Phage Cocktail Development against Aeromonas salmonicida subsp. salmonicida Strains Is Compromised by a Prophage.

Aquaculture is a rapidly growing food production sector. Fish farmers are experiencing increasing problems with antibiotic resistance when fighting against pathogenic bacteria such as Aeromonas salmonicida subsp. salmonicida, the causative agent of furunculosis. Phage therapy may provide an alternative, but effective use must be determined. Here, we studied the inhibition of A. salmonicida subsp. salmonicida strains by five phages (HER98 [44RR2.8t.2], HER110 [65.2], SW69-9, L9-6 and Riv-10) used individually or as combinations of two to five phages. A particular combination of four phages (HER98 [44RR2.8t.2], SW69-9, Riv-10, and HER110 [65.2]) was found to be the most effective when used at an initial multiplicity of infection (MOI) of 1 against the A. salmonicida subsp. salmonicida strain 01-B526. The same phage cocktail is effective against other strains except those bearing a prophage (named Prophage 3), which is present in 2/3 of the strains from the province of Quebec. To confirm the impact of this prophage, we tested the effectiveness of the same cocktail on strains that were either cured or lysogenized with Prophage 3. While the parental strains were sensitive to the phage cocktail, the lysogenized ones were much less sensitive. These data indicate that the prophage content of A. salmonicida subsp. salmonicida can affect the efficacy of a cocktail of virulent phages for phage therapy purposes.

Occurrence, diversity and temperature-dependent growth kinetics of Aeromonas spp. in lettuce.

Bagged, pre-cut and prewashed lettuce products are marketed as ready to eat. This concept poses a food safety concern, due to lack of efficient hurdles to eliminate possible microbial contaminants from the fresh produce and/or the processing itself. Aeromonas spp. are potential foodborne pathogens that are frequently isolated from lettuce. High counts of, e.g., A. hydrophila have been found in retail ready-to-eat (RTE) vegetable salads. The aim of this study was to assess the general microbiological quality, the occurrence and diversity of potential human pathogenic mesophilic Aeromonas spp. of retail RTE lettuce products. Additionally, temperature-dependent growth kinetic parameters of Aerobic Plate Counts (APC) and Aeromonas spp. in one selected RTE lettuce product, rocket lettuce, were quantified by performing storage experiments at 4 °C, 8 °C and 12 °C. The Aeromonas isolates were further characterized regarding pathogenic traits and phylogenetic relationship. The overall hygienic quality of the lettuce products was unsatisfactory, as 60% of the products had an APC level higher than 7.0 log CFU/g. Presumptive Aeromonas spp. were detected in 52% of the samples, levels ranging from approximately 2.0-6.0 log CFU/g. Significantly lower counts of APC and Aeromonas spp. were found in uncut and unwashed products. Presumptive Aeromonas spp. were able to proliferate in rocket lettuce stored at 4 °C (µmax = 0.39 ± 0.06/d and µmax = 0.43 ± 0.05/d for lettuce from producers A and B, respectively), and µmax was approximately 2× higher at 8 °C and 3× higher at 12 °C. Eighty-four percent of the collected isolates were identified as A. media, based on partial gyrB sequencing. Additionally A. salmonicida and A. bestiarum were detected. The pathogenic potential in this material was high, most of the isolates harbored at least one of the toxin genes, act, ast, alt.

Characterization of co-culture of Aeromonas and Pseudomonas bacterial biofilm and spoilage potential on refrigerated grass carp (Ctenopharyngodon idellus).

Aeromonas and Pseudomonas are important bacterial species involved in spoilage of refrigerated freshwater fish. In this study, 10 Aeromonas and seven Pseudomonas bacterial strains were isolated from spoiled grass carp and identified. Twelve of seventeen bacterial strains showed high potential of biofilm formation and 14 of 17 can produce extracellular protease. In order to explore the spoilage capacity of dual-species, the sterile grass carp fillets were inoculated with mono- and dual-species of Aeromonas salmonicida and Pseudomonas azotoformans strains. The results revealed significantly higher levels of the total viable count and total volatile basic nitrogen in dual-species as compared to mono-species from day 6. The higher contents of histamine, cadaverine and serious degradation in muscles tissue were also observed in dual-species after 10 days of storage. Results of in vitro experiments showed that the co-culture of A. salmonicida and P. azotoformans significantly increased the bacterial maximum growth rate, promoted the biofilm formation and improved the spoilage capacity of bacterial strains. This study has revealed that the co-culture of Aeromonas and Pseudomonas bacterial strains accelerated spoilage process of grass carp and increased biofilm formation. It indicates that the mixed-cultures of spoilage micro-organisms pose a huge threat to food industry.

Effect of grape seed extract on quality and microbiota community of container-cultured snakehead (Channa argus) fillets during chilled storage.

Herein, the effects of grape seed extract (GSE) on the microflora and biochemical changes of container cultured snakehead (Channa argus) fillets during 11 days of chilled storage were investigated. The sensory analysis, the total number of viable colonies, the total amount of volatile basic nitrogen, and k-value analysis revealed that GSE retarded the deterioration of snakehead fillets. The degradation of inosine 5'-monophosphate and the accumulation of inosine and hypoxanthine in the GSE group were slower than these in the control group. Moreover, GSE treatment effectively decreased the accumulation of putrescine, cadaverine, and histamine. Illumina-MiSeq high throughput sequencing results showed that GSE inhibited the growth of Aeromonas on snakehead fillets. Based on the microbial enumeration, sensory analysis, and k-value, GSE prolonged the shelf life of fillets for 3 days, suggesting its potential for snakehead fillets preservation.

Bacterial and archaeal compositions and influencing factors in soils under different submergence time in a mercury-sensitive reservoir.

Soils in the water-level-fluctuating zone (WLFZ) of Three Gorges Reservoir (TGR) inundated by water for different periods of time are confirmed to have disparate characteristics to mercury (Hg), and thus it is of great significance to further investigate microbial compositions and influencing factors. The objective of this study was to compare bacterial and archaeal richness, α-diversities and compositions, as well as affecting variables, especially Hg concentrations, among soils under different submergence time-SI (inundated soil), SS (semi-inundated soil), SN(non-inundated soil) and SSe (sediment)-based on high throughput sequencing. Results showed that sediment had significantly higher bacterial and archaeal richness and α-diversities than the other soil types. Anaerolinea and Aeromonas, as well as Altiarchaeales, Nitrosoarchaeum, and Methanosarta were dominant in SSe, while sharply decreasing in the other soil types, with significant difference among groups. An unclassified genus in SCG critically predominating in SI, SS and SN, drastically reduced in SSe, with extremely significant difference among groups. Bathyarchaeota and Nitrososphaera, both dominating in SSe, decreased dramatically and almost vanished in SI and SN. All the variables except pH posed a significant positive effect on bacterial and archaeal compositions in SSe, while opposite effect in the other three soil types. MeHg and THg concentrations had relatively weaker effects on microbial compositions comparing to variables like NH4+, CEC, OM and SO42+.

Dynamic characteristics of AHLs-secreting strain Aeromonas sp. A-L2 and its bioaugmentation during quinoline biodegradation.

AIMS: In order to probe a more environmentally friendly method of pollutant treatment based on microbial bioaugmentation and quorum sensing (QS) effects. METHODS AND RESULTS: The dynamic characteristics and QS effects of the acylated homoserine lactones (AHLs)-secreting strain Aeromonas sp. A-L2 (A-L2), which was isolated from the activated sludge system, was discussed. According to the liquid chromatography-mass spectrometry results, N-butyryl-homoserine lactone (C4-HSL) and N-hexanoyl-homoserine lactone (C6-HSL) were the major AHLs secreted by strain A-L2, and the swarming of strain Ochrobactrum sp. LC-1 (LC-1) was induced by these compounds. The extracellular polymeric substance secretion of the strain LC-1 was mainly led by C6-HSL, and the biofilm formation ability was mainly influenced by C6-HSL or C4-HSL (60 µg l-1 ). The optimal AHLs secretion conditions of strain A-L2 were also studied. Drawing support from the AHLs-secreting strain A-L2 during quinoline degradation by strain LC-1, the degradation time was greatly shortened. CONCLUSIONS: Hence, AHLs-secreting strain A-L2 can be useful as an AHLs continuous supplier during bioaugmentation and pollutant biodegradation. SIGNIFICANCE AND IMPACT OF THE STUDY: The bioaugmentation process of strain A-L2 on quinoline biodegradation based on QS effects would lay a certain theoretical and practical significance for large-scale applications.

Effects of phytic acid and lysozyme on microbial composition and quality of grass carp (Ctenopharyngodon idellus) fillets stored at 4 °C.

This study investigated the effect of phytic acid and lysozyme on the microbial composition and quality of grass carp (Ctenopharyngodon idellus) fillets stored at 4 °C. The control, 0.5 mg/mL lysozyme-treated fillets (T1), 0.5 mg/mL phytic acid-treated fillets (T2) and 0.25 mg/mL lysozyme + 0.25 mg/mL phytic acid-treated fillets (T3) were evaluated based on sensory assessment, biogenic amines, ATP-related compounds, total volatile basic nitrogen (TVB-N), and total viable counts (TVC). Changes in microbial composition were analyzed using high-throughput sequencing. Results showed that phytic acid and lysozyme treatment delayed the decrease in sensory scores, reduced the rate of degradation of IMP to Hx, inhibited the growth of microorganisms, and attenuated the increase in TVB-N and putrescine. Phytic acid exhibited better preservation effects than lysozyme and their combination was more effective than using either alone. High-throughput sequencing showed that Acinetobacter and Kocuria were the predominant bacteria in fresh grass carp, but Pseudomonas rose rapidly with storage time; Pseudomonas, Shewanella, and Aeromonas constituted the main spoilage bacteria of grass carp fillets. Lysozyme treatment significantly reduced the proportion of Shewanella and Acinetobacter, and phytic acid and the combination of phytic acid and lysozyme significantly reduced the proportion of Pseudomonas in spoiled grass carp fillets.

Single-strain regression analysis evaluating disc potencies of flumequine and enrofloxacin for testing Aeromonas sobria and Vibrio anguillarum.

Two quite different disc contents are used for antimicrobial susceptibility testing of two fluoroquinolone drugs, flumequine and enrofloxacin, in the disc diffusion test, 30 and 5 µg, respectively. Using the SRA method, single-strain regression analysis, we studied the impact of disc content when testing two relevant bacterial species, Aeromonas sobria and Vibrio anguillarum. There were no major differences between the antimicrobial regression lines for the two species. Wild-type strains produced acceptable zones of inhibition over a wide range of disc contents. The flumequine 30 µg disc should be lowered in its drug content. No rational reasons for choosing so different disc contents for the two antimicrobials were apparent. At present, the choice of disc content for new antimicrobials are outside the realm of clinical microbiologists. It is recommended that reference authorities, such as EUCAST, CLSI and USCAST, are consulted for the choice of disc contents in the future.

Nutritional interference for phenotypic biofilm quantification in Aeromonas spp. isolates containing the fla gene.

The Aeromonas genus has several virulence factors associated with the development of diseases in aquatic organisms, leading to losses in aquaculture. One of these factors is the flagella's formation which allows the biofilm's formation that provides the microorganisms a greater pathogenicity, greater protection to certain substances such as antibiotics. The aim of the study was to verify the presence of the fla gene, related to biofilm production in isolates of Aeromonas spp. from fishes and also to determine the best quantification condition of phenotypic biofilm production in vitro. Polymerase Chain Reactions were performed to obtain the amplification of the region comprising the fla gene. To determine the best condition for the production biofilm, the microplate adhesion test was carried out under different concentrations of TSB broth and it combined with glucose. Of the 43 isolates of Aeromonas spp. analyzed, 28 were positive for the fla gene and, in the quantification of the biofilm, all these were able to form biofilm in the TSB broth without dilution and without addition of glucose, being this the best condition tested. It was observed that the isolates of Aeromonas spp. analyzed have potential for biofilm formation, and hence potential for virulence.

Application of Illumina-MiSeq high throughput sequencing and culture-dependent techniques for the identification of microbiota of silver carp (Hypophthalmichthys molitrix) treated by tea polyphenols.

This study evaluated the antimicrobial effects of tea polyphenols (TP) on changes in microbiota composition and quality attributes in silver carp fillets stored at 4 °C. During storage, TP treatment was found to be effective in enhancing sensory quality, inhibiting microbial growth, and attenuating chemical quality deterioration. Meanwhile, the composition of microbiota of silver carp fillets was investigated using culture-dependent and culture-independent methods. Initially, compared to the control, TP obviously decreased the relative abundance of Aeromonas, which allowed Acinetobacter and Methylobacterium to become the dominant microbiota in TP treated fillets on day 0. The controls, 0.5% TP-treated fillets, and 1% TP-treated fillets were rejected by sensory panelists on days 8, 12, and 12, respectively. At the time of sensory rejection, Aeromonas, followed by Acinetobacter and Pseudomonas, became the main spoilers in the control on day 8. However, TP treatment inhibited the growth of Aeromonas and Acinetobacter significantly. Consequently, Aeromonas followed by Pseudomonas and Shewanella became the predominant microbiota in all TP-treated fillets on day 12. Therefore, TP improved the quality of fillets during chilled storage, which was mainly due to their modulating effects on microbiota that resulted in the change in pattern and process of spoilage in fillets.

Aeromonas shuberti as a cause of multi-organ necrosis in internal organs of Nile tilapia, Oreochromis niloticus.

A disease with white spots in internal organs of Nile tilapia occurred in Zhanjiang, southern China. Multiple, white nodules, 0.8-2.2 mm in diameter, were scattered throughout the liver, spleen and kidney of diseased fish. Signs of nodules reproduced after artificial infection with the isolated strain. Isolated bacteria were Gram-negative, facultative anaerobic, motile, short rod-shaped, with a length of 1.2-2.2 µm. Morphological and biochemical tests, as well as phylogenetic analysis, all strongly indicated that the isolate from tilapia is identical to Aeromonas schubertii (A. schubertii) which temporary named LF1708 strain. Antibiotic sensitivity assays showed the LF1708 is sensitive to 24 of 27 tested antibiotics. Pathogenicity test revealed that the isolate at the dose of 3.75 × 106 CFU/g killed 100% of experimental tilapia within 2 days and the dose of 1 × 107 CFU/g killed 100% of experimental zebrafish within 1 day. Histopathology of diseased tilapia infected with A. schubertii showed numerous necrotic lesions widely distributed in spleen, liver and kidney, and infiltration with a large number of bacteria. To our knowledge, this was the first report that associated A. schubertii with mortality in tilapia.

Effects of chitosan oligosaccharides on microbiota composition of silver carp (Hypophthalmichthys molitrix) determined by culture-dependent and independent methods during chilled storage.

This study evaluated the effects of chitosan oligosaccharides (COS) on the changes in quality and microbiota of silver carp fillets stored at 4 °C. During storage, 1% (w/v) COS treated samples maintained good quality, as evidenced by retarding sensory deterioration, inhibiting microbial growth, attenuating the production of total volatile basic nitrogen, putrescine, cadaverine and hypoxanthine, and delaying degradation of inosine monophosphate and hypoxanthine ribonucleotide. Meanwhile, variability in the predominant microbiota in different samples was investigated by culture-dependent and -independent methods. Based on sensory analysis, shelf-life of silver carp fillets was 4 days for the control and 6 days for COS treated samples. Meanwhile, Pseudomonas, followed by Aeromonas, Acinetobacter, and Shewanella were dominated in the control samples at day 4 and contributed to fish spoilage at day 6. However, COS inhibited the growth of Pseudomonas, Aeromonas, and Shewanella significantly. Consequently, Acinetobacter followed by Pseudomonas became the predominant microbiota in COS treated samples at day 6. With the growth of Pseudomonas, COS treated samples were spoiled at day 8. Therefore, COS improved the quality of fillets and prolonged the shelf life of silver carp fillets by 2 days during chilled storage, which was mainly due to their modulating effects on microbiota.

Environmental biodegradation of halophenols by activated sludge from two different sewage treatment plants.

Halophenols make a group of aromatic compounds that are resistible to biodegradation by environmental microorganisms. In this study, the biodegradation of 4-bromo-, 4-chloro- and 4-fluorophenols was studied with two types of activated sludges (from a small rural plant and from a bigger municipal plant) as an inoculum. Because of their wide use, surfactants are present in the wastewater and inhibitors enhance the biodegradation of different pollutants; the influence of natural surfactants on halophenols' biodegradation was also tested. Both types of activated sludge contained bacterial strains which were active in the halophenols' biodegradation process. The coexistence of surfactants and halophenols in the wastewater does not prevent microorganisms from effective halophenols' biodegradation. Moreover, surfactants can enhance the effectiveness of halophenols' removal from the environment. Different cell surface modifications of two isolated bacterial strains were observed in the same system of halophenols with or without surfactants. Halophenols and surfactants may also induce changes in bacteria cell surface properties.

Characterization of the microbiota in lightly salted bighead carp (Aristichthys nobilis) fillets stored at 4 °C.

The microbiota of unsalted and salted (dry-cured with 2% salt) bighead carp (Aristichthys nobilis) fillets during storage at 4 °C were identified by 16S rRNA gene analysis. Eleven genera were present in the initial microbiota of bighead carp fillets, where Acinetobacter, Aeromonas and Kocuria were the dominant bacteria. As storage time progressed, the microbial composition of both unsalted and salted fillets became less diverse. Additionally, differences in microbiota were observed between these two treatments. For unsalted bighead carp fillets, Aeromonas became the dominant genus at the end of storage and Pseudomonas was found less commonly. For salted fillets, Pseudomonas was the only bacteria identified at the end of storage.

Long-Term Bacterial Dynamics in a Full-Scale Drinking Water Distribution System.

Large seasonal variations in microbial drinking water quality can occur in distribution networks, but are often not taken into account when evaluating results from short-term water sampling campaigns. Temporal dynamics in bacterial community characteristics were investigated during a two-year drinking water monitoring campaign in a full-scale distribution system operating without detectable disinfectant residual. A total of 368 water samples were collected on a biweekly basis at the water treatment plant (WTP) effluent and at one fixed location in the drinking water distribution network (NET). The samples were analysed for heterotrophic plate counts (HPC), Aeromonas plate counts, adenosine-tri-phosphate (ATP) concentrations, and flow cytometric (FCM) total and intact cell counts (TCC, ICC), water temperature, pH, conductivity, total organic carbon (TOC) and assimilable organic carbon (AOC). Multivariate analysis of the large dataset was performed to explore correlative trends between microbial and environmental parameters. The WTP effluent displayed considerable seasonal variations in TCC (from 90 × 103 cells mL-1 in winter time up to 455 × 103 cells mL-1 in summer time) and in bacterial ATP concentrations (<1-3.6 ng L-1), which were congruent with water temperature variations. These fluctuations were not detected with HPC and Aeromonas counts. The water in the network was predominantly influenced by the characteristics of the WTP effluent. The increase in ICC between the WTP effluent and the network sampling location was small (34 × 103 cells mL-1 on average) compared to seasonal fluctuations in ICC in the WTP effluent. Interestingly, the extent of bacterial growth in the NET was inversely correlated to AOC concentrations in the WTP effluent (Pearson's correlation factor r = -0.35), and positively correlated with water temperature (r = 0.49). Collecting a large dataset at high frequency over a two year period enabled the characterization of previously undocumented seasonal dynamics in the distribution network. Moreover, high-resolution FCM data enabled prediction of bacterial cell concentrations at specific water temperatures and time of year. The study highlights the need to systematically assess temporal fluctuations in parallel to spatial dynamics for individual drinking water distribution systems.

High Prevalence of Plasmid-Mediated Quinolone Resistance and IncQ Plasmids Carrying qnrS2 Gene in Bacteria from Rivers near Hospitals and Aquaculture in China.

Effluents from hospital and aquaculture are considered important sources of quinolone resistance. However, little information is available on the impact of this effluent on nearby rivers. In this study, 188 ciprofloxacin-resistant bacterial isolates obtained from rivers near hospitals and aquaculture were screened for plasmid-mediated quinolone resistance (PMQR) genes. Species identification, antibiotic susceptibility testing, and PMQR gene transferability assessment were conducted for PMQR-positive bacteria. Representative qnrS2-encoding plasmids were subsequently sequenced using a primer-walking approach. In total, 44 isolates (23.4%) were positive for qnr genes (16 qnrB2, 3 qnrS1, and 25 qnrS2) and 32 isolates (17.0%) were positive for aac(6')-Ib-cr. Other PMQR genes were not detected. The qnrB2 and aac(6')-Ib-cr genes had a higher prevalence in aquaculture samples than in hospital samples, and were significantly associated with Enterobacteriaceae (p < 0.05). In contrast, the prevalence of qnrS2 was not site-related, but was significantly associated with Aeromonas spp. (p < 0.05). All PMQR isolates were resistant to three or more classes of antibiotics. Eleven qnrS2-harboring plasmids from Aeromonas spp., including a novel conjugative plasmid pHP18, were selected for sequencing. These plasmids were small in size (6,388-16,197 bp) and belonged to the IncQ or IncU plasmid family, with qnrS2 being part of a mobile insertion cassette. Taken together, our findings suggest that aquaculture is a possible source for aac(6')-Ib-cr and qnrB2 dissemination, and demonstrate the ubiquity of qnrS2 in aquatic environments. Finally, Aeromonas spp. served as vectors for qnrS2 with the help of IncQ-type plasmids.

Evaluation of different conditions and culture media for the recovery of Aeromonas spp. from water and shellfish samples.

AIMS: To perform a comparative study for determining the optimum culture method (direct plating or enrichment) and medium (ampicillin dextrin agar (ADA), starch ampicillin agar (SAA), bile salts irgasan brilliant green modified (BIBG-m)) for recovering Aeromonas species from water and shellfish samples. METHODS AND RESULTS: By direct culture, Aeromonas was detected in 65% (13/20) of the water samples and in 54·5% (6/11) of the shellfish samples. However, when a pre-enrichment step was included, the number of positive water samples increased to 75% (15/20) and the ones of shellfish to 90·1% (10/11). The enriched culture significantly favoured (P < 0·05) the isolation of Aeromonas allosaccharophila from water, Aeromonas salmonicida from shellfish and Aeromonas caviae from both types of samples. The most specific (P < 0·05) culture medium for detecting Aeromonas from water was ADA. However, no differences were observed in the case of shellfish samples (P > 0·05). Isolation of Aeromonas media from water was favoured (P < 0·05) in the ADA medium, while SAA enhanced (P < 0·05) the isolation of Aer. salmonicida from shellfish. CONCLUSIONS: The culture method and medium used influenced the recovery of some Aeromonas species from water and shellfish samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This fact should be considered in future prevalence studies to avoid overestimating the above mentioned Aeromonas species.

Quality Changes and Biogenic Amines Accumulation of Black Carp (Mylopharyngodon piceus) Fillets Stored at Different Temperatures.

Postmortem quality changes of black carp (Mylopharyngodon piceus) fillets stored at 20, 4, and 0°C (in ice) were determined in terms of pH value, K value, total volatile basic nitrogen, free amino acids, biogenic amines, drip loss, electrical conductivity (EC), sensory score, and microbial growth. The results showed that black carp fillets could maintain a good quality for 2, 9, and 12 days when stored at 20, 4, and 0°C, respectively. Pseudomonads, Aeromonas, and Enterobacteriaceae were the main spoilage bacteria in black carp. Tryptamine, 2-phenylethylamine, putrescine, cadaverine, and tyramine increased significantly (P < 0.05) during storage at the three temperatures, but not spermidine and spermine, among which tyramine and putrescine were the main biogenic amines in black carp fillets. A significantly higher concentration of histamine (132.05 mg/kg on the third day) was detected in the samples stored at 20°C (P < 0.01) than at 4 and 0°C (0.62 to 3.28 mg/kg) throughout storage, indicating storage of samples at 20°C favored the formation of histamine. The accumulations of tyramine, cadaverine, and histamine were highly correlated with the productions of tyrosine, lysine, and histidine, respectively. Correlations between EC and sensory, physical, chemical, and microbial parameters at the three storage temperatures showed that EC could be used as a better quality indicator to assess the overall quality of fish stored at 4 and 0°C (low temperature) than at 20°C.

Characterization of N-acyl homoserine lactones (AHLs) producing bacteria isolated from vacuum-packaged refrigerated turbot (Scophthalmus maximus) and possible influence of exogenous AHLs on bacterial phenotype.

Quorum sensing (QS) is a cell-to-cell communication mechanism through which microbial cells communicate and regulate their wide variety of biological activities. N-acyl homoserine lactones (AHLs) are considered to be the most important QS signaling molecules produced by several Gram-negative bacteria. The present study aimed to screen the AHLs-producing bacteria from spoiled vacuum-packaged refrigerated turbot (Scophthalmus maximus) by biosensor assays, and the profiles of AHLs produced by these bacteria were determined using reversed-phase thin-layer chromatography (RP-TLC) and gas chromatography-mass spectrometry (GC-MS). Effects of exogenous AHLs and QS inhibitor (QSI) on the phenotypes (i.e., extracellular proteolytic activity and biofilm formation) of the AHLs-producing bacteria were also evaluated. Our results demonstrated that eight out of twenty-two isolates were found to produce AHLs. Three of the AHLs-producing isolates were identified as Serratia sp., and the other five were found to belong to the family of Aeromonas. Two isolates (i.e., S. liquefaciens A2 and A. sobria B1) with higher AHLs-producing activities were selected for further studies. Mainly, RP-TLC and GC-MS analysis revealed three AHLs, i.e., 3-oxo-C6-HSL, C8-HSL and C10-HSL were produced by S. liquefaciens A2, while five AHLs, i.e., C4-HSL, C6-HSL, C8-HSL, C10-HSL, and C12-HSL, were produced by A. sobria B1. Moreover, production of AHLs in both bacterial strains were found to be density-dependent, and the AHLs activity reached a maximum level in their middle logarithmic phase and decreased in the stationary phase. The addition of exogenous AHLs and QSI decreased the specific protease activity both of the Serratia A2 and Aeromonas B1. Exogenous AHLs inhibited the biofilm formation of Serratia A2 while it enhanced the biofilm formation in Aeromonas B1. QSI inhibited the specific protease activity and biofilm formation in both bacterial strains.