β-Lactam Resistance Genes: Characterization,Epidemiology,and First Detection of bla CTX-M-1 and bla CTX-M-14 in Salmonella spp. Isolated from Poultry in Brazil—Brazil Ministry of Agriculture's Pathogen Reduction Program

Introduction

Food-borne salmonellosis is an important public health problem worldwide and is a major cause of illness in both developed and developing countries such as Brazil.^22,25,26,31 Most of the human Salmonella infections occur through the consumption of contaminated food of animal origin, including poultry, beef, pork, eggs, and milk.^2,10

In Brazil, the Ministry of Agriculture (MAPA) is responsible for the supervision and inspection of food production. In 2004, MAPA launched the Pathogen Reduction Program, with one of its aims being to monitor and reduce Salmonella contamination in poultry slaughtering establishments. ²³

In the past two decades, there has been a rapid increase in human Salmonella infections caused by isolates producing extended-spectrum beta-lactamase (ESBL) and/or plasmid-mediated AmpC enzymes.^7,37 Since food of animal origin is the source of most human salmonellosis, the food production system is a likely origin for the increase in antibiotic-resistant bacteria seen in human infections. ²⁴

In uncomplicated cases of human salmonellosis, antibiotics are not required; however, antibiotics are lifesaving in invasive infections. Invasive Salmonella infections occur with greater frequency in children, the elderly, and other immunocompromised individuals. ²⁰ These serious infections are usually treated with either extended-spectrum cephalosporin or ciprofloxacin. ²⁰

The rise in β-lactam-resistant Salmonella is limiting the therapeutic options for cases of invasive salmonellosis. To combat the increasing antibiotic resistance among food-borne pathogens, many nations, including Brazil, have sought to limit the use of antibiotics as agricultural growth promoters. In this case, subtherapeutic doses of antibiotics are fed to farm animals to promote weight gain. It is thought that this application of antibiotics contributes to the increase in drug resistance in general and ESBL-based resistance among Salmonella spp. in particular. ²¹ The prevalence of Salmonella spp. isolated from food-producing animals, carrying ESBLs coding genes, such as bla_CTX-M and AmpC bla_CMY-2, has now reached high proportions, leading to even greater concerns. ³³

Molecular methods, such as ribotyping, sequencing (for both typing and detection of resistance genes), or microarray approaches, can be applied in food-borne pathogen studies to elucidate epidemiology and resistance mechanisms. These data allow the tracking of both the resistant microorganisms and the resistance genes themselves, as they spread within an agricultural production system through the food chain and in subsequent human disease.

Objective

The aims of this study are to determine the occurrence of β-lactam resistance among Salmonella isolates from the Brazil Ministry of Agriculture's Pathogen Reduction Program, to catalog and quantify the prevalence of the resistance genes causing the β-lactam resistance, and to obtain substrain-level characterization of the isolates to identify potential cases of clonal population.

Materials and Methods

Results

Automated ribotyping and serotype assignment

Automated ribotyping was performed on the 55 isolates, subjects of this study, and the results were used to assign presumptive serotypes based on the ribogroups generated by the restriction enzyme PvuII. ¹ A total of 31 distinct ribogroups were obtained from the 55 isolates tested (Table 2).

The most frequent inferred serotype was Salmonella ser Heidelberg (n = 16, 29.1%). Of these, 11 were isolated in 2009 from two cities located in Rio Grande do Sul state. Of these, seven shared a single ribogroup (222-202-5). The other five samples were isolated in 2011, four of these from one city in Paraná State and one from a city in São Paulo State. These five samples were divided among four ribogroups. All the states from which serotype Heidelberg was isolated are located along the coast in the southern part of Brazil (Fig. 1).

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FIG. 1.

Map of Brazil, showing the three regions and the locations where the 55 samples reported in this study were isolated. South region: PR, Paraná (n = 20); RS, Rio Grande do Sul (n = 13); SC, Santa Catarina (n = 2); Southeast region: SP, São Paulo (n = 3). Midwest region: GO, Goiás (n = 9); MT, Mato Grosso (n = 2); MS, Mato Grosso do Sul (n = 6). Matching symbols show various locations where clonal samples occurred. Salmonella ser Heidelberg/222-202-5 (n = 7). Salmonella ser Heidelberg/222-400-2 (n = 2). Salmonella ser Heidelberg/222-215-1 (n = 2). Salmonella ser Minnesota/222-208-8 (n = 3). Salmonella ser Schwarzengrund/Bredeney/222-208-4 (n = 5). Salmonella ser Typhimurium/222-205-5 (n = 6). Salmonella ser Agona/222-213-6 (n = 2). For further information about the isolates refer to Table 2.

The serotype Salmonella ser Typhimurium was inferred to nine isolates (16.4%) from six distinct cities in five separate states. Of these nine isolates, six were isolated in 2005 and shared a single ribogroup (222-205-5) (Fig. 1).

The third most common serogroup was Salmonella ser Schwarzengrund/Bredeney, assigned to six isolates (10.9%). These six isolates were all from the same year (2009) and the same state, Paraná, but from four different cities (Table 2). Five of the six isolates share a single ribogroup (222-208-4) (Fig. 1).

The next most frequent serotype was Salmonella ser Minnesota, with five isolates (9.1%), being isolated over 4 years (2008–2011) from four cities, in three distinct states, Goiás, Mato Grosso do Sul, and Rio Grande do Sul (Fig. 1). Three of the isolates share the same ribogroup, despite being isolated from three different states and in 3 different years (2009–2011).

Thirteen serotypes had only a single representative among the 55 isolates studied. The description of all serotypes and ribogroups and the respective place of isolation can be seen in Table 2.

Discussion

The finding that 10.0% Salmonella spp. isolates collected between 2004 and 2011 show ampicillin resistance and 5.2% possessed extended-spectrum resistance to ceftriaxone as well indicates that Salmonella spp. resistant to one of the two preferred therapeutic options for invasive Salmonella spp. cases, which are extended-spectrum cephalosporin or ciprofloxacin, is present at significant levels in the Brazilian poultry production system. The bla_CTX-M-2 resistance gene appears to have entered multiple times into Salmonella spp. as it is found distributed among multiple distinct ribogroups and presumptive serotypes. The data are consistent with several discreet instances of clonal emergence over the collection interval (2004–2011). In all these instances of clonal occurrence, the resistance marker was bla_CTX-M-2. This resistance marker is frequently carried on plasmids that likely readily pass between a number of Enterobacteriaceae. ³

Our findings show that the earliest clonal emergence chronologically is seen in the data from 2005 isolates, where five Salmonella ser Typhimurium isolates all belong to the same ribogroup and all carried bla_CTX-M-2 resistance genes. These five specimens were isolated from four cites in two adjacent states with over 300 miles, separating the poultry operations where they were isolated (Fig. 1). In 2013, the CDC reported an outbreak of Salmonella ser Typhimurium, distributed through 39 states in the United States. ¹⁰ In 2009, Fernandes et al. reported 10 genetically related isolates of Salmonella ser Typhimurium carrying bla_CTX-M-2 isolated from human and poultry sources in two distant cities in Brazil. One possible explanation of the widely disseminated nature of these outbreaks is that broiler breeding stock and other products used in poultry production, as well as the carcasses themselves, are often exchanged across the national production system territory and can also be internationally traded, enabling the spread of multidrug-resistant isolates and transfer of mobile genetic elements carrying resistance genes.^16,24,36 Once in the food production system, the spread of drug-resistant isolates from food sources into the human population is well documented.^5,29,40

Our data show that two additional apparent clonal emergences occurred in 2009, where seven isolates were assigned to serotype Heidelberg and had the same ribogroup (222-202-5). All of them were bearing the bla_CTX-M-2 resistance gene and were isolated from two cities in a single state, Rio Grande do Sul, during 2009 (Fig. 1). Salmonella ser Heidelberg has been known to be very likely to cause outbreaks, like the two recently reported by the CDC, where more than 520 people were infected through 25 states, from 2013 to 2014. The isolates collected in this outbreak from the infected people were multidrug resistant. The outbreak strains of Salmonella ser Heidelberg are known to be resistant to several regularly prescribed antibiotics. Even though these antibiotics are not typically used to treat Salmonella bloodstream infections or other severe Salmonella infections, antibiotic resistance can also be associated with increased risk of hospitalization in infected patients. ¹⁰

The first report of Salmonella ser Schwarzengrund carrying bla_CTX-M-2 gene from poultry farms and carcasses in Brazil was made by Silva et al. in 2013, where 11 genetically related samples were isolated in 2008 from poultry farms in the states of Santa Catarina and Paraná, both located in the Southern region. ³⁴ In our study, five isolates of Salmonella ser Schwarzengrund/Bredeney, isolated in 2009, belonged to a single ribogroup (222-202-4) and all carried the bla_CTX-M-2 resistance gene and were collected from four distinct cities in Paraná, which is one of the states described in the study by Silva et al. ³⁴ There is a possibility that these isolates could be related to those described by Silva et al., however, this hypothesis is yet to be tested. ³⁴

Acquisition of resistance markers can be driven by the use of antibiotics, particularly by oral administration, ³⁸ which activates mobile genetic elements and enhances the spread of resistance genes between the gut bacteria, even those of different species. Once a resistance gene has entered the bacteria, continued use of the antibiotic to which it confers resistance provides a selective advantage to the bacteria carrying the gene driving further expansion of the clone. Our data indicate that in Brazilian poultry operations, the bla_CTX-M-2 gene has been detected in Salmonella spp., likely on multiple occasions. This is indicated by its presence in multiple genetic backgrounds (ribogroups) and the fact that in several instances clonal regional occurrence of the resistant bacteria ensued.

Since interspecies transfer of microbial resistance genes occurs among intestinal Enterobacteriaceae,^7,11,27 it is perhaps not surprising that others have found recent evidence of other bla_CTX-M-2- and bla_CTX-M-8-bearing Enterobacteriaceae in poultry from Brazil.^9,17,28,31 A 2008 study found that 40% of chicken breasts imported from Brazil to the United Kingdom carried E. coli bearing the bla_CTX-M-2 gene, indicting a high prevalence for this resistance gene in E. coli in Brazilian poultry. ³⁶ The apparent reservoir of extended-spectrum β-lactam resistance genes in Brazilian poultry is of concern and should be addressed. Children, the elderly, and others with weakened defenses are at risk of significant morbidity by organisms bearing these resistance genes. A previous report of Salmonella ser Typhimurium isolates producing bla_CTX-M-2 identified from pediatric patients and poultry sources in two distant cities in Brazil, between 2003 and 2004, showed high genetic similarity among the clinical and poultry isolates and also characterized the presence of bla_CTX-M-2 gene located on transferable plasmids. ¹⁶ This underscores that drug-resistant Salmonella originating from food was causing infections among populations at high risk for invasive infection.

Despite being included in the initial single PCR screening, the bla_TEM gene was not detected before in six samples. It was subsequently detected in the samples by the microarray assay. A similar occurrence was reported in 2014 by Cunningham et al., who described that 42 isolates were found to contain resistance genes not originally known to be present, but were confirmed subsequently. This can be explained by the microarray format, which allows the investigation of a large range of genetic targets simultaneously. ¹⁴

Our findings show that several bla_CTX-M variants were present in multiple Salmonella strains (various serotypes and ribogroups) in many states in Brazil. Our finding of several distinct clusters of genetically similar isolates indicates multiple discreet instances in which clonal emergence of resistant isolates between poultry production establishments has occurred. It also bears mention that this study is the first report of Salmonella sp carrying a bla_CTX-M-1 and bla_CTX-M-14 gene isolated from poultry in Brazil.