Species Identification and Antimicrobial Resistance of Coagulase-Negative Staphylococci Isolated from the Meat of Sea Fish Ivana Regecov´a, Monika Pipov´a, Pavlina Jevinov´a, Katar´ına Maruˇskov´a, Vladim´ır Kmeť, and Peter Popelka
Seventy-eight isolates of staphylococci obtained from the meat of Theragra chalcogramma, Scomber scombrus, and Clupea harengus were identified and tested in this study. 16S rDNA sequence specific for the genus Staphylococcus was detected in all isolates with the help of PCR method. All of 78 isolates were coagulase-negative, and DNAse activity was only confirmed in 4 of them. The following species of staphylococci were identified using MALDI-TOF mass spectrometry: S. warneri (52%), S. epidermidis (33%), S. haemolyticus (6.4%), S. pasteuri (3.8%), S. sciuri (1.2%), S. capitis (1.2%), and S. hominis (1.2%). Antimicrobial resistance to 7 antibiotics was determined in each isolate with the help of agar dilution method. In general, resistance against ampicillin was observed in majority of isolates (87%). On the contrary, the best sensitivity of CoNS was determined to gentamicin (96%). Only 1 S. warneri strain showed resistance to cefoxitin. Furthermore, 83% of staphylococcal isolates were simultaneously resistant to 2 or more antibiotics.
Abstract:
Keywords: antibiotic, fish, nuclease, resistance, Staphylococcus
This study confirmed the need of monitoring antimicrobial resistance in coagulase-negative staphylococci not only in the meat of slaughter animals but also in retail marine fish. The results showed that MALDITOF mass spectrometry is useful, accurate, and rapid method for species identification of food pathogens including Staphylococcus spp.
Introduction The prophylactic use of antibiotics in fish farms has led to a rise in the number of resistant bacteria (Cabello 2006). Aquaculture promotes the production of various sizes and types of aquatic organisms, and the use of antibiotics and drugs in the fish industry is complicated because of the need to administer the compounds usually direct into the water where the organisms live. In addition, some countries, such as Norway, utilize natural structures like fjords for fish farming and, for this reason, there are concerns about the wastes that collect in the fjord bottoms, which can lead to contamination of the environment with antibiotics and subsequently to the emergence of resistant bacteria in environment (FAO/NACA/WHO 1997). Strikingly, these bacteria can transfer the resistance genes to human pathogens (Rhodes and others 2000a). The selection pressure applied by the antibiotics used in clinics and agriculture (Martin and Liras 1989) caused an increase of antimicrobial resistance in fish pathogens (Davies and others 1999; Rhodes and others 2000a,b; Schmidt and others 2000; Sørum 2000; L’Abee-Lund and Sørum 2001; Sørum 2006) resulting in the risk of possible passage of both resistant bacteria and their determinants of antimicrobial resistance to various bacteria of terrestrial animals and human beings, including pathogens (Cabello 2006). The use of antimicrobial agents is not the only
MS 20130840 Submitted 6/20/2013, Accepted 2/4/2014. Authors Regecov´a, Pipov´a, Jevinov´a, Maruskov´a and Popelka are with Dept. of Food Hygiene and Technology, Univ. of Veterinary Medicine and Pharmacy, Komensk´eho 73, 041 81, Koˇsice, Slovak Republic. Author Kmet’ is with Inst. of Animal Physiology, Slovak ˇ esovej 4-6, 040 01, Koˇsice, Slovak Republic. Direct Academy of Sciences Koˇsice, Solt´ inquiries to author Regecov´a (E-mail: [email protected]).
R C 2014 Institute of Food Technologists
doi: 10.1111/1750-3841.12429 Further reproduction without permission is prohibited
cause of the occurrence and spread of antimicrobial mono- and multiresistance. Spontaneous mutations of food bacteria and spread of resistant bacterial strains in the absence of selective pressure may also contribute to an increase of antimicrobial resistance among food bacteria (B´ıreˇs and others 2008). In recent years, the food chain is one of the main routes of transmission of resistant bacteria between animal and human populations (Mass 1986; Ito and others 2003). In the case of primary contamination, slaughter animals are carriers of multiresistant strains. The food chain can also be contaminated secondarily at the time of processing, transporting, and storage of raw materials or finished products. Thus, resistant bacteria may come from both human and animal sources (Bardoˇn and others 2007). Recently, an increased resistance is also observed among strains of Staphylococcus spp., this resulting in several diseases, some of them being fatal. Staphylococci are not part of the normal fish microflora (Huss 1988). Their presence on fish is an indication of (a) postharvest contamination due to poor personnel hygiene and (b) disease in fish (Huss 1988; Austin and Austin 2007). In addition, CoNS in aquaculture mainly serves as a reservoir of resistance genes that can be transferred to bacteria in aquatic environments, or further disseminated and ultimately end up in human pathogens miscellaneous (Perreten and others 1998; WHO 2006). The level of antimicrobial resistance varies significantly among species of staphylococci. As reported by Drozenov´a and Petr´aˇs (2000), a high proportion of resistant strains occurs in species and subspecies that are the most common causative agents of nosocomial infections (S. epidermidis, S. hominis subsp. hominis, S. haemolyticus, and S. hominis subsp. novobiosepticus). Transferable resistance, which also occurs in the genus Staphylococcus, does not respect any phylogenetical, geographical, or ecological limits. Genes
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Practical Application:
Staphylococci in sea fish . . . of resistance are distributed horizontally among bacteria of land animals, fish, and human populations (B´ıreˇs and others 2009). However, no data have yet been presented regarding antimicrobial resistance of CoNS from retail fish (Hammad and others 2012). Moreover, no studies aimed at species identification of CoNS isolates from marine fish with the help of MALDI-TOF mass spectrometry are available. Therefore, this study is focused on both species identification and determination of antimicrobial resistance in coagulase-negative staphylococci isolated from the meat of Atlantic mackerel, Alaska pollock, and Atlantic herring.
Materials and Methods
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Isolation of staphylococci Staphylococci were isolated from samples of frozen fish meat as follows: 5 samples of Alaska pollock (Theragra chalcogramma) originating from fishing area FAO 61 (China), 4 samples of Atlantic mackerel (Scomber scombrus) originating from fishing area FAO 27 (Ireland), and 6 samples of Atlantic herring (Clupea harengus) originating from fishing area FAO 27 (Norway). Samples were taken aseptically from whole fish meat after its defrosting, removal of the consumer packaging, and the skin of fish in accordance with the requirements of STN ISO 3100-2 (1999). Basic and further subsequent decimal dilutions were prepared according to STN EN ISO 6887-3/01 (2003). Staphylococci were isolated from fish samples in accordance with STN EN ISO 6888-1/A1 (2004). The first 2 decimal dilutions were spread in an amount of 0.1 mL on the surface of Baird-Parker medium (Oxoid, Basingstoke, Hampshire, U.K.). Inoculated media were incubated at 37 °C for 24 to 48 h. Based on their characteristic appearance, both typical and atypical colonies of staphylococci were further identified. Plates containing 15 to 150 colonies were used for this purpose.
CCM 4223 served as positive control. Reaction mixture in a volume of 50 μL contained 1 μL genomic DNA; 10 mmol/L Tris-HCl (pH 8.8); 3 mmol/L MgCl2; 200 μmol/L dNTP; 12,5 pmol/L of each primer, and 1U Taq DNA polymerase (Ecoli s.r.o., Bratislava, Slovakia). The PCR protocol was as follows: initial denaturation at 94 °C for 3 min, 30 cycles consisting of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s. Final extension at 72 °C for 4 min followed the last cycle. PCR products were separated in a 2% agarose gel stained TM with Goldview Nucleic acid stain (Beijing SBS Genetech Co. LTD, Beijing, China) and visualized using the DNR Bio Imaging R Sytem (MiniBIS Pro , Jerusalem, Israel). The species of staphylococci were subsequently identified with the help of MALDI BioTyper(TM) system (Bruker Daltonics, Billerica, Mass., U.S.A.) based on protein “fingerprints” measured by MALDI-TOF mass spectrometry. As staphylococci are Grampositive bacteria, lysates of bacterial cells had to be prepared according to the instruction of producer (Bruker Daltonics 2008) prior to their identification.
Determination of susceptibility to selected antibiotics Susceptibility of staphylococcal isolates to 7 antibiotics was determined by the agar dilution method according to the procedure described by CLSI document M07-A8 (2009). M¨ueller-Hinton agar was used as the test medium (Hi-Media, Mumbai, Maharashtra India). Test plates containing different concentrations of erythromycin (0.25; 0.5; 1.0; 2.0; 4.0; 8.0 mg/L); tetracycline (2.0; 4.0; 8.0; 16.0 mg/L); ampicillin (0.25; 0.5; 1.0 mg/L); gentamicin (2.0; 4.0; 8.0; 16.0 mg/L); cefoxitin (2.0; 4.0; 8.0; 16.0; 32.0 mg/L); oxacillin (0.125; 0.25; 0.5; 1.0; 2.0; 4.0; 8.0 mg/L); and penicillin (0.06; 0.125; 0.25; 0.5 mg/L) were used for the determination of minimum inhibitory concentrations (MICs). Drops of 24-h bacterial suspensions adjusted to the 0.5 McFarland turbidity standard were placed in parallel on the surface of each test plate. Inoculated plates were then incubated at 37 °C for 24 h. After incubation, the lowest concentrations of antibiotics that inhibited the visible growth of staphylococcal strains were determined. Results were evaluated according to CLSI document M100-S20 (2010).
Determination of plasma-coagulase activity Colonies of staphylococci were individually reinoculated into test tubes containing 2 mL of Brain Heart Infusion Broth (Oxoid). After 18 to 24 h of incubation at 37 °C, 0.1 mL of each bacterial culture was added to another test tube with 1 mL of reconstituted freeze-dried rabbit plasma (Stafylo PK, Imuna Pharm, Sˇ ariˇssk´e Michal’any, Slovakia). Inoculated test tubes were incubated at 37 Results and Discussion °C. Formation of coagulum was considered as a positive reaction. The bacterial community associated with fish is generally related Results were evaluated after 1, 2, 3, 6, and 24 h. with the features of the aquatic habitat, such as salinity and the bacterial load in the water (Cahill 1990). Retail fish microflora can be Determination of nuclease activity influenced by the level of hygiene, where the role of food handlers Each isolate was inoculated on the surface of DNase agar in preparation of fish is crucial in determining the hygienic sta(Oxoid) where the production of thermostable deoxyribonucle- tus of the final product, with poor handling resulting in increased ase was confirmed after a 24-h-incubation at 37 °C. Colonies likelihood of contamination by human-borne microbes including producing DNase hydrolyzed the deoxyribonucleic acid (DNA) multidrug-resistant and/or enterotoxigenic bacteria (Hammad and contained in the medium. When the medium was flooded and others 2012). The dominance of Staphylococcus spp. among groups acidified with 1 N hydrochloric acid, the DNA precipitated, the of sea fish bacterial isolates was also confirmed by Hashizume and medium became turbid and clear zones appeared around DNase- others (2005). positive colonies. As seen in Table 1, 78 strains of staphylococci were isolated from samples of frozen meat of Alaska pollock, Atlantic mackerel, and Genus and species identification Atlantic herring in this study, all of them being coagulase-negative. The total genomic DNA was isolated from staphylococcal Strains of CoNS can cause serious nosocomial infections. Such strains as described by Hein and others (2005) and further tested strains frequently possess various virulence factors including anby PCR method according to Strommenger and others (2003). timicrobial resistance and the ability to form a biofilm. Correct Primers 16S1 (5-CAGCTCGTGTCGTGAGATGT) and 16S2 identification and typing of CoNS is therefore important from (5-AATCATTTGTCCCACCTTCG) rDNA (Generi Biotech both clinical and epidemiological points of view (Chomouck´a s.r.o., Hradec Kr´alov´e, Czech Republic) typical for the genus 2009). DNA sequence-based species identification is currently the Staphylococcus were used for PCR. The reference strain S. aureus most accurate method for CoNS identification and is considered M2 Journal of Food Science r Vol. 00, Nr. 0, 2014
Staphylococci in sea fish . . . Table 1–Number of staphylococcal species in isolates from sea Table 2–Number of resistant (R), intermediately susceptible (I), fish. and susceptible (S) isolates of staphylococci from the meat of sea fish. Species Alaska Atlantic Atlantic of pollock mackerel herring Total Alaska Atlantic Atlantic staphylococci (n = 27) (n = 24) (n = 27) (n = 78) pollock mackerel herring Total (n = 27) (n = 24) (n = 27) (n = 78) S. warneri 12 13 16 41 S. epidermidis 13 11 2 26 R I S R I S R I S R I S S. haemolyticus – – 5 5 Penicillin 10 – 17 19 – 5 18 – 9 47 – 31 S. pasteuri 1 – 2 3 Ampicillin 24 – 3 19 – 5 25 – 2 68 – 10 S. sciuri 1 – – 1 Tetracycline 4 4 19 1 5 18 – 5 22 5 14 59 S. capitis – – 1 1 Oxacillin 2 – 25 15 – 9 15 – 12 32 – 46 S. hominis ssp. Hominis – – 1 1 Erythromycin 19 8 – 17 6 1 15 10 2 51 24 3 Gentamicin – 3 24 – – 24 – – 27 – 3 75 Cefoxitin – 1 26 – 2 22 1 2 24 1 5 72
They found that most S. epidermidis, S. warneri, S. Hominis, and S.haemolyticus strains tested produced DNAse. Cunha and others (2006) reported the production of DNase in 9 isolates of S. epidermidis and 2 isolates of S. haemolyticus out of 117 CoNS isolates tested. However, the authors did not confirm any DNase activity in S. warneri isolates that is in contrast to our study, where the DNase activity was detected in 3 isolates of S. warneri and 1 isolate of S. epidermidis.
Testing of antimicrobial resistance All of 41 S. warneri isolates showed the most frequent resistance against ampicillin (95%) and did not show any resistance against gentamicin. Resistance to cefoxitin was confirmed in the only strain of S. warneri isolated from the meat of Atlantic herring (Table 2). Thirty-four S. warneri isolates were resistant to at least 2 antibiotics tested. Simultaneous resistance to 2, 3, 4 (penicillin, erythromycin, oxacillin, and ampicillin), or 5 (penicillin, ampicillin, tetracycline, erythromycin, and oxacillin) out of 7 antibiotics tested was confirmed in 17, 12, 4, and 1 isolate of S. warneri, respectively. Similarly, Gil and others (2000) isolated 3 strains of S. warneri from Oncorhynchus mykiss, these being resistant to ampicillin and penicillin. In the study of Hammad and others (2012), resistance to ampicillin in S. warneri isolates was combined with simultaneous resistance to another 4 antibiotics (erythromycin, gentamicin, oxacillin, and tetracycline). Abrahim and others (2010) confirmed resistance to oxacillin in 2 strains of S. warneri gained from 269 isolates of freshwater fish. Moreover, the resistance to this antibiotic was also confirmed in 1 isolate of S. haemolyticus and 3 isolates of S. epidermidis. During the last decade, S. epidermidis and S. haemolyticus have gained increasing attention as nosocomial pathogens in humans. They cause postoperative infections as common complication in surgery that can result in intensive postoperative care, prolonged hospitalization, increased costs, and mortality (Santschi 2006). Moreover, S. epidermidis has been reported previously as a fish pathogen in some marine and freshwater fish in Japan, Taiwan, and Greece (Kusuda and Sugiyama 1981; Varvarigos 2001). It may be present in the fish throughout the year, but the disease is induced by a sudden rise in water temperatures or other stress factors in the aquatic environment. It usually appears in the spring and causes problems throughout the summer (Varvarigos 2001). Twenty-six S. epidermidis isolates from the meat of 3 sea fish showed resistance to tetracycline, erythromycin, and oxacillin. However, resistance to ampicillin was confirmed most frequently. Moreover, majority of S. epidermidis isolates from the meat of Atlantic mackerel (10 strains) showed resistance to penicillin. Kubilay and Uluk¨oy (2004) also reported upon the first isolation of S. Vol. 00, Nr. 0, 2014 r Journal of Food Science M3
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as the gold standard (Zadoks and Watts 2009). The presence of 16S rDNA sequence typical for Staphylococcus spp. was confirmed in all 78 isolates from sea fish. Even though the 16S rDNA gene is considered to be highly conserved with limited discriminatory power especially in closely related staphylococcal species (Heikens and others 2005; Ghebremedhin and others 2008). Therefore, MALDI-TOF mass spectrometry based on bacterial protein profiles was used for species identification of staphylococci. Sample preparation and its analysis require only several minutes that make this method an attractive alternative to previous time-consuming conventional biochemical tests (Carbonnelle and others 2007). MALDI-TOF MS of intact bacterial cells was already used for the detection and identification of 24 different foodborne pathogens and food spoilage bacteria including staphylococci (Mazzeo and others 2006). The genus Staphylococcus usually shows a spectrum with 50 to 75 signals of 1.8 to 11 kDa (Smole and others 2002). Bal´azˇ ov´a (2010) reported that except for the genus-specific signal with the value of 9625 Da, typical signals for individual staphylococcal species can be visually distinguished. Typical signal values for S. aureus are 3442, 4304, and 6888 Da; for S. hominis 4828 Da and for S. haemolyticus 3230 Da. Based on these signals, 7 species were identified among 78 isolates of staphylococci (Table 1) as follows: S. warneri (52%), S. epidermidis (33%), S. haemolyticus (6.4%), S. pasteuri (3.8%), S. sciuri (1.2%), S. capitis (1.2%), and S. hominis (1.2%). Hammad and others (2012) obtained 10 isolates of MRSA and MR-CoNS from 10 samples (5%, 10/200) of fish products collected from 10 shops (40%, 10/25) belonging to 4 supermarket chains. Isolates were identified as S. aureus (n = 5), S. haemolyticus (n = 2), S. warneri (n = 2), and S. pasteuri (n = 1). Abrahim and others (2010) investigated freshwater fish and the environment of fish markets of Northern Greece. A total of 269 samples were examined, consisting of 71 rainbow trouts (Oncorhynchus mykiss), 65 gibel carps (Carassius gibelio), and 133 environmental samples swabbed from various surfaces at fish markets. Staphylococcus spp. was isolated from 27% of samples, and 16%, 9%, 0.7%, and 0.4% of samples inspected were positive for S. aureus, S. epidermidis, S. warneri, S. hominis subsp. hominis, and S. haemolyticus, respectively. Another study also confirmed the presence of CoNS in sea fish (Rodrigues and others 2003), these being identified as S. epidermidis, S. hominis, S. warneri, S. haemolyticus, S. simulans, and S. saprophyticus. DNase activity is important to distinguish between pathogenic staphylococci and nonpathogenic resident flora. For pathogenesis, DNase is as important as coagulase (Pfaller and Herwaldt 1988). However, this test is not entirely reliable as an indicator of pathogenicity because it has been estimated that about 18% of coagulase-negative staphylococci show DNase activity (Quinn and others 1999). Production of DNase by coagulase-negative staphylococci was also confirmed by Lambe and others (1990).
Staphylococci in sea fish . . . Table 3–Phenotypes of mono-, multi-, and polyresistance among Similar results have also been published by Ozaktas and others staphylococcal isolates from the meat of sea fish. (2012). In their study, the resistance against ampicillin was about Anttibiotics Amp Ery Pen-Amp Pen-Ery Ery-Amp Ery-Oxa Oxa-Amp Pen-Ery-Amp Pen-Ery-Oxa Pen-Tet-Amp Pen-Oxa-Amp Ery-Oxa-Amp Pen-Ery-Oxa-Amp Pen-Tet-Ery-Amp Pen-Tet-Ery-Amp-Oxa Pen-Ery-Oxa-Amp-Cef Total
Alaska pollock
Atlantic mackerel
5 3 3 0 10 0 0 3 0 1 0 0 0 1 1 0 27
1 0 1 1 1 1 2 5 2 0 4 0 5 1 0 0 24
Atlantic 92%. Resistance to β-lactams is dominant in coagulase-positive herring Staphylococcus species isolated from fish products. V´azquez-S´anchez 2 2 2 0 4 0 0 4 0 0 8 5 1 0 0 1 27
Pen: penicillin, Amp: ampicillin, Oxa: oxacillin, Ery: erythromycin, Tet: tetracycline, Cef: cefoxitin.
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epidermidis from gilthead sea bream (Sparus aurata) reared in the southwest of Turkey and its multiresistance to penicillin, ampicillin, erythromycin, and oxacillin. The presence of S. haemolyticus was only confirmed in frozen meat of Atlantic herring (Table 1). These 5 isolates showed multiresistance to erythromycin, oxacillin, and ampicillin. Similarly, Hammad and others (2012) reported that S. haemolyticus strains isolated from fresh fish meat were multiresistant to ampicillin, oxacillin, tetracycline, and gentamicin. Simultaneous resistance to erythromycin and ampicillin was also confirmed in S. sciuri isolated from Alaska Pollock, while S. capitis and S. hominis spp. hominis isolates from the meat of Atlantic herring showed mainly monoresistance to erythromycin. As reported by Lina and others (1999), erm(A) and erm(C) are the most frequent determinants of resistance to erythromycin in Staphylococcus spp. Macrolides have been known for 15 decades, and, since the introduction of erythromycin into therapy, a number of these molecules have been developed for clinical use. For years, these antibiotics have represented a major alternative to the use of penicillins and cephalosporins for the treatment of infections due to Gram-positive microorganisms. However, the worldwide development of macrolide resistance with wide variations, according to both the country and the bacterial species, has sometimes constrained to limit the use of these antibiotics to certain indications. Although the evolution of the macrolide class has been marked, in the 1990s, by the development of semisynthetic macrolides with improved pharmacokinetics and tolerability, these new derivatives have proved unable to overcome erythromycin resistance (Denis and others 1999). As to our study, resistance to erythromycin was also confirmed in 3 isolates of S. pasteuri. Among them, multiresistance to 4 antibiotics (penicillin, oxacillin, ampicillin, and erythromycin) was observed in 1 isolate from Alaska pollock meat. Moreover, 2 S. pasteuri isolates from the meat of Atlantic herring showed multiresistance to 3 antibiotics (penicillin, oxacillin, and ampicillin). These results cannot be compared with another published data because they are not yet available. As seen in Table 2, all staphylococci isolated from the meat of seawater fish showed the best sensitivity to gentamicin (96%; 75 isolates) and cefoxitin (92%; 72 isolates). On the other hand, resistance to ampicillin occurred most frequently (87%; 68 isolates). M4 Journal of Food Science r Vol. 00, Nr. 0, 2014
and others (2012) reported the highest resistance of S. aureus isolates against penicillin. Albuquerque and others (2007) isolated S. aureus from fish stalls and hands, nasal, and oral cavities of fish handlers. As reported, all S. aureus isolates were also resistant to ampicillin. Moreover, multidrug resistance was confirmed in 44% of isolates. Multiresistant coagulase-negative staphylococci isolates were also found in our study (Table 3). Multiresistance against 2 antibiotics was confirmed in 25 isolates (32%), against 3 antibiotics in 30 isolates (38.5%), against 4 antibiotics in 8 isolates (10%), and against 5 antibiotics in 2 isolates (2.5%) out of 78. These results correspond with findings of Resch and others (2008) and Perreten and others (1997). They confirmed that strains of coagulase-negative staphylococci isolated from fish products are often resistant to 1 or several antibiotics, thus becoming a source of gene-encoded antimicrobial resistance.
Conclusion The results of this study confirmed the occurrence of mono- and multiresistant coagulase-negative staphylococci in the meat of 3 marine fish and stressed the importance of species identification in CoNS, as the rate of antimicrobial resistance differed significantly among species of staphylococci tested. The study also testified to an alarming increase of antimicrobial resistance in staphylococcal isolates from seafood in the last decade. Although CoNS in fish is not a food poisoning issue, fish may serve as indicators of the spread of antibiotic resistant bacteria/genes in the environment. Therefore, the reduction of the use of antibiotics in aquaculture, permanent control of the food chain aimed at the presence of both causative agents of infections and drug-resistant bacteria, adopting preventive measures related to environmental hygiene as well as regular monitoring of actual antimicrobial resistance are effective tools to prevent the spread of antimicrobial resistance worldwide.
Acknowledgment This study was supported by the project VEGA 1/0067/13 and KEGA 010 UVLF-4/2011.
Author Contributions I. Regecov´a staphylococci; identified by MALDI BioTyper (TM ) system, collected test data and drafted the manuscript. M. Pipov´a isolated staphylococci, designed the study, and drafted the manuscript. P. Jevinov´a detected antimicrobial resistance in staphylococci. K. Maruˇskov´a detected 16S rDNA sequence in staphylococci by PCR. V. Kmeť staphylococci identified by MALDI BioTyper (TM ) system. P. Popelka collected samples and detected nuclease activity in staphylococci.
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Bull Eur Ass Fish Pathol 24(3):137. Kusuda R, Sugiyama A. 1981. Studies on characters of Staphylococcus epidermidis isolated from diseased fishes – 1. On the morphological, biological, and biochemical properties. Fish Pathol 16:15–24. L’Abee-Lund TM, Sørum H. 2001. Class 1 integrons mediate antibiotic resistance in the fish pathogen Aeromonas salmonicida worldwide. Microb Drug Resist 7(3):263–72. Lambe Jr. DW, Ferguson KP, Keplinger JL, Gemmell CG, Kalbfleisch JH. 1990. Pathogenicity of Staphylococcus lugdunensis, Staphylococcus schleiferi, and three other coagulase-negative staphylococci in a mouse model and possible virulence factors. Can J Microbiol 36(7):455–63. Lina G, Quaglia A, Reverdy ME, Leclercq R, Vandenesch F, Etienne J. 1999. Distribution of genes encoding resistance to macrolides, incosamides, and streptogramins among staphylococci. Antimicrob Agents Chemother 43(5):1062–6.
Martin MF, Liras P. 1989. Organization and expression of genes involved in the biosynthesis of antibiotics and other secondary metabolites. Annu Rev Microbiol 43:173– 206. Mass WK. 1986. Antibiotics in laboratory medicine. 2nd ed. New York: Williams and Wilkins. 899 pp. Mazzeo MF, Sorrentino A, Gaita M, Cacace G, Di Stasio M, Facchiano A, Comi G, Malorni A, Siciliano RA. 2006. Matrix-assisted laser desorption ionization-time of flight mass spectrometry for the discrimination of food-borne microorganisms. Appl Environ Microbiol 72(2):1180–9. Ozaktas T, Taskin B, Gozen AG. 2012. High level multiple antibiotic resistance among fish surface associated bacterial populations in non-aquaculture freshwater environment. Water Res 46(19):6382–90. Perreten V, Giampa N, Schuler-Schmid U, Teuber M. 1998. Antibiotic resistance genes in coagulase-negative staphylococci isolated from food. System Appl Microbiol 21(1):113– 20. Perreten V, Schwarz F, Cresta L, Boeglin M, Dasen G, Teuber M. 1997. Antibiotic resistance spread in food. Nature 389:801–2. Pfaller MA, Herwaldt LA. 1988. Laboratory, clinical and epidemiological aspects of coagulase negative staphylococci. Microbiology 1(3):281–99. Quinn PJ, Carter ME, Markey B, Carter GR. 1999. Clinical veterinary microbiology. London, UK: Elsevier Ltd. 651 pp. Rhodes G, Huys G, Swings J, McGann P, Hiney M, Smith P, Pickup RW. 2000a. Distribution of oxytetracycline resistance plasmids between aeromonads in hospital and aquaculture environments: implication of Tn1721 in dissemination of the tetracycline resistance determinant Tet A. Appl Environ Microbiol 66(9):3883–90. Rhodes LD, Grayson TH, Alexander SM, Strom MS. 2000b. Description and characterization of IS994, a putative IS3 family insertion sequence from the salmon pathogen, Renibacterium salmoninarum. Gene 244(1–2):97–107. Resch M, Nagel V, Hertel C. 2008. Antibiotic resistance of coagulase-negative staphylococci associated with food and used in starter cultures. Intl J Food Microbiol 127(1–2):99– 104. Rodrigues MJ, Ho P, Lopez-Caballero ME, Vaz-Pires P, Nunes ML. 2003. Characterization and identification of microflora from soaked cod and respective salted raw materials. Food Microbiol 20(4):471–81. doi: 10.1016/S0740-0020(02)00086. Santschi EM. 2006. Prevention of postoperative infections in horses. Vet Clin North Am Equine Pract 22(2):323–34. Schmidt AS, Bruun MS, Dalsgaard I, Pedersen K, Larsen JL. 2000. Occurrence of antimicrobial resistance in fish-pathogenic and environmental bacteria associated with four Danish rainbow trout farms. Appl Environ Microbiol 66(11):4908– 15. Smole SC, King LA, Leopold PE, Arbeit RD. 2002. Sample preparation of Gram-positive bacteria for identification by matrix assisted laser desorption/ionization time-of-flight. J Microbiol Methods 48(2–3):107–15. Sørum H. 2000. Farming of Atlantic salmon – an experience from Norway. Acta Vet Scand 93:129–34. Sørum H. 2006. Antimicrobial drug resistance in fish pathogens. In: Aarestrup FM, editor. Antimicrobial resistance in bacteria of animal origin. Washington, D.C.: American Society for Microbiology Press. p 213–38. ´ STN EN ISO 6887-3/01. 2003. Mikrobiol´ogia potrav´ın a krm´ıv. Uprava analytick´ych vzoriek, ˇ pr´ıprava z´akladnej suspenzie a desaťn´asobn´ych rieden´ı na mikrobiologick´e sk´uˇsanie. Casť 3: ˇSpecifick´e pokyny na u´ pravu r´yb a ryb´ıch v´yrobkov. Bratislava, SR: SUTN. ´ 16 pp. STN EN ISO 6888-1/A1. 2004. Mikrobiol´ogia potrav´ın a krm´ıv. Horizont´alna met´oda stanoveˇ nia poˇctu koagul´azopozit´ıvnych stafylokokov (Staphylococcus aureus a ďalˇsie druhy). Casť 1: ´ Met´oda s pouˇzit´ım Bairdovho-Parkerovho agarov´eho m´edia. Bratislava, SR: SUTN. 14 pp. STN ISO 3100-2. 1999. M¨aso a m¨asov´e v´yrobky. Odber vzoriek a pr´ıprava analytick´ych vzoriek. ˇ ´ Casť 2: Pr´ıprava analytick´ych vzoriek na mikrobiologick´e sk´uˇsanie. Bratislava, SR: SUTN. 10 pp. Strommenger B, Kettlitz C, Werner G, Witte W. 2003. Multiplex PCR assay for simultaneous detection of nine clinically relevant antibiotic resistance genes in Staphylococcus aureus. J Clin Microbiol 41(9):4089–94. Varvarigos P. 2001. Gram-positive cocco-bacteria (Micrococcaceae, Streptococcaceae) causing systemic disease in intensively farmed fish. VETCARE Veterinary Services to Aquaculture and Distribution of Fish Health Products. Athens, Greece. http://www.vetcare.gr/ Gram_positive_cocci.htm. Last update May 25th, 2001. V´azquez-S´anchez D, L´opez-Cabo M, Sa´a-Ibusquiza P, Rodr´ıguez-Herrera JJ. 2012. Incidence and characterization of Staphylococcus aureus in fishery products marketed in Galicia (Northwest Spain). Intl J Food Microbiol 157(2):286–96. WHO. 2006. Antimicrobial use in aquaculture and antimicrobial resistance. Report of a Joint FAO/OIE/WHO Expert Consultation on Antimicrobial use in Aquaculture and Antimicrobial Resistance. Seoul, Republic of Korea, 13–16 June 2006, Geneva, WHO, 97 pp (available at ftp://ftp.fao.org/ag/agn/food/aquaculture_rep_13_16june2006.pdf). Zadoks RN, Watts JL. 2009. Species identification of coagulase-negative staphylococci: genotyping is superior to phenotyping. Vet Microbiol 134(1–2):20–8.
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Staphylococci in sea fish . . .
Seventy-eight isolates of staphylococci obtained from the meat of Theragra chalcogramma, Scomber scombrus, and Clupea harengus were identified and tested in this study. 16S rDNA sequence specific for the genus Staphylococcus was detected in all isolates with the help of PCR method. All of 78 isolates were coagulase-negative, and DNAse activity was only confirmed in 4 of them. The following species of staphylococci were identified using MALDI-TOF mass spectrometry: S. warneri (52%), S. epidermidis (33%), S. haemolyticus (6.4%), S. pasteuri (3.8%), S. sciuri (1.2%), S. capitis (1.2%), and S. hominis (1.2%). Antimicrobial resistance to 7 antibiotics was determined in each isolate with the help of agar dilution method. In general, resistance against ampicillin was observed in majority of isolates (87%). On the contrary, the best sensitivity of CoNS was determined to gentamicin (96%). Only 1 S. warneri strain showed resistance to cefoxitin. Furthermore, 83% of staphylococcal isolates were simultaneously resistant to 2 or more antibiotics.
Abstract:
Keywords: antibiotic, fish, nuclease, resistance, Staphylococcus
This study confirmed the need of monitoring antimicrobial resistance in coagulase-negative staphylococci not only in the meat of slaughter animals but also in retail marine fish. The results showed that MALDITOF mass spectrometry is useful, accurate, and rapid method for species identification of food pathogens including Staphylococcus spp.
Introduction The prophylactic use of antibiotics in fish farms has led to a rise in the number of resistant bacteria (Cabello 2006). Aquaculture promotes the production of various sizes and types of aquatic organisms, and the use of antibiotics and drugs in the fish industry is complicated because of the need to administer the compounds usually direct into the water where the organisms live. In addition, some countries, such as Norway, utilize natural structures like fjords for fish farming and, for this reason, there are concerns about the wastes that collect in the fjord bottoms, which can lead to contamination of the environment with antibiotics and subsequently to the emergence of resistant bacteria in environment (FAO/NACA/WHO 1997). Strikingly, these bacteria can transfer the resistance genes to human pathogens (Rhodes and others 2000a). The selection pressure applied by the antibiotics used in clinics and agriculture (Martin and Liras 1989) caused an increase of antimicrobial resistance in fish pathogens (Davies and others 1999; Rhodes and others 2000a,b; Schmidt and others 2000; Sørum 2000; L’Abee-Lund and Sørum 2001; Sørum 2006) resulting in the risk of possible passage of both resistant bacteria and their determinants of antimicrobial resistance to various bacteria of terrestrial animals and human beings, including pathogens (Cabello 2006). The use of antimicrobial agents is not the only
MS 20130840 Submitted 6/20/2013, Accepted 2/4/2014. Authors Regecov´a, Pipov´a, Jevinov´a, Maruskov´a and Popelka are with Dept. of Food Hygiene and Technology, Univ. of Veterinary Medicine and Pharmacy, Komensk´eho 73, 041 81, Koˇsice, Slovak Republic. Author Kmet’ is with Inst. of Animal Physiology, Slovak ˇ esovej 4-6, 040 01, Koˇsice, Slovak Republic. Direct Academy of Sciences Koˇsice, Solt´ inquiries to author Regecov´a (E-mail: [email protected]).
R C 2014 Institute of Food Technologists
doi: 10.1111/1750-3841.12429 Further reproduction without permission is prohibited
cause of the occurrence and spread of antimicrobial mono- and multiresistance. Spontaneous mutations of food bacteria and spread of resistant bacterial strains in the absence of selective pressure may also contribute to an increase of antimicrobial resistance among food bacteria (B´ıreˇs and others 2008). In recent years, the food chain is one of the main routes of transmission of resistant bacteria between animal and human populations (Mass 1986; Ito and others 2003). In the case of primary contamination, slaughter animals are carriers of multiresistant strains. The food chain can also be contaminated secondarily at the time of processing, transporting, and storage of raw materials or finished products. Thus, resistant bacteria may come from both human and animal sources (Bardoˇn and others 2007). Recently, an increased resistance is also observed among strains of Staphylococcus spp., this resulting in several diseases, some of them being fatal. Staphylococci are not part of the normal fish microflora (Huss 1988). Their presence on fish is an indication of (a) postharvest contamination due to poor personnel hygiene and (b) disease in fish (Huss 1988; Austin and Austin 2007). In addition, CoNS in aquaculture mainly serves as a reservoir of resistance genes that can be transferred to bacteria in aquatic environments, or further disseminated and ultimately end up in human pathogens miscellaneous (Perreten and others 1998; WHO 2006). The level of antimicrobial resistance varies significantly among species of staphylococci. As reported by Drozenov´a and Petr´aˇs (2000), a high proportion of resistant strains occurs in species and subspecies that are the most common causative agents of nosocomial infections (S. epidermidis, S. hominis subsp. hominis, S. haemolyticus, and S. hominis subsp. novobiosepticus). Transferable resistance, which also occurs in the genus Staphylococcus, does not respect any phylogenetical, geographical, or ecological limits. Genes
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Practical Application:
Staphylococci in sea fish . . . of resistance are distributed horizontally among bacteria of land animals, fish, and human populations (B´ıreˇs and others 2009). However, no data have yet been presented regarding antimicrobial resistance of CoNS from retail fish (Hammad and others 2012). Moreover, no studies aimed at species identification of CoNS isolates from marine fish with the help of MALDI-TOF mass spectrometry are available. Therefore, this study is focused on both species identification and determination of antimicrobial resistance in coagulase-negative staphylococci isolated from the meat of Atlantic mackerel, Alaska pollock, and Atlantic herring.
Materials and Methods
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Isolation of staphylococci Staphylococci were isolated from samples of frozen fish meat as follows: 5 samples of Alaska pollock (Theragra chalcogramma) originating from fishing area FAO 61 (China), 4 samples of Atlantic mackerel (Scomber scombrus) originating from fishing area FAO 27 (Ireland), and 6 samples of Atlantic herring (Clupea harengus) originating from fishing area FAO 27 (Norway). Samples were taken aseptically from whole fish meat after its defrosting, removal of the consumer packaging, and the skin of fish in accordance with the requirements of STN ISO 3100-2 (1999). Basic and further subsequent decimal dilutions were prepared according to STN EN ISO 6887-3/01 (2003). Staphylococci were isolated from fish samples in accordance with STN EN ISO 6888-1/A1 (2004). The first 2 decimal dilutions were spread in an amount of 0.1 mL on the surface of Baird-Parker medium (Oxoid, Basingstoke, Hampshire, U.K.). Inoculated media were incubated at 37 °C for 24 to 48 h. Based on their characteristic appearance, both typical and atypical colonies of staphylococci were further identified. Plates containing 15 to 150 colonies were used for this purpose.
CCM 4223 served as positive control. Reaction mixture in a volume of 50 μL contained 1 μL genomic DNA; 10 mmol/L Tris-HCl (pH 8.8); 3 mmol/L MgCl2; 200 μmol/L dNTP; 12,5 pmol/L of each primer, and 1U Taq DNA polymerase (Ecoli s.r.o., Bratislava, Slovakia). The PCR protocol was as follows: initial denaturation at 94 °C for 3 min, 30 cycles consisting of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s. Final extension at 72 °C for 4 min followed the last cycle. PCR products were separated in a 2% agarose gel stained TM with Goldview Nucleic acid stain (Beijing SBS Genetech Co. LTD, Beijing, China) and visualized using the DNR Bio Imaging R Sytem (MiniBIS Pro , Jerusalem, Israel). The species of staphylococci were subsequently identified with the help of MALDI BioTyper(TM) system (Bruker Daltonics, Billerica, Mass., U.S.A.) based on protein “fingerprints” measured by MALDI-TOF mass spectrometry. As staphylococci are Grampositive bacteria, lysates of bacterial cells had to be prepared according to the instruction of producer (Bruker Daltonics 2008) prior to their identification.
Determination of susceptibility to selected antibiotics Susceptibility of staphylococcal isolates to 7 antibiotics was determined by the agar dilution method according to the procedure described by CLSI document M07-A8 (2009). M¨ueller-Hinton agar was used as the test medium (Hi-Media, Mumbai, Maharashtra India). Test plates containing different concentrations of erythromycin (0.25; 0.5; 1.0; 2.0; 4.0; 8.0 mg/L); tetracycline (2.0; 4.0; 8.0; 16.0 mg/L); ampicillin (0.25; 0.5; 1.0 mg/L); gentamicin (2.0; 4.0; 8.0; 16.0 mg/L); cefoxitin (2.0; 4.0; 8.0; 16.0; 32.0 mg/L); oxacillin (0.125; 0.25; 0.5; 1.0; 2.0; 4.0; 8.0 mg/L); and penicillin (0.06; 0.125; 0.25; 0.5 mg/L) were used for the determination of minimum inhibitory concentrations (MICs). Drops of 24-h bacterial suspensions adjusted to the 0.5 McFarland turbidity standard were placed in parallel on the surface of each test plate. Inoculated plates were then incubated at 37 °C for 24 h. After incubation, the lowest concentrations of antibiotics that inhibited the visible growth of staphylococcal strains were determined. Results were evaluated according to CLSI document M100-S20 (2010).
Determination of plasma-coagulase activity Colonies of staphylococci were individually reinoculated into test tubes containing 2 mL of Brain Heart Infusion Broth (Oxoid). After 18 to 24 h of incubation at 37 °C, 0.1 mL of each bacterial culture was added to another test tube with 1 mL of reconstituted freeze-dried rabbit plasma (Stafylo PK, Imuna Pharm, Sˇ ariˇssk´e Michal’any, Slovakia). Inoculated test tubes were incubated at 37 Results and Discussion °C. Formation of coagulum was considered as a positive reaction. The bacterial community associated with fish is generally related Results were evaluated after 1, 2, 3, 6, and 24 h. with the features of the aquatic habitat, such as salinity and the bacterial load in the water (Cahill 1990). Retail fish microflora can be Determination of nuclease activity influenced by the level of hygiene, where the role of food handlers Each isolate was inoculated on the surface of DNase agar in preparation of fish is crucial in determining the hygienic sta(Oxoid) where the production of thermostable deoxyribonucle- tus of the final product, with poor handling resulting in increased ase was confirmed after a 24-h-incubation at 37 °C. Colonies likelihood of contamination by human-borne microbes including producing DNase hydrolyzed the deoxyribonucleic acid (DNA) multidrug-resistant and/or enterotoxigenic bacteria (Hammad and contained in the medium. When the medium was flooded and others 2012). The dominance of Staphylococcus spp. among groups acidified with 1 N hydrochloric acid, the DNA precipitated, the of sea fish bacterial isolates was also confirmed by Hashizume and medium became turbid and clear zones appeared around DNase- others (2005). positive colonies. As seen in Table 1, 78 strains of staphylococci were isolated from samples of frozen meat of Alaska pollock, Atlantic mackerel, and Genus and species identification Atlantic herring in this study, all of them being coagulase-negative. The total genomic DNA was isolated from staphylococcal Strains of CoNS can cause serious nosocomial infections. Such strains as described by Hein and others (2005) and further tested strains frequently possess various virulence factors including anby PCR method according to Strommenger and others (2003). timicrobial resistance and the ability to form a biofilm. Correct Primers 16S1 (5-CAGCTCGTGTCGTGAGATGT) and 16S2 identification and typing of CoNS is therefore important from (5-AATCATTTGTCCCACCTTCG) rDNA (Generi Biotech both clinical and epidemiological points of view (Chomouck´a s.r.o., Hradec Kr´alov´e, Czech Republic) typical for the genus 2009). DNA sequence-based species identification is currently the Staphylococcus were used for PCR. The reference strain S. aureus most accurate method for CoNS identification and is considered M2 Journal of Food Science r Vol. 00, Nr. 0, 2014
Staphylococci in sea fish . . . Table 1–Number of staphylococcal species in isolates from sea Table 2–Number of resistant (R), intermediately susceptible (I), fish. and susceptible (S) isolates of staphylococci from the meat of sea fish. Species Alaska Atlantic Atlantic of pollock mackerel herring Total Alaska Atlantic Atlantic staphylococci (n = 27) (n = 24) (n = 27) (n = 78) pollock mackerel herring Total (n = 27) (n = 24) (n = 27) (n = 78) S. warneri 12 13 16 41 S. epidermidis 13 11 2 26 R I S R I S R I S R I S S. haemolyticus – – 5 5 Penicillin 10 – 17 19 – 5 18 – 9 47 – 31 S. pasteuri 1 – 2 3 Ampicillin 24 – 3 19 – 5 25 – 2 68 – 10 S. sciuri 1 – – 1 Tetracycline 4 4 19 1 5 18 – 5 22 5 14 59 S. capitis – – 1 1 Oxacillin 2 – 25 15 – 9 15 – 12 32 – 46 S. hominis ssp. Hominis – – 1 1 Erythromycin 19 8 – 17 6 1 15 10 2 51 24 3 Gentamicin – 3 24 – – 24 – – 27 – 3 75 Cefoxitin – 1 26 – 2 22 1 2 24 1 5 72
They found that most S. epidermidis, S. warneri, S. Hominis, and S.haemolyticus strains tested produced DNAse. Cunha and others (2006) reported the production of DNase in 9 isolates of S. epidermidis and 2 isolates of S. haemolyticus out of 117 CoNS isolates tested. However, the authors did not confirm any DNase activity in S. warneri isolates that is in contrast to our study, where the DNase activity was detected in 3 isolates of S. warneri and 1 isolate of S. epidermidis.
Testing of antimicrobial resistance All of 41 S. warneri isolates showed the most frequent resistance against ampicillin (95%) and did not show any resistance against gentamicin. Resistance to cefoxitin was confirmed in the only strain of S. warneri isolated from the meat of Atlantic herring (Table 2). Thirty-four S. warneri isolates were resistant to at least 2 antibiotics tested. Simultaneous resistance to 2, 3, 4 (penicillin, erythromycin, oxacillin, and ampicillin), or 5 (penicillin, ampicillin, tetracycline, erythromycin, and oxacillin) out of 7 antibiotics tested was confirmed in 17, 12, 4, and 1 isolate of S. warneri, respectively. Similarly, Gil and others (2000) isolated 3 strains of S. warneri from Oncorhynchus mykiss, these being resistant to ampicillin and penicillin. In the study of Hammad and others (2012), resistance to ampicillin in S. warneri isolates was combined with simultaneous resistance to another 4 antibiotics (erythromycin, gentamicin, oxacillin, and tetracycline). Abrahim and others (2010) confirmed resistance to oxacillin in 2 strains of S. warneri gained from 269 isolates of freshwater fish. Moreover, the resistance to this antibiotic was also confirmed in 1 isolate of S. haemolyticus and 3 isolates of S. epidermidis. During the last decade, S. epidermidis and S. haemolyticus have gained increasing attention as nosocomial pathogens in humans. They cause postoperative infections as common complication in surgery that can result in intensive postoperative care, prolonged hospitalization, increased costs, and mortality (Santschi 2006). Moreover, S. epidermidis has been reported previously as a fish pathogen in some marine and freshwater fish in Japan, Taiwan, and Greece (Kusuda and Sugiyama 1981; Varvarigos 2001). It may be present in the fish throughout the year, but the disease is induced by a sudden rise in water temperatures or other stress factors in the aquatic environment. It usually appears in the spring and causes problems throughout the summer (Varvarigos 2001). Twenty-six S. epidermidis isolates from the meat of 3 sea fish showed resistance to tetracycline, erythromycin, and oxacillin. However, resistance to ampicillin was confirmed most frequently. Moreover, majority of S. epidermidis isolates from the meat of Atlantic mackerel (10 strains) showed resistance to penicillin. Kubilay and Uluk¨oy (2004) also reported upon the first isolation of S. Vol. 00, Nr. 0, 2014 r Journal of Food Science M3
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as the gold standard (Zadoks and Watts 2009). The presence of 16S rDNA sequence typical for Staphylococcus spp. was confirmed in all 78 isolates from sea fish. Even though the 16S rDNA gene is considered to be highly conserved with limited discriminatory power especially in closely related staphylococcal species (Heikens and others 2005; Ghebremedhin and others 2008). Therefore, MALDI-TOF mass spectrometry based on bacterial protein profiles was used for species identification of staphylococci. Sample preparation and its analysis require only several minutes that make this method an attractive alternative to previous time-consuming conventional biochemical tests (Carbonnelle and others 2007). MALDI-TOF MS of intact bacterial cells was already used for the detection and identification of 24 different foodborne pathogens and food spoilage bacteria including staphylococci (Mazzeo and others 2006). The genus Staphylococcus usually shows a spectrum with 50 to 75 signals of 1.8 to 11 kDa (Smole and others 2002). Bal´azˇ ov´a (2010) reported that except for the genus-specific signal with the value of 9625 Da, typical signals for individual staphylococcal species can be visually distinguished. Typical signal values for S. aureus are 3442, 4304, and 6888 Da; for S. hominis 4828 Da and for S. haemolyticus 3230 Da. Based on these signals, 7 species were identified among 78 isolates of staphylococci (Table 1) as follows: S. warneri (52%), S. epidermidis (33%), S. haemolyticus (6.4%), S. pasteuri (3.8%), S. sciuri (1.2%), S. capitis (1.2%), and S. hominis (1.2%). Hammad and others (2012) obtained 10 isolates of MRSA and MR-CoNS from 10 samples (5%, 10/200) of fish products collected from 10 shops (40%, 10/25) belonging to 4 supermarket chains. Isolates were identified as S. aureus (n = 5), S. haemolyticus (n = 2), S. warneri (n = 2), and S. pasteuri (n = 1). Abrahim and others (2010) investigated freshwater fish and the environment of fish markets of Northern Greece. A total of 269 samples were examined, consisting of 71 rainbow trouts (Oncorhynchus mykiss), 65 gibel carps (Carassius gibelio), and 133 environmental samples swabbed from various surfaces at fish markets. Staphylococcus spp. was isolated from 27% of samples, and 16%, 9%, 0.7%, and 0.4% of samples inspected were positive for S. aureus, S. epidermidis, S. warneri, S. hominis subsp. hominis, and S. haemolyticus, respectively. Another study also confirmed the presence of CoNS in sea fish (Rodrigues and others 2003), these being identified as S. epidermidis, S. hominis, S. warneri, S. haemolyticus, S. simulans, and S. saprophyticus. DNase activity is important to distinguish between pathogenic staphylococci and nonpathogenic resident flora. For pathogenesis, DNase is as important as coagulase (Pfaller and Herwaldt 1988). However, this test is not entirely reliable as an indicator of pathogenicity because it has been estimated that about 18% of coagulase-negative staphylococci show DNase activity (Quinn and others 1999). Production of DNase by coagulase-negative staphylococci was also confirmed by Lambe and others (1990).
Staphylococci in sea fish . . . Table 3–Phenotypes of mono-, multi-, and polyresistance among Similar results have also been published by Ozaktas and others staphylococcal isolates from the meat of sea fish. (2012). In their study, the resistance against ampicillin was about Anttibiotics Amp Ery Pen-Amp Pen-Ery Ery-Amp Ery-Oxa Oxa-Amp Pen-Ery-Amp Pen-Ery-Oxa Pen-Tet-Amp Pen-Oxa-Amp Ery-Oxa-Amp Pen-Ery-Oxa-Amp Pen-Tet-Ery-Amp Pen-Tet-Ery-Amp-Oxa Pen-Ery-Oxa-Amp-Cef Total
Alaska pollock
Atlantic mackerel
5 3 3 0 10 0 0 3 0 1 0 0 0 1 1 0 27
1 0 1 1 1 1 2 5 2 0 4 0 5 1 0 0 24
Atlantic 92%. Resistance to β-lactams is dominant in coagulase-positive herring Staphylococcus species isolated from fish products. V´azquez-S´anchez 2 2 2 0 4 0 0 4 0 0 8 5 1 0 0 1 27
Pen: penicillin, Amp: ampicillin, Oxa: oxacillin, Ery: erythromycin, Tet: tetracycline, Cef: cefoxitin.
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epidermidis from gilthead sea bream (Sparus aurata) reared in the southwest of Turkey and its multiresistance to penicillin, ampicillin, erythromycin, and oxacillin. The presence of S. haemolyticus was only confirmed in frozen meat of Atlantic herring (Table 1). These 5 isolates showed multiresistance to erythromycin, oxacillin, and ampicillin. Similarly, Hammad and others (2012) reported that S. haemolyticus strains isolated from fresh fish meat were multiresistant to ampicillin, oxacillin, tetracycline, and gentamicin. Simultaneous resistance to erythromycin and ampicillin was also confirmed in S. sciuri isolated from Alaska Pollock, while S. capitis and S. hominis spp. hominis isolates from the meat of Atlantic herring showed mainly monoresistance to erythromycin. As reported by Lina and others (1999), erm(A) and erm(C) are the most frequent determinants of resistance to erythromycin in Staphylococcus spp. Macrolides have been known for 15 decades, and, since the introduction of erythromycin into therapy, a number of these molecules have been developed for clinical use. For years, these antibiotics have represented a major alternative to the use of penicillins and cephalosporins for the treatment of infections due to Gram-positive microorganisms. However, the worldwide development of macrolide resistance with wide variations, according to both the country and the bacterial species, has sometimes constrained to limit the use of these antibiotics to certain indications. Although the evolution of the macrolide class has been marked, in the 1990s, by the development of semisynthetic macrolides with improved pharmacokinetics and tolerability, these new derivatives have proved unable to overcome erythromycin resistance (Denis and others 1999). As to our study, resistance to erythromycin was also confirmed in 3 isolates of S. pasteuri. Among them, multiresistance to 4 antibiotics (penicillin, oxacillin, ampicillin, and erythromycin) was observed in 1 isolate from Alaska pollock meat. Moreover, 2 S. pasteuri isolates from the meat of Atlantic herring showed multiresistance to 3 antibiotics (penicillin, oxacillin, and ampicillin). These results cannot be compared with another published data because they are not yet available. As seen in Table 2, all staphylococci isolated from the meat of seawater fish showed the best sensitivity to gentamicin (96%; 75 isolates) and cefoxitin (92%; 72 isolates). On the other hand, resistance to ampicillin occurred most frequently (87%; 68 isolates). M4 Journal of Food Science r Vol. 00, Nr. 0, 2014
and others (2012) reported the highest resistance of S. aureus isolates against penicillin. Albuquerque and others (2007) isolated S. aureus from fish stalls and hands, nasal, and oral cavities of fish handlers. As reported, all S. aureus isolates were also resistant to ampicillin. Moreover, multidrug resistance was confirmed in 44% of isolates. Multiresistant coagulase-negative staphylococci isolates were also found in our study (Table 3). Multiresistance against 2 antibiotics was confirmed in 25 isolates (32%), against 3 antibiotics in 30 isolates (38.5%), against 4 antibiotics in 8 isolates (10%), and against 5 antibiotics in 2 isolates (2.5%) out of 78. These results correspond with findings of Resch and others (2008) and Perreten and others (1997). They confirmed that strains of coagulase-negative staphylococci isolated from fish products are often resistant to 1 or several antibiotics, thus becoming a source of gene-encoded antimicrobial resistance.
Conclusion The results of this study confirmed the occurrence of mono- and multiresistant coagulase-negative staphylococci in the meat of 3 marine fish and stressed the importance of species identification in CoNS, as the rate of antimicrobial resistance differed significantly among species of staphylococci tested. The study also testified to an alarming increase of antimicrobial resistance in staphylococcal isolates from seafood in the last decade. Although CoNS in fish is not a food poisoning issue, fish may serve as indicators of the spread of antibiotic resistant bacteria/genes in the environment. Therefore, the reduction of the use of antibiotics in aquaculture, permanent control of the food chain aimed at the presence of both causative agents of infections and drug-resistant bacteria, adopting preventive measures related to environmental hygiene as well as regular monitoring of actual antimicrobial resistance are effective tools to prevent the spread of antimicrobial resistance worldwide.
Acknowledgment This study was supported by the project VEGA 1/0067/13 and KEGA 010 UVLF-4/2011.
Author Contributions I. Regecov´a staphylococci; identified by MALDI BioTyper (TM ) system, collected test data and drafted the manuscript. M. Pipov´a isolated staphylococci, designed the study, and drafted the manuscript. P. Jevinov´a detected antimicrobial resistance in staphylococci. K. Maruˇskov´a detected 16S rDNA sequence in staphylococci by PCR. V. Kmeť staphylococci identified by MALDI BioTyper (TM ) system. P. Popelka collected samples and detected nuclease activity in staphylococci.
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Staphylococci in sea fish . . .
