Drug resistance, AmpC-β-lactamase and extended-spectrum β-lactamase-producing Enterobacteriaceae isolated from fish and shrimp

Marília Viana Albuquerque de Almeida Ítalo Mendes Cangussú Antonia Leonadia Siqueira de Carvalho Izabelly Linhares Ponte Brito Renata Albuquerque Costa About the authors

ABSTRACT

The present study aims to detect the production of extended-spectrum beta-lactamases (ESBL) by enterobacteria isolated from samples of fresh shrimp and fish obtained from the retail trade of the city of Sobral, Ceará State, Brazil. All bacterial isolates were submitted to identification and antimicrobial susceptibility testing using aminopenicillin, beta-lactamase inhibitors, carbapenem, 1st, 2nd, 3rd and 4th generation cephalosporins, and monobactam. Three types of beta-lactamases - ESBL, AmpC and KPC - were investigated. 103 strains were identified, and the most frequent species in shrimp and fish samples was Enterobacter cloacae (n = 54). All the strains were resistant to penicillin and more than 50% of the isolates were resistant to ampicillin and cephalothin. Resistance to three 3rd generation cephalosporins (cefotaxime, ceftriaxone and ceftazidime) and one fourth generation cephalosporin (cefepime) was detected in two isolates of E. cloacae from shrimp samples. Phenotypic detection of AmpC was confirmed in seven strains. The ESBL was detected in two strains of E. cloacae from shrimp samples. No strain showed KPC production. These data can be considered alarming, since food (shrimp and fish) may be carriers of enterobacteria resistant to drugs of clinical interest.

Enterobacter cloacae; Beta-lactam antibiotics; Food contamination; Antibiotic resistance; Food safety; Food-borne infections

INTRODUCTION

Enterobacteria are often associated with drug resistance and contamination of fish and shrimp11. Nguyen Do P, Nguyen TA, Le TH, Tran NM, Ngo TP, Dang VC, et al. Dissemination of extended-spectrum β-lactamase- and AmpC β-lactamase-producing Escherichia coli within the food distribution system of Ho Chi Minh City, Vietnam. BioMed Res Int. 2016;2016:8182096.. Food contamination with multidrug-resistant bacteria, particularly extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, is considered a potential source for the wide dissemination of ESBL-producing bacteria in communities22. Le QP, Ueda S, Nguyen TN, Dao TV, Van Hoang TA, Tran TT, et al. Characteristics of extended-spectrum β-lactamase-producing Escherichia coli in retail meats and shrimp at a local market in Vietnam. Foodborne Pathog Dis. 2015;12:719:25..

Beta-lactam antibiotics are one of the main groups used to combat Gram-negative and Gram-positive bacteria and account for 60% of the antibiotics used worldwide for treatment of infectious diseases33. Öztürk H, Ozkirimli E, Özgür A. Classification of Beta-lactamases and penicillin binding proteins using ligand-centric network models. Plos One. 2015;10:e0117874.. This group is characterized by the presence of the beta-lactam ring, which provides not only the mechanism of action, but also a low direct toxicity; since that acts on the cell wall and this is not present in human cells44. Suarez C, Gudiol F. Antibióticos betalactámicos. Enferm Infecc Microbiol Clin. 2009;27:116-29.. One of the major mechanisms of resistance presented by enterobacteria is the production of beta-lactamases. Several groups of these enzymes are widespread among almost all pathogenic species of Gram-negative bacteria. The main beta-lactamase producers are Escherichia coli, Klebsiella pneumoniae, Proteus, Providencia and Enterobacter55. Lago A, Fuentefria SR, Fuentefria DB. Enterobactérias produtoras de ESBL em Passo Fundo, Estado do Rio Grande do Sul, Brasil. Rev Soc Bras Med Trop. 2010;43:430-4.,66. Laudy AE, Osińska P, Namysłowska A, Zajac O, Tyski S. Modification of the susceptibility of Gram-negative rods producing ESBLS to beta-Lactams by the efflux phenomenon. Plos One. 2015;10:e0119997..

ESBL are encoded by genes located on chromosomes and/or mobile genetic elements such as plasmids, transposons and integrons, carrying resistance genes to other classes of antibiotics. The wide range of genes encoding the beta-lactamase are related to this mobility. The isolation of ESBL producing multi-resistant strains is a major cause of therapy failure in humans, leading to a considerable increase in morbidity of bacterial infections77. Silva KC, Lincopan N. Epidemiologia das betalactamases de espectro estendido no Brasil: impacto clínico e implicações para o agronegócio. J Bras Patol Med Lab. 2012;48:91-9..

The emergence of drug-resistant bacteria is a public health problem and threatens the effectiveness of drug treatments88. van Bijnen EM, Paget J, de Lange-de Klerk ES, den Heijer CD, Versporten A, Stobberingh EE, et al. Antibiotic exposure and other risk factors for antimicrobial resistance in nasal commensal Staphylococcus aureus: an ecological study in 8 european countries. Plos One. 2015;10:e0135094.. One of the factors contributing to the spread of this problem is the indiscriminate use of antimicrobials in animal production, which favors the selection of resistant bacteria with the potential to spread in the community through direct contact and consumption of contaminated food99. Colello R, Etcheverría AI, Di Conza JA, Gutkind GO, Padola NL. Antibiotic resistance and integrons in shiga toxin-producing Escherichia coli (STEC). Braz J Microbiol. 2015;46:1-5..

Considering that drug-resistant Enterobacteriaceae are associated with food-borne infections via seafood consumption, this study aims to detect the production of ESBLs by enterobacteria isolated from fish and shrimp.

MATERIAL AND METHODS

Enterobacteriaceae isolation

Enterobacteria were isolated from samples of farmed shrimp Litopenaeus vannamei and farmed fish Oreochromis niloticus obtained in the retail trade in the city of Sobral, Ceará State, Brazil). Shrimp (n=5) and fish (n=5) samples, consisting of 500 g each, were placed in sterile bags, packed in boxes with ice and transported to the Núcleo de Bioprospecção e Experimentação Molecular Aplicada (NUBEM) of the Instituto Superior de Teologia Aplicada (INTA Faculty). The time between the sample collection and samples processing did not exceed one hour. All samples were weighed (50 g) and each sample was inoculated in 450 mL of Lactose Broth (Difco, USA) and incubated at 35 °C for 48 h. An inoculum of Lactose Broth growth was plated on MacConkey Agar (Difco, USA) and Brilliant Green Bile Agar (Difco, USA), incubated at 35 °C for 24 h. After the incubation period, 103 colonies were isolated in order to perform morphotypes and biochemical characterization.

Morphotypes and biochemical characterization

All isolates were submitted to Gram staining and identified through their phenotypic profile using the Vitek 2 Gram-negative test card1010. Crowley E, Bird P, Fisher K, Goetz K, Boyle M, Benzinger MJ Jr, et al. Evaluation of the VITEK 2 Gram-negative (GN) microbial identification test card: collaborative study. J AOAC Int. 2012;95:778-85. (bioMérieux, France) in an automated instrument for identification (Vitek® 2).

Antibiogram

The bacterial isolates (n = 103) were maintained in Tryptone Soy Agar (Difco) until the completion of the antibiogram test. The antimicrobial susceptibility profile was performed by disk diffusion technique using Mueller-Hinton Agar (MH-Difco, USA)1111. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial disk susceptibility tests; approved standard. 11th ed. Wayne: CLSI; 2012., and the following antimicrobials disks (Laborclin, Brazil) were tested: Ampicillin 10 μg, Amoxicillin/clavulanate 20/10 μg, Aztreonam 30 μg, Cefuroxime 30 μg, Cefepime 30 μg, Cefoxitin 30 μg, Cefotaxime 30 μg, Ceftriaxone 30 μg, Cefaclor 30 μg, Cefpodoxime 10 μg, Ceftazidime 30 μg, Imipenem 10 μg, Meropenem 10 μg. All strains were diluted in 0.85% saline to obtain turbidity equivalent to McFarland scale 0.5 and aliquots were seeded with swabs on MH agar plates, with subsequent application of antibiotic disks. Plates were incubated at 35 °C and interpretation of inhibition halos was made according to the CLSI1111. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial disk susceptibility tests; approved standard. 11th ed. Wayne: CLSI; 2012..

AmpC phenotypic detection

AmpC production was confirmed by the disk approximation test and performed with isolates of the CESP group (Citrobacter spp., Enterobacter spp., Serratia spp., Providencia spp.) susceptible to cefoxitin. To perform this technique, a cefoxitin disk (30 µg) (Laborclin, Brazil) was placed in a 20 mm plate center (center to center) away from a ceftriaxone dis-k (30 µg) (Laborclin, Brazil) and ceftazidime disk (30 µg) (Laborclin, Brazil). The plate was incubated at 35 °C for 18-24 h. Cefoxitin acted as an inducer of the AmpC enzyme and the positivity was considered when the flattening halo around the ceftriaxone and/or ceftazidime disk was observed1212. Leão-Vasconcelos LS, Lima AB, Costa DM, Rocha-Vilefort LO, Oliveira AC, Gonçalves NF, et al. Enterobacteriaceae isolates from the oral cavity of workers in a brazilian oncology hospital. Rev Inst Med Trop São Paulo. 2015;57:121-7..

ESBL phenotypic detection

The phenotypic screening for ESBL production was carried out through antibiogram for isolates identified as E. coli, K. pneumoniae and K. oxytoca with five substrates (Laborclin discs, Brazil): aztreonam 30 µg, cefotaxime 30 µg, cefpodoxime 10 µg, ceftazidime 30 µg, ceftriaxone 30 µg. Strains resistant to at least one of the antimicrobial agents used in the screening were used to confirmatory test for synergism approach disk or double-disk synergism1212. Leão-Vasconcelos LS, Lima AB, Costa DM, Rocha-Vilefort LO, Oliveira AC, Gonçalves NF, et al. Enterobacteriaceae isolates from the oral cavity of workers in a brazilian oncology hospital. Rev Inst Med Trop São Paulo. 2015;57:121-7.. In this test, amoxicillin/clavulanic acid disk (20 mg/10 mg) (Laborclin, Brazil) was placed in the center of the plate and 20 mm (center to center) away from an aztreonam disk (30 µg) and a ceftazidime disk (30 mcg). The plate was incubated at 35 oC for 18-24 h. The test was considered positive when an increase or distortion of any inhibition zone of marker and amoxicillin/clavulanic acid disk1212. Leão-Vasconcelos LS, Lima AB, Costa DM, Rocha-Vilefort LO, Oliveira AC, Gonçalves NF, et al. Enterobacteriaceae isolates from the oral cavity of workers in a brazilian oncology hospital. Rev Inst Med Trop São Paulo. 2015;57:121-7..

KPC phenotypic detection

For the KPC detection test, isolates with resistance to some third-generation cephalosporins (ceftazidime, ceftriaxone or cefotaxime) and carbapenem (imipenem or meropenem) were submitted to the modified Hodge test. To carry out this test, an inoculum of E. coli ATCC 25922 was seeded on the surface of a Mueller-Hinton Agar (Difco, USA) plate and a 10-μg meropenem disk (Laborclin, Brazil) was placed in the center of the plate. Thus, three to five newly cultured (24 h) colonies from the sample were seeded from the center of the meropenem disk to the periphery of the Petri dish in order to draw an imaginary line of 20 to 25 mm. After incubation at 35 °C for 16-20 h the test was considered positive when there was growth of E. coli ATCC® at 25922 in the inhibition zone of meropenem1212. Leão-Vasconcelos LS, Lima AB, Costa DM, Rocha-Vilefort LO, Oliveira AC, Gonçalves NF, et al. Enterobacteriaceae isolates from the oral cavity of workers in a brazilian oncology hospital. Rev Inst Med Trop São Paulo. 2015;57:121-7..

RESULTS

Table 1 shows the diversity of 103 enterobacteria strains among the isolates from shrimp (L. vannamei) and fish (O. niloticus). E. cloacae (n = 54) was the most frequently isolated species from shrimp and fish samples, followed by K. pneumoniae (n = 22) and E. coli (n = 14).

Table 1
- Enterobacteria species isolated from shrimp Litopenaeus vannamei and fish Oreochromis niloticus

It was observed that all strains were resistant to penicillin and more than 50% of the isolates were resistant to ampicillin and cephalothin. Resistance to third (cefotaxime, ceftriaxone and ceftazidime) and fourth-generation (cefepime) cephalosporins was detected in two isolates of E. cloacae from shrimp (Table 2).

Table 2
- Distribution of the enterobacteria strains (n=103) according to beta-lactam resistance

AmpC phenotypic detection was only confirmed in E. cloacae (n = 3) and C. freundii (n = 2) from shrimp and in E. cloacae (n = 2) and C. braakii (Table 3). ESBL was detected in two strains of E. cloacae from shrimp. No strain showed KPC production.

Table 3
- Number of enterobacteria strains according to the expression of AmpC beta-lactamase

DISCUSSION

In the present study, 49 (89%) of the shrimp isolates and 41 (83%) of the fish isolates corresponded to E. cloacae, K. pneumoniae and E coli species (Table 1). The isolation of enterobacteria from shrimp and fish has already been reported1313. Bakr WM, El Sayed AM, El Shamy HA, Amine AE. Is it safe to eat raw seafood? Prevalence of Salmonella in some seafood products sold in Alexandria markets. J Egypt Public Health Assoc. 2013;88:115-20.,1414. Sousa DA, Mulder KC, Nobre KS, Parachin NS, Franco OL. Production of a polar fish antimicrobial peptide in Escherichia coli using an ELP-intein tag. J Biotechnol. 2016;234:83-9., indicating fecal contamination, since these microorganisms are not part of the normal microbiota of these animals.

E. cloacae has already been isolated from aquatic organisms1515. Wei F, Ma T, Gong X, Zhang N, Bao B. Identification of tetrodotoxin-producing bacteria from goby Yongeichthys criniger. Toxicon. 2015;104:46-51.,1616. Snuossi M, Trabelsi N, Ben Taleb S, Dehmeni A, Flamini G, De Feo V. Laurus nobilis, Zingiber officinale and Anethum graveolens essential oils: composition, antioxidant and antibacterial activities against bacteria isolated from fish and shellfish. Molecules. 2016;21:e1414. and stands out as a human pathogen present in nosocomial outbreaks1717. Davin-Regli A, Pagès JM. Enterobacter aerogenes and Enterobacter cloacae; versatile bacterial pathogens confronting antibiotic treatment. Front Microbiol. 2015;6:392.. In addition, E. cloacae with the ability to produce biofilm was isolated from fish and seafood, a fact that favors resistance to antibiotics1616. Snuossi M, Trabelsi N, Ben Taleb S, Dehmeni A, Flamini G, De Feo V. Laurus nobilis, Zingiber officinale and Anethum graveolens essential oils: composition, antioxidant and antibacterial activities against bacteria isolated from fish and shellfish. Molecules. 2016;21:e1414..

The presence of K. pneumoniae in fish and shrimp samples (Table 1) serves as an alert and the health risks presented by these strains should not be underestimated. Some studies corroborate the present investigation since they reported the isolation of these species from fish, seafood and other types of food1818. Marathe NP, Gaikwad SS, Vaishampayan AA, Rasane H, Shouche YS, Gade WN. Mossambicus tilapia (Oreochromis mossambicus) collected from water bodies impacted by urban waste carries extended-spectrum beta-lactamases and integron-bearing gut bacteria. J Biosci. 2016;41:341-6.,1919. Guo Y, Zhou H, Qin L, Pang Z, Qin T, Ren H, et al. Frequency, antimicrobial resistance and genetic diversity of Klebsiella pneumoniae in food samples. Plos One. 2016;11:e0153561..

The third most frequently isolated microorganism in the present study was E. coli (Table 1) and it is known that this species has been associated with food-borne infections and was isolated from fish and aquatic environments. It is important to note that fish contaminated by this bacterium is a risk factor for consumers2020. Cho TJ, Kim NH, Kim SA, Song JH, Rhee MS. Survival of foodborne pathogens (Escherichia coli O157:H7, Salmonella Typhimurium, Staphylococcus aureus, Listeria monocytogenes, and Vibrio parahaemolyticus) in raw ready-to-eat crab marinated in soy sauce. Int J Food Microbiol. 2016;238:50-5.

21. Kim HW, Hong YJ, Jo JI, Ha SD, Kim SH, Lee HJ, et al. Raw ready-to-eat seafood safety: microbiological quality of the various seafood species available at fishery, hyper, and online market. Lett Appl Microbiol. 2017;64:27-34.
-2222. Kaktcham PM, Temgoua JB, Ngoufack Zambou F, Diaz-Ruiz G, Wacher C, Pérez-Chabela ML. Quantitative analyses of the bacterial microbiota of rearing environment, tilapia and common carp cultured in earthen ponds and inhibitory activity of its lactic acid bacteria on fish spoilage and pathogenic bacteria. World J Microbiol Biotechnol. 2017;33:32..

In addition, some studies have reported the presence of C. freundii2323. Gallani SU, Sebastião FA, Valladão GM, Boaratti AZ, Pilarski F. Pathogenesis of mixed infection by Spironucleus sp. and Citrobacter freundii in freshwater angelfish Pterophyllum scalare. Microb Pathog. 2016;100:119-23. and C. braakii2424. Nawaz M, Khan AA, Khan S, Sung K, Steele R. Isolation and characterization of tetracycline-resistant Citrobacter spp. from catfish. Food Microbiol. 2008;25:85-91. in fish, corroborating data of the present study. This genus, generally regarded as a commensal bacterium of the human intestinal microbiota, can cause opportunistic infections. It has been associated with nosocomial, urinary and respiratory infections2525. Rezaei M, Akya A, Elahi A, Ghadiri K, Jafari S. The clonal relationship among the Citrobacter freundii isolated from the main hospital in Kermanshah, west of Iran. Iran J Microbiol. 2016;8:175-80..

In the present study, a strain of R. planticola (Table 1) was also isolated from a fish sample. This species has already been isolated from herbs, vegetables, water and soil, but rarely from animals. This species is responsible for infections such as pneumonia, bacteremia in cancer patients or in those with a depressed immune system. R. planticola converts histidine to histamine and may produce symptoms of poisoning when undercooked seafood is consumed2626. Lam PW, Salit IE. Raoultella planticola bacteremia following consumption of seafood. Can J Infect Dis Med Microbiol. 2014;25:e83-4.,2727. Sękowska A. Raoultella spp. - clinical significance, infections and susceptibility to antibiotics. Folia Microbiol (Praha). 2017;62:221-7..

P. stuartii, another bacterium isolated from fish sample (Table 1), usually causes opportunistic infections and is associated with nosocomial outbreaks2828. Mao YC, Chang CL, Huang YC, Su LH, Lee CT. Laboratory investigation of a suspected outbreak caused by Providencia stuartii with intermediate resistance to imipenem at a long-term care facility. J Microbiol Immunol Infect. In Press 2016.. Khunthongpan et al.2929. Khunthongpan S, Sumpavapol P, Tanasupawat S, Benjakul S, H-Kittikun A. Providencia thailandensis sp. nov., isolated from seafood processing wastewater. J Gen Appl Microbiol. 2013;59:185-90. isolated this strain from processed wastewater from seafood.

Also in this sense, occurrence of a S. marcescens (Table 1) strain was observed in a fish sample. This bacterium is an important opportunistic pathogen associated with serious infections. Recently, in Brazil, Cayô et al.3030. Cayô R, Leme RC, Streling AP, Matos AP, Nodari CS, Chaves JR, et al. Serratia marcescens harboring SME-4 in Brazil: a silent threat. Diagn Microbiol Infect Dis. 2017;87:357-8. reported an outbreak of septic shock associated with this strain. S. marcescens has been isolated from frozen shrimp and fish and may be associated with fish deterioration3131. Noor Uddin GM, Larsen MH, Christensen H, Aarestrup FM, Phu TM, Dalsgaard A. Identification and antimicrobial resistance of bacteria isolated from probiotic products used in shrimp culture. Plos One. 2015;10:e0132338..

Incidence of beta-lactam-resistant Enterobacteriaceae in aquatic organisms (shrimp and fish) observed in the present study (Table 2) may be related to the indiscriminate use of antibacterial drugs in the culture. In the present study, more than 50% of the strains were resistant to ampicillin. Some enterobacteria are naturally resistant to aminopenicillins or may acquire beta-lactam resistance genes, which results in ampicillin resistance2626. Lam PW, Salit IE. Raoultella planticola bacteremia following consumption of seafood. Can J Infect Dis Med Microbiol. 2014;25:e83-4.,2727. Sękowska A. Raoultella spp. - clinical significance, infections and susceptibility to antibiotics. Folia Microbiol (Praha). 2017;62:221-7..

Fifty-four (52.4%) strains presented resistance to cephalothin (Table 2). These data are in agreement with findings in the literature, since strains isolated from fish have been showing resistance to this first-generation cephalosporin3232. Kang CH, Shin Y, Jeon H, Choi JH, Jeong S, So JS. Antibiotic resistance of Shewanella putrefaciens isolated from shellfish collected from the West Sea in Korea. Mar Pollut Bull. 2013;76:85-8.,3333. Xu, X Cheng J, Wu Q, Zhang J, Xie T. Prevalence, characterization, and antibiotic susceptibility of Vibrio parahaemolyticus isolated from retail aquatic products in North China. BMC Microbiol. 2016;16:32.. In addition, cefoxitin- and cefuroxime-resistant bacteria have also been isolated from food3434. Chajęcka-Wierzchowska W, Zadernowska A, Nalepa B, Sierpińska M, Laniewska-Trokenheim L. Retail ready-to-eat food as a potential vehicle for Staphylococcus spp. harboring antibiotic resistance genes. J Food Prot. 2014;77: 993-8.,3535. Kumar Y, Gupta N, Vaish VB, Gupta S. Distribution trends & antibiogram pattern of Salmonella enterica serovar Newport in India. Indian J Med Res. 2016;144:82-6..

In the present study, resistant strains of third and fourth-generation cephalosporins were detected (Table 2). These data are alarming, since these drugs are used in clinical practice. Liu et al.3636. Liu LT, Wan LH, Song XH, Xiong Y, Jin SJ, Zhou LM. Relevance of class 1 integrons and extended-spectrum beta-lactamases in drug-resistant Escherichia coli. Mol Med Rep. 2013;8:1251-5. detected enterobacteria with resistance to third-generation cephalosporins, suggesting that resistance was associated with class 1 integrons. Resistance to cefepime has been reported in strains of hospital origin3737. Alyamani EJ, Khiyami AM, Booq RY, Majrashi MA, Bahwerth FS, Rechkina E. The occurrence of ESBL-producing Escherichia coli carrying aminoglycoside resistance genes in urinary tract infections in Saudi Arabia. Ann Clin Microbiol Antimicrob. 2017;16:1..

The present study also demonstrated that 3 (2.9%) of the isolates were resistant to aztreonam (Table 3). Bacteria resistant to this drug have been isolated from shrimp farms3838. Costa RA, Araújo RL, Souza OV, Vieira RH. Antibiotic-resistant Vibrios in farmed shrimp. BioMed Res Int. 2015;2015:505914.. On the other hand, resistance to imipenem, that has also being observed in the present research (Table 2), has been reported in the literature in clinical isolates3939. Razavi NH, Ardebili A, Mardaneh J. Systematic review of antimicrobial resistance of clinical Acinetobacter baumannii isolates in Iran: an update. Microb Drug Resist. In Press 2017..

AmpC beta-lactamase is associated with the bacterial ability to resist cephalosporins and beta-lactamase inhibitors. In the present research, this enzyme was detected in the species E. cloacae, C. freundii and C. braakii (Table 3). A survey carried out in a Vietnamese fish market revealed a high contamination rate of shrimp (49.1%) and fish (36.6%) by enterobacteria. The authors showed that all strains of the Enterobacteriaceae family showed to be AmpC enzyme carriers and showed resistance to at least 6 classes of antimicrobials, including beta-lactams. The presence of these multiresistant multi-resistant strains is a concern for public health and suggests that the use of antimicrobial agents in fish culture is to be strictly controlled11. Nguyen Do P, Nguyen TA, Le TH, Tran NM, Ngo TP, Dang VC, et al. Dissemination of extended-spectrum β-lactamase- and AmpC β-lactamase-producing Escherichia coli within the food distribution system of Ho Chi Minh City, Vietnam. BioMed Res Int. 2016;2016:8182096..

In the present study, ESBL was detected in two strains of E. cloacae from shrimp, only. Le et al.22. Le QP, Ueda S, Nguyen TN, Dao TV, Van Hoang TA, Tran TT, et al. Characteristics of extended-spectrum β-lactamase-producing Escherichia coli in retail meats and shrimp at a local market in Vietnam. Foodborne Pathog Dis. 2015;12:719:25. detected E. coli producing ESBL in shrimp. The authors warn of the severity of these results, since this fact requires monitoring and effort by the monitoring agencies to control the spread of ESBL-producing bacteria in the community.

Janecko et al.4040. Janecko N, Martz SL, Avery BP, Daignault D, Desruisseau A, Boyd D, et al. Carbapenemo-resistant Enterobacter spp. in retail seafood imported from Southeast Asia to Canada. Emerg Infec Dis. 2016;22:1675-7. detected blaNDM-1 and blaIMI-type, resistance genes encoding beta-lactamases in E. cloacae and E. aerogenes isolated from fish and shrimp. This research suggests that, in addition to the probable fish contamination by water, the presence of beta-lactamases may be associated with fish exposure during transport.

In the present study, no strain expressed KPC. This fact may be related to the limitation of the phenotypic test used (Hodge test), which, although already used for KPC detection1212. Leão-Vasconcelos LS, Lima AB, Costa DM, Rocha-Vilefort LO, Oliveira AC, Gonçalves NF, et al. Enterobacteriaceae isolates from the oral cavity of workers in a brazilian oncology hospital. Rev Inst Med Trop São Paulo. 2015;57:121-7., is not recommended by CLSI to detect this enzyme. In addition, the lack of KPC expression may also be related to the high number of strains sensitive to monobactam and carbapenens. KPC is an extended-spectrum beta-lactamase capable of hydrolyzing carbapenems and other beta-lactam antibiotics. Their detection is crucial because they are often associated with total or partial antibiotic resistance4141. Thomson KS. Extended-spectrum-beta-lactamase, AmpC, and carbapenemoase issues. J Clin Microbiol. 2010;48:1019-25.. This enzyme has already been detected in seafood isolates that showed cross-resistance to penicillins, cephalosporins, monobactam and carbapenem4141. Thomson KS. Extended-spectrum-beta-lactamase, AmpC, and carbapenemoase issues. J Clin Microbiol. 2010;48:1019-25.,4242. Mangat CS, Boyd D, Janecko N, Martz SL, Desruisseau A, Carpenter M, et al. Characterization of VCC-1, a novel Ambler class A carbapenemase from Vibrio cholerae isolated from imported retail shrimp sold in Canada. Antimicrob Agents Chemother. 2016;60:1819-25..

The results indicate that aquatic organisms (fish and shrimp) may constitute sources of enteric bacteria resistant to beta-lactams. These data reveal the need to establish legislation regulating the use of antimicrobials in aquaculture activities, and it is suggested that sanitary authorities urgently sanction control measures for use of antimicrobials in fish and shrimp farming. It is also emphasized that this study presents as a limitation the absence of information about the genetic characterization of strains by PCR, therefore, there is no data of distribution of the genes-blaCTX-M, blaTEM and blaSHV among the 103 isolates.

ACKNOWLEDGMENTS

The first author received a postgraduate scholarship from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior.

REFERENCES

  • 1
    Nguyen Do P, Nguyen TA, Le TH, Tran NM, Ngo TP, Dang VC, et al. Dissemination of extended-spectrum β-lactamase- and AmpC β-lactamase-producing Escherichia coli within the food distribution system of Ho Chi Minh City, Vietnam. BioMed Res Int. 2016;2016:8182096.
  • 2
    Le QP, Ueda S, Nguyen TN, Dao TV, Van Hoang TA, Tran TT, et al. Characteristics of extended-spectrum β-lactamase-producing Escherichia coli in retail meats and shrimp at a local market in Vietnam. Foodborne Pathog Dis. 2015;12:719:25.
  • 3
    Öztürk H, Ozkirimli E, Özgür A. Classification of Beta-lactamases and penicillin binding proteins using ligand-centric network models. Plos One. 2015;10:e0117874.
  • 4
    Suarez C, Gudiol F. Antibióticos betalactámicos. Enferm Infecc Microbiol Clin. 2009;27:116-29.
  • 5
    Lago A, Fuentefria SR, Fuentefria DB. Enterobactérias produtoras de ESBL em Passo Fundo, Estado do Rio Grande do Sul, Brasil. Rev Soc Bras Med Trop. 2010;43:430-4.
  • 6
    Laudy AE, Osińska P, Namysłowska A, Zajac O, Tyski S. Modification of the susceptibility of Gram-negative rods producing ESBLS to beta-Lactams by the efflux phenomenon. Plos One. 2015;10:e0119997.
  • 7
    Silva KC, Lincopan N. Epidemiologia das betalactamases de espectro estendido no Brasil: impacto clínico e implicações para o agronegócio. J Bras Patol Med Lab. 2012;48:91-9.
  • 8
    van Bijnen EM, Paget J, de Lange-de Klerk ES, den Heijer CD, Versporten A, Stobberingh EE, et al. Antibiotic exposure and other risk factors for antimicrobial resistance in nasal commensal Staphylococcus aureus: an ecological study in 8 european countries. Plos One. 2015;10:e0135094.
  • 9
    Colello R, Etcheverría AI, Di Conza JA, Gutkind GO, Padola NL. Antibiotic resistance and integrons in shiga toxin-producing Escherichia coli (STEC). Braz J Microbiol. 2015;46:1-5.
  • 10
    Crowley E, Bird P, Fisher K, Goetz K, Boyle M, Benzinger MJ Jr, et al. Evaluation of the VITEK 2 Gram-negative (GN) microbial identification test card: collaborative study. J AOAC Int. 2012;95:778-85.
  • 11
    Clinical and Laboratory Standards Institute. Performance standards for antimicrobial disk susceptibility tests; approved standard. 11th ed. Wayne: CLSI; 2012.
  • 12
    Leão-Vasconcelos LS, Lima AB, Costa DM, Rocha-Vilefort LO, Oliveira AC, Gonçalves NF, et al. Enterobacteriaceae isolates from the oral cavity of workers in a brazilian oncology hospital. Rev Inst Med Trop São Paulo. 2015;57:121-7.
  • 13
    Bakr WM, El Sayed AM, El Shamy HA, Amine AE. Is it safe to eat raw seafood? Prevalence of Salmonella in some seafood products sold in Alexandria markets. J Egypt Public Health Assoc. 2013;88:115-20.
  • 14
    Sousa DA, Mulder KC, Nobre KS, Parachin NS, Franco OL. Production of a polar fish antimicrobial peptide in Escherichia coli using an ELP-intein tag. J Biotechnol. 2016;234:83-9.
  • 15
    Wei F, Ma T, Gong X, Zhang N, Bao B. Identification of tetrodotoxin-producing bacteria from goby Yongeichthys criniger. Toxicon. 2015;104:46-51.
  • 16
    Snuossi M, Trabelsi N, Ben Taleb S, Dehmeni A, Flamini G, De Feo V. Laurus nobilis, Zingiber officinale and Anethum graveolens essential oils: composition, antioxidant and antibacterial activities against bacteria isolated from fish and shellfish. Molecules. 2016;21:e1414.
  • 17
    Davin-Regli A, Pagès JM. Enterobacter aerogenes and Enterobacter cloacae; versatile bacterial pathogens confronting antibiotic treatment. Front Microbiol. 2015;6:392.
  • 18
    Marathe NP, Gaikwad SS, Vaishampayan AA, Rasane H, Shouche YS, Gade WN. Mossambicus tilapia (Oreochromis mossambicus) collected from water bodies impacted by urban waste carries extended-spectrum beta-lactamases and integron-bearing gut bacteria. J Biosci. 2016;41:341-6.
  • 19
    Guo Y, Zhou H, Qin L, Pang Z, Qin T, Ren H, et al. Frequency, antimicrobial resistance and genetic diversity of Klebsiella pneumoniae in food samples. Plos One. 2016;11:e0153561.
  • 20
    Cho TJ, Kim NH, Kim SA, Song JH, Rhee MS. Survival of foodborne pathogens (Escherichia coli O157:H7, Salmonella Typhimurium, Staphylococcus aureus, Listeria monocytogenes, and Vibrio parahaemolyticus) in raw ready-to-eat crab marinated in soy sauce. Int J Food Microbiol. 2016;238:50-5.
  • 21
    Kim HW, Hong YJ, Jo JI, Ha SD, Kim SH, Lee HJ, et al. Raw ready-to-eat seafood safety: microbiological quality of the various seafood species available at fishery, hyper, and online market. Lett Appl Microbiol. 2017;64:27-34.
  • 22
    Kaktcham PM, Temgoua JB, Ngoufack Zambou F, Diaz-Ruiz G, Wacher C, Pérez-Chabela ML. Quantitative analyses of the bacterial microbiota of rearing environment, tilapia and common carp cultured in earthen ponds and inhibitory activity of its lactic acid bacteria on fish spoilage and pathogenic bacteria. World J Microbiol Biotechnol. 2017;33:32.
  • 23
    Gallani SU, Sebastião FA, Valladão GM, Boaratti AZ, Pilarski F. Pathogenesis of mixed infection by Spironucleus sp. and Citrobacter freundii in freshwater angelfish Pterophyllum scalare. Microb Pathog. 2016;100:119-23.
  • 24
    Nawaz M, Khan AA, Khan S, Sung K, Steele R. Isolation and characterization of tetracycline-resistant Citrobacter spp. from catfish. Food Microbiol. 2008;25:85-91.
  • 25
    Rezaei M, Akya A, Elahi A, Ghadiri K, Jafari S. The clonal relationship among the Citrobacter freundii isolated from the main hospital in Kermanshah, west of Iran. Iran J Microbiol. 2016;8:175-80.
  • 26
    Lam PW, Salit IE. Raoultella planticola bacteremia following consumption of seafood. Can J Infect Dis Med Microbiol. 2014;25:e83-4.
  • 27
    Sękowska A. Raoultella spp. - clinical significance, infections and susceptibility to antibiotics. Folia Microbiol (Praha). 2017;62:221-7.
  • 28
    Mao YC, Chang CL, Huang YC, Su LH, Lee CT. Laboratory investigation of a suspected outbreak caused by Providencia stuartii with intermediate resistance to imipenem at a long-term care facility. J Microbiol Immunol Infect. In Press 2016.
  • 29
    Khunthongpan S, Sumpavapol P, Tanasupawat S, Benjakul S, H-Kittikun A. Providencia thailandensis sp. nov., isolated from seafood processing wastewater. J Gen Appl Microbiol. 2013;59:185-90.
  • 30
    Cayô R, Leme RC, Streling AP, Matos AP, Nodari CS, Chaves JR, et al. Serratia marcescens harboring SME-4 in Brazil: a silent threat. Diagn Microbiol Infect Dis. 2017;87:357-8.
  • 31
    Noor Uddin GM, Larsen MH, Christensen H, Aarestrup FM, Phu TM, Dalsgaard A. Identification and antimicrobial resistance of bacteria isolated from probiotic products used in shrimp culture. Plos One. 2015;10:e0132338.
  • 32
    Kang CH, Shin Y, Jeon H, Choi JH, Jeong S, So JS. Antibiotic resistance of Shewanella putrefaciens isolated from shellfish collected from the West Sea in Korea. Mar Pollut Bull. 2013;76:85-8.
  • 33
    Xu, X Cheng J, Wu Q, Zhang J, Xie T. Prevalence, characterization, and antibiotic susceptibility of Vibrio parahaemolyticus isolated from retail aquatic products in North China. BMC Microbiol. 2016;16:32.
  • 34
    Chajęcka-Wierzchowska W, Zadernowska A, Nalepa B, Sierpińska M, Laniewska-Trokenheim L. Retail ready-to-eat food as a potential vehicle for Staphylococcus spp. harboring antibiotic resistance genes. J Food Prot. 2014;77: 993-8.
  • 35
    Kumar Y, Gupta N, Vaish VB, Gupta S. Distribution trends & antibiogram pattern of Salmonella enterica serovar Newport in India. Indian J Med Res. 2016;144:82-6.
  • 36
    Liu LT, Wan LH, Song XH, Xiong Y, Jin SJ, Zhou LM. Relevance of class 1 integrons and extended-spectrum beta-lactamases in drug-resistant Escherichia coli. Mol Med Rep. 2013;8:1251-5.
  • 37
    Alyamani EJ, Khiyami AM, Booq RY, Majrashi MA, Bahwerth FS, Rechkina E. The occurrence of ESBL-producing Escherichia coli carrying aminoglycoside resistance genes in urinary tract infections in Saudi Arabia. Ann Clin Microbiol Antimicrob. 2017;16:1.
  • 38
    Costa RA, Araújo RL, Souza OV, Vieira RH. Antibiotic-resistant Vibrios in farmed shrimp. BioMed Res Int. 2015;2015:505914.
  • 39
    Razavi NH, Ardebili A, Mardaneh J. Systematic review of antimicrobial resistance of clinical Acinetobacter baumannii isolates in Iran: an update. Microb Drug Resist. In Press 2017.
  • 40
    Janecko N, Martz SL, Avery BP, Daignault D, Desruisseau A, Boyd D, et al. Carbapenemo-resistant Enterobacter spp. in retail seafood imported from Southeast Asia to Canada. Emerg Infec Dis. 2016;22:1675-7.
  • 41
    Thomson KS. Extended-spectrum-beta-lactamase, AmpC, and carbapenemoase issues. J Clin Microbiol. 2010;48:1019-25.
  • 42
    Mangat CS, Boyd D, Janecko N, Martz SL, Desruisseau A, Carpenter M, et al. Characterization of VCC-1, a novel Ambler class A carbapenemase from Vibrio cholerae isolated from imported retail shrimp sold in Canada. Antimicrob Agents Chemother. 2016;60:1819-25.

Publication Dates

  • Publication in this collection
    2017

History

  • Received
    10 Mar 2017
  • Accepted
    25 July 2017
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