine efficacy [J]. Epidemiology and infection, 2017, 145(7): 1326-38.
Sisay T, Zerihun A. Diversity of Mannheimia haemolytica and pasteurella trehalosi serotypes from apparently healthy sheep and abattoir specimens in the highlands of Wollo, North East Ethiopia [J]. Veterinary research communications, 2003, 27(1): 3-14.
Mombeni E G, Gharibi D, Ghorbanpoor M, Jabbari A R, Cid D. Molecular characterization of Mannheimia haemolytica associated with ovine and caprine pneumonic lung lesions [J]. Microbial Pathogenesis, 2021, 153.
Snyder E, Credille B. Mannheimia haemolytica and Pasteurella multocida in Bovine Respiratory Disease How Are They Changing in Response to Efforts to Control Them? [J]. Veterinary Clinics of North America-Food Animal Practice, 2020, 36(2): 253-+.
Girma S, Getachew L, Beyene A, Tegegne D T, Tesgera T, Debelo M, et al. Identification of serotypes of Mannheimia haemolytica and Pasteurella multocida from pneumonic cases of sheep and goats and their antimicrobial sensitivity profiles in Borana and Arsi zones, Ethiopia [J]. Scientific Reports, 2023, 13(1).
Fraser C M, Eisen J A, Salzberg S L. Microbial genome sequencing [J]. Nature, 2000, 406(6797): 799-803.
Bruger E L, Marx C J. A decade of genome sequencing has revolutionized studies of experimental evolution [J]. Current Opinion in Microbiology, 2018, 45: 149-55.
Jiao Z, Jiang J, Meng Y, Wu G, Tang J, Chen T, et al. Immune Cells in the Spleen of Mice Mediate the Inflammatory Response Induced by Mannheimia haemolytica A2 Serotype [J]. Animals : an open access journal from MDPI, 2024, 14(2).
Christensen H, Bisgaard M, Menke T, Liman M, Timsit E, Foster G, et al. Prediction of Mannheimia haemolytica serotypes based on whole genomic sequences [J]. Veterinary Microbiology, 2021, 262.
Confer A W, Ayalew S. Mannheimia haemolytica in bovine respiratory disease: immunogens, potential immunogens, and vaccines [J]. Animal health research reviews, 2018, 19(2): 79-99.
Andrés-Lasheras S, Zaheer R, Klima C, Sanderson H, Polo R O, Milani M R M, et al. Serotyping and antimicrobial resistance of Mannheimia haemolytica strains from European cattle with bovine respiratory disease [J]. Research in veterinary science, 2019, 124: 10-2.
Timsit E, Hallewell J, Booker C, Tison N, Amat S, Alexander T W. Prevalence and antimicrobial susceptibility of Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni isolated from the lower respiratory tract of healthy feedlot cattle and those diagnosed with bovine respiratory disease [J]. Veterinary Microbiology, 2017, 208: 118-25.
Shanthalingam S, Goldy A, Bavananthasivam J, Subramaniam R, Batra S A, Kugadas A, et al. PCR ASSAY DETECTS MANNHEIMIA HAEMOLYTICA IN CULTURE-NEGATIVE PNEUMONIC LUNG TISSUES OF BIGHORN SHEEP (OVIS CANADENSIS) FROM OUTBREAKS IN THE WESTERN USA, 2009-2010 [J]. Journal of Wildlife Diseases, 2014, 50(1): 1-10.
Klima C L, Zaheer R, Briggs R E, Mcallister T A. A multiplex PCR assay for molecular capsular serotyping of Mannheimia haemolytica serotypes 1, 2, and 6 [J]. Journal of Microbiological Methods, 2017, 139: 155-60.
Dassanayake R P, Shanthalingam S, Herndon C N, Lawrence P K, Frances Cassirer E, Potter K A, et al. Mannheimia haemolytica serotype A1 exhibits differential pathogenicity in two related species, Ovis canadensis and Ovis aries [J]. Vet Microbiol, 2009, 133(4): 366-71.
García-Alvarez A, Fernández-Garayzábal J F, Chaves F, Pinto C, Cid D. Ovine Mannheimia haemolytica isolates from lungs with and without pneumonic lesions belong to similar genotypes [J]. Vet Microbiol, 2018, 219: 80-6.
Garduño Rafael A, Garduño E, Hoffman Paul S. Surface-Associated Hsp60 Chaperonin of Legionella pneumophila Mediates Invasion in a HeLa Cell Model [J]. Infection and Immunity, 1998, 66(10): 4602-10.
Cozens D, Sutherland E, Lauder M, Taylor G, Berry C C, Davies R L. Pathogenic Mannheimia haemolytica Invades Differentiated Bovine Airway Epithelial Cells [J]. Infect Immun, 2019, 87(6).
Kabanov D S, Prokhorenko I R. Structural analysis of lipopolysaccharides from Gram-negative bacteria [J]. Biochemistry (Moscow), 2010, 75(4): 383-404.
Step D L, Confer A W J f a p. Mannheimia haemolytica– and Pasteurella multocida–Induced Bovine Pneumonia [J]. 2009.
Yang F, Zhou Y, Chen P, Cai Z, Yue Z, Jin Y, et al. High-Level Expression of Cell-Surface Signaling System Hxu Enhances Pseudomonas aeruginosa Bloodstream Infection [J]. Infection and Immunity, 2022, 90(10): e00329-22.
Highlander S K. Molecular genetic analysis of virulence in Mannheimia (pasteurella) haemolytica [J]. Frontiers in bioscience : a journal and virtual library, 2001, 6: D1128-50.
Mahboob S, Ullah N, Farhan Ul Haque M, Rauf W, Iqbal M, Ali A, et al. Genomic characterization and comparative genomic analysis of HS-associated Pasteurella multocida serotype B:2 strains from Pakistan [J]. BMC Genomics, 2023, 24(1): 546.
Klima C L, Zaheer R, Cook S R, Booker C W, Hendrick S, Alexander T W, et al. Pathogens of bovine respiratory disease in North American feedlots conferring multidrug resistance via integrative conjugative elements [J]. J Clin Microbiol, 2014, 52(2): 438-48.
Credille B. Antimicrobial resistance in Mannheimia haemolytica: prevalence and impact [J]. Animal health research reviews, 2020, 21(2): 196-9.
Eidam C, Poehlein A, Leimbach A, Michael G B, Kadlec K, Liesegang H, et al. Analysis and comparative genomics of ICEMh1, a novel integrative and conjugative element (ICE) of Mannheimia haemolytica [J]. The Journal of antimicrobial chemotherapy, 2015, 70(1): 93-7.
Snyder E R, Alvarez-Narvaez S, Credille B C. Genetic characterization of susceptible and multi-drug resistant Mannheimia haemolytica isolated from high-risk stocker calves prior to and after antimicrobial metaphylaxis [J]. Vet Microbiol, 2019, 235: 110-7.
Kehrenberg C, Salmon S A, Watts J L, Schwarz S. Tetracycline resistance genes in isolates of Pasteurella multocida, Mannheimia haemolytica, Mannheimia glucosida and Mannheimia varigena from bovine and swine respiratory disease: intergeneric spread of the tet(H) plasmid pMHT1 [J]. The Journal of antimicrobial chemotherapy, 2001, 48(5): 631-40.
Roberts M C. Tetracycline resistance determinants: mechanisms of action, regulation of expression, genetic mobility, and distribution [J]. FEMS microbiology reviews, 1996, 19(1): 1-24.
Ramirez M S, Tolmasky M E. Aminoglycoside modifying enzymes [J]. Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy, 2010, 13(6): 151-71.
Timsit E, Hallewell J, Booker C, Tison N, Amat S, Alexander T W. Prevalence and antimicrobial susceptibility of Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni isolated from the lower respiratory tract of healthy feedlot cattle and those diagnosed with bovine respiratory disease [J]. Vet Microbiol, 2017, 208: 118-25.
Zong B, Liu W, Zhang Y, Wang X, Chen H, Tan C. Effect of kpsM on the virulence of porcine extraintestinal pathogenic Escherichia coli [J]. FEMS Microbiology Letters, 2016, 363(21).
Benz R, Piselli C, Potter A A. Channel Formation by LktA of Mannheimia (Pasteurella) haemolytica in Lipid Bilayer Membranes and Comparison of Channel Properties with Other RTX-Cytolysins [J]. 2019, 11(10): 604.
Bavananthasivam J, Shanthalingam S, Kugadas A, Raghavan B, Batra S, Srikumaran S. β-Hemolysis May Not Be a Reliable Indicator of Leukotoxicity of Mannheimia haemolytica Isolates [J]. Toxins, 2018, 10(5).
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0 [J]. Molecular biology and evolution, 2013, 30(12): 2725-9.
Humphries R, Bobenchik April M, Hindler Janet A, Schuetz Audrey N. Overview of Changes to the Clinical and Laboratory Standards Institute Performance Standards for Antimicrobial Susceptibility Testing, M100, 31st Edition [J]. Journal of Clinical Microbiology, 2021, 59(12): 10.1128/jcm.00213-21.
Alexander T W, Cook S R, Yanke L J, Booker C W, Morley P S, Read R R, et al. A multiplex polymerase chain reaction assay for the identification of Mannheimia haemolytica, Mannheimia glucosida and Mannheimia ruminalis [J]. Vet Microbiol, 2008, 130(1-2): 165-75.
Zhou X, Ren L, Meng Q, Li Y, Yu Y, Yu J. The next-generation sequencing technology and application [J]. Protein & Cell, 2010, 1(6): 520-36.
Bankevich A, Nurk S, Antipov D, Gurevich A A, Dvorkin M, Kulikov A S, et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing [J]. Journal of computational biology : a journal of computational molecular cell biology, 2012, 19(5): 455-77.
Boetzer M, Pirovano W. Toward almost closed genomes with GapFiller [J]. Genome biology, 2012, 13(6): R56.
Seemann T. Prokka: rapid prokaryotic genome annotation [J]. Bioinformatics (Oxford, England), 2014, 30(14): 2068-9.
Grant J R, Stothard P. The CGView Server: a comparative genomics tool for circular genomes [J]. Nucleic acids research, 2008, 36(Web Server issue): W181-4.
Jolley K A, Bray J E, Maiden M C J. Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications [J]. Wellcome open research, 2018, 3: 124.
Chen L, Zheng D, Liu B, Yang J, Jin Q. VFDB 2016: hierarchical and refined dataset for big data analysis--10 years on [J]. Nucleic acids research, 2016, 44(D1): D694-7.
Mcarthur A G, Waglechner N, Nizam F, Yan A, Azad M A, Baylay A J, et al. The comprehensive antibiotic resistance database [J]. Antimicrobial agents and chemotherapy, 2013, 57(7): 3348-57.

No competing interests reported.

supplementaryfile.docx

Download PDF

Editorial decision: Revision requested
21 Aug, 2024
Editor assigned by journal
19 Aug, 2024
Submission checks completed at journal
19 Aug, 2024
First submitted to journal
17 Aug, 2024

You are reading this latest preprint version

Isolation, phenotypical and genotypical characterization of Mannheimia haemolytica serotype A5 strain associated with pneumonic Pasteurellosis

Status:

Version 1

Abstract

Background

Results

Conclusion

Figures

Background

Results

Isolation and identification of M. haemolytica

16S rRNA and sequence evolution analysis

Growth trends of MH-1

Antibiotic susceptibility testing of MH-1

Pathogenicity test of MH-1 inoculated mice

Whole-genome basic information of MH-1

Annotation of virulence genes

Annotation of drug resistance genes

Pan-genome analysis

Discussion

Conclusions

Methods

Sample collection

Isolation and phenotypic identification

16S rRNA sequencing

Growth curve measurement

Antibiotic susceptibility testing

Pathogenicity to mice

Whole-genome sequencing

Prediction of virulence genes, resistance genes

Pan-genome analysis

Declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Funding

Author Contribution

Acknowledgements

Availability of data and materials

References

Additional Declarations

Supplementary Files

Status:

Version 1