Abstract
As Salmonella is a common foodborne pathogen, the present study aimed to determine the distribution of Salmonella enterica serotypes isolated during 2011–2012 from poultry, swine, cattle, and processed food in Portugal, and to characterize the antimicrobial susceptibility and the extended-spectrum β-lactamases (ESBLs). Results were also compared with data obtained before the implementation of the National Control Program in Poultry and the ban of antimicrobial agents in animal feed in the European Union (EU). A total of 14 serotypes were identified, from 258 isolates recovered, with Salmonella Typhimurium (32.6%, n=84) and Salmonella Enteritidis (10.1%, n=26) being the most common. Salmonella Enteritidis in poultry was less frequent than in previous studies, which might be associated with the implementation of the National Control Program for Salmonella in poultry. Nevertheless, other serotypes seem to occupy this biological niche, and may be more common in human salmonellosis in the future. The majority of isolates (70.2%, n=181) were resistant to at least one class of antimicrobial agent and exhibited higher frequency of resistance to tetracycline (47.7%, n=123) and ampicillin (36.0%, n=93), with Salmonella Typhimurium being the more resistant serotype. Resistance to fluoroquinolones was shown in 8% (n=21) of isolates, a lower value compared to data obtained before 2004. ESBLs producers Salmonella Typhimurium bla CTX-M-1 and Salmonella Enteritidis bla SHV-12 were isolated from swine and poultry, respectively. The bla CTX-M-1 and bla SHV-12 genes were carried on conjugative plasmids of IncHI2replicon types and IncI1, respectively. This was the first report of a bla CTX-M-1 in Salmonella Typhimurium in Portugal. Overall, the results revealed changes in animal origin Salmonella serotypes, mainly emerging serotypes, in frequency of resistance, and in occurrence of ESBLs-producing Salmonella. The control measures taken by the EU seem to have some impact on the resistance rate of some antibiotics such as quinolones. The emergence of ESBLs and its potential spread among animal reservoirs and the food chain highlight the continuous antimicrobial surveillance at the animal level.
Introduction
F
This study aimed to assess the distribution and antimicrobial susceptibility of S. enterica serotypes isolated from livestock samples and processed food of animal origin, and to characterize the ESBLs. Results were also compared with data obtained before the ban of antimicrobial agents in animal feed in EU, and the implementation of a National Control Program in poultry (NPCS, 2012).
Materials and Methods
Strain collection and bacterial identification
Between June 2011 and December 2012, 258 isolates of Salmonella spp. were recovered from poultry (n=144), swine (n=98), and cattle (n=16), in central Portugal. The isolation was performed in a wide range of samples from biologic samples such as blood, fur, neck, liver, feces, and carcasses, and processed food such as meat cuts, hamburgers, or sausages. The isolation was made according to standard methods (ISO 6579/A1, 2007). Briefly, 10 g of sample was suspended in buffered peptone water (Merck, Darmstadt, Germany) (1:10). The suspension was homogenized in a Stomacher (90 s), incubated at 37°C for 18±2 h, after which 0.1 mL and 1.0 mL, respectively, were inoculated in Rappaport-Vassiliadis medium containing Soya peptone (RVS broth) (Oxoid, Cambridge, UK) and in Muller-Kauffmann tetrathionate/novobiocin broth (MKTTn broth) (Merck). The RVS broth was incubated at 41.5±1°C for 24 h±3 h and the MKTTn broth at 37°C±1°C for 24 h±3 h. In a second stage, one loop of each selective enrichment broth was streaked onto the surface of two selective solid media: Hektoen and xylose-lysine-deoxycholate agar (Oxoid, Cambridge, UK). Finally, isolates of presumptive Salmonella (one or two colonies from each sample) were confirmed by means of biochemical tests followed by serotyping according to the White-Kauffmann/Le Minor scheme (Grimont et al., 2007).
Susceptibility testing and ESBLs production
The minimal inhibitory concentrations (MICs) were evaluated using a commercial microdilution broth method with preprepared microplates with a concentration range to each antibiotic (Microscan Panel, Siemens, West Sacramento, CA), according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2013). The microplates used included 33 antimicrobial agents (ampicillin, piperacillin, mezlocillin, cefazolin, ceftazidime, cefotaxime, cefpodoxime, cefepime, cefuroxime, cefoxitin, ampicillin/sulbactam, amoxicillin/clavulanate, cefotaxime/clavulanate, ceftazidime/clavulanate, piperacillin/tazobactam, imipenem, meropenem, ertapenem, aztreonam, norfloxacin, ciprofloxacin, moxifloxacin, levofloxacin, gentamicin, amikacin, tobramycin tetracycline, colistin, chloramphenicol, nitrofurantoin, tigecycline, fosfomycin, and trimethoprim/sulfamethoxazole). The susceptibility breakpoints were interpreted accordingly to CLSI guidelines (CLSI, 2013), while breakpoints of colistin and moxifloxacin were analyzed by the European Committee on Antimicrobial Susceptibility Testing guidelines (EUCAST, 2010). Multidrug resistance was defined as the resistance shown at least to three classes of antibiotics. The double-disc synergy test was additionally used in the isolates that showed resistance to the third- generation cephalosporins and susceptibility to the β-lactamases inhibitors, suggesting ESBL production (Giriyapur et al., 2011).
Polymerase chain reaction (PCR) and gene sequencing
Presence of TEM-type, CTX-M-type, and SHV-type β-lactamase genes was tested by PCR in isolates with ESBL phenotype (Mendonça et al., 2007), as well as the insertion sequence ISEcp1 in CTX-M–positive strains, using a forward primer in the ISEcp1 gene and reverse primer in the bla
CTX-M gene. Presence of mobile genetic elements was assessed by amplification of conserved segments in integrons of classes 1, 2, and 3 (Lévesque et al., 1995). Nucleotide sequencing positive amplicons was performed by Macrogen (Amsterdam, The Netherlands). Sequences were edited by BioEdit Software (Ibis Biosciences, Carlsbad, CA) and identified using BLASTn (
Typing of plasmids
Plasmids were recovered using the QIAGEN Plasmid Mini Kit (Quiagen, Hilden, Germany). The PCR-based Inc/rep typing method was used for the identification of the replicon type of plasmids. It consists of five multiplex-PCRs recognizing three different replicon types and three simplex PCRs for K, F, and B/O replicon types (Carattoli et al., 2005).
Conjugation experiments
Conjugation assays were used to assess the transfer of resistance in ESBL-positive strains, using Escherichia coli strain J53 as recipient (resistant to azide sodium, AziR). Briefly, conjugation experiments were performed in trypticase-soy (TS) broth at a ratio donor/recipient of 1:10 with 10,000×g at 37°C overnight. Selection was performed on MacConkey agar plates containing 1 mg/L of cefotaxime. Lactose-positive colonies were cultured in TS agar containing cefotaxime (1 mg/L) and sodium azide (10 mg/L). The antimicrobial susceptibility of donor strains, recipient E. coli J53, and transconjugants was performed by standard disc diffusion method on Mueller-Hinton agar (Oxoid, Wesel, Germany) following the CLSI recommendations. The MICs for transconjugants, donor strains, and recipient strain were determined by E-test (Biomérieux, Marcy l'Étoile, France).
Results
Diversity of serotypes according to their origin
Among the 258 Salmonella isolates recovered, a total of 14 serotypes were identified, with Salmonella Typhimurium being the most prevalent serotype (32.6%, n=84) followed by Salmonella Enteritidis (10.1%, n=26) (Table 1). Salmonella Typhimurium was mainly isolated from swine samples (58.6%, n=17) and from swine-origin processed food (50.7%, n=35), but was also present in samples from poultry (9.9%, n=11) and cattle (33.3%, n=2). Salmonella Typhimurium represented 48.5% of poultry-food products. Isolates from poultry (n=111) presented 14 different serotypes, with Salmonella Enteritidis (20.7%, n=23) being the most prevalent followed by Salmonella Havana (18.1%). In cattle isolates (n=6), 33.3% (n=2) of Salmonella Typhimurium were identified and 16.7% (n=1) Salmonella Infantis were identified. In processed-food isolates (n=112), Salmonella Typhimurium represent 48.2% (n=54) of isolates, Salmonella Infantis 12.5% (n=14), and Salmonella Enteritidis 2.7% (n=3).
Not determinated serotype.
Antimicrobial resistance of Salmonella spp. isolates
Antimicrobial susceptibility patterns among the isolates according to their origin are shown in Table 2. One hundred eighty one (70.2%) isolates were resistant to at least 1 antimicrobial agent tested. Almost half of the isolates (47.7%, n=123) were resistant to tetracycline and 36.0% (n=93) showed resistance to ampicillin.
Pansusceptible.
All strains were susceptible to ertapenem, imipenem, meropenem, fosfomycin, tigecycline, and amikacin.
The highest frequency levels of resistance were observed for isolates of swine (n=29), namely, to tetracycline (89.7%, n=26), ampicillin (65.5%, n=19), and chloramphenicol (13.7%, n=4); only 1 isolate was pansusceptible. Fifty-three (47.7%) isolates of poultry (n=111) were pansusceptible, and 2 (33.3%) isolates of cattle (n=6) revealed pansusceptibility.
From a total of 112 isolates of processed food, 28 (25%, n=112) showed pansusceptibility. Resistance levels were higher to tetracycline (72.7%, n=24) in isolates of poultry-derivate food and lower in isolates of swine-food products (68.1%, n=47), while resistance to ampicillin was similar in the 2 types of processed food (56.5% [n=39] in swine food and 54.5% [n=18] in poultry products). Resistance to chloramphenicol was observed in 20 isolates (30.0%) of products of swine origin. Multidrug resistance was observed in 6.3% (n=7), 24.1% (n=7), 16.6% (n=1), and 29.5% (n=33) of poultry, swine, cattle, and processed food isolates, respectively.
Antimicrobial resistance patterns of Salmonella serotypes are presented in Table 3. Salmonella Typhimurium (n=84) was the more resistant serotype, with 39.3% (n=33) of isolates multidrug resistant, and showing resistance to tetracycline (88.1%, n=74) and ampicillin (79.8%, n=67). Salmonella Enteritidis (n=26) presented more frequency of resistance to colistin (88.5%, n=23) and ampicillin (15.4%, n=4), with 11.5% (n=3) of isolates being multidrug resistant. Salmonella Infantis (n=20) presented more frequency of resistance to tetracycline (35%, n=7) and to trimethoprim/sulfamethoxazole (15%, n=3), with multidrug resistance being reported in 10% (n=2) of isolates. NTS serotypes not determined (n=86) presented high resistance levels to tetracycline (47.7%, n=41) and ampicillin (22.1%, n=19), with 11.6% (n=10) of isolates being multidrug resistant. Strains of Salmonella Newport, Salmonella Agona, Salmonella Patel, Salmonella Altona, Salmonella Brandenburg, Salmonella London, and Salmonella Heidelberg were pansusceptible.
Serotypes Newport, Agona, Altona, London. Branderburg, Patel, and Heidelberg.
Serotypes not determined.
Pansusceptible.
PCR and DNA sequencing of ESBL producers
Salmonella Typhimurium Sal25 and Salmonella Enteritidis Sal353 were resistant to several β-lactams, including extended-spectrum cephalosporins, and the former also showed resistance to aminoglycosides. Sal25 and Sal353 strains were resistant to cefotaxime (MIC ≥256 mg/L and MIC 12 mg/L, respectively) and ceftazidime (MIC 8 mg/L and MIC ≥256 mg/L, respectively). Both strains showed a typical double-disc synergy that suggested ESBL production. Nucleotide sequencing of the amplicons identified the bla CTX-M-1 and bla TEM-1 resistance genes in Salmonella Typhimurium Sal25 and the bla SHV-12 resistance gene in Salmonella Enteritidis Sal353.
Class 1 integrons were detected in both strains Sal25 and Sal353. The nucleotide sequencing of class 1 integron from the aminoglycoside-resistant Sal25 strain showed only the aadA1 gene, which confers resistance to streptomycin and spectinomycin.
Conjugation assays
Resistance to β-lactams was transferred from Salmonella Sal25 and Sal353 to E. coli J53 recipient strain by conjugation. Transconjugants and the parental strain Sal25 were positive for ISEcp1 sequence, located upstream of bla CTX-M-1 gene. The incompatibility group of ESBL's plasmid was identified as IncHI2 in Sal25 strain and IncI1 in Sal353 strain. Class 1 integrons were also transferred, confirmed by PCR.
Discussion
In Europe, more than 100,000 salmonellosis human cases are reported each year (EFSA, 2012). In Portugal, little information is available on antimicrobial resistance of Salmonella. The main objective of this study was to assess the distribution and antimicrobial susceptibility of S. enterica serotypes isolated from animals and processed food, and to characterize the ESBLs. Moreover, analysis of the results allowed us to compare with data obtained before the antimicrobial agents were banned in animal feed in EU in 2006, and the implementation of the National Control Program for Salmonella in poultry.
Salmonella Typhimurium and Salmonella Enteritidis are reported as the main sources of salmonellosis (EFSA, 2012). Here, they represented 42.7% (n=110) of the isolates (Table 1), suggesting that other serotypes, usually referred to as being less common, might becoming important as a cause of human salmonellosis.
Salmonella Typhimurium was the major serotype isolated in swine (58.6%, n=17), which is in concordance with other reports (EFSA, 2012). However, we found a higher frequency of this serotype when compared with previous Portuguese studies (Clemente et al., 2013; Da Silva and Carneiro, 2006; DGV, 2007). Salmonella Enteritidis was the prevalent serotype in poultry (20.7%, n=23), while it was present in 44% and 78% in isolates collected in 1999 and 2003/2004, respectively (Antunes et al., 2003; Da Silva and Carneiro, 2006). A recent study showed a reduction of Salmonella Enteritidis in poultry as well (32.8%) (Clemente et al., 2013). The administration of Salmonella vaccine as part of the National Program for Control for Salmonella implemented after 2008 might be associated with the reduction in this host and the emergence of others serotypes, including Typhimurium. Our data show an increase of Salmonella Typhimurium in poultry comparing with previous reports (Antunes et al., 2003; Da Silva and Carneiro, 2006; Clemente et al., 2013), suggesting an occupation of a new biological niche. Salmonella Typhimurium was also the serotype with the greatest frequency and diversity for antimicrobial resistance, which is a public health concern. The results obtained, and compared with previous data, indicated that the control measures to decrease the Salmonella dissemination in poultry have been effective, but they should be extended to swine (Rasschaert et al., 2012). Salmonella serotypes Infantis, Mbandaka, Newport, Virchow, Derby, and Agona have been consistently in the top 10 most frequently reported serotypes in Europe, although not so common as Typhimurium and Enteritidis (EFSA, 2012). All are known to induce gastroenteritis in a broad range of unrelated host species (Eurosurveillance Editorial Team, 2012), and our results revealed an emergence of these serotypes, especially in poultry, which might be more common in the near future (Clemente et al., 2013).
In Portugal, the use of antimicrobial agents in animal therapeutics increased to 180 tonnes (US 198.4 tons) in 2010 (DGV, 2010). A high rate of resistance to ampicillin and tetracycline was observed, especially in isolates from processed food (Table 2) (ampicillin and tetracycline were the main antimicrobial agents used in animal therapeutic agents in Portugal) (DGV, 2010). The high frequency of resistance to ampicillin and tetracycline had been described in uncooked food and in foods ready for consumption (Da Silva et al., 2006; EFSA, 2012; Clemente et al., 2013), which may compromise effective treatment with these antimicrobials in case of infection (Hohmann, 2001). A decrease in the frequency of fluoroquinolone resistance was observed when compared to the resistance reported previously (Antunes et al., 2003; Da Silva and Carneiro, 2006), after the legalization of the use of this antimicrobial class in veterinary use (Threlfall et al., 1997). This finding might be associated with the ban of antimicrobial agents as growth promoters in animal feed in 2006 in the EU countries (Guardabassi et al., 2006). However, resistant Salmonella strains continued to be isolated, demonstrating that the control measures instituted at feed level may be insufficient to minimize the emergence of resistant strains. Like in human therapy, there must be continuous surveillance and an evaluation of the need for and careful choice of antimicrobial agents in livestock.
In this study, the prevalence of ESBLs was low. In Portugal, ESBL-producing E. coli is relatively common in animals (Ramos et al., 2013), but scarce in Salmonella isolates (Clemente et al., 2013). A Salmonella Typhimurium strain produced bla TEM-1 and the bla CTX-M-1 ESBL, associated with IncHI2 type plasmid, while the bla SHV-12 was identified in a Salmonella Enteritidis strain. Recently, the bla CTX-M-1 gene was found in Salmonella also isolated from animal and human samples in France, but in an IncI1 conjugative plasmid (Cloeckaert et al., 2010). In Portugal, ESBLs in strains of animal origin, such as CTX-M-9 and CTX-M-15 enzymes, were identified in E. coli clinical strains and were associated with IncHI2 and IncFII plasmid groups, respectively (Novais et al., 2006; Coque et al., 2008). The European spread of the bla CTX-M-9 in clinical E. coli and S. enterica strains is largely due to dissemination of plasmids of the IncHI2 group (Guardabassi et al., 2006; Novais et al., 2006). Plasmids of the IncHI2 group were also associated with the dissemination of the bla CTX-M-2 gene in animal reservoirs (Fernández et al., 2007). An IncHI2 replicon associated with bla CTX-M-9 in a clinical isolate of Salmonella Bovismorbificans was reported in Portugal (Antunes et al., 2013). To the best of our knowledge, Salmonella Typhimurium with IncHI2 plasmids carrying bla CTX-M-1 have not been described so far in Portugal. Its spread by conjugation was demonstrated, which is a public health concern. Overall, IncHI2 replicon-type plasmids seem to have a role in the dissemination of CTX-M-type ESBLs among reservoirs in animals.
We also reported the first bla SHV-12 in Salmonella Enteritidis, one of the most common sources of human gastroenteritis (EFSA, 2012). This ESBL has been reported worldwide in other Salmonella serotypes (Miriagou et al., 2004; Morosini et al., 2010; Clemente et al., 2013). Despite the low number of ESBL-producing Salmonella encountered, this study confirmed the importance of food-producing animals and food products as important reservoirs of Salmonella isolates carrying ESBLs-encoding genes, which can easily be transferred to humans through the food chain or from direct contact with animals infected, or even transferred to other human pathogens (Liebana et al., 2013).
In conclusion, our data revealed changes in animal-origin Salmonella serotypes and antimicrobial resistance rate, and occurrence of ESBLs-producing Salmonella. Through the years, a decrease of resistance to some antimicrobial agents such as fluoroquinolones was observed, suggesting that fluoroquinolones may be considered a good alternative against Salmonella human infection. In contrast, there is an emergence of ESBLs-producing strains that can compromise foodborne Salmonella infection treatment. A Salmonella control program reduced the Enteritidis serotype in poultry, but Typhimurium seems to adapt to these hosts, since it is the serotype more resistant to antimicrobial agents, and the Salmonella control program should be extended to other animals. The control measures taken by the EU seem to have some impact in the frequency of resistance. However, data from a continuous monitoring of food production is of critical importance, and will contribute to the evaluation of strategies for prevention and control of antimicrobial resistance.
Footnotes
Acknowledgments
R. Figueiredo was supported by the grant SFRH/BD/78833/2011 and N. Mendonça by the grant from SFRH/BPD/45815/2008 Fundação para a Ciência e a Tecnologia, Lisbon, Portugal. This work was supported financially by the PTDC/AGR-ALI/113953/2009 project from Fundação para a Ciência e a Tecnologia and by PEst-OE/SAU/UI0177/2014 of the Centre for Pharmaceutical Studies, University of Coimbra, Portugal.
Disclosure Statement
No competing financial interests exist.
Author Contributions
All authors contributed equally to this work.