Quinolones were introduced for veterinary use in various countries, and subsequent use has been followed by the development of quinolone resistance in bacteria of food‐producing animals and consequently transmits the resistant zoonotic bacteria to humans [29]. In many countries, FQs are drug of first choice for prescription in acute gastrointestinal symptoms caused by *Salmonella* infection, and resistance to this drug group has often been described, particularly to NAL [15]. In a study performed between 1996 and 2003, *Salmonella* isolates were investigated for quinolone susceptibility; the results revealed that NAL and CIP resis‐ tances were 1.6 and 7%, respectively. A significant upward trend in resistance was observed for NAL from 0.4% in 1996 to 2.3% in 2003 [30]. In Germany, an increase in the frequency of NAL‐resistant *Salmonella* strains was discovered after the approval and use of ENR [31]. Concurrent increase in resistance was observed in France among *Salmonella* isolates from animals and humans, and the same clones were determined among the different hosts [32]. In the United Kingdom, also in Spain, the incidence of NAL‐resistant *Salmonella* illnesses in humans was increased followed the introduction for veterinary use of FQs in 1993 [33, 34]. A study from Denmark and Taiwan described the emergence of salmonellosis caused by multidrug and quinolone‐resistant *S*. Typhimurium DT104 linked to a swine herd and the subsequent spread of those isolates to humans [35–37]. In European countries, similar associa‐ tions between FQ resistance development in *Salmonella* infecting humans and retail poultry products have been described. Therefore, the FQ‐resistant *Salmonella* in poultry has reached alarming proportions in some countries [38]. In the United States, there was an increase in the proportion of FQ resistance development in *Salmonella* infections following the first approved use of FQs in food‐producing animals in 1995 [39].

The data indicate that it would be reasonable to assume that the veterinary usage of FQs will have made a remarkable contribution to FQ resistance in human *Salmonella* infections.

## **4. The potential impact on human health**

FQ resistance in *Salmonella* is clearly associated with FQ use in food‐producing animals, and foodborne infections caused by such resistant bacteria are well investigated in human. FQ resistance in *S.* Typhimurium DT104 has been associated with increased hospitalization, more frequent and longer illness, treatment failures, and a higher risk of death [40]. Many studies also investigated that infections with multidrug‐resistant *Salmonella* were associated with longer hospitalization and a higher death rate than infections with susceptible isolates [41–43]. Previous study has found a 3.15 times increased mortality when patients infected with NAL‐resistant *S.* Typhimurium compared to patients infected with susceptible isolates [44]. For treatment of the infections with FQ‐resistant *Salmonella,* alternative antimicrobials are the third or fourth generation cephalosporin. Nevertheless, it should be considered con‐ traindications for treatment of uncomplicated non‐typhoidal *Salmonella* infection because FQ treatment can induce prolonged excretion of *Salmonella* and increased frequency of relapses [45]. However, for patients at risk such as immunocompromised, severely infected and elderly, FQs are considered first choice drugs and effective in reducing the disease length if the treatment starts early in the infection.

## **5. The potential impact on animal health**

FQs are highly potent antimicrobial agents rapidly absorbed after oral administration and have a long half‐life and widespread distribution to most body tissues, which made them suitable for using in herd treatment of food‐producing animals. FQs are effective for serious infections in food‐producing animals such as systematic gastroenteritis and severe respira‐ tory diseases and are also used to treat urinary tract, skin, and soft‐tissue infections caused by Gram‐negative or some Gram‐positive aerobic bacteria. Moreover, they also have potential for treatment of infections caused by *Mycoplasma*, *Mycobacterium*, *Chlamydia*, *Ehrlichia*, and *Rickettsia*. However, documentation about authorized dosages and the effectiveness of FQs to treat all these infections in animals have not been determined on the base of the pharmaco‐ kinetic and pharmacodynamics properties. Sufficient knowledge about the selecting optimal dose and duration of FQs could help to develop appropriate dosing regimens to maximize the clinical efficacy, avoid therapeutic failure, and decrease the selection of resistance which would ensure for the benefit of animals and their future use.

However, the potential clinical disadvantage associated with FQ use was a rapid selection for resistance. Several pathogenic bacteria of food‐producing animals have been investigated the increasing of resistance to FQs following the introduction of ENR [46]. If FQ resistance emerges in animal pathogenic bacteria, this may result in treatment failure and increased mortality. This is a risk for poor animal welfare conditions and will result in economical losses. Consequently, for some animal infectious diseases, antimicrobial therapeutic use will be complicated if FQs lose their efficacy. As described in a previous study, multidrug‐resis‐ tant *S.* Typhimurium infections in veal calves were resistant to most conventionally used antimicrobials and also resistant to ENR resulted in a mortality exceeding 90%. FQs are also considered effective in other infections such as pneumonia, neonatal diarrhea, and mastitis caused by Gram‐negative organisms in piglets and calves. However, there were insufficient data to support the animal health or welfare problems when diseases cannot be treated result from FQ resistance during treatment.

The data indicate that it would be reasonable to assume that the veterinary usage of FQs will

FQ resistance in *Salmonella* is clearly associated with FQ use in food‐producing animals, and foodborne infections caused by such resistant bacteria are well investigated in human. FQ resistance in *S.* Typhimurium DT104 has been associated with increased hospitalization, more frequent and longer illness, treatment failures, and a higher risk of death [40]. Many studies also investigated that infections with multidrug‐resistant *Salmonella* were associated with longer hospitalization and a higher death rate than infections with susceptible isolates [41–43]. Previous study has found a 3.15 times increased mortality when patients infected with NAL‐resistant *S.* Typhimurium compared to patients infected with susceptible isolates [44]. For treatment of the infections with FQ‐resistant *Salmonella,* alternative antimicrobials are the third or fourth generation cephalosporin. Nevertheless, it should be considered con‐ traindications for treatment of uncomplicated non‐typhoidal *Salmonella* infection because FQ treatment can induce prolonged excretion of *Salmonella* and increased frequency of relapses [45]. However, for patients at risk such as immunocompromised, severely infected and elderly, FQs are considered first choice drugs and effective in reducing the disease length if

FQs are highly potent antimicrobial agents rapidly absorbed after oral administration and have a long half‐life and widespread distribution to most body tissues, which made them suitable for using in herd treatment of food‐producing animals. FQs are effective for serious infections in food‐producing animals such as systematic gastroenteritis and severe respira‐ tory diseases and are also used to treat urinary tract, skin, and soft‐tissue infections caused by Gram‐negative or some Gram‐positive aerobic bacteria. Moreover, they also have potential for treatment of infections caused by *Mycoplasma*, *Mycobacterium*, *Chlamydia*, *Ehrlichia*, and *Rickettsia*. However, documentation about authorized dosages and the effectiveness of FQs to treat all these infections in animals have not been determined on the base of the pharmaco‐ kinetic and pharmacodynamics properties. Sufficient knowledge about the selecting optimal dose and duration of FQs could help to develop appropriate dosing regimens to maximize the clinical efficacy, avoid therapeutic failure, and decrease the selection of resistance which

However, the potential clinical disadvantage associated with FQ use was a rapid selection for resistance. Several pathogenic bacteria of food‐producing animals have been investigated the increasing of resistance to FQs following the introduction of ENR [46]. If FQ resistance emerges in animal pathogenic bacteria, this may result in treatment failure and increased mortality. This is a risk for poor animal welfare conditions and will result in economical

have made a remarkable contribution to FQ resistance in human *Salmonella* infections.

**4. The potential impact on human health**

120 Current Topics in Salmonella and Salmonellosis

the treatment starts early in the infection.

**5. The potential impact on animal health**

would ensure for the benefit of animals and their future use.

## **6. The current state of knowledge of quinolone resistance mechanisms**

FQs are strong inhibitors of bacterial enzymes, which are necessary enzymes associated in major biological processes including DNA replication [47–49]. In prokaryotes, DNA is known as a double helix because there are two strands that intertwine around each other. However, additional complexity comes from the further twisting (supercoiling) of the double‐strand structure to put the double helix under torsion stress [50]. This supercoiling process that enables the long strands of DNA is condensed into compact supercoils permitting large amounts of DNA to be packed into the cell [51].

Topoisomerase I and topoisomerase II enzymes are enzymes that regulate the overwinding or underwinding of DNA and control the level of twisting within DNA. Topoisomerase I removes the number of negative supercoils, in contrast to topoisomerase II, which intro‐ duces negative supercoils that facilitate the unwinding of the over‐twisted DNA and can further change the DNA topology into an under‐twisted DNA [50]. DNA gyrase and DNA topoisomerase IV are type II topoisomerase comprising 2 A subunits and 2 B subunits enco‐ ded by the *gyrA* and *gyrB* genes or 2 C subunits and 2 E subunits encoded by the *parC* and *parE* genes, respectively [52]. DNA gyrase and topoisomerase IV have distinct roles although both enzymes have homologous action to relax positively supercoiled DNA. DNA gyrase decatenates replicating DNA by introducing negative supercoils into relaxed DNA while topoisomerase IV unlinks the newly replicated daughter chromosomes during cell division [52–54].

FQs are direct inhibitors of bacterial DNA synthesis by inhibiting two enzymes, DNA gyrase and topoisomerase IV, which have important roles in DNA replication. The quinolones bind to these enzymes with DNA to form drug‐enzyme‐DNA complexes (known as a ternary complex) subsequently induces double‐strand DNA breaks and blocks replication, therefore, results in damage to bacterial DNA and bacterial cell death [55–58]. However, the primary target enzyme, either DNA gyrase or topoisomerase IV, of FQs varies depending on the bacte‐ rial species. The preferential target of FQs in Gram‐negative bacteria is DNA gyrase, whereas in Gram‐positive microorganisms, topoisomerase IV is the primary target [58].

Resistance to quinolones occurs by different ways. The major mechanisms of bacterial resis‐ tance to FQs are altered target enzymes, expression of an active efflux, and altered membrane permeability.

#### **6.1. Target‐site mutation**

The main mechanism of FQ resistance is due to mutation in target genes (*gyrA*, *gyrB*, *parC*, and *parE*) that encode the primary and secondary target enzymes of these drugs. The mutations in quinolone resistance‐determining region (QRDR) of target genes alter the target enzyme conformation by amino acid substitutions and subsequently decrease in the drug binding affinity of the target enzyme, leading to FQ resistance [59–62].

In *Salmonella*, quinolone resistance was firstly investigated in the *gyrA* gene coding for the A subunit of gyrase. Mutations associated with FQ resistance in GyrA have been clustered between amino acids 67 and 106, termed the QRDR region. Amino acid substitutions of GyrA at Ser83 (to Phe, Tyr, or Ala) or at Asp87 (to Gly, Asn, or Tyr) are most usually identified in NAL‐resistant *Salmonella* strains. Previous studies have observed that single point mutation in QRDR of *gyrA* led to reduced sensitivity to CIP in *Salmonella* isolates [63]. Similar decreasing in CIP susceptibility was also found in three amino acid mutations of *parC* at Ser67 (to Cys), Arg76 (to Cys), and Cys80 (to Arg) in *S.* Enteritidis [64, 65]. Nevertheless, less frequently, the previous study detected novel mutations inside QRDR of GyrA at codon Asp72, Asp82, and Ala119 and also outside the QRDR [66]. Moreover, in another studies, the authors found double mutations in GyrA at both Ser83 and Asp87 in *S.* Typhimurium DT204 [67] and a single mutation at Asp87 (to Tyr) in all *Salmonella* strains [68] showing high‐level resistance to FQs. A *gyrB* gene mutation has also been observed in a quinolone‐resistant *S*. Typhimurium at Ser463 (to Tyr) [69].

These target‐site mutations show that different mutations of FQ‐resistant *Salmonella* isolates can result in very different resistance levels of quinolones, and this is not the same for all strains and all resistance mutations. Therefore, amino acid substitutions in topoisomerases are inadequate to clarify the level of resistance to quinolones in *S. enterica*. Nevertheless, it remains to be investigated what the specific role of these mutations on quinolone resistance in *Salmonella*.

#### **6.2. Transmissible quinolone‐resistance mechanisms**

Plasmid‐mediated quinolone resistance (PMQR) genes on mobile genetic elements are able to reduce susceptibility of quinolone or FQ antimicrobials. The PMQR gene, *qnr*, encodes a pen‐ tapeptide repeat motif protein (Qnr) that protects the target enzyme DNA gyrase and topoi‐ somerase IV by blocking the quinolone inhibition [70]. Recently, several Qnr proteins were investigated in Enterobacteriaceae (QnrA, QnrB, QnrC, QnrD, QnrS) [71, 72]. A recent study reported six variants of *qnrB* genes in *Salmonella* and *E. coli* isolates of human and animal isolates [73]. Nonetheless, the prevalence of *qnrS* genes is higher than the other *qnr* genes in *Salmonella*. A study from different European countries investigated a *qnrS* gene in 10% of the *Salmonella* isolates [73]. Moreover, *qnrS* gene has been identified in non‐typhoidal *Salmonella* clinical isolate from the USA [74]. The *qnrD* gene also has been investigated in eight different *Salmonella* serovars from 13 European countries [73].

Another plasmid‐encoded quinolone resistance determinant is a variant of an aminogly‐ coside acetyl transferase gene, *aac(6*'*)‐Ib‐cr*, which is able to acetylate the amino nitrogen on the piperazinyl substituent in aminoglycoside, and FQ drug classes lead to decreased susceptibility of these drugs [75–77]. However, the variant enzyme is not able to acetylate moxifloxacin and levofloxacin due to the absence of a piperazinyl substituent at position C‐7. Recently, this *aac(6*'*)‐Ib‐cr* gene has been reported in *Salmonella* isolated from chickens in China [78]. Plasmid‐mediated quinolone resistance determinants in *Salmonella* isolated from food‐producing animals are serious public health concern. Continuous surveillance of quinolone resistance determinants at national and international levels needs for limiting the dissemination of quinolone‐resistant *Salmonella* strains.

#### **6.3. Membrane permeability**

**6.1. Target‐site mutation**

122 Current Topics in Salmonella and Salmonellosis

in *Salmonella*.

The main mechanism of FQ resistance is due to mutation in target genes (*gyrA*, *gyrB*, *parC*, and *parE*) that encode the primary and secondary target enzymes of these drugs. The mutations in quinolone resistance‐determining region (QRDR) of target genes alter the target enzyme conformation by amino acid substitutions and subsequently decrease in the drug binding

In *Salmonella*, quinolone resistance was firstly investigated in the *gyrA* gene coding for the A subunit of gyrase. Mutations associated with FQ resistance in GyrA have been clustered between amino acids 67 and 106, termed the QRDR region. Amino acid substitutions of GyrA at Ser83 (to Phe, Tyr, or Ala) or at Asp87 (to Gly, Asn, or Tyr) are most usually identified in NAL‐resistant *Salmonella* strains. Previous studies have observed that single point mutation in QRDR of *gyrA* led to reduced sensitivity to CIP in *Salmonella* isolates [63]. Similar decreasing in CIP susceptibility was also found in three amino acid mutations of *parC* at Ser67 (to Cys), Arg76 (to Cys), and Cys80 (to Arg) in *S.* Enteritidis [64, 65]. Nevertheless, less frequently, the previous study detected novel mutations inside QRDR of GyrA at codon Asp72, Asp82, and Ala119 and also outside the QRDR [66]. Moreover, in another studies, the authors found double mutations in GyrA at both Ser83 and Asp87 in *S.* Typhimurium DT204 [67] and a single mutation at Asp87 (to Tyr) in all *Salmonella* strains [68] showing high‐level resistance to FQs. A *gyrB* gene mutation

has also been observed in a quinolone‐resistant *S*. Typhimurium at Ser463 (to Tyr) [69].

These target‐site mutations show that different mutations of FQ‐resistant *Salmonella* isolates can result in very different resistance levels of quinolones, and this is not the same for all strains and all resistance mutations. Therefore, amino acid substitutions in topoisomerases are inadequate to clarify the level of resistance to quinolones in *S. enterica*. Nevertheless, it remains to be investigated what the specific role of these mutations on quinolone resistance

Plasmid‐mediated quinolone resistance (PMQR) genes on mobile genetic elements are able to reduce susceptibility of quinolone or FQ antimicrobials. The PMQR gene, *qnr*, encodes a pen‐ tapeptide repeat motif protein (Qnr) that protects the target enzyme DNA gyrase and topoi‐ somerase IV by blocking the quinolone inhibition [70]. Recently, several Qnr proteins were investigated in Enterobacteriaceae (QnrA, QnrB, QnrC, QnrD, QnrS) [71, 72]. A recent study reported six variants of *qnrB* genes in *Salmonella* and *E. coli* isolates of human and animal isolates [73]. Nonetheless, the prevalence of *qnrS* genes is higher than the other *qnr* genes in *Salmonella*. A study from different European countries investigated a *qnrS* gene in 10% of the *Salmonella* isolates [73]. Moreover, *qnrS* gene has been identified in non‐typhoidal *Salmonella* clinical isolate from the USA [74]. The *qnrD* gene also has been investigated in eight different

Another plasmid‐encoded quinolone resistance determinant is a variant of an aminogly‐ coside acetyl transferase gene, *aac(6*'*)‐Ib‐cr*, which is able to acetylate the amino nitrogen on the piperazinyl substituent in aminoglycoside, and FQ drug classes lead to decreased

affinity of the target enzyme, leading to FQ resistance [59–62].

**6.2. Transmissible quinolone‐resistance mechanisms**

*Salmonella* serovars from 13 European countries [73].

The membrane permeability and the ability of FQs to enter the bacterial cells are an important determinant of the potency of these drugs that have intracellular targets [79]. The outer‐ membrane proteins (OMPs) of Gram‐negative bacteria consist of pore‐forming outer‐mem‐ brane proteins which serve as a particular barrier for the entry of hydrophilic molecules into the cell. It has been shown that CIP (hydrophilic quinolones) preferentially entry into the cells via porin pathway [80]. Down‐regulation of OMPs results in reduced FQ susceptibility in FQ‐resistant isolates of different species [81–84]. Very few researches have investigated on alterations of OMP expression or the role of lipopolysaccharide composition in quinolone‐ resistant *Salmonella* isolates [68, 85–89]. The lengthening of the O chains has been studied in quinolone‐resistant *Salmonella* that could also lead to a lower level in the permeability of the outer membrane [85]. The previous studies have found the lack of OmpF porin expression result from SoxS up‐regulates micF transcription in quinolone‐resistant *Salmonella* strains [86–88, 90]. However, it remains unclear whether such alterations contributed to signifi‐ cant reduction of outer‐membrane permeability and reduced susceptibility of quinolones in *Salmonella* isolates.

#### **6.4. Efflux**

Chromosomal multidrug efflux pumps are capable of actively removing FQs and a broad range of antimicrobial agents from the bacterial cell and are mostly encoded by chromo‐ somal genes. These efflux systems consist of different classes of transporters such as the resistance nodulation division (RND) family of tripartite transporters of Gram‐negative pathogens [91, 92]. These systems are mainly responsible for the intrinsic pattern of reduced susceptibility to FQs and other antimicrobial agents but are also responsible for increased resistance resulting from derepression of the transporter. Previous studies showed the evi‐ dence for the participation of active efflux in quinolone‐resistant *Salmonella* isolates [85, 93]. It was concluded that the overproduction of the AcrAB‐TolC efflux pump appeared prior to *gyrA* mutations in *in vitro* selected quinolone‐resistant *Salmonella* mutants [85]; therefore, the AcrAB‐TolC efflux system is the major mechanism that involved in quinolone resis‐ tance in *S*. Typhimurium DT104 strains. However, both target gene mutations and active efflux mediated by AcrAB*‐*TolC are necessary to obtain high‐level FQ resistance for *S*. Typhimurium DT204 strains [94]. Nevertheless, there is no direct evidence to demonstrate the role of the AcrAB‐TolC efflux system in quinolone‐resistant *Salmonella*; therefore, sub‐ stantial work remains to be done in order to understand the role of efflux and its regulation in *Salmonella*.

#### **6.5. The fitness costs**

Mechanisms associated with high‐level FQ resistance are multiple mutations in the type II topoisomerase‐encoding genes and the over‐expression of multidrug resistance efflux pumps. The presence of mutations in these structural or regulatory genes not only increases resistance to quinolones but also affects fitness costs such as reduced growth rates and virulence of the bacterial cell in a lack of antibiotic selective pressure [95–99]. However, maintenance of resis‐ tance can arise through the development of second‐site compensatory mutations that restore fitness and virulence without loss of resistance [100].

The fitness cost of the genes responsible for quinolone resistance traits has not been fully elucidated in high‐level FQ‐resistant *Salmonella*. Nevertheless, results from previous stud‐ ies suggest that high‐level CIP resistance mechanisms in *Salmonella* lead to restrictive condi‐ tions of fitness costs and minimizing the emergence and spread of highly resistant clones in the absence of drug selection pressure [101, 102]. As demonstrated in previous study [103], high‐level CIP‐resistant *S*. Enteritidis *in vitro* derived mutants in the absence of antibiotic selective pressure result in compensatory evolution favoring a reversion back to a more sensi‐ tive phenotype associated with lesser fitness costs, rather than the compensatory mutations that would restore resistance. However, under *in vivo* conditions, a previous study has found that chromosomal mutations of *S*. Typhimurium that confer resistance to NAL, streptomy‐ cin, or rifampicin decrease growth rate and ability to colonize in mice rather than a rever‐ sion to the susceptible phenotype and restore virulence [104]. In contrast to the high‐level FQ resistance, an intermediate level of resistance to CIP of *S*. Typhimurium mutants apparently favored a partial reversion to a susceptible level and a normal growth rate with successfully colonized the gut of chickens, rather than the acquisition of resistance to FQs [101].

Quinolone resistance of non‐typhoidal *Salmonella* is complicated. The understanding of the various mechanisms of quinolone resistance, the fitness costs of each *Salmonella* strain, and the interplay between different quinolone resistance mechanisms has increased in recent years. Increased resistance to quinolones could be selected under a wide range of selective conditions even in the absence of quinolone selective pressure. Therefore, minimizing the emergence and spread of quinolone resistance will not be as simple as limiting the use of these drugs.

## **7. To decrease the emergence and spread of quinolone resistance**

FQs are intensively used in animal production and have allowed better treatment of several animal infectious diseases. The risks of the overuse and misuse of FQs in food‐animal produc‐ tion can contribute to higher levels of resistance in human *Salmonella* infections. Therefore, the FQ resistance of *Salmonella* should be taken into account and prevented as resistant bacteria or resistance genes may be transferred to humans through the food chain. Given the importance of FQ resistance as a global health concern, many researchers have reviewed the existing sci‐ entific literatures and developed guidelines to limit all compounds of FQ use, including use in food‐producing animals.

Priority setting of agendas for research on minimizing the emergence of FQ resistance in *Salmonella* is needed to identify missing scientific data and to specify research designs and methods to address these resistance problems in food‐producing animals and human medi‐ cine. The priorities identified by the research agenda must include contributions by different experts in basic genetics and microbiology sciences, veterinary medicine, human medicine, public health organization, social sciences, economics sciences, and public policy.

Furthermore, sufficient research funding for minimizing the FQ resistance of *Salmonella* in human and food‐producing animals has likely contributed to the adequate scientific evidence which necessary for informing public health decisions. Given the scale of the FQ resistance problem and the demonstrated role of FQ uses in food‐producing animals in this public health crisis, adequate support for research specific to the role of food‐producing animal uses of FQs in the development of resistance must be a national priority.

Urgently address complex barriers that limit the quality of data on the use of FQs in food‐produc‐ ing animals and human medicine. Currently, such data from human and veterinary medicine are provided on a voluntary basis, and the methods used to collect, analyze, and report are not standardized because of political, economic, and social barriers. Effective surveillance of FQ use in food‐producing animals and humans is a key first step toward for estimating the full scope of FQ resistance in *Salmonella*. Despite increasingly widespread recognition that FQ use in food‐pro‐ ducing animals is a major factor of human infections with FQ‐resistant *Salmonella*, there remains a significant need for scientific evidence of the FQ use practices that affect the human health risk.

## **8. Conclusion**

**6.5. The fitness costs**

124 Current Topics in Salmonella and Salmonellosis

fitness and virulence without loss of resistance [100].

Mechanisms associated with high‐level FQ resistance are multiple mutations in the type II topoisomerase‐encoding genes and the over‐expression of multidrug resistance efflux pumps. The presence of mutations in these structural or regulatory genes not only increases resistance to quinolones but also affects fitness costs such as reduced growth rates and virulence of the bacterial cell in a lack of antibiotic selective pressure [95–99]. However, maintenance of resis‐ tance can arise through the development of second‐site compensatory mutations that restore

The fitness cost of the genes responsible for quinolone resistance traits has not been fully elucidated in high‐level FQ‐resistant *Salmonella*. Nevertheless, results from previous stud‐ ies suggest that high‐level CIP resistance mechanisms in *Salmonella* lead to restrictive condi‐ tions of fitness costs and minimizing the emergence and spread of highly resistant clones in the absence of drug selection pressure [101, 102]. As demonstrated in previous study [103], high‐level CIP‐resistant *S*. Enteritidis *in vitro* derived mutants in the absence of antibiotic selective pressure result in compensatory evolution favoring a reversion back to a more sensi‐ tive phenotype associated with lesser fitness costs, rather than the compensatory mutations that would restore resistance. However, under *in vivo* conditions, a previous study has found that chromosomal mutations of *S*. Typhimurium that confer resistance to NAL, streptomy‐ cin, or rifampicin decrease growth rate and ability to colonize in mice rather than a rever‐ sion to the susceptible phenotype and restore virulence [104]. In contrast to the high‐level FQ resistance, an intermediate level of resistance to CIP of *S*. Typhimurium mutants apparently favored a partial reversion to a susceptible level and a normal growth rate with successfully

colonized the gut of chickens, rather than the acquisition of resistance to FQs [101].

spread of quinolone resistance will not be as simple as limiting the use of these drugs.

**7. To decrease the emergence and spread of quinolone resistance**

but only used as life‐saving therapeutic treatment of individual sick animals.

Quinolone resistance of non‐typhoidal *Salmonella* is complicated. The understanding of the various mechanisms of quinolone resistance, the fitness costs of each *Salmonella* strain, and the interplay between different quinolone resistance mechanisms has increased in recent years. Increased resistance to quinolones could be selected under a wide range of selective conditions even in the absence of quinolone selective pressure. Therefore, minimizing the emergence and

FQs are intensively used in animal production and have allowed better treatment of several animal infectious diseases. The risks of the overuse and misuse of FQs in food‐animal produc‐ tion can contribute to higher levels of resistance in human *Salmonella* infections. Therefore, the FQ resistance of *Salmonella* should be taken into account and prevented as resistant bacteria or resistance genes may be transferred to humans through the food chain. Given the importance of FQ resistance as a global health concern, many researchers have reviewed the existing sci‐ entific literatures and developed guidelines to limit all compounds of FQ use, including use in food‐producing animals. FQs should be banned for all preventive use and mass medication, Infections in humans with quinolone‐resistant *Salmonella* resulted in increased risk of hospi‐ talization and mortality. FQs are efficient and valuable antimicrobials in some serious animal indications because FQs are the only alternative available. Therefore, if FQs lose their ability for the treatment of animal diseases, the therapeutic effect of some diseases will be compli‐ cated and may result in poor animal welfare and economical losses. Recently, it is now critical that food‐producing animal use of FQs be recognized as one of the major contributors to the development of resistant *Salmonella* strains that result in life‐threatening human infections and included as part of the strategy to control the public health crisis of FQ resistance.

## **Author details**

Siriporn Kongsoi<sup>1</sup> , Chie Nakajima2,3 and Yasuhiko Suzuki2,3\*

\*Address all correspondence to<EMAIL_ADDRESS>

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**Provisional chapter**

## *Salmonella* **in Wastewater: Identification, Antibiotic Resistance and the Impact on the Marine Environment** *Salmonella* **in Wastewater: Identification, Antibiotic Resistance and the Impact on the Marine Environment**

Abdellah El Boulani, Rachida Mimouni, Hasna Mannas, Fatima Hamadi and Nouredine Chaouqy Hasna Mannas, Fatima Hamadi and Nouredine Chaouqy Additional information is available at the end of the chapter

Abdellah El Boulani, Rachida Mimouni,

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67298

#### **Abstract**

Many coastal cities around the world discharge their wastewaters into the marine environment. These wastewaters contain a high variety of pathogenic microorganisms that would have a role in the contamination of this ecosystem and may have potential risks for public health and environment. Using an environmental approach, we inves‐ tigate the presence of *Salmonella* in wastewater treatment plants and its presence after the treatment in its receiving marine environment. In this environmental approach, we provide information about the inefficiency of wastewater treatment to remove *Salmonella*, especially that wastewater is considered as a good tank of high diversity of *Salmonella* serotypes. The identified *Salmonella* serotypes in the receiving marine environment almost coincide with those identified in wastewater. This characterization of *Salmonella* strains from wastewater and marine environment involves the direct impact of municipal wastewater discharges on this environment. Antibiotic susceptibility tests reveal gener‐ ally the presence of multiresistant *Salmonella* strains in wastewater, which usually end up in the marine environment and may have a significant risk on the public health.

**Keywords:** antibiotic susceptibility, marine environment, *Salmonella*, Wastewater, wastewater treatment

## **1. Introduction**

Many major coastal cities around the world discharge their wastewaters into the marine envi‐ ronment. Wastewater may consist of a combination of domestic, industrial and agricultural effluent. Generally, wastewater treatment plants (WWTPs) are mostly designed to effectively remove fecal bacteria, some chemical pollutants but are not provided to eliminate pathogenic

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

microorganisms [1, 2]. Furthermore, most of these treatment systems remain imperfect and continue to reject varied concentrations of enteric bacteria in the environment [1–5].

Pathogenic bacteria become normally more harmful to environment and humans when they acquire resistance to antibiotics. Moreover, many studies [5–7] confirmed the detection of antibiotic concentrations in wastewater that treatment systems generally fail to eliminate. Therefore, when there is a contact between bacteria and antibiotics traces, the wastewater could be an ideal environment to the exchange of genetic material between bacteria and consequently the acquisition of antibiotic resistance [5, 6, 8, 9].

The *Salmonella* genus is one of the pathogen bacteria that could be conveyed to the environmen‐ tal waters through sewage pollution. Therefore, the determination of the presence of *Salmonella*, its levels and its antibiotic resistance at the same time in wastewaters and environmental waters are crucial and are required to assess the related health risks.

Previous works [1, 9–13] have studied the presence of *Salmonella* in wastewater but they stay insufficient to provide enough information on the diversity and the antibiotic resistance pro‐ files of *Salmonella*. This makes transmission of *Salmonella* via wastewater, its impact on the environment and the origin of contamination poorly understood. In Morocco, *Salmonella* in wastewater is rarely discussed and its bibliography is very scarce. In this context, we are cur‐ rently leading the first study about *Salmonella* in the southern region of Morocco. This work is focusing on the identification, serotyping and antibiotic susceptibility of *Salmonella* strains isolated from Agadir wastewater treatment plant.

The aim of this chapter is first to provide qualitative information on the capacity of wastewater treatment process to eliminate *Salmonella* considered among the most pathogenic bacteria eas‐ ily transmissible in water. The second aim is to present the most *Salmonella* serotypes isolated from wastewater and marine environment and the possible impact of wastewater discharges on marine environment.

## **2. WWTPs removal efficiency of** *Salmonella*

Wastewater treatment plants know a growing pressure which leads to the evacuation of untreated or insufficiently treated effluents into the environment. Thus, the efficient removal of pathogenic bacteria in wastewater is a crucial task because wastewater discharges can significantly increase the contamination of surface water and lead to water and seafood‐ borne infections. Generally, an optimal wastewater treatment process can attain 90–99% of reduction of microbial load [2, 14, 15]. However, in some cases, the reduction level decreases are mostly due to a nonrespective operating mode or due to bad or inadequate plant maintenance.

Several studies have shown the presence of *Salmonella* in wastewater before and after treatment even if treatment processes are different [1, 9, 10, 13]. This could be explained by the fact that WWTPs are mostly designed just to reduce microbial load but are not conceived specifically to completely eliminate the pathogenic bacteria.

An activated sludge treatment process remains unable to totally remove *Salmonella* [9, 12]. Koivunen et al. have also shown that activated sludge process coupled with phosphorus pre‐ cipitation followed by a secondary settling or coupled with denitrification‐nitrification fails to remove *Salmonella* from municipal wastewater [1].

microorganisms [1, 2]. Furthermore, most of these treatment systems remain imperfect and

Pathogenic bacteria become normally more harmful to environment and humans when they acquire resistance to antibiotics. Moreover, many studies [5–7] confirmed the detection of antibiotic concentrations in wastewater that treatment systems generally fail to eliminate. Therefore, when there is a contact between bacteria and antibiotics traces, the wastewater could be an ideal environment to the exchange of genetic material between bacteria and

The *Salmonella* genus is one of the pathogen bacteria that could be conveyed to the environmen‐ tal waters through sewage pollution. Therefore, the determination of the presence of *Salmonella*, its levels and its antibiotic resistance at the same time in wastewaters and environmental waters

Previous works [1, 9–13] have studied the presence of *Salmonella* in wastewater but they stay insufficient to provide enough information on the diversity and the antibiotic resistance pro‐ files of *Salmonella*. This makes transmission of *Salmonella* via wastewater, its impact on the environment and the origin of contamination poorly understood. In Morocco, *Salmonella* in wastewater is rarely discussed and its bibliography is very scarce. In this context, we are cur‐ rently leading the first study about *Salmonella* in the southern region of Morocco. This work is focusing on the identification, serotyping and antibiotic susceptibility of *Salmonella* strains

The aim of this chapter is first to provide qualitative information on the capacity of wastewater treatment process to eliminate *Salmonella* considered among the most pathogenic bacteria eas‐ ily transmissible in water. The second aim is to present the most *Salmonella* serotypes isolated from wastewater and marine environment and the possible impact of wastewater discharges

Wastewater treatment plants know a growing pressure which leads to the evacuation of untreated or insufficiently treated effluents into the environment. Thus, the efficient removal of pathogenic bacteria in wastewater is a crucial task because wastewater discharges can significantly increase the contamination of surface water and lead to water and seafood‐ borne infections. Generally, an optimal wastewater treatment process can attain 90–99% of reduction of microbial load [2, 14, 15]. However, in some cases, the reduction level decreases are mostly due to a nonrespective operating mode or due to bad or inadequate plant

Several studies have shown the presence of *Salmonella* in wastewater before and after treatment even if treatment processes are different [1, 9, 10, 13]. This could be explained by the fact that WWTPs are mostly designed just to reduce microbial load but are not conceived specifically to

continue to reject varied concentrations of enteric bacteria in the environment [1–5].

consequently the acquisition of antibiotic resistance [5, 6, 8, 9].

are crucial and are required to assess the related health risks.

isolated from Agadir wastewater treatment plant.

**2. WWTPs removal efficiency of** *Salmonella*

completely eliminate the pathogenic bacteria.

on marine environment.

138 Current Topics in Salmonella and Salmonellosis

maintenance.

Until now, no study has been conducted on the elimination of *Salmonella* by infiltration percolation treatment process through sand. However, a current study conducted by our research team (unpublished data) showed the inefficiency of this treatment system to remove *Salmonella*. Thus, treated wastewater still contains *Salmonella* which continues to be detected at the treatment plant exit.

To get an efficient reduction or a total removal of *Salmonella*, a tertiary wastewater treatment is needed. Indeed, Koivunen et al. show that tertiary treatment by rapid sand contact filter allows a very good efficiency to remove *Salmonella.* Consequently, *Salmonella* was not found in wastewaters after tertiary filtration [1]. Furthermore, a current study conducted by our team showed that tertiary treatment by UV irradiation applied just on a portion of the secondary treated effluents (which are intended for the irrigation of golf courses) allows total elimination of *Salmonella* (unpublished data).

## **3. Microbiological risks associated with** *Salmonella* **in wastewater**

Wastewater is known to be a common vehicle for the transport and transmission of *Salmonella* serovars and is able to pollute environment and infect humans. Indeed, several outbreaks and contaminations have been related to *Salmonella* in wastewater through irrigation of crops [16–18], infiltration and transport in soil to groundwater [19–21] or to its discharge into the marine environment [22, 23]. In this last case, although some pathogens are natu‐ rally present in the aquatic environment, *Salmonella* may be introduced through animal or human fecal and sewage pollution [24].

The risk of contamination linked to *Salmonella* is enhanced by its ability to survive lon‐ ger in the environment. Indeed, contamination of groundwater, environmental water and soil by *Salmonella* is due to its ability to survive very long time in these environments [25]. Furthermore, contaminations related to *Salmonella* in seawater and seafood are increased by the capacity of *Salmonella* to survive in relatively high salt conditions [26]. Thus, the per‐ sistence of wastewater discharges in coastal waters may increase the load of *Salmonella* in marine environment, and therefore increase the higher incidence of seawater and seafood‐ borne infections.

## **4. Identification methods of** *Salmonella*

Various methods have been developed for the subtyping of *Salmonella*. Each of them has its advantages and drawbacks in terms of cost, speed, robustness, and sensitivity [27]. The choice of identification techniques of bacteria is generally done according to the objectives of identifying and the available means. Biochemical techniques are usually used to isolate and identify only species. For further characterization, determination of the origins and the relationships between different isolates, many other accurate methods are needed.

Conventional serotyping using somatic and flagellar *Salmonella* antisera is the most frequently used reference method for serotyping *Salmonella* isolated from wastewater [1, 9, 28, 29]. This method is based on the direct agglutination technique blade, involving *Salmonella* strains with different antisera to identify variants of the somatic O and flagellar H antigens. Serotyping is generally performed according to the antigenic formulae of Kauffmann‐White‐Le Minor scheme [30]. The main limitation of this technique is that not all O serotypes are included in Polyvalent O antisera which *Salmonella* species should agglutinate with [31]. A combina‐ tion between conventional serotyping and the ribosomal spacer‐heteroduplex polymorphism (RS‐HP) methods was also used to characterize *Salmonella* strains from wastewater [13]. This technique based on the PCR amplification of the intergenic spacer region between the 16S and 23S rRNA genes can produce amplicon profiles allowing the discrimination of species at both serotype and intraserotype levels [13].

PFGE allows a high discrimination and it is usually used for outbreak investigations and it is also widely used for characterizing epidemic *Salmonella* strains. The capacity of PFGE to differ‐ entiate strains of bacterial pathogens makes it a standard method used to assess the epidemic spread of infectious diseases and to trace *Salmonella* outbreaks. However, even if it is reproduc‐ ible and discriminatory, some strains of *Salmonella* cannot be typed by PFGE [27]. Recently, matrix‐assisted laser desorption/ionization time of light mass spectrometry (MALDI‐TOF MS) has been used for the identification of *Salmonella.* It is also utilized to discriminate *Salmonella Typhi* from other *Salmonella* serovars [32]. Despite its rapidity and simplicity, the preparation of MALDI‐TOF requires more modifications and improvements to available protocols before being adopted as an autonomous method [32]. Other molecular methods developed as alter‐ natives to conventional serotyping (MLST, MLVA, SNP, and molecular typing with composite microarrays) seem successful. However, these methods do not provide exactly similar results like those obtained by the current reference method which is agglutination serotyping [27].

## **5.** *Salmonella* **serotypes isolated from wastewater**

As reported, wastewater is an ideal tank of *Salmonella* strains. Furthermore, identified *Salmonella* showed generally high variability of serotypes. The major serotypes isolated in wastewater from different countries are presented in **Table 1**.

Data concerning *Salmonella* serotyping (**Table 1**) show high heterogeneity of serotypes iso‐ lated from wastewater. This high diversity can be explained by the variety of the origin of effluents carrying these *Salmonella* strains. The most frequent serotypes identified in France wastewater were Newport, Saintpaul, and Brandenburg [13]. Indeed, Serotypes Mbandaka, Virchow, Hadar, Indiana, Infantis, Saintpaul, and Senftenberg are commonly isolated from poultry farms. Also, Typhimurium and Indiana serotypes are generally isolated in human pathology and can derive from healthy carriers [13]. Espigares et al. have also explained this diversity by the variety of the origin of *Salmonella* which may be of human or animal origin [9]. *Salmonella* Agona, Saintpaul, Virchow, and Corvallis are the most frequent serotypes identi‐ fied in Finnish wastewater [1]. According to Koivunen et al., this diversity may be reported to the size of the population suggesting, therefore, that larger populations produce larger spec‐ tra of serovars [1]. However, despite these explanations, the origin of this diversity remains unknown. The large variability in serovars identified in USA wastewater indicates multiple sources of the isolates. This variability may be due to fecal shedding in clinical salmonel‐ losis or to animal agriculture [33]. Our current study (not shown data) concerning isolated *Salmonella* from wastewater in Agadir, a coastal city in Morocco, show high variability of serotypes. Thus, among 52 *Salmonella* strains we identified 18 different serotypes. *Salmonella Muenster* was the major serotype which showed a high incidence with 14 isolates followed by *Salmonella Infantis*, *Salmonella Senftenberg*, *Salmonella Montevideo*, and *Salmonella Kentucky* as the most frequent serotypes identified*.* This high diversity of serotypes in Agadir wastewater can be explained by its various origins including domestic and industrial effluents especially those coming from poultry slaughterhouse and fish processing plants very widespread in the city.

**Table 1.** *Salmonella* serotypes isolated from wastewater.

and the available means. Biochemical techniques are usually used to isolate and identify only species. For further characterization, determination of the origins and the relationships between

Conventional serotyping using somatic and flagellar *Salmonella* antisera is the most frequently used reference method for serotyping *Salmonella* isolated from wastewater [1, 9, 28, 29]. This method is based on the direct agglutination technique blade, involving *Salmonella* strains with different antisera to identify variants of the somatic O and flagellar H antigens. Serotyping is generally performed according to the antigenic formulae of Kauffmann‐White‐Le Minor scheme [30]. The main limitation of this technique is that not all O serotypes are included in Polyvalent O antisera which *Salmonella* species should agglutinate with [31]. A combina‐ tion between conventional serotyping and the ribosomal spacer‐heteroduplex polymorphism (RS‐HP) methods was also used to characterize *Salmonella* strains from wastewater [13]. This technique based on the PCR amplification of the intergenic spacer region between the 16S and 23S rRNA genes can produce amplicon profiles allowing the discrimination of species at both

PFGE allows a high discrimination and it is usually used for outbreak investigations and it is also widely used for characterizing epidemic *Salmonella* strains. The capacity of PFGE to differ‐ entiate strains of bacterial pathogens makes it a standard method used to assess the epidemic spread of infectious diseases and to trace *Salmonella* outbreaks. However, even if it is reproduc‐ ible and discriminatory, some strains of *Salmonella* cannot be typed by PFGE [27]. Recently, matrix‐assisted laser desorption/ionization time of light mass spectrometry (MALDI‐TOF MS) has been used for the identification of *Salmonella.* It is also utilized to discriminate *Salmonella Typhi* from other *Salmonella* serovars [32]. Despite its rapidity and simplicity, the preparation of MALDI‐TOF requires more modifications and improvements to available protocols before being adopted as an autonomous method [32]. Other molecular methods developed as alter‐ natives to conventional serotyping (MLST, MLVA, SNP, and molecular typing with composite microarrays) seem successful. However, these methods do not provide exactly similar results like those obtained by the current reference method which is agglutination serotyping [27].

As reported, wastewater is an ideal tank of *Salmonella* strains. Furthermore, identified *Salmonella* showed generally high variability of serotypes. The major serotypes isolated in

Data concerning *Salmonella* serotyping (**Table 1**) show high heterogeneity of serotypes iso‐ lated from wastewater. This high diversity can be explained by the variety of the origin of effluents carrying these *Salmonella* strains. The most frequent serotypes identified in France wastewater were Newport, Saintpaul, and Brandenburg [13]. Indeed, Serotypes Mbandaka, Virchow, Hadar, Indiana, Infantis, Saintpaul, and Senftenberg are commonly isolated from poultry farms. Also, Typhimurium and Indiana serotypes are generally isolated in human pathology and can derive from healthy carriers [13]. Espigares et al. have also explained this

different isolates, many other accurate methods are needed.

**5.** *Salmonella* **serotypes isolated from wastewater**

wastewater from different countries are presented in **Table 1**.

serotype and intraserotype levels [13].

140 Current Topics in Salmonella and Salmonellosis

## **6.** *Salmonella* **serotypes isolated from a wastewater receiving marine environment**

In coastal cities, the marine environment is usually the final end of treated and untreated sewage. **Table 2** shows some studies throughout the world carried in this regard.

**Table 2.** *Salmonella* serotypes isolated from marine environment.

As reported in wastewater, data from marine environment also reveal a large diversity of *Salmonella* serotypes. However, the origin of these serovars remains little known. Following to data in **Tables 1** and **2**, it appears that the most serotypes found in wastewater are also isolated in the surrounding marine environment. Furthermore, studies from France [13] and Mexico [29] showed that Newport and Typhimurium were respectively the major *Salmonella* serotypes isolated in wastewater. These serotypes were also the same major serotypes isolated from marine environment [13, 29]. Serovar Typhimurium has been shown to be the most common serovar isolated from marine environment in different parts of the world [13, 29, 34–36]. It was suggested previously that this serotype shows an excellent adaptation to the marine water stress after passing through wastewater [37]. In Agadir, study conducted by our team [38] in marine environment has identified *S. Muenster* as the major serotype (13 strains among 46 isolates). This high occurrence of serotype Muenster coincides with its high inci‐ dence in Agadir wastewater. Therefore, these findings reveal that wastewater discharges into the marine environment could be the main source of contamination and the principal origin of *Salmonella* strains found in this environment.

## **7. Antimicrobial resistance of** *Salmonella* **isolated from wastewater**

Urban wastewater treatment plants (UWTPs) are suspected to be among the main anthropo‐ genic sources for antibiotics, antibiotic resistant genes (ARGs) and antibiotic resistant bac‐ teria (ARB) spread into the environment [14, 39, 40]. The presence of antibiotics in effluents is generally due to incomplete metabolism in humans or because of the mismanagement of unused antibiotics [4, 41].

Generally, bacteria in wastewater are in permanent contact with various elements including antibiotics and other chemical products. Furthermore, antibiotic resistance was positively cor‐ related with the occurrence of antibiotic residues. In addition, a relationship between antibi‐ otic residues, bacterial community structure and antibiotic resistance was demonstrated [42]. Therefore, the elimination of these antibiotics from wastewater is a major concern. Sorption and hydrolysis could be a degradation pathway that may lead to the removal of antibiotics in wastewater [39]. However, in previous reports, it has been demonstrated that UV radiation is not effective to remove antibiotics [43]. This indicates that photolysis is not an important mechanism for degradation of these compounds in wastewater. A combination of physico‐ chemical and biological treatment and the optimization of their operating conditions might prove an effective removal increase of persistent antibiotic residues in wastewater [39].

According to previous studies [6, 44, 45], ARB have been detected widely in wastewater samples and comparatively to surface water, higher proportion of ARB was reported in raw and treated wastewater than in surface water [4, 40]. It has also been reported that *Salmonella* strains show a high proportion among other multidrug resistant bacteria [46]. According to these studies, conditions in wastewater treatment plants (WWTPs) seem to be favorable for the proliferation of ARB.

As reported in wastewater, data from marine environment also reveal a large diversity of *Salmonella* serotypes. However, the origin of these serovars remains little known. Following to data in **Tables 1** and **2**, it appears that the most serotypes found in wastewater are also isolated in the surrounding marine environment. Furthermore, studies from France [13] and Mexico [29] showed that Newport and Typhimurium were respectively the major *Salmonella* serotypes isolated in wastewater. These serotypes were also the same major serotypes isolated from marine environment [13, 29]. Serovar Typhimurium has been shown to be the most common serovar isolated from marine environment in different parts of the world [13, 29, 34–36]. It was suggested previously that this serotype shows an excellent adaptation to the marine water stress after passing through wastewater [37]. In Agadir, study conducted by our team [38] in marine environment has identified *S. Muenster* as the major serotype (13 strains among 46 isolates). This high occurrence of serotype Muenster coincides with its high inci‐ dence in Agadir wastewater. Therefore, these findings reveal that wastewater discharges into the marine environment could be the main source of contamination and the principal origin

**Country France Spain Mexico Morocco USA**

Serotypes Virchow Hadar Othmarschen Hadar Bardo

References [13] [34] [29] [35, 36, 38] [47]

Cerro Anatum Stanley Altona Adelaide Newport Enteritidis Galiema Anatum Agona Typhimurium Goldcoast Bulovka Corvallis Arizona

> London Tonev Senftenberg Hartford Newport Subaru Typhimurium Newport Typhimurium Typhimurium Kentucky Poona

> > Vejle London Reading Winnipeg Mbandaka Typhimurium Muenster Oakland Blockley Labadi

**7. Antimicrobial resistance of** *Salmonella* **isolated from wastewater**

Urban wastewater treatment plants (UWTPs) are suspected to be among the main anthropo‐ genic sources for antibiotics, antibiotic resistant genes (ARGs) and antibiotic resistant bac‐ teria (ARB) spread into the environment [14, 39, 40]. The presence of antibiotics in effluents

of *Salmonella* strains found in this environment.

**Table 2.** *Salmonella* serotypes isolated from marine environment.

142 Current Topics in Salmonella and Salmonellosis

Antibiotic resistance (ABR) of *Salmonella* isolated from wastewater has been discussed in some studies. A wide variety of antibiotic resistance patterns of *Salmonella* serotypes have been found in USA wastewater [33]. Indeed, 86% of 647 *Salmonella* isolates were susceptible, 4% were monoresistant and 10% were multiresistant. *Salmonella* serovars multiresistant to at least four antibiotics were *Salmonella Braenderup*, Derby, Edinburg, Hadar, Saintpaul, Typhimurium, Uganda and Virchow. A resistance to the third generation of cephalosporins considered as one of the first drugs of choice to treat human salmonellosis has also been reported [33]. From Finnish wastewater [1], among 197 isolates, 44% of the *Salmonella* strains were resistant to antimicrobial agents, almost 20% were multiresistant. The most multiresistant serotype was *Salmonella Saintpaul* (to 6 antibiotics). Moreover, 32% of strains were resistant to nalidixic acid which can indicates the reduction of sensitivity to ciprofloxacin considered as the selected drug in severe infections [1]. The most resistant serotype isolated in wastewater from Spain was *Salmonella Hadar* with a pattern of multiresistance to six antibiotics [9]. In Morocco, among 42 strains isolated from wastewater, 19 (45.2%) were resistant at least to two antibiotics. The most multiresistant serotype was *S. Typhimurium* followed by *S. Hadar* and *S. Senftenberg*. *S. Typhimurium* was resistant to ampicillin, chloramphenicol, streptomycin, sulfametoxazole/ trimetoprim, and tetracycline. Serovars Give, Newport and Anatum were susceptible to all used antibiotics [28]. Antibiotic susceptibility test (ATS) of *Salmonella* serotypes from Agadir wastewater treatment plant carried out by our team showed that among 52 isolates 23% of strains were multiresistant. ATS was carried out by the disk diffusion method, with a panel of 32 antimicrobial drugs (Bio‐Rad) and interpreted according to the EUCAST clinical guide‐ lines (http://www.eucast.org/clinical\_breakpoints/). Multiresistant serovars were Chester, Kentucky, and Typhimurium. The most multiresistant serotype identified in this study was Typhimurium with a pattern of multiresistance to 18 antibiotics.

All these data showed that wastewater is a tank of a wide variety of *Salmonella enterica* serovars and ABR patterns. Other data showed that multiresistant bacteria have been detected extensively in wastewater samples which pass through WWTPs and arrive to the receiving environment [14, 40, 44]. Consequently, this problem is of great concern for the wastewater management of coastal cities because multiresistant bacteria have become a significant public health problem. The caused infec‐ tion is much more difficult to be treated because the panel of effective antibiotics will be reduced.

## **8. Conclusions**

## **Acknowledgements**

We wish to thank the Institute Pasteur (IP) of Paris, France for help. We are grateful to the Laboratory of Microbiology, National Health Security Food Office (ONSSA) Agadir, Morocco for their excellent technical assistance. We thank also AGROTECH Agadir for their cooperation.

## **Author details**

Abdellah El Boulani1 , Rachida Mimouni1 \*, Hasna Mannas1 , Fatima Hamadi1 and Nouredine Chaouqy2

\*Address all correspondence to<EMAIL_ADDRESS>
1 Faculty of Sciences, Laboratory of Microbial Biotechnology & Plant Protection, University Ibn Zohr, Agadir, Morocco

2 Laboratory of Microbiology, National Health Security Food Office (ONSSA), Agadir, Morocco

## **References**

All these data showed that wastewater is a tank of a wide variety of *Salmonella enterica* serovars and ABR patterns. Other data showed that multiresistant bacteria have been detected extensively in wastewater samples which pass through WWTPs and arrive to the receiving environment [14, 40, 44]. Consequently, this problem is of great concern for the wastewater management of coastal cities because multiresistant bacteria have become a significant public health problem. The caused infec‐ tion is much more difficult to be treated because the panel of effective antibiotics will be reduced.

‐ Conventional wastewater treatment without efficient tertiary treatment, like filtration or dis‐ infection by UV, is generally insufficient and consequently constitutes a risk for public health. ‐ Wastewater is an ideal tank of high diversity of *Salmonella* serotypes and ABR patterns

‐ The presence of the same major *Salmonella* serovars simultaneously in sewage and in the marine environment confirms that the principal source of contamination of marine envi‐

‐ Improvement in the efficiency of treating antibiotics residues in WWTPs is the first line of defense against the potential ecological impacts of these chemicals in the environment. ‐ On the one hand, wastewater treatment must adopt effective methods for treatment, and on the other hand, there should be a rigorous approach for surveillance and monitoring effluents

We wish to thank the Institute Pasteur (IP) of Paris, France for help. We are grateful to the Laboratory of Microbiology, National Health Security Food Office (ONSSA) Agadir, Morocco for their excellent technical assistance. We thank also AGROTECH Agadir for their cooperation.

\*, Hasna Mannas1

1 Faculty of Sciences, Laboratory of Microbial Biotechnology & Plant Protection, University

2 Laboratory of Microbiology, National Health Security Food Office (ONSSA), Agadir,

, Fatima Hamadi1

and Nouredine

which usually end up in the marine environment in coastal cities.

before and after treatment and discharge into the environment.

, Rachida Mimouni1

\*Address all correspondence to<EMAIL_ADDRESS>
ronment by *Salmonella* is wastewater discharges.

**8. Conclusions**

144 Current Topics in Salmonella and Salmonellosis

**Acknowledgements**

**Author details**

Chaouqy2

Morocco

Abdellah El Boulani1

Ibn Zohr, Agadir, Morocco

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**3. Antibiotic resistance among companion livestock**

MRSA was identified for very first time in 1972, from mastitis-affected cows and then MRSA from human origin was isolated from dairy cows [37]. MRSA has been found in almost all tame animals since its first observation. Major associative factor for MRSA found to be wound infections and post-operative skin infections, especially in dogs, cats, horses, and rabbits [38, 39]. In one of the study of milk-producing bovines in Mathura reported that MRSA in India was shown to be *S. aureus* was found in 33.75% of subclinical mastitis and clinical mastitis cases among cattle than buffaloes [40]. Tiwari *et al.* (2015) published *S. aureus* among different specimens from pigs, buffalo, dogs, goats, sheep, camels, and horses with different skin disorders of all companion livestock's [41]. *S. aureus* isolates from healthy cow milk samples were found to be resistant to erythromycin (75%), penicillin (100%), and amoxicillin (100%), but susceptible to cloxacillin (100%), neomycin (100%) oxacillin (100%), and ciprofloxacin (83.33%) [42]. MRSA study by Kumar *et al.* (2017) was shown to be penicillin and oxytetracycline resistant (88% each), cefoxitin resistant (75%), cotrimoxazole resistant (62%), and amoxyclav resistant (62%) in antimicrobial susceptibility screening conducted in study on 136 skin and nasal samples collected from cows and buffaloes. Vancomycin resistance was discovered in 3 (16.7%) MRSA isolates from buffalo isolates [43].

### **3.1 Transmission of antimicrobial resistance from livestock to humans**

Livestock's colonization with antimicrobial resistance organisms and human infections due to drug resistant organisms are appearing as a potential threat worldwide. MRSA is now regarded as an emerging zoonotic agent. Over the last

### *Antimicrobial Resistance in Staphylococci Special Emphasis on Methicillin Resistance… DOI: http://dx.doi.org/10.5772/intechopen.99830*

two decades, MRSA has been noticed in the population. Novel MRSA strain known as livestock-associated MRSA (LA-MRSA) has been discovered in farmers and fodder producing animals. [44, 45]. In previously literature, MRSA sequence form was known as non-typable MRSA (NT-MRSA) lacking property of typed using pulsed field gel electrophoresis(PFGE) with SmaI. ST398 isolated as a prevalent sequence type in the agricultural animals, especially pigs and veal calves, as well as persons who worked with these animals [46]. Carriers of MRSA are at risk of developing an MRSA (wound) infection following skin damage (scarification) or surgery. The mode of MRSA transmission from animal to human is frequently inferred from parallel genetic observations including isolates obtained from *in-vitro* experiments [47, 48]. Another study revealed that incorporation of mecA gene encoding penicillin-binding protein 2a into the *Staphylococcal* cassette chromosome *mec* (SCCmec), are considered as factors in the drug resistance of *S. aureus* strains. There is evidence that methicillin-sensitive *S. aureus* strains inherited the SCC mec factor from coagulase-negative staphylococcal strains and subsequently became methicillin-resistant [49].

Reports on the carriage rate of pigs and the farmers associated with those pigs in Netherland divulge a high isolation of MRSA ST398 whereas the same sequence lineage has been observed in the dirt samples from the pig propagation space and eatable samples in Austria. In China, LA MRSA ailment is considered as an professional pitfall for farm workers, LAMRSA in the animal handlers may ranges from region to region i.e., 19.2% in Taiwan, 5.5% from Malaysia and 15% in China [50, 51]. Globally, MRSA percentage increased by 20% in all WHO sectors while 80% increase in few countries [52].

Factors - The emergence and spread of LA-MRSA is attributed to antimicrobial resistance upsurge. However hygiene along with under-described or poorly investigated factors i.e., farm dimensions, cultivation system, use of bioinoculants, and in-feed microelements also contributing the emergence of AMR [53]. Other than *S. aureus* some species of Coagulase negative staphylococci (CoNS) may also be transferred from animals to humans in contact with them, evidence found from a study (Jangra et al. 2018) that revealed some pathogenic strains of Coagulase negative staphylococci (*Staphylococcus warneri* and *Staphylococcus scuiri*) that are announced as a particular pathogen linked with animal origin however their isolation from the human subjects associated with them were also be detected. These results further strengthening the probability for the transmission of such pathogenic species from the domesticated animals [54].


**5.5 Cogeneration technology**

There is few biomass based-co-generation plants in the country. These include sugar processing plants: Tanganyika Planting Company (TPC), Kilombero Sugar Company (KSC), Mtibwa Sugar Estates (MSE) and Kagera Sugar Company, Tanwat (Tanning) and Sao Hill Sawmill have waste that can be used in generating electricity. Table 13 shows electricity generated from cogeneration technology.

Fig. 9. The 3D Concept of the Steam –Electrical Generator Prototype (Source DIT R&PGS-2011)

The highlighted technologies have the potential to contribute to rural energy electrification in the country in the near future. Some of the technologies are still under development. Wind and solar technologies are already in application. These technologies need improvement. Tertiary, higher learning institutions and research centres in the county have the role of improving these technologies.

Table 13. Electricity Generated from Cogeneration Technology (Source: MEM 2011)

With all of the technologies already in place and some underdevelopment, there are still many challenges and barriers that need attention. Some of the barriers are presented in section 6.