*Review* **Human** *mecC***-Carrying MRSA: Clinical Implications and Risk Factors**

#### **Carmen Lozano \*, Rosa Fernández-Fernández, Laura Ruiz-Ripa, Paula Gómez , Myriam Zarazaga and Carmen Torres**

Area of Biochemistry and Molecular Biology, University of La Rioja, 26006 Logroño, Spain; rosa.fernandez.1995@gmail.com (R.F.-F.); laura\_ruiz\_10@hotmail.com (L.R.-R.); paula\_gv83@hotmail.com (P.G.); myriam.zarazaga@unirioja.es (M.Z.); carmen.torres@unirioja.es (C.T.)

**\*** Correspondence: carmen.lozano@unirioja.es; Tel.: +34-941-299-752

Received: 18 September 2020; Accepted: 19 October 2020; Published: 20 October 2020

**Abstract:** A new methicillin resistance gene, named *mecC*, was first described in 2011 in both humans and animals. Since then, this gene has been detected in different production and free-living animals and as an agent causing infections in some humans. The possible impact that these isolates can have in clinical settings remains unknown. The current available information about *mecC*-carrying methicillin resistant *S. aureus* (MRSA) isolates obtained from human samples was analyzed in order to establish its possible clinical implications as well as to determine the infection types associated with this resistance mechanism, the characteristics of these *mecC*-carrying isolates, their possible relation with animals and the presence of other risk factors. Until now, most human *mecC*-MRSA infections have been reported in Europe and *mecC*-MRSA isolates have been identified belonging to a small number of clonal complexes. Although the prevalence of *mecC*-MRSA human infections is very low and isolates usually contain few resistance (except for beta-lactams) and virulence genes, first isolates harboring important virulence genes or that are resistant to non-beta lactams have already been described. Moreover, severe and even fatal human infection cases have been detected. *mecC*-carrying MRSA should be taken into consideration in hospital, veterinary and food safety laboratories and in prevention strategies in order to avoid possible emerging health problems.

**Keywords:** *Staphylococcus aureus*; methicillin resistance; human infection; CC130

#### **1. Introduction**

*Staphylococcus aureus* is an opportunistic pathogen that causes high morbidity and mortality. This microorganism is able to cause diverse diseases that range from having a relatively minor impact, such as a skin infection, to serious and life-threatening episodes, such as endocarditis, pneumonia or sepsis. The impact of *S. aureus* is enhanced by its great capacity to develop and acquire resistance to various antimicrobial agents. Among the antibiotic resistance of *S. aureus*, methicillin resistance mediated by the *mecA* gene is highly relevant as this mechanism provides this bacterium resistance to almost all beta-lactam antibiotics, seriously limiting therapeutic options [1,2]. Recently, the World Health Organization (WHO) outlined the greatest threats in terms of antimicrobial resistance and methicillin-resistant *S. aureus* (MRSA) was classified as a high-priority microorganism. For many years, MRSA infections were only reported in hospitals, with it being considered to be a nosocomial pathogen (hospital-associated MRSA or HA-MRSA). In the 1990s, community-associated MRSA (CA-MRSA) cases in healthy humans without any connection to healthcare settings started to be described and, nowadays, the distinction between CA-MRSA and HA-MRSA seems to be disappearing [3,4].

For the last two decades, a third epidemiological group known as livestock-associated MRSA (LA-MRSA) has been described. *S. aureus* has been considered to be an important zoonotic agent with

a great capacity to cause infections in different animal species and in humans. Various studies have suggested that there is a high specificity of the different genetic lineages of *S. aureus* for the host [5]. However, many cases of clones related to animals have been detected and have caused infections in humans [6,7]. Presently, different clonal lineages associated with LA-MRSA have been described and, among these, the clonal complex (CC) CC398 stands out (Table S1). CC398 is related to production animals, mainly pigs, and has been detected worldwide [8]. Infection cases have been identified in humans, both in contact and without contact with animals [9–11]. In addition to CC398, there are other clonal complexes associated with animals such as CC5 in birds, CC9 in pigs, CC97 in cattle or CC133 in small ruminants [12–15].

Remarkably, a new methicillin resistance gene (*mecA*LGA251, which shares only 70% similarity to *mecA* (Figure S1)) was first described in 2011 in both humans and animals [16,17]. Initially these strains were associated with dairy cows and these animals were considered to be a possible reservoir [16]. Since then, this gene has been detected in different production and free-living animals and as an agent causing infection in some humans [8,18]. This new gene was named *mecC* since *mecB* had previously been described in macrococci, but not in staphylococcal species [19]. Worryingly, *mecB* has been recently identified in *S. aureus* and future studies should determine the potential risk that this entails [20]. In the case of MRSA isolates carrying the *mecC* gene (*mecC*-MRSA isolates), these isolates have already been identified as belonging to diverse clonal lineages such as CC130, CC49, sequence type (ST) 151, ST425, CC599 or CC1943 and in very different hosts, including its detection in environmental samples [8,21–23]. There are different theories about the origin of the *mecC* gene and the possible impact that these isolates can have in clinical settings. In this review, the objective was to describe current knowledge about *mecC* detection in humans and its possible clinical implications, as well as to determine the infection types associated with this resistance mechanism, the characteristics of these *mecC*-carrying isolates, their possible relationship with animals and the presence of other risk factors.

#### **2. Detection of** *mecC***-MRSA Isolates in Humans**

#### *2.1. Human Studies Related to mecC-MRSA*

Although the *mecC* gene was initially discovered in an isolate from bulk milk in England, the first human *mecC*-MRSA isolates were also identified in that same study [16]. These human isolates were obtained from patients from the United Kingdom and Denmark. Moreover, in a publication from the same year, two human MRSA isolates carrying this new resistance gene were independently identified in Ireland [17].

Since then, several retrospective and prospective studies using human *S. aureus* isolates/samples were carried out in order to search for *mecC*-MRSA isolates (Tables 1 and 2) [16,18,24–74]. Most of these studies were performed in European countries (Tables 1 and 2), and the UK and Denmark were the countries in which the highest levels of *mecC*-MRSA isolates were detected [16,24,25,39,41].



**Table 1.** Human studies related to *mecC*-MRSA isolates in which prevalence can be estimated

1

*Microorganisms* **2020**, *8*, 1615


**Table 1.** *Cont.*

 Case reports were also analyzed in some other studies but, in this table, only results from prevalence studies are included. CC, clonal complex; ST, sequence type; IEC, immune evasion cluster; 4 L, resistant to lincosamides (CLI, clindamycin); M, resistant to macrolides (ERY, erythromycin); Q, resistant to fluoroquinolones (CIP, ciprofloxacin, MFL, moxifloxacin, NOR, norfloxacin); S, susceptible to all non-beta lactam agents tested. UK, United Kingdom.



**Table 2.** Studies performed on humans, in which *mecC*-MRSA isolates were sought but not detected.

1Screening: isolates obtained in epidemiological studies for colonization

 detection.

Unfortunately, the design of these studies was very different, which complicates any comparison of the data obtained. Importantly, the criteria chosen for the selection of the initial isolates/samples varied significantly. While all *S. aureus* isolates were collected in some studies [29,38,45], only MRSA isolates were included in others [24,26–28,32,33,36,41,42]. Moreover, several studies were more restrictive and only used isolates that showed characteristics suspected of carrying the *mecC* gene such as *spa* types associated with *mecC*-positive clonal lineages previously described, *mecA*-negative MRSA isolates, isolates with antimicrobial susceptibility suspected to be *mecC*-positive or *pvl*-negative MRSA isolates [25,26,37,69] (Table S1). In any case, the *mecC*-MRSA human prevalence detected in most of the studies was very low. Several studies did not identify any *mecC*-positive *S. aureus* among included human isolates/samples (Table 2) [45–74]. In studies in which this gene was detected (Table 1), the prevalence identified, considering the total number of isolates/samples included, was < 1% in most of the cases [24,27–29,32,33,37,40–43], similar to that identified in the first study in which *mecC* was discovered (approximately 0.04%) [16]. In a few studies, the prevalence was > 1% but, in all of these, only a small number of initial isolates (<400 isolates) was used; this may be the reason for the high prevalence value obtained (up to 6.3%) [25,26,36,38,39]. Recently, a meta-analysis of the prevalence of *mecC*-MRSA, based on previously published results, estimated the prevalence of *mecC*-MRSA in the human subgroup at 0.004% (95% CI = 0.002–0.007), and the prevalence in the animal subgroup to be 0.098% (95% CI = 0.033–0.174) [75].

#### *2.2. mecC-MRSA Human Case Reports*

A total of 61 human case reports associated with *mecC*-MRSA isolates has been described (Table 3) [17,36,37,45,76–81]. Although *mecC*-positive isolates have been identified in Asia, Europe, and Oceania [21,82,83] in different hosts, all human case reports were described in European countries (Table 3). This was to be expected considering that the majority of the papers in which *mecC*-MRSA has been detected in both animals and humans, as well as in environmental samples, have been focused on countries on this continent [8,21–23].

In 4 of the 61 human case reports, *mecC*-MRSA was only identified in screen swabs (for colonization detection), with it not being related to the cause of the patient's admission [36,37,45], and the clinical information was not indicated in another two case reports [17]. In the remaining 56 studies, *mecC*-MRSA isolates were related to (number of cases): skin and wound infections (47 cases) [37,76,79,81], joint and bone infections (3 cases) [37,77,78], respiratory infections (2 cases) [76] and bacteremia (2 cases) [37,80]. Taking into consideration the type of samples in which *mecC*-positive isolates have been detected in humans (Tables 1–4), most *mecC* human cases were implicated in skin or wound infections. However, the detection of *mecC*-MRSA isolates in other types of samples such as blood, sputum or urine is remarkable (Table 4). Pertinently, some serious infections have been described, such as severe bone infections [78], nosocomial pneumonia [33] and bacteremia [16,24,80], in some cases ending with the death of the patient [37].

*Microorganisms* **2020**, *8*, 1615


**Table3.**Human*mecC*MRSAcasereports.

sequence type; 4 IEC, immune evasion cluster. 5 L, resistant to lincosamides;

 M, resistant to macrolides; S, susceptible to all non-beta lactam agents tested.


**Table 4.** Type of sample/infection in which *mecC*-MRSA isolates have been identified among human patients.

<sup>1</sup> In screen swab: all samples in which was clearly indicated that they did not cause infection were included. However, in several studies it was not indicated whether samples were screen samples or if these samples were taken in infection sites. <sup>2</sup> In human case reports, only one isolate from the most representative infection sample was considered.

#### **3. Risk Factors for** *mecC***-MRSA Infection**

#### *3.1. Contact with Animals*

Since the first description of the *mecC* gene, contact with animals has been considered to be a risk factor for *mecC-*MRSA infection or carriage for several reasons [16,17]. This gene was identified in isolates belonging to CC130, and this clonal complex was predominantly detected among methicillin-susceptible *S. aureus* (MSSA) isolates from bovine sources [17]. Moreover, the discovery of this gene in isolates obtained from dairy cows suggested that these animals might provide a reservoir of this resistance mechanism [16]. Thereby, in some of the studies carried out since then, information about the possible contact of patients with animals was indicated (Tables 1 and 3). Many studies found out that most of the patients lived in rural areas or areas with a high density of farms [18,24,26,29,36,76,79]. In this sense, four studies indicated patient contact with livestock or farm animals [18,24,76,78,81], two referred to only contact with pets [38,45], two patients had no contact with animals and the authors did not have a plausible explanation for the detection of these isolates [37,80], one patient was a veterinarian [33], and in several studies this information was not indicated [16,17,27,30,31,41,77]. Interestingly, *mecC*-MRSA transmission between animals and humans was demonstrated in two human infection cases by whole genome sequencing. Specific clusters including isolates from each human infection case and their own livestock were detected. Thus, human and animal isolates from the same farm only differed by a small number of SNPs [18]. These findings highlight the role of livestock as a potential reservoir for *mec*C-MRSA.

#### *3.2. mecC-MRSA Carriage in Humans*

*S. aureus* shows a great capacity to colonize the skin and nares of hosts, being able to last over time and cause opportunistic infections [84,85]. *mecC*-MRSA isolates were identified as commensals in several prevalence and case report studies (see screen swab in Tables 1–4). At least 54 *mecC-*MRSA positive isolates were obtained from screen swabs, mainly from the nose, but also from throat and groin sites. Moreover, isolates obtained from other types of samples could also be considered as commensals, as in one human case report in Spain in which the isolate recovered from the urine of one patient was considered as a colonizer since the patient did not present urinary symptoms [37].

*mecC*-MRSA isolates implicated in both colonization and infection were obtained from the same patient in some studies [18,37]. Indeed, one patient with bacteremia due to an *mecC*-MRSA isolate also presented nasal colonization by the same *mec*C-MRSA isolate (with the same genetic characteristics) [18]. These results corroborated the importance of colonization being the previous step, which enables isolates causing severe disease. Interestingly, in another bacteremia case in which the patient died, a household transmission between grandfather and grandson was detected, with the grandson being colonized by the same isolate [37]. Nevertheless, in other studies, *mecC-*MRSA isolates were not identified as colonizers from patients with *mecC* infections [81], and it has been suggested that *mecC*-MRSA isolates might be worse colonizers and less contagious in humans than *mecA*-MRSA isolates [76]. In the study carried out in Sweden, only two out of the patient's 27 family members were positive for *mecC*-MRSA isolates and the median time for *mecC* carriage was 21 days [76].

#### *3.3. Patient Age*

Most of the patients described in case reports (Table 3) were middle-aged or elderly [17,36,37,45,77–80], except two patients: one of them was a 34 year-old farm worker with high contact with animals who presented a superficial skin lesion [81], and the other was a healthy 3 year-old child [37]. The average age of patients with *mecC*-MRSA detected in Denmark during 2007–2011 was 51 [24] and the average detected in Sweden in 2005–2014 was 60 [76]. In the Danish study, CA-MRSA *mecC* patients were significantly older than other CA-MRSA cases, indicating that *mecC*-MRSA seems to have a different origin and epidemiology to typical CA-MRSA [24].

#### *3.4. Underlying Chronic Disease*

Remarkably, in the 45 human cases detected in Sweden, most patients had some kind of underlying chronic disease (diabetes mellitus, cancer, autoimmune diseases or atherosclerotic diseases), or an existing skin lesion [76]. Infection by *mecC*-MRSA of wounds has also been suggested by others [79]. Moreover, *mecC-*MRSA infections were identified in patients with primary pathologies (diabetes, myelodysplastic syndrome, peripheral arterial occlusion disease, etc.) in one study in Austria [38], and in a patient with an urothelial carcinoma in Spain [80]. Unfortunately, information about other underlying diseases of *mecC*-MRSA positive patients is missing in most of the papers.

#### **4. Characterization of** *mecC***-MRSA Human Isolates**

#### *4.1. Clonal Lineages of mecC-MRSA of Human Origin*

As in other hosts, most of the *mecC-*MRSA isolates obtained from human samples belonged to CC130 (Tables 1 and 3) (Figure 1). Other clonal complexes identified were CC49, CC425, CC599, CC1943 and CC2361 [16,24,25,29–31,38,40,41,76] (Table 1) (Figure 1). Worryingly, it has been hypothesized that SCC*mec* XI (the SCC element that contains the *mecC* gene) might have the potential to be transferred to other *S. aureus* clonal lineages due to the fact that it is bounded by integration site sequence repeats and that it has intact site specific recombination components [16] (Table S1 and S2). Until now, *mecC*-MRSA CC130 isolates have been identified in all countries in which clonal lineages were determined and it was the unique CC detected in Spain, France, Ireland, Slovenia and Switzerland [17,37,45,77–79,81] (Figure 1). Remarkably, in France and Spain there were several human infection reports, but in all of them the *mecC*-MRSA isolates belonged to CC130 (Table 3). After CC130, the clonal complexes CC1943 and CC599 were the most widely detected in humans, being identified in four and three countries respectively [16,25,29,31,38,41] (Figure 1). Conversely, CC49 was only described in one study in Belgium [29]. While CC49, CC130, CC425, CC599 and CC1943 were also identified in *mecC*-MRSA isolates from a non-human origin, CC2361 has been only described in humans so far [24,76]. Thus, CC130 was described in farm, domestic and wild animals and in food samples; CC49 in horses and small mammals, CC425 in wild animals and food, CC599 in pets and farm animals and CC1943 in pets [8].

**Figure 1.** Clonal complexes (CCs) detected in *mecC*-MRSA human isolates.

A large variety of *spa* types was detected among the human *mecC*-MRSA isolates (Figure 2). The most predominant *spa* type was t843, which is associated with CC130 and was identified in a total of 260 human isolates. This *spa* type was detected in all countries in which human *mecC*-MRSA isolates were detected, except in Switzerland [45]. Other *spa* types were also described in several countries. Some of them were identified only in two countries, this is the case of t792, t1773, t5930, t6293, t6386, t7485, t7734, t7945, t7946, t7947 and t9397, but others were more widely spread as t978, t1535, t1736, t3391 or t6220 (Figure 2). Although there is a strong association among *spa* types and MLST clonal complexes [86], some *spa* types were associated with different clonal complexes. Two isolates obtained in screen swabs from two patients in two different hospitals from England presented the *spa* type t11706 [40]; one of these isolates belonged to ST1245 (CC130) and the other one to ST425 (CC425). Moreover, the *spa* types t978, t2345, t3391 and t8835 were associated in some studies with CC1943 [16,25,29], and in others with CC2361 [24,76]. Nevertheless, the founders of both clonal complexes, ST1943 and ST2361, are Single Locus Variant (SLV) of each other (and only differ at the *aroE* allele), which could explain these results (Table S1).

**Figure 2.** *spa* types detected in *mecC*-MRSA isolates of humans. Colors indicate the clonal complexes associated with each *spa* type: green CC49, red CC130, blue CC425, purple CC599, orange CC1943, black CC2361. The number of isolates of each *spa* type is indicated in parentheses (to calculate the number of isolates in human case reports, only one isolate from each *spa* type and each patient was considered)

#### *4.2. Treatment and Antimicrobial Resistance Profile of mecC-MRSA Human Isolates*

Most of the human *mecC*-MRSA isolates detected were susceptible to all non-beta-lactam antimicrobials tested (Tables 1 and 3). This is in accordance with results obtained in *mecC*-MRSA isolates from other origins [21]. In one study performed in Spain, isolates using this criterion were selected in order to identify *mecC*-MRSA or CA-MRSA isolates [69]. Although *mecC*-MRSA was not detected, this resistance phenotype was a valuable marker for Panton-Valentine Leukocidin (PVL)-producer isolates (Table S1). Nevertheless, the low prevalence of *mecC*-MRSA isolates in this region could be responsible for this result and the use of this phenotype to suspect the presence of the *mecC* mechanism should not be discarded.

The most important problem for treating *mecC*-MRSA infections is that these isolates must be correctly identified. Although *mecC* isolates are considered to be MRSA, these isolates sometimes show borderline susceptibility results for oxacillin or cefoxitin, being identified as MSSA if the *mecA* gene is only tested [44]. This could lead to the implementation of inappropriate therapies. Resistance development to other antimicrobial agents could be added to this, considering the capacity of *S. aureus* to acquire resistance to various antimicrobial agents. Some *mecC*-MRSA isolates detected in humans have shown resistance to non-beta-lactam antimicrobials [24,33,40,41,76] (Table 1). Fluoroquinolone resistance was identified in two isolates in Germany [33] and in one isolate in Denmark [24]. Macrolide and lincosamide resistance was also detected in some studies: one erythromycin resistant isolated in the UK [41] and one erythromycin and clindamycin resistant isolate in Sweden [76] and England [40]. Regarding resistance mechanisms, in two studies carried out in Ireland, the gene *sdrM*, which encodes a multidrug efflux pump related to norfloxacin resistance and *tet* efflux related to tetracycline resistance, were identified in one and two *mecC-*MRSA CC130 isolates, respectively [17,26]. Although there has only been one study, whose objective was to compare different diagnostic tests, human *mecC*-MRSA isolates resistant to several antimicrobial families have been

detected [87]. The presence of resistance to non beta-lactam agents in *mecC*-MRSA isolates significantly limits our therapeutic options.

#### *4.3. Virulence of mecC-MRSA Human Isolates*

The search for virulence genes in human *mecC*-MRSA isolates has been highly variable from one study to another. In any case, for the moment, the most common virulence genes detected have been *hla*, *hld*, *hlb*, *edinB*, *lukED*, *cap8* or *ica*, with these genes being highly associated with CC130 [17,26,31,33,36,38,41]. Fortunately, no *mecC*-MRSA isolates carrying the PVL genes were detected. However, other clonal lineages associated with animals have been able to acquire this virulence factor [88]. For this, their detection in *mecC*-MRSA isolates cannot be ruled out in the future. Significantly, some pyrogenic toxin superantigen (PTSAg) genes have been detected in *mecC*-MRSA isolates [29,31,38,41]. These genes might be related to specific clonal lineages such as CC599, CC1943 or CC2361. Thus, the gene *tst* encoding the toxic shock syndrome toxin has been found in three CC1943 isolates (two harboring *sec* gene and one containing *seg* and *sei* genes) [29,41], in three CC599 isolates (two of them positive for *sel* gene and one for *sec* and *sel*) [31,38] and in one *sec, seg, sei, sel, sen, seo* and *seu* positive CC2361 isolate [31] (Table S1).

#### **5.** *mecC***-MRSA Problem: What is its Origin? Is It an Emerging Problem?**

The oldest known *mecC*-MRSA isolate, dated in 1975, was detected in the retrospective study performed by García-Álvarez et al. [16] This isolate was identified in a human blood sample from Denmark and its detection suggested a possible human origin for the *mecC* gene [16]. Later, in two other retrospective studies also carried out in Denmark, two *mecC*-positive isolates were identified in samples dated in 1975 [24,25], both were also identified in human blood samples [24,25]. However, in two of these studies, the oldest sample studied was obtained in 1975, so the presence of older isolates cannot be ruled out [16,25]. With respect to the remaining retrospective studies in which the presence of the *mecC* gene was sought, the dates of the samples were much later than these three studies, with them being isolates obtained from the year 2000 and later (Table 1). Regarding the earliest reported *mecC*-MRSA isolate in other hosts, 1975 also seems to be the key date [89,90]. Therefore, this resistance mechanism might have been present for over 45 years.

Moreover, this resistance mechanism is highly associated with CC130 since most of the *mecC*-MRSA isolates belong to this clonal lineage. A human-to-bovine host-jump of CC130, which occurred ∼5429 years ago, has been suggested [91]. The time and host in which CC130 MSSA isolates acquired the *mecC* resistance gene remain unknown today. In order to establish a possible human or animal origin for the detected *mecC*-MRSA isolates in human samples, several studies analyzed the presence of IEC (immune evasion cluster) genes [17,24,26,31,33,36,41,42,78,81] (Tables 1 and 3). In all cases, human *mecC*-MRSA isolates were negative for *sak*, *chp* and *scn* except for one ST1945 (CC130) isolate obtained from a screen swab from a patient in the UK that was positive for *sak* and *scn* (IEC type E), suggesting a possible human origin [41]. Nevertheless, it has been suggested that IEC type E might be a conserved part of ST1945 since *mecC* MRSA ST1945 isolates from wild animals also showed IEC type E in several studies in Spain [41,63,92].

The newest human *mecC*-MRSA isolates detected so far in Europe were obtained in 2015, one of them in Germany [27], and the other in England [42]. Both strains showed similarities to those identified in the first studies [16,17] and both belonged to CC130. Nevertheless, after phylogenetic analysis, the strain identified in England seemed not to be highly related to any of the published sequenced *mecC*-MRSA CC130 isolates [42]. Despite the non-description of *mecC*-MRSA isolates in humans in the last 5 years in Europe, data provided by previous studies have detected an increasing tendency in the prevalence of *mecC*-positive isolates [24], indicating that surveillance in detecting this resistance mechanism must be maintained. The lack of detection could be due to the low prevalence of this resistance mechanism and/or problems in *mecC* diagnostic methods. Important difficulties in the detection of *mecC*-MRSA isolates have been indicated [44,93]. It has been shown that various clinical tests used in hospital labs might have failed to identify 0 to 41% of *mecC*-MRSA isolates [93]. It is important to optimize and develop new testing protocols and redefine currently available phenotypic testing methods [44]. In this regard, several commercial PCR-based tests that detect *mecC* and *mecA* genes have been developed. Moreover, recently, *mecA*/*mecC* MRSA isolates from cattle have been described [83]. The possible clinical impact of isolates carrying both genes is currently unknown.

#### **6. Implications in Veterinary and Food Safety**

Although this review is focused on the human health implications of *mecC*-MRSA isolates, the effects that these isolates can have on veterinary medicine should not be forgotten. *mecC*-MRSA isolates causing infections in domestic animals have been identified in several studies [8,94,95]. However, this resistance gene seems to be most frequently detected in livestock animals including cattle, sheep and rabbits [8,21]. Although *mecC*-MRSA rarely causes clinical disease in these food-producing animals, there are reports of bovine mastitis in several countries [96,97]. As observed in humans, most of the *mecC*-MRSA isolates detected in other hosts also belong to CC130, with the characteristics of these animal *mecC*-MRSA isolates being very similar to those detected in humans [8,21].

On the other hand, the presence of *mecC*-MRSA in dairy animals is highly relevant since it could be a route of entry of these isolates into the food chain. Indeed, milk samples have been identified carrying *mecC*-MRSA [8] with the consequent risk of colonization for food handlers. In this case, it is worth highlighting one of the clinical cases included in this review in which the patient was a cheese producer [81]. *mecC*-MRSA zoonotic transmission has been demonstrated in some studies [18], with the correct prevention, detection and control measures in veterinary and food safety laboratories being necessary.

#### **7. Conclusions and Future Problems Associated with** *mecC*

Currently, the prevalence of human *mecC-*MRSA infections is very low. However, *mecC*-MRSA isolate transmission between different hosts indicates the great capacity of these isolates for spreading. There is a wide range of reservoirs in wild, livestock and companion animals and zoonotic transmission of these isolates could increase the number of *mecC-*MRSA human clinical cases. Moreover, SCC*mec* XI might have the potential to be transferred to other clonal lineages in the future. Their transfer to more virulent and better-adapted human clones would be deeply troubling. Worryingly, the *mecC* gene has already been detected in clonal lineages in which important virulence genes were identified (CC599, CC1943 or CC2361) or in which IEC was described (ST1945-CC130). Moreover, *mecC*-MRSA isolates resistant to non-beta lactams have been detected. Acquisition of non-beta lactam resistance by *mecC*-MRSA isolates significantly limits our therapeutic options. *mecC*-MRSA should be taken into consideration in hospital and veterinary laboratories and in food safety institutions, and prevention strategies must be implemented in order to avoid possible emerging health problems.

#### **Supplementary Materials:** Supplementary Materials are available online at http://www.mdpi.com/2076-2607/8/ 10/1615/s1.

**Author Contributions:** C.L. contributed to the search of articles and to their tabulation and classification into different categories. She also contributed to the design and the analysis of the review and to the writing of the paper. R.F.-F., L.R.-R., P.G. and M.Z. helped in the general review of the manuscript. C.T. contributed with project funding, and with the original idea and the design of the manuscript and reviewed the initial version of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by project SAF2016-76571-R of the Agencia Estatal de Investigación (AEI) and the Fondo Europeo de Desarrollo Regional (FEDER) of EU. Laura Ruiz-Ripa has a pre-doctoral fellowship from the Universidad de La Rioja. Rosa Fernández-Fernández has a predoctoral fellowship from the Ministerio de Ciencia, Innovación y Universidades of Spain (FPU18/05438).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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## *Review* **No Change, No Life? What We Know about Phase Variation in** *Staphylococcus aureus*

**Vishal Gor 1,\*, Ryosuke L. Ohniwa <sup>2</sup> and Kazuya Morikawa 2,\***


**Abstract:** Phase variation (PV) is a well-known phenomenon of high-frequency reversible geneexpression switching. PV arises from genetic and epigenetic mechanisms and confers a range of benefits to bacteria, constituting both an innate immune strategy to infection from bacteriophages as well as an adaptation strategy within an infected host. PV has been well-characterized in numerous bacterial species; however, there is limited direct evidence of PV in the human opportunistic pathogen *Staphylococcus aureus*. This review provides an overview of the mechanisms that generate PV and focuses on earlier and recent findings of PV in *S. aureus*, with a brief look at the future of the field.

**Keywords:** *Staphylococcus aureus*; phase variation

#### **1. Introduction**

The Gram-positive human commensal *Staphylococcus aureus* is an opportunistic pathogen that imposes a major health and economic burden on a global scale [1]. *S. aureus* can colonize multiple sites of the human body, but the primary niche of commensal colonization is the anterior nares, with various other skin surfaces making up secondary niches. There are three main carrier-patterns of *S. aureus* amongst healthy individuals: persistent carriers (~20%), intermittent carriers (~30%), and non-carriers (~50%) [2]. Nasal carriage of *S. aureus* has been linked to a higher chance of contracting infection [2]. *S. aureus* is responsible for an astounding diversity of infections. It is the leading cause of infective endocarditis, osteoarticular infections, and surgical site infections and *S. aureus* bacteraemia is also prevalent [3,4]. *S. aureus* can also cause pneumonia and other respiratory infections, particularly in people living with cystic fibrosis [3]. Furthermore, *S. aureus* is supremely adept at colonizing alien surfaces within the body and is often responsible for infections associated with catheters, cannula, artificial heart valves, and prosthetic joints [3]. This diverse range of infections is enabled by a vast arsenal of virulence factors that are ready to be deployed in a variety of host environments [5,6]. Of particular concern is *S. aureus'* rapid development of antibiotic resistance. Methicillin Resistant *S. aureus* (MRSA) has broad-spectrum resistance against the β-lactam group of antibiotics and is a global danger with clones existing in both nosocomial and community settings [7]. MRSA is also a problem in the livestock sector, where it can co-infect both animals and humans [8]. The infamous development of antibiotic resistance, coupled with its worrying genetic plasticity, has earned *S. aureus* a place in the ESKAPE group of pathogens: a collection of bacteria that represent paradigms of acquisition, development, and transfer of antibiotic resistance [9]. Thus, to better combat this dangerous pathogen it is vitally important to study adaptation mechanisms of *S. aureus*.

Another particular trait of *S. aureus* that makes it notoriously difficult to combat in the clinical setting is phenotypic heterogeneity. An example of this is the phenomenon of persister cells, where sub-populations of *S. aureus* gain a resistance phenotype against antibiotic treatment resulting from arrested growth [10]. Persister cells may be generated in numerous ways, one of which is the formation of Small Colony Variants (SCVs) that are

**Citation:** Gor, V.; Ohniwa, R.L.; Morikawa, K. No Change, No Life? What We Know about Phase Variation in *Staphylococcus aureus*. *Microorganisms* **2021**, *9*, 244. https://doi.org/10.3390/ microorganisms9020244

Academic Editor: Rajan P. Adhikari Received: 31 December 2020 Accepted: 23 January 2021 Published: 25 January 2021

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characterized by auxotrophy for various compounds involved in the electron transport chain and slow growth, allowing them to escape the effects of many antibiotics [11,12]. Importantly, these populations do not acquire conventional resistance mechanisms against the antibiotics. This heterogenous phenomenon has severe clinical implications and is thought to be a significant cause of antibiotic treatment failure and chronic recurrent infections [13].

Heterogeneity is not limited to antibiotic resistance. As we discuss in this review, diverse traits, including pathogenicity factors, have recently been recognized as having sub-population patterns of expression. The scientific point-of-view has been increasingly focused on such heterogenous phenomena, yet progress is still in the relatively early stages and much work remains to be done. In this review, we summarize the information regarding bacterial Phase Variation (PV), a mechanism of high-frequency reversible gene expression switching (Section 2) and collate the known examples of *S. aureus* PV into one source (Section 3) to aid in future studies on heterogeneity in *S. aureus* (Section 4).

#### **2. Bacterial Phase Variation**

#### *2.1. Background of Phase Variation*

All living organisms are faced with the constant challenge of maintaining fitness in order to survive and reproduce, and this is no less true for bacterial species. Bacteria are under constant onslaught from fluctuations in their local environment, infection from bacteriophages, and (in the case of pathogenic bacteria) attack from their infected host. Although bacteria possess robust mechanisms of classical gene regulation that allow them to respond to extracellular changes (e.g., Bacterial Two-Component Systems), these alone may be unable to cope with the constant barrage of fluctuating pressures they face. These selective pressures are often focused on bacterial external proteins which form the first line of contact with the outside environment and this has led to development of what have been termed "contingency loci" [14,15]. Contingency loci are hypermutable genes that generate genetic and phenotypic variation allowing bacterial populations to survive unpredictable pressures. This hypermutability is conferred by the phenomena of Phase Variation (PV) and antigenic variation.

PV is a reversible gene expression switch that can alter expression between an ON and an OFF state and occurs through several genetic and epigenetic mechanisms [16]. It is characterized by high frequencies, usually exceeding 1 × <sup>10</sup>−<sup>5</sup> variants per total number of cells [17,18] which is orders of magnitude above the typical frequencies of spontaneous mutations (10−<sup>6</sup> to 10−<sup>8</sup> per cell per generation) [18]. Depending on the method of calculation, the frequency of PV may describe not only rate of the PV mechanisms but also the growth of the phase variants themselves. Antigenic variation is related to PV and occurs through similar mechanisms. However, rather than alternating between an ON and OFF state, antigenic variation mechanisms generate variations in the sequence of surface proteins resulting in the expression of different forms and structures of the antigenic proteins on the cell surface [17–19]. As such, due to the similar nature of the mechanisms involved, antigenic variation will not be separately addressed in this review.

As mentioned above, genes subject to PV often encode for cell-surface associated features such as adhesins, liposaccharide synthesis enzymes, and pili [20–22] but can also encode for virulence factors and secreted proteins such as iron acquisition machinery [23,24]. The collection of phase variable loci in a bacterial species is referred to as the "phasome" [16] and generally includes genes which are involved in bottlenecks experienced by the bacterial population. This is most clearly seen in pathogenic bacteria which undergo constant challenge from host immunity during the infection process. For example, PV mediated shutdown of liposaccharide synthesis genes in the invasive pathogen *Haemophilus influenzae* confers protection against neutrophil-mediated immune clearance but is detrimental in other environments [22,25,26]. In another example, PV in *Salmonella typhimurium* flagellae can modulate their antigenic properties and allow for evasion from host immunity [27].

It is likely that the original role of PV was as a mechanism of innate immunity against bacteria's greatest enemy: bacteriophages [28]. Although bacteriophages exist in exaggerated abundance relative to their bacterial hosts, their host range is often limited to just a few specific strains of a given bacterial species [29]. Thus, there is a constant cyclical arms race between bacteria and bacteriophages in order to stay one step ahead of each other [30], and PV plays an important role in both sides of this war. An example can once again be found in liposaccharide synthesis genes of *H. influenzae* in which PV can result in a switch from a sensitive to resistant phenotype against the HP1c1 phage [31]. On the other hand, PV in the *Escherichia coli* phage Mu causes a switch in expression between two sets of tail fibers resulting in modulation of the host specificity [32,33] with similar phenomena identified in other phages [34].

Considering the above information, it can be inferred that genetic loci susceptible to PV would be found in abundance amongst bacterial species that experience population bottlenecks. Typically, such bottlenecks often occur during the infectious process which imposes limits onto the bacterial population size. These bottlenecks reduce genetic diversity at a time when variation is most beneficial, and PV offers a solution to this hurdle and indeed, several pathogenic bacteria have been documented to undergo PV [18].

While PV is, by definition, a stochastic process, it occurs through several discrete mechanisms. Broadly speaking, mechanisms of generating PV can be discriminated into genetic and epigenetic mechanisms [16] both of which will be addressed in Sections 2.2 and 2.3 respectively.

#### *2.2. Genetic Mechanisms of PV*

There are three genetic mechanisms of PV which shall be discussed in the following chapters: Variation in length of DNA Short Sequence Repeats (SSRs) [35–37], DNA inversion [38], and DNA recombination [39,40].

#### 2.2.1. Variation in Length of DNA Short Sequence Repeats (SSRs)

SSRs are homo- or hetero-nucleotide repeats in DNA that are highly prone to insertion/deletion (indel) errors due to Slipped-Strand Mispairings (SSMs) during DNA replication [35–37]. SSRs can be as complex as repeating units of tetranucleotides or as simple as a straight homonucleotide run. Indels in SSRs can result in frameshifts that largely have an ON↔OFF effect on protein function or gene expression (by resulting in abrupt termination of translation or inhibition of RNA polymerase binding, respectively Figure 1A) but can have an alternative gradation effect on gene expression as well. For example, alterations in the length of a dinucleotide TA10 tract in the promoter regions of the divergently transcribed *hifA* and *hifB* genes controlling fimbriae expression in *H. influenzae* can either significantly affect *hif* expression (TA10→TA9) or only moderately affect it (TA10→TA11) [41]. The evolution of the mutability of SSR tracts is largely driven by a combination of environmental and molecular drivers. The environmental drivers include factors such as the aforementioned population bottlenecks arising during infection processes. These bottleneck conditions exert a primary selective pressure for phenotypes that can survive them, e.g., a population that can shut down the expression of a surface protein that is targeted by host immunity. The necessity to survive this recurrent primary selection serves as a secondary layer of selection for plasticity of the gene itself.

The molecular factors are intrinsic to SSR tracts and include the DNA replication and the Mismatch Repair (MMR) [42]. The discriminating factors of SSRs can be broadly delineated into two groups: the composition of the repeating nucleotide unit (i.e., a homonucleotide or a heteronucleotide repeat) and the tract length. These in turn are differentially affected by the DNA replication and MMR machinery. Amongst these proteins are the DNA polymerase enzymes which include the polymerase responsible for the construction of new DNA strands (DNA polymerase III) as well as the polymerase responsible for DNA repair (DNA polymerase I). Studies have shown that these polymerases have an inherent frequency of generating addition/deletion errors when constructing new DNA strands [43,44]. Following DNA replication, any errors are corrected by the MMR machinery which is a suite of Mut proteins that target and fix errors in a strand specific manner. Inactivation of components from either of these suites of proteins results in a hypermutable phenotype and can lead to SSR alteration e.g., [45]. Additionally, the hypermutable phenotype that results from loss of the MMR machinery is directly responsible for genetic variability of bacteria and mutator phenotypes play an important role in bacterial adaptation [46]. For example, both *S. aureus* and *Pseudomonas aeruginosa* isolated from the lungs of people suffering from cystic fibrosis are commonly associated with antibiotic resistance caused by hypermutability [47–49]. Interestingly, while both the MMR machinery and DNA polymerases are involved in SSR evolution, they do not appear to be fully redundant. Several studies have shown that MMR is more responsible for variability of homonucleotide SSRs, especially for those which exceed eight nucleotides in length, whereas DNA polymerase I is exclusively responsible for mutations in heteronucleotide SSRs [50–52]. This could have evolutionary implications for the mechanisms of generating SSRs. For example, *H.influenza* is enriched with tetra-nucleotide SSRs [51] whose expansion/contraction is affected by DNA polymerase I. Furthermore, evidence suggests that the frequency of DNA polymerase I mediated errors differs between the leading and the lagging strands of newly synthesized DNA, implying that the direction of genes in the chromosome can also dictate the type of SSR that would evolve in them [53]. Lastly, an interesting study carried out by Lin et al. investigated the distribution of SSRs within the genomes of several bacterial species. They found that in many pathogenic species, SSRs were enriched towards the N-termini of protein coding sequences increasing the probability of frameshifts resulting in non-functional proteins [54,55]. This further suggests that bacteria have evolved SSRs in a manner to provide maximal PV.

#### 2.2.2. DNA Inversion

DNA inversion was the first documented example of PV, though the mechanism was not known at the time the phenomenon was documented [38] (Figure 1B). It involves recognition of inverted repeat (IR) sequences by invertase enzymes and subsequent enzyme-mediated inversion of the DNA. An elaborate study was carried out by Jiang and colleagues who developed an algorithm to search published bacterial genome datasets for IR sequences that might be phase variable [56]. Not only did they identify that IR sequences were enriched in host-associates species (implying a benefit of PV during commensalism or infection) but they also discovered three antibiotic resistance genes regulated by invertible promoters: a macrolide resistance gene, a multidrug resistance cassette conferring resistance to macrolides and cephalosporins, and a cationic antibacterial peptide resistance operon [56]. The presence of antibiotics influenced the switch from an OFF to an ON state for these genes. Some of the invertible promoters seem to be located on genetic elements homologous to those conveyable by horizontal gene transfer mechanisms, raising the worrying possibility that these resistance gene switches can be transferred to other species [56].

#### 2.2.3. DNA Recombination

Homologous recombination provides a pathway for DNA re-arrangement and subsequent PV. Events arising from recombination mechanisms are often due to DNA deletions, and thus tend to be in a one way ON→OFF direction. However, gene duplication or transfer events can often occur to balance out the accumulation of inactive variants in the population. A well characterized example of recombination mediated variation occurs in the *Neisseria gonorrhoea* pilus organelle, which is essential for full infectivity and natural transformation. *N. gonorrhoea* contains a *pilE* gene that encodes for a pilin protein that is the major component of the pilus, but also contains several silent *pilS* alleles several Kb away [39]. RecA-dependent recombination events can unidirectionally transfer large sections of the *pilS* allele into *pilE*, thus creating an OFF variant [40,57] (Figure 1C). The *N. gonorrhoea* pilus also undergoes PV by SSM-mediated variation in the length of a poly(G)

tract in the *pilC* gene (which encodes for the adhesive tip of the pilus [58]) resulting in ON↔OFF switching [59,60].

**Figure 1.** Genetic mechanisms of Phase Variation. A cartoon depicting the three main genetic mechanisms of Phase variation (PV). (**A**) Slipped-Strand Mispairing events within Short Sequence Repeats (SSR) result in expression (green tick mark) of truncated dysfunctional proteins (if SSR is in the CDS) or inhibition (red cross) of transcription by preventing RNA polymerase/transcription factor binding or by other mechanisms. For example, an interesting method of PV-mediated transcriptional control is shown by Danne et al. who demonstrate SSR alterations upstream of the *pilA* locus of *Streptococcus gallolyticus* can destabilize a premature transcription-terminating stem loop [61]. (**B**) Site-specific inversion is carried out by recombinases that recognize inverted repeat regions (Inverted Repeat Left/Right IRL/IRR) and flip the DNA sequence in between them. If a promoter region (e.g., pB) lies within the sequence flanked by the inverted repeats this leads to shut down of gene expression. (**C**) RecA-mediated DNA recombination of *N. gonorrhoea pilS* into *pilE* results in the formation of new *pilE* variants. Both *pilS* and *pilE* contain variable regions (depicted in green and orange, respectively) interspersed with conserved regions (white) while *pilE* has a further 5' conserved region (dark orange) and a promoter to initiate transcription.

#### *2.3. Epigenetic Mechanisms of Phase Variation*

An epigenetic trait has been defined as a heritable phenotype resulting from modified gene expression that is not due to any alterations in the DNA sequence of the chromosome [62,63]. In prokaryotes, DNA methylation occurs mainly at the nucleotide adenine although studies have shown that cytosine methylation can also occur [64–66]. DNA methylation usually occurs at specific target sites and is carried out either by methyltransferases that are part of dedicated Restriction–Modification (RM) systems or by orphan methyltransferases. A well-studied methylase responsible for bacterial epigenetic regulation of

PV is the DNA Adenine Methyltransferase (DAM) which is an orphan methyltransferase of the gammaproteobacterial family that is specific for GATC sites [64]. Methylation of DNA represses transcription, and thus PV can result if there are GATC sites within a gene promoter which also binds transcription factors, causing mutually exclusive binding competition between the transcription factor(s) and DAM. If there are numerous GATC sites within a promoter region then the mutually exclusive competition can result in differential methylation patterns of the promoter region resulting in switching between an ON and OFF state. A paradigm of this sort of PV is established by a series of intriguing reports studying the *pap* operon of *E. coli* and the *opvAB* operon of *Salmonella enterica* [21,67–69].

#### *2.4. Combined Mechanisms of Phase Variation*

There is growing evidence that shows that many bacterial species undertake a combined approach for PV to maximize the ability to generate rapid and diverse variation. This strategy involves generating PV through genetic mechanisms in genes of RM systems that can modify the transcriptome of the cell via epigenetic control. Such systems are referred to as "phasevarions" as they control phase-variable regulons [70] and are immensely powerful weapons in the arsenal of pathogens.

The earliest phasevarions identified are controlled by Type III RM systems. PV occurs in SSRs in the *mod* gene resulting in ON↔OFF variation and altered methylation states [71,72]. Strikingly, analyses of known Type III system sequences indicate that at least 20% of these systems contain SSRs and could potentially be phasevarions [73]. Furthermore, *mod* genes are highly conserved, with variation occurring mainly in the DNA recognition domain. This allows *mod* genes to exist within the species as multiple alleles, each of which controls distinct phasevarions [16].

There is some evidence of a Type II RM regulated phasevarion detected in *Campylobacter jejuni*, and gene expression patterns were detectably different upon RNAseq analysis, though no direct link to any altered phenotype was reported [74].

PV in Type I systems largely occurs through DNA inversion in the *hsdS* gene, creating multiple allelic variants of the specificity protein of the Type I system resulting in different gene targets upon PV [75]. An example of a Type I RM phasevarions can be seen in variable capsular expression controlling virulence in *Streptococcus pneumonia* [16,76].

In theory, phasevarions must be also seen by PV in other regulators of gene expression, such as transcription factors. Some examples of these are described in Section 3.

#### **3. Known Examples of Phase Variation in** *S. aureus*

*S. aureus* is an opportunistic pathogen that has claimed several distinct niches in the human body, thus being subject to a variety of different conditions and stresses against which it has accumulated diverse colonisation and pathogenicity factors. As such, it is primed to exploit the phenomenon of PV; however, there have been surprisingly few documented reports of PV examples, possibly due to a link between heterogenous phenotypes and PV not being made. This section (Section 3) will outline reports that have identified PV, or PV-like mechanisms, in *S. aureus*. Reports detailing heterogeneity arising from non-PV mechanisms will not be discussed as they have been detailed elsewhere [77].

Perhaps the best studied example of PV in *S. aureus* relates to its ability to form biofilms. A report from the early 1990s identified phase variation in the production of an extracellular polysaccharide coat (or "slime") whose production could be reversibly switched across generations of the same lineage with variants easily distinguishable by differential colony morphology on Congo Red Agar [78]. From there, the topic took on multiple approaches from various groups. Tormo and colleagues identified that expression of the Bap protein (a major surface component involved in biofilm formation that promotes primary attachment as well as intercellular adhesion) was phase variable [79]. However, although they confirmed that Bap-negative variants did not express the *bap* gene, they could not detect any sequence alterations, suggesting that the exact mechanism of PV was either indirect or occurred through epigenetic means. Investigating from another

direction, Valle and colleagues discovered that *IS256* transposition can also result in biofilm PV by disrupting the *sarA* regulator and *icaC* [80]. The *icaADBC* operon encodes for genes involved in the synthesis of poly N-acetylglucosamine exopolysaccharide and its deacylated variant polysaccharide intercellular adhesion (PNAG/PIA) [81](other major extracellular components involved in biofilm formation that also have roles in immune evasion [82]). Interestingly, they further discovered that there is a connection between this variation and the global stress sigma factor σB, as a σ<sup>B</sup> deletion mutant has significantly higher *IS256* copies and transposition frequencies [80]. However, there are yet more layers to this example. In 2003, Jefferson and colleagues discovered a 5-nucleotide SSR (TATTT) in the promoter region of the *ica* operon, whose expansion/contraction affected the binding of *ica* regulatory elements and shut down PNAG/PIA production [83]. In a subsequent study, Brooks and Jefferson discovered that there are further SSRs present in the operon in the form of tetranucleotide repeats within the *icaC* ORF (Open Reading Frame), and SSM events in those also reversibly control PNAG/PIA production [81]. Finally, an elaborate mechanism for the *icaC* SSM expansion has been proposed. The *icaC* tetranucleotide SSR can stably form a so called "mini dumbbell structure" by folding back on itself and making a small loop [84]. It has been proposed that if such a structure were to form during DNA replication it would increase the frequency of SSM events, resulting in expansion of the SSR [84].

Surface proteins are theoretically particularly prone to PV, and one of the first conclusively identified PV events occurs in the extracellular MapW protein, which may have functions in immune evasion based on its high degree of similarity with Major Histocompatibility Complex Class II molecules [85]. The *mapW* gene has a poly(A) tract that results in premature termination of translation. A change in the poly(A) tract can shift the reading frame and result in full-length protein being transcribed [85]. This example of PV varies the length of the protein product rather than switching between an ON↔OFF state, though it is yet to be confirmed if both the truncated and full-length protein have distinct functions.

A PV-like system was also found in the regulation of natural transformation just short of a decade ago [86]. The finding of staphylococcal natural transformation has implications regarding its rapid acquisition of antibiotic resistance genes, but intriguingly it was demonstrated that only a subset of the population can enter a competence state. The genes necessary for entering the competence state are controlled by the alternative sigma factor σ<sup>H</sup> [87] and the transcription factor ComK that synergistically works with σ<sup>H</sup> [88]. It was found that two independent mechanisms control σ<sup>H</sup> expression in *S. aureus* [86]. The translation of the σ<sup>H</sup> mRNA is likely repressed through the action of an inverted repeat loop in the 5' UTR, and the still elusive de-repression mechanism allows σ<sup>H</sup> expression in subpopulations. In addition, as a second genetic mechanism, at low frequencies (~≤10<sup>−</sup>5) *sigH* undergoes a gene duplication event with downstream genes, effectively replacing the native 5' UTR, and thus lifting repressive control [86]. This event is reversible and reverts to the native chromosomal structure at a frequency of 10-2. Although the frequency of duplication is lower than that commonly associated with PV, the reversible nature of the mechanism coupled with the contingency-like nature of the *sigH* locus [89] allows for a justification of this phenomenon being discussed under the umbrella of PV.

A very recent study in our lab uncovered another example of PV in *S. aureus*, one with potentially far-reaching implications. Upon investigating the phenomenon of hemolytic heterogeneity commonly observed in *S. aureus*, we identified PV-controlled reversible shutdown of the central virulence regulatory system, the Accessory Gene Regulator (Agr) system [90]. PV occurred via two distinct mechanisms: the first was a duplication and inversion event within the ORF of *agrC* (which encodes for the sensor component of the Agr TCS) and the second involved alteration in the length of homonucleotide SSRs within the *agrA* ORF (which encodes for the TCS response regulator). The second mechanism was also identified in a single clinical isolate, and although we were unable to determine the clinical significance of this findings (owing to a minor frequency of clinical revertant strains), the results demonstrated that *S. aureus* has a phasevarion under control of the Agr

system. Furthermore, a study that investigated *S. aureus* dermal colonization in children identified that chronic colonizers tended to have a mutationally inactivated Agr system. Importantly, we found that two of their samples (out of four) had frameshifts resulting from alterations in homonucleotide SSRs within the *agrC* and agrA ORFs [91]. The implications of this suggest that this phasevarion could come into play as a cryptic insurance strategy against host-mediated immune attack and may possibly even allow *S. aureus* to manipulate host phagocytic cells and use them as a Trojan horse to disseminate itself within the host (Figure 2). This is of particular consideration as under certain conditions bacterial infections can be established by a single surviving cell [92].

**Figure 2.** Phase Variation in *Staphylococcus aureus*. A cartoon depicting known PV in *S. aureus* and its roles. Double headed arrows indicate reversible PV events. (**A**) PV of Bap (Biofilm Associated Protein) (unclear mechanism) and of the *ica* (Intracellular Adhesion) operon (transposon insertion and SSR alteration) reversibly affects the biofilm-forming capability. (**B**) Phase variation of the Agr (Accessory Gene Regulator) system may have multiple possible roles [90]. It could serve as a cryptic insurance strategy against host immune attack, allowing phagocytosed revertant cells to activate their reverted Agr system to survive within a "Trojan Horse" (Top). It may also aid in the proper structuring of biofilms, as revertant cells and their progeny can activate their Agr system in structured non-planktonic architecture and the resultant exoproteins form channels in the biofilm for circulation of waste and nutrients as well as facilitating the exodus of cells from the biofilm [93] (Bottom). (**C**) PVlike expression is one of two independent expression mechanisms known for the alternative sigma factor σH. Either mechanism allows for a subpopulation of cells to express the competence machinery and undergo natural transformation. (**D**) PV of the cell-wall associated MapW (MHC class II Analog Protein) may have multiple impacts [94]. MapW is involved in bacterial aggregation and may lead to biofilm formation; It is also implicated in the adherence to the host matrix and in-dwelling medical devices (e.g., cannulas); MapW has immunomodulatory effects and seems to suppress T-cells and their recruitment, though the exact mechanism remains elusive; MapW is important in adherence to, and internalization by, host non-phagocytic cells (e.g., epithelial cells). (Clockwise from top).

#### **4. Future Perspectives**

There is mounting evidence of PV being important in the evolution and adaptation of bacteria, with roles ranging from their arms race with phages to their pathogenic proficiency. *S. aureus* is an important global pathogen that can survive in a variety of different niches within the human body using its arsenal of virulence factors. Considering this, *S. aureus* should be primed to exploit PV in its lifestyle yet there remain few documented cases of clear PV phenomena. However, it is very possible that cases reminiscent of PV have been overlooked in the past and may be worth further investigating. For example, a study carried out by Aarestrup and colleagues as far back as 1999 [95] documented heterogenous expression of the alpha and beta hemolysins of *S. aureus* amongst strains that carried the hemolytic genes. This could be due to PV shutdown of these hemolysins in the nonexpressing strains. We recently identified non-hemolytic clinical isolates that could revert hemolytic activity without any change in their Agr phenotype [96], which could support the idea that the heterogenous hemolytic phenotypes observed by Aarestrup arise from PV.

With modern developments in genetic and experimental technology, the way we can go about investigating phenomena such as PV has drastically changed. This is no more clearly demonstrated by Jiang et al. who mined genome databases for inverted repeat regions as a primary screen for potentially phase variable genes [56]. In an attempt to gain similar preliminary insight, we screened the genome of the highly virulent communityacquired Methicillin-Resistant strain MW2 for genes that contained homonucleotide SSRs of adenine and thymine that are 6 nucleotides or longer within the ORF and the putative promoter region of the gene. We focused on homonucleotide tracts as three cases of PV in *S. aureus* have been demonstrated to occur through homonucleotide tract-length alteration (*mapW* and 2 discrete SSRs in *agrA*, [85,90]). Our initial results were astonishing. More than 700 genes contained at least one Poly(A) or Poly(T) SSR, with a substantial number containing 3 to 4 SSRs, and one gene containing a stunning 26 SSRs (Figure S1). These initial data corroborate an extensive study carried out by Orsi et al. who identified that Poly(A)/Poly(T) tracts are overrepresented in numerous bacterial genomes [54]. Interestingly, we noticed a greater abundance of Poly(A) tracts compared to Poly(T) in coding sequences. This is corroborated by Orsi et al., who found a similar result when looking at tracts longer than 6 nucleotides in length, though the difference reduced with shorter tracts. Surprisingly however, although Orsi et al. identified that these SSRs are predominantly located near the 5' end of the CDS (coding sequence), we only observed this distribution pattern for Poly(T) SSRs, not Poly(A) (Figure 3). Taken together, PV in *S. aureus* may be severely under-reported, which is understandable as most PV may not be noticeable through conventional bulk analysis and elaborate experimental systems may be needed to observe the PV-mediated switch in gene expression. Intriguingly, approximately 13% of genes (from those with known function) that contained Poly(A) or Poly(T) SSRs were essential genes. If these genes are indeed subject to PV, it raises a question as to what circumstances or trade-offs could possibly merit the shutdown of essential genes as advantageous. Transcriptional slippage at Poly(A)/Poly(T) tracts can lead to a population

of mRNAs with varying tract length, some of which may contain the correct number of nucleotides for the entire CDS to be in-frame [97]. This could enable low-level expression of essential genes that have been shut down by PV. Alternatively, the phenomenon of biological hysteresis [98], wherein functional proteins are inherited by daughter cells during splitting from the mother cell, could support daughter cells for a short period without further de novo synthesis, potentially giving chances of further changes to the SSR.

**Figure 3.** Location distribution of homonucleotide SSRs in *S. aureus*. A graph showing the location-dependent frequency of Poly(A)/(T) SSRs ≥6 nucleotides in length. A 100-nucleotide region upstream of all Open Reading Frames (ORFs) was also screened for SSRs. While Poly(T) SSRs are relatively enriched towards the 5' of the ORF as reported [54], Poly(A) SSRs ≥6 nucleotides in length were found to be evenly distributed across the ORF. In contrast, the abundance of both SSR types is evenly matched in the region immediately upstream of the ORF.

> The aim of this review is to stimulate interest in identifying PV in *S. aureus* and to increase attention to an area of study that warrants more investigation with modern technological approaches. Here, we have described a comprehensive understanding of the mechanisms of PV and the scope of the discoveries yet to be made in *S. aureus.* Further investigations focusing on PV in *S. aureus* are sure to lead to exciting new information, and the more we learn of the ingenious adaptation mechanisms this important pathogen employs during its infectious process, the better we will be equipped in dealing with it.

> **Supplementary Materials:** The following are available online at https://www.mdpi.com/2076-260 7/9/2/244/s1, Figure S1: Homonucleotide SSRs in S. aureus MW2.

> **Author Contributions:** Conceptualization, V.G. and K.M.; sequence analysis, R.L.O.; writing original draft preparation, V.G.; writing—review and editing, V.G. and K.M.; visualization, V.G., R.L.O. and K.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was partly supported by JSPS KAKENHI grant number 18H02652.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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