**5. Antibiotic resistance**

#### **5.1. Beta-lactam resistance**

#### *5.1.1. Penicillin resistance*

The first beta-lactam antibiotic penicillin G was discovered in 1928 by Alexander Fleming and the drug was used in human as chemotherapeutic agent in 1941 [59]. The antibiotic was potent against Gram positive pathogens [60] and a power weapon against Staphylococcal infections. However, first reports of *S. aureus* strains that were resistant to penicillin appeared after a year of its clinical use [30]. Such penicillin-resistant isolates carried a plasmid gene, *blaZ* which encoded a beta-lactamase enzyme, referred to as penicillinase [33, 34]. The enzyme is capable of cleaving the beta-lactam ring of penicillin resulting inactivation of the antibiotic [31, 32].

The emergence and spread of penicillinase-mediated resistance in *S. aureus* is referred to as first wave of resistance. This has spread in alarm proportions and became pandemic in the 1960s. About 80% of both community and hospital acquired *S. aureus* isolates were resistant to penicillin by late 1960s [33, 49]. By early 2000s, more than 90% of Staphylococcal isolates produced penicillinase enzyme irrespective of their community or hospital origin [34].

#### *5.1.2. Methicillin resistance*

As discussed earlier, the penicillinase resistance in *S. aureus* was countered by the discovery of methicillin, penicillinase-stable semisynthetic penicillin. The drug was introduced into clinics in 1961 and subsequently strains showing methicillin resistance (MRSA) was reported in the same year [35]. After the initial report, MRSA clones spread rapidly across the world but restricted to nosocomial settings. This is referred to as second wave of beta-lactam resistance in *S. aureus* [40]. As discussed earlier, methicillin resistance was mediated by the presence of *mecA* gene. The therapeutic outcome of MRSA infections was worse than methicillin sensitive *S. aureus* (MSSA) due to the underlying comorbid factors such as old age, immune suppression and, importantly, lack of effective antibiotics to treat MRSA, which were often multidrug resistant [34]. The rise in MRSA infections in hospitals resulted in high morbidity and mortality and increase in cost of health care [61, 62].

The third wave of beta-lactam resistance in *S. aureus* began with reports of MRSA infections in community in early 1990s. As discussed earlier, these strains were phenotypically and genetically distinct from MRSA isolates from hospitalized patients, resulting in definitions of HA-MRSA and CA-MRSA [51, 53]. In the last decade, community MRSA strains invaded the hospital settings and the difference between HA and CA MRSA is now blurred [58].

#### **5.2. Quinolones resistance**

be a CA-MRSA which marked the arrival of CA-MRSA into nosocomial settings [57]. Since then, hospital outbreaks of *S. aureus* strains which are phenotypically and genotypically CA-MRSA, have been reported many parts of the world [55]. Entry of CA-MRSA into hospitals blurred the differences between CA-MRSA and HA-MRSA. The increased reports of CA-MRSA outbreaks

**Common names for specific MRSA clones**

ST5 EMRSA-3 SCCmecI

ST5 HDE288/Pediatric clone SCCmecVI

ST239 Brazilian/Hungarian clone SCCmecIII

ST8 AUS-2 and Aus-3 SCCmecII

ST8 USA500 and EMRSA-2-6 SCCmecIV

CC22 ST22 EMRSA-15 International clone,

CC30 ST36 USA200 and EMRSA-16 Single most abundant

CC45 ST45 USA600 and Berlin SCCmecII

**Table 2.** The lineages of common HA-MRSA (based on Ref. [49]).

ST239 EMRSA-1 Eastern Australian

ST8 Irish-1 Common nosocomial

ST5 USA800/Pediatric clone Prevalent in Argentina,

ST247 Iberian clone and EMRSA-5 Descendant of COL-type

clone

CC8 ST250 Archiac First MRSA clone

**Comment**

SCCmecII

SCCmecIV

Most common US health care-associated MRSA,

Colombia, United States,

identified, COL strain as an example; SCCmecI

strains, SCCmecIII

SCCmecIII

States

epidemic clone of 1980s,

isolate in the 1990s in Europe and the United

prominent in Europe and Australia, SCCmecIV

cause of MRSA infections in UK; second most common cause of MRSA infections in US hopsitals in 2003, SCCmecII

in hospital suggest that CA-MRSA may eventually displace HA-MRSA in hospitals [58].

**Clonal complex Molecular sequence** 

**type**

CC5 ST5 USA100 and NewYork/Japan

Nalidixic acid, the prototype quinolone and the second generation quinolones (e.g. ciprofloxacin and norfloxacin) are predominately active towards Gram negative bacteria while third generation (e.g. levofloxacin) and fourth generation (e.g. moxifloxacin, gemifloxacin) quinolones exhibited improved and greater activity against Gram-positive bacteria [63–65]. Quinolones exert their antibacterial action by inhibiting bacterial topoisomerases (topoisomerase IV and DNA Gyrase), which are essential for relieving DNA super coiling and separation of concatenated DNA strands [66]. The resistance to quinolones in *S. aureus* arises in stepwise manner, due to point mutations primarily in GrlA subunit of topoisomerase IV and GyrA subunit of Gyrase. Additional mechanism by which *S. aureus* become resistant to quinolones is by expression of NorA efflux pumps [67].

The quinolone resistance in *S. aureus* is mostly associated with methicillin resistance though the mechanism of resistance and encoding genes are altogether different from each other. This could be due to higher usage of quinolones in hospital settings where the HA-MRSA prevalence is high resulting in selection of quinolone resistance [68–70]. In year 2008, the fluoroquinolone resistance among MRSA isolates implicated in acute bacterial skin and skin structure infections (ABSSSIs) in hospitals was at 70.3%. Due to such high level of quinolone resistance among MRSA in hospital settings, even third- and fourth-generation quinolones have not been considered for treatment of MRSA [71]. With respect to CA-MRSA, though they were susceptible to non-beta-lactam antibiotics including quinolones, the scenario has changed in recent years, with the rise in incidence of CA-MRSA infections which were multi-drug resistant [72].

#### **5.3. Vancomycin resistance**

Vancomycin, a glycopeptide antibiotic, was discovered from a microbial source (*Streptomyces orientalis*) in 1952. The drug was approved for clinical use in 1958; however, it was eclipsed by methicillin and other anti-staphylococcal penicillins which were considered less toxic than vancomycin and equally efficacious against penicillin-resistant Staphylococci [73]. Beginning early 1980s, there was sudden increase in vancomycin usage due to rise in HA-MRSA infections and emergence of pseudomembranous enterocolitis cause by *Clostridium difficile* in hospitalized patients [73–75]. Clinical efficacy of vancomycin efficacy in treatment of MRSA infections was well established over the period of time, thus the drug emerged as workhorse anti-MRSA drug [76].

#### *5.3.1. Vancomycin intermediate S. aureus*

The antibacterial activity of vancomycin is mediated by its binding to the C-terminal D-Ala-D-Ala residue of the peptidoglycan precursor, and formation of non-covalent complex, thereby, prevents the use of the precursor in bacterial cell wall synthesis [77, 78]. Three decades after its introduction into clinics, no clinical resistance to vancomycin was reported. The first report of a MRSA strain showing reduced susceptibility to vancomycin was reported in 1997. The vancomycin MIC against this strain (Mu50) was 8 mg/L, thus, designated as intermediate sensitive category. The strain had thickened cell wall when observed under electron microscopy and did not carry *vanA* or *vanB* genes as found in vancomycin-resistant enterococci (VRE) [79]. Subsequently, there were more reports of clinical infections due to MRSA strains with decreased vancomycin susceptibility similar to that of Mu50 strain. The *S. aureus* strains with a MIC range of 4–8 mg/L are referred to as vancomycin intermediate *S. aureus* (VISA). There were strains, which showed vancomycin MIC of 2 mg/L but had subpopulation with vancomycin MIC of 4–8 mg/L. These strains are referred to as hetero VISA (hVISA) [80, 81].

The genetic basis of emergence of VISA appears complex. The genetic analysis of VISA strains identified mutations in determinants that control the biosynthesis of bacterial cell wall and/ or mutations in the ribosomal gene rpoB [82]. The increased MRSA infection in hospitals has led to extensive use of vancomycin resulting in the selection of MRSA strains with reduced vancomycin susceptibility [83]. The study on prevalence of hVISA and VISA has met with the problem of accurate detection of decreased susceptibility to vancomycin. Different diagnostic methods showed variable sensitivity and specificity leading to contradictory reports in prevalence [80, 84–86]. During 2010–2014, the prevalence rates of hVISA and VISA among MRSA strain were at 7.01% and 7.93%, respectively [87]. The emergence and increased incidence of hVISA and VISA has limited the therapeutic use of vancomycin in the treatment of MRSA infections in hospital. However, by optimizing the dose regimen and drug delivery, thereby, achieving the desired blood plasma concentration which would give the clinical efficacy is the way forward in preserving the clinical utility of vancomycin [88, 89].

#### *5.3.2. Vancomycin-resistant S. aureus*

third generation (e.g. levofloxacin) and fourth generation (e.g. moxifloxacin, gemifloxacin) quinolones exhibited improved and greater activity against Gram-positive bacteria [63–65]. Quinolones exert their antibacterial action by inhibiting bacterial topoisomerases (topoisomerase IV and DNA Gyrase), which are essential for relieving DNA super coiling and separation of concatenated DNA strands [66]. The resistance to quinolones in *S. aureus* arises in stepwise manner, due to point mutations primarily in GrlA subunit of topoisomerase IV and GyrA subunit of Gyrase. Additional mechanism by which *S. aureus* become resistant to qui-

The quinolone resistance in *S. aureus* is mostly associated with methicillin resistance though the mechanism of resistance and encoding genes are altogether different from each other. This could be due to higher usage of quinolones in hospital settings where the HA-MRSA prevalence is high resulting in selection of quinolone resistance [68–70]. In year 2008, the fluoroquinolone resistance among MRSA isolates implicated in acute bacterial skin and skin structure infections (ABSSSIs) in hospitals was at 70.3%. Due to such high level of quinolone resistance among MRSA in hospital settings, even third- and fourth-generation quinolones have not been considered for treatment of MRSA [71]. With respect to CA-MRSA, though they were susceptible to non-beta-lactam antibiotics including quinolones, the scenario has changed in recent years, with the rise in incidence of CA-MRSA infections which were

Vancomycin, a glycopeptide antibiotic, was discovered from a microbial source (*Streptomyces orientalis*) in 1952. The drug was approved for clinical use in 1958; however, it was eclipsed by methicillin and other anti-staphylococcal penicillins which were considered less toxic than vancomycin and equally efficacious against penicillin-resistant Staphylococci [73]. Beginning early 1980s, there was sudden increase in vancomycin usage due to rise in HA-MRSA infections and emergence of pseudomembranous enterocolitis cause by *Clostridium difficile* in hospitalized patients [73–75]. Clinical efficacy of vancomycin efficacy in treatment of MRSA infections was well established

The antibacterial activity of vancomycin is mediated by its binding to the C-terminal D-Ala-D-Ala residue of the peptidoglycan precursor, and formation of non-covalent complex, thereby, prevents the use of the precursor in bacterial cell wall synthesis [77, 78]. Three decades after its introduction into clinics, no clinical resistance to vancomycin was reported. The first report of a MRSA strain showing reduced susceptibility to vancomycin was reported in 1997. The vancomycin MIC against this strain (Mu50) was 8 mg/L, thus, designated as intermediate sensitive category. The strain had thickened cell wall when observed under electron microscopy and did not carry *vanA* or *vanB* genes as found in vancomycin-resistant enterococci (VRE) [79]. Subsequently, there were more reports of clinical infections due to MRSA strains with decreased vancomycin susceptibility similar to that of Mu50 strain. The *S. aureus* strains with a MIC range of 4–8 mg/L are referred to as

over the period of time, thus the drug emerged as workhorse anti-MRSA drug [76].

nolones is by expression of NorA efflux pumps [67].

multi-drug resistant [72].

**5.3. Vancomycin resistance**

*5.3.1. Vancomycin intermediate S. aureus*

*S. aureus* strains which are referred to as hVISA and VISA are not considered resistant based on vancomycin susceptibility breakpoint (vancomycin MIC of 8 mg/L) defined by clinical laboratory standards institute (CLSI). Unlike VRE, these strains do not carry *vanA* or *vanB* type of genes to confer resistance to vancomycin. In 2002, first report of a *S. aureus* strain showing vancomycin MIC of >128 mg/L was published. The strain was methicillin resistant and carried *vanA* gene which was responsible for high-level resistance to vancomycin [90]. This report was followed by sporadic incidences of isolation of *S. aureus* strains with resistance to vancomycin [91]. All these strains showed high vancomycin MIC (>8 mg/L) and are referred to as vancomycin-resistant *S. aureus* (VRSA).

VRSA strains carried copies of the transposon Tn*1546*, which was acquired from vancomycin-resistant *Enterococcus faecalis*. The transposon which mediates the VanA-type resistance, encodes a dehydrogenase (VanH), which reduces pyruvate to D-Lac, and the VanA ligase, which catalyzes the formation of an ester bond between D-Ala and D-Lac. The resulting D-Ala-D-Lac depsipeptide replaces the D-Ala-D-Ala dipeptide in peptidoglycan synthesis, a substitution that decreases the affinity of the molecule for vancomycin and other glycopeptide antibiotic, teicoplanin, considerably [92, 93].

#### **5.4. Resistance to other antibiotics**

Since HA-MRSA strains are often MDR phenotype, drugs such as sulphonamides, tetracyclines, aminoglycosides, chloramphenicol and clindamycin were sidelined due to lack of activity, while vancomycin remained the mainstay of therapy. Resistance to sulphonamides and trimethoprim [94], tetracyclines [95–97], aminoglycosides [98–100], chloramphenicol [101] and clindamycin [102], occurring in *S. aureus* especially among MRSA was widely reported.
