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Serotypes and virulence genes of Pseudomonas aeruginosa isolated from mink and its pathogenicity in mink
Microbial Pathogenesis 139 (2020) 103904
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Serotypes and virulence genes of Pseudomonas aeruginosa isolated from mink and its pathogenicity in mink
T
Zhu Qiana,b,c,1, Peng Huia,b,c,1, Li Hana,b,c, Yang Ling-zhic,d, Zhang Bo-shuna,b,c, Zhu Jiec,d, Guo Wan-lia,b,c, Wang Nanc,d, Jiang Shi-jina,b,c, Xie Zhi-jinga,b,c,∗ a
Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Agricultural University, Taian City, Shandong Province, 271018, China b College of Veterinary Medicine, Shandong Agricultural University, Taian City, Shandong Province, 271018, China c Shandong Provincial Engineering Technology Research Center of Animal Disease Control and Prevention, Shandong Agricultural University, Taian City, Shandong Province, 271018, China d Shandong Binzhou Wohua Biotech Co.,LTD, Binzhou City, Shandong Province, 256600, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Pseudomonas aeruginosa Mink Serotype Virulence genes Infection route
In this study, 20 P. aeruginosa strains were isolated from 112 farmed mink exhibiting hemorrhagic pneumonia in mideastern Shandong province, China. Serotype G (18/20) was the dominant serotype among the isolates with prevalence in mink, followed by serotype B (1/20), serotype C (1/20). The 9 virulence-associated genes of P. aeruginosa were tested using PCR. The prevalence of the virulence genes for the isolates were algD 95% (19/20), plcH 85% (17/20), exoY 80% (16/20), aprA 75% (15/20), lasB 70% (14/20), exoS 65% (13/20), exoT 60% (12/ 20) and toxA 60% (12/20), respectively. The 20 isolates were negative for exoU gene. The isolates exhibited multidrug resistance and cross resistance, using antimicrobial disc susceptibility assays. The animal experiments demonstrated that LD50% of the P.aeruginosa–CS–2 in the intratracheally challenged mink was 2.2 × 107.0 CFU, and 6.8 × 104.0 CFU in the intraperitoneally challenged mink. It implied that both the inoculation doses and the routes of inoculation could have influences on the pathogenicity of P. aeruginosa in mink. Therefore, the evolutionary and epidemiological surveillance of P. aeruginosa in mink should be further strengthened for public health.
1. Introduction Pseudomonas aeruginosa (P. aeruginosa) thrives in moist and wet conditions and is able to utilize a wide range of organic compounds, which is a prevalent opportunistic human pathogen and one of the most important causative agents of hospital-acquired nosocomial infections, characteristically in immunocompromised individuals [1,2]. In the context of a breakdown in host defenses, P. aeruginosa infected a plethora of tissues, causing both acute and chronic infections [3]. And P. aeruginosa also caused serious harms to dogs, cats and fur-bearing animals, such as mink, foxes and raccoon dogs [4]. The still water, unclean cages, water cups and feed troughs has been a reservoir for P. aeruginosa, serving as foci for the dissemination of the organism in common-source outbreaks [1,5]. The multidrug resistant P. aeruginosa was becoming prevalent and difficult to be clinically treated [6,7]. The resistance was coordinated
by a variety of virulence genes, contributing to its persistence and pathogenicity in vivo [8]. Some of virulent factors assisted bacterial establishment and colonization on the surface of the host, others expedited invasion of numerous tissues [9]. The type III secretion system (T3SS) of P. aeruginosa injected toxic effector proteins directly into the cytosol of host cells, subverting host cell defense and signaling systems [10,11]. The secreted toxins, such as exoenzyme S (exoS), exoenzyme T (exoT), exoenzyme U (exoU) and exoenzyme Y (exoY), were referred to as effector proteins [12]. The exoS, exoT and exoU contributed to virulence of P. aeruginosa in the pathogenicity of lung diseases [13]. Elastase B (lasB) could degrade mucins and surfactant proteins that promoted bacterial clearance [14]. Alkaline protease A (aprA) was thought to modulate the host response and prevent bacterial clearance by degrading proteins of the host immune system, including Tumor necrosis factor-α (TNF-α) and complement factors [15]. The lasB of P. aeruginosa acted in concert with aprA to prevent flagellin-mediated
∗
Corresponding author. College of Veterinary Medicine, Shandong Agricultural University, Taian City, Shandong Province, 271018, China. E-mail address: [email protected] (X. Zhi-jing). 1 The authors contributed equally to this work. https://doi.org/10.1016/j.micpath.2019.103904 Received 27 August 2019; Received in revised form 26 November 2019; Accepted 28 November 2019 Available online 01 December 2019 0882-4010/ © 2019 Elsevier Ltd. All rights reserved.
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immune recognition [14]. P. aeruginosa also contained of the other virulence factors, including Phospholipase C (plcH), GDP-mannose dehydrogenase (algD), exotoxin A (toxA) and so on [9,16,17]. Mink was the only animal known to be susceptible to acute, contagious, and fatal lung infections caused by P. aeruginosa [5,18]. Once established within the lungs, P. aeruginosa usually causes episodic bouts of pneumonia that lead to progressive irreversible lung injury and ultimately death [19]. The simultaneous determination of serogroups and virulence factors was of interest for the efficacy of surveillance of infections associated with P. aeruginosa [9]. Here, the objectives of the study were to clarify serotypes, virulence genes and antimicrobial susceptibility of the P. aeruginosa isolates from the mink experiencing respiratory diseases. Furthermore, animal experiments were performed to clarify the pathogenicity of P. aeruginosa to mink.
Table 1 Primers used for amplification of the virulence genes of P. aeruginosa. Gene
Primer
Sequence (5′–3′)
Size of product (bp)
Reference
exoT
F R F R F R F R F R F R F R F R F R
CAATCATCTCAGCAGAACCC TGTCGTAGAGGATCTCCTG GCACGTGGTCATCCTGATGC TCCGTAGGCGTCGACGTAC ATCAGCATCTTTGGTTTGGG TGTGGCGTTCGGACTTCT CAGACCCTGACCCACGAGAT CATTGCCCTTCAACCCG TATCGACGGTCATCGTCAGGT TTGATGCACTCGACCAGCAAG TGAAGACTTTCCGTGGCAC CATCCTCAGGCGTACATCC GGTAACCAGCTCAGCCACA TGCCTTCCCAGGTATCGT CGTCTCCTACCTGATTCCCG GCACCTTCATGTACAGCTTGTG GATTCCATCACAGGCTCG CTAGCAATGGCACTAATCG
1159
[21]
608
[22]
plcH algD aprA exoY exoS toxA
2. Materials and methods
lasB
2.1. P. aeruginosa isolation exoU
During June to July 2016, 112 lung samples of the mink exhibiting respiratory diseases were collected in mideastern Shandong province, China, which were used for bacterial isolation according to standard clinical microbiologic methods. In brief, the samples were isolated in Luria-Bertani (LB) nutrient agar plates and agar plates with 5% sheep blood using a spreading technique, respectively. Plates were cultured for 18–24 h and observed for suspected colonies of P. aeruginosa, which were identified by colony pigmentation, grape-like odor, growth at 42 °C. The isolates were identified by the 16 S ribosomal RNA (16 S rRNA) gene, and the specific primers, 27 F: 5′-AGAGTTTGATCCTGGC TCAG-3′ and 1492 R: 5′-GGTTACCTTGTTACGACTT-3’ [20]. The polymerase chain reaction (PCR) products were extracted from agarose gels, and sequenced in Sangon Biological (Shanghai) Co., Ltd (Shanghai, China). The nucleotide sequences of 16 S rRNA genes were submitted to GenBank, and GenBank accession numbers were MG547323MG547342.
346 445 1035
[21]
398 301 413 3308
[21]
(30 μg), ciprofloxacin (5 μg), ofloxacin (5 μg), clindamycin (2 μg) and imipenem (10 μg). 2.5. Pathogenicity of P. aeruginosa isolate in mink To elucidate the pathogenicity of the P. aeruginosa isolates, the animal experiments were carried out on 70 healthy American mink (twomonth-old), which were divided into 14 groups on average. According to serotype and virulence genes of P. aeruginosa, P.aeruginosa–CS–2 was selected for animal experiments. The mink in the 1–6 groups were lightly anesthetized with ketamine chloride and intratracheal inoculation with P.aeruginosa–CS–2 of 108.0 Colony-Forming Units (CFU), 107.0 CFU, 106.0 CFU, 105.0 CFU, 104.0 CFU and 103.0 CFU, respectively, and the mink intraperitoneally inoculated in the 8–13 groups, the mink in Group 7 intratracheally inoculated with 0.9% NaCl solution and the mink in Group 14 intraperitoneally inoculated with 0.9% NaCl solution, serving as the control group, respectively. The animals were raised separately and fed twice a day on a commercial meat-based diet. Water was available all day. Clinical symptoms of the mink were monitored and recorded daily for 15 days. The tissues, including lung, spleen, liver, kidney, lymph node, cerebrum and heart, were collected from the dead mink or euthanized on day 15 postinfection (p.i.), rapidly immersed in 10% neutral formalin buffer to prevent autolysis for the detection of histological lesions. The 50% lethal dose (LD50%) of P. aeruginosa in mink was titrated using Reed and Muench Method. In brief, the calculation method reads as follows: Distance ratio = (higher than 50% death score 50%)/(higher than 50% death percentage - lower than 50% death score), Logarithm of LD50% = logarithm higher than 50% virus dilution + distance ratio × logarithm of dilution coefficient. This study was carried out in accordance with the recommendations of Guidelines of Animal Care and Use, Animal Care and Use Committee of Shandong Agricultural University. The protocol (SDAUA-2017-37) was approved by the Animal Care and Use Committee of Shandong Agricultural University.
2.2. Serotypes of P. aeruginosa isolates The isolates were serotyped using the slide agglutination with P. aeruginosa Antisera Kit (Denka Seiken Co. Ltd., Tokyo, Japan). In brief, confirming an agglutination according to the procedure, using polyvalent sera, then when an agglutination occurs with a polyvalent serum, confirming agglutination by the same procedure using each monovalent grouping serum which is included in the polyvalent serum. 2.3. Virulence gene assays The genomic DNA was extracted using Bacteria Genomic DNA Kit (CWBIO). Nine virulence genes in P. aeruginosa, were screened using PCR, including exoT, plcH, algD, aprA, exoY, exoS, toxA, lasB and exoU genes. The specific primers and the length of PCR products were shown in Table 1. The PCR conditions used were available upon request. The PCR products were extracted from agarose gels, and sequenced in Sangon Biological (Shanghai) Co., Ltd (Shanghai, China). The nucleotide sequences of the corresponding genes were submitted to GenBank, and GenBank accession numbers were MG506925-MG507043. 2.4. Antimicrobial susceptibility assays
3. Results and discussion Antimicrobial susceptibility of 16 antimicrobials for the P. aeruginosa isolates was performed using disk diffusion method, and the results were interpreted according to the Clinical and Laboratory Standards Institute [23]. The 16 antimicrobial agents included penicillin (10 μg), erythrocin (15 μg), streptomycin (10 μg), gentamicin (10 μg), amikacin (30 μg), tobramycin (10 μg), kanamycin (30 μg), cefoperazone (75 μg), ceftazidime (30 μg), ceftriaxone (30 μg), cefotaxime (30 μg), cefepime
3.1. P. aeruginosa isolation, serotypes and virulence genes In the study, 20 P. aeruginosa strains were isolated from the mink exhibiting respiratory diseases, named as P.aeruginosa–CS–1 to P.aeruginosa–CS–20, and the prevalence of P. aeruginosa in the sampled mink was 17.9% (20/112). The 16 S rRNA genes of the isolates shared 2
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Table 2 The serotypes and virulent genes of the 20 P.aeruginosa isolates. Strains
PA–CS–1 PA–CS–2 PA–CS–3 PA–CS–4 PA–CS–5 PA–CS–6 PA–CS–7 PA–CS–8 PA–CS–9 PA–CS–10 PA–CS–11 PA–CS–12 PA–CS–13 PA–CS–14 PA–CS–15 PA–CS–16 PA–CS–17 PA–CS–18 PA–CS–19 PA–CS–20 P
Virulence genes
Serotypes
Severity degree of infection
exoT
plcH
algD
aprA
exoY
exoS
toxA
lasB
exoU
G
B
C
+ + – – + + + + – + – – + + + – + – – + 60%
+ + + + + + + + – + + + + + + + + – + – 85%
+ + + + + + + + + + + – + + + + + + + + 95%
+ + + + + + + + + – + + + – – + + + – – 75%
+ + + + + + + + – + – + + + + + + – + – 80%
+ + – – – + + + + + + + + – + – + + – – 65%
+ + – + + + + + – – – + + + + + – – – – 60%
– + – + + + + – – + + + + + + – + + + – 70%
– – – – – – – – – – – – – – – – – – – – 0%
+ + + + + + + + + + – – + + + + + + + + 90%
– – – – – – – – – – – + – – – – – – – – 5%
– – – – – – – – – – + – – – – – – – – – 5%
3 3 2 2 2 2 2 2 1 2 2 2 3 2 3 1 2 2 1 1
Note: PA, P.aeruginosa; +, positive; −, negative; P, prevalence; 1, mild infection; 2, moderate infection; 3, severe infection.
(1/20). All strains were sensitive to cefoperazone, ceftriaxone, cefepime and imipenem. The isolates in the mink exhibiting severe hemorrhagic pneumonia had the higher antibiotic resistance rates than the other isolates. Furthermore, it appears that there was no correlation between serotype and antibiotic resistance. The findings provide the information available to veterinarians on rational antimicrobial treatment therapies.
99%–100% identity with the reference P. aeruginosa strains, furtherly confirming that the isolates belong to P. aeruginosa. Eighteen (90%) of the 20 P. aeruginosa isolates belonged to serotype G using slide agglutination, one (5%) serotype B and one (5%) serotype C (Table 2). It demonstrated that P. aeruginosa serotype G was prevalent in the mink farms, playing an important role in mink hemorrhagic pneumonia. Interestingly, P.aeruginosa–CS–11 and P.aeruginosa–CS–20 were isolated from the same farm, but P.aeruginosa–CS–11 belonged to serotype B and P.aeruginosa–CS–20 belonged to serotype G. It implied that the different serotypes could co-circulate in the same farm. The interactions between different serotypes need to be further explored. The prevalence of virulence genes in the P. aeruginosa isolates was shown in Table 2, and algD gene was 95% (19/20), plcH 85% (17/20), exoY 80% (16/20), aprA 75% (15/20), lasB 70% (14/20), exoS 65% (13/20), exoT 60% (12/20) and toxA 60% (12/20). The 20 isolates were negative for exoU gene using PCR. Based on sequence analysis, the virulence gene contents were different among different P. aeruginosa strains. Pathogenicity of the bacterium completely depends on its virulence factors [24]. In the study, the algD, plcH, lasB, aprA toxA, exoS, exoY, and exoT genes were prevalent in the P. aeruginosa isolates. The algD gene had the highest incidence in the isolates, which could be at the origin of the conversion of P. aeruginosa strains to a mucoid phenotype overproducing alginates [9]. Every isolate expressed at least one protein of the type III secretion system protein (T3SS+), an important virulence determinant in P. aeruginosa. And previous studies suggested that failure to eradicate P. aeruginosa in patients with ventilator-associated pneumonia (VAP) might be linked to T3SS, whereas eradication was achieved in patients with undetectable levels of T3SS+ [25]. ToxA was an important virulence factor of P. aeruginosa in clinical infections and the most virulent factor among other factors produced by P. aeruginosa [26].
3.3. Pathogenicity of P. aeruginosa isolates in mink During days 2–8 p.i., some of the challenged animals exhibited clinical symptoms, including lethargy, dullness, dyspnea, anorexia, coarse fur, sneezing and coughing. Some of the inoculated mink died from P. aeruginosa infection, reaching a peak on days 3–5 p.i., and showing lung hemorrhage, splenic hematoma, pericardial effusion, lymph node enlargement, and liver hemorrhage and swelling. The challenged mink developed histologic lesions, including lung bleeding and congesting, spleen bleeding and congesting, liver congesting and steatosis, and heart hemorrhage. The control mink showed no clinical signs. The survived mink were weak, but resumed intake and clinically recovered. As shown in Table 4, the data demonstrated the dose-dependent pathogenicity of P. aeruginosa in challenged mink via the intratracheally or intraperitoneally routes of infections. LD50% (2.2 × 107.0 CFU) of P.aeruginosa–CS–2 in the intratracheally challenged mink was higher than LD50% (6.8 × 104.0 CFU) in the intraperitoneally challenged mink. It implied that different infection routes could affect the pathogenicity of P. aeruginosa in mink. The studies reported that P. aeruginosa strains with multiple virulence gene contents should be different in pathogenesis [9–12]. In this study, the isolates carried different virulence gene contents and should be different in pathogenicity to mink, such as PA–CS–12 without algD gene, PA–CS–19 without exoT, aprA, exoS, toxA, and exoU genes, and PA–CS–20 solely with exoT and algD genes. In the further study, the influence of virulence gene contents on pathogenicity of P. aeruginosa will be investigated.
3.2. Antimicrobial susceptibility of P. aeruginosa isolates Among the 20 P.aeruginosa isolates, multidrug resistance and cross resistance were commonly found (Table 3), which might be associated with widespread use of antimicrobial agents. All the isolates were resistant to penicillin, erythrocin and clindamycin. A high incidence of resistance was found to kanamycin (12/20). A low percentage of isolates was resistant to tobramycin, amikacin, cefotaxime and ofloxacin
4. Conclusions The study implied that P. aeruginosa was prevalent in mink, with multidrug resistance and cross resistance. The virulence gene profiles among the P. aeruginosa isolates were varieties. Both the infective doses 3
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Table 3 The antimicrobial resistance of the 20 P.aeruginosa isolates. Strains
Antimicrobial resistance
PA–CS–1 PA–CS–2 PA–CS–3 PA–CS–4 PA–CS–5 PA–CS–6 PA–CS–7 PA–CS–8 PA–CS–9 PA–CS–10 PA–CS–11 PA–CS–12 PA–CS–13 PA–CS–14 PA–CS–15 PA–CS–16 PA–CS–17 PA–CS–18 PA–CS–19 PA–CS–20
PNC
ERY
Strep
GEN
TOB
AMK
Kan
CPZ
CAZ
CTRX
CTX
cefepime
CPFX
OFX
CLDM
imipenem
R R R R R R R R R R R R R R R R R R R R
R R R R R R R R R R R R R R R R R R R R
R R S R I I I I R I I I I I I I I I S I
I I S S I I I I S S S I S I S I I S S I
S S S S S S S S S S S S S S R S I S S S
S I S S S S S I S S S S S S S S S R S S
R R S I R R I R I R S I R R R R R I S S
S S S S S S S S S S S S S S S S S S S S
S S I S S S S I S S S S S S S S S S S S
S S S S S S S S S S S S S S S S S S S S
R S S S I I I S I S I I I I S I I S S S
S S S S S S S S S S S S S S S S S S S S
S S S S S S S S S S S S I S S S S S S S
S S S S S S S S S S S S R S S S S S S S
R R R R R R R R R R R R R R R R R R R R
S S S S S S S S S S S S S S S S S S S S
Note: PNC, penicillin; ERY, erythrocin; Strep, streptomycin; GEN, gentamicin; AMK, amikacin; TOB, tobramycin; Kan, kanamycin; CPZ, cefoperazone; CAZ, ceftazidime; CTRX, ceftriaxone; CTX, cefotaxime; CPFX, ciprofloxacin; OFX, ofloxacin; CLDM, clindamycin; S, sensitivity; I, intermediate susceptibility; R, resistance.
Acknowledgements
Table 4 Mink mortality in each group at the observation end point. IR
Group
Dose (CFU/mL)
Dead
Total
M (D/T)
IT
1 2 3 4 5 6 7 8 9 10 11 12 13 14
108.0 107.0 106.0 105.0 104.0 103.0 0 108.0 107.0 106.0 105.0 104.0 103.0 0
3 2 2 1 0 0 0 5 4 3 2 0 0 0
5 5 5 5 5 5 5 5 5 5 5 5 5 5
3/5 2/5 2/5 1/5 0/5 0/5 0/5 5/5 4/5 3/5 2/5 0/5 0/5 0/5
IP
We thank the staff of College of Veterinary Medicine, Shandong Agricultural University, in Taian, Shandong Province, China. And we also thank the staff of Fur Animal Disease Inspection Institute, Weifang, Shandong, China.
Glossary algD GDP-mannose dehydrogenase aprA Alkaline protease A CFU Colony-forming units exoS Exoenzyme S exoT Exoenzyme T exoU Exoenzyme U exoY Exoenzyme Y lasB Elastase B LB Luria-Bertani LD50% 50% lethal dose P. aeruginosa Pseudomonas aeruginosa
Note: IR, Inoculation route; IT, Intratracheal inoclution; IP, intraperitoneal inoclution; M(D/T), Mortality (Dead/Total).
and the infective routes would play a role in the pathogenicity of P. aeruginosa in mink. It should be in mind that different strains of P. aeruginosa might be different in pathogenicity to mink. The findings contribute to prevent the epidemic of Pseudomonas aeruginosa in mink, which was significant for public health. How virulence gene contents influence pathogenicity of P. aeruginosa need to be further explored, and the complete interaction mechanism among virulence genes of P. aeruginosa will undoubtedly be considered in the future.
PCR Polymerase chain reaction p.i. Postinfection plcH Phospholipase C 16 S rRNA 16 S ribosomal RNA T3SS Type III secretion system T3SS+ Type III secretion system protein TNF-α Tumor necrosis factor toxA Exotoxin A VAP Ventilator-associated pneumonia
CRediT authorship contribution statement Zhu Qian: Methodology, Validation, Visualization, Writing - review & editing. Peng Hui: Investigation, Writing - original draft. Li Han: Validation. Yang Ling-zhi: Data curation. Zhang Bo-shun: Investigation. Zhu Jie: Data curation. Guo Wan-li: Investigation. Wang Nan: Supervision. Jiang Shi-jin: Resources. Xie Zhi-jing: Conceptualization, Resources, Funding acquisition.
Funding This work was supported by Key Project of Chinese National Programs for Research and Development (2016YFD0501005), China; Shandong Modern Agricultural Technology & Industry System (SDAIT21-07), China; Natural Science Foundation of Shandong Province (ZR2017MC054), China; and Funds of Shandong “Double Tops” Program.
Declaration of competing interest The authors declare that they have no competing financial interests. 4
Microbial Pathogenesis 139 (2020) 103904
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Serotypes and virulence genes of Pseudomonas aeruginosa isolated from mink and its pathogenicity in mink
T
Zhu Qiana,b,c,1, Peng Huia,b,c,1, Li Hana,b,c, Yang Ling-zhic,d, Zhang Bo-shuna,b,c, Zhu Jiec,d, Guo Wan-lia,b,c, Wang Nanc,d, Jiang Shi-jina,b,c, Xie Zhi-jinga,b,c,∗ a
Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Agricultural University, Taian City, Shandong Province, 271018, China b College of Veterinary Medicine, Shandong Agricultural University, Taian City, Shandong Province, 271018, China c Shandong Provincial Engineering Technology Research Center of Animal Disease Control and Prevention, Shandong Agricultural University, Taian City, Shandong Province, 271018, China d Shandong Binzhou Wohua Biotech Co.,LTD, Binzhou City, Shandong Province, 256600, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Pseudomonas aeruginosa Mink Serotype Virulence genes Infection route
In this study, 20 P. aeruginosa strains were isolated from 112 farmed mink exhibiting hemorrhagic pneumonia in mideastern Shandong province, China. Serotype G (18/20) was the dominant serotype among the isolates with prevalence in mink, followed by serotype B (1/20), serotype C (1/20). The 9 virulence-associated genes of P. aeruginosa were tested using PCR. The prevalence of the virulence genes for the isolates were algD 95% (19/20), plcH 85% (17/20), exoY 80% (16/20), aprA 75% (15/20), lasB 70% (14/20), exoS 65% (13/20), exoT 60% (12/ 20) and toxA 60% (12/20), respectively. The 20 isolates were negative for exoU gene. The isolates exhibited multidrug resistance and cross resistance, using antimicrobial disc susceptibility assays. The animal experiments demonstrated that LD50% of the P.aeruginosa–CS–2 in the intratracheally challenged mink was 2.2 × 107.0 CFU, and 6.8 × 104.0 CFU in the intraperitoneally challenged mink. It implied that both the inoculation doses and the routes of inoculation could have influences on the pathogenicity of P. aeruginosa in mink. Therefore, the evolutionary and epidemiological surveillance of P. aeruginosa in mink should be further strengthened for public health.
1. Introduction Pseudomonas aeruginosa (P. aeruginosa) thrives in moist and wet conditions and is able to utilize a wide range of organic compounds, which is a prevalent opportunistic human pathogen and one of the most important causative agents of hospital-acquired nosocomial infections, characteristically in immunocompromised individuals [1,2]. In the context of a breakdown in host defenses, P. aeruginosa infected a plethora of tissues, causing both acute and chronic infections [3]. And P. aeruginosa also caused serious harms to dogs, cats and fur-bearing animals, such as mink, foxes and raccoon dogs [4]. The still water, unclean cages, water cups and feed troughs has been a reservoir for P. aeruginosa, serving as foci for the dissemination of the organism in common-source outbreaks [1,5]. The multidrug resistant P. aeruginosa was becoming prevalent and difficult to be clinically treated [6,7]. The resistance was coordinated
by a variety of virulence genes, contributing to its persistence and pathogenicity in vivo [8]. Some of virulent factors assisted bacterial establishment and colonization on the surface of the host, others expedited invasion of numerous tissues [9]. The type III secretion system (T3SS) of P. aeruginosa injected toxic effector proteins directly into the cytosol of host cells, subverting host cell defense and signaling systems [10,11]. The secreted toxins, such as exoenzyme S (exoS), exoenzyme T (exoT), exoenzyme U (exoU) and exoenzyme Y (exoY), were referred to as effector proteins [12]. The exoS, exoT and exoU contributed to virulence of P. aeruginosa in the pathogenicity of lung diseases [13]. Elastase B (lasB) could degrade mucins and surfactant proteins that promoted bacterial clearance [14]. Alkaline protease A (aprA) was thought to modulate the host response and prevent bacterial clearance by degrading proteins of the host immune system, including Tumor necrosis factor-α (TNF-α) and complement factors [15]. The lasB of P. aeruginosa acted in concert with aprA to prevent flagellin-mediated
∗
Corresponding author. College of Veterinary Medicine, Shandong Agricultural University, Taian City, Shandong Province, 271018, China. E-mail address: [email protected] (X. Zhi-jing). 1 The authors contributed equally to this work. https://doi.org/10.1016/j.micpath.2019.103904 Received 27 August 2019; Received in revised form 26 November 2019; Accepted 28 November 2019 Available online 01 December 2019 0882-4010/ © 2019 Elsevier Ltd. All rights reserved.
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immune recognition [14]. P. aeruginosa also contained of the other virulence factors, including Phospholipase C (plcH), GDP-mannose dehydrogenase (algD), exotoxin A (toxA) and so on [9,16,17]. Mink was the only animal known to be susceptible to acute, contagious, and fatal lung infections caused by P. aeruginosa [5,18]. Once established within the lungs, P. aeruginosa usually causes episodic bouts of pneumonia that lead to progressive irreversible lung injury and ultimately death [19]. The simultaneous determination of serogroups and virulence factors was of interest for the efficacy of surveillance of infections associated with P. aeruginosa [9]. Here, the objectives of the study were to clarify serotypes, virulence genes and antimicrobial susceptibility of the P. aeruginosa isolates from the mink experiencing respiratory diseases. Furthermore, animal experiments were performed to clarify the pathogenicity of P. aeruginosa to mink.
Table 1 Primers used for amplification of the virulence genes of P. aeruginosa. Gene
Primer
Sequence (5′–3′)
Size of product (bp)
Reference
exoT
F R F R F R F R F R F R F R F R F R
CAATCATCTCAGCAGAACCC TGTCGTAGAGGATCTCCTG GCACGTGGTCATCCTGATGC TCCGTAGGCGTCGACGTAC ATCAGCATCTTTGGTTTGGG TGTGGCGTTCGGACTTCT CAGACCCTGACCCACGAGAT CATTGCCCTTCAACCCG TATCGACGGTCATCGTCAGGT TTGATGCACTCGACCAGCAAG TGAAGACTTTCCGTGGCAC CATCCTCAGGCGTACATCC GGTAACCAGCTCAGCCACA TGCCTTCCCAGGTATCGT CGTCTCCTACCTGATTCCCG GCACCTTCATGTACAGCTTGTG GATTCCATCACAGGCTCG CTAGCAATGGCACTAATCG
1159
[21]
608
[22]
plcH algD aprA exoY exoS toxA
2. Materials and methods
lasB
2.1. P. aeruginosa isolation exoU
During June to July 2016, 112 lung samples of the mink exhibiting respiratory diseases were collected in mideastern Shandong province, China, which were used for bacterial isolation according to standard clinical microbiologic methods. In brief, the samples were isolated in Luria-Bertani (LB) nutrient agar plates and agar plates with 5% sheep blood using a spreading technique, respectively. Plates were cultured for 18–24 h and observed for suspected colonies of P. aeruginosa, which were identified by colony pigmentation, grape-like odor, growth at 42 °C. The isolates were identified by the 16 S ribosomal RNA (16 S rRNA) gene, and the specific primers, 27 F: 5′-AGAGTTTGATCCTGGC TCAG-3′ and 1492 R: 5′-GGTTACCTTGTTACGACTT-3’ [20]. The polymerase chain reaction (PCR) products were extracted from agarose gels, and sequenced in Sangon Biological (Shanghai) Co., Ltd (Shanghai, China). The nucleotide sequences of 16 S rRNA genes were submitted to GenBank, and GenBank accession numbers were MG547323MG547342.
346 445 1035
[21]
398 301 413 3308
[21]
(30 μg), ciprofloxacin (5 μg), ofloxacin (5 μg), clindamycin (2 μg) and imipenem (10 μg). 2.5. Pathogenicity of P. aeruginosa isolate in mink To elucidate the pathogenicity of the P. aeruginosa isolates, the animal experiments were carried out on 70 healthy American mink (twomonth-old), which were divided into 14 groups on average. According to serotype and virulence genes of P. aeruginosa, P.aeruginosa–CS–2 was selected for animal experiments. The mink in the 1–6 groups were lightly anesthetized with ketamine chloride and intratracheal inoculation with P.aeruginosa–CS–2 of 108.0 Colony-Forming Units (CFU), 107.0 CFU, 106.0 CFU, 105.0 CFU, 104.0 CFU and 103.0 CFU, respectively, and the mink intraperitoneally inoculated in the 8–13 groups, the mink in Group 7 intratracheally inoculated with 0.9% NaCl solution and the mink in Group 14 intraperitoneally inoculated with 0.9% NaCl solution, serving as the control group, respectively. The animals were raised separately and fed twice a day on a commercial meat-based diet. Water was available all day. Clinical symptoms of the mink were monitored and recorded daily for 15 days. The tissues, including lung, spleen, liver, kidney, lymph node, cerebrum and heart, were collected from the dead mink or euthanized on day 15 postinfection (p.i.), rapidly immersed in 10% neutral formalin buffer to prevent autolysis for the detection of histological lesions. The 50% lethal dose (LD50%) of P. aeruginosa in mink was titrated using Reed and Muench Method. In brief, the calculation method reads as follows: Distance ratio = (higher than 50% death score 50%)/(higher than 50% death percentage - lower than 50% death score), Logarithm of LD50% = logarithm higher than 50% virus dilution + distance ratio × logarithm of dilution coefficient. This study was carried out in accordance with the recommendations of Guidelines of Animal Care and Use, Animal Care and Use Committee of Shandong Agricultural University. The protocol (SDAUA-2017-37) was approved by the Animal Care and Use Committee of Shandong Agricultural University.
2.2. Serotypes of P. aeruginosa isolates The isolates were serotyped using the slide agglutination with P. aeruginosa Antisera Kit (Denka Seiken Co. Ltd., Tokyo, Japan). In brief, confirming an agglutination according to the procedure, using polyvalent sera, then when an agglutination occurs with a polyvalent serum, confirming agglutination by the same procedure using each monovalent grouping serum which is included in the polyvalent serum. 2.3. Virulence gene assays The genomic DNA was extracted using Bacteria Genomic DNA Kit (CWBIO). Nine virulence genes in P. aeruginosa, were screened using PCR, including exoT, plcH, algD, aprA, exoY, exoS, toxA, lasB and exoU genes. The specific primers and the length of PCR products were shown in Table 1. The PCR conditions used were available upon request. The PCR products were extracted from agarose gels, and sequenced in Sangon Biological (Shanghai) Co., Ltd (Shanghai, China). The nucleotide sequences of the corresponding genes were submitted to GenBank, and GenBank accession numbers were MG506925-MG507043. 2.4. Antimicrobial susceptibility assays
3. Results and discussion Antimicrobial susceptibility of 16 antimicrobials for the P. aeruginosa isolates was performed using disk diffusion method, and the results were interpreted according to the Clinical and Laboratory Standards Institute [23]. The 16 antimicrobial agents included penicillin (10 μg), erythrocin (15 μg), streptomycin (10 μg), gentamicin (10 μg), amikacin (30 μg), tobramycin (10 μg), kanamycin (30 μg), cefoperazone (75 μg), ceftazidime (30 μg), ceftriaxone (30 μg), cefotaxime (30 μg), cefepime
3.1. P. aeruginosa isolation, serotypes and virulence genes In the study, 20 P. aeruginosa strains were isolated from the mink exhibiting respiratory diseases, named as P.aeruginosa–CS–1 to P.aeruginosa–CS–20, and the prevalence of P. aeruginosa in the sampled mink was 17.9% (20/112). The 16 S rRNA genes of the isolates shared 2
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Table 2 The serotypes and virulent genes of the 20 P.aeruginosa isolates. Strains
PA–CS–1 PA–CS–2 PA–CS–3 PA–CS–4 PA–CS–5 PA–CS–6 PA–CS–7 PA–CS–8 PA–CS–9 PA–CS–10 PA–CS–11 PA–CS–12 PA–CS–13 PA–CS–14 PA–CS–15 PA–CS–16 PA–CS–17 PA–CS–18 PA–CS–19 PA–CS–20 P
Virulence genes
Serotypes
Severity degree of infection
exoT
plcH
algD
aprA
exoY
exoS
toxA
lasB
exoU
G
B
C
+ + – – + + + + – + – – + + + – + – – + 60%
+ + + + + + + + – + + + + + + + + – + – 85%
+ + + + + + + + + + + – + + + + + + + + 95%
+ + + + + + + + + – + + + – – + + + – – 75%
+ + + + + + + + – + – + + + + + + – + – 80%
+ + – – – + + + + + + + + – + – + + – – 65%
+ + – + + + + + – – – + + + + + – – – – 60%
– + – + + + + – – + + + + + + – + + + – 70%
– – – – – – – – – – – – – – – – – – – – 0%
+ + + + + + + + + + – – + + + + + + + + 90%
– – – – – – – – – – – + – – – – – – – – 5%
– – – – – – – – – – + – – – – – – – – – 5%
3 3 2 2 2 2 2 2 1 2 2 2 3 2 3 1 2 2 1 1
Note: PA, P.aeruginosa; +, positive; −, negative; P, prevalence; 1, mild infection; 2, moderate infection; 3, severe infection.
(1/20). All strains were sensitive to cefoperazone, ceftriaxone, cefepime and imipenem. The isolates in the mink exhibiting severe hemorrhagic pneumonia had the higher antibiotic resistance rates than the other isolates. Furthermore, it appears that there was no correlation between serotype and antibiotic resistance. The findings provide the information available to veterinarians on rational antimicrobial treatment therapies.
99%–100% identity with the reference P. aeruginosa strains, furtherly confirming that the isolates belong to P. aeruginosa. Eighteen (90%) of the 20 P. aeruginosa isolates belonged to serotype G using slide agglutination, one (5%) serotype B and one (5%) serotype C (Table 2). It demonstrated that P. aeruginosa serotype G was prevalent in the mink farms, playing an important role in mink hemorrhagic pneumonia. Interestingly, P.aeruginosa–CS–11 and P.aeruginosa–CS–20 were isolated from the same farm, but P.aeruginosa–CS–11 belonged to serotype B and P.aeruginosa–CS–20 belonged to serotype G. It implied that the different serotypes could co-circulate in the same farm. The interactions between different serotypes need to be further explored. The prevalence of virulence genes in the P. aeruginosa isolates was shown in Table 2, and algD gene was 95% (19/20), plcH 85% (17/20), exoY 80% (16/20), aprA 75% (15/20), lasB 70% (14/20), exoS 65% (13/20), exoT 60% (12/20) and toxA 60% (12/20). The 20 isolates were negative for exoU gene using PCR. Based on sequence analysis, the virulence gene contents were different among different P. aeruginosa strains. Pathogenicity of the bacterium completely depends on its virulence factors [24]. In the study, the algD, plcH, lasB, aprA toxA, exoS, exoY, and exoT genes were prevalent in the P. aeruginosa isolates. The algD gene had the highest incidence in the isolates, which could be at the origin of the conversion of P. aeruginosa strains to a mucoid phenotype overproducing alginates [9]. Every isolate expressed at least one protein of the type III secretion system protein (T3SS+), an important virulence determinant in P. aeruginosa. And previous studies suggested that failure to eradicate P. aeruginosa in patients with ventilator-associated pneumonia (VAP) might be linked to T3SS, whereas eradication was achieved in patients with undetectable levels of T3SS+ [25]. ToxA was an important virulence factor of P. aeruginosa in clinical infections and the most virulent factor among other factors produced by P. aeruginosa [26].
3.3. Pathogenicity of P. aeruginosa isolates in mink During days 2–8 p.i., some of the challenged animals exhibited clinical symptoms, including lethargy, dullness, dyspnea, anorexia, coarse fur, sneezing and coughing. Some of the inoculated mink died from P. aeruginosa infection, reaching a peak on days 3–5 p.i., and showing lung hemorrhage, splenic hematoma, pericardial effusion, lymph node enlargement, and liver hemorrhage and swelling. The challenged mink developed histologic lesions, including lung bleeding and congesting, spleen bleeding and congesting, liver congesting and steatosis, and heart hemorrhage. The control mink showed no clinical signs. The survived mink were weak, but resumed intake and clinically recovered. As shown in Table 4, the data demonstrated the dose-dependent pathogenicity of P. aeruginosa in challenged mink via the intratracheally or intraperitoneally routes of infections. LD50% (2.2 × 107.0 CFU) of P.aeruginosa–CS–2 in the intratracheally challenged mink was higher than LD50% (6.8 × 104.0 CFU) in the intraperitoneally challenged mink. It implied that different infection routes could affect the pathogenicity of P. aeruginosa in mink. The studies reported that P. aeruginosa strains with multiple virulence gene contents should be different in pathogenesis [9–12]. In this study, the isolates carried different virulence gene contents and should be different in pathogenicity to mink, such as PA–CS–12 without algD gene, PA–CS–19 without exoT, aprA, exoS, toxA, and exoU genes, and PA–CS–20 solely with exoT and algD genes. In the further study, the influence of virulence gene contents on pathogenicity of P. aeruginosa will be investigated.
3.2. Antimicrobial susceptibility of P. aeruginosa isolates Among the 20 P.aeruginosa isolates, multidrug resistance and cross resistance were commonly found (Table 3), which might be associated with widespread use of antimicrobial agents. All the isolates were resistant to penicillin, erythrocin and clindamycin. A high incidence of resistance was found to kanamycin (12/20). A low percentage of isolates was resistant to tobramycin, amikacin, cefotaxime and ofloxacin
4. Conclusions The study implied that P. aeruginosa was prevalent in mink, with multidrug resistance and cross resistance. The virulence gene profiles among the P. aeruginosa isolates were varieties. Both the infective doses 3
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Table 3 The antimicrobial resistance of the 20 P.aeruginosa isolates. Strains
Antimicrobial resistance
PA–CS–1 PA–CS–2 PA–CS–3 PA–CS–4 PA–CS–5 PA–CS–6 PA–CS–7 PA–CS–8 PA–CS–9 PA–CS–10 PA–CS–11 PA–CS–12 PA–CS–13 PA–CS–14 PA–CS–15 PA–CS–16 PA–CS–17 PA–CS–18 PA–CS–19 PA–CS–20
PNC
ERY
Strep
GEN
TOB
AMK
Kan
CPZ
CAZ
CTRX
CTX
cefepime
CPFX
OFX
CLDM
imipenem
R R R R R R R R R R R R R R R R R R R R
R R R R R R R R R R R R R R R R R R R R
R R S R I I I I R I I I I I I I I I S I
I I S S I I I I S S S I S I S I I S S I
S S S S S S S S S S S S S S R S I S S S
S I S S S S S I S S S S S S S S S R S S
R R S I R R I R I R S I R R R R R I S S
S S S S S S S S S S S S S S S S S S S S
S S I S S S S I S S S S S S S S S S S S
S S S S S S S S S S S S S S S S S S S S
R S S S I I I S I S I I I I S I I S S S
S S S S S S S S S S S S S S S S S S S S
S S S S S S S S S S S S I S S S S S S S
S S S S S S S S S S S S R S S S S S S S
R R R R R R R R R R R R R R R R R R R R
S S S S S S S S S S S S S S S S S S S S
Note: PNC, penicillin; ERY, erythrocin; Strep, streptomycin; GEN, gentamicin; AMK, amikacin; TOB, tobramycin; Kan, kanamycin; CPZ, cefoperazone; CAZ, ceftazidime; CTRX, ceftriaxone; CTX, cefotaxime; CPFX, ciprofloxacin; OFX, ofloxacin; CLDM, clindamycin; S, sensitivity; I, intermediate susceptibility; R, resistance.
Acknowledgements
Table 4 Mink mortality in each group at the observation end point. IR
Group
Dose (CFU/mL)
Dead
Total
M (D/T)
IT
1 2 3 4 5 6 7 8 9 10 11 12 13 14
108.0 107.0 106.0 105.0 104.0 103.0 0 108.0 107.0 106.0 105.0 104.0 103.0 0
3 2 2 1 0 0 0 5 4 3 2 0 0 0
5 5 5 5 5 5 5 5 5 5 5 5 5 5
3/5 2/5 2/5 1/5 0/5 0/5 0/5 5/5 4/5 3/5 2/5 0/5 0/5 0/5
IP
We thank the staff of College of Veterinary Medicine, Shandong Agricultural University, in Taian, Shandong Province, China. And we also thank the staff of Fur Animal Disease Inspection Institute, Weifang, Shandong, China.
Glossary algD GDP-mannose dehydrogenase aprA Alkaline protease A CFU Colony-forming units exoS Exoenzyme S exoT Exoenzyme T exoU Exoenzyme U exoY Exoenzyme Y lasB Elastase B LB Luria-Bertani LD50% 50% lethal dose P. aeruginosa Pseudomonas aeruginosa
Note: IR, Inoculation route; IT, Intratracheal inoclution; IP, intraperitoneal inoclution; M(D/T), Mortality (Dead/Total).
and the infective routes would play a role in the pathogenicity of P. aeruginosa in mink. It should be in mind that different strains of P. aeruginosa might be different in pathogenicity to mink. The findings contribute to prevent the epidemic of Pseudomonas aeruginosa in mink, which was significant for public health. How virulence gene contents influence pathogenicity of P. aeruginosa need to be further explored, and the complete interaction mechanism among virulence genes of P. aeruginosa will undoubtedly be considered in the future.
PCR Polymerase chain reaction p.i. Postinfection plcH Phospholipase C 16 S rRNA 16 S ribosomal RNA T3SS Type III secretion system T3SS+ Type III secretion system protein TNF-α Tumor necrosis factor toxA Exotoxin A VAP Ventilator-associated pneumonia
CRediT authorship contribution statement Zhu Qian: Methodology, Validation, Visualization, Writing - review & editing. Peng Hui: Investigation, Writing - original draft. Li Han: Validation. Yang Ling-zhi: Data curation. Zhang Bo-shun: Investigation. Zhu Jie: Data curation. Guo Wan-li: Investigation. Wang Nan: Supervision. Jiang Shi-jin: Resources. Xie Zhi-jing: Conceptualization, Resources, Funding acquisition.
Funding This work was supported by Key Project of Chinese National Programs for Research and Development (2016YFD0501005), China; Shandong Modern Agricultural Technology & Industry System (SDAIT21-07), China; Natural Science Foundation of Shandong Province (ZR2017MC054), China; and Funds of Shandong “Double Tops” Program.
Declaration of competing interest The authors declare that they have no competing financial interests. 4
Microbial Pathogenesis 139 (2020) 103904
Z. Qian, et al.
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