Protective efficacy evaluation of immunogenic protein AHA_3793 of Aeromonas hydrophila as vaccine candidate for largemouth bass Micropterus salmoides

Aeromonas hydrophila is a Gram-negative pathogen that can infect various fish,including largemouth bass(Micropterus salmoides),which have caused huge economic losses.In present study,largemouth bass anti-A.hydrophila antibodies were produced,then a highly immunogenic outer membrane proteins,AHA_3793,was identified by combined western blotting and mass spectrometry analysis.Moreover,AHA_3793 was expressed,and its immunogenicity was further verified by western blotting.Subsequently,the protective efficacy of AHA_3793 were evaluated in largemouth bass.The results showed that rAHA_3793 could produce a relative percentage survival(RPS)of 61.76% for largemouth bass against A.hydrophila challenge.ELISA analysis showed the specific serum antibodies of largemouth bass against rAHA_3793 and A.hydrophila in vaccinated group in weeks 4 and 5 after immunization were significantly higher than those in control group,which suggested that rAHA_3793 induced production of specific serum antibodies against rAHA_3793 and A.hydrophila.The qRT-PCR analysis showed that expressions of CD4-2 and MHC Ⅱα were also significantly up-regulated after immunization.These results collectively demonstrated that rAHA_3793 could induce a strong humoral immune response of largemouth bass,and then produce high immune protection ef fects against A.hydrophila infection.

Aeromonas hydrophila infection in acute myeloid leukemia: A case report

Rationale:Aeromonas hydrophila,a bacterium commonly found in a variety of soils,aquatic habitats,and other natural environments,is known for causing infections and hemorrhagic diseases in both aquatic and terrestrial animals.Patients with leukemia,when infected by Aeromonas hydrophila following chemotherapy,face an increased risk of developing necrotizing fasciitis.Patient concerns:A 48-year-old male with a history of acute myeloid leukemia presented with swelling and pain in his right hand after being stabbed by a grass carp two days ago.The injury occurred on the fifth day after his last chemotherapy session when he was accidentally punctured in the right thumb and ring finger by a carp while fishing.Two days following the injury,the skin isurrounding the wound turned black immediately,while the rest of his hand was reddened,swollen,and bleeding.Diagnosis:Aeromonas hydrophila infection in the soft tissue of the right hand.Interventions:Intravenous antibiotics and local debridement.Outcomes:The patient experienced an alleviation of systemic poisoning symptoms,effective control of the local wound infection.Lessons:It is important for physicians to appreciate the potential for highly unusual and life-threatening infections in patients with acute myeloid leukemia.Early diagnosis and prompt treatment can prevent the development of necrotizing fasciitis and save patients’lives.

Effects of Compound Probiotics on Intestine and Intestinal Aeromonas hydrophila of Fish

[Objective] This study was conducted to develop compound probiotics that can be used as the alternatives of chemical drugs and antibiotics in aquaculture. [Method] Different concentrations of EM （effective microorganisms） and Bacillus amyloliquefaciens were mixed at a ratio of 1：1, and sprayed on fish feed. The growth of intestinal villi of the fishes that had been fed with the feed mixed with compound probiotics for three months was observed; meanwhile, the content of in- testinal Aeromonas hydrophila was detected by real-time quantitative PCR. ｜Result] The compound probiotics promoted the development of intestinal villi, and inhibited the growth of A. hydrophila, and the effects were also concentration dependent. However, the compound probiotics did not increase the thickness of the intestinal serous layer, muscular layer and submucosal layer. [Conclusion] The compound probiotics we prepared can be used and popularized in aquiculture as it can inhibit the growth of A. hydrophila.

Protection of Carassius auratus Gibelio against infection by Aeromonas hydrophila using specific immunoglobulins from hen egg yolk

Specific immunoglobulin (IgY) from egg yolk against Aeromonas hydrophila was produced by immunization of White Leghorn hens with formalin-killed whole cells of A. hydrophila. ELISA test using A. hydrophila as the coating antigen revealed that the specific antibody titer started to increase in the egg yolk at the 13th day post-immunization (P/N=2.18), reached the peak at the 56th day (P/N=13.82), and remained at high level until day 133 (P/N=7.03). The antibody was purified by saturated ammonium sulphate with a recovery rate of 63.5%. The specific IgY inhibited the growth of A. hydrophila at a concentration of 1.0 mg/ml during the 18 h incubation. Pre-treatment of polyploid gibel carps Carassius auratus Gibelio with specific IgY had a protection rate of 60% (6/10) against challenge with A. hydrophila, while none of the fishes in the control groups receiving sterile phosphate buffered saline (PBS) or non-specific IgY survived the challenge. Treatment of fishes with the specific IgY 4 h after the challenge also had lower mortality (70%, 7/10), a 30% reduction against the control PBS or non-specific IgY groups (10/10). These results indicate that specific IgY antibodies could be obtained easily from hens immunized with an inactivated A. hydrophila and could provide a novel alternative approach to control of diseases in fishes caused by this organism.

Cloning,expression in Escherichia coli,and enzymatic properties of laccase from Aeromonas hydrophila WL-11

A strain WL-11 with high laccase activity was isolated from activated sludge collected from the effluent treatment plant of a textile and dyeing industry. It was identified as Aeromonas hydrophila by physiological test and 16S rDNA sequence analysis. A gene encoding of laccase from a newly isolated Aeromonas hydrophila WL-11 was cloned and characterized. Nucleotide sequence analysis showed an open reading frame of 1605 bp encoding a polypeptide comprised of 534 amino acids. The primary structure of the enzyme predicted the structural features characteristic of other laccases, including the conserved regions of four histidine-rich copper-binding sites. The predicted amino acid sequence showed a high homology （more than 60%） with bacterial laccases in the genome and protein databases and the highest degree of similarity （61% identity） was observed with the multicopper oxidase of KlebsieUa sp. 601. When expressed in Escherichia coli, the recombinant enzyme was overproduced in the cytoplasm as soluble and active form. The purified enzyme had an optimum pH of 2.6 and 8.0 for ABTS （2,2＇-azino-bis（3-ethylbenzthiazolinesulfonic acid） and DMP （2,6-dimethoxyphenol）, respectively. The kinetic study on ABTS revealed a higher affinity of this enzyme to this substrate than DMP.

Antimicrobial Susceptibility and Characterization of Outer Membrane Proteins of Aeromonas hydrophila Isolated in China

Aeromonas hydrophila isolates from clinical cases （n=43） were tested against 8 antimicrobial agents and typed by outer membrane protein （OMP） pattern by using sodium dodecyl sulfate gel electrophoresis. All isolates were resistant to ampicillin （MICs, ≥16 μg mL-1） and sulfamonomethoxine （MICs≥64 μg mLl）, but susceptible to norfloxacin （MICs,≤0.5 μg mL-1）. There was a high incidence of resistance to erythromycin （90.70%） and tylosin （93.02%）, while a low incidences of resistance to ciprofloxacin （2.33%）, enrofloxacin （2.33%） and florfenicol （4.65%）. Six different outer membrane protein patterns were found among 34 isolates by analyzing proteins in the range of 22 to 50 kDa, other than 9 isolates with their respective profiles. The strains with the similar OMP profiles had similar resistances. Compared with the other strains from the same OMP patterns, NB-1, A.Pun and MR-1 had lacked the proteins in the range of 30 to 45 kDa and their resistance to florfenicol substantially increased. It is speculated that the outer membrane protein changes might correlate with decreased susceptibility to florfenicol in the three strains. Some strains which showed completely identical OMP types had a little difference in their resistance to fluoroquinolones, indicating that there might be other factors that were involved in the antimicrobial resistance of A. hydrophila.

Molecular Characterization and Virulence Genes of Aeromonas hydrophila Isolated from the Chinese Giant Salamander (Andrias davidianus)

The Chinese giant salamander(Andrias davidianus) is the largest living amphibian in the world. Aeromonas hydrophila strain L602 was isolated from A. davidianus. The 16S rDNA gene of this isolate was amplified using PCR,and the phylogenetic tree was constructed by the neighbor-joining method. Four virulence genes(aerA,aha1,hly and alt) of A. hydrophila were amplified by PCR and drug resistances were tested using Kirby-Bauer disk diffusion method. The results showed that the length of this 16S rDNA sequence was 1453 bp,which showed 99% homology with A. hydrophila. The GenBank accession number was JX155398. Phylogenetic analysis indicated it grouped together with A. hydrophila. Four virulence genes were all detected,indicating that strain L602 was highly virulent. This stain was resistant to four antibiotics(vibramycin,furazolidone,ampicillin and erythromycin),while it was insensitive to streptomycin. Furthermore,this strain was susceptible to six antibiotics(sulfafurazole,ciprofloxacin,penbritin,norfloxacin,florfenicol and enrofloxacin). This study will help to validate the classification and virulence of pathogenic bacteria in amphibians.

Non-specific immune response of bullfrog Rana catesbeiana to intraperitoneal injection of bacterium Aeromonas hydrophila

Non-specific immune response of bullfrog Rana catesbeiana to pathogenic Aeromonas hydrophila was studied to 60 individuals in two groups.Each bullfrog in bacterium-injected group was injected intraperitoneally(i.p.) with 0.2 ml bacterial suspension at a density of 5.2 × 106 CFU/ml,while each one in control group injected i.p.with 0.2 ml sterile saline solution(0.85%,w/v).Three bullfrogs in both groups were sampled at 0,1,3,7,11,15 and 20 days post-injection(dpi) for the evaluation of non-specific immune parameters.It was observed that intraperitoneal injection of A.hydrophila significantly increased the number of leucocytes and that of NBT-positive cells in peripheral blood.Significant increases in serum bactericidal activity and serum acid phosphatase activity were also observed in the bacterium-injected frogs when compared with those in the control group.However,a significant reduction was detected in vitro in phagocytosis activity of peripheral blood phagocytes.No significant difference in changes in the number of peripheral erythrocytes,serum superoxide dismutase(SOD) activity,and lysozyme activity was detected between the two groups.It is suggested that bullfrogs may produce a series of non-specific immune reactions in response to the A.hydrophila infection.

Molecular Typing of Aeromonas hydrophila Isolated from Common Carp in Northeast China

A total of 59 isolates of Aeromonas hydrophila were collected from common carp suffering from freshwater fish hemorrhage disease in 13 fishing grounds in the northeast China, and their phenotypic and genetic characteristics were investigated. All of the isolates were identified as A. hydrophila by traditional biochemical method and yielded a 686-bp DNA fragment of the 16S rDNA gene in the PCR experiments. Collected strains were also evaluated for their susceptibility to 17 different antibiotics. The isolates showed an even trend of the resistance and sensitivity to drugs, highly sensitive to antibiotics, such as Levofloxacin, PolymyxinB, Ofloxacin and resistant to antibiotics, such as Bristopen, Lincomycin, Ampicillin, Teicoplanin. Evaluation of genetic diversity was performed on all isolates by molecular typing with enterobacterial repetitive intergenic consensus （ERIC-PCR） method. The results showed that three different types, i.e. type I, ]I, and type ~I, were found in 59 isolates and type III accounted for a large proportion of 54.84%. There was no dominant difference between the tendency of the isolates of Heilongjiang Province and Jilin Province in these three types, which showed DI 〉 I 〉 I, while the isolates of Liaoning Province showed III 〉 II 〉 I. The percentage of different types in different provinces varied in each other; however, they didn＇t show any obvious regional or cluster-specific branches. In conclusion, the ability to distinguish Aeromonas hydrophila strains from diseased common carp with ERIC-PCR would be useful for epidemiological investigation and population genetic analysis of this pathogen in China.

In Vitro Antibacterial Effect of Effective Microorganisms( EM) on Aeromonas hydrophila

In order to understand in vitro antibacterial effect of antibiotics and EM on Aeromonas hydrophila,14 isolates of A. hydrophila were isolated from diseased fish and aquaculture in the study. PCR amplification results of aerolysin gene( Aero gene) of isolates indicated that 7 out of 14 isolates were pathogenic A.hydrophila. In vitro antibacterial test indicated that 14 isolates were resistance to penicillin G,ampicillin,cephradine,cefazolin,tetracycline,terimethoprim,lincomycin and cephalexin,but showed high sensitivity to cefotaxime and furazolidone. In vitro antibacterial effect of EM on 14 isolates of A. hydrophila was positively correlated with concentration,and EM with the concentration greater than 60% had antibacterial effect on 14 isolates.

Preliminary Studies of Aeromonas hydrophila Diseases in Rana spinosa David and Whole Bacteria Inactivated Vaccine Preparation Method

[Objective]In order to carriy on effective medication and immune prevention and control,pathogen and etiology of a Rana spinosa David disease in a Rana spinosa David breeding company in Zhejiang was researched from 2009 to 2010. [Methods]Conventional bacteria separation and purification technology was used to purify pathogenic bacteria,regression infection and toxicity test was used to determine its virulence,physiological and biochemical and molecular biology identification was carried out to the the pathogen,k- b paper method was adopted for the susceptibility test,normal paraffin wax flaking technology was used for pathology observations,finally,the inactivation conditions of pathogenic bacteria was explored. [Results]The pathogenic bacteria separated from sick Rana spinosa David body had Median Lethal Concentration（ LC 50） of 5.62 ×105 cfu / ml; the strain was identified as Aeromonas hydrophila by ATB bacteria identification instrument and 16 s rRNA sequence analysis（ GenBank login number： HQ322682）; histopathology observation results showed that the disease had main symptoms of the liver and kidney damage,inflammatory cells increase. Susceptibility test showed that the strain was highly sensitive to gentamicin,norfloxacin,florfenicol,enrofloxacin etc. [Conclusions]1% formaldehyde could well inactivate the pathogenic bacteria at 60 ℃ for 24 h,which would provide technical reference for whole bacteria inactivated vaccine preparation.

Emerging role of vitamin D_(3)in alleviating intestinal structure injury caused by Aeromonas hydrophila in grass carp(Ctenopharyngodon idella)

Bacterial pathogens destroy the structural integrity of functional organs in fish,leading to severe challenges in the aquaculture industry.Vitamin D_(3)(VD_(3))prevents bacterial infections and strengthens immune system function via vitamin D receptor(VDR).However,the correlation between VD_(3)/VDR and the structural integrity of functional organs remains unclarified.This study aimed to investigate the influence of VD_(3)supplementation on histological characteristics,apoptosis,and tight junction characteristics in fish intestine during pathogen infection.A total of 540 healthy grass carp(257.24±0.63 g)were fed different levels of VD_(3)(15.2,364.3,782.5,1,167.9,1,573.8,and 1,980.1 IU/kg)for 70 d.Subsequently,fish were challenged with Aeromonas hydrophila,a pathogen that causes intestinal inflammation.Our present study demonstrated that optimal supplementation with VD_(3)(1)alleviated intestinal structural damage,and inhibited oxidative damage by reducing levels of oxidative stress biomarkers;(2)attenuated excessive apoptosisrelated death receptor and mitochondrial pathway processes in relation to p38 mitogen-activated protein kinase signaling(P<0.05);(3)enhanced tight junction protein expression by inhibiting myosin light chain kinase signaling(P<0.05);and(4)elevated VDR isoform expression in fish intestine(P<0.05).Overall,the results demonstrated that VD_(3)alleviates oxidative injury,apoptosis,and the destruction of tight junction protein under pathogenic infection,thereby strengthening pathogen defenses in the intestine.This finding supports the rationale for VD_(3)intervention as an essential practice in sustainable aquaculture.

Biosorption and biotransformation of crystal violet by Aeromonas hydrophila DN322p

DN322p, an offspring of Aeromonas hydro- phila DN322, has the capacity to adsorb and decolorize triphenylmethane dyes in wastewater simultaneously. As a common triphenylmethane dye, crystal violet （CV） was chosen to test the decolorization characteristics of DN322p. Within 0.5h, the strain DN322p adsorbed a large amount of CV, producing a deep-colored cell pellet and colorless supernatant. The colors of the cell pellet and supematant lightened over time. The supernatant and dichloromethane extract of the cell pellet both showed conspicuous CV and leuco CV （LCV） characteristic absorbance peaks at 590 nm and 260 nm, respectively, in the UV-vis spectral analysis. This finding indicated that the DN322p cells can adsorb the two dyes. A 99% （w/w） decolorization rate was achieved within 2.5 h with shaking at 30℃ for 50mg CV.L-1. High Performance Liquid Chromatography （HPLC） analysis of the dichloromethane extract of the supernatant and cell pellet confirmed that CV was mainly converted into its leuco form. Dead cells had a similar adsorption capacity with living cells. About 90% of CV in the dye solution （50mg-L-1） was removed by autoclaved cells with an optical delnsity at 600 nm （OD600） above 1.0.

Production of Poly(3-Hydroxybutyrate-co-3-Hydroxyhexanoate) Using Aeromonas hydrophila 4AK4 Grown in Mixed Carbon Source

Aeromonas hydrophila 4AK4 was grown on mixed substrates of soybean oil and lauric acid for the production of polyhydroxyalkanoate copolymer consisting of 3 hydroxybutyrate (3HB) and 3 hydroxyhexanoate (3HHx). A maximal poly(3 hydroxybutyrate co 3 hydroxyhexanoate) (PHBHHx) content of 49.13% in dry cells was obtained in a shake flask culture. PHBHHx of 6.26 g/L was produced in a fermentation experiment over 48 h on a sole carbon source containing 100 g/L soybean oil, while 12.40 g/L PHBHHx was produced on a mixed carbon source containing 80 g/L soybean and 20 g/L lauric acid over the same period of time, resulting in a polyhydroxyalkanoate (PHA) productivity of 0.25 g/(L·h). The results show that mixed carbon sources are suitable for industrialized production of PHBHHx from A. hydrophila 4AK4, as the mixed carbon sources also overcome the foaming problem that occurs when lauric acid is employed as a sole carbon source in PHBHHx production.

Time-resolved and multi-tissue RNAseq provides new insights on the immune responses of European eels following infection with Aeromonas hydrophila

No studies have systematically compared time-resolved and multi-tissue innate immune responses for European eels(Anguilla anguilla)with Aeromonas hydrophila infection by a high throughput method.Here,we challenged European eels with A.hydrophila(infected)or PBS(control).A low fatality rate(16.1%)was observed for the infected group at 2 d post-infection(dpi).Then we examined transcriptional profiles of intestines,livers and spleens from 12 European eels with/without infection by A.hydrophila at 6,18 and 36 h post-infection(hpi).The results showed that the most differentially expressed genes(DEGs)were found in the spleen at 6 hpi(7569 DEGs),followed by the intestine at 6 hpi(3129 DEGs)and the liver at 18 hpi(2722 DEGs).Only 540,41 and 130 DEGs were found in the spleen,liver,and intestine at 36 hpi,respectively.The comparison of DEG numbers and enriched KEGG pathways suggested a consistent time-course immune response in these tissues.A similar enrichment of pattern recognition receptors-related pathways including Toll-like receptors,NOD-like receptors and RIG-I-like receptors was found in the liver and spleen,but not in the intestine.Among immune-related DEGs,62 paralogue pairs were found,and the expression trends of most paralogues were consistent.Some paralogues of immune-related DEGs had unique domains.Furthermore,large clusters representing similar tissue were shown for the intestine and spleen.A different co-expression network involved in cytokine signal transduction and interaction existed between the liver and spleen.This study has provided time-resolved and multi-tissue transcriptome characteristics of A.anguilla in response to A.hydrophila infection.

Production of Poly(3-Hydroxybutyrate-co-3-Hydroxyhexanoate) by Aeromonas hydrophila and Recombinant Escherichia coli

Aeromonas hydrophila (A. hydrophila) 4AK4 produced poly(3 hydroxybutyrate co 3 hydroxyhexanoate) (PHBHHx) with an almost constant 3 hydroxyhexanoate (3HHx) content of 10%15% from lauric acid and/or soybean oil. Both A. hydrophila 4AK4 and recombinant Escherichia coli (E. coli) JMU193 (pBH32) produced PHBHHx with controllable 3HHx content when fed lauric acid and another co substrate. With glucose or gluconate as the co substrate, the 3HHx content in the copolyester produced by A. hydrophila 4AK4 was reduced slightly from 12% to 9%. However, the 3HHx content in the copolyester produced by E. coli JMU193 (pBH32) was significantly reduced from 9% to 2% with fructose as the co substrate. These results show that regulation of 3HHx content in PHBHHx can be achieved using genetically engineered E. coli.

Correlation between the distribution pattern of virulence genes and virulence of Aeromonas hydrophila strains

Nine strains of Aeromonas hydrophila isolated from diseased fish or soft-shelled tortoise were tested for the presence of three virulence genes including the genes encoding aerolysin,hemolysin,and extracellular serine protease(i.e.,aerA,hlyA,and ahpA,respectively).These genes were investigated using polymerase chain reaction(PCR)with specific primers for each gene.And the pathogenicities to Carrassius auratus ibebio of these strains were also assayed.PCR results demonstrated that the distribution patterns of aerA,hlyA,and ahpA were different in these strains.6/9 of A.hydrophila strains were aerA positive,8/9 of strains hlyA positive,7/9 of strains ahpA positive,respective-ly.However,the assay for pathogenesis showed that two strains(A.hydrophila XS91-4-1 and C2)were strong virulent,two strains(A.hydrophila ST78-3-3 and 58-20-9)avirulent and the rest middle virulent was to the fish.In conclusion,there are significant correlation between the distribution pattern of the three virulence genes and the patho-genicity to Carrassius auratus ibebio.All strong virulent A.hydrophila strains were aerA+hlyA+ahpA+genotype,and all aerA+hlyA+ahpA+strains were virulent.Strains with the genotype of aerA−hlyA−ahpA+have middle pathogenicity.In the present study,we found for the first time that all A.hydrophila isolated from the ahpA positive were virulent to Carrassius auratus ibebio.Additionally,there was a positive correlation between the virulence of A.hydrophila and the presence of aerA and ahpA.

Edwardsiella tarda and Aeromonas hydrophila isolated from diseased Southern flounder(Paralichthys lethostigma)are virulent to channel catfish and Nile tilapia

Objective:To identify bacteria isolated from diseased Southern flounder and determine whether they are virulent to channel catfish and Nile tilapia.Methods:Gram-negative bacteria isolates were recovered from five tissues of diseased Southern flounder(Paralichthys lethostigma).The isolates were subjected to biochemical and molecular identification followed by virulence study in fish.Results:Based on biochemical analysis,the 25 isolates were found to share homologies with either Edwardsiella tarda(E.tarda)or Aeromonas hydrophila(A.hydrophila).Based on sequencing results of partial 16S rRNA gene,15 isolates shared 100%identities with the 16S rRNA sequence of previously identified E.tarda strain TX1,whereas the other 10 isolates shared 100%identities with the 16S rRNA sequence of previously identified A.hydrophila strain An4.When healthy fish were exposed to flounder isolate by intracoelomic injection,the LD50 values of flounder isolate E.tarda to channel catfish or Nile tilapia[(10±2)g]were 6.1×10^(4)and 1.1×10^(7)CFU/fish,respectively,whereas that of flounder isolate A.hydrophila to channel catfish and Nile tilapia were 1.4×10^(7)and 5.6×10^(7)CFU/fish,respectively.Conclusions:This is the first report that E.tarda and A.hydrophila isolated from diseased Southern flounder are virulent to catfish and tilapia.

Protective effect of probiotic diets on haematobiochemical and histopathology changes of Mystus montanus(Jerdon 1849)against Aeromonas hydrophila

Objectives:To evaluate the protective effct of probiotic diets on haemotobiochemical and histopathology changes of Mystus montanus against Aeromonas hydrophila(A.hydrophila).Methods:Three experimental groups of fish were fed with a diet supplemented with Lactobacillus acidophilus(L.acidophilus)(Sporolac),comprising about 0.1 g,0.2 g and 0.3 g.Control group of fish were fed without L.acidophilus.After 60 d of feeding the fishes in experimental group were injected with 1 mL of A.hydrophila and were supplemented with probiotic diets.The control group fishes were injected with 1 mL of physiological saline solution alone.Results:Blood samples were collected for haematobiochemical analysis,while samples of the liver,and gills were examined for path histology after 7 d of infection.The result showed that the growth parameters,weight gain,specific growth rate were better in infected group maintained on the probiotic diet compared to those in control group.The haematology parameters,erythrocyte sedimentation rate,red blood cell,white blood cell,total serum protein,Mg^(2+),Ca^(2+),Cl,glucose,cholesterol and total immunoglobulin concentration and the pathohistology of the liver,gills were better in the infected fish maintained on the probiotic diet than those in the group fed the control diet.Conclusions:The result of the present study showed that L.acidophilus is useful as a probiotic agent in Mystus montanus against A.hydrophila.

Monitoring the mode of action of synthetic and natural biocides against Aeromonas hydrophila by Raman spectroscopy and chemometrics

We have investigated the mode of action of synthetic biocides,(2-(thiocyanomethylthio)benzothiazole(TCMTB),dichlorophen,(commonly used in leather industry for preservation)and natural biocides,oregano and eucalyptus oils,on Aeromonas hydrophila using Raman spectroscopy in collaboration with multivariate analysis and 2D correlation spectroscopy to evaluate whether Raman spectra acquired contained valuable information to study the action of biocides on bacterial cells.The growth of A.hydrophila in clear and outer edge zone of inhibition differ in their reaction with different biocides,which allows us to highlight the differences as a characteristic of two kinds of bacteria.Such classification helps identify oregano oil as the most effective biocide by altering clear and outer edge zone of bacteria.Standard disk diffusion assay method was used for screening biocide bacteria interactions and later analysed by Raman spectroscopy.The paper also presents the introduction of TCMTB and oregano oil into leather processing stages to examine and determine the antimicrobial effect as an application to real-world setting.Therefore,we conclude that Raman spectroscopy with appropriate computational tools constitutes a powerful approach for screening biocides,which provide solutions to all the industries using biocides including leather industry,considering the potentially harmful effect of biocides to humans and the environment.