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1.
World J Microbiol Biotechnol ; 35(8): 127, 2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-31375931

RESUMO

Aeromonas hydrophila is a Gram-negative bacterium that causes serious infections in aquaculture and exhibits significant multidrug resistance. The LysR-type transcriptional regulator (LTTR) family proteins are a well-known group of transcriptional regulators involved in diverse physiological functions. However, the role of LTTRs in the regulation of bacterial resistance to antibiotics is still largely unknown. In this study, to further investigate the role of four putative LTTR family proteins (A0KIU1, A0KJ82, A0KPK0, and A0KQ63) in antibiotic resistance in A. hydrophila, their genes were cloned and overexpressed in engineered Escherichia coli. After the optimization of experimental conditions including incubation time, temperature, and IPTG concentration, these proteins were successfully purified, and their specific antibodies against mice were obtained. Using western blot analysis, we found that these LTTR family proteins were downregulated in A. hydrophila following antibiotic treatment, indicating that they may be involved in the regulation of antibiotic resistance. Additionally, minimum inhibitory concentration (MIC) assays of chloramphenicol (CM), chlortetracycline (CTC), ciprofloxacin (CF), furazolidone (FZ), and balofloxacin (BF) in E. coli showed that overexpression of these LTTRs led to increased sensitivity to several antibiotics. To further validate their functional role in antibiotic resistance, we demonstrated that bacteria with loss of A0KQ63 (ΔAHA_3980) exhibited multi-drug resistance properties. Our results indicate that these LTTR family proteins may play an important role in the antibiotic resistance of A. hydrophila, and the that underlying mechanisms controlling antibiotic resistance should be further investigated.


Assuntos
Aeromonas hydrophila/efeitos dos fármacos , Aeromonas hydrophila/genética , Farmacorresistência Bacteriana , Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Fatores de Transcrição/metabolismo , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Western Blotting , Clonagem Molecular , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Deleção de Genes , Expressão Gênica , Perfilação da Expressão Gênica , Genes Bacterianos , Camundongos , Testes de Sensibilidade Microbiana , Fatores de Transcrição/análise , Fatores de Transcrição/genética
2.
Nat Commun ; 10(1): 2900, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31263098

RESUMO

The alpha helical CytolysinA family of pore forming toxins (α-PFT) contains single, two, and three component members. Structures of the single component Eschericia coli ClyA and the two component Yersinia enterolytica YaxAB show both undergo conformational changes from soluble to pore forms, and oligomerization to produce the active pore. Here we identify tripartite α-PFTs in pathogenic Gram negative bacteria, including Aeromonas hydrophila (AhlABC). We show that the AhlABC toxin requires all three components for maximal cell lysis. We present structures of pore components which describe a bi-fold hinge mechanism for soluble to pore transition in AhlB and a contrasting tetrameric assembly employed by soluble AhlC to hide their hydrophobic membrane associated residues. We propose a model of pore assembly where the AhlC tetramer dissociates, binds a single membrane leaflet, recruits AhlB promoting soluble to pore transition, prior to AhlA binding to form the active hydrophilic lined pore.


Assuntos
Aeromonas hydrophila/metabolismo , Toxinas Bacterianas/química , Proteínas Hemolisinas/química , Proteínas Citotóxicas Formadoras de Poros/química , Aeromonas hydrophila/química , Aeromonas hydrophila/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Cristalografia por Raios X , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo
3.
Ecotoxicol Environ Saf ; 180: 309-316, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31102840

RESUMO

Triclosan (TCS) is a biocide commonly used in household and personal care items to prevent the microbial growth and is currently considered as an emerging pollutant. It has a ubiquitous distribution which can substantially contribute towards antimicrobial resistance. The present study was designed to evaluate the effect of different concentrations of TCS exposure on the antibiotic sensitivity of aquatic bacteria. Aeromonas hydrophila ATCC® 49140™ and Edwardsiella tarda ATCC® 15947™ exposed to TCS for short (30 min) and long duration (serial passages). The agar-disc diffusion assay during the serial passages of TCS exposure and subsequent exposure withdrawal showed clinically insignificant changes in the zone of inhibition for six selected antibiotics in both bacterial strains at all exposure concentrations. Four folds concentration-dependent increase in the minimum inhibitory concentrations (MICs) of TCS was observed in both the strains following TCS exposure. Similarly, a concentration-dependent increase in the MICs of oxytetracycline (OTC) up to 4 folds in A. hydrophila, and up to 8 folds in E. tarda, was also documented during the TCS exposure. In all the cases, withdrawal of TCS exposure effectively reduced the MICs of TCS and OTC in blank passages suggesting a decline in acquired resistance. The frequencies of mutation during 30 min TCS exposure for E. tarda and A. hydrophila ranged between >10-6 and 10-7 levels. Nevertheless, the TCS exposure did not cause any detectable mutation on the fabV gene of A. hydrophila indicating that the TCS may elicit phenotypic adaptation or other resistance mechanism. Although the reduction in MICs due to exposure withdrawal did not restore the bacterial susceptibility up to the initial level, the study proved that the reduced TCS use could significantly help reduce the antimicrobial-resistance and cross-resistance in pathogenic bacteria.


Assuntos
Aeromonas hydrophila/efeitos dos fármacos , Antibacterianos/farmacologia , Desinfetantes/toxicidade , Farmacorresistência Bacteriana , Edwardsiella tarda/efeitos dos fármacos , Triclosan/toxicidade , Aeromonas hydrophila/genética , Relação Dose-Resposta a Droga , Farmacorresistência Bacteriana/genética , Edwardsiella tarda/genética , Humanos , Testes de Sensibilidade Microbiana , Fatores de Tempo
4.
J Fish Dis ; 42(6): 895-904, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30919989

RESUMO

Survival in host macrophages is an effective strategy for pathogenic bacteria to spread. Aeromonas hydrophila has been found to survive in fish macrophages, but the mechanisms remain unknown. In this paper, the roles and possible mechanisms of IcmF in bacterial survival in fish macrophages were investigated. First, a stable silencing strain icmF-RNAi was constructed by shRNA and RT-qPCR confirmed the expression of icmF was down-regulated by 94.42%. The expression of Hcp, DotU and VgrG was also decreased in icmF-RNAi. The intracellular survival rate of the wild-type strain was 92.3%, while the survival rate of icmF-RNAi was only 20.58%. The escape rate of the wild-type strain was 20%, while that of the icmF-RNAi was only 7.5%. Further studies indicated that the expression of icmF can significantly affect the adhesion, biofilm formation, motility and acid resistance of A. hydrophila, but has no significant effect on the growth of A. hydrophila even under the stress of H2 O2 . The results indicated that IcmF of A. hydrophila not only acts as a structural protein which participates in virulence-related characteristics such as bacterial motility, adhesion and biofilm formation, but also acts as a key functional protein which participates in the interaction between bacteria and host macrophages.


Assuntos
Aeromonas hydrophila/genética , Proteínas de Bactérias/genética , Peixes/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Macrófagos/microbiologia , Adesinas Bacterianas/genética , Aeromonas hydrophila/patogenicidade , Animais , Biofilmes/crescimento & desenvolvimento , Doenças dos Peixes/microbiologia , Peixes/imunologia , Inativação Gênica , Infecções por Bactérias Gram-Negativas/imunologia , Viabilidade Microbiana , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Virulência/genética
5.
Int J Infect Dis ; 81: 73-80, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30690211

RESUMO

BACKGROUND: Vibrio vulnificus, Aeromonas hydrophila, methicillin-resistant Staphylococcus aureus (MRSA), group A Streptococcus, and group B Streptococcus are commonly detected causative agents of necrotizing fasciitis (NF) in Chia-Yi Chang Gung Memorial Hospital. The aim of this study was to develop and evaluate a multiplex PCR method for the simultaneous detection of five of the most important human pathogens involved in NF by using a novel combination of species-specific genes. METHODS: The samples used were collected from 99 patients with surgically confirmed NF of the extremities who were hospitalized consecutively between June 2015 and November 2017. Two sets of blood and tissue samples were collected from all patients; one set was sent to a microbiology laboratory for bacterial identification and the other set was sent to an immunohistochemistry laboratory for PCR amplification. RESULTS: The multiplex PCR results for the blood samples showed negative findings. The multiplex PCR results for the tissue specimens showed 28 positive findings. Fourteen (87.5%) of the 16 V. vulnificus culture-positive tissue specimens, six (75%) of the eight A. hydrophila culture-positive tissue specimens, and four (100%) of the four MRSA culture-positive tissue specimens were positive by PCR. Similarly, two (100%) of the group A Streptococcus and two (100%) of the group B Streptococcus were PCR-positive. CONCLUSIONS: The accuracy rate of the multiplex PCR presenting positive results in these culture-positive tissue samples was 87.5% (28/32). This suggests that multiplex PCR of tissue specimens may be a useful and rapid diagnostic tool for the detection of these lethal microorganisms in patients with NF.


Assuntos
Primers do DNA/genética , Fasciite Necrosante/diagnóstico , Infecções por Bactérias Gram-Negativas/diagnóstico , Reação em Cadeia da Polimerase Multiplex , Infecções Estafilocócicas/diagnóstico , Infecções Estreptocócicas/diagnóstico , Vibrioses/diagnóstico , Aeromonas hydrophila/genética , Aeromonas hydrophila/isolamento & purificação , Fasciite Necrosante/complicações , Humanos , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/métodos , Sensibilidade e Especificidade , Streptococcus agalactiae/genética , Streptococcus agalactiae/isolamento & purificação , Streptococcus pyogenes/genética , Streptococcus pyogenes/isolamento & purificação , Vibrio vulnificus/genética , Vibrio vulnificus/isolamento & purificação
6.
FEMS Microbiol Lett ; 366(1)2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30596975

RESUMO

Defined organic waste products are ideal and sustainable secondary feedstocks for production organisms in microbial biotechnology. Chitin from mycelia of fungal fermentation processes represents a homogeneous and constantly available waste product that can, however, not be utilised by typical bacterial production strains. Therefore, enzymes that degrade chitin within fungal mycelia have to be identified and expressed in production organisms. In this study, chitin-degrading bacteria were enriched and isolated from lake water with mycelia of Aspergillus tubingensis as sole organic growth substrate. This approach yielded solely strains of Aeromonas hydrophila. Comparison of the isolated strains with other A. hydrophila strains regarding their chitinolytic activities on fungal mycelia identified strain AH-1N as the best enzyme producer. From this strain, a chitinase (EC:3.2.1.14) was identified by peptide mass fingerprinting. Heterologous expression of the respective gene combined with mass spectrometry showed that the purified enzyme was capable of releasing chitobiose from fungal mycelia with a higher yield than a well-described chitinase from Serratia marcescens. Expression of the newly identified chitinase in biotechnological production strains could be the first step for making fungal mycelium accessible as a secondary feedstock. Additionally, the enrichment strategy proved to be feasible for identifying strains able to degrade fungal chitin.


Assuntos
Aeromonas hydrophila/enzimologia , Quitina/metabolismo , Quitinases/genética , Quitinases/metabolismo , Microbiologia Industrial , Micélio/enzimologia , Aeromonas hydrophila/genética , Biotecnologia
7.
Lett Appl Microbiol ; 68(3): 212-218, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30609084

RESUMO

Hundreds of farmed Siamese crocodiles (Crocodylus siamensis) died during July 2016 at a farm in Wenchang, Hainan, China. In two necropsied crocodiles, we observed symptoms of dermatorrhagia, hepatomegaly and hepatic congestion. Pulmonitis was diagnosed by pulmonary congestion and pulmonary fibrinous exudate. Septicaemia was diagnosed by isolation of three Aeromonas species from blood and visceral tissues; A. dhakensis, A. hydrophila and A. jandaei were identified by biochemical and molecular tests. We used a zebrafish model to determine the half-maximal lethal dose (LD50 ), and A. dhakensis was found to be the most virulent species, with an LD50 of 8·91 × 105 CFU per ml. The results of a drug sensitivity test indicated that these species were sensitive to 11 antibiotics. This is the first report of A. dhakensis, A. hydrophila and A. jandaei being isolated from a mixed infection in Siamese crocodiles. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we isolated three species of Aeromonas (A. dhakensis, A. hydrophila and A. jandae) from farmed Siamese crocodiles with fatal fibrinous pneumonia and septicaemia. This is the first description of a mixed infection with three Aeromonas species among captive crocodilians.


Assuntos
Aeromonas hydrophila/classificação , Aeromonas hydrophila/isolamento & purificação , Jacarés e Crocodilos/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Pneumonia/microbiologia , Sepse/microbiologia , Aeromonas hydrophila/efeitos dos fármacos , Aeromonas hydrophila/genética , Animais , Antibacterianos/farmacologia , China , Coinfecção , DNA Girase/genética , RNA Polimerases Dirigidas por DNA/genética , Água Doce , Testes de Sensibilidade Microbiana , Fator sigma/genética , Peixe-Zebra/microbiologia
8.
J Med Microbiol ; 67(11): 1535-1537, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30289383

RESUMO

Several species of Aeromonas produce the enzyme CphA metallo-ß-lactamase. This study describes an isolate of Aeromonas hydrophila harbouring an acquired gene encoding the carbapenemase GES-24. This isolate was obtained from an inpatient in Okinawa, Japan, with no apparent record of travelling overseas. The minimum inhibitory concentrations of carbapenems against this isolate were 8 µg ml-1 for imipenem and 16 µg ml-1 for meropenem. Recombinant GES-24 hydrolyzed all of the tested ß-lactams, including imipenem and meropenem. The genomic environment surrounding blaGES-24 was intI1-blaGES-24-aac(6')-IIc-qacEdelta1-sulI-orfX-tetR-tetE. This is the first report of A. hydrophila producing a GES-type carbapenemase.


Assuntos
Aeromonas hydrophila/efeitos dos fármacos , Aeromonas hydrophila/genética , Carbapenêmicos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Bactérias Gram-Negativas/epidemiologia , beta-Lactamases/genética , Aeromonas hydrophila/enzimologia , Aeromonas hydrophila/isolamento & purificação , Idoso de 80 Anos ou mais , Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/efeitos dos fármacos , Bile/microbiologia , DNA Bacteriano/genética , Feminino , Genes Bacterianos , Genoma Bacteriano , Infecções por Bactérias Gram-Negativas/microbiologia , Hospitalização , Humanos , Japão/epidemiologia , Testes de Sensibilidade Microbiana , Análise de Sequência de DNA , beta-Lactamases/biossíntese , beta-Lactamases/efeitos dos fármacos
9.
Gene ; 672: 156-164, 2018 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-29906530

RESUMO

The success of the pathogenic bacteria is partly attributable to their ability to thwart host innate immune responses, which includes resisting the antimicrobial functions of macrophages. And reactive oxygen species (ROS) is one of the most effective antimicrobial components of macrophages to kill invading bacteria. Our previous studies found that Aeromonas hydrophila can survive in fish macrophages, which suggested that this bacterium might take fish macrophages as their shelters to resist drug killings and other immune damage. But how A. hydrophila survive in host macrophages remains unknown. Since KatG has been reported to have not only catalase activity but also peroxidase and peroxynitritase activity, the amino acid sequence and protein structure of KatG was analyzed in this study, the function of KatG in A. hydrophila survival in and escape from host macrophages was also carried out. The bioinformatics analysis displayed that KatG of A. hydrophila B11 showed >93% homologous to that of KatG in other Aeromonas. KatG of A. hydrophila was stable silenced by shRNA and RT-qPCR confirmed the expression of KatG in KatG-RNAi was significantly reduced. The survival rate of intracellular KatG-RNAi decreased by 80% compared to that of the wild type strain B11, while the intracellular ROS level of the macrophages that phagocytosed KatG-RNAi increased 65.9% when compared to that of the macrophages phagocytosed wild-type strain. The immune escape rate of A. hydrophila decreased by 85% when the expression of KatG was inhibited. These results indicated that (1) The amino acid sequence and protein structure of KatG of A. hydrophila is conserved; (2) KatG helped A. hydrophila to survive in fish macrophages by eliminating the harm of intracellular H2O2 and inhibiting intracellular ROS levels increased; (3) A small portion of intracellular A. hydrophila could escape from host macrophages for further infection, in this process KatG also played important role.


Assuntos
Aeromonas hydrophila/imunologia , Proteínas de Bactérias/genética , Catalase/genética , Doenças dos Peixes/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Macrófagos/microbiologia , Aeromonas hydrophila/enzimologia , Aeromonas hydrophila/genética , Sequência de Aminoácidos , Animais , Células Cultivadas , Doenças dos Peixes/imunologia , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Técnicas de Silenciamento de Genes , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/microbiologia , Evasão da Resposta Imune , Macrófagos/imunologia , Viabilidade Microbiana , Modelos Moleculares , Estrutura Terciária de Proteína , Espécies Reativas de Oxigênio/metabolismo , Tilápia/imunologia , Tilápia/microbiologia
10.
Appl Microbiol Biotechnol ; 102(16): 7083-7095, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29862449

RESUMO

The type VI secretion system (T6SS) has been considered as a crucial factor in bacterial competition and virulence. The hemolysin co-regulated protein (Hcp) is the hallmark of T6SS. The secretion of Hcp in Aeromonas hydrophila Chinese epidemic strain NJ-35 indicated a functional T6SS. In this study, three copies of the hcp gene were identified in the genome of strain NJ-35. We targeted these Hcp family proteins for generating deletion mutants. These mutants showed varying levels in Hcp production, the interaction with other bacteria or eukaryotic cells, and bacterial virulence. Hcp1 was necessary for T6SS assembly and played a predominant role in the bacterial competition; Hcp2 negatively functioned in the biofilm formation and bacterial adhesion and was more involved in the A. hydrophila virulence in zebrafish and survival against the predation of Tetrahymena, and Hcp3 positively influenced the biofilm formation and bacterial adhesion. These findings illustrate that the T6SS of A. hydrophila NJ-35 is active, and the three Hcp family proteins take part in different processes in environmental adaptation and virulence of this bacterium. This study will provide valuable insights into our understanding of microbial interactions and thus contribute to a broader effort to manipulate these interactions for therapeutic or environmental benefit.


Assuntos
Aeromonas hydrophila/metabolismo , Aeromonas hydrophila/patogenicidade , Proteínas de Bactérias/genética , Proteínas Hemolisinas/genética , Virulência/genética , Aeromonas hydrophila/genética , Animais , Aderência Bacteriana/genética , Biofilmes , China , Microbiologia Ambiental , Deleção de Sequência
11.
J Appl Microbiol ; 124(6): 1629-1637, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29453863

RESUMO

AIMS: Aeromonas hydrophila has been isolated from various fish species in Egypt and is known to carry virulence and antimicrobial resistance genes, which pose a risk for public health. The aim of the present study is to report, for the first time, the infection of mullet (Mugil cephalus) with A. hydrophila and to clarify the potential association between antimicrobial resistance and virulence traits encoded in A. hydrophila. METHODS AND RESULTS: In this study, the occurrence of A. hydrophila in marketed mullet and the antimicrobial resistance phenotypes of these isolates were determined. Aeromonas hydrophila isolates were screened for the presence of virulence and ß-lactam resistance genes; the correlation between both gene groups was also investigated. The infection rate of examined mullet with A. hydrophila was 37% (50/135). The highest antimicrobial resistance was detected to cefoxitin (100%), followed by ampicillin (84%), ceftazidime (56%) and cefotaxime (40%). Only 4% of the isolates were resistant to erythromycin; 6% were resistant to both gentamicin and kanamycin with no resistance to ciprofloxacin. Variable frequencies of virulence and ß-lactam resistance genes were evident from PCR, where aerA and blaTEM predominated. The study also indicated a general weak positive correlation (R = 0·3) between both virulence and ß-lactam resistance genes. Some of the studied virulence genes (e.g. aerA:hlyA and hlyA:ast) were found to correlate positively. CONCLUSIONS: The presence of virulence and resistance genes in A. hydrophila from food sources poses a serious threat to public health. To our knowledge, this is the first report describing the occurrence of A. hydrophila in mullet and highlighting the coexistence of virulence and ß-lactam resistance genes encoded by these bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: These data provide insights into the potential association of antimicrobial resistance and virulence genes in A. hydrophila from marketed mullet in Egypt, which could pose threats to humans even if a weak positive correlation exists between both genes.


Assuntos
Aeromonas hydrophila , Doenças dos Peixes/microbiologia , Infecções por Bactérias Gram-Negativas/microbiologia , Smegmamorpha/microbiologia , Resistência beta-Lactâmica/genética , Aeromonas hydrophila/efeitos dos fármacos , Aeromonas hydrophila/genética , Aeromonas hydrophila/patogenicidade , Animais , Antibacterianos/farmacologia , Egito , Genes Bacterianos/genética , Reação em Cadeia da Polimerase , Virulência/genética
12.
J Vet Med Sci ; 80(3): 421-426, 2018 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-29367518

RESUMO

Aeromonas hydrophila is an opportunistic pathogen of a variety of aquatic animals that displays extreme diversity in drug resistance, phenotypes, virulence genes, and virulence. In this study, eight pathogenic A. hydrophila strains were isolated from diseased Amur sturgeons and investigated for their sensitivity to select antibiotics, their phenotype, virulence genes, and virulence. According to the phylogenetic analysis of the DNA gyrase subunit B protein, the eight isolates formed a single branch in the A. hydrophila group. The antibiotics ceftazidime, cefuroxime, cefoperazone, cefotaxime, ceftriaxone, aztreonam, and cefepime appeared effective against them. All of the isolates possessed the virulence genes for aerolysin, flagellin, heat-stable cytotonic enterotoxin, heat-labile cytotonic enterotoxin, hemolysin, and elastase, while only one isolate, HZ8, possessed the gene for lateral flagella. The cytolytic enterotoxin and lipase genes were present in all isolates, except in ZJ10 and ZJ12. Enterobacterial repetitive intergenic consensus sequence PCR indicated that the eight A. hydrophila isolates could be divided into four types. Isolates YW2, TR3, HZ8 and ZJ10, each representing a different type, were selected for challenge experiments. The challenge tests revealed that isolate HZ8 had the lowest lethal dose, causing 50% mortality at 2.30 × 104 colony forming units (cfu)/ml. The isolate ZJ10 had the highest LD50, 1.25 × 106 cfu/ml. Knowledge of the characteristics of the A. hydrophila isolates obtained from Amur sturgeon will be beneficial in developing potential disease control strategies.


Assuntos
Aeromonas hydrophila/patogenicidade , Doenças dos Peixes/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Aeromonas hydrophila/efeitos dos fármacos , Aeromonas hydrophila/genética , Aeromonas hydrophila/isolamento & purificação , Animais , Antibacterianos/farmacologia , Peixes/microbiologia , Genes Bacterianos/genética , Infecções por Bactérias Gram-Negativas/microbiologia , Testes de Sensibilidade Microbiana/veterinária , Filogenia , Virulência
13.
Microb Pathog ; 116: 135-145, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29355702

RESUMO

Aeromonas hydrophila is a freshwater-dwelling zoonotic bacterium that has economic importance in aquaculture. In the past decade, Aeromonas hydrophila has become increasingly important because of its emergence as a food-borne zoonotic pathogen that is resistant to different treatment regimes. Being an aquatic bacterium, Aeromonas hydrophila is frequently subjected to several stressful environmental conditions, including changes in temperature, acidic pH and starvation that challenge its survival. To cope with these stressful conditions, like every cell, A. hydrophila possesses stress response mechanisms, such as alternative sigma factors, two-component systems, heat shock proteins, cold shock proteins, and acid tolerance response systems that eventually lead the fittest to survive. Moreover, the establishment of genetic variations among the strains related to environmental stress is also of great concern. This review presents the understandings based on inter-strain variations and stress response behavior of A. hydrophila that are important to control the increasing outbreaks of this bacterium in both human populations and aquaculture.


Assuntos
Aeromonas hydrophila/fisiologia , Exposição Ambiental , Estresse Fisiológico , Adaptação Biológica , Adaptação Fisiológica , Aeromonas hydrophila/genética , Concentração de Íons de Hidrogênio , Temperatura Ambiente
14.
Dev Comp Immunol ; 82: 49-54, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29317232

RESUMO

Following the Aeromonas hydrophila aerolysin, various aerolysin-like pore-forming proteins have been identified from bacteria to vertebrates. We have recently reported the mechanism of receptor recognition and in vitro pore-formation of a zebrafish aerolysin-like protein Dln1/Aep1. However, the physiological function of Aep1 remains unknown. Here we detected that aep1 gene is constitutively expressed in various immune-related tissues of adult zebrafish; and moreover, its expression is significantly up-regulated upon bacterial challenge, indicating its involvement in antimicrobial infection. Pre-injection of recombinant Aep1 into the infected zebrafish greatly accelerated the clearance of bacteria, resulting in significantly increased survival rate. Meanwhile, the induced expression of cytokines such as interleukin IL-1ß and tumor necrosis factor TNF-α in zebrafish upon injection of recombinant Aep1 suggested that Aep1 may be a pro-inflammatory protein that triggers the antimicrobial immune responses. However, compared to the overproduction of these cytokines in the infected zebrafish, pre-injection of Aep1 could significantly reduce the expression level of these cytokines, accompanying with a reduced bacterial load. Moreover, the expression profiles through the developmental stages of zebrafish demonstrated that aep1 is activated at the very early stage prior to the maturation of adaptive immune system. Altogether, our findings proved that Aep1 is an innate immune molecule that prevents the bacterial infection.


Assuntos
Toxinas Bacterianas/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/imunologia , Imunidade Adaptativa/genética , Aeromonas hydrophila/genética , Animais , Toxinas Bacterianas/genética , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Imunidade Inata , Mediadores da Inflamação/metabolismo , Interleucina-1beta/metabolismo , Filogenia , Proteínas Citotóxicas Formadoras de Poros/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteínas de Peixe-Zebra/genética
15.
J Mol Microbiol Biotechnol ; 28(5): 225-235, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30783060

RESUMO

Three different polyhydroxyalkanoate (PHA) synthase genes (Ralstonia eutropha H16, Aeromonas sp. TSM81 or Aeromonas hydrophila ATCC7966 phaC) were introduced into the chromosome of two Pseudomonas strains: a native medium-chain-length 3-polyhydroxyalkanoate (PHAMCL) producer (Pseudomonas sp. LFM046) and a UV-induced mutant strain unable to produce PHA (Pseudomonas sp. LFM461). We reported for the first time the insertion of a chromosomal copy of phaC using the transposon system mini-Tn7. Stable antibiotic marker-free and plasmid-free recombinants were obtained. Subsequently, P(3HB-co-3HAMCL) was produced by these recombinants using glucose as the sole carbon source, without the need for co-substrates and under antibiotic-free conditions. A recombinant harboring A. hydrophila phaC produced a terpolyester composed of 84.2 mol% of 3-hydroxybutyrate, 6.3 mol% of 3-hydroxyhexanoate, and 9.5 mol% of 3-hydroxydecanoate from only glucose. Hence, we were successful in increasing the industrial potential of Pseudomonas sp. LFM461 strain by producing PHA copolymers containing 3HB and 3HAMCL using an unrelated carbon source, for the first time in a plasmid- and antibiotic-free bioprocess.


Assuntos
Plasmídeos/genética , Poli-Hidroxialcanoatos/biossíntese , Poli-Hidroxialcanoatos/genética , Pseudomonas/genética , Pseudomonas/metabolismo , Ácido 3-Hidroxibutírico/metabolismo , Aciltransferases/genética , Aeromonas/genética , Aeromonas hydrophila/genética , Antibacterianos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caproatos/metabolismo , Cromossomos Bacterianos , Meios de Cultura/química , Cupriavidus necator/genética , Ácidos Decanoicos/metabolismo , Glucose/metabolismo , Mutação , Pseudomonas/enzimologia , Transformação Bacteriana
16.
Microb Biotechnol ; 10(6): 1400-1411, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28840964

RESUMO

Phasins are unusual amphiphilic proteins that bind to microbial polyhydroxyalkanoate (PHA) granules in nature and show great potential for various applications in biotechnology and medicine. Despite their remarkable diversity, only the crystal structure of PhaPAh from Aeromonas hydrophila has been solved to date. Based on the structure of PhaPAh , homology models of PhaPAz from Azotobacter sp. FA-8 and PhaPTD from Halomonas bluephagenesis TD were successfully established, allowing rational mutagenesis to be conducted to enhance the stability and surfactant properties of these proteins. PhaPAz mutants, including PhaPAz Q38L and PhaPAz Q78L, as well as PhaPTD mutants, including PhaPTD Q38M and PhaPTD Q72M, showed better emulsification properties and improved thermostability (6-10°C higher melting temperatures) compared with their wild-type homologues under the same conditions. Importantly, the established PhaP homology-modelling approach, based on the high-resolution structure of PhaPAh , can be generalized to facilitate the study of other PhaP members.


Assuntos
Aeromonas hydrophila/genética , Proteínas de Bactérias/genética , Lectinas de Plantas/genética , Poli-Hidroxialcanoatos/metabolismo , Aeromonas hydrophila/química , Aeromonas hydrophila/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Dados de Sequência Molecular , Mutagênese , Lectinas de Plantas/química , Lectinas de Plantas/metabolismo , Estabilidade Proteica
17.
Biofouling ; 33(7): 580-590, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28685594

RESUMO

The quorum sensing (QS) phenomenon regulates a myriad of pathogenic traits in the biofilm forming fish pathogen, Aeromonas hydrophila. Blocking the QS mechanism of A. hydrophila is a novel strategy to prevent disease in fish. This study evaluated the effect of tannic acid, a QS inhibitor, on A. hydrophila-associated QS regulated phenomena. A streaking assay with Chromobacterium violaceum (CVO26) reported the presence of N-acyl homoserine lactone (AHL) in A. hydrophila, which was confirmed by HPLC and GC-MS analysis. Tannic acid-treated A. hydrophila showed a considerable reduction in violacein production, blood haemolysis activity and the pattern of swarming motility. Biofilm formation was significantly reduced (p < 0.001) (up to 95%), after tannic acid treatment for 48 h. Analysis by qRT-PCR revealed significant downregulation (p < 0.001) of AhyI and AhyR transcripts in A. hydrophila after tannic acid treatment. Co-stimulation of Catla catla with A. hydrophila and tannic acid attenuated pathogen-induced skin haemorrhages and increased the relative survival rate up to 86.6%. The study provides a mechanistic basis of tannic acid as a QS blocker and indicates its therapeutic potential against A. hydrophila-induced pathogenesis.


Assuntos
Aeromonas hydrophila/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Cyprinidae/microbiologia , Percepção de Quorum/efeitos dos fármacos , Taninos/farmacologia , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , Aeromonas hydrophila/genética , Aeromonas hydrophila/patogenicidade , Aeromonas hydrophila/fisiologia , Animais , Proteínas de Bactérias/genética , Chromobacterium/fisiologia , Relação Dose-Resposta a Droga , Regulação para Baixo , Virulência/efeitos dos fármacos
18.
Fish Shellfish Immunol ; 68: 1-9, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28676336

RESUMO

The iron-regulated outer membrane protein (OMP) of Aeromonas hydrophila is an effective vaccine candidate, but its intrinsic functional components are largely unknown. In this study, we compared the differentially expressed sarcosine-insoluble fractions of A. hydrophila in iron-limited and normal medium using tandem mass tag labeling-based quantitative proteomics, and identified 91 upregulated proteins including 21 OMPs and 83 downregulated proteins including 10 OMPs. Subsequent bioinformatics analysis showed that iron chelate transport-related proteins were enriched in increasing abundance, whereas oxidoreductase activity and translation-related proteins were significantly enriched in decreasing abundance. The proteomics results were further validated in selected altered proteins by Western blotting. Finally, the vaccine efficacy of five iron-related recombinant OMPs (A0KGW8, A0KFG8, A0KQ46, A0KIU8, and A0KQZ1) that were increased abundance in iron-limited medium, were evaluated when challenged with virulent A. hydrophila against zebrafish, suggesting that these proteins had highly efficient immunoprotectivity. Our results indicate that quantitative proteomics combined with evaluation of vaccine efficacy is an effective strategy for screening novel recombinant antigens for vaccine development.


Assuntos
Aeromonas hydrophila/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Vacinas Bacterianas/imunologia , Doenças dos Peixes/prevenção & controle , Regulação Bacteriana da Expressão Gênica , Ferro/metabolismo , Peixe-Zebra , Aeromonas hydrophila/genética , Animais , Proteínas da Membrana Bacteriana Externa/metabolismo , Vacinas Bacterianas/genética , Western Blotting/veterinária , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/prevenção & controle , Infecções por Bactérias Gram-Negativas/veterinária , Proteômica
19.
PLoS One ; 12(7): e0179549, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28708867

RESUMO

Enrofloxacin is the most commonly used antibiotic to control diseases in aquatic animals caused by A. hydrophila. This study conducted de novo transcriptome sequencing and compared the global transcriptomes of enrofloxacin-resistant and enrofloxacin-susceptible strains. We got a total of 4,714 unigenes were assembled. Of these, 4,122 were annotated. A total of 3,280 unigenes were assigned to GO, 3,388 unigenes were classified into Cluster of Orthologous Groups of proteins (COG) using BLAST and BLAST2GO software, and 2,568 were mapped onto pathways using the Kyoto Encyclopedia of Gene and Genomes Pathway database. Furthermore, 218 unigenes were deemed to be DEGs. After enrofloxacin treatment, 135 genes were upregulated and 83 genes were downregulated. The GO terms biological process (126 genes) and metabolic process (136 genes) were the most enriched, and the terms for protein folding, response to stress, and SOS response were also significantly enriched. This study identified enrofloxacin treatment affects multiple biological functions of A. hydrophila. Enrofloxacin resistance in A. hydrophila is closely related to the reduction of intracellular drug accumulation caused by ABC transporters and increased expression of topoisomerase IV.


Assuntos
Aeromonas hydrophila/genética , Farmacorresistência Bacteriana/genética , Fluoroquinolonas/farmacologia , Transcriptoma/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Aeromonas hydrophila/efeitos dos fármacos , Aeromonas hydrophila/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Análise por Conglomerados , DNA Topoisomerase IV/genética , DNA Topoisomerase IV/metabolismo , Bases de Dados Genéticas , Regulação para Baixo/efeitos dos fármacos , Enrofloxacina , Sequenciamento de Nucleotídeos em Larga Escala , RNA Bacteriano/química , RNA Bacteriano/isolamento & purificação , RNA Bacteriano/metabolismo , Análise de Sequência de RNA , Regulação para Cima/efeitos dos fármacos
20.
Fish Shellfish Immunol ; 66: 11-25, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28476666

RESUMO

Outbreaks of infectious diseases in common carp Cyprinus carpio, a major cultured fish in northern regions of China, constantly result in significant economic losses. Until now, information proteomic on immune defence remains limited. In the present study, a profile of intestinal mucosa immune response in Cyprinus carpio was investigated after 0, 12, 36 and 84 h after challenging tissues with Aeromonas hydrophila at a concentration of 1.4 × 108 CFU/mL. Proteomic profiles in different samples were compared using label-free quantitative proteomic approach. Based on MASCOT database search, 1149 proteins were identified in samples after normalisation of proteins. Treated groups 1 (T1) and 2 (T2) were first clustered together and then clustered with control (C group). The distance between C and treated group 3 (T3) represented the maxima according to hierarchical cluster analysis. Therefore, comparative analysis between C and T3 was selected in the following analysis. A total of 115 proteins with differential abundance were detected to show conspicuous expressing variances. A total of 52 up-regulated proteins and 63 down-regulated proteins were detected in T3. Gene ontology analysis showed that identified up-regulated differentially expressed proteins in T3 were mainly localised in the hemoglobin complex, and down-regulated proteins in T3 were mainly localised in the major histocompatibility complex II protein complex. Forty-six proteins of differential abundance (40% of 115) were involved in immune response, with 17 up-regulated and 29 down-regulated proteins detected in T3. This study is the first to report proteome response of carp intestinal mucosa against A. hydrophila infection; information obtained contribute to understanding defence mechanisms of carp intestinal mucosa.


Assuntos
Aeromonas hydrophila/imunologia , Carpas , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Imunidade Inata/genética , Aeromonas hydrophila/genética , Animais , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Mucosa Intestinal/imunologia , Proteoma , Proteômica
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