**3. Novel perspectives of** *Staphylococcus* **associated with food safety**

#### **3.1. Antimicrobial resistance mediated by MGEs**

Antibiotics, as compounds or substances that kill or inhibit the growth of microorganisms, have been regarded as one of the greatest contributions to medicine and humanity and used to treat a wide range of infectious diseases caused by bacteria for both animals and human beings [5, 165]. Abuse of existing antibiotics contributes to the spread of antibiotic resistance and poses a predicament for the treatment of several bacterial infections, including therapy for individuals with food poisoning. In China, as one of the currently worst areas for antibiotics abuse, the annual prescription of antibiotics, including both clinical and veterinary treatment, is approaching 140 grams per person and has been roughly estimated to be 10 times higher per capita than that in United Kingdom [6, 7, 163, 164]. From a retrospective study conducted on 1,739 *Staphylococcus* isolates from a hospital in Guangzhou, China, from 2001 to 2010, antimicrobial resistance of tested drugs (exclusively for teicoplanin and vancomycin) was commonly observed among the isolates examined, with high resistance rates for β-lactamases (94.0% and 73.7% for penicillin and oxacillin, respectively) and resistance percentages for cefoxitin, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, gentamicin, trimetho‐ prim-sulfamethoxazole, and tetracycline ranging from 83.9% to 19.4% [165]. As a consequence, antibiotic resistance in microorganisms still remains one of the core concerns in global public health, with methicillin-resistant staphylococci (MRS) strains representing one important group, commonly considered as "super bugs" [7]. Since their first discovery in 1961, MRS (including MRSA and MRCNS) have become among the most prevalent pathogens causing nosocomial infections throughout the world [13, 16, 17, 19, 166]. With the first report of an MRSA-mediated gastrointestinal illness outbreak [174], MRS strains have been considered a major contributor to both health-care-associated and foodborne illnesses. MRS had been identified from contamination of various food samples, such as milk, pork, chicken, veal, beef, turkey, and lamb meat [168–170] as well as in food production animals, such as cattle, chickens, pigs, and cows and are closely connected with the newly discovered MRSA designated as livestock-associated MRSA (LA-MRSA) [171–173]. Carriage of MRS strains in a wide variety of food and food production livestock may not be limited to only food hazard but also poses a significant occupational risk for the industrial staff, such as handlers, asymptomatic carriers, and uncolonized individuals. MRS strains show resistance to nearly all β-lactam antibiotics and commonly multiple other drugs due to the *mecA* and other resistance genes carried by an MGE designated as staphylococcal cassette chromosome *mec* (SCC*mec*). Additionally, the role of integrons as a mobile genetic mechanism in horizontal transfer of antibiotic resistance has also been well established [174–177].

#### *3.1.1. SCCmec*

As one of the major foodborne infectious pathogens, *S. aureus* (in particular, MRSA) has been considered to be a potential "super bug," posing a challenge to hospital infection control and a threat to global food safety. Due to the carriage of the *mecA* gene encoding a novel specific penicillin-binding protein (PBP2a), which exhibits a decreased binding affinity to antibiotics, MRSA presents resistance to virtually all β-lactam antibiotics [178]. Evolution from methicillinsusceptible *S. aureus* (MSSA) to MRSA occurred with the acquisition of a genomic island, the staphylococcal cassette chromosome (SCC*mec*). Harboring *mec*A and a large number of functional and regulatory genes, SCC*mec* is an MGE present in *Staphylococcus* species. With accurate excision and integration mediated by site-specific recombinase genes *ccrA* and *ccrB*, SCC*mec* is capable of integration into the bacterial chromosome, leading to the rapid spread of antimicrobial resistance (to β-lactam or other antibiotics) among staphylococcal strains.

MRSA was found shortly after the common use of methicillin, which was first licensed to treat penicillin-resistant *S. aureus* infections in Britain in 1959 [179], resulting in outbreaks of MRSA occurring worldwide. In the 1980s, an extraordinary large chromosomal DNA segment greater than 30 kb carrying *mec* was found to contain no allelic equivalence in MSSA strains by direct chromosome analysis of MRSA strains; this region was designated as *mec* DNA [180–183]. In 1987, the sequence of *mecA* gene cloned from a Japanese MRSA strain was determined [184,

185]. In consideration of the threat caused by this pathogen, the characteristics of MRSA were studied and SCC*mec* was found to be an MGE in MRSA. Additionally, the types of SCC*mec* were found to be genetically diverse. In 1999, the cloning and determination of the structure of the entire *mec* DNA sequence from a Japanese *S. aureus* strain N315 (first isolated in 1982) was reported [186]. Based on the structure, it was reported that *mec* DNA was a novel genomic element designated as staphylococcal cassette chromosome *mec* (SCC*mec*) driven by two sitespecific recombinase genes referred to as cassette chromosome recombinases A (*ccrA*) and cassette chromosome recombinases B (*ccrB*) [178]. This was the first time that SCC*mec* was defined, and *ccrA* and *ccrB* were proposed as a novel set of recombinases, defining a new family of staphylococcal genomic elements. After the discovery of SCC*mec*, various types of SCC*mec* were continuously found by scientists around the world. In 2001, the identification of two additional types of SCC*mec* was isolated in other countries of the world and designated as type II SCC*mec* (found in N315) [187]. The two additional types of SCC*mec* were type I found in NCTC10442, which is the first MRSA isolate in England in 1961, and type III found in 85/2082 isolated in New Zealand in 1985. In 2002, a novel type of SCC*mec* designated as type IV was identified from CA-MRSA strains [188]. According to the foundation of type IV SCC*mec*, a new type of SCC*mec* designated as VI, which was originally mistaken as type IV SCC*mec*, was explored [189, 190]. In 2004, type V SCC*mec* was found in the chromosome of a CA-MRSA strain (WIS [WBG8318]) isolated in Australia [191]. Since 2008, novel types of SCC*mec* were found globally. Types VII and VIII SCC*mec* were identified in MRSA strains JCSC6082 (a Swedish isolate) [192] and C10682 (a Canadian isolate) [193], respectively. Novel types of SCC*mec* designated as types IX and X were identified in MRSA strains JCSC6943 and JCSC6945, respectively [194]. The latest type of SCC*mec* to be classified was designated as type XI; it was discovered in the MRSA strain LGA251 genome with a divergent *mec*A homologue (*mec*ALGA251) [195].

#### *3.1.1.1. Structure and types*

As a major member of the SCC family and a carrier for gene exchange in staphylococci strains, SCC*mec* is located near the replication origin of the *Staphylococcus* chromosome and inserted at *attB* site located at the 3 end of a novel ORF with unclear function (*orfX*). Typical SCC*mec* comprise 3 basic genetic elements: (i) *Ccr* complex, composed of two site-specific recombinase genes (*ccrA* and *ccrB*), and surrounding ORFs. Via site-specific recombination mediated by *ccrA* and *ccrB*, multiple antibiotic resistance and heavy metal resistance genes are capable of insertion into SCC*mec*. SCC*mec* is further integrated to the staphylococcal chromosome by accurate excision and integration, leading to adaption of the bacterial host to different environments and pressure of antibiotic selection. According to the different types of *ccrA* and *ccrB*, *Ccr* complex was classified into 8 allotypes: type 1 for *ccrA1* and *ccrB1*, type 2 for *ccrA2* and *ccrB2*, type 3 for *ccrA3* and *ccrB3*, type 4 for *ccrA4* and *ccrB4*, type 5 for *ccrC1*, type 6 for *ccrA5* and *ccrB3*, type 7 for *ccrA1* and *ccrB6*, and type 8 for *ccrA1* and *ccrB3*. (ii) *Mec* complex harboring *mecA* and related regulatory genes. Based on the regulatory genes located upstream and downstream of *mecA* and the difference of insertion sequences, *mec* complex was classified to five classes: class A carried the integrated *mecI-mecR1-mecA-IS431* structure, class B carried devoid the *IS1272-∆mecR1-mecA-IS431* structure that contains integration insertion sequence, class C carried the *IS431-mecA-∆mecR1-IS431* structure that contains two copies of insertion sequence *IS431*, class D carried the *IS431-mecA-∆mecR* structure, and class E carried the *blaZmecALGA251-mecR1LGA251-mecILGA251* structure. Class C *Mec* complex was divided into two different class designations: class C1 (two *IS431*s were arranged in the same direction) and class C2 (two *IS431*s were arranged in the opposite direction) by the inserted direction of *IS431*. (iii) A junkyard region (J region) is located between *Ccr* complex and *Mec* complex. According to its location in SCC*mec*, J region was classified into J1 (also known as L-C region), J2 (also known as C-M region), and J3 (also known as M-R region) region, located at the upstream of *ccr* gene complex and downstream of *Ccr* complex and the upstream of *Mec* complex and downstream of *Mec* complex, respectively.

Up to date, SCC*mec* elements are classified into types I to XI based on the nature of *Ccr* and *Mec* complex and are further classified into different subtypes in accordance with diverse J region. Types and subtypes of SCC*mec* are described in detail as follows: (i) Type I SCC*mec* was first discovered and had a long history dating back to the 2000s. The characteristic MRSA strain carrying type I SCC*mec* was identified, designated as NCTC10442, which was recovered in the United Kingdom in 1961, representing the first MRSA strain [187]. Type I SCC*mec*, carrying class B *Mec* complex and type 1 *Ccr* complex, carried a *pls* regulator in J1 region. A subtype within type I SCC*mec* was designated as IA, containing a plasmid pUB110 located in J3 region [202]. According to the nomenclature proposed in 2006 [271], type I SCC*mec* was designated as 1B.1.1 and its subtype IA was designated as 1B.1.2. (ii) The characteristic MRSA strain carrying type II SCC*mec* had been identified and designated as N315, which was first isolated in 1982 and discovered in 1999 [178, 186, 196, 197]. Type II SCC*mec* harbored class A *Mec* complex and type 2 *Ccr* complex. In J3 region, an integrated copy of staphylococcal plasmid pUB110 was found and a *kdp* regulator was found in J1 region. A number of subtypes were designated as IIA, IIB, IIC, IID, IIE, and IIb and a variant in type II SCC*mec* in consider‐ ation with the difference of J1 and J3 regions. According to the nomenclature reported in 2006 [196], type II SCC*mec* was named 2A.1.1 and type IIb was designated as 2A.2. IIA, IIB, IIC, IID, and IIE were designated as 2A.3.1, 2A.3.2, 2A.3.3, 2A.3.4, and 2A.3.5, respectively. The variant of type II was designated as 2A.1.2. (iii) A Zelanian isolate designated as 85/2082 first isolated in 1985 was found carrying type III SCC*mec*, which was first discovered in 2001 together with type I SCC*mec* [196] and was known as the representative MRSA strain of type III SCC*mec* until now. Type III SCC*mec* carries class A *Mec* complex, type 3 *Ccr* complex, and an integrated copy of plasmid pT181 encoding tetracycline and mercury resistance in J3 region. Regarding the difference within the J3 region, there were several subtypes in type III SCC*mec* designated as IIIA and IIIB and two variants designated as IIIC and IIID. According to the 2006 nomenclature [196], type III SCC*mec* was designated as 3A.1.1, IIIA was designated as 3A.1.2, and IIIB was designated as 3A.1.3. (iv) The two commonly characteristic MRSA strains carrying type IV SCC*mec*, which was first discovered in 2002, were designated as CA05 (JCSC1986) and 8/6-3p (JCSC1978) [188]. Type IV SCC*mec* was found to have a unique combination of class B *Mec* complex and type 2 *Ccr* complex, and transposon Tn4001 was found in J3 region of type IV SCC*mec*. Diversity in subtypes of type IV SCC*mec* was obtained, including IVa, IVb, IVc, IVd, IVE, IVF, IVA, IVg, IVh, IVi, IVj, and IV1. Based on the 2006 nomenclature [196], IVa, IVb, IVc, and IVd were designated as 2B.1.1, 2B.2.1, 2B.3.1, and 2B.4, respectively. IVE was named 2B. 3.3, IVF was designated as 2B.2.2, and IVA was designated as 2B.N.2. IVg, IVh, IVi, IVj, and IVk were designated as 2B.5.1, 2B.6.1, 2B.7.1, and 2B.8.1, and IV1 was designated as 2B.new.1. (v) The CA-MRSA strain WIS (JCSC3624) isolated in Australia was the characteristic MRSA strain carrying type V SCC*mec* [191], which carried class C2 *Mec* complex and type 5 *Ccr* complex. No subtype had been found so far within the group of type V SCC*mec*. According to the nomenclature proposed in 2006 [196], type V SCC*mec* was designated as 5C.1. (vi) In 2001, type VI SCC*mec* was first identified from a pediatric MRSA clone named HDE288, which was first reported in 1992 [189, 190]. Type VI SCC*mec* carries a class B *Mec* complex and type 4 *Ccr* complex. Until now, no subtype of type VI SCC*mec* has been found, which was designated as 4B according to the 2006 nomenclature [196]. (vii) CA-MRSA strain designated as JCSC6082 (p5747/2002) was isolated in 2002 [198] and identified to carry a type VII SCC*mec* in 2008 [192]. Type VII SCC*mec* carries a class C1 *Mec* complex that was different from class C2 *Mec* complex carried by type V SCC*mec* and type 5 *Ccr* complex. There was no subtype of type VII SCC*mec* reported. According to the nomenclature put forward in 2006 [196], type VII SCC*mec* was designated as 5C1. (viii) Type VIII SCC*mec* was first identified from a Canadian MRSA strain designated as C10682 isolated in 2003 [193]. It harbored a novel combination of class A *Mec* complex and type 4 *Ccr* complex. No subtype of type VIII SCC*mec* has been found currently. According to the nomenclature proposed in 2006 [196], type VIII SCC*mec* was designated as 4A. (ix) Type IX SCC*mec* was first reported to be identified in a MRSA strain designated as JCSC6943 isolated from a Thailand participant [194]. It was found carrying class C2 *Mec* complex and type 1 *Ccr* complex. No subtype of type IX SCC*mec* was found so far. According to the nomenclature put forward in 2006 [196], type IX SCC*mec* was designated as 1C2. (x) Together with type IX SCC*mec*, type X SCC*mec* carried class C1 *Mec* complex and novel type 7 *Ccr* complex was identified in a Canadian MRSA strain designated as JCSC6945 [194]. So far, no subtype of type X SCC*mec* has been found. According to the 2006 nomenclature [196], type X SCC*mec* was designated as 7C1. (xi) Type XI SCC*mec* isolated from MRSA strain LGA254 in southwest England of 2007 was a novel type different from other SCC*mec* with carriage of distinct class E *Mec* complex and type 8 *Ccr* complex. No subtype was found in type XI SCC*mec*, and according to the 2006 nomenclature, it was designated as 8E [195, 196, 199].

A thorough understanding of the molecular epidemiology and evolution of MRSA may aid in the further identification, control, prevention, and therapy of *Staphylococcus*-mediated human diseases, necessitating SCC*mec* typing as an essential tool for discrimination of different types and subtypes. Currently, there are several SCC*mec* typing methods available for the global evolutionary study of MRSA, with multiplex PCR as the major and widely used methodology. Multiplex PCR was first developed in 1988 and put in use to distinguish different types and subtypes of SCC*mec* in 2002 [198, 200]. In various multiplex PCR strategies, specificity of primer design has been the major concern determining the application of SCC*mec* typing. The multiplex PCR assay described in 2006 [201] was applicable for unique and specific typing of types and subtypes I, II, III, IVa, IVb, IVc, IVd, and V, respectively. After years of validation, this multiplex PCR strategy had been demonstrated to be a rapid, simple, and feasible method for SCC*mec* typing and serves as a useful tool for further prevention and control of *Staphylo‐ coccus*-mediated infections by clinicians and epidemiologists. However, with emergence of novel SCC*mec* (11 types and various subtypes to date), inclusive and novel SCC*mec* typing methodologies are desperately required.

#### *3.1.1.2. Prevalence and occurrence*

As different types and subtypes of SCC*mec* have been verified to influence the multidrug resistance and the antimicrobial MIC of β-lactam, a thorough understanding of the prevalence of SCC*mec* may aid in the further identification, control, prevention, and therapy of *Staphylo‐ coccus*-mediated human diseases. Consequently, surveillance of SCC*mec* has been performed globally in past decades. As the first identified type, type I SCC*mec* was nonpredominant in the 1970s, which was reported in a limited number of areas, including Brazil, Iran, Japan, Philippines, Spain, Switzerland, and the United States [202–210]. Type II SCC*mec* had been commonly found in Japan, Korea (occasionally in China), and the United States [206, 207, 211– 215] and occasionally detected in Algeria, Brazil, China, Iran, Turkey, and Thailand [205, 216– 221]. Type III SCC*mec* has been most frequently found among HA-MRSA and remains the predominant type in many countries or areas including Asia (China, Hong Kong, Iran, Malaysia, Singapore, Taiwan, and Thailand), Europe (Poland, Portugal, and Turkey), and South America (Brazil) [205, 208–214, 217, 220, 222–231]. Types IV and V have been implicated as CA-MRSA-associated SCC*mec*. A large number of variants (subtypes) have been reported within type IV, which is also the predominant type in Algeria, Brazil, Denmark, Korea, New Zealand, Portugal, Philippines, Sweden, Switzerland, Spain, and the United States [202–204, 209, 219, 221, 231–238]. Other types of SCC*mec* are rarely detected and reported [166]. Accord‐ ing to our preliminary studies, from 2001 to 2006 in Guangzhou, analysis of the distribution of SCC*mec* type in 262 *Staphylococcus* strains demonstrated that the classic nosocomial SCC*mec* type (I–III) dominated among the tested strains, and none of the tested strain carried type IV or V. For MRSA strains, 3 and 198 strains belonged to SCC*mec* types II and III, respectively, with 8 strains untypeable. For MRCNS strains, 9, 24, and 12 strains were classified as SCC*mec* types I, II, and III respectively, with 8 strains untypeable. From a retrospective study conducted on 1,739 *Staphylococcus* isolates from a local hospital in Guangzhou from 2001 to 2010, SCC*mec* typing was performed on 263 randomly selected MRSA strains. Type III SCC*mec* was most frequently observed with an identification rate of 94.7% [249/263], with type II detected in 4 isolates (one individual isolate in 2001, 2002, 2005, and 2008, respectively) and 10 untypeable MRSA strains were recorded [165]. However, diversity in SCC*mec* types had been obtained from SCC*mec* surveillance of MRSA from another medical setting in Guangzhou from 2009 to 2012, as types I, II, III, IIIA, IV, V, and VI SCC*mec* carriage were found to be 17.6%, 56.8%, 6.2%, 10.7%, 4.1%, and 2.1%, respectively.

#### *3.1.2. Other resistance determinants in Staphylococcus*

Aside from SCC*mec*, the role of integrons as a mobile genetic mechanism in the horizontal transfer of antimicrobial genes or determinants among microorganisms has been recently well characterized, established, and documented, which may contribute to the broad distribution and spread of antibiotic resistance and ultimate emergence and unleashing of "super bugs" [174–177]. A complete and functional integron platform comprises three elements: (i) the integrase gene (*intI*) encoding an integrase, (ii) a proximal primary recombination site *attI*, and (iii) a promoter gene (Pc) functionally demonstrated for all integrons [240]. Several classes of integrons have been identified and distinguished by differences and divergence in the *intI* sequences, and integron classes 1 to 3 are so-called multiresistant integron (RIs) with a capability of acquiring identical gene cassettes [173]. Class 4 integron is considered to be a distinct type of integron and termed super integron (SI), which was found on the small chromosome of *Vibrio cholerae* and known to be an integral component of various γ-proteo‐ bacterial genomes [17, 241, 242]. As a direct result of the linkage to Tn402-like transposons and associated with Tn3 transposon family (Tn21 or Tn1696), the class 1 integron platform has been the most ubiquitous among microbes and remains the focus of numerous studies, with a large variety of clinical Gram-negative organisms and a few Gram-positive bacteria reported to harbor this integron class [243–245]. The first observation of class 1 integron within *Staphylo‐ coccus* spp. was reported in 2004, with species including *Staphylococcus lentus*, *Staphylococcus nepalensis*, and *Staphylococcus xylosus* [246]. In Guangzhou, class 1 integrons were commonly found in MRSA strains (31.6%, 83/263) during 2001 to 2010, with decreasing identification rates observed [6, 7, 165, 166, 247]. From 2001 to 2004 in Guangzhou, the detection rate of class 1 integron for MRSA and MRCNS was 51.7% [46/89] and 56.6% [30/53], respectively [6, 13, 165, 247, 248]. From 2007 to 2010, class 1 integron was found in MRS isolates based on a series of studies of systematic integron investigation in hundreds of staphylococci strains from 2001 to 2006 [165, 247, 248]. Nevertheless, only 38.3% [46/120] of MRSA isolates carried class 1 integron. Undoubtedly, the commonly detected integron-based antimicrobial resistance mechanisms have contributed to the evolution of the resistance of MRSA and may further lead to dissem‐ ination of new waves of "super bugs." Class 2 integron has an organization similar to that of class 1 but is associated with the Tn7 transposon family [174, 249]. Class 3 integron contains a comparable structure to that of class 2 integron and up to date has only been found in a limited number of microorganisms, including *Pseudomonas*, *Alcaligenes*, *Serratia marcescens*, and *Klebsiella pneumoniae* [249–252]. Class 4 integron harbors hundreds of gene cassettes encoding adaptations that extend beyond antibiotic resistance and pathogenicity [253]. The remaining classes of integrons may also contain antibiotic resistance gene cassettes, but knowledge of their worldwide prevalence remains limited [240, 254]. As a genetic element existing in 9% of bacteria and representatives from a broad range of phyla and environments, integrons play a core role in antibiotic resistance among clinical organisms and contribute to the evolution and adaption of bacteria.

#### *3.1.3. Mobility and evolution of MGEs in staphylococci*

As a commonly found MGE with an antibiotic resistance gene (*mecA*) and site-specific recombinase genes (*ccrA* and *ccrB*), SCC*mec* has been classified into 11 types, various subtypes, and variants and plays a core role in antibiotic resistance, molecular epidemiology, and evolution of staphylococci. Through recognition of recombination sites (*attB*, *attSCC*, *attI1*, *attC*, secondary sites, etc.) and via this site-specific recombination event, MGEs are capable of capturing foreign genes. The mobility of MGEs is defined as being associated with mobile DNA elements (transposons or plasmids) and antibiotic resistance genes in addition to having a small array size and substantial heterogeneity in recombination sites [187, 229] From Southern hybridization analysis in preliminary studies, 58 staphylococci isolates were found to harbor one copy of class 1 integron on the chromosomal instead of plasmid DNA compared with their frequent location on plasmids for facilitation of conjugative-mediated transfer [13]. As natural capture systems and assembly platforms, MGEs in *Staphylococcus* (SCC*mec* or integrons system) allow bacteria to incorporate foreign genes and convert them to functional proteins by ensuring the correct expression. Despite affinity for self-transposition, integron systems are commonly associated with the transposons and conjugative plasmids serving as vehicles for the intra- and interspecies transmission of genetic material as well as gene cassettes capable of mobilizing to other integrons or to secondary sites in the bacterial genome [255]. This event has been regarded as a key mechanism in the dissemination and spread of resistance genes responsible for the swift spread of resistance genes and the rapid evolution of resistance to a wide range of unrelated antibiotics among diverse bacteria [251, 256]. Any ORF existing in the environmental "gene pool" is conceivably capable of being structured into the bacterial genome through the recombination platforms, and MGEs consequently have the potentially limitless capacity to exchange and stockpile functional genes, which enables rapid adaptation to selective pressure and may ultimately endow additional fitness and advantage to the bacterial host. In addition, a vast number of MGEs (such as conjugative plasmids, transposons, insertion sequences, and even entire chromosome) and the captured genes comprise the vast reservoirs of integrons and lead to the longstanding concept of a single massive "gene pool" that is available and temporally shared among bacteria [73]. The common observation of MGEs in microorganisms from the general environment and its enormous sequence diversity detected from such microbes, as well as various products unrelated to antibiotic resistance, strongly suggests that MGEs are ancient genomic structural elements and have played a general role in evolution and adaptation for a considerable period of time [43].

As a genomic island (G island) and MGE demarcated by a pair of DRs and inverted repeats, SCC*mec* has a set of site-specific recombinase genes (*ccrA* and *ccrB*) required for its movement and is inserted at the 3 end of *orfX* and located adjacent to the replication origin [220]. In the chromosome of staphylococci, SCC*mec* may have evolved from a primordial mobile element SCC, into which the *mec* complex was inserted. However, the function of the putative SCC*mec* may not be limited as the conveyer of antimicrobial resistance (mediated by *mec* complex) alone, and this MGE may serve as a vehicle for the exchange of useful genes for the better survival for staphylococci in various environments. In addition, SCC*mec* is a general genetic information exchange system of staphylococci with *ccrA* and *ccrB* involved in the recombination events (integration and excision), which plays a significant role in the evolution of *Staphylococcus*. MGEs serve as the reservoir for various genes and possess the function of interspecies genetic exchange. It is interesting to speculate whether multiple MGEs carried by staphylococci would speed up the rate of gene exchange or genome evolution, although these hypotheses require further investigation. From previous surveys, the influence of carriage of multiple MGEs on antimicrobial resistance had been investigated in MRSA. The presence of multiple MGEs was found to be strongly correlated with antimicrobial resistance, including erythromycin, gentamicin, tetracycline, and trimethoprim-sulfamethoxazole, which further limits the therapeutic options for deep-seated *Staphylococcus* infection and diseases. For treatment of complicated *Staphylococcus* infections, gentamicin is commonly prescribed by many clinicians in combination with vancomycin due to enhanced efficacy based on syner‐ gistic antibacterial activity [257]. For penicillin-allergic patients, erythromycin has been frequently used. As the first choice for suspected CA-MRSA cutaneous infections, trimetho‐ prim-sulfamethoxazole has also been commonly used in combination with rifampin for MRSA in carriers despite the high recurrence (up to 50%) and frequent emergent resistance of this organism.

Up to date, the most known functional genes carried by MGES are found to encode resistance to the oldest groups of antibiotics (such as tetracycline, streptomycin, and spectinomycin) that have been discontinued in clinical settings for decades but still available in veterinary practice. Although the indiscriminate use of these older antibiotics is no longer occurring in the clinical setting, their use in veterinary medicine may contribute to a novel and significant concern in food safety. Abuse of antibiotics leads to the emergence of antibiotic resistance and poses a predicament for the future treatment of bacterial infection, with MGEs undoubtedly facilitat‐ ing the rapid spread and dissemination of a vast number of resistance genes among microor‐ ganisms.

#### *3.1.4. Livestock-Associated MRSA (LA-MRSA)*

As a common pathogen for both clinical medicine and food safety, MRSA was first reported as hospital associated before the 1990s and thus designated as HA-MRSA. Since the 1990s, CA-MRSA strains have increasingly been reported among groups of patients with no apparent connection to hospitals. It is noteworthy that a large number of such CA-MRSA-infected patients or carriers were pediatric associated. Aside from HA-MRSA and CA-MRSA, LA-MRSA has been recently documented and is known to be more persistent in food products from swine and cattle [258], which is also responsible for pneumonia, endocarditis, and necrotizing fasciitis by LA-MRSA carriers [259]. Nowadays, LA-MRSA acts as an increasing risk for public health and a challenge to livestock farming and related food products. LA-MRSA was mostly found among animals (particularly pigs) and humans with frequent contact to livestock farming or livestock food products [260–262]. After the first isolation of MRSA from livestock (cows with mastitis) [263], a extremely limited number of reports were focused on LA-MRSA. However, after an initial LA-MRSA case occurred in humans, described in 2005 [262], LA-MRSA have been the focus of numerous recent studies. In 2007, a transmission of MRSA (ST1, spa-type t127) between cows and humans was reported, verifying the transmis‐ sion between animals and humans [264]. Afterwards, different types of LA-MRSA have been continuously discovered globally, and the prevalence and occurrence of LA-MRSA vary significantly in different areas. In Europe and America, the majority of LA-MRSA strains belong to sequence type (ST) 398, whereas ST9 is frequently discovered in Asia [265–268]. Both livestock and humans are potential carriers of LA-MRSA, and individuals working in animal clinics and livestock production environments with direct contact or exposed to MRSApositive animals or ingestion of the MRSA-positive livestock food products have an increased risk of becoming MRSA carriers [269, 270]. A high risk of animal to human transmission of ST398 was found to result from direct association between animal and/or human MRSA carriages in the farm setting [261, 269, 271–273] despite much lower occurrence of transmission between humans by LA-MRSA and that of HA-MRSA [274–276].

As a clone of typical LA-MRSA, ST398-LA-MRSA has been responsible for serious infections and outbreaks worldwide [277, 278]. Containing various spa-types, ST398-LA-MRSA strains are mostly found to carry type IV or V SCC*mec*, which are nontypeable by standard PFGE using *SmaI* digestion due to protection from digestion by the presence of a restriction/ methylation system [271, 279, 280]. According to the virulent properties of ST398 strains, most animal-associated ST398-LA-MRSA strains lack the major virulence factors in staphylococci, such as Panton-Valentine leukocidin (PVL), TSS toxin 1, and exfoliative toxins [281]. However, various resistance genes commonly present in staphylococci of human and animal origins are also recovered in ST398-LA-MRSA strains, including the β-lactamase gene cluster *blaZ-blaIblaR*, the tetracycline resistance genes *tetM* and *tetK*, the macrolide-lincosamide-streptogramin B (MLSB) resistance genes *ermA*, *ermB*, and *ermC*, the lincosamide resistance gene *lnuA*, and arrays *aacA-aphD* or *aadD* for resistance to gentamicin-tobramycin-kanamycin or kanamycinneomycin, respectively [281–285]. In addition, novel resistance genes were also discovered in ST398-LA-MRSA strains, such as *dfrK* (trimethoprim resistance), ABC transporter genes *vgaC* and *vgaE* (pleuromutilin-lincosamide-eptogramin A resistance), and *apmA* (apramycin resistance) [286].

Emergence, spread, and dissemination of ST398-LA-MRSA from animals, as well as its transmission between humans and animals, strongly suggest that the antimicrobial resistance caused by veterinary antibiotic abuse poses a hazard to both humans and animals regarding food safety challenges associated with animal origins.

#### **3.2. Viable Putative but Nonculturable (VPNC)**

In nature, bacteria exist in various states such as normal growth state, dead state, dormant state, and VPNC state, which was first reported in 1986 [287]. Differing significantly from the "starvation survival" state, VPNC state [previously known as viable but nonculturable (VBNC)] is a specific state under which bacteria remain alive but fail to form a colony on routine bacteriological media that normally support their growth. Consequently, routine bacteriolog‐ ical detection methodology fails to detect the VPNC bacteria. However, given the right conditions, bacteria in the VPNC state remain active and can "resuscitate" to the normal state. Hence, the VPNC food spoilage or pathogenic bacteria are considered to be a stealth source of contamination, posing a significant concern for traditional surveillance and control method‐ ologies of foodborne pathogens.

#### *3.2.1. Induction and resuscitation*

Entering into the VPNC state is considered to be a survival mechanism for nonsporulation bacteria under a number of harsh environmental conditions, which is described in detail as follows: [1] Nutrient starvation [288]. Without essential nutrients, bacterial growth and metabolism may be terminated and thus enter the death-like status. Nutrient starvation, such as the absence of carbon source or nitrogen source, which is an extreme condition for the growth of bacteria, can induce the VPNC state. [2] Extreme temperature [289]. The appropriate temperature for typical bacterial growth ranges from 20°C to 37°C, and termination of growth usually occurs under extremely high or low temperature. The temperature of 4°C or -20°C, at which bacteria stop growth and metabolism, is frequently used for induction of VPNC state. The combination of nutrient starvation and low temperature has also been widely applied as an induction condition. [3] pH value [290]. Most microorganisms grow in neutral and slightly acidic or alkaline pH conditions. Strong acidity or alkalinity may lead to bacterial death-like states, which has been occasionally used to induce the VPNC state. [4] Salinity [291]. As an extreme condition for bacterial growth, high salinity has been found to enable the entering of VPNC state. [5] Osmotic stress [291]. Extremely high osmotic stress was reported to be applied for VPNC state induction. [6] Oxygen availability [292, 293]. In an aerobic environment, anaerobic bacteria would enter into the VPNC state and the absence of oxygen would induce the VPNC state of aerobic bacteria and vice versa. [7] Existence of heavy metals [294, 295]. [8] Common food preservatives (cryopreservation, vacuum preservation, etc.). Currently, numerous bacteria are reported to have the ability to enter into VPNC state, such as *Salmonel‐ la* spp. [287, 296, 297], *Enterococcus* spp. [298–300], *Vibrio* spp. [301–309], *Campylobacter* spp. [310], *Pseudomonas* spp. [307, 311–313], *Shigella* spp. [314, 315], *Lactobacillus* spp. [316, 318], *Escherichia coli* [313, 316], and *Staphylococcus* spp. [318–324]. Furthermore, it has been well established and documented that bacteria in VPNC state can resuscitate and regain cultura‐ bility when provided with appropriate conditions [308, 325, 326]. A variety of processes, including elevation of temperature gradually or directly [308], heat shock treating [325], adding nutrients [326], and adding organic matter (Tween-20, Tween-80, catalase, sodium pyruvate, etc.) were found to be applicable for resuscitation from the VPNC to normal state. The resuscitated bacteria are comparatively similar to their exponential-phase bacterial counterparts.

Currently, only 2 species of *Staphylococcus*, *S. aureus*, and *S. epidermidis*, were capable of entry into VPNC state [318–324]. In 2009, formation of VPNC *S. aureus* by radiation was reported for the first time, representing the first evidence of *Staphylococcus* cells entering the VPNC state [318]. One year later, induction of VPNC state by starvation of the *Staphylococcus* cells at low temperature (4°C) was also obtained [319]. Resuscitation of *S. aureus* strain under VPNC state was induced by temperature upshift (from 4°C to 22°C) or rich medium supplemented with sodium pyruvate [319, 320]. The prevention of resuscitation was observed by deficiencies in catalase or superoxide dismutase, indicating the relation of VPNC formation of *S. aureus* to oxidative stress [319, 320], constituting the initial studies on the mechanism of the formation and resuscitation of *S. aureus* in VPNC state. In addition, *S. aureus* cells in biofilm were found to enter into a VPNC state under antibiotic pressure (vancomycin or quinupristin/dalfopristin) [320, 321], suggesting that central venous catheter (CVC) or medical implant-associated biofilms may be potential reservoirs for *S. aureus* and *S. epidermidis* in the VPNC state [323]. Thus, both biofilm formation and VPNC induction may augment clinical challenges associated with antibacterial treatment options. *S. epidermidis* biofilms were reported to enter into the VPNC state when grown in excess glucose presumably due to accumulation of acidic com‐ pounds as the degradation products of glucose metabolism. This process was counteracted by high extracellular levels of calcium and magnesium added to the culture medium allowing modulation of the proportions of VPNC bacteria within *S. epidermidis* biofilms [324]. Although the induction and resuscitation of *Staphylococcus* cells in VPNC state has been verified, relatively little is known with respect to inducing and resuscitating condition, necessitating further investigation into this fascinating bacterial survival strategy.

#### *3.2.2. Characteristics and mechanisms*

Remaining metabolically or physiologically active, bacteria in VPNC state maintain cell integrity but exhibit dwarfing, which contribute to protect against a wide variety of stressors. The maintenance of metabolic activity and continuous gene expression under VPNC state [327, 328] indicates that potentially ingested bacteria may still be capable of causing foodborne illnesses. Such microorganisms also possess the capacity to regain culturability *in vivo* [329], exhibiting high ATP level, membrane potential [298], and retained plasmids, presenting higher autolytic capability than exponentially growing cells. The outer membrane protein profile also alters with entry into VPNC state [330]. Due to the diversity of VPNC bacteria, various characteristics among different species of microorganisms are being discovered worldwide. Regarding the mechanism of the VPNC state, the up- or down-regulation of genes and proteins associated with VPNC status compared to the exponential phase and the resuscitated status is considered to be potential factors for entering and exiting of VPNC state. However, it is currently unclear as to which genes are essential for these processes.

As for the pathogenicity of *Staphylococcus* cells under VPNC state, the viable cell numbers and gene expression had been found to remain constant in VPNC state by examination of epi‐ fluorescence microscopy, flow cytometry, and reverse transcription-PCR (RT-PCR) [320, 321]. This finding implied that *S. aureus* cells are likely still pathogenic in VPNC state and thus pose a significant concern on its threat to food safety.

#### *3.2.3. Detection and identification*

VPNC pathogenic bacteria are considered to be a threat to public health and food safety due to incapability of detection by the "gold standard" methodology for identification of foodassociated microorganisms. Hence, the development, evaluation, validation, and further application of rapid and accurate detection methodology for VPNC bacteria are considered to be the leading concerns for the surveillance of bacterial cells in VPNC state as well as further understanding of the mechanisms on their survival and persistence in the extreme environ‐ ment. The conventional detection method for VPNC bacteria was the combination of acridine orange direct count (AODC) (for total bacterial cell number counting), bright-field microscopy with nalidixic acid (for metabolically active cell number counting), and plate counting (for determination of culturability). The occurrence of entry of bacterial cells into VPNC state was validated and confirmed when colony counts were totally depleted on culture plates (with no observed colonies), which was designated as nonculturable, whereas the total bacterial and metabolically active cells still remained countable. Despite the limited application of nalidixic acid on Gram-negative microorganisms, the novel LIVE/DEAD Bacterial Viability Kit with requirement on differential fluorescence was employed for the detection of both Gram-positive and Gram-negative bacteria [331]. In consideration of the carcinogenesis and expense of fluorescence substances, the development of molecular assays, such as random amplified polymorphism DNA and RT-PCR [328], was recently applied to identify bacterial cells in the VPNC state. As *Staphylococcus* species were concerned, an immunosensing system using impedance spectroscopy measurements was recently developed and applied for rapid verification and quantification of *S. aureus* cells in the VPNC state [322], with high sensitivity and specificity obtained.

In conclusion, foodborne pathogens, especially *S. aureus* strains, which contain various virulence genes, are capable of forming VPNC state and resuscitating into active and patho‐ genic state under specific conditions, posing a significant threat to food safety. The "farm to table" process includes food ingredients, processing, transportation, and storage, which involves a large variety of conditions. A number of such conditions (such as low temperature during refrigeration) may be sufficient for entry into the VPNC state, complicating the use of routine diagnostics by resulting in high "false-negative" rates of pathogen detection. However, once resuscitation occurs under proper conditions, foodborne pathogens remain active and virulent, which thus are highly likely to cause food poisoning outbreaks.

#### **4. Concluding remarks**

Ingestion of food is the major (although, not only) way for human beings to obtain nutrient substances for basic living; therefore, the quality and safety of food have recently become a major concern. Considered to be an expanding global problem and leading topic in public health, food safety is no longer limited to foodborne illnesses but has been extended to all safety issues associated with "farm to table" food approaches. In the past decade, a large number of worldwide cases or reports have been available regarding food containing unheal‐ thy, harmful, or toxic substances (other than food poisoning outbreaks). Foodborne microor‐ ganisms, previously limited to pathogenic bacteria and toxic substances produced in food, have played a critical role in food safety. However, now due to diversity in the genus and species of microbes, variety of mechanisms on the regulation of growth and survival, and complexity of ecosystem involving polymicrobial interaction and environmental factors, a number of novel microbial issues associated with food safety have been recently acknowl‐ edged. Microorganisms may very well be capable of surviving the journey from farm to table via various evasion mechanisms at various food processing stages, including source (antimi‐ crobial resistance caused by the use of drugs in veterinary medicine or livestock feed), processing (formation of biofilm and further survival of bacterial elimination), storage (formation of VPNC state and "false-negative" detection), and even after cooking (production of heat-stable toxins that remain active despite elimination of host bacteria). The contributions of aforementioned and novel evasion mechanisms with respect to food safety undoubtedly require further investigation in vitro and in vivo for improved diagnostic and decontamination procedures.
