Between 30 and 90% of the antibiotics given for human or veterinary use are excreted essentially unchanged (Sarmah et al., 2006). These compounds can be both persistent and mobile, and are often not removed during sewage treatment (Watkinson et al., 2007; Le-Minh et al., 2010; Zuccato et al., 2010). Antibiotics are also released in high concentrations from facilities where antibiotics are produced (Li et al., 2009, 2010), and are disseminated during application of manure to agricultural land (Chee-Sanford et al., 2009; Heuer et al., 2011). The consequences of pollution with antibiotics, particularly for aquatic systems, are being actively examined (Taylor et al., 2011; Lupo et al., 2012), and there is a call for development of policies to reduce the release of antibiotics and bacteria via human waste streams (Baquero et al., 2008).

Waste streams that release antibiotics into the environment also disseminate antibiotic resistance genes and mobile DNA elements, which should similarly be regarded as pollutants emanating from human activity (Pruden et al., 2006; Martinez, 2009a; Storteboom et al., 2010; Graham et al., 2011). The abundance of resistance genes in soils has been increasing since the introduction of antibiotics in the 1940s (Knapp et al., 2009), in parallel with the increasing concentrations of more conventional chemical pollutants.

Resistance genes and complex mobile elements are commonly reported from wastewater and sewage treatment plants (Tennstedt et al., 2003; Zhang et al., 2009; Pellegrini et al., 2011). Wastewater is regarded as a hotspot for interactions between mobile elements and for their lateral transfer between pathogens, commensals, and

environmental bacteria (Schlüter et al., 2008; Moura et al., 2010). Resistome and mobilome elements are not necessarily removed by water treatment (Graham et al., 2011; Drudge et al., 2012), allowing resistance genes to be used as markers of human influence on aquatic ecosystems (Pei et al., 2006; Pruden et al., 2006; Storteboom et al., 2010).

Domestic and agricultural animals are a source of significant quantities of antibiotics and resistance genes.  The long term fate of antibiotics and resistance genes is difficult to predict without quantitative measurements over appropriate timescales (Chee-Sanford et al., 2009). However, it is clear that antibiotic resistant bacteria will increase in abundance, lateral transfers to soil organisms will occur, and resistance genes will be sequestered by diverse elements of the mobilome (Heuer et al., 2011).

## **CONCLUSION**

Human use of antibiotics for medicine and agriculture may have consequences beyond their intended applications. Large quantities of antibiotics now emanate from human waste streams, as do the xenogenetic elements fixed in human ecosystems by antibiotic selection. Much more attention needs to be paid to the origins and fates of such pollutants (Allen et al., 2010). The antibiotic revolution may be having effects across the entire microbial biosphere (Martinez, 2009b), changing the basal rate of bacterial evolution, altering the composition of the resistome and mobilome, and promoting lateral transfer of mobile genetic elements (Couce and Blázquez, 2009; Gillings and Stokes, 2012). Antibiotic contamination promotes the fixation and mobilization of resistance genes between environmental and clinical microbiota (Kristiansson et al., 2011), and resistance genes are now widely spread through the biosphere (Martinez, 2009a; Stokes and Gillings, 2011).

Above all, we need to address the antibiotic resistance problem from a broader evolutionary and ecological perspective (Aminov and Mackie, 2007; Baquero et al., 2009; Fajardo et al., 2009). The ability of natural selection to shape species and communities is the same for microorganisms as it is for larger species (Gillings and Stokes, 2012), and the ecological theory of community assembly developed for multicellular organisms can be applied to the microbiome (Costello et al., 2012). The risk associated with the environmental spread of resistance genes with known adverse consequences for human welfare has had little attention, nor has the potential for pollution with antibiotics to widely affect the global microbiome. In comparison, the potential escape of resistance gene markers used in the generation of genetically modified plants has been the subject of considerable research (Martinez, 2012). It is time to pay more attention to the bioactive molecules that humans release into the environment.

#### **ACKNOWLEDGMENTS**

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Aspects of the author's work are supported by the Australian Research Council, the National Health and Medical Research Council of Australia and Macquarie University.

## **REFERENCES**

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Stokes, H. W. (2009b). Mobilization of a Tn402-like class 1 integron with a novel cassette array via flanking miniature inverted-repeat transposable element-like structures. *Appl. Environ. Microbiol.* 75, 6002–6004.

the 'pre-antibiotic' era. *Nature* 302, 725–726.

a multidrug-resistant phenotype. *Int. J. Antimicrob. Agents* 37, 156–161.

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the evolution of bacterial cooperation and virulence. *Curr. Biol.* 19, 1683–1691.

*toyocaensis* NRRL15009. *Proc. Natl. Acad. Sci. U.S.A.* 99, 8962–8967.

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ribosome. *Chem. Biol.* 11, 1307– 1316.

*Natl. Acad. Sci. U.S.A.* 95, 6578– 6583.

mechanisms: drug efflux across two membranes. *Mol. Microbiol.* 37, 219–225.

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 25 October 2012; paper pending published: 11 December 2012; accepted: 03 January 2013; published online: 22 January 2013.*

*Citation: Gillings MR (2013) Evolutionary consequences of antibiotic use for the resistome, mobilome, and microbial pangenome. Front. Microbio. 4:4. doi: 10.3389/fmicb.2013.00004*

*This article was submitted to Frontiers in Antimicrobials, Resistance and Chemotherapy, a specialty of Frontiers in Microbiology.*

*Copyright © 2013 Gillings. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any thirdparty graphics etc.*

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**REVIEW ARTICLE** published: 14 March 2013 doi: 10.3389/fmicb.2013.00048

## "Stormy waters ahead": global emergence of carbapenemases

## *Gopi Patel1 and Robert A. Bonomo2,3,4,5,6\**

1 Department of Medicine, Mount Sinai School of Medicine, New York, NY, USA

2 Research Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, OH, USA

3 Division of Infectious Diseases and HIV Medicine, University Hospitals Case Medical Center, Cleveland, OH, USA

4 Department of Medicine, Case Western Reserve School of Medicine, Cleveland, OH, USA

5 Department of Molecular Biology and Microbiology, Case Western Reserve School of Medicine, Cleveland, OH, USA

6 Department of Pharmacology, Case Western Reserve School of Medicine, Cleveland, OH, USA

#### *Edited by:*

Fiona Walsh, Agroscope Changins-Wädenswil, Switzerland

*Reviewed by:*

Charles W. Knapp, University of Strathclyde, UK Yoshikazu Ishii, Toho University School of Medicine, Japan

#### *\*Correspondence:*

Robert A. Bonomo, Research Service, Louis Stokes Cleveland Department of Veteran Affairs Medical Center, 10701 East Boulevard, Cleveland, OH 44106, USA.

e-mail<EMAIL_ADDRESS>
Carbapenems, once considered the last line of defense against of serious infections with Enterobacteriaceae, are threatened with extinction. The increasing isolation of carbapenem-resistant Gram-negative pathogens is forcing practitioners to rely on uncertain alternatives. As little as 5 years ago, reports of carbapenem resistance in Enterobacteriaceae, common causes of both community and healthcare-associated infections, were sporadic and primarily limited to case reports, tertiary care centers, intensive care units, and outbreak settings. Carbapenem resistance mediated by β-lactamases, or carbapenemases, has become widespread and with the paucity of reliable antimicrobials available or in development, international focus has shifted to early detection and infection control. However, as reports of Klebsiella pneumoniae carbapenemases, New Delhi metallo-βlactamase-1, and more recently OXA-48 (oxacillinase-48) become more common and with the conveniences of travel, the assumption that infections with highly resistant Gramnegative pathogens are limited to the infirmed and the heavily antibiotic and healthcare exposed are quickly being dispelled. Herein, we provide a status report describing the increasing challenges clinicians are facing and forecast the "stormy waters" ahead.

**Keywords: carbapenemases, NDM-1, KPC, OXA-48, metallo-β-lactamases, CHDL**

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Carbapenems are potent and broad-spectrum β-lactam antibiotics traditionally reserved for the treatment of the most serious infections (El-Gamal and Oh, 2010). The emergence and dissemination of carbapenem-resistant Gram-negative pathogens including *Pseudomonas aeruginosa*, *Acinetobacter baumannii*, and Enterobacteriaceae is a significant contributor to patient morbidity and mortality (Patel et al., 2008; Schwaber et al., 2008; Lautenbach et al., 2009, 2010; Marchaim et al., 2011). Despite radical efforts in infection control (Schwaber et al., 2011) and improvements in rapid molecular diagnostics (Centers for Disease Control and Prevention, 2009; Nordmann et al., 2012c), carbapenem-resistant Gram-negative bacilli remain a formidable threat as few antimicrobial agents are reliably active and very little is expected to be available in the near future.

Clinicians hold that the increasing prevalence of extendedspectrum β-lactamases (ESBLs) among *Klebsiella pneumoniae* and *Escherichia coli* in the 1980s and 1990s contributed to the increased consumption of carbapenems. Experience implied that delayed administration of carbapenems in at-risk patients led to poor clinical outcomes (Paterson and Bonomo, 2005; Endimiani and Paterson, 2007). Thus, carbapenems (i.e., imipenem, meropenem, ertapenem, and doripenem) became vital tools in the treatment of healthcare-associated and severe community-acquired infections. Despite heavy reliance on these agents, carbapenem resistance in Enterobacteriaceae, common causes of both community and healthcare-associated infections, remained rare until the past decade.

Carbapenem resistance among Gram-negative bacteria results from one or more of the following mechanisms: (i) hyperproduction or derepression of Ambler class C β-lactamases (AmpC β-lactamases) or ESBLs (e.g., sulfhydryl variable (SHV), temoneira (TEM), cefotaxime (CTX-M) type β-lactamases) with loss or alteration in outer membrane porins; (ii) augmented drug efflux; (iii) alterations in penicillin binding proteins (PBPs); (iv) carbapenemase production (Patel and Bonomo, 2011). Carbapenemases belong to three molecular classes of β-lactamases, Ambler class A, B, and D (Ambler, 1980; Bush and Jacoby, 2010). Our aim is to provide a status report of the molecular diversity and epidemiology of carbapenemases as well as current and future therapeutics. The increasing public safety concerns associated with organisms harboring these enzymes has created significant turmoil. Regrettably, the situation is critical and our patients are in peril.

### **AMBLER CLASS A CARBAPENEMASES**

Few Ambler class A β-lactamases demonstrate carbapenemhydrolyzing activity and, up until a decade ago, these were rarely recovered. Class A carbapenemases include: *K. pneumoniae* carbapenemase (KPC), Guiana extended-spectrum (GES), nonmetallo-carbapenemase-A (Nmc-A)/imipenem-resistant (IMI), *Serratia marcescens* enzyme (SME), serratia fonticola carbapenemase (SFC), and BIC β-lactamases (**Table 1**; Walther-Rasmussen and Høiby, 2007). With the notable exception of KPCs, the clinical isolation of these types of carbapenemases is relatively limited.

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#### **Table 1 | Class A carbapenemases\*.**

\* Adapted from Walther-Rasmussen and Høiby (2007).

†Environmental isolates.

‡KPC-1 was later found to be the same enzyme as KPC-2 (Higgins et al., 2012a).

§Chromosomal expression of blaKPC−2 has been described in P. aeruginosa (Villegas et al., 2007).

Non-metallo-carbapenemase-A is a chromosomal carbapenemase originally isolated from *Enterobacter cloacae* in France (Nordmann et al., 1993). Currently, reports of this particular β-lactamase are still rare (Pottumarthy et al., 2003; Castanheira et al., 2008; Osterblad et al., 2012). IMI-1 was initially recovered from the chromosome of an *Enterobacter cloacae* isolate in the southwestern USA (Rasmussen et al., 1996). A variant of IMI-1, IMI-2, has been identified on plasmids isolated from environmental strains of *Enterobacter asburiae*in USA rivers (Aubron et al., 2005).

SME-1 (*S. marcescens* enzyme) was originally identified in an isolate of *S. marcescens* from a patient in London in 1982 (Yang et al., 1990). SME-2 and SME-3 were subsequently isolated in the USA, Canada, and Switzerland (Naas et al., 1994; Queenan et al., 2000, 2006; Deshpande et al., 2006b; Poirel et al., 2007; Carrer et al., 2008). Chromosomally encoded SME-type carbapenemases continue to be isolated at a low frequency in North America (Deshpande et al., 2006a,b; Fairfax et al., 2011; Mataseje et al., 2012). Both SFC-1 and BIC-1 are chromosomal serine carbapenemases recovered from environmental isolates. The former from a *S. fonticola* isolate in Portugal (Henriques et al., 2004) and the latter from *Pseudomonas fluorescens* isolates recovered from the Seine River (Girlich et al., 2010).

The GES-type β-lactamases are acquired β-lactamases recovered from *P. aeruginosa*, Enterobacteriaceae, and *A. baumannii* (Poirel et al., 2000a; Castanheira et al., 2004a). The genes encoding these β-lactamase have often, but not exclusively, been identified within class 1 integrons residing on transferable plasmids (Bonnin et al., 2013; Walther-Rasmussen and Høiby, 2007). GES-1 has a similar hydrolysis profile to other ESBLs, although they essentially spare monobactams. Several GES β-lactamases are described with six (i.e., GES-2, GES-4, GES-5, GES-6, GES-11, and GES-14), demonstrating detectable carbapenemase activity in the setting of amino acid substitutions at their active sites (specifically at residue 104 and 170; Walther-Rasmussen and Høiby, 2007; Kotsakis et al., 2010). These GES-type carbapenemases have been described in Europe, South Africa, Asia, and the Middle East (Poirel et al., 2002; Jeong et al., 2005; da Fonseca et al., 2007; Moubareck et al., 2009; Bonnin et al., 2011, 2013).

Currently, most carbapenem resistance among Enterobacteriaceae in the USA and Israel is attributed to plasmid-mediated expression of a KPC-type carbapenemase (Endimiani et al., 2009b; Nordmann et al., 2009; Gupta et al., 2011; Schwaber et al., 2011). KPC-producing Enterobacteriaceae are considered endemic to Greece along with other carbapenemases, specifically VIM-type [Verona integron-encoded metallo-β-lactamases (MBLs); Canton et al., 2012]. KPCs efficiently hydrolyze carbapenems as well as penicillins, cephalosporins, and aztreonam and are not overcome *in vitro* by clinically available β-lactamase inhibitors (i.e., clavulanic acid, sulbactam, tazobactam – in fact these are hydrolyzed).

Carbapenem resistance secondary to KPC production was first described in a *K. pneumoniae* recovered in North Carolina in 1996 (Yigit et al., 2001). To date 12 KPC subtypes (KPC-2 to KPC-13; Robledo et al., 2008; Kitchel et al., 2009a; Navon-Venezia et al., 2009; Wolter et al., 2009; Gregory et al., 2010) have been reported with the vast majority of analyzed isolates expressing either KPC-2 or KPC-3.

The *bla*KPC gene has been mapped to a highly conserved Tn*3*-based transposon, Tn*4401* (**Figure 1A**), and five isoforms of Tn*4401* are described (Naas et al., 2008; Cuzon et al., 2010; Kitchel et al., 2010). Plasmids carrying *bla*KPC are of various sizes and many carry additional genes conferring resistance to fluoroquinolones and aminoglycosides thus limiting the antibiotics available to treat infections with KPC-producing pathogens (Endimiani et al., 2008; Rice et al., 2008). *bla*KPC has rarely been mapped to a chromosomal location (Villegas et al., 2007; Castanheira et al., 2009).

A predominant strain of *K. pneumoniae* appears responsible for outbreaks and the international spread of KPC-producing*K. pneumoniae* (Woodford et al., 2008; Kitchel et al., 2009a; Samuelsen et al., 2009). Congruent pulsed-field gel electrophoresis (PFGE) patterns also suggest a clonal relationship between outbreakassociated strains of KPC-producing *K. pneumoniae* recovered from different areas that are endemic (Navon-Venezia et al., 2009; Woodford et al., 2011). The Centers for Disease Control and Prevention (CDC) performed PFGE and multilocus sequence typing (MLST) on isolates submitted to their reference laboratory from 1996 to 2008. A dominant PFGE pattern was observed and noted to be of a specific MLST type, ST 258 (Kitchel et al., 2009a). A second sequence type, ST 14, was common in institutions in the Midwest (Kitchel et al., 2009b). These findings implied that certain strains of *K. pneumoniae* may be more apt to obtain and retain the *bla*KPC gene. Another study, however, analyzing 16 KPC-2 producing *K. pneumoniae* isolates from different geographic regions demonstrated diverse PFGE patterns and MLST types. This included four

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#### **FIGURE 1 | Basic genetic construct of select carbapenemase genes. (A)** Schematic representation of Tn4401 type of transposon associated with blaKPC which includes a transposase gene (tnpA), a resolvase gene (tnpR), as well as insertion sequences, ISKpn6 and ISKpn7 (Cuzon et al., 2010). **(B)** The blaNDM−1 construct demonstrates ISAba125 insertion sequence(s) upstream

of the blaNDM−1 and a novel bleomycin resistance gene, bleMBL, downstream (Dortet et al., 2012). **(C)** blaOXA−48 is often mapped to a Tn1999 composite transposon where it is bracketed between two copies of the same insertion sequence, IS1999. Downstream of blaOXA−48 lies a lysR gene which encodes for a regulatory protein (Poirel et al., 2012b).

different MLST types in Colombia (ST 14, ST 337, ST 338, and ST 339) and two in Israel (ST 227 and ST 340). Although this study analyzed a smaller number of isolates, these findings suggest that the global propagation of KPC-2 is more complicated than the successful expansion of a fixed number of clones (Cuzon et al., 2010; Qi et al., 2011). More recently, a study evaluating the MLST types associated with widespread KPC-2 production in *K. pneumoniae* in Greece suggested that although ST 258 predominates at least 10 additional sequence types were found to carry *bla*KPC−2. Of note three (i.e., ST 147, ST 323, and ST 383) carried both KPC-2 as well as genes encoding VIM-type MBLs (Giakkoupi et al., 2011; Woodford et al., 2011). A retrospective study in Cleveland documented the presence of ST 36 in a long-term care facility for children (Viau et al., 2012).

*Klebsiella pneumoniae* carbapenemases-production can confer variable levels of carbapenem resistance with reported minimum inhibitory concentrations (MICs) ranging from susceptible to ≥16 μg/mL. Analysis of isolates displaying high-level carbapenem resistance demonstrated that increased phenotypic resistance may be due to increased *bla*KPC gene copy number or the loss of an outer membrane porin, OmpK35 and/or OmpK36. The highest level of imipenem resistance was seen with isolates lacking both porins and with augmented KPC enzyme production (Kitchel et al., 2010).

## **AMBLER CLASS B CARBAPENEMASES: METALLO-β-LACTAMASES**

Class B β-lactamases (**Table 2**) are referred to as MBLs and require a metal ion, usually zinc, for β-lactam hydrolysis (Walsh et al., 2005). Due to the dependence on Zn2+, catalysis is inhibited in the presence of metal-chelating agents like ethylenediaminetetraacetic acid (EDTA). MBL expression in Gram-negative bacteria confers

#### **Table 2 | Metallo-β-lactamases.**

resistance to penicillins, cephalosporins, and carbapenems. MBLs are not inhibited by the presence of commercially available β-lactamase inhibitors and susceptibility to monobactams (e.g., aztreonam) appears to be preserved in the absence of concomitant expression of other resistance mechanisms (e.g., ESBL production). The more geographically widespread MBLs include IMP (imipenem-resistant), VIM, and New Delhi metallo-β-lacta mase (NDM).

Chromosomal MBLs were the first to be identified and are the cause of carbapenem resistance observed in *Bacillus cereus*, *Aeromonas* spp., and *Stenotrophomonas maltophilia* (Walsh et al., 2005). However, of growing concern are the "mobile" MBLs that have been reported since the mid-1990s. Although most frequently found in carbapenem-resistant isolates of *P. aeruginosa* and occasionally *Acinetobacter* spp., there is growing isolation of these enzymes in Enterobacteriaceae.

Prior to the description of NDM-1, frequently detected MBLs include IMP-type and VIM-type with VIM-2 being the most prevalent. These MBLs are embedded within a variety of genetic structures, most commonly integrons. When these integrons are associated with transposons or plasmids they can readily be transferred between species.

In 1991, IMP-1, a plasmid-mediated MBL, was identified in an isolates of *S. marcescens* from Japan (Ito et al., 1995). Since then plasmid-mediated carbapenem resistance secondary to IMP-1 spread widely in Japan, Europe, Brazil, and other parts of Asia and in several species of Gram-negative bacilli including *Acinetobacter* spp. and Enterobacteriaceae. At the present time, 42 variants of IMP have been identified with most cases of IMP-mediated carbapenem resistance being reported from Asia and among *P. aeruginosa* (Bush and Jacoby, 2010).

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\*Single report of SPM-1 in Europe linked to healthcare exposure in Brazil (Salabi et al., 2010).

A more commonly recovered MBL is the VIM-type enzyme. VIM-1 was first described in Italy in 1997 in *P. aeruginosa* (Lauretti et al., 1999). VIM-2 was next discovered in southern France in *P. aeruginosa* cultured from a neutropenic patient in 1996 (Poirel et al., 2000b). Although originally thought to be limited to non-fermenting Gram-negative bacilli, VIM-type MBLs are being increasingly identified in Enterobacteriaceae as well (Giakkoupi et al., 2003; Kassis-Chikhani et al., 2006; Morfin-Otero et al., 2009; Canton et al., 2012). To date, 37 variants of VIM have been described with VIM-2 being the most common MBL recovered worldwide.

Other more geographically restricted MBLs include SPM-1 (Sao Paulo MBL), which has been associated with hospital outbreaks in Brazil (Toleman et al., 2002; Rossi, 2011); GIM-1 (German imipenemase) isolated in carbapenem-resistant *P. aeruginosa* isolates in Germany (Castanheira et al., 2004b); SIM-1 (Seoul imipenemase) isolated from *A. baumannii* isolates in Korea (Lee et al., 2005); KHM-1 (Kyorin Health Science MBL) isolated from a *C. freundii* isolate in Japan (Sekiguchi et al., 2008); AIM-1 (Australian imipenemase) isolated from *P. aeruginosa* in Australia (Yong et al., 2007); DIM-1 (Dutch imipenemase) isolated from a clinical *P. stutzeri* isolate in the Netherlands (Poirel et al., 2010c); SMB-1 (*S. marcescens* MBL) in *S. marcescens* in Japan (Wachino et al., 2011); TMB-1 (Tripoli MBL) in *Achromobacter xylosoxidans* in Libya (El Salabi et al., 2012), and FIM-1 (Florence imipenemase) from a clinical isolate of *P. aeruginosa* in Italy (Pollini et al., 2012). With the notable exception of SPM-1, these MBLs have remained confined to their countries of origin (Salabi et al., 2010).

NDM-1 was first identified in 2008. Due to its rapid international dissemination and its ability to be expressed by numerous Gram-negative pathogens, NDM is poised to become the most commonly isolated and distributed carbapenemase worldwide. Initial reports frequently demonstrated an epidemiologic link to the Indian subcontinent where these MBLs are endemic (Kumarasamy et al., 2010). Indeed, retrospective analyses of stored isolates suggest that NDM-1 may have been circulating in the subcontinent as early as 2006 (Castanheira et al., 2011). Despite initial controversy, the Balkans may be another area of endemicity for NDM-1 (Struelens et al., 2010; Jovcic et al., 2011; Livermore et al., 2011c; Halaby et al., 2012). Sporadic recovery of NDM-1 in the Middle East suggests that this region may be an additional reservoir (Poirel et al., 2010a, 2011d; Nordmann et al., 2011; Ghazawi et al., 2012).

Like KPCs, the conveniences of international travel and medical tourism have quickly propelled this relatively novel MBL into a formidable public health threat. Gram-negative bacilli harboring *bla*NDM have been identified worldwide with the exception of Central and South America.

NDM-1 was first identified in Sweden in a patient of Indian descent previously hospitalized in India (Yong et al., 2009). The patient was colonized with a *K. pneumoniae* and an *E. coli* carrying *bla*NDM−1 on transferable plasmids. In the UK, an increase in the number of clinical isolates of carbapenem-resistant Enterobacteriaceae was seen in both 2008 and 2009. A UK reference laboratory reported that at least 17 of 29 patients found to be harboring NDM-1 expressing Enterobacteriaceae had a

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history of recent travel to the Indian subcontinent with the majority having been hospitalized in those countries (Kumarasamy et al., 2010).

European reports suggest that horizontal transfer of *bla*NDM−1 exists within hospitals outside endemic areas. Of overwhelming concern are the reported cases without specific contact with the healthcare system locally or in endemic areas suggesting autochthonous acquisition (Kumarasamy et al., 2010; Kus et al., 2011; Arpin et al., 2012; Borgia et al., 2012; Nordmann et al., 2012b).

Surveillance of public water supplies in India indicates that exposure to NDM-1 may be environmental. Walsh et al. (2011) analyzed samples of public tap water and seepage water from sites around New Delhi. The results were disheartening in that *bla*NDM−1 was detected by PCR in 4% of drinking water samples and 30% of seepage samples. In this survey, carriage of *bla*NDM−1 was noted in 11 species of bacteria not previously described, including virulent ones like *Shigella boydii* and *Vibrio cholerae*.

The rapid spread of NDM-1 highlights the fluidity and rapidity of gene transfer between bacterial species. Although *bla*NDM−1 was initially and repeatedly mapped to plasmids isolated from carbapenem-resistant *E. coli* and *K. pneumoniae*, reports of both plasmid and chromosomal expression of *bla*NDM−1 has been noted in other species of Enterobacteriaceae as well as *Acinetobacter* spp. and *P. aeruginosa* (Moubareck et al., 2009; Bogaerts et al., 2010; Bonnin et al., 2011; Nordmann et al., 2011; Patel and Bonomo, 2011). Recently, bacteremia with a NDM-1 expressing *V. cholerae* has been described in a patient previously hospitalized in India colonized with a variety of Enterobacteriaceae previously known to be capable of carrying plasmids with *bla*NDM−1 (Darley et al., 2012).

In contrast to KPCs, the presence of a dominant clone among *bla*NDM−1 carrying isolates remains elusive (Poirel et al., 2011c). NDM-1 expression in *E. coli* has been noted among sequence types previously associated with the successful dissemination of other β-lactamases including ST 101 and ST 131 (Mushtaq et al., 2011). Mushtaq et al. (2011) analyzed a relatively large group of *bla*NDM−1 expressing *E. coli* from the UK, Pakistan, and India in order to potentially identify a predominant strain responsible for the rapid and successful spread of NDM-1. The most frequent sequence type identified was ST 101. Another study examining a collection of carbapenem-resistant Enterobacteriaceae from India demonstrates the diversity of strains capable of harboring *bla*NDM−1. Carriage of *bla*NDM−1 was confirmed in 10 different sequence types of *K. pneumoniae* and 5 sequence types of *E. coli* (Lascols et al., 2011). This multiplicity was confirmed in a study looking at a collection of *bla*NDM−1 expressing Enterobacteriaceae from around the world (Poirel et al., 2011c). Of most concern is that NDM-1 has been identified in *E. coli* ST 131, the strain of *E. coli* credited with the global propagation of CTX-M-15 ESBLs (Mushtaq et al., 2011; Peirano et al., 2011; Pfeifer et al., 2011b; Woodford et al., 2011). Similar to KPCs, NDM-1 expression portends variable levels of carbapenem resistance and there is often concomitant carriage of a myriad of resistance determinants including other βlactamases and carbapenemases as well as genes associated with resistance to fluoroquinolones and aminoglycosides (Nordmann et al., 2011).

NDM-1 shares the most homology with VIM-1 and VIM-2. It is a 28-kDa monomeric protein that demonstrates tight binding to both penicillins and cephalosporins (Zhang and Hao, 2011). Binding to carbapenems does not appear to be as strong as other MBLs, but hydrolysis rates appear to be similar. Using ampicillin as a substrate, allowed for detailed characterization of the interactions between NDM's active site and β-lactams as well as improved evaluation of MBLs unique mechanism of β-lactam hydrolysis. More recent crystal structures of NDM-1 reveal the molecular details of how carbapenem antibiotics are recognized by dizinc-containing MBLs (King et al., 2012).

To date, NDM-1 remains the most common NDM variant isolated. Seven variants (NDM-1 to NDM-7) exist (Kaase et al., 2011; Nordmann et al., 2012a). It is currently held that *bla*NDM−1 is a chimeric gene that may have evolved from *A. baumannii* (Toleman et al., 2012). Contributing to this theory is the presence of complete or variations of the insertion sequence, IS*Aba125*, upstream to the *bla*NDM−1 gene in both Enterobacteriaceae and *A. baumannii* (Pfeifer et al., 2011a; Poirel et al., 2011a; Dortet et al., 2012; Toleman et al., 2012). This insertion sequence has primarily been found in *A. baumannii*.

A recent evaluation of the genetic construct associated with *bla*NDM−1 (**Figure 1B**) has lead to the discovery of a new bleomycin resistance protein, BRPMBL. Evaluation of 23 isolates of *bla*NDM−1/2 harboring Enterobacteriaceae and *A. baumannii* noted that the overwhelming majority of them possessed a novel bleomycin resistance gene, *ble*MBL (Dortet et al., 2012). Co-expression of *bla*NDM−1 and *ble*MBL appear to be mediated by a common promoter (*P*NDM−1) which includes portions of IS*Aba125*. It is postulated that BRPMBL expression may contribute some sort of selective advantage allowing NDM-1 to persist in the environment.

A contemporary evaluation of recently recovered NDM-1 producing *A. baumannii* isolates from Europe demonstrates that *bla*NDM−1 and *bla*NDM−2 genes are situated on the same chromosomally located transposon, Tn*125* (Bonnin et al., 2012). Dissemination of *bla*NDM in *A. baumannii* seems be due to different strains carrying Tn*125* or derivatives of Tn*125* rather than plasmid-mediated or clonal (Bonnin et al., 2013; Poirel et al., 2012a).

## **CARBAPENEM-HYDROLYZING CLASS D β-LACTAMASES**

Oxacillinases comprise a heterogeneous group of class D βlactamases which are able to hydrolyze amino- and carboxypenicillins (Poirel et al., 2010b). The majority of class D β-lactamases are not inhibited by commercially available β-lactamase inhibitors but are inhibited *in vitro* by NaCl. Over 250 types of oxacillinases are reported with a minority demonstrating low levels of carbapenem-hydrolyzing activity. This select group of enzymes is also referred to as the carbapenem-hydrolyzing class D βlactamases (CHDLs; **Table 3**). CHDLs have been identified most frequently in *Acinetobacter* spp., however, there has been increasing isolation among Enterobacteriaceae, specifically OXA-48 (oxacillinase-48; Lascols et al., 2012; Mathers et al., 2012).

With the exception of OXA-163 (Poirel et al., 2011b), CHDLs efficiently inactivate penicillins, first generations cephalosporins, and β-lactam/β-lactamase inhibitor combinations, but spare extended-spectrum cephalosporins. Carbapenem hydrolysis efficiency is lower than that of other carbapenemases, including the MBLs, and often additional resistance mechanisms are expressed in organisms demonstrating higher levels of phenotypic carbapenem resistance. These include expression of other carbapenemases, alterations in outer membrane proteins (e.g., CarO, OmpK36; Perez et al., 2007; Gülmez et al., 2008; Pfeifer et al., 2012), increased transcription mediated by *IS* elements functioning as promoters, increased gene copy number, and amplified drug efflux (Poirel and Nordmann, 2006; Perez et al., 2007). Many subgroups of CHDLs have been described. We will focus on those found in*A. baumannii* and Enterobacteriaceae: OXA-23 and OXA-27; OXA-24/40, OXA-25, and OXA-26; OXA-48 variants; OXA-51, OXA-66, OXA-69; OXA-58, and OXA-143.

CHDLs can be intrinsic or acquired. *A. baumannii* does have naturally occurring but variably expressed chromosomal CHDLs, OXA-51, OXA-66, and OXA-69 (Brown et al., 2005; Héritier et al., 2005b). For the most part, in isolation the phenotypic carbapenem resistance associated with these oxacillinases is low. However, levels of carbapenem resistance appear to be increased in the presence of specific insertion sequences promoting gene expression (Figueiredo et al., 2009; Culebras et al., 2010). Additional resistance to extended-spectrum cephalosporins can be seen in the setting of co-expression of ESBLs and/or other carbapenemases (Castanheira et al., 2011; Mathers et al., 2012; Pfeifer et al., 2012; Voulgari et al., 2012; Potron et al., 2013).

The first reported "acquired" oxacillinase with appreciable carbapenem-hydrolyzing activity was OXA-23. OXA-23, or ARI-1, was identified from an *A. baumannii* isolate in Scotland in 1993 (the isolate was first recovered in 1985; Paton et al., 1993). Subsequently, OXA-23 expression has been reported worldwide (Mugnier et al., 2010) and both plasmid and chromosomal carriage of *bla*OXA−23 are described. The OXA-23 group includes OXA-27, found in a single *A. baumannii* isolate from Singapore (Afzal-Shah et al., 2001). With the exception of an isolate of *Proteus mirabilis* identified in France in 2002, this group of β-lactamases has been exclusively recovered from *Acinetobacter* species (Bonnet et al., 2002). Increased expression of OXA-23 has been associated with the presence of upstream insertion sequences (e.g., IS*Aba1* and IS*Aba4*) acting as strong promoters (Corvec et al., 2007).

Another group of CHDLs include OXA-24/40, OXA-25, and OXA-26 (Bou et al., 2000b; Afzal-Shah et al., 2001). OXA-24 and OXA-40 differ by a few amino acid substitutions and OXA-25 and OXA-26 are point mutation derivatives of OXA-40 (Afzal-Shah et al., 2001). Although primarily linked with clonal outbreaks in Spain and Portugal (Bou et al., 2000a; Lopez-Otsoa et al., 2002; Da Silva et al., 2004; Acosta et al., 2011), OXA-24/40 β-lactamases has been isolated in other European countries and the USA (Lolans et al., 2006). OXA-40 was in fact the first CHDL documented in the USA (Lolans et al., 2006).

OXA-58 has also only been detected in *Acinetobacter* spp. initially identified in France (Héritier et al., 2005a; Poirel et al., 2005), OXA-58 has been associated with institutional outbreaks and has been recovered from clinical isolates of *A. baumannii* worldwide (Coelho et al., 2006; Mendes et al., 2009; Gales et al., 2012).

As civilian and military personnel began returning from Afghanistan and the Middle East, practitioners noted increasing

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#### **Table 3 | Carbapenem-hydrolyzing class D β-lactamases.**

\*Single isolate described in France.

recovery of *A. baumannii* from skin and soft tissue infections. Drug resistance was associated with expression of both OXA-23 and OXA-58 (Hujer et al., 2006; Scott et al., 2007; Perez et al., 2010b). Many isolates carrying the *bla*OXA−58 gene concurrently carry insertion sequences (e.g., IS*aba1*, IS*Aba2*, or IS*Aba3*) associated with increased carbapenemase production and thus higher levels of carbapenem resistance. In one report increased gene copy number was also associated with a higher level of enzyme production and increased phenotypic carbapenem resistance (Bertini et al., 2007).

Spread of OXA-type carbapenemases among *A. baumannii* appears to be clonal and in depth reviews of the molecular epidemiology and successful dissemination of these clones have been published (Woodford et al., 2011; Zarrilli et al., 2013). Two MLST schemes with three loci in common exist for *A. baumannii* – the PubMLST scheme (Bartual et al., 2005) and the Pasteur scheme (Diancourt et al., 2010). Both schemes assign different sequence types into clonal complexes (CC). Sequence types and CC from both schemes can be further categorized into the international (European) clones I, II, and III. It should be noted, however, that the molecular taxonomy of *A. baumannii* continues to evolve (Higgins et al., 2012a). OXA-23 producing *A. baumannii* predominantly belong to international clones I and II with a notable proportion being part of CC92 (PubMLST; Mugnier et al., 2010; Adams-Haduch et al., 2011). Similarly, *A. baumannii* isolates associated with epidemic spread of OXA-24/40 in Portugal and Spain appear are incorporated in international clone II (Da Silva et al., 2004; Grosso et al., 2011) and ST 56 (PubMLST; Acosta et al., 2011). OXA-58 expressing *A. baumannii* have been associated with international clones I, II, and II and a variety of unrelated sequence types (Di Popolo et al., 2011; Gogou et al., 2011).

OXA-48 was originally identified in a carbapenem-resistant isolate of *K. pneumoniae* in Turkey (Poirel et al., 2004c). Early reports suggested that this enzyme was geographically restricted to Turkey. In the past few years, however, the enzyme has been recovered from variety of Enterobacteriaceae and has successfully circulated outside of Turkey with reports of isolation in the Middle East, North Africa, Europe (Carrer et al., 2010), and most recently the USA (Lascols et al., 2012; Mathers et al., 2012). The Middle East and North Africa may be secondary reservoirs for these CHDLs (Hays et al., 2012; Poirel et al., 2012c). Indeed, the introduction of OXA-48 expressing Enterobacteriaceae in some countries has been from patients from the Middle East or Northern Africa (Decre et al., 2010; Adler et al., 2011; Poirel et al., 2011e; Canton et al., 2012). In the USA, the first clinical cases were associated with ST 199 and ST 43 (Mathers et al., 2012).

At least six OXA-48 variants (e.g., OXA-48, OXA-162, OXA-163, OXA-181, OXA-204, and OXA-232) have been identified. OXA-48 is by far the most globally dispersed and its epidemiology has been recently reviewed (Poirel et al., 2012c). Unlike KPCs and NDM-1 which have been associated with a variety of plasmids, a single 62 kb self-conjugative IncL/M-type plasmid has contributed to a large proportion of the distribution of *bla*OXA−48 in Europe (Potron et al., 2011a). Sequencing of this plasmid (pOXA-48a) notes that *bla*OXA−48 had been integrated through the acquisition of a Tn*1999* composite transposon (**Figure 1C**; Poirel et al., 2012b) *bla*OXA−48 appears to be associated with a specific insertion sequence, IS*1999* (Poirel et al., 2004c, 2012b). A variant of Tn*1999*, Tn*1999.2*, has been identified among isolates from Turkey and in Europe (Carrer et al., 2010; Potron et al., 2011a). Tn*1999.2* harbors an IS*1R* element within the IS*1999*. OXA-48 appears to have the highest affinity for imipenem of the CHDLs specifically those harboring *bla*OXA−48 within a Tn*1999.2* composite transposon (Docquier et al., 2009). Three isoforms of the Tn*1999* transposon have been described (Giani et al., 2012).

Although much of the spread of OXA-48 is attributed to a specific plasmid, outbreak evaluations demonstrate that a variety of strains have contributed to dissemination of this emerging carbapenemase in *K. pneumoniae*. The same *K. pneumoniae* sequence type, ST 395, harboring *bla*OXA−48 was identified in Morocco, France, and the Netherlands (Cuzon et al., 2011; Potron et al., 2011a). ST 353 was associated with an outbreak of OXA-48 producing *K. pneumoniae* in London (Woodford et al., 2011) and ST 221 with an outbreak of OXA-48 in Ireland (Canton et al., 2012). OXA-48 production in *K. pneumoniae*, like KPC-expressing *K. pneumoniae*, has also been associated with ST 14 (Poirel et al., 2004c) and a recent outbreak in Greece was associated with ST 11 (Voulgari et al., 2012).

*bla*OXA−48 is remarkably similar to *bla*OXA−54, a β-lactamase gene intrinsic to *Shewanella oneidensis* (Poirel et al., 2004a).

OXA-163, a single amino acid variant of OXA-48, was identified in isolates of *K. pneumoniae* and *Enterobacter cloacae* from Argentina and is unique in that it has activity against extendedspectrum cephalosporins (Poirel et al., 2011b). OXA-163 also has been identified in Egypt, which has a relatively prevalence of OXA-48, in patients without epidemiologic links toArgentina (Abdelaziz et al., 2012).

OXA-181 was initially identified among carbapenem-resistant Enterobacteriaceae collected from India (Castanheira et al., 2011). OXA-181 differs from OXA-48 by four amino acids, however, appears to be nestled in an entirely different genetic platform. The *bla*OXA−181 gene has been mapped to a different group of plasmids, the ColE family, and has been associated with an alternative insertion sequence, IS*Ecp1*. The latter insertion sequence has been associated with the acquisition of other β-lactamases including CTX-M-like ESBLs. Like, OXA-48, it appears that OXA-181 may have evolved from a waterborne environmental species *Shewanella xiamenensis* (Potron et al., 2011b).

OXA-204 differs from OXA-48 by a two amino acid substitution. It was recently identified in a clinical *K. pneumoniae* isolate from Tunisia (Potron et al., 2013). Its genetic construct appears to be similar to that of OXA-181. OXA-232 was recently identified among *K. pneumoniae* isolates in France (Poirel et al., 2012c).

OXA-143 is a novel plasmid-borne carbapenem-hydrolyzing oxacillinase recovered from clinical *A. baumannii* isolates in Brazil (Higgins et al., 2009). Information regarding its significance and prevalence continues to evolve (Antonio et al., 2010;Werneck et al., 2011; Mostachio et al., 2012).

## **AVAILABLE AGENTS AND DRUGS IN DEVELOPMENT**

Few antimicrobials are currently available to treat infections with carbapenemase-producing Gram-negative bacteria. Carriage of concurrent resistance determinants can result in decreased susceptibility non-β-lactams including the fluoroquinolones and aminoglycosides thus further compromising an already limited antimicrobial arsenal. What frequently remains available are the polymyxins (including colistin), tigecycline, and fosfomycin but susceptibilities to these agents are unpredictable (Falagas et al., 2011).

The reintroduction of polymyxins, both polymyxin B and colistin overlaps with the evolution of carbapenem resistance among Gram-negative bacilli. The clinical "resurgence" of these agents is well documented (Falagas and Kasiakou, 2005; Li et al., 2006a; Landman et al., 2008). Some experts advocate for the use of polymyxins in combination with other agents like rifampicin (Hirsch and Tam, 2010; Urban et al., 2010). *In vitro* evaluations of different combinations including carbapenems, rifamycins, and/or tigecycline demonstrate variable results (Bercot et al., 2011; Biswas et al., 2012; Deris et al., 2012; Jernigan et al., 2012). Most evaluations of the clinical outcomes or "effectiveness" of combination therapies have been retrospective (Qureshi et al., 2012; Tumbarello et al., 2012). Prospective clinical trials evaluating the superiority of colistin-based combination therapy over monotherapy are in their infancy. A real interest in combination therapy persists due to the concern of hetero-resistance (Li et al., 2006b; Poudyal et al., 2008; Lee et al., 2009; Yau et al., 2009; Meletis et al., 2011).

Early evaluations of the glycylcycline, tigecycline, demonstrated favorable *in vitro* activity against ESBL-producing Enterobacteriaceae and specific isolates of carbapenem-resistant *A. baumannii* and Enterobacteriaceae (Bratu et al., 2005; Fritsche et al., 2005; Noskin, 2005; Castanheira et al., 2008; Wang and Dowzicky, 2010). Tigecycline remains untested in prospective trials and reports of resistance are increasing (Navon-Venezia et al., 2007; Anthony et al.,2008;Wang and Dowzicky, 2010; Sun et al., 2012). The role of tigecycline in treating primary bloodstream infections or urinary tract infections remains undefined due less than therapeutic concentrations of drug achieved in the serum (Rodvold et al., 2006) and urine (Satlin et al., 2011). We also note that meta-analyses of pooled data from trials evaluating the use of tigecycline for a variety of indications suggest there is a excess mortality associated with the use of tigecycline over comparator regimens (Cai et al., 2011; Tasina et al., 2011; Yahav et al., 2011;Verde and Curcio, 2012). However, in the absence of other tested regimens tigecycline may be an appropriate or perhaps the only therapeutic option.

Growing resistance to both the polymyxins and tigecycline has resulted the revisiting of older drugs including chloramphenicol, nitrofurantoin, and temocillin (Livermore et al., 2011d). Fosfomycin is also one of these earlier antibiotics being reassessed

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(Falagas et al., 2008). In an *in vitro* evaluation of 68 KPCexpressing *K. pneumoniae* isolates, fosfomycin demonstrated *in vitro* activity against 87% of tigecycline and/or polymyxin nonsusceptible isolates and 83% of isolates that were resistant to both (Endimiani et al., 2010b). Fosfomycin may be a potential therapeutic option for patients infected with carbapenemase-producing Enterobacteriaceae if the infection is localized to the genitourinary tract. Unfortunately, fosfomycin does not demonstrate reliable activity against non-urinary pathogens. Fosfomycin demonstrated activity against only 30.2% of 1693 multidrug-resistant (MDR) *P. aeruginosa* isolates and 3.5% of 85 MDR *A. baumannii* isolates (Falagas et al., 2009). The individual studies included in this review did not employ uniform MDR definitions or consistent susceptibility breakpoints. Moreover, access to the parenteral fosfomycin is limited and the threshold for resistance is low (Rodriguez-Rojas et al.,2010; Karageorgopoulos et al.,2012). Concerns regarding the emergence of resistance have lead to an increasing interest in the utility of combination therapy (Michalopoulos et al., 2010; Bercot et al., 2011; Souli et al., 2011).

Few agents are in the advanced stages of development with demonstrable *in vitro* activity against carbapenemase-producing organisms. These include β-lactamase inhibitors, aminoglycoside derivatives, polymyxin derivatives, and novel monobactams and monobactams-β-lactamase inhibitor combinations.

Avibactam, or NXL104, is a β-lactamase inhibitor which has been tested in combination with ceftazidime, ceftaroline, and aztreonam against several carbapenemase-producing Enterobacteriaceae with impressive decreases in MICs (Livermore et al., 2008, 2011b; Endimiani et al., 2009a; Mushtaq et al., 2010c). Cephalosporin–avibactam combinations do not inhibit MBLs. Avibactam in combination with aztreonam, however, does seem to demonstrate activity against isolates harboring a variety of carbapenem resistance mechanisms including MBLs (Livermore et al., 2011b). Regrettably, the avibactam and aztreonam combination is not currently in clinical trials. The combination of ceftazidime–avibactam has been evaluated against collections of non-fermenting Gram-negative pathogens and its role remains undefined (Mushtaq et al., 2010b). In some evaluations of ceftazidime non-susceptible isolates of *P. aeruginosa* decrease MICs were noted with the addition of avibactam (Mushtaq et al., 2010b; Walkty et al., 2011; Crandon et al., 2012; Levasseur et al., 2012). The combinations of ceftaroline– avibactam and ceftazidime–avibactam are currently in clinical trials.

Methylidene penems (penem-1 and penem-2) are β-lactamase inhibitors and appear to be potent inhibitors of KPC-2 (Papp-Wallace et al., 2010). The combination of cefepime with penem-1 demonstrated lower cefepime MICs in 88.1% of the 42 KPCproducing *K. pneumoniae* isolates evaluated (Endimiani et al., 2010a). MK-7655 is a novel β-lactamase being evaluated in combination with imipenem against carbapenem-resistant Gramnegative bacilli (Hirsch et al., 2012).

ME1071, formerly CP3242 (Bassetti et al., 2011), is a maleic acid derivative that competitively inhibits MBLs. Earlier studies demonstrated concentration-dependent decreases in carbapenem MICs in MBL-producing *P. aeruginosa* (Ishii et al., 2010), *A. baumannii*, and select Enterobacteriaceae (Shahid et al., 2009) A

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contemporary pre-clinical evaluation of ME1071 in combination with various type 2 carbapenems (i.e., biapenem, doripenem, meropenem, imipenem) confirms remarkable decreases in the carbapenem MICs for Enterobacteriaceae and *A. baumannii* harboring IMP, VIM, and NDM-type MBLs (Livermore et al., 2013). Irrespective of the candidate carbapenem, ME1071 activity against NDM MBLs was less than that of VIM-type and IMP-type MBLs. Of note, biapenem was the carbapenem with the lowest baseline MICs to the MBLs, but it is commercially unavailable in many countries including the USA. Other MBL-specific inhibitors are in pre-clinical development (Chen et al., 2012).

Plazomicin (ACHN-490) is an aminoglycoside derivative with potent activity against some carbapenem-resistant Gram-negative bacilli (Zhanel et al., 2012). Studies have noted that susceptibilities to aminoglycosides vary among KPC-producing *K. pneumoniae*. In one evaluation, 48% of 25 tested isolates were susceptible to amikacin, 44% to gentamicin, and 8% to tobramycin. Plazomicin demonstrated an MIC90 significantly lower than that of amikacin (Endimiani et al., 2009c). *In vitro* studies also indicate that depending on the aminoglycoside resistance mechanisms present, Plazomicin may have activity against select isolates of *P. aeruginosa* and *A. baumannii* (Aggen et al., 2010; Landman et al., 2011). Susceptibility to plazomicin in the setting of resistance to other aminoglycosides appears to be dependent on the mechanism of aminoglycoside resistance (Livermore et al., 2011a).

NAB739 and NAB7061 are polymyxin derivatives that may be less nephrotoxic than commercially available polymyxins. In a small *in vitro* study, NAB739 displayed activity against nine carbapenemase-producing polymyxin-susceptible isolates of Enterobacteriaceae (Vaara et al., 2010). A contemporary evaluation of NAB739 demonstrated higher MICs compared to those of polymyxin B in a collection of polymyxin-susceptible and non-susceptible Enterobacteriaceae, *P. aeruginosa*, and *A. baumannii* (Vaara et al., 2012). NAB7061 when used in combination with rifampicin or clarithromycin demonstrated synergistic activity against seven strains of carbapenemase-producing Gramnegative bacilli including one polymyxin-resistant strain (Vaara et al., 2010). It remains unclear what role these agents will play in the setting the increasing burden of infections with carbapenemase-producing Enterobacteriaceae.

The activity of the siderophore monosulfactam, BAL30072, has been against non-fermenting carbapenemase-producing Gramnegative bacilli (Page et al., 2010). In one study, susceptibility to BAL30072 was noted in 73% of 200 isolates of carbapenemaseproducing *A. baumannii*, the majority of which were of the same OXA-23 producing clone (Mushtaq et al., 2010a). In that same study, smaller percentages of susceptibility were noted in a selection of carbapenem-resistant *Burkholderia cepacia* and *P. aeruginosa* isolates. Recent evaluations of BAL30072 confirm that there may be a role for this agent in the treatment of resistant *A. baumannii* infections (Russo et al., 2011; Higgins et al., 2012b). BAL 30376 is a combination of a siderophore monobactam with clavulanic acid. In two studies, this combination demonstrated reasonable *in vitro* activity against CHDL, including OXA-48, and MBLs but not KPCs (Livermore et al., 2010; Page et al., 2011).

## **CONCLUDING REMARKS**

In the last 5 years, we have witnessed the global spread of carbapenem resistance among Gram-negative organisms. The notion that multidrug resistance among these pathogens is limited to isolated outbreaks among the critically ill has met the ultimate challenge with NDM-1 (Kumarasamy et al., 2010). The conveniences of travel and medical tourism have introduced resistance mechanisms across states, countries, and even continents at an alarming rate (Rogers et al., 2011; van der Bij and Pitout, 2012). Rates of resistance in some countries may be underestimated due to the lack of organized reporting structures and limited resources. Long-term healthcare facilities are now recognized reservoirs for the continued propagation of MDR organisms (Urban et al., 2008; Aschbacher et al., 2010; Perez et al., 2010a; Ben-David et al., 2011; Prabaker et al., 2012; Viau et al., 2012).

Until the introduction of accurate, affordable, and readily accessible diagnostics and reliably effective antimicrobials a major focus remains containment and eradication of these organisms within the healthcare environment. Many cite a "bundle" type approach that includes administrative support, active surveillance, antimicrobial stewardship, and augmented infection control practices (Centers for Disease Control and Prevention, 2009; Schwaber et al., 2011; Snitkin et al., 2012). Just as with drug development (Tillotson, 2010), the future savings of investing in prevention is not as tangible as the immediate capital investment required to allot appropriate resources including advanced laboratory platforms, experienced laboratory personnel, dedicated nursing staff, and infection control personnel (Bilavsky et al., 2010). Expanding these efforts to non-acute healthcare settings is recommended to begin to stem the evolving pandemic of carbapenem resistance (Gupta et al., 2011).

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The prudent use of antibiotics is essential in combating the continuing evolution of resistance (Marchaim et al., 2012). This may be even more crucial in areas where non-prescription antimicrobial use is common and continues to be unregulated. In an age where multidrug resistance is so widespread, even the appropriate use of broad-spectrum antibiotics has contributed to our current state.

Research funding and support for the description of resistance mechanisms, validation of current infection control practices, and antimicrobial development must be prioritized. Institutions supporting infection control, state of the art microbiology laboratories, and antimicrobial stewardship programs should receive recognition and incentives for their foresight. Despite these continuing challenges, considerable progress has been made to identify at-risk populations and to describe resistance determinants. Collaborative efforts (Kitchel et al., 2009a; Struelens et al., 2010; Canton et al., 2012) have led to a better understanding and awareness of the epidemiology and the contribution of antimicrobial use and the environment to the propagation of antimicrobial resistance. These joint efforts have proven crucial for the propagation of information about carbapenemases. Continuing to encourage these partnerships is imperative in the ongoing struggle against antimicrobial resistance and to prevent antimicrobials from essentially becoming obsolete.

## **ACKNOWLEDGMENTS**

This work was supported in part by the Veterans Affairs Merit Review Program (to Robert A. Bonomo), the National Institutes of Health (grants R01-A1063517 and RO3-A1081036 to Robert A. Bonomo), and the Geriatric Research Education and Clinical Center VISN 10 (to Robert A. Bonomo).

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 19 December 2012; paper pending published: 07 January 2013; accepted: 20 February 2013; published online: 14 March 2013.*

*Citation: Patel G and Bonomo RA (2013) "Stormy waters ahead": global emergence of carbapenemases. Front. Microbiol. 4:48. doi: 10.3389/fmicb.2013.00048*

*This article was submitted to Frontiers in Antimicrobials, Resistance and Chemotherapy, a specialty of Frontiers in Microbiology.*

*Copyright © 2013 Patel and Bonomo. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any thirdparty graphics etc.*

**REVIEW ARTICLE** published: 12 March 2013 doi: 10.3389/fmicb.2013.00047

## The multifaceted roles of antibiotics and antibiotic resistance in nature

## *Saswati Sengupta1, Madhab K. Chattopadhyay2 and Hans-Peter Grossart 3,4\**

1 Independent, Hyderabad, India

2 Centre for Cellular and Molecular Biology (Council of Scientific and Industrial Research), Hyderabad, India

3 Limnology of Stratified Lakes, Leibniz Institute of Freshwater Ecology and Inland Fisheries, Stechlin, Germany

4 Institute for Biochemistry and Biology, Potsdam University, Potsdam, Germany

#### *Edited by:*

Fiona Walsh, Agroscope Changins-Wädenswil, Switzerland

#### *Reviewed by:*

Jun Lin, The University of Tennessee, USA

Eddie Cytryn, Agricultural Research Organization, Volcani Agriculture Research Center, Israel

#### *\*Correspondence:*

Hans-Peter Grossart, Limnology of Stratified Lakes, Leibniz Institute of Freshwater Ecology and Inland Fisheries, Alte Fischerhuette 2, 16775 Stechlin, Germany. e-mail<EMAIL_ADDRESS>
Antibiotics are chemotherapeutic agents, which have been a very powerful tool in the clinical management of bacterial diseases since the 1940s. However, benefits offered by these magic bullets have been substantially lost in subsequent days following the widespread emergence and dissemination of antibiotic-resistant strains.While it is obvious that excessive and imprudent use of antibiotics significantly contributes to the emergence of resistant strains, antibiotic resistance is also observed in natural bacteria of remote places unlikely to be impacted by human intervention. Both antibiotic biosynthetic genes and resistance-conferring genes have been known to evolve billions of years ago, long before clinical use of antibiotics. Hence it appears that antibiotics and antibiotics resistance determinants have some other roles in nature, which often elude our attention because of overemphasis on the therapeutic importance of antibiotics and the crisis imposed by the antibiotic resistance in pathogens. In the natural milieu, antibiotics are often found to be present in sub-inhibitory concentrations acting as signaling molecules supporting the process of quorum sensing and biofilm formation. They also play an important role in the production of virulence factors and influence host–parasite interactions (e.g., phagocytosis, adherence to the target cell, and so on). The evolutionary and ecological aspects of antibiotics and antibiotic resistance in the naturally occurring microbial community are little understood. Therefore, the actual role of antibiotics in nature warrants in-depth investigations. Studies on such an intriguing behavior of the microorganisms promise insight into the intricacies of the microbial physiology and are likely to provide some lead in controlling the emergence and subsequent dissemination of antibiotic resistance. This article highlights some of the recent findings on the role of antibiotics and the genes that confer resistance to antibiotics in nature.

**Keywords: antibiotics, sub-inhibitory concentration, quorum sensing, virulence, stress response, antibiotic resistance, antibiotic paradox**

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## **INTRODUCTION**

The term "antibiotics" was first coined by the American microbiologist Selman Waksman and his colleagues to describe chemical substances produced by microorganisms and having antagonistic effects on the growth of other microorganisms. It excluded synthetic antimicrobials (sulfur drugs) and biological products of non-microbial origin having antagonistic effects on bacteria. Though antibiotics were introduced into the clinical practice only in the middle of the last century, the use of microorganisms for the management of microbial infections in ancient Egypt, Greece, China, and some other places of the world is well-documented. The modern era of antibiotics started with the serendipitous discovery of penicillinfrom the culture filtrate of afungus, *Penicillium notatum* by Alexander Fleming in 1928 (Fleming, 1929).

In the present scenario, antibiotics available in the market are either produced by microbial fermentation or are derived via semi-synthetic route using the existing antibiotic backbone structure. They are classified into different chemically defined

groups. Antibiotics target bacterial physiology and biochemistry, causing microbial cell death or the cessation of growth. A significant number of these antibiotics affect cell walls or membranes (e.g., β-lactam and glycopeptides), while several others exert their antibacterial activity by targeting protein synthetic machinery via interaction with ribosomal subunits and these include antibiotics such as macrolides, chloramphenicol, tetracycline, linezolid, and aminoglycosides. Other "mechanistic" groups include molecules which interfere with the nucleic acid synthesis [e.g., fluoroquinolones (FQ) and rifampin], while some others exert their effects by interfering with the metabolic pathways (e.g., sulphonamides and folic acid analog) or by disruption of the bacterial membrane structure (e.g., polymyxins, daptomycin, and others).

A surge of discovery of several such antibacterial and antifungal antibiotics accompanied with a new generation of semi-synthetic drugs initially led to euphoria that any infectious disease could be successfully controlled using antibiotics. However, emergence and

propagation of bacterial strains, resistant to almost all the therapeutically useful antibiotics during the past few decades revealed the limitation of the wonder drugs. Though imprudent and excessive use of antibiotics is highlighted as a major causative factor behind the setback, it is evident by this time that antibiotic resistance does not call for exposure of the organisms to antibiotics. It is also found that genes involved in the biosynthesis of antibiotics and antibiotic resistance evolved thousands of years before antibiotics were introduced into the clinical practice. Hence both antibiotic and its resistance determinants have some other role in bacterial physiology.

#### **ROLE OF ANTIBIOTICS IN NATURE**

Antibiotics in the biosphere are produced by microorganisms as secondary metabolites at a concentration much lower than the therapeutic dose. Waksman was convinced that antibiotics play "no real part in modifying or influencing living processes that occur in nature" (Waksman, 1961) though there is evidence to the contrary (Gullberg et al., 2011). Antibiotics are produced in the late stages of microbial stationary growth phase, decoupled from the doubling time, implying that they are not indispensable for sustenance of life of the producer organism. However, it is also a fact that the production of an antibiotic is a multistep process that involves a number of genes. Hence it does not seem tenable that such a complex anabolic process has been sustained through evolution without having any obvious purpose to serve. Recent studies reveal that antibiotics do have some specific effects on the natural milieu of the microbes while they assume an entirely different role as antibacterial agents in the dose used in therapeutics. This dual role of antibiotics remains a riddle. In the natural habitat, the diverse microbial communities exist as a multi-cellular network. They constitute a huge reservoir of different metabolic activities and have to evolve continuously to encounter the constant environmental threat and different selection pressure. Survival is most often a major challenge because of the limiting nutrients. Under nutrient starvation, the organisms start secreting secondary metabolites, which are a diverse array of low molecular weight organic molecules, known as the parvome (Davies, 2009). The total number of parvome detected so far is at least an order of magnitude larger than the number of bacteria in the biosphere. Bacteria of the phylum Actinobacteria (Ventura et al., 2007), a huge taxonomic group of diverse genera with a high genomic guanine–cytosine (GC) content, produce millions of complex bioactive small molecules. The ecological role of the majority of these small molecules in nature remains largely unknown. Amongst these molecules, only afraction has been identified to have antibiotic activity and has been extensively studied only from the therapeutic point of view whereas the significance of antibiotics in nature still remains a mystery. The prevailing confusion about its role in nature has been dealt by Julian Davis in an article titled: "are antibiotics naturally antibiotic?" (Davies, 2006).

#### **ROLE OF ANTIBIOTICS IN INTERSPECIES COMPETITION**

Antibiotics are critical to the producer organisms in the natural environment as they are needed both for survival and competitive advantage. Thus antibiotics are widely perceived as an arsenal of the producer microorganisms that they use against other naturally occurring cohabiting microorganisms and eliminate these competing bacteria for the purpose of "empire building" in the microbial community (Davies, 1990). The concept of the empire building role of antibiotics stemmed from the soil borne nature of the antibiotic-producing microorganisms. In certain instances like plant–microbe interactions, the antibiotic-producing organisms have indeed been found to secrete antibiotics to eliminate the competing bacteria in the vicinity. *Pseudomonas fluorescens* 2-79 (NRRL B-15132) colonizing the rhizosphere of wheat, secrete the antibiotic, phenazine which inhibits the growth of *Gaeumannomyces graminis* var. *tritici* thus suppressing a major root and crown disease of wheat and barley (Thomashow and Weller, 1988). Few other ecological examples of probable antibiotic function of these secondary metabolites in nature include the fungus-growing ants, which carry an antibiotic-producing actinomycete (*Pseudonocardia* sp.) on their cuticle specifically for biocontrol of the fungal garden parasite, *Escovopsis* sp. (Currie et al., 1999; Cafaro and Currie, 2005). Another example is the antibiotic-producing strain *Streptomyces diastatochromogenes* strain PonSSII controlling the growth of *Streptomyces scabies* strain RB4, the causative agent of potato scab (Neeno-Eckwall et al., 2001).

#### **ROLE OF ANTIBIOTICS AS SIGNALING MOLECULES**

During primordial development, the cells instead of antagonizing each other remained in a communal harmony to share an evolutionary advantage and the symbiotic coexistence of microbes in biofilms, in lichens and in a metabolically quiescent state is well-documented (Davies, 2006). Chemical communication, an essential feature in such mixed populations, helps the microorganisms coordinate with each other in an orchestrated fashion. Probably the small molecule antibiotics act as a liaison in the communication between the microbes. It has been postulated that antibiotics in the early stages of biochemical evolution had some functions mediated by their interaction with primitive macromolecule receptors. Later on, many of these low molecular weight interacting partners were employed to antagonize the original receptor sites in macromolecular structure thus imparting the antibiosis property. Such a switchover in the role of the primordial effector molecule has been hypothesized in a schematic biochemical pathway by J. Davies (Davies, 1990). During the past seven decades, the scientists were mostly preoccupied with studying the antibiotic property of these small molecules because of its overwhelming success in pharmacotherapy. Only during the recent past antibiotics were implicated in several other physiological phenomena ranging from control of transcription and translation to growth of the producer organism in the microbial communities. From a broader perspective of evolutionary and ecological point of view, antibiotics, apart from having a growth inhibitory activity, appear to have a role in intra- and inter-domain communication in various ecosystems. Under normal physiological condition, microorganisms in their marine and soil habitats produce antibiotics at sub-minimum inhibitory concentration (sub-MIC). Despite having evidence that resistant bacteria representing a low fraction in a sample were enriched at sub-inhibitory concentrations of three clinically useful antibiotics (Gullberg et al., 2011), antibiotics are believed not to display any antibiotic activity

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at concentrations lower than 1/100 of the minimum inhibitory concentrations (Davies, 2006). Instead, many antibiotics are known to possess biological activities other than the inhibitory role at such a low concentration and have major effects on global transcription pattern. Antibiotics such as erythromycin (an inhibitor of translation) and rifampicin (an inhibitor of transcription), at low concentrations, could modulate global bacterial transcription pattern in a random promoter library construct of *Salmonella typhimurium*having *lux asthe*reporter gene. In response to the low concentration of different antibiotics, a variety of promoters were activated, including those involved in virulence, metabolic, and adaptive functions. However, transcription was markedly reduced in antibiotic-resistant hosts but the mutants defective in stress responses such as *rec* and *lex* (SOS), *dnaJ* and Δ*dnaKJ* (heat shock response), and Δ*relA* Δ*spoT* (universal stress response) could not prevent antibiotic-induced modulation of transcription pattern at sub-inhibitory concentration (Goh et al.,2002). Antibiotics having different binding sites on the ribosome (chloramphenicol, aminoglycosides, macrolides, and tetracycline), or intruding into the process of cell-wall synthesis (some β-lactams and fosfomycin) were reported to alter the transcription pattern under different circumstances. So it has been postulated that the transcription machinery must be capable of sensing these subtle variations in the conformation or stoichiometry of small molecule antibiotics to respond to specific up- or downregulation.

These small molecules have a significant role in the dynamics of bacterial communities in nature, thus contributing to both competitive and interactive responses. Inhibition occurs when high concentrations are attained, transcriptional changes occur at low concentrations (Davies et al., 2006). This dose-dependent dual role, defined as hormesis, is not exclusively associated with antibiotics but also found in different other biomolecules (Davies et al., 2006; Calabrese, 2009). The dual role of antibiotics has been confirmed by the detection of a variety of bioactivities of several antibiotics. Some β-lactam antibiotics were shown to stimulate the glutamate transporter-1 (GLT1) both in cell culture and in animal models (Rothstein et al., 2005). Thiostrepton, a potent protein synthesis inhibitor which binds to 23S RNA, was found to act as a transcription inducer at a concentration lower than the inhibitory concentration (Murakami et al., 1989). Lincomycin, which terminates the peptide bond elongation, could stabilize certain mRNAs like the *bla* mRNA (Matsushita et al., 1989). Puromycin, in spite of having a chain termination activity in polypeptide synthesis, can affect nucleic acid synthesis, mediated by its aminonucleoside moiety (Yarmolinsky and Haba, 1959). Edeine, apart from its inhibitory role in protein synthesis, has specific effects on nucleic acid synthesis (Kurylo-Borowska and Szer, 1972). Many of the socalled antibiotics have been shown to have sex pheromone activity and found to stimulate bacterial conjugation by several orders of magnitude (Evans and Dyke, 1988). Sub-MICs of antibiotics are thus known to induce extensive transcriptional changes in bacteria (Tsui et al., 2004; Yim et al., 2006a). In the majority of cases the underlying mechanisms of the different ecological role of the antibiotics at sub-inhibitory concentration still remain to be elucidated.

A second level of signaling via antibiotics has also been documented in different organisms. Cationic peptides, which have a broad range of antimicrobial activities against most Gramnegative and Gram-positive bacteria, fungi, enveloped viruses, and eukaryotic parasites, also have a broad spectrum of proinflammatory and anti-inflammatory effects on the host immune system. Apart from their antimicrobial activity, these molecules also act as transcription modulators. Cecropin, an insect-derived cationic peptide, was shown to alter the transcription pattern of 26 genes when added to *Escherichia coli* (Hong et al., 2003). Some cationic peptides of microbial- and mammalian origin (polymyxins B and E, cattle indolicidin and human LL-37) were found to activate the two component *pmrA–pmrB* regulatory operon, which regulates the resistance to polymyxin B and cationic antimicrobial peptides in *Pseudomonas aeruginosa* (McPhee et al., 2003). Inhibitory effect of several antibiotics on sporulation of bacteria is well-known (Ochi and Freese, 1983; Babakhani et al., 2012).

### **ROLE OF ANTIBIOTICS IN VIRULENCE**

Some antibiotics, at concentrations below the MIC, are able to modulate the expression of some genes associated with virulence. Up- and downregulation of the transcription of virulence and motility genes were induced by the RNA polymerase inhibitor, rifampicin (Yim et al., 2006b). Sub-inhibitory concentration of several other antibiotics such as metronidazole, vancomycin, clindamycin, and linezolid could induce the early transcription of *tcdA* (*toxin A*) and *tcdB* (*toxin B*), the major virulence factorsencoding genes of *Clostridium difficile*, a nosocomial pathogen responsible for diarrhea, during exponential phase growth of the organism (Gerber et al., 2008). Imipenems, at sub-inhibitory concentration, were shown to induce β-lactamase and production of alginate during biofilm formation in *Pseudomonas aeruginosa*. Sub-inhibitory concentration of an aminoglycoside antibiotic tobramycin induced biofilm formation in *Pseudomonas aeruginosa* and *Escherichia coli*(Hoffman et al.,2005). On the other hand, sub-inhibitory level of the macrolide antibiotic azithromycin was shown to elicit the exactly opposite phenomenon like suppression of the alginate overproduction and biofilm formation in *Pseudomonas aeruginosa* (Ichimiya et al., 1996). These contradictory observations suggest that the molecular targets of different antibiotics for the execution of their inhibitory effects could be similar but they might differ extensively while functioning as signaling molecules (Aminov, 2009). This postulation is substantiated by the fact that tobramycin at sub-inhibitory concentrations does not bind the ribosome (the usual target for its role as an antibiotic), rather it targets the aminoglycoside response regulator gene (arr) which is responsible for the induction of biofilm-specific aminoglycoside resistance. The gene is also predicted to encode an inner-membrane phosphodiesterase acting on the substrate, cyclic di-guanosine monophosphate (c-di-GMP), a bacterial second messenger that regulates cell surface adhesiveness (Hoffman et al., 2005).

The influence of sub-inhibitory doses of several antibiotics on the expression of the gene encoding the *Staphylococcus aureus* alpha-toxin (*hla*), a major virulence factor of the pathogen, revealed that glycopeptide antibiotics had no effect whereas βlactams induced a strong expression of *hla*. On the other hand, the macrolide erythromycin and several aminoglycosides reduced, while FQs slightly stimulated *hla* expression and an almost

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complete inhibition of alpha-toxin expression was obtained using clindamycin. Furthermore, methicillin enhanced Hla production of both MSSA (methicillin-sensitive *Staphylococcus aureus*) and MRSA (methicillin-resistant *Staphylococcus aureus*; Ohlsen et al., 1998). The sub-inhibitory concentration of ceftazidime (βlactam) and ciprofloxacin (FQ) appeared to be effective in decreasing the expression of a range of quorum sensing (QS)-regulated virulence factors (Skindersoe et al., 2008).

Genomic rearrangements in pathogenic strains of *Escherichia coli* and *Shigella* following exposure to sub-inhibitory concentrations of kanamycin and ampicillin was demonstrated (Pedro et al., 2011). Some of the rearrangements may engender genotypes with increased virulence. SOS response, induced by ciprofloxacin, was found to stimulate transfer of pathogenicity Island in *Staphylococcus aureus* (Ubeda et al., 2005). Thus antibiotic used for the treatment of clinical infections may lead to the dissemination of virulence in some cases.

#### **ROLE OF ANTIBIOTICS IN HOST–PARASITE INTERACTION**

Antibiotics have also been found to modulate host immune cell response, mostly mediated by cytokines. There are ample evidences of sub-inhibitory concentrations of antibiotics interference with processes of host–parasite interactions such as phagocytosis, adherence, and virulence. Antibiotics promote phagocytosis by altering the surface properties of pathogens. Many clinical isolates of *Enterococcus faecium* are resistant to neutrophil-mediated phagocytosis but exposure of vancomycin-susceptible *Enterococcus faecium* to quinupristin/dalfopristin at concentrations both at either sub-inhibitory or suprainhibitory concentrations promoted susceptibility to subsequent PMN (polymorphonuclear leucocytes)-mediated phagocytosis. Vancomycin-resistant strains on the contrary exhibited very little change in their binding toward PMNs after antibiotic pretreatment (Herrera-Insúa et al., 1997). Use of amoxicillin for the treatment of *Haemophilus influenzae* in acute otitis media caused upregulation in the expression of cytokines, interleukin (IL)-6, tumor necrosis factor-α, and IL-10 (Melhus, 2001).

#### **ROLE OF ANTIBIOTICS IN SOS AND DNA REPAIR GENE EXPRESSION**

The SOS response, a regulatory network present in most bacteria is induced in response to DNA damage and has been shown to promote stress-induced mutagenesis, virulence, and dissemination of antibiotic resistance specifically by horizontal gene transfer (HGT). Beaber et al. (2004) showed that ciprofloxacin induced transfer of the *Vibrio cholerae* SXT integrative conjugative element (ICE) was controlled by SOS through RecA-dependent cleavage of the SXT encoded repressor SetR. SXT is a 100-kb ICE derived from *V. cholerae* that encodes genes for the resistance toward chloramphenicol, sulfamethoxazole, trimethoprim, and streptomycin (Beaber et al., 2004). Even the induction of the CTX prophage which encodes the cholera toxin is regulated by the SOS repressor LexA in conjunction with the phage repressor rstR which is also regulated by LexA (Quinones et al., 2005). In *V. cholerae*, the recombination-mediated excision of integron cassette was induced by SOS in response to different antibiotics (Guerin et al., 2009). On the other hand, SOS-independent recombination between divergent sequences either by RecBCD or RecFOR pathways was demonstrated in *Escherichia coli* by quinolones (DNAdamaging antibiotics) induced emergence of drug resistance (López et al., 2007).

Fluoroquinolones at sub-MIC have been shown to upregulate both the SOS and the methyl mismatch repair (MMR) pathway genes in *Staphylococcus aureus* with different SOS response and DNA repair-associated promoter-*lux* gene fusion construct (Mesak et al., 2008). Upregulation of several genes involved in the SOS response such as *umuD*, *lexA*, *sbmC*, and *dinP* was demonstrated in *Salmonella* using sub-inhibitory concentrations of quinolones. In addition, transcriptional regulators, genes putatively associated with membrane integrity (*spr*), virulence (*sicA*), and metabolism (*plsB*) were also affected (Yim et al., 2011). On the contrary, it has also been shown that exposure of *Streptococcus pneumoniae* to quinolones, aminoglycosides, and penicillin at sub-inhibitory concentrations may result in increased mutability, without the apparent involvement of SOS-response components (Henderson-Begg et al., 2006; Cortes et al., 2008).

SOS response also enhances frequency of mutations. Hence antibiotics which induce SOS response, contribute to the increase in the frequency of resistant strains. Exposure to ciprofloxacin was found to increase in the frequency of rifampicin-resistant mutants of *Streptococcus pneumoniae* almost by fivefold (Henderson-Begg et al., 2006). Carbapenem resistance in *Pseudomonas aeruginosa* was also found to be enhanced in presence of the same antibiotic almost in the same order (Tanimoto et al., 2008).

Thus low antibiotic concentration regulates the transcription of different genes in target bacteria while increasing higher concentration elicits a stress response and even higher concentration is lethal. Therefore, it is apparent that these small molecule at a sub-inhibitory concentration participate in a new form of signaling network by binding to different cytoplasmic macromolecular receptors such as ribosomes, and those involved in DNA replication, RNA transcription machinery, and cell-wall biosynthesis (Ryan and Dow, 2008). These receptors were also identified earlier as the inhibitory targets for the bioactive molecules at higher concentration. The cellular targets for the lethal and sub-inhibitory concentration sometimes also differ thus implying that antibiotics are multi-ligand-specific sensor molecules targeting different ligands in a concentration-dependent manner (Hoffman et al., 2005).

#### **ANTIBIOTICS AND QUORUM SENSING**

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Quorum sensing is a mode of bacterial communication in response to release of chemical messenger which regulate bacterial gene expression (Bassler, 1999). Secondary metabolites are activated during slow growth in the stationary phase and trigger the transition from primary to secondary metabolism (Bibb, 2005). This transition is a complex process and involves many signals, including those by small signaling molecules called γbutyrolactones (also known as autoregulatory factor or A-factor). These signaling molecules, found mainly in *Streptomyces* species, are considered "bacterial hormones" or the QS factor. The QS signaling network initiates a set of metabolic changes leading to a number of events including morphological differentiation and synthesis of secondary metabolites such as antibiotics. The γbutyrolactones constitute the major group of QS molecules in

bacteria. The first γ-butyrolactone, A-factor, was identified in *Streptomyces griseus* in 1967 (Hsiao et al., 2009).

Biosynthesis of γ-butyrolactones is not well understood, but seems to be mediated by γ-butyrolactone synthase AfsA. The Afactor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactones), at a certain critical concentration, binds to cytoplasmic receptor protein, a dimer of ArpA,which primarily acts as repressors by binding to the promoter region of adpA (A-factor-responsive transcriptional activator, a key pleiotropic regulator). The binding of the A factor dissociates ArpA from the promoter, thus facilitating the transcription and translation of adpA. Subsequently, AdpA binds an upstream activation sequence to initiate the transcription of strR (a pathway-specific regulatory gene responsible for transcription of other streptomycin biosynthetic genes cluster). strR is a pathway-specific regulator which acts as a transcriptional activator and induces transcription of most of the streptomycin biosynthetic genes by binding multiple sites in the gene cluster (Retzlaff and Distler, 1995). Thus onset of streptomycin biosynthesis is initiated by the QS factor, γ-butyrolactones. The gene *aph*D, encoding a major streptomycin resistance determinant streptomycin-6-phosphotransferase, is also under the control of the *adp*A-dependent promoter. The binding to γ-butyrolactones thus induces expression of the target genes (transcription factors) many of which are involved in regulation of specific antibiotic biosynthesis clusters. Onset of streptomycin biosynthesis is initiated by the QS factor, γ-butyrolactones (Retzlaff and Distler, 1995). The expression of the gene *aph*D, encoding a major streptomycin resistance determinant streptomycin-6 phosphotransferase, is also a QS-mediated event. Each receptor protein is highly specific for its cognate γ-butyrolactone. In *Erwinia carotovora* carbapenem biosynthesis is regulated by a classical autoinducer *N*-(3-oxohexanoyl)-L-homoserine. In another cephamycin C-producing bacterium, *Streptomyces clavuligerus*, antibiotic synthesis is regulated at the primary regulatory level by γ-butyrolactone (Liras et al., 2008).

Some 10–11 probable γ-butyrolactone synthates, all from the *Streptomyces* genus were found while 37–42 putative γ-butyrolactone receptors were found in the genome of different other bacteria apart from *Streptomyces*. Thus, it is believed that many of the cellular activities including the antibiotic production is under the control of a well-orchestrated QS signaling network mediated by a small population of γ-butyrolactone producers acting as a signaling factor for a diverse population of signal recipient (Aminov, 2009).

Structural similarity between antibiotic and intracellular signaling molecule has also been evidenced in several cases. The quinolones produced by *Pseudomonas aeruginosa* have a wide range of activities starting from antibiotics to autoinducers. 2- Heptyl-3-hydroxy-4-quinolone (pseudomonas quinolone signal; PQS), belonging to the family of 2-alkyl-4-quinolones (AQs), was previously described for their antimicrobial activities. Later on, it was found that PQS is integrated within an intricate QS circuit and plays an important role in *Pseudomonas aeruginosa* pathogenesis by regulating the production of diverse virulence factors including elastase, pyocyanin, and LecA lectin in addition to affecting biofilm formation (Dubern and Diggle, 2008; Heeb et al., 2011). Several other autoinducers like *N*-(3-oxododecanoyl) homoserine lactone and its tetrameric acid degradation product in *Pseudomonas aeruginosa* have antibacterial properties against Gram-positive bacteria (Kaufmann et al., 2005). *N*-(3-oxododecanoyl) homoserine lactone and other 3-oxo series homoserine lactones with 8, 10, and 14 length carbon chains were shown to inhibit growth of the Gram-positive *Staphylococcus aureus* (Qazi et al., 2006).

## **ANTIBIOTIC RESISTANCE, THE TIP OF THE ICEBERG**

In the late 1960s, the unprecedented successes of early antibiotic therapies led US Surgeon General William H. Stewart to make the famous declaration: "it is time to close the book on infectious diseases and declare the war against pestilence won" (Spellberg et al., 2008). The euphoria did not last long and the magic bullets started losing the efficacy because of the steady emergence of antibioticresistant pathogens simultaneously with their widespread use. Following the success of penicillin in controlling bacterial infection among the soldiers during the Second World War, emergence of penicillin-resistant strain was evidenced in the 1940s. By 1960, it assumed the shape of a pandemic problem. New β-lactam antibiotics were introduced into the clinical practice to restrain the problem. Simultaneously bacterial strains resistant to them came into being, a phenomenon dubbed as β-lactamase cycle. The first case of MRSA was identified in UK in 1961 (Johnson, 2011) and the first report on MRSA in the United States came in 1968. Now it is prevalent all over the world. MRSA is actually resistant to the entire class of penicillin-like antibiotics called β-lactams. Presence of an enzyme called New Delhi metallo-β-lactamase (NDM-1) in some Gram-negative bacteria (notably *Escherichia coli* and *Klebsiella pneumoniae*) makes them resistant to virtually all β-lactams including carbapenems, which are most often considered the last line of defense against multidrug-resistant pathogens (Kumarasamy et al., 2010). Isolation of bacterial strains producing extended spectrum β-lactamase (ESBL) resistant to third generation of cephalosporins and monobactams is also reported from time to time (Kurihara et al., 2013).

The glycopeptide antibiotic vancomycin was introduced in clinical practice in 1958 for the treatment of methicillin resistance in both *Staphylococcus aureus* and coagulase-negative staphylococci. Vancomycin resistance was so difficult to induce that it was believed to be very unlikely to occur in a clinical setting. Until the late 1980s, the glycopeptide antibiotic vancomycin was considered the drug of last resort for treatment of diseases caused by Grampositive bacteria such as *Enterococcus faecalis*, MRSA, *Streptococcus pneumoniae*, and *Clostridium difficile* (Cunha, 1995). However, vancomycin resistance was first reported in coagulase-negative staphylococci in 1979 and 1983 (Srinivasan et al., 2002). At present vancomycin-resistant enterococci (VRE) pose a major challenge in therapeutics. Multidrug-resistant tuberculosis (caused by *Mycobacterium tuberculosis*) has assumed an epidemic proportion in some parts of the world. Multidrug-resistant strains of *M. tuberculosis*, now known to be present in 50 countries, heighten the threat posed by untreatable and fatal human tuberculosis.

Because of the rapid dissemination of antibiotic resistance in pathogens, many of the antibiotics, which were highly effective earlier, became obsolete during the past few decades. The efficacy of antibiotic treatment is on the wane as a result of the emergence and

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dissemination of antibiotic resistance amongst the pathogens. This warrants the discovery of newer and more promising antibiotics with long-term efficacy against various life threatening infections. However, the steady discovery of antibiotics witnessed during 1940–1980 could not be sustained. In the last two decades, only one new antibiotic class viz the oxazolidinone was introduced in the year 1990 and all other entrants were just the variation of the existing one (Raghunath, 2008). Eventually, clinical management of antibiotic-resistant superbugs is assuming the shape of a gruesome problem because of the depleting antibiotic reserve (Norrby et al., 2005). The widespread apprehension that we might be heading to a situation similar to the pre-antibiotic era cannot be dismissed as a far-stretched imagination.

Imprudent and excessive use of antibiotics is highlighted as a major causative factor behind the setback. The idea dubbed as Antibiotic Paradox by Levy (1992) is corroborated by numerous evidences. In 1967, the first penicillin-resistant *Streptococcus pneumonia* was observed in Australia, and after 7 years another case of penicillin-resistant *Streptococcus pneumonia* was reported from the U.S. in a patient with pneumococcal meningitis (Doern et al., 2001). In 1980, 3–5% of *Streptococcus pneumonia* was estimated to be penicillin-resistant and by 1998, 34% of the *Streptococcus pneumonia* sampled was resistant to penicillin (Doern et al., 2001). Antibiotic resistance in other organisms reflects the same trend as observed in *Streptococcus pneumonia* against penicillin. Tetracycline resistance by normal human intestinal flora exploded from 2% in the 1950s to 80% in the 1990s (Shoemaker et al., 2001). Kanamycin, an antibiotic used in the 1950s, has become clinically obsolete as a result of the prevalence of kanamycin-resistant bacteria (e.g., *M. tuberculosis*, *Campylobacter jejuni*).

On the other hand, it is also a fact that antibiotic resistance is observed even in bacteria isolated from totally uninhabited and thinly populated places, where they are unlikely or least likely to come in contact with the antibiotics (Chattopadhyay and Grossart, 2010). It is evident that antibiotic resistance is an outcome of evolution and pre-exposure of microorganisms to antibiotics is not a pre-requisite for emergence of resistance. In a systematic analysis, the conjugative plasmids occurring in several bacteria belonging to Enterobacteriaceae obtained from Murray collection were found to belong to the same incompatibility group as those present in contemporary Enterobacteriaceae (Datta and Hughes, 1983). It was shown that emergence and spread of conjugative plasmids, which are known to be a major vehicle for transmission of resistance, were not fostered by the widespread use of antibiotics, rather these plasmids evolved by inclusion of resistance-conferring genes in pre-existing plasmids (Datta and Hughes, 1983; Hughes and Datta, 1983).

Despite intensive investigations continuing for the last few decades in a global scale, no significant achievement has been made so far in the prevention and control of resistance development. A recent database reveals the existence of more than 20,000 potential resistance genes of nearly 400 different types in the available bacterial genome sequences. The fact that the number of functional resistance determinants in pathogens is much smaller (Liu and Pop, 2009) provides no relief since the new generation of therapeutically useful antibiotics are reaching the market only in trickle.

The World Health Organization (WHO) has long recognized anti microbial resistance (AMR) as a growing global health threat, and the World Health Assembly, through several resolutions over two decades, has called upon Member States and the international community to take measures to curtail the emergence and spread of AMR.

## **EMERGENCE OF RESISTANCE PHENOTYPE**

Antibiotic resistance is known to occur both in the pre-antibiotic and antibiotic-era. The pre-antibiotic era constitutes the time before the introduction of sulphonamides in 1930. It is well established that resistance phenotype and the antibiotics in the pre-antibiotic era coexisted in the natural environment without facilitating the process of selection of the deadly resistant pathogens.

The antibiotic era started following its discovery of antibiotics and their use in different spheres of life. The use of high concentration of lethal dose of antibiotics as a consequence of human activity led to a major change in innate functional role to give rise to the emergence of antibiotic-resistant pathogens within a short span of time. Antibiotic resistance genes, which were once involved in other cellular functions before human intervention, have been subsequently selected for the resistance phenotype with increased use of antibiotics. They have been mobilized from the environmental genomic reservoirs, with the rapid dissemination into taxonomically divergent commensal and pathogenic bacteria. Metagenomic approach in conjunction with a pipeline for the *de novo* assembly of short-read sequence data from functional selections (termed PARFuMS) has shown the evidence of lateral transmission of five different antibiotic resistance genes along with different non-coding region as well as multiple mobilization sequences from environmental reservoir of soil bacteria to the clinical pathogens (Forsberg et al., 2012). Thus HGT or lateral gene transfer, which contributes significantly to the evolution, maintenance, and transmission of virulence in pathogenic bacteria, also plays a pivotal role in dissemination of antibiotic resistance conferring genes from the environment in clinical settings (Colomer-Lluch et al., 2011).

Epigenetic inheritance-based evolution of antibiotic resistance genes has been reported in an isogenic population of *Escherichia coli*, exposed to gradually increasing concentration of different antibiotics such as ampicillin, nalidixic acid, and tetracycline. The high frequency of survival on low antibiotic concentration could not be accounted for by the occurrence of random spontaneous mutation. Instead it suggested that the antibiotic resistance genes were acquired by epigenetic inheritance. High reversion of this resistance phenotype further proved that it was indeed a case of epigenetic inheritance which does not impart a stable phenotype due to maintenance of certain chromatin configuration or DNA methylation state (Adam et al., 2008).

There are ample evidences to suggest that anthropogenic factors (use of antibiotics by human both for therapeutic and non-therapeutic purposes and disposal of the unused antibiotic formulations) inflict significant changes on the natural flora of bacteria. But the actual concentrations of antibiotics (and other pharmaceuticals) that bacteria in the natural population is exposed to, remain unknown in most of the cases (Kümmerer, 2003, 2004).

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Its role in the emergence of antibiotic resistance still remains highly controversial (Bhullar et al., 2012).

In therapeutics, the pathogens are challenged with an overwhelmingly high concentration of a single or few antibiotics whereas the soil borne microbes exist in a complex microenvironment and encounter a number of simultaneously occurring stress factors in a network of multiple interactions. Probably, these interactions have a nullifying effect on each other thus favoring the evolutionary selection of antibiotic sensitivity over resistance (Chait et al., 2011). Therefore, the dynamics of antibiotic resistance in clinical settings is believed to be inflicted by anthropocentricfactors and is quite differentfrom naturally occurring resistance. Simulation of these natural niches may reveal the dynamics by which antibiotic resistance is disseminated across the microbial population.

#### **IMPLICATION OF SUBLETHAL CONCENTRATIONS OF ANTIBIOTICS IN DISSEMINATION OF RESISTANCE**

In nature, sub-inhibitory concentrations of antibiotics are probably encountered by bacteria more frequently than inhibitory concentrations. Most often bacteria are exposed to antibiotics at a sublethal concentration in certain clinical situations such as incomplete treatment of an infection, patient non-compliance, and reduced or limited drug accessibility to certain tissues (e.g., bone or cerebrospinal fluid; Bryskier, 2005). In the colon of a person taking antibiotics, sub-inhibitory concentrations would be experienced by colonic bacteria during the initial phase of treatment and at the end of treatment. Thus host tissues are exposed to a range of drug concentration starting from higher to a subinhibitory concentration. Therefore, micro-niches within the host, such as epidermis, lungs, and joints, may attain significantly lower drug concentrations than the plasma (Rybak, 2006). Outside clinical settings, sub-inhibitory condition is established because of the use of manure from livestock whose feed is supplemented with antibiotics and in consequence multiple drugs in a very low level find their way both to the soil and aqueous environment.

Due to such imprudent use of antibiotics, bacteria are quite likely to experience sublethal levels of antibiotics which have high implications in the spread of multidrug resistance. There is evidence that low level of antibiotics gives rise to mutagenesis in a wider range of antibacterial resistance genes and drug efflux systems resulting in multidrug resistance (Girgis et al., 2009). Subinhibitory concentrations of β-lactams were found to enhance the transfer of tetracycline resistance plasmids in *Staphylococcus aureus* by up to 1,000-fold (Barr et al., 1986). Ampicillin treatment of *Escherichia coli* was associated with the formation of norfloxacinresistant isolates with mutations in *gyr*A, *gyr*B, or the *acr*AB promoter (PacrAB) and kanamycin-resistant isolates with mutations in *rpsL* or *arcA* (A and B). It has been hypothesized that low level of bactericidal antibiotics give rise to reactive oxygen species (ROS) which leads to DNA damage-induced mutations thus facilitating the emergence of multidrug-resistant bacteria (Kohanski et al., 2010). Induction of prophage in animal feed by antibiotics was evidenced. It may contribute to dissemination of antibiotic resistance (Allen et al., 2011). Induction of shigella toxin-encoding bacteriophage by ciprofloxacin and enhanced Shiga toxin (Stx) production from *Escherichia coli* O157:H7 was demonstrated *in* *vitro* and *in vivo*. Hence antibiotic-induction of phage may also contribute to increased virulence (Zhang et al., 2000).

In many cases, exposure to a low-dose antibiotic is associated with mobile element-mediated dissemination of antibiotic resistance genes through HGT which is known to be enhanced by sub-MIC concentrations of tetracycline (Celli and Trieu-Cuot, 1998). Tetracycline at a sub-inhibitory concentration in the mating medium substantially enhanced Tn916 mediated conjugal transfer to the recipient *Bacillus thuringiensis* subsp. *israelensis* (Showsh and Andrews, 1992). Spread of antibiotic resistance genes between human colonic *Bacteroides* spp. is mediated by self-transmissible elements known as conjugative transposons (CTns). The exposure of donor *Bacteroides* cells to low concentration of tetracycline appeared to be a pre-requisite for the excision of the CTnDOT family of CTns from the chromosome and conjugal transfer of the excised elements. Virtually no transfer occurs without the tetracycline induction of donor cells (Stevens et al., 1993; Whittle et al., 2002). Low concentrations of tetracycline might increase the likelihood of HGT of integrated mobile elements, such as CTnDOT and NBU1. At sub-inhibitory tetracycline concentration, the excision is not associated with growth phase (Song et al., 2005). Mobilization of coresident non-conjugative plasmids (from *Bacteroides* strain to *Bacteroides* strain or *Escherichia coli*) by chromosomally encoded tetracycline conjugal elements (Valentine et al., 1988) was enhanced by 20–10,000-folds when the donor was pre-grown in a sub-inhibitory concentration of tetracycline (1 μg/ml). The similar stimulatory effect of tetracycline on conjugation transfer was also demonstrated for the conjugative transposon Tn925 (Torres et al., 1991).

These *in vitro* observations were validated *in vivo* in gnotobiotic mice where tetracycline at a sublethal concentration in drinking water could induce the frequency of conjugative transfer of the transposon Tn1545 from *Enterococcus faecalis* to *Listeria monocytogenes* in the digestive tract and there was an approximately 10-fold increase in the transposition (Doucet-Populaire et al., 1991). In gnotobiotic rats, selection for the resistant phenotype was the major factor causing higher numbers of Tn916 transconjugants in the presence of tetracycline (Bahl et al., 2004). Therefore, the enhancement of conjugal transfer of antibiotic resistance-carrying transposons in the presence of sub-inhibitory concentration of antibiotics is not only an *in vitro* phenomenon but also takes place in the human intestinal microbiome of animal models.

Adverse effect of some antibiotics on bacterial motility was reported earlier (Molinari et al., 1992; Kawamura-Sato et al., 2000). However, sub-inhibitory concentration of tobramycin was observed to increase motility of *Pseudomonas aeruginosa* and enhance the expression of virulence determinants of the organism (Linares et al., 2006).

### **ANTIBIOTIC RESISTANCE AND ITS ROLE IN NATURE**

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Antibiotic resistance can be defined in different ways: from the microbiological point of view, the resistance is defined as a phenotype which makes the microorganism less susceptible than other members of the same species irrespective of any level of resistance. On the contrary, when the resistance reaches a certain critical level so as to interfere with the pharmacotherapy of a clinical problem

caused by the bacterium, it is called clinical resistance (Cantón and Morosini, 2011).

In the present scenario, acquired clinical resistance of pathogens is a major concern in the healthcare management which results from genetic changes involving mutation or HGT. Besides acquired resistance dealt with in this article, some other types of antibiotic resistances are also observed in bacteria. For example in some cases tolerance of a bacterium to a certain antibiotic is dependent on its metabolic state. It exhibits susceptibility to the antibiotic in growing state but become resistant to the same antibiotic in stationary phase. The micro-environments in a biofilm also contribute to the differential susceptibility of bacteria embedded in different parts of the polysaccharide matrix. Swarming, a phenomenon induced in some bacteria in response to nitrogen limitation, confers resistance to some antibiotics (Martinez and Rojo, 2011). Resistance to a variety of antibiotics was observed in swarming cells of some bacteria. This observation together with the resistance observed in biofilm clearly indicates that antibiotic resistance is a manifestation of social behavior of bacteria (Lai et al., 2009). Accumulation of the alarmone (p) ppGpp was also reported to modulate antibiotic resistance. Involvement of this alarmone in persistence (resistance occurring in a small fraction of naturally occurring bacteria) is postulated (Jayaraman, 2009). This type of resistance, known as phenotypic resistance, significantly contributes to some problems in clinical management of some bacterial infections.

The most ancient resistance also known as intrinsic resistance (Sheldon, 2005) is species-specific and results from impermeability of the cells to the antibiotic or lack of target of an antibiotic to a certain organism. However, besides these passive factors, intrinsic resistance is also caused by some antibiotic-detoxifying determinants encoding chromosomally encoded β-lactamases, efflux pumps or target-protecting proteins. This antibiotic resistance is widespread in nature and this huge intrinsic resistome of bacteria consists of genes of varied phylogenetic origin This intrinsic resistome has a high degree of non-specificity and many of the proteins encoded by these genes are involved in the basic physiological functions in the natural ecosystem as demonstrated in *Pseudomonas aeruginosa*, the novel nosocomial Gram-negative pathogen of environmental origin (Fajardo et al., 2008). The presence of antibiotics acts as a switch in physiological function to the antibiotic-resistance phenotype. These resistance genes persist in bacteria even in antibiotic-free environment thus further substantiating their alternative role in bacterial physiology. Tet(L) and Tet(K), the specific tetracycline c-resistance determinants are known to catalyze Na+ and K+ exchange for protons (Krulwich et al., 2001). Tet(L), a tetracycline-efflux transporter, and MdfA, a multidrug-efflux transporter, both responsible for alkali tolerance offer a striking example in support of this hypothesis (Krulwich et al., 2005). In *M. tuberculosis* the P55 efflux pump plays an important role in oxidative stress response and *in vitro* cell growth in addition to contributing to its intrinsic resistance phenotype (Ramón-García et al., 2009).

It is obvious that bacteria evolve with antibiotic resistance determinants get selective advantage over their antibiotic-sensitive counterparts in presence of antibiotics. However, some of the multidrug resistance determinants confer resistance not only to antibiotics but also to a number of structurally unrelated compounds viz, ethidium bromide, quaternary ammonium compounds, the DNA-intercalating mutagen acridine, the anionic detergent sodium dodecyl sulfate and uncouplers. Hence they appear to have a greater role in bacterial physiology. They also confer resistance to some chemical substances produced by the host, e.g., bile acids. It is speculated that antibiotic resistance is an offshoot of some hitherto undefined physiological roles played by the determinants in these bacteria (Martinez and Rojo, 2011).

Most antibiotic-producing strains carry genes encoding resistance to the antibiotics that they produce (Hopwood, 2007; Tahlan et al., 2007) and these genes are usually found in the same gene cluster as the antibiotic biosynthesis pathway genes (Benveniste and Davies, 1973; Martin and Liras, 1989). The proximity of the antibiotic resistance gene with the antibiotic biosynthetic gene as evidenced in *Streptomyces coelicolor* (where the genes encoding export proteins are embedded in the actinorhodin biosynthesis pathway) led to the postulation that these resistance determinants could have some role in the biosynthetic pathway of antibiotics (Allen et al., 2010). Antibiotic resistance genes are frequently present even in non-producer strains (Yamashita et al., 1985). In consequence, bacteria have evolved with a diverse pool of genes (the "resistome") that protect them against the therapeutic dose of antibiotics. Gene orthologous to these have been identified on mobile genetic elements in resistant pathogens in clinical settings. These genes that make up this environmental resistome have the potential to be transferred to pathogens and indeed there is some evidence that at least some clinically relevant resistance genes have originated in environmental microbes (Wright, 2010).

## **ANCIENT ORIGIN OF ANTIBIOTIC RESISTANCE GENES**

It is well-established documented that the antibiotic resistance is a long-evolved trait in prokaryotes and the diverse pool of resistant genes co-evolved with antibiotics in non-clinical (natural) environment much before these have been used in human therapy. Most of the antibiotic producer organisms carry the respective antibiotic resistance genes and both the resistance and the antibiotic biosynthesis pathway genes are usually found in the same gene cluster (Allen et al., 2010).

In the evolutionary scale many antibiotics or their structurally related precursors are believed to be as old as amino acids. It has been suggested that antibiotics are more than 500 million years old, dating back to the Cambrian period and probably evolved at the same time vertebrate fish emerged (Baltz, 2008). The components of the antibiotics are believed to be even older and the postulation is substantiated by the occurrence of non-protein amino acids of peptide antibiotics in meteorites and other primordial sources (Johnson et al., 2008). Julian Davies at the University of British Columbia proposes that antibiotics could be some of the oldest biomolecules (Amábile-Cuevas, 2003).

Metagenomic analyses of ancient DNA from 30,000-yearold to Beringian permafrost sediments dating back to the late Pleistocene age demonstrated the presence of different antibiotic resistance genes encoding resistance to β-lactam, tetracycline, and glycopeptide antibiotics (D'Costa et al., 2011). Structural and functional studies of one of these ancient resistance genes, the *vanA*

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(vancomycin resistance operon) that encodes an ATP dependent D-alanyl-D-lactate ligase confirmed that it is essentially indistinguishable from VanA ligase associated with the recently discovered vancomycin resistance in the clinic.

Very recently, Lechuguilla Cave in New Mexico, cut off from the human activity for over four million years, was found to harbor culturable microbiomes, highly resistant to antibiotics. Some strains were found to be resistant to as many as 14 different commercially available antibiotics. Resistance was detected to a wide range of structurally different antibiotics including daptomycin, an antibiotic of last resort in the treatment of drug resistant Gram-positive pathogens. Enzyme-mediated mechanisms (e.g., glycosylation, phosphorylation) leading to resistance against both natural and semi-synthetic antibiotics were also discovered in some ancient bacteria. Characterization of macrolide kinase obtained from one of these resistant organisms revealed it to be related to a known family of kinases circulating in drug resistant pathogens at present (Bhullar et al., 2012).

In another study, molecular analysis of a metagenomic library from the cold-seep sediments of the deep sea Edison seamount (about 10,000 years old) also demonstrated the presence of TEMtype ESBLs (TEM-1 and TEM-116) suggesting that β-lactam resistance in microorganisms is likely to be present prior to the modern antibiotic era, and the diversity of TEM β-lactamases is not a recent phenomenon, rather it is the result of a very ancient evolution (Song et al., 2005). Therefore, despite the fact that increase in the use of an antibiotic is directly associated with increase in the frequency of resistant strains, it is obvious that exposure to antibiotics is not a pre-requisite for the emergence of resistance. Even the diversification of the antibiotic resistance appears to occur much before the human intervention as evidenced by the occurrence of the TEM-type ESBL in the 10,000 year-old sediments of the Edison seamount. But the prevalence of the resistance bacteria having TEM-1 and TEM-116 was as low as 0.3% (25 of 8,823) and 0.06% (5 of 8,823), respectively (Allen et al., 2009). Recent investigations revealed that the origin of both antibiotic resistance and the biosynthetic genes date back to the ancient period much before the human civilization began. Both antibiotics and the resistance phenotype existed together in nature for a long time.

There are many other cases of occurrence of antibiotic resistance genes in apparently antibiotic-free environments. The metagenomic studies of remote Alaskan soil revealed the presence of divergent β-lactamase genes and a bifunctional β-lactamase (Allen et al., 2009). These β-lactamases are more closely related to ancestral homologs compared to those isolated in clinical settings and are capable of conferring resistance on *Escherichia coli* despite the evolutionary distance. In another example of antibiotic resistance in environments not impacted by antibiotics, the phenotype of more than 60% of the Enterobacteriaceae isolates from a pristine freshwater environment was found to be multidrugresistant. An example of this evidence exists in the two strains of *Citrobacter freundii* that were collected prior to the antibiotic era (the 1920s) which carried ampC β-lactamase genes. The encoded AmpC β-lactamases were as active as the recent plasmid borne AmpC β-lactamases (class C) that were found in the antibiotic era (Barlow and Hall, 2002). Phylogenetic analysis of the genetically diversified serine β-lactamases suggests an ancient root for the antibiotic resistance genes (Hall and Barlow, 2003).

Even the Murray collection (procured before and after the introduction of antibiotics, i.e., between 1917 and 1952) exhibited ampicillin and tetracycline resistance in 11 out of 433 enterobacterial strains and the resistance was non-conjugative. These studies reveal that the determinants of antibiotic resistance existed in nature much before the human intervention. Millions of years ago, antibiotics and antibiotic biosynthetic pathways evolved suggesting that both antibiotics and the resistance genes are very ancient (Wright and Poinar, 2012). The presence of multidrug-resistant organisms even in this pristine environment reinforces the idea that the antibiotic resistome is ancient and omnipresent in the microbial genome.

## **CAN THE DOOMSDAY BE POSTPONED?**

In keeping with a policy package recommended by WHO in 2002 emphasis was given on the availability of proper facilities to ensure rapid testing of antibiotic resistance, regular documentation and sharing of the surveillance data, uninterrupted access to quality medical service, regulation on the sale of antibiotics, development of new diagnostic tests, and novel antimicrobials. But the problem continues to be unabated till now (Leung et al., 2011).

Recently, scientists (Marraffini and Sontheimer, 2008) from the Northwestern University have discovered a specific DNA sequence, called a CRISPR (clustered, regularly interspaced, short palindromic repeat) locus, which confer prokaryotes a type of acquired immunity against the acquisition of resistance genes by encoding a sequence-specific defense mechanism against bacteriophages and thus conferring an immunity toward HGT. Members of recently potentially virulent strains of enterococcal have been shown to lack complete CRISPR loci. Expression of the CRISPR interference has also been found to prevent the transfer of antibiotic resistance and virulent genes, not only *in vitro*, but also *in vivo*, during a pneumococcal infection (Bikard et al., 2012). "If this mechanism could be manipulated in a clinical setting, it would provide a means to limit the spread of antibiotic resistance genes and virulence factors in *Staphylococcus* and other bacterial pathogens," hopes Erik Sontheimer, Associate Professor at the Weinberg College of Arts and Sciences.

The new Eco-Evo drugs and strategies target prevention of the evolution and emergence of resistant bacteria in the environment. This might be achieved by using inhibitors against four P's viz, penetration (by using 7-valent conjugated vaccine, PC7 to prevent colonization of resistant serotype in human), promiscuity (using broad host range conjugation inhibitor), plasticity (recombinase inhibitor), and persistence (decontamination of high risk clones or mobile genetic elements) of resistant bacteria. This strategy appears implementable in view of the fact that preventive measures recommended in various steps are mostly adoptable. However, their long-term impact on the environment warrants rigorous testing before implementation (Baquero et al., 2011). In order to combat the looming crisis, a number of different other approaches have been adopted. Pharmaceutical companies are tapping new sources (samples obtained from tropical rain forests, myxobacteria, marine bacteria, extremophilic bacteria) for novel antibiotics. Development of antimicrobials against bacterial molecules,

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which were not targeted earlier (e.g., bacteria DNA polymerase III, the cell division protein FtsZ, fibronectin binding proteins) is aimed at. Genes indispensable for survival of pathogens are shortlisted in search of new targets. In another strategy, molecular techniques are being used to clone the antibiotic biosynthetic genes cluster of a strain into a different strain, ultimately to get a hybrid molecule with antibiotic activity. In another approach called directed evolution, libraries are developed with randomly mixed and matched gene clusters obtained from different antibiotic producer strains. These libraries are transformed into protoplasts and recombinants are screened for improved spectrum of antibiotic activity. Large number of new compounds synthesized on a solid support, are screened for activity in another technique called combinatorial chemistry. These new strategies are expected to generate new compounds with novel antimicrobial activities (Strohl, 1997).

Several secondary approaches like reduction of antibiotic consumption, preservation of existing therapeutics, development of new antibiotics, and development of new strategies like sensitization of pathogens (Toney et al., 1998) have been adopted. The formulation, augmentin, a combination of β-lactamase inhibitor (clavulanic acid) and amoxicillin, is being successfully used against a variety of penicillin-resistant pathogens.

#### **CONCLUSION**

It is apparent that antibiotics are endowed with multifaceted activities and the cell modulates their role. The dual nature of antibiotic acting both as signaling molecule as well as growth inhibitory molecule suggests that antibiotic resistance genes have

#### **REFERENCES**

L. (2012). Fidaxomicin inhibits spore production in *Clostridium difficile*. *Clin. Infect. Dis.* 55(Suppl. 2), S162– S169.

to co-evolve with antibiotics to safe guard the producer organism from antibiotic threat.

While it is obvious that dissemination of resistance-conferring genes has been facilitated following introduction of antibiotics into the clinical practice, we are yet to get sufficient evidence to conclude that human intervention is the only contributing factor behind the spread of resistance. Until and unless we generate required amount of information to fill up the gap of knowledge in this aspect, we cannot expect to devise measures which would effectively arrest the progress of the crisis.

Antibiotic resistance is a complex and continually evolving problem. A vast body of information has been engendered during the past 70 years on the antibiotic resistance and its dissemination. Comparative genomics, molecular genetics, combinatorial chemistry, and structural biology are being applied to explore new antibiotics but still the struggle is on. Several secondary approaches like reduction of antibiotic consumption, preservation of existing therapeutics, development of new antibiotics, and development of new strategies like sensitization of antibiotic-resistant organisms using QS inhibitors have been adopted but without much success.

Relatively rare genes that happened to confer antibiotic resistance were once involved in other cellular functions, but were selected for the resistance phenotype and mobilized from the environmental genomic reservoirs, with the rapid dissemination into taxonomically divergent commensal and pathogenic bacteria. This process was very rapid on an evolutionary scale and HGT, mediated by mobile genetic elements, played a prominent role in it. So it would be wise to plan a strategy to control the process of HGT to control subsequent transfer of resistance genes to pathogens.

*aureus*. *J. Antimicrob. Chemother.* 17, 409–413.

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acquisition during in vivo bacterial infection. *Cell Host Microbe* 12, 177–186.

resistance in nature? *Nat. Chem. Biol.* 8, 2–5.

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T., and Ohta, M. (2000). Effect of subinhibitory concentrations of macrolides on expression of flagellin in *Pseudomonas aeruginosa* and *Proteus mirabilis*. *Antimicrob. Agents Chemother.* 44, 2869–2872.