The Occurrence of Bacteria of the Genus Aeromonas spp . in Oreochromis niloticus ( Tilapia ) and in the Water of Amateur Sport Fish Ponds and Sensitiveness to Antimicrobials

Since amateur fishing in fishponds has been on the increase in Brazil, there is a great concern on the microbiological quality of fish. One hundred and thirty-eight samples were collected during the dry and rainy periods in ten fish farms. Aeromonas spp. counts and tests for sensitiveness to antimicrobials were performed, coupled to the physical and chemical analyses of water. Tests revealed that 70% of samples were contaminated by Aeromonas spp., with water averaging 2.92 Log CFU/100 mL during the rainy period and 3.16 Log CFU/100 mL during the dry one. Fish contaminated by Aeromonas spp. averaged 2.58 Log CFU/100 mL during the rainy period and 3.53 Log CFU/100 mL during the dry one. Aeromonas spp. samples were multi-resistant to 2 or 8 antimicrobials in 62.5% of the samples. Ampicillin was the antimicrobial with the highest resistance percentage rate. Results showed that fish bred in amateur fish farms constituted a health risk for the population.


Introduction
Fish farming is currently one of the most relevant food sources, with high growth rates.Fish production has increased significantly in Brazil and worldwide.The wave of amateur and recreational fishing on fish farms is definitely on the increase, with the Nile tilapia featuring the most farmed fresh water fish [1] A. D. S. Silva et al.According to IBAMA Ordinance 136/1998, a fish farm may be defined as a company which establishes fish tanks or ponds for the commercial exploitation of amateur fishing [2].
Fish farming is a profitable activity in the country.However, several environmental issues, such as an increase in the population growth of specific bacteria of natural water microbiota, wastes from feeding, and a possible proliferation of microorganisms resistant to antimicrobials used indiscriminately, have been detected.Several bacteria related to fish pathology have been reported, among which may be mentioned Aeromonas spp., a pathogenic bacterium harmful to fish and humans [3].
Aeromonas spp.strains are renowned for their increased capacity to acquire and exchange resistance genes with antimicrobials.In fact, there is a strong relationship between aquaculture, Aeromonas spp.diversity and resistance to antimicrobials.Robust link clues exist between prophylactic and systemic use of antimicrobials in aquaculture and the spread of resistance against antimicrobials [4].
The genus Aeromonas has pathogenic strains for aquatic animals.They have been pinpointed in the literature as the main etiological agents, causing infection in fish and jeopardizing other aquatic animals, with great economic loss for Brazilian fish farms.Several bacteria of the genus have been identified as pathogenic to public health.Epidemiological data have shown that food consumption, contact with and intake of contaminated water are the main transmission means of the pathogen and are described as the main etiological agents in human gastroenteritis [5] [6].However, ANVISA's RDC 12/2001 does not establish any limits for the microorganism in food [7] Aeromonas spp.has been described as an opportunist pathogen worldwide, especially in developing countries, associated with traveller's diarrhea.Although these pathogens mainly inhabit aquatic environments, they may be found in animals, and have been related to several human and animal infections [8].Reports on human contamination by several species, particularly Aeromonas hydrophila, A. caviae and A. veronii biovar sobria, are extant worldwide.The former is associated to watery diarrhea and dysentery [9].New infection cases have been reported in 2010, caused by wounds and by gastroenteritis, associated to two new species, A. sanarellii and A. taiwanensis [10].
It has been proved that bacteria of the genus Aeromonas are pathogenic microorganisms which harm humans and animals.Their importance to public health is due to their high virulence and resistance to antimicrobials, observed in an increasing number of cases, in Brazil and worldwide, underscoring the occurrence of cases of gastroenteritis caused by the microorganisms, vectored by contaminated food and water.Since few research works on Aeromonas have been undertaken in amateur fishing ponds in the state of Bahia, Brazil, studies on the occurrence of Aeromonas spp.and their sensitiveness to antimicrobial agents in water and in fish retrieved from fishing ponds in the towns of Amélia Rodrigues, Ubaíra, Amargosa, São Felipe, Conceição do Almeida, Dom Macedo

Materials and Methods
The amateur sporting fish ponds selected for current research lie in the towns of Amélia Rodrigues, Amargosa, São Felipe, Conceição do Almeida, Dom Macedo Costa, Santo Antônio de Jesus, Ubaíra and in the island of Itaparica, all in the state of Bahia, Brazil.Main criterion for selection consisted of fishing ponds with Oreochromis niloticus adults since the Nile tilapia is the most farmed fresh water fish in the state of Bahia [1].
Sample collection occurred between March and November 2017 during two periods, namely, the dry and rainy periods.For this purpose, 500-mL water samples from the water surface were collected from four equidistant sites of each pond, in up to three ponds in each fish farm, totaling 48 samples, from 60 fishing ponds.Sterilized flasks were dipped in the water, opened, filled with water, conditioned in isothermal boxes with ice, and transported to the Laboratory of Animal Parasitology and Microbiology of the Universidade Federal do Recôncavo da Bahia.Microorganisms were identified and counted within 24 h of incubation within a single stage.Two fish samples, collected weekly, were retrieved from up to three ponds, totaling 90 fish samples.Fish were caught by fishing lines or nets used on the fish farms.They were slaughtered by thermal shock (thermonarcosis) [11] and samples were conditioned in refrigerated thermal boxes and transported to the laboratory.
The fish were eviscerated and 25 g of each sample were weighed, to which were added 225 mL of peptone water 0.1% for the first dilution.Serial dilutions were performed until dilution 10 −6 [9].The fish were prepared for processing following methodologies by Silva et al. [12] and Apha [13].All procedures were undertaken for each sample retrieved from different fish farms.
Aeromonas Isolation Agar (Base), supplemented by Aeromonas Selective Supplement, was employed to analyze Aeromonas spp.Since the medium contains ampicillin, the former becomes selective.Ampicillin is employed for the selective and differential isolation of Aeromonas hydrophila from clinical and environmental samples.In fact, it is the medium employed for fast and simultaneous identification of the species Aeromonas, Plesiomonas, Proteus and Enterobacteriaceae, or rather, it is employed as an overall medium to investigate en- teric diseases.
The pour plate method was employed to count the microorganisms Aeromonas spp. [9] [12].Serial dilution, up to dilution 10 4 , was performed after the preparation of the sample.The incubation process occurred posterior to serial dilution.Plating was done in triplicate.Plates were inverted for incubation and colonies with a dark green color, opaque, featuring dark hubs, were positive for Aeromonas ssp.Aeromonas spp.isolates were counted with a colony counter (Phoenix counter CE 550A) from plates with 25 -250 colonies [9] [15].

Results
Table 1 shows results of colony forming units in Log CFU/100mL of contamination by Aeromonas spp. in fish pond water under analysis.Forty-eight samples were contaminated by Aeromonas spp., ranging between 1.17 and 4.  Further, pH rates ranged between 5.00 and 7.11, mean 6.07 during the rainy season and 6.04 during the dry season.Consequently, 87% (42/48) of samples complied with standards.Color also complied with current legislation, whilst DO reached 87.5% (43/48) of samples in the two periods and failed to comply with legislation.In current analysis, BOD reached 93.8% (45/48) of samples, or rather, it failed to comply with limits (up to 5 mg/L O 2 ) established by CONAMA [16].
ANOVA statistical assessment showed a possible significant variation among  1).After the elaboration of a matrix for Spearman's correlation, parameters with significance p ≤ 0.01 and p ≤ 0.05 were chosen.Figure 2 shows variations of each parameter.

Discussion
Fish have an important role in human diet and a per capita rise in fish consump-    researchers when they were collecting the samples.Contamination also occurs due to cesspits and sewerage issues from small inns on the farms and other similar types of human activity lacking a sewage system.Data have been corroborated by other researches which also described contamination of fish ponds by Aeromonas spp.[9] [19] [20].
Water contamination is particularly high during the rainy season and alters the physical and chemical characteristics of water and the microbiological quality of fish.Increase in rain water and a greater load of dirt in the water are the causes of high pathogen rates.Natural factors, such as rock dissolution, photosynthesis and household sewerage affect pH.Increase in rain volume causes a rise in pH through the dilution of dissolved compounds [21].
There has been an increase in the number of contamination cases in animal-derived food by Aeromonas spp. in fresh or sea water.According to Resolution by CONAMA 375/05 [16] on the quality of fresh water for fish farming and fishing activities (class 2), thermotolerant coliforms should not exceed 1000 (3 log) in 80% or more in at least 6 samples collected during the year [16].Although official parameters for Aeromonas spp.are lacking, coliform rates in the water of the fish farms analyzed are a real public health issue [22].Health legislation in several countries determines limits in the quantity of Aeromonas spp. in drinking and mineral water.Bacteria have been frequently detected in public supply water and even in bottled mineral water [22] [23] [24].
Ribeiro et al. [25] state that limits for Aeromonas spp. in mineral water at the source and in bottled water were provisionally determined in Italy, in 1997, or rather, 10 UFC/100 mL and 100 UFC/100 mL respectively.
According to ANVISA'S RDC n. 12, published on the 2nd January 2001, the microbiological standards for "in natura" and thawed fish are: positive staphylococci coagulase/g.from 10 Salmonella spp.(absence in 25 g) [7].Although Brazilian legislation fails to give standards, high Aeromonas spp.levels indicate deficient hygiene and sanitary conditions, coupled to pathogenic bacteria with harmful species, such as Aeromonas hydrophila, A. caviae, A. veronii biovar sobria and A. hydrophila, associated with aqueous diarrhea and dysentery [9].
Data from the Ministry of Health have shown that animal-derived food, such as fish, triggers outbreaks of Food and Water Borne Diseases (FWBD).Water is the natural habitat of these bacteria and an important source of food contamination.Aeromonas spp. in food is a predominant feature in fish consumption, even though it has been studied in swine, chickens and humans.It may occur in the excrement of infected animals and sick people who handle food [24] [26] [27].Neyts et al. [28] detected Aeromonas spp. in 72% of fish and shrimps.Data corroborate those in current analysis.
It has also been perceived that the fish farms analyzed either lacked proper water management or there was no management at all, with the consequent water and fish contamination.Lack of water management may cause disease and even death in fish.Due to faulty knowledge and inadequate man power, producers employ antimicrobials indiscriminately, causing the proliferation of resistant or multi-resistant bacteria to antimicrobials [19].According to Souza [34], there is very scanty information on fish farming management and its consequences on water quality and on the health of fish in fish farms.
Aeromonas spp. is usually isolated from patients suffering from traveller's diarrhea.Hofe et al. [35] detected the pathogen as the cause of traveller's diarrhea in 18 (2%) out of 863 patients.A study on the outbreak of diarrhea in the town of São Bento do Una PE Brazil, in 2004, revealed that 114 (19.5%) out of the 2170 registered cases were caused by Aeromonas spp.[36].
There was a high positive correlation for DO and pH and for pH and BOD, which have a direct relationship with the maintenance of water life, in the case of aerobic respiration processes (as in DO) and in the maintenance of an environment that favors important chemical reactions for life (as in pH).There was also an expected positive correlation between color and turbidity, since increase in water turbidity is caused by suspended matter, such as clay, silt, organic and inorganic matter, soluble organic compounds.Increase in turbidity increases apparent color [37].
The co-relationship between pH and BOD revealed negative correlation at p ≤ 0.01, where a pH increase is a decrease of BOD in water, perhaps caused by water acidification through a fermentation process produced by bacterial growth and evidenced in current study [38]  data were also reported by other authors [40].
Positive correlations at significance level p ≤ 0.01 between Aeromonas spp. in fish and BOD revealed that the latter increases when Aeromonas spp.increases.BOD results in current assay indicate contamination in fish pond water and explain the high levels of these microorganisms in water and fish.In fact, BOD indicates organic matter in water and is defined as the concentration of dissolved oxygen required to stabilize levels of organic matter in water [41].Baron et al. [42] registered that Aeromonas spp.bacteria acquire the mechanisms of antimicrobial resistance and may accompany the dissemination of antimicrobial resistance in water.The literature has few interpretation criteria for Aeromonas spp. in antimicrobial susceptibility tests.EUCAST has no epidemiological rate for Aeromonas spp. to interpret Minimum Inhibitory Concentrations (MIC).The only interpretation criteria available are the clinical breakpoints of CLSI guidelines adapted from Enterobacteriaceae.
Several research works have shown the importance of Aeromonas spp.resistant to antimicrobials from fish farming and suggest that high multi-resistant levels indicate the possibility of horizontal diffusion of resistant genes.They also suggest that, since Aeromonas spp.are reported in the environment, the exchange of plasmids among them may be facilitated, with an increase in the frequency of multi-resistant samples [9] [43] [44].
Bacteria of the genus Aeromonas are pathogenic for humans and are a risk to their health.Results in current assay reveal that evaluated fish farms have high Aeromonas spp.contamination rates.In fact, water is an important disseminator of Aeromonas spp.Food, especially animal-derived, should be well monitored for the prevention of infections [43] [44].
Hirsch et al. [45] underscored that 43% of analyzed isolates had multiple resistance to antimicrobials (MRAM) for two or more out of the nine drugs under analysis.Data indicate imminent risk due to isolation of samples which are potentially pathogenic to humans and to the isolates' multi-resistant profile.
Aeromonas strains are known for their increasing capacity to acquire and exchange resistant genes to antimicrobials.There is a strong co-relationship between fish farming, Aeromonas diversity and resistance to antimicrobials.There are robust clues for bonds between prophylactic and systemic use of antimicrobials in fish farming and the spread of resistance against antimicrobials.In fact, the above factors may influence results since the indiscriminate use of antimicrobials has been detected in several fish farms [4] [44].
The contamination in fish and water reported in current assay is a potential infection vector by Aeromonas spp.The indiscriminate and unauthorized use of antimicrobials directly affects the development of bacteria with a multi-resistance profile against antimicrobials, such as the contamination of the environment.Aeromonas spp.are frequently isolated in animal-derived food and in fish farms with high resistance percentage to several antimicrobials of groups A, B and C, registered in current study and in others by Silva et al. [9], Peixoto et al. [27], Baron et al. [42], Hirsch et al. [45] and Nagar et al. [4].

Figure 1 .
Figure 1.(a) (b): Microbiological analyses.Mean of results for Aeromonas spp. in Log, Most Probable Number (Log MPN/100 mL) in water and fish of fish ponds on fish farms in towns of Bahia and Island of Itaparica, state of Bahia, Brazil, between March and November 2017.
greatest positive and significant correlations (p < 0.01) occurred between counts of total coliforms for fish and color and turbidity counts (r = 0.62**) and for pH and DO (r = 0.61**).The above demonstrated that a level increase or decrease of any variable triggered a proportional increase or decrease of the other.Physical and chemical factors (pH and DO) also showed a negative correlation with significance rate at p ≤ 0.01 (r = 0.47**), featuring an inversely proportional relationship.In other words, an increase in one of the variables triggered a decrease in the other.In current study, Spearman's correlations were not significant between Aeromonas spp. in the water and in fish, although there were positive correlations at significance levels p ≤ 0.01 between BOD and Aeromonas spp. in fish (r = 0.51**).Aeromonas spp.isolates from the fish ponds under analysis revealed high resistance rates against the 13 antimicrobials tested.Resistance and multi-resistance percentages against antibiotics indicate threats against human health and therefore urgency to discover alternative effective antibiotics.The sensitiveness and multi-resistance profile of 109 isolates included the evaluation of 13 antibiotics by the disc diffusion method.In fact, they represent different classes of antibiotics usually employed in the treatment of infections in animals and humans.Twenty-seven out of the 109 isolates showed resistance against 1 antimicrobial, whilst 45 isolates proved to be multi-resistant against 2 to 8 antimicrobials.The interpretation of the sensitivity profile complied with CLSI M45-A (2006) by halo measuring (Table farms (Prop), Number of samples (N), Cefalotin (CFL), Cefuroxine (CRX), Ciprofloxacin (CIP), Gentamicin (GEN), Imipenem (IPM), Sulfamethoxazole = Trimitopim (SUT), Meropenem (MER) Frequency (%) of resistant isolates (R), with intermediary resistance (I) and sensitivity (S).

Figure 3 .
Figure 3. Multi-resistance profile of Aeromonas sp.isolates of water and fish samples from fish ponds on fish farms in towns of Bahia and Island of Itaparica, state of Bahia, Brazil, between March and November 2017.

Figure 4 .
Figure 4. Multi-resistance profile of Aeromonas spp.isolates.Number of antimicrobials to which they offered resistance in water and fish samples in towns of Bahia and Island of Itaparica, state of Bahia, Brazil, between March and November 2017.

Table 1 .
Minimum and maximum rates, mean, standard deviation, analysis of variance ANOVA (AV) and Shapiro-Wilks normality test (W) for Aeromonas spp. in water and fish assessed in ponds on fish farms in towns of the state of Bahia and on the Island of Itaparica, state of Bahia, Brazil, between March and November 2017.
Results showed that 70.3% of samples had an intermediate resistance to at least one antimicrobial under analçysis (Table2 and Table 3).
bials (Figure3and Figure4).Fish farms P2, P5, P7, P8 and P9 out of the ten evaluated had a higher percentage rate of samples which were resistant to antimicrobials.Highest percentage (75%) occurred in P9.DOI: 10.4236/fns.2019.101007 Figure 2. Statistical analyses.Spearman Co-relationship of Colony Forming Units (Log CFU) in water and fish from fish ponds on fish farms in towns of Bahia and Island of Itaparica, state of Bahia, Brazil, between March and November 2017.Table2.Antibiotics tested-frequency of resistant isolates, with intermediary resistance and sensitive (%) in water and fish samples in towns of Bahia and Island of Itaparica, state of Bahia, Brazil, between March and November 2017.

Table 3 .
Continuation: Antibiotics tested-frequency of resistant isolates, with intermediary resistance and sensitive (%), in water and fish samples in towns of Bahia and Island of Itaparica, state of Bahia, Brazil, between March and November 2017.
tion has been reported in Brazil and worldwide.Nevertheless, intensive industrial and agricultural growth may contaminate natural and artificial aquatic environments.It may affect not only the health of fish but triggers concern on food safety when fish is consumed by humans.It is a well-known fact that fish and DOI: 10.4236/fns.2019.101007 3NMP/100 ml −1 (maximum 3 Log CFU/g) and