Archives of Clinical Microbiology

Keywords

ESBL; E. coli; Pseudomonas spp.; Mackerel frozen fishes; Resistance

Introduction

Over the past few decades, research has shown that the nutrients and minerals in seafood such as fish can bring about a possible change in the development and reproduction of brain and has emphasized the role of seafood in the function of the human body. Fish is also a source of vitamin A, and is needed for healthy skin and clear eyes, and vitamin D, which is needed to help the body absorb calcium for strong bones and teeth. Products from fishery, which are of enormous relevance for the nutrition of humans globally and contribute fairly to well-being can act as a source of food-borne pathogens and may be a possible source of infection [1]. The bacterial flora of marine fish, debris and sea water has been analyzed all over the globe and diseases caused by bacteria are mainly due to contaminated water and sea foods. The largest groups of pathogenic bacteria in fish are Gram-negative, aerobic and/or facultative anaerobic bacteria. The most common treatment for bacterial infections is the beta-lactam antimicrobial agents. The persistent exposure of bacterial strains to a multitude of beta-lactams has induced dynamic and continuous production and mutation of β- lactamases in these bacteria, expanding their activity even against the newly developed β-lactam antibiotics. These enzymes are known as extended-spectrum beta-lactamases (ESBLs) [2]. Extended-spectrum beta-lactamases (ESBL) are enzymes that confer resistance to most beta-lactam antibiotics, including 2nd and 3rd generation cephalosporins, penicillins, and the monobactam (aztreonam). Infections with ESBLproducing organisms have been associated with poor outcomes. These enzymes (Extended Spectrum Beta-lactamases) open the beta-lactam ring, making the antibiotics to be inactive [3]. Treatment of infectious diseases has become a worldwide problem due to the resistance of pathogenic organisms to antibiotics. There is an increase use/misuse of antibiotics in human medicine, agriculture and veterinary which are contributing to it. There is heightened increase of antibiotic resistance in bacteria that cause either community infections or nosocomial infections; of particular interest are the multidrug resistant pathogens, e.g. Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae, vancomycin-resistance Enterococcus, and extensive drugresistant Mycobacterium tuberculosis [4]. This study is therefore designed to isolate, phenotypically characterize, and determine the antibiotic resistance patterns of extended spectrum betalactamase (ESBL)-producing Escherichia coli and Pseudomonas species from retail imported mackerel frozen fishes sold in Abakaliki metropolis.

Materials and Methods

Study area

This study was conducted in Abakpa and Kpirikpiri market area in Abakaliki, the state capital of Ebonyi State. Ebonyi State is located in the South Eastern part of Nigeria. It is bounded by Enugu State in the West, Cross River in the East, Abia state in the South, and Benue state in the North. It is between longitude 7°30’ and latitude 60°45’ E. In the 2006 population and housing census, Ebonyi State had an estimated population of 2.3 million people.

Sample collection

One hundred samples of mackerel frozen fishes were collected from various markets in Abakaliki, using sterile containers. The samples were labeled accordingly and transported within 2 hr in an ice-packed container to microbiology laboratory unit of Ebonyi State University, Abakaliki for bacteriological analysis.

Sample preparation, culturing, and identification of isolates

Mackerel frozen fish preparations were made by cutting fish sample from the gill and intestine region with a sterile knife. The cut samples were crushed in a sterile mortar using pestle. From the crushed sample, one ml volume was measured out and homogenized in a clean, dry sterile test tube containing nine milliliters of peptone water. Preparation of serial dilutions was done and the crushed sample was diluted from 101 to 1010 for each fish sample [1]. The diluted crushed frozen fish samples were incubated at 37°C for 18-24 h. Turbid tubes with signify growth was cultured on eosin methylene blue (EMB) agar and MacConkey agar for the isolation of E. coli, and cetrimide agar for the isolation of Pseudomonas spp. The isolates were further identified and characterized using standard microbiological techniques [5].

Preliminary determination of ESBL-producing E. coli

All the bacterial pathogens (E. coli and Pseudomonas spp) were screened for the production of ESBL. Single antibiotic discs containing cefotaxime (30 μg), ceftazidime (30 μg), cefepime (30 μg), and aztreonam (30 μg) were placed aseptically at a distance of 30 mm apart on a Mueller-Hinton (MH) agar (Oxoid, UK) plates that was previously inoculated with standardized inoculums of the test bacteria. The plate was allowed for 30 minutes for pre-diffusion of antibiotics and was incubated for 18-24 h at 37°C. After the incubation, zones of inhibition were measured in millimeter using a metre rule and recorded. ESBL production was suspected if any of the test bacteria showed reduced susceptibility or is resistant to any of the antibiotics used for the screening studies according to the CLSI guidelines [6].

Double disk synergy test (DDST)

ESBL production was confirmed on the bacterial isolates by double disk synergy test [6]. DDST was performed as a standard disk diffusion assay on Mueller-Hilton (MH) agar (Oxoid, UK) plates in line with CLSI criteria [7]. Sterile swab sticks were dipped into bacterial suspension (standardized to 0.5 McFarland turbidity equivalent) with reduced susceptibility to the 2nd and 3rd generation cephalosporins. Antibiotic discs of amoxicillinclavulanic acid (30 μg) was placed at the center of the MH agar plate, and antibiotic discs containing cefotaxime (30 μg) and ceftazidime (30 μg) was each placed at a distance of 15 mm (centre to centre) from the central disk, amoxycillin/clavulanic acid (30 μg). The plate was incubated at 37°C for 18-24 h. A zone of inhibition ≥5 mm in diameter of either cefotaxime and ceftazidime tested in combination with amoxicillin/clavulanic acid against its zone when tested alone confirms ESBL production phenotypically [6,8].

Antimicrobial susceptibility test

Antimicrobial susceptibility test of the ESBL-producing E. coli isolates was done using the standard Kirby-Bauer disk diffusion [9]. The ESBL-producing inoculum was prepared by suspending the freshly grown bacteria in 5 ml sterile nutrient broth and its turbidity adjusted to 0.5 McFarland standards. The antimicrobial susceptibility testing was performed on Mueller-Hinton agar using the following antibiotics; amoxicilin (AMC, 10μg), cefepime (FEP, 30 μg), cefuroxine sodium (CXM, 30 μg), ceftriazone (CRO, 30 μg), ciprofloxacin (CIP, 5 μg), chloramphenicol (C, 30 μg), ticarcilline/clavanic acid (TIM, 85 μg), piperacillin (PRL, 100 μg) and tobramycin (TOB, 10 μg).The plates were incubated aerobically at 37°C for 18-24 h. The zones of inhibition were measured with a metre rule and the results were recorded and interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [7-12].

Determination of multiple-antibiotic resistance index (MARI)

This was calculated using the method described by Chitanand et al. [10]:

MARI=a/b

Where, a=Total number of resistance scored; b=Total number of antibiotics tested.

Results and Discussion

Mackerel fishes can be exposed to pathogenic microorganisms during, before and after harvest; also during packaging, transportation, storage, and selling by handlers and consumers which in all is a health hazard to the consumer/public health. Infection of mackerel fishes by microbes may be as a result of human or animal wastes inundated with antibiotic resistant bacteria in the pond where these fishes are reared. This study reports the presence of Pseudomonas spp. and ESBLproducing E. coli from retail imported mackerel frozen fishes sold in Abakaliki metropolis, Ebonyi state Nigeria. A total of 100 mackerel frozen fish samples were collected from two selected markets (Abakpa and kpirikpiri) in Abakaliki and were analyzed for the isolation of extended spectrum beta-lactamaseproducing E. coli and Pseudomonas spp. Out of the 100 mackerel frozen fishes sampled from the two markets; a total of 69 Pseudomonas spp. and 21 E. coli were isolated (Tables 1 and 2). The result of ESBL screening showed that all the Pseudomonas spp. isolated were ESBL-negative while 7 (33%) out of the 21 E. coli isolated were ESBL-positive (Tables 3-6). Four (57.1%) out of the 7 E. coli isolates from Abakpa market screened for confirmatory ESBL-production were positive while 3 (75%) were positive from the 4 E. coli isolates from kpirikpiri market (Tables 7 and 8).

No. of samples	No. of Isolate	Prevalence of isolate in samples (%)
60 from Abakpa	44 (63.8%)	73
40 from Kpirikpiri	25 (36.2%)	63

Table 1: Frequency of Pseudomonas species in mackerel frozen fish samples.

No. of samples	No. of Isolate	Prevalence of isolate in samples (%)
60 from Abakpa	13 (61.9%)	22
40 from Kpirikpiri	8 (38.1%)	20

Table 2: Frequency of E. coli in mackerel frozen fish samples.

Antibiotics used	Total number of isolate	Sensitivity (N (%))	Resistance (N (%))
Cefepime	44	44 (100)	0 (0)
Aztreonam	44	44 (100)	0 (0)
Cefotaxine	44	44 (100)	0 (0)
Ceftazidime	44	44 (100)	0 (0)

Table 3: Preliminary screening for ESBL production by Pseudomonas spp. isolated from mackerel frozen fishes sold in Abakpa market.

Antibiotics used	Total number of isolate	Sensitivity (N (%))	Resistance (N (%))
Cefepime	25	25 (100)	0 (0)
Aztreonam	25	25 (100)	0 (0)
Cefotaxine	25	25 (100)	0 (0)
Ceftazidime	25	25 (100)	0 (0)

Table 4: Preliminary screening for ESBL production by Pseudomonas spp. isolated from mackerel frozen fishes sold in Kpirikpiri market.

Antibiotics used	Total number of isolate	Sensitivity (N (%))	Resistance (N (%))
Cefepime	13	7 (54)	6 (46)
Aztreonam	13	10 (77)	3 (23)
Cefotaxime	13	6 (46)	7 (54)
Ceftazidime	13	8 (62)	5 (38)

Table 5: Preliminary screening for ESBL production by E. coli isolated from mackerel frozen fishes sold in Abakpa market.

Antibiotics used	Total number of isolate	Sensitivity (N (%))	Resistance (N (%))
Cefepime	8	4 (50.0)	4 (50.0)
Aztreonam	8	7 (87.5)	1 (12.5)
Cefotaxine	8	4 (50.0)	4 (50.0)
Ceftazidime	8	5 (62.5)	3 (27.5

Table 6: Preliminary screening for ESBL production by E. coli isolated from mackerel frozen fishes sold in Kpirikpiri market.

No. of isolates screened	ESBL-ve (N)	ESBL+ve (N)
7	3 (42.9%)	4 (57.1%)

Table 7: Confirmatory screening for ESBL-producing E. coli isolated from mackerel frozen fishes sold in Abakpa market.

No. of isolates screened	ESBL -ve (N)	ESBL +ve (N)
4	1 (25%)	3 (75%)

Table 8: Confirmatory screening for ESBL-producing E. coli isolated from mackerel frozen fishes sold in Kpirikpiri market.

Our result on the isolation of ESBL-producing Escherichia coli in this study is in agreement with the work carried out by Elhadi et al. [6] in Saudi Arabia who reported the presence of ESBLproducing E. coli in mackerel fishes. He further showed that mackerel fishes might be the possible reservoirs for ESBLproducing E. coli and may contribute to the dissemination and transfer of beta-lactamasegenes to humans through the food chain. ESBL-producing E. coli has also been reported in fishes by Sivaraman et al. [13] in India.

Our study is also in concord with the work of Eze et al. [7] in Nsukka, Nigeria who reported that frozen mackerel fish could be contaminated by a lot of microorganisms which include Escherichia coli and Pseudomonas species. Our result on antimicrobial susceptibility test showed that the isolated ESBLproducing E. coli from the two markets exhibited varying levels of susceptibilities and resistances to the test antibiotics. The ESBL-producing E. coli isolates from Abakpa market were completely resistant (100%) to ceftriaxone, chloramphenicol, amoxicillin, cefuroxine sodium, ticarcillin/clavanic acid, cefepime, and piperacillin (Table 9).

S/N	Antibiotics	Total number of isolate	Sensitivity (N (%))	Resistance (N (%))
1	Ceftriaxone	4	0 (0)	4 (100)
2	Chloramphenicol	4	0 (0)	4 (100)
3	Tobramycin	4	2 (50)	2 (50)
4	Ciprofloxacin	4	4 (100)	0 (0)
5	Amoxicillin	4	0 (0)	4 (100)
6	Cefuroxin sodium	4	0 (0)	4 (100)
7	Ticarcillin/clavanic acid	4	0 (0)	4 (100)
8	Cefepime	4	0 (0)	4 (100)
9	Piperacine	4	0 (0)	4 (100)

Table 9: Antibiotic Susceptibility Patterns of ESBL-Producing E. coli isolated from mackerel frozen fishes sold in Abakpa market.

They also showed resistance to tobramycin (50%) but were completely susceptibility (100%) to ciprofloxacin. This is in agreement with the report of Elhadi et al. [6] who in their work reported that the least frequency of resistance was observed for ciprofloxacin while the highest frequency of resistance was observed for piperacillin and ceftriazone. The ESBL-producing E. coli isolates from kpirikpiri market were completely resistant (100%) to ceftriaxone, amoxicillin, cefuroxime sodium, ticarcillin/clavanic acid, cefepime, and piperacillin and strangely to ciprofloxacin (Tables 10-12). Some level of susceptibility (33%) was observed for chloramphenicol and tobramycin. The result of the multiple antibioticresistance index (MARI) showed that the isolated ESBL-producing E. coli in our study had an average MARI of 0.84. This depicts the multi-drug resistance traits of the ESBL-producing E. coli isolates as they were resistant to at least two different classes of antibiotics such as the carbapenems, penicillins, aminoglycosides, and fluoroquinolones.

S/N	Antibiotics	Total number of isolate	Sensitivity (N (%))	Resistance (N (%))
1	Ceftriaxone	3	0 (0)	3 (100)
2	Chloramphenicol	3	1 (33)	2 (67)
3	Tobramycin	3	1 (33)	2 (67)
4	Ciprofloxacin	3	0 (0)	3 (100)
5	Amoxicillin	3	0 (0)	3 (100)
6	Cefuroxin sodium	3	0 (0)	3 (100)
7	Ticarcillin/clavanic acid	3	0 (0)	3 (100)
8	Cefepime	3	0 (0)	3 (100)
9	Piperacine	3	0 (0)	3 (100)

Table 10: Antibiotic Susceptibility patterns of ESBL-producing E. coli isolated from mackerel frozen fishes sold in Kpirikpiri market.

S/N	Organism	MARI Value
1	E. coli	0.89
2	E. coli	0.89
3	E. coli	0.78
4	E. coli	0.89
Total		3.45; Average=0.86

Table 11: MARI values for the ESBL-producing E. coli strains isolated from mackerel frozen fishes sold in Abakpa market.

S/N	Organism	MARI Value
1	E. coli	0.89
2	E. coli	0.67
3	E. coli	0.89
Total		2.45; Average=0.82

Table 12: MARI values for the ESBL-producing E. coli strains isolated from mackerel frozen fishes sold in Kpirikpiri market

Conclusion

The results of our study have shown that some frozen mackerel fishes are heavily contaminated with Pseudomonas spp. and ESBL-producing E. coli pathogens that can cause infections in humans via the consumption of undercooked or illprocessed fishes mostly those who consume the gills and intestine of these fishes. Those who cut fishes at home for preparation and fish sellers in the market, who often cut these fishes for their buyers without washing their hands properly, can serve as potential reservoirs for the transmission of these pathogens which eventually leads to serious public health consequences. It is therefore imperative that strict hygienic practices be maintained when handling fishes for consumption. Also, proper monitoring and evaluation of environment, especially where fishes are sold, is very important to ensure that their wastes are properly disposed. Concerted effort to monitor the storage conditions of these imported fishes is also vital to curb or reduce the spread of food-borne illnesses/diseases.

Conflict of Interest Statement

The authors declare that there are no conflicts of interest.

Funding

This work was supported by the Russian Science Foundation (Grant # 18-15-00254).

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