**5. Drug resistance**

190 Salmonella – A Dangerous Foodborne Pathogen

green agar, in the analysis of fish meal. Allen et al. (1991) reported that the sensitivity of Rappaport Vassiliadis medium depended on the substrate in the detection of *Salmonella* spp. in high moisture foods, compared with tetrathionate or selenite cystine broth. Eckner et al. (1992) added novobiocin to tetrathionate selective enrichment and increased the incubation

The conventional technique for the detection of the microorganism includes the following steps: preenrichment, selective enrichment, isolation and selection, biochemical characterization, serological characterization and final identification. This technique requires at least four days for a negative result and six to seven days for the identification and confirmation of positive samples (Soumet et al*.,* 1997). The presence of *Salmonella* has

New methodologies, such as immunological tests, have been proposed as alternatives for direct detection of this pathogen. For example, ELISA (Enzyme-linked Immunosorbent Assay) was used by Loguercio et al. (2002). Immunoenzymatic technology may be combined with other rapid methods in order to decrease total assay time. Luk et al. (1997) combined a digoxigenin-based ELISA with the polymerase chain reaction (PCR) to detect amplified r*fb*S, a lipopolysaccharide gene of *Salmonella* spp.; in this case, preenrichment

Other types of assays have also been used: techniques based on molecular biology, such as nucleic acid hybridization or PCR, which was used by Flôres et al. (2003); and tests based on metabolism measurements (impedance and radiometry) (Franco & Landgraf, 1996). Ribotyping is the most recent addition to the automated identification of bacteria. The RiboPrinterTM Microbial Characterization System is based on the highly conserved nature of the rRNA operon. Ribotyping provides a reproducible method by which rRNA and polymorphic fragments can be compared with a database for identification of genus, species and strain (Grimont & Grimont, 1986). The system is almost completely automated, requiring only picking up the colonies, suspending them in buffer and submitting them to heat treatment in a special carrier. Once heated, the sample is placed in the device, which automatically lyses the bacteria, releasing DNA; digests it with restriction enzymes; transfers the sample to agarose gel; and separates restricted fragments by electrophoresis. DNA fragments separated by size are then transferred to a nylon membrane, which is hybridized with a chemically-labeled and treated DNA

Resulting stained bands are then photographed, and the image is stored in the computer database and compared with other images in it. The database for this system is less comprehensive than that of other automated systems, but it still adequate for *Salmonella*  spp. The system would, however, be invaluable in epidemiological studies related to

Serotyping is an important epidemiological tool that complements the identification of *Salmonella*, making it possible to determine the prevalence/emergence or to show trends of a given serovar in different geographical regions, as well as to identify outbreaks, and discover sources of infection and routes of transmission. Serotyping is based on the Kauffmann & White

The somatic structure is identified based on the recognition of the serovars, which are represented by uppercase letters. For example, group A (O:2), group B (O:4); group C1 (O:6,7), group C2 (O:6,8,20), group D (O:9), group E1 (O:3,10), group E2 (O:3,15), group E4 (O:1,3,19), etc. Some factors identify the antigenic group, for example, O:4, O:9. Other

classification and involves the identification of somatic and flagellar antigens.

temperature to 42ºC.

was no longer than 16 hours.

antibody/alkaline phosphatase conjugate.

(HACCP) incidents.

to be determined in at least 25g or mL of sample.

Microbial resistance is related to strains of microorganisms that are able to multiply in the presence of concentrations of antimicrobial compounds even higher than those given as therapeutic doses to humans. Development of resistance is a natural phenomenon that followed the introduction of antimicrobial agents in clinical practice. The irrational and widespread use of these agents has added to the problem, and resistance rates vary from place to place, depending on the local use of antibiotics.

One of the major concerns of the poultry industry is maintaining the sanitary status of the herds. In the incubators where birds are born, there is an attempt to reduce contamination to minimum levels in all phases of the process. Lack of contact with natural biota soon after birth interferes with the normal development of bird intestines (Silva, 2000). Generally, antimicrobial substances (antibiotic or chemotherapic agents), called growth promoters, are used in the feed from the first day of life to the moment of slaughter of the birds, respecting the recommended withdrawal period (Mota, 1996). These growth promoters improve performance because they "modulate" intestinal microbiota and improve feed efficiency.

Suppliers of growth promoters guarantee that these substances are not absorbed through the intestinal walls and are shed in feces, where they are quickly biodegraded. Thus, they do not leave residues in the animal, and do not pose risks to human health or the environment (Mota, 1996). However, consumers are constantly concerned on the possible risks that antimicrobial resistance poses to human health.

In veterinary medicine, antimicrobial agents are used in therapy, metaphylaxis, prophylaxis, and as growth promoters (Scharwz et al., 2001). The use of subtherapeutic doses of antibiotics as growth promoters is a public health problem, because many resistant microorganisms may transfer resistance to microorganisms found in bird feces. This kind of use may be responsible for selective pressure that generates resistant bacteria, a current, worldwide-spread, public health problem, due to the risk of dissemination of pathogens and transfer of resistance genes, via food chain, to pathogenic and commensal microorganisms of humans, decreasing the treatment options for infections (Medeiros, 2011).

Since antimicrobials started to be widely used by humans at the end of the 1940s, the emergence of resistant strains was observed in most bacterial species, and against all drugs available (Flemming, 2005). The use of antimicrobials, combined with improvements in

Important Aspects of Salmonella in the Poultry Industry and in Public Health 193

(Hakanen et al*.*, 2001) observed increased antimicrobial resistance in strains of *Salmonella* Enteritidis isolated from travelers after they came back from Asian countries where quinolones were used indiscriminately. There was an increase from 3.9% to 23.5% in the resistance to fluoroquinolones in samples analyzed between 1995 and 1999 in Finland. These facts, suggest that drug resistance genes may be associated with virulence, or that humans strains have an improved resistance profile compared with *Salmonella* of animal origin, making the whole situation even more concerning from a public health viewpoint. The frequency and extent of *Salmonella* resistance to antimicrobials vary based on the use of antibiotics in humans and animals, and on ecological differences in the epidemiology of *Salmonella* infections (McDermott, 2006). Globally, *Salmonella* exhibits extensive resistance profiles which have been associated both with higher rates of morbidity and mortality and the use of antimicrobials in food-producing animals (Angulo et al., 2004). Antibiotics suppress normal intestinal microbiota, breaking its protective effect, increasing the competitive advantage of antibiotic-resistant *Salmonella*, and favoring the occurrence of

Salmonellosis surveillance has been described all over the world, specially after the emergence of strains resistant to multiple antibiotics, making control and treatment even more difficult. The WHO observed an alarming increase in the number of strains of *Salmonella* resistant to antibiotics due to the abusive use in intensive animal raising (Eurosurveillance, 1997). This finding is a concern for surveillance and environmental control organisms, once the use of antibiotics in animal feed as growth promoters

Antibiotics may be either bactericidal or bacteriostatic agents. Bactericidal agents cause changes incompatible with bacterial survival, whereas bacteriostatic agents inhibit bacterial growth and reproduction, without immediately killing microorganisms (Tavares, 2001). The mechanism of action of antibiotics is essentially related to interference with cell wall synthesis. Cell wall constitution varies in Gram-positive or Gram-negative bacteria, leading to differences in permeability to drugs. Antibiotics that affect the permeability of the cytoplasmic membrane are similar to cationic detergents, due to the presence of basic

Insertion of antibiotic molecules disorganizes the membrane, producing leakage of cell components and death. Antibiotics that interfere with DNA replication generate toxic products that get inserted in the DNA molecule, breaking it up and preventing its synthesis. Others compounds loosen the DNA spiral structure, making it larger and breaking the bacterial cell. Agents that affect protein synthesis act on the ribosome, inhibiting protein

Some bacterial species are considered naturally resistant to antibacterial compounds (primary resistance), because only concentrations that would be unviable *in vivo* would affect them. Under continuous exposure to antimicrobials, microorganisms show acquired resistance (secondary) caused by the development of new mechanisms of defense (Fuchs &

Resistance mechanisms may emerge because of changes in bacterial DNA, or biochemical mechanisms of molecule production, reactions and behaviors, which may be transmissible or not to the daughter cells. Resistance is observed when an antibiotic is administered to patients who are carriers of sensitive, mutant strains. Antimicrobials eliminate microorganisms that are sensitive, "selecting" the ones that are resistant. The rate of emergence of mutant strains is highly variable, and the mutation process may occur quickly

contributes for the emergence of resistant and pathogenic strains (Pinto, 2000).

synthesis by different mechanisms (Tavares, 2001; Trabulsi & Alterthum, 2008).

groups (NH3 +) in a lateral chain of the fatty acid.

salmonellosis (Eley, 1994).

Wannmacher, 1999).

sanitation, nutrition and immunization, has lead to a dramatic decrease in deaths and a major gain in human life expectancy (WHO, 2002). However, with the increased use of antimicrobials, antimicrobial resistance has emerged as one of the greatest threats to the safety of human health (WHO, 2007), and as a most pressing problem for public health, animal health and food safety authorities (Tenover, 2006; Marchese & Schito, 2007).

The increase in antimicrobial resistance has narrowed the potential uses of antibiotics for the treatment of infections in humans and animals (Angulo et al., 2004). As a striking example, the CDC estimated that the total of methicillin-resistant *Staphylococcus* Infections (MRSI) in US hospitals and communities have increased from 2 % in 1974 to almost 63% in 2004 (CDC, 2010).

In the US, more than 40% of the antibiotics produced are used in animal feed. This nontherapeutic use of antibiotics is a way to promote the selection of a growing number of resistant bacteria (Levy, 1998). As more strains responsible for poultry infections become resistant to therapeutic drugs, these compounds become less available for human treatments. Similarly, with *Salmonella* being an important cause of foodborne diarrheal disease in humans 10/12, the reduction in the number of antibiotics available for effective treatment of *Salmonella-*related infections in humans and animals has become a serious concern (Angulo et al., 2004).

In Europe, besides this concern with resistance, several recent public health episodes were branded on the mind of the consumers. Among them, the connection between eggs and *Salmonella* Enteritidis, BSE/"mad cow disease" and cattle meat and, more recently, avian flu in Asia. Therefore, zoonoses and restricted use of additives and antimicrobials as growth promoters in feeds, together with the occurrence of resistant microorganisms, have become an important challenge in the control of detrimental microorganisms found in the digestive system of birds.

There is a consensus in several countries that the indiscriminate use of antimicrobials in animal production is one of the causes of the increased resistance to antimicrobials. Human infections are more severe when a strain of a given microorganism is resistant to the drug of choice for its treatment. The use of antimicrobials may stimulate the selection of resistant bacteria in this ecosystem. Human pathogens and resistant genes may cross species and ecosystems by contact with, or consumption of contaminated food and water (Kelley et al., 1998). Due to the little knowledge on single, multiple or cross-resistance mechanisms in microorganisms that are highly pathogenic to humans, the WHO has recommended careful use and restrictions to antimicrobials in animal production (WHO, 2001).

Before *Salmonella* Enteritidis outbreaks related to traditional drugs in Europe, different antibiotics – such as nitrofurazone, furazolidone, novobiocine and tetracyclines - were used in drinking water and in feed offered to poultry. In Brazil, tetracyclines, penicillins, chloramphenicol, sulphonamides, furazolidone, nitrofurazone and avoparcin were banned as additives in animal feed in 1998. However, the use of several other drugs is still allowed: 3-nitro acid, arsanilic acid, avilamycin, colistine sulfate, enramycin, flavomycin, lincomycin, spiramycin, tylosin sulfate and zinc bacitracin.

Extensive use of quinolones in birds was made possible by very flexible prescription regulations, use of generic, lower cost drugs in feed and water, and, without a doubt, because of the efficiency of these agents against *Salmonella*. The use of fluoroquinolones, which have a similar mechanism of action, followed quinolones (Rossi, 2005).

Strains of *Salmonella* Enteritidis may become resistant because of the indiscriminate use of drugs in their country of origin, imports of foodstuffs contaminated with bacteria carrying resistance genes, or infected people returning from international trips. Finnish researchers

sanitation, nutrition and immunization, has lead to a dramatic decrease in deaths and a major gain in human life expectancy (WHO, 2002). However, with the increased use of antimicrobials, antimicrobial resistance has emerged as one of the greatest threats to the safety of human health (WHO, 2007), and as a most pressing problem for public health,

The increase in antimicrobial resistance has narrowed the potential uses of antibiotics for the treatment of infections in humans and animals (Angulo et al., 2004). As a striking example, the CDC estimated that the total of methicillin-resistant *Staphylococcus* Infections (MRSI) in US hospitals and communities have increased from 2 % in 1974 to almost 63%

In the US, more than 40% of the antibiotics produced are used in animal feed. This nontherapeutic use of antibiotics is a way to promote the selection of a growing number of resistant bacteria (Levy, 1998). As more strains responsible for poultry infections become resistant to therapeutic drugs, these compounds become less available for human treatments. Similarly, with *Salmonella* being an important cause of foodborne diarrheal disease in humans 10/12, the reduction in the number of antibiotics available for effective treatment of *Salmonella-*related infections in humans and animals has become a serious

In Europe, besides this concern with resistance, several recent public health episodes were branded on the mind of the consumers. Among them, the connection between eggs and *Salmonella* Enteritidis, BSE/"mad cow disease" and cattle meat and, more recently, avian flu in Asia. Therefore, zoonoses and restricted use of additives and antimicrobials as growth promoters in feeds, together with the occurrence of resistant microorganisms, have become an important challenge in the control of detrimental microorganisms found in the digestive

There is a consensus in several countries that the indiscriminate use of antimicrobials in animal production is one of the causes of the increased resistance to antimicrobials. Human infections are more severe when a strain of a given microorganism is resistant to the drug of choice for its treatment. The use of antimicrobials may stimulate the selection of resistant bacteria in this ecosystem. Human pathogens and resistant genes may cross species and ecosystems by contact with, or consumption of contaminated food and water (Kelley et al., 1998). Due to the little knowledge on single, multiple or cross-resistance mechanisms in microorganisms that are highly pathogenic to humans, the WHO has recommended careful

Before *Salmonella* Enteritidis outbreaks related to traditional drugs in Europe, different antibiotics – such as nitrofurazone, furazolidone, novobiocine and tetracyclines - were used in drinking water and in feed offered to poultry. In Brazil, tetracyclines, penicillins, chloramphenicol, sulphonamides, furazolidone, nitrofurazone and avoparcin were banned as additives in animal feed in 1998. However, the use of several other drugs is still allowed: 3-nitro acid, arsanilic acid, avilamycin, colistine sulfate, enramycin, flavomycin, lincomycin,

Extensive use of quinolones in birds was made possible by very flexible prescription regulations, use of generic, lower cost drugs in feed and water, and, without a doubt, because of the efficiency of these agents against *Salmonella*. The use of fluoroquinolones,

Strains of *Salmonella* Enteritidis may become resistant because of the indiscriminate use of drugs in their country of origin, imports of foodstuffs contaminated with bacteria carrying resistance genes, or infected people returning from international trips. Finnish researchers

use and restrictions to antimicrobials in animal production (WHO, 2001).

which have a similar mechanism of action, followed quinolones (Rossi, 2005).

spiramycin, tylosin sulfate and zinc bacitracin.

animal health and food safety authorities (Tenover, 2006; Marchese & Schito, 2007).

in 2004 (CDC, 2010).

concern (Angulo et al., 2004).

system of birds.

(Hakanen et al*.*, 2001) observed increased antimicrobial resistance in strains of *Salmonella* Enteritidis isolated from travelers after they came back from Asian countries where quinolones were used indiscriminately. There was an increase from 3.9% to 23.5% in the resistance to fluoroquinolones in samples analyzed between 1995 and 1999 in Finland.

These facts, suggest that drug resistance genes may be associated with virulence, or that humans strains have an improved resistance profile compared with *Salmonella* of animal origin, making the whole situation even more concerning from a public health viewpoint.

The frequency and extent of *Salmonella* resistance to antimicrobials vary based on the use of antibiotics in humans and animals, and on ecological differences in the epidemiology of *Salmonella* infections (McDermott, 2006). Globally, *Salmonella* exhibits extensive resistance profiles which have been associated both with higher rates of morbidity and mortality and the use of antimicrobials in food-producing animals (Angulo et al., 2004). Antibiotics suppress normal intestinal microbiota, breaking its protective effect, increasing the competitive advantage of antibiotic-resistant *Salmonella*, and favoring the occurrence of salmonellosis (Eley, 1994).

Salmonellosis surveillance has been described all over the world, specially after the emergence of strains resistant to multiple antibiotics, making control and treatment even more difficult. The WHO observed an alarming increase in the number of strains of *Salmonella* resistant to antibiotics due to the abusive use in intensive animal raising (Eurosurveillance, 1997). This finding is a concern for surveillance and environmental control organisms, once the use of antibiotics in animal feed as growth promoters contributes for the emergence of resistant and pathogenic strains (Pinto, 2000).

Antibiotics may be either bactericidal or bacteriostatic agents. Bactericidal agents cause changes incompatible with bacterial survival, whereas bacteriostatic agents inhibit bacterial growth and reproduction, without immediately killing microorganisms (Tavares, 2001).

The mechanism of action of antibiotics is essentially related to interference with cell wall synthesis. Cell wall constitution varies in Gram-positive or Gram-negative bacteria, leading to differences in permeability to drugs. Antibiotics that affect the permeability of the cytoplasmic membrane are similar to cationic detergents, due to the presence of basic groups (NH3 +) in a lateral chain of the fatty acid.

Insertion of antibiotic molecules disorganizes the membrane, producing leakage of cell components and death. Antibiotics that interfere with DNA replication generate toxic products that get inserted in the DNA molecule, breaking it up and preventing its synthesis. Others compounds loosen the DNA spiral structure, making it larger and breaking the bacterial cell. Agents that affect protein synthesis act on the ribosome, inhibiting protein synthesis by different mechanisms (Tavares, 2001; Trabulsi & Alterthum, 2008).

Some bacterial species are considered naturally resistant to antibacterial compounds (primary resistance), because only concentrations that would be unviable *in vivo* would affect them. Under continuous exposure to antimicrobials, microorganisms show acquired resistance (secondary) caused by the development of new mechanisms of defense (Fuchs & Wannmacher, 1999).

Resistance mechanisms may emerge because of changes in bacterial DNA, or biochemical mechanisms of molecule production, reactions and behaviors, which may be transmissible or not to the daughter cells. Resistance is observed when an antibiotic is administered to patients who are carriers of sensitive, mutant strains. Antimicrobials eliminate microorganisms that are sensitive, "selecting" the ones that are resistant. The rate of emergence of mutant strains is highly variable, and the mutation process may occur quickly

Important Aspects of Salmonella in the Poultry Industry and in Public Health 195

Plasmids are circular, extrachromosomal DNA molecules found in many bacterial species, and in some eukaryotes. They replicate separately or together with the host cell, and are passed on to the daughter cells. Plasmids may removed from the cell by different stress conditions, such as changes in temperature, presence of some stains or lack of certain nutrients. They are not essential to the cell, but may confer some selective advantages: they may have information for the degradation of certain substrates, resistance antibiotics or heavy metals. Plasmids may self-replicate independently of chromosomal replication, and may occur in variable numbers. Sex factors (F factor), antibiotic resistance (R factor), N2-

Antimicrobial resistance is one of the most important problems for human and veterinary medicine, and it is recognized by the WHO as an important public health problem (Rossi,

There was a significant increase in the occurrence of *Salmonella* Enteritidis in poultry carcasses from 2000 to 2005 in the US. Studies in Brazil between 2000 and 2009 show the predominance of this serovar in poultry. More than half of the strains were resistant to multiple antibiotics, and *Salmonella* Enteritidis was the only serovar that showed different degrees of resistance to all antimicrobial compounds. Studies carried out with *Salmonella* Heidelberg demonstrated that all strains showed multiple resistance, including marked resistance to third generation cephalosporins. In the past years in the US, increased resistance to ceftiofur was observed in poultry strains. In 1997, resistance to this antibiotic

During decades, ampicillin, chloramphenicol and trimetoprim-sulfametoxazole were the most frequent antimicrobials used in salmonellosis treatment. However, the increase in the number of strains resistant to these drugs reduced their used in medical practice. Consequently, fluoroquinolones became the main antimicrobials used in the treatment of

Resistance to *Salmonella* transmitted by contaminated foods of animal origin is undesirable, but it is an inevitable consequence of the use of antimicrobials in animals used in food production (Threlfall et al., 2002). Bacterial resistance is a natural process, but it should and can be prevented with the rational use of antimicrobials in animal production. Therefore, it is very important to follow the evolution of resistance in order to use efficient methods for

Prevention and control programs for infections caused by paratyphoid *Salmonellae* aim at protecting the health of the birds, ensure the safety of the consumers, and strengthen the reliability of the poultry production chain. In the case of *Salmonella*, measures recommended for prevention and control are not specific due to the large number of species and their complex epidemiological behavior. Similarly, variability in the implementation of these measures depends on the requisites determined by the international market, or the

In the past 10 years, there have been important outbreaks of emerging foodborne diseases all over the world. These outbreaks showed sanitary authorities of the countries affected that there is an increasing need for measures to prevent the risk of transmission. This led the Food and Agriculture Organization (FAO) to create the WTO, which motivated countries to review their innocuousness policies, rules and strategies to ensure that the food consumed

fixation (Trabulsi & Alterthum, 2008) are example of plasmids.

was 1.6%, and in 2003, 7.4% (Medeiros, 2011).

human infections (Souza et al., 2010).

*Salmonella* control.

**6. Prevention and control** 

adaptation of the industry to the chronogram of production.

2005).

in some cases, and slowly and gradually in other cases, taking years to appear. Some cells may present random genetic changes that may lead to resistance to a given antibiotic (Decamp & Moriarty, 2006). The process is called single resistance when the bacterium is resistant to only one drug; multiple resistance, when it is simultaneously resistant to two or more drugs (Tavares, 2001).

According to Claus (1988), mechanisms of antimicrobial resistance may involve chromosomal DNA, by means of mutations; or may be due to the acquisition of extrachromosomal DNA (by means of gene transduction, transformation or conjugation).

Mutations occur by chromosome swapping. These changes may be random, or caused by physical and/or chemical agents, and the process may be caused or not by exposure to antimicrobial agents. Many microorganisms isolated before the use of antibiotics showed mutations, and were not sensitive to antibiotics when these were discovered. Antimicrobials are not necessarily responsible for mutations, but they have an important role in the selection of resistant strains. Commonly, the genetic change that causes the resistance in a microorganism is generated by genes transported in extrachromosomal plasmids (Claus, 1988).

In the transduction process, a bacteriophage transfers, from a resistant to a sensitive bacterium, extrachromosomal bacterial DNA incorporated in its protein. The previously sensitive bacterium, then, will acquire resistance and transfer it to its daughter cells. This mechanism is easily observed in *Staphylococcus aureus* strains that acquired resistance to penicillins.

Transformation occurs when bacteria that are sensitive to one substance incorporate the DNA with genes that encode resistance, that are found in the environment. These bacteria, then, become resistant to one or more antimicrobials. Some bacteria, in certain growth phases, are able to excrete DNA to the environment.

Conjugation is caused by a passage of genes (R factors) from a resistant to a sensitive bacterium by attachment to a sex pilus. The R factor may contain resistance information against several antimicrobials. Conjugation and production of the sex pilus requires intervention of another group of genes, called transference factor. Without them, the process is not carried out. The R determinant complex, plus the resistance transfer factor, are known as R factor. R factor is important to Gram-negative bacteria, specially enterobacteria. *Escherichia coli*, *Salmonella*, *Shiguella*, *Klebsiella* and *Pseudomonas aeruginosa* are among microorganisms capable of transferring this type of resistance to sensitive bacteria. This resistance mechanism has been observed in relation to tetracyclines, chloramphenicol, sulfonamides, penicillins and aminoglycosides.

All these genetic alterations give rise to several biochemical changes in bacterial metabolism. Resistance to antibiotics may be carried out by three basic mechanisms produced by these changes (Strohl et al., 2004): decreased absorption or increase efflux of the antibiotic; change in the target site of the antibiotic, and acquisition of the ability to break or modify the antibiotic.

Acquired resistance to antibiotics is a necessary gain, or temporary or permanent change of bacterial genetic information. Most resistance genes are found in plasmids, which may be swapped with chromosomal elements. Acquired resistance is caused by mutations in the bacterial chromosome (which leads to the emergence of resistance genes in a sensitive bacterium), or by the transfer of resistance genes from one cell to another, with DNA fragments with these genes being inserted in the receptor cell. Both types of resistance, mutation (chromosomal) and transferable (plasmidial) may be found in the same bacterium (Tavares, 2001).

in some cases, and slowly and gradually in other cases, taking years to appear. Some cells may present random genetic changes that may lead to resistance to a given antibiotic (Decamp & Moriarty, 2006). The process is called single resistance when the bacterium is resistant to only one drug; multiple resistance, when it is simultaneously resistant to two or

According to Claus (1988), mechanisms of antimicrobial resistance may involve chromosomal DNA, by means of mutations; or may be due to the acquisition of extrachromosomal DNA (by means of gene transduction, transformation or conjugation). Mutations occur by chromosome swapping. These changes may be random, or caused by physical and/or chemical agents, and the process may be caused or not by exposure to antimicrobial agents. Many microorganisms isolated before the use of antibiotics showed mutations, and were not sensitive to antibiotics when these were discovered. Antimicrobials are not necessarily responsible for mutations, but they have an important role in the selection of resistant strains. Commonly, the genetic change that causes the resistance in a microorganism is generated by genes transported in extrachromosomal

In the transduction process, a bacteriophage transfers, from a resistant to a sensitive bacterium, extrachromosomal bacterial DNA incorporated in its protein. The previously sensitive bacterium, then, will acquire resistance and transfer it to its daughter cells. This mechanism is

Transformation occurs when bacteria that are sensitive to one substance incorporate the DNA with genes that encode resistance, that are found in the environment. These bacteria, then, become resistant to one or more antimicrobials. Some bacteria, in certain growth

Conjugation is caused by a passage of genes (R factors) from a resistant to a sensitive bacterium by attachment to a sex pilus. The R factor may contain resistance information against several antimicrobials. Conjugation and production of the sex pilus requires intervention of another group of genes, called transference factor. Without them, the process is not carried out. The R determinant complex, plus the resistance transfer factor, are known as R factor. R factor is important to Gram-negative bacteria, specially enterobacteria. *Escherichia coli*, *Salmonella*, *Shiguella*, *Klebsiella* and *Pseudomonas aeruginosa* are among microorganisms capable of transferring this type of resistance to sensitive bacteria. This resistance mechanism has been observed in relation to tetracyclines, chloramphenicol,

All these genetic alterations give rise to several biochemical changes in bacterial metabolism. Resistance to antibiotics may be carried out by three basic mechanisms produced by these changes (Strohl et al., 2004): decreased absorption or increase efflux of the antibiotic; change in the target site of the antibiotic, and acquisition of the ability to

Acquired resistance to antibiotics is a necessary gain, or temporary or permanent change of bacterial genetic information. Most resistance genes are found in plasmids, which may be swapped with chromosomal elements. Acquired resistance is caused by mutations in the bacterial chromosome (which leads to the emergence of resistance genes in a sensitive bacterium), or by the transfer of resistance genes from one cell to another, with DNA fragments with these genes being inserted in the receptor cell. Both types of resistance, mutation (chromosomal) and transferable (plasmidial) may be found in the same bacterium

easily observed in *Staphylococcus aureus* strains that acquired resistance to penicillins.

phases, are able to excrete DNA to the environment.

sulfonamides, penicillins and aminoglycosides.

break or modify the antibiotic.

(Tavares, 2001).

more drugs (Tavares, 2001).

plasmids (Claus, 1988).

Plasmids are circular, extrachromosomal DNA molecules found in many bacterial species, and in some eukaryotes. They replicate separately or together with the host cell, and are passed on to the daughter cells. Plasmids may removed from the cell by different stress conditions, such as changes in temperature, presence of some stains or lack of certain nutrients. They are not essential to the cell, but may confer some selective advantages: they may have information for the degradation of certain substrates, resistance antibiotics or heavy metals. Plasmids may self-replicate independently of chromosomal replication, and may occur in variable numbers. Sex factors (F factor), antibiotic resistance (R factor), N2 fixation (Trabulsi & Alterthum, 2008) are example of plasmids.

Antimicrobial resistance is one of the most important problems for human and veterinary medicine, and it is recognized by the WHO as an important public health problem (Rossi, 2005).

There was a significant increase in the occurrence of *Salmonella* Enteritidis in poultry carcasses from 2000 to 2005 in the US. Studies in Brazil between 2000 and 2009 show the predominance of this serovar in poultry. More than half of the strains were resistant to multiple antibiotics, and *Salmonella* Enteritidis was the only serovar that showed different degrees of resistance to all antimicrobial compounds. Studies carried out with *Salmonella* Heidelberg demonstrated that all strains showed multiple resistance, including marked resistance to third generation cephalosporins. In the past years in the US, increased resistance to ceftiofur was observed in poultry strains. In 1997, resistance to this antibiotic was 1.6%, and in 2003, 7.4% (Medeiros, 2011).

During decades, ampicillin, chloramphenicol and trimetoprim-sulfametoxazole were the most frequent antimicrobials used in salmonellosis treatment. However, the increase in the number of strains resistant to these drugs reduced their used in medical practice. Consequently, fluoroquinolones became the main antimicrobials used in the treatment of human infections (Souza et al., 2010).

Resistance to *Salmonella* transmitted by contaminated foods of animal origin is undesirable, but it is an inevitable consequence of the use of antimicrobials in animals used in food production (Threlfall et al., 2002). Bacterial resistance is a natural process, but it should and can be prevented with the rational use of antimicrobials in animal production. Therefore, it is very important to follow the evolution of resistance in order to use efficient methods for *Salmonella* control.
