Acquired antibiotic resistance genes: an overview.

In this review an overview is given on antibiotic resistance (AR) mechanisms with special attentions to the AR genes described so far preceded by a short introduction on the discovery and mode of action of the different classes of antibiotics. As this review is only dealing with acquired resistance, attention is also paid to mobile genetic elements such as plasmids, transposons, and integrons, which are associated with AR genes, and involved in the dispersal of antimicrobial determinants between different bacteria.

Introduction

The discovery and production of (synthetic) antibiotics in the first half of the previous century has been one of medicine’s greatest achievements. The use of antimicrobial agents has reduced morbidity and mortality of humans and contributed substantially to human’s increased life span. Antibiotics are, either as therapeutic or as prophylactic agents, also widely used in agricultural practices.

The first discovered antimicrobial compound was penicillin (Flemming, 1929) a β-lactam antibiotic. Soon after this very important discovery, antibiotics were used to treat human infections starting with sulfonamide and followed by the aminoglycoside streptomycin and streptothricin (Domagk, 1935; Schatz and Waksman, 1944). Nowadays numerous different classes of antimicrobial agents are known and they are classified based on their mechanisms of action (Neu, 1992). Antibiotics can for instance inhibit protein synthesis, like aminoglycoside, chloramphenicol, macrolide, streptothricin, and tetracycline or interact with the synthesis of DNA and RNA, such as quinolone and rifampin. Other groups inhibit the synthesis of, or damage the bacterial cell wall as β-lactam and glycopeptide do or modify, like sulfonamide and trimethoprim, the energy metabolism of a microbial cell.

Upon the introduction of antibiotics it was assumed that the evolution of antibiotic resistance (AR) was unlikely. This was based on the assumption that the frequency of mutations generating resistant bacteria was negligible (Davies, 1994). Unfortunately, time has proven the opposite. Nobody initially anticipated that microbes would react to this assault of various chemical poisons by adapting themselves to the changed environment by developing resistance to antibiotics using such a wide variety of mechanisms. Moreover, their ability of interchanging genes, which is now well known as horizontal gene transfer (HGT) was especially unexpected. Later on it was discovered that the emergence of resistance actually began before the first antibiotic, penicillin, was characterized. The first β-lactamase was identified in Escherichia coli prior to the release of penicillin for use in medical practice (Abraham and Chain, 1940). Besides β-lactams, the aminoglycoside–aminocyclitol family was also one of the first groups of antibiotics to encounter the challenges of resistance (Wright, 1999; Bradford, 2001). Over the years it has been shown by numerous ecological studies that (increased) antibiotic consumption contributes to the emergence of AR in various bacterial genera (MARAN, 2005, 2007; NethMap, 2008). Some examples of the link between antibiotic dosage and resistance development are the rise of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). The initial appearance of MRSA was in 1960 (Jevons et al., 1963), whereas VRE were first isolated about 20 years ago (Uttley et al., 1988). Over the last decades they have remained a reason for concern, but additional public health threats in relation to resistant microorganisms have also arisen (see for example Cantón et al., 2008; Goossens, 2009; Allen et al., 2010).

Bacteria have become resistant to antimicrobials through a number of mechanisms (Spratt, 1994; McDermott et al., 2003; Magnet and Blanchard, 2005; Wright, 2005):

Permeability changes in the bacterial cell wall which restricts antimicrobial access to target sites,

Active efflux of the antibiotic from the microbial cell,

Enzymatic modification of the antibiotic,

Degradation of the antimicrobial agent,

Acquisition of alternative metabolic pathways to those inhibited by the drug,

Modification of antibiotic targets,

Overproduction of the target enzyme.

These AR phenotypes can be achieved in microorganisms by chromosomal DNA mutations, which alter existing bacterial proteins, through transformation which can create mosaic proteins and/or as a result of transfer and acquisition of new genetic material between bacteria of the same or different species or genera (Spratt, 1994; Maiden, 1998; Ochman et al., 2000).

There are numerous examples of mutation based resistance. For example, macrolide resistance can be due to nucleotide(s) base substitutions in the 23S rRNA gene. However, a similar resistance phenotype may also result from mutations within the ribosomal proteins L4 and L22 (Vester and Douthwaite, 2001). Single nucleotide polymorphisms (SNPs) can be the cause for resistance against the synthetic drugs quinolones, sulfonamides, and trimethoprim (Huovinen et al., 1995; Hooper, 2000; Ruiz, 2003) and mutations within the rpsL gene, which encodes the ribosomal protein S12, can result in a high-level streptomycin resistance (Nair et al., 1993). A frame shift mutation in the chromosomal ddl gene, encoding a cytoplasm enzyme d-Ala–d-Ala ligase, can account for glycopeptides resistance (Casadewall and Courvalin, 1999).

Acquired Resistance

This review deals with the description of acquired resistance against several classes of antibiotics. For each class the development of resistance is summarized along with the mechanisms of action. Furthermore an extensive summary is given of the resistance mechanisms and resistance genes involved.

Aminoglycoside

History and action mechanism

The aminoglycoside antibiotics initially known as aminoglycosidic aminocyclitols are over 60 years old (Siegenthaler et al., 1986; Begg and Barclay, 1995). In the early 1940s the first aminoglycoside discovered was streptomycin in Streptomyces griseus (Schatz and Waksman, 1944). Several years later, additional aminoglycosides were characterized from other Streptomyces species; neomycin and kanamycin in 1949 and 1957, respectively. Furthermore, in the 1960s gentamicin was recovered from the actinomycete Micromonospora purpurea. Because most aminoglycosides have been isolated from either Streptomyces or Micromonospora a nomenclature system has been set up based on their source. Aminoglycosides that are derived from bacteria of the Streptomyces genus are named with the suffix “-mycin,” while those which are derived from Micromonospora are named with the suffix “-micin.”

The first semi-synthetic derivatives were isolated in the 1970s. For example netilmicin is a derivative of sisomicin whereas amikacin is derived from kanamycin (Begg and Barclay, 1995; Davies and Wright, 1997).

Aminoglycosides are antimicrobials since they inhibit protein synthesis and/or alter the integrity of bacterial cell membranes (Vakulenko and Mobashery, 2003). They have a broad antimicrobial spectrum. Furthermore, they often act in synergy with other antibiotics as such it makes them valuable as anti-infectants.

Resistance mechanisms

Several aminoglycoside resistance mechanisms have been recognized; (I) Active efflux (Moore et al., 1999; Magnet et al., 2001), (II) Decreased permeability (Hancock, 1981; Taber et al., 1987), (III) Ribosome alteration (Poehlsgaard and Douthwaite, 2005), (IV) Inactivation of the drugs by aminoglycoside-modifying enzymes (Shaw et al., 1993). Intrinsic mechanisms, i.e., efflux pumps and 16S rRNA methylases but also chromosomal mutations can cause the first three resistance properties. In recent years acquired 16S rRNA methylases appear to have increased in importance (Galimand et al., 2005; Doi and Arakawa, 2007; Table 1). The first gene identified of a plasmid-mediated type of aminoglycoside resistance was armA (Galimand et al., 2003). To date five additional methylases have been reported, i.e., npmA, rmtA, rmtB, rmtC, and rmtD (Courvalin, 2008; Doi et al., 2008). Data regarding the 16S rRNA methylase genes are accumulated and provided at the website: www.nih.go.jp/niid/16s_database/index.html.

Table 1

Acquired Aminoglycoside resistance genes.

Gene name	Mechanism	Length (nt)	Accession number or reference	Coding region	Genera
aac(2′)-Ia	ACT	537	L06156	264..800	Providencia
aac(2′)-Ib	ACT	588	U41471	265..852	Mycobacterium
aac(2′)-Ic	ACT	546	U72714	373..918	Mycobacterium
aac(2′)-Id	ACT	633	U72743	386..1018	Mycobacterium
aac(2′)-Ie	ACT	549	NC_011896	3039059..3039607	Mycobacterium
aac(3)-I	ACT	465	AJ877225	5293..5757	Pseudomonas
aac(3)-Ia	ACT	534	X15852	1250..1783	Acinetobacter, Escherichia, Klebsiella, Salmonella, Serratia, Streptomyces
aac(3)-Ib	ACT	531	L06157	555..1085	Pseudomonas
aac(3)-Ib-aac(6′)-Ib	ACT	1,005	AF355189	1435..2439	Pseudomonas
aac(3)-Ic	ACT	471	AJ511268	1295..1765	Pseudomonas
aac(3)-Id	ACT	477	AB114632	104..580	Proteus, Pseudomonas, Salmonella, Vibrio
aac(3)-Ie	ACT	477	AY463797	8583..9059	Proteus, Pseudomonas, Salmonella, Vibrio
aac(3)-If	ACT	465	AY884051	61..525	Serratia, Pseudomonas
aac(3)-Ig	ACT	477	CP000282	2333620..2334096	Saccharophagus
aac(3)-Ih	ACT	459	CP000490	509912..510370	Paracoccus
aac(3)-Ii	ACT	459	CP000356	638262..638720	Sphingopyxis
aac(3)-Ij	ACT		CP000155		Hahella
aac(3)-Ik	ACT	444	BX571856	765853..766296	Staphylococcus
aac(3)-IIa	ACT	861	X51534	91..951	Acinetobacter, Enterobacter, Escherichia, Klebsiella, Pseudomonas, Salmonela
aac(3)-IIb	ACT	810	M97172	656..1465	Serratia
aac(3)-IIc	ACT	861	X54723	819..1679	Escherichia
aac(3)-IId	ACT	861	EU022314	1..861	Escherichia
aac(3)-IIe	ACT	861	EU022315	1..861	Escherichia
aac(3)-IIIa	ACT	816	X55652	1124..1939	Pseudomonas
aac(3)-IIIb	ACT	738	L06160	984..1721	Pseudomonas
aac(3)-IIIc	ACT	840	L06161	106..945	Pseudomonas
aac(3)-IVa	ACT	786	X01385	244..1029	Escherichia
aac(3)-Va
aac(3)-Vb
aac(3)-VIa	ACT	900	M88012	193..1092	Enterobacter, Escherichia, Salmonella
aac(3)-VIIa	ACT	867	M22999	493..1359	Streptomyces
aac(3)-VIIIa	ACT	861	M55426	466..1326	Streptomyces
aac(3)-IXa	ACT	846	M55427	274..1119	Micromonospora
aac(3)-Xa	ACT	855	AB028210	2711..3565	Streptomyces
aac(6′)	ACT	441	AY553333	1392..1832	Pseudomonas
aac	ACT	555	AJ628983	1985..2539	Pseudomonas
aac(6′)	ACT	402	DQ302723	81..482	Pseudomonas
aac(6′)	ACT	555	EU912537	2092..2646	Pseudomonas
aac(6′)-Ia	ACT	558	M18967	757..1314	Citrobacter, Escherichia, Klebsiella, Shigella
aac(6′)-Ib	ACT	606	M21682	380..985	Klebsiella, Proteus, Pseudomonas
aac(6′)-Ib-cr	ACT	519	EF636461	1124..1642	Enterobacter, Escherichia, Klebsiella, Pseudomonas, Salmonella
aac(6′)-Ic	ACT	441	M94066	1554..1994	Serratia
aac(6′)-Id	ACT	450	X12618	905..1354	Klebsiella
aac(6′)-Ie
aac(6′)-If	ACT	435	X55353	279..713	Enterobacter
aac(6′)-Ig	ACT	438	L09246	544..981	Acinetobacter
aac(6′)-Ih	ACT	441	L29044	352..792	Acinetobacter
aac(6′)-Ii	ACT	549	L12710	169..717	Enterococcus
aac(6′)-Ij	ACT	441	L29045	260..700	Acinetobacter
aac(6′)-Ik	ACT	438	L29510	369..806	Acinetobacter
aac(6′)-Il	ACT	522	Z54241	530..1051	Acinetobacter, Citrobacter
aac(6′)-Im	ACT	537	AF337947	1215..1751	Escherichia
aac(6′)-In	ACT	573	Wu et al. (1997)		Citrobacter
aac(6′)-Iq	ACT	552	AF047556	127..678	Klebsiella, Salmonella
aac(6′)-Ir	ACT	441	AF031326	1..441	Acinetobacter
aac(6′)-Is	ACT	441	AF031327	1..441	Acinetobacter
aac(6′)-It	ACT	441	AF031328	1..441	Acinetobacter
aac(6′)-Iu	ACT	441	AF031329	1..441	Acinetobacter
aac(6′)-Iv	ACT	441	AF031330	1..441	Acinetobacter
aac(6′)-Iw	ACT	441	AF031331	1..441	Acinetobacter
aac(6′)-Ix	ACT	441	AF031332	1..441	Acinetobacter
aac(6′)-Iy	ACT	438	AF144880	3452..3979	Salmonella
aac(6′)-Iz	ACT	462	AF140221	390..851	Stenotrophomonas
aac(6′)-Iaa	ACT	438	NC_003197	1707358..1707795	Salmonella
aac(6′)-Iad	ACT	435	AB119105	1..435	Acinetobacter
aac(6′)-Iae	ACT	552	AB104852	1935..2486	Pseudomonas, Salmonella
aac(6′)-Iaf	ACT	552	AB462903	1200..1751	Pseudomonas
aac(6′)-Iai	ACT	567	EU886977	544..1110	Pseudomonas
aac(6′)-I30	ACT	555	AY289608	1524..2078	Salmonella
aac(6′)-31	ACT	519	AJ640197	2474..2992	Acinetobacter
aac(6′)-32	ACT	555	EF614235	2247..2801	Pseudomonas
aac(6′)-33	ACT	555	GQ337064	1203..1757	Pseudomonas
aac(6′)-IIa	ACT	555	M29695	707..1261	Aeromonas, Klebsiella, Pseudomonas, Salmonella
aac(6′)-IIb	ACT	543	L06163	532..1074	Pseudomonas
aac(6′)-IIc	ACT	582	AF162771	62..643	Enterobacter, Klebsiella, Pseudomonas
aac(6′)-IId
aac(6′)-III
aac(6′)-IV	ACT	435	X55353	279..713	Enterobacter
aac(6′)-aph(2″)	NUT	1,440	M13771	304..1743	Enterococcus, Lactobacillus, Staphylococcus, Streptococcus
aacA29	ACT	381	AY139599	768..1148	Unknown
aacA43	ACT	564	HQ247816	639..1202	Klebsiella
aadA1	NUT	972	X02340	223..1194	Acinetobacter, Aeromonas, Enterobacter, Escherichia, Klebsiella, Proteus, Pseudomonas, Salmonella, Shigella, Vibrio
aadA1b	NUT	792	M95287	3320..4111	Pseudomonas, Serratia
aadA2	NUT	780	X68227	166..945	Acinetobacter, Aeromonas, Citrobacter, Enterobacter, Escherichia, Klebsiella, Proteus, Pseudomonas, Salmonella, Shigella, Staphylococcus, Vibrio, Yersinia
aadA3	NUT	792	AF047479	1296..2087	Escherichia
aadA4	NUT	789	Z50802	1306..2094	Acinetobacter, Aeromonas, Escherichia, Pseudomonas,
aadA5	NUT	789	AF137361	64..852	Acinetobacter, Aeromonas, Escherichia, Pseudomonas, Salmonella, Shigella, Staphylococcus, Vibrio
aadA6	NUT	846	AF140629	61..906	Pseudomonas
aadA7	NUT	798	AF224733	32..829	Escherichia, Salmonella, Vibrio
aadA8	NUT	792	AF326210	1..792	Klebsiella, Vibrio
aadA8b	NUT	792	AM040708	1174..1965	Escherichia
aadA9	NUT	837	AJ420072	26773..27609	Corynebacterium
aadA10	NUT	834	U37105	2807..3640	Pseudomonas
aadA11	NUT	846	AY144590	1..846	Pseudomonas, Riemerella
aadA12	NUT	792	AY665771	1..792	Escherichia, Salmonella, Yersinia
aadA13	NUT	798	AY713504	1..798	Escherichia, Pseudomonas, Yersinia
aadA14	NUT	786	AJ884726	540..1325	Pasteurella
aadA15	NUT	792	DQ393783	1800..2591	Pseudomonas
aadA16	NUT	846	EU675686	3197..4042	Escherichia, Klebsiella, Vibrio
aadA17	NUT	792	FJ460181	774..1565	Aeromonas
aadA21	NUT	792	AY171244	47..838	Salmonella
aadA22	NUT	792	AM261837	74..865	Escherichia, Salmonella
aadA23	NUT	780	AJ809407	119..898	Salmonella
aadA24	NUT	780	AM711129	1264..2043	Escherichia, Salmonella
aadC	NUT	477	V01282	225..701	Staphylococcus
aadD	NUT	771	AF181950	3176..3946	Staphylococcus
ant(2″)-Ia	NUT	543	X04555	1296..1829	Acinetobacter, Enterobacter, Escherichia, Klebsiella, Proteus, Pseudomonas, Salmonella, Serratia, Shigella, Vibrio
ant(3″)-Ih-aac(6′)-IId	NUT-ACT	1,392	AF453998	3555..4946	Serratia
ant(4′)-Ib	NUT	771	AJ506108	209..979	Bacillus
ant(4′)-IIa	NUT	759	M98270	145..903	Pseudomonas
ant(4′)-IIb	NUT	756	AY114142	1061..1816	Pseudomonas
ant(6)-Ia	NUT	909	AF330699	22..930	Enterococcus, Staphylococcus
ant(6)-Ib	NUT	858	FN594949	27482..28339	Campylobacter
ant(9)-Ia	NUT	783	X02588	331..1113	Enterococcus, Staphylococcus
ant(9)-Ib	NUT	768	M69221	271..1038	Enterococcus, Staphylococcus
aph(2″)-Ia
aph(2″)-Ib	PHT	900	AF337947	272..1171	Enterococcus, Escherichia
aph(2″)-Ic	PHT	921	U51479	196..1116	Enterococcus
aph(2″)-Id	PHT	906	AF016483	131..1036	Enterococcus
aph(2″)-Ie	PHT	906	AY743255	131..1036	Enterococcus
aph(3′)-Ia	PHT	816	J01839	1162..1977	Escherichia, Klebsiella, Pseudomonas, Salmonella
aph(3′)-Ib	PHT	816	M20305	779..1594	Escherichia
aph(3′)-Ic	PHT	816	X625115	410..1225	Acinetobacter, Citrobacter, Escherichia, Klebsiella, Salmonella, Serratia, Yersinia
aph(3′)-Id	PHT	816	Z48231	820..1635	Escherichia
aph(3′)-IIa	PHT	795	X57709	1..795	Escherichia, Pseudomonas, Salmonella
aph(3′)-IIb	PHT	807	X90856	388..1194	Pseudomonas
aph(3′)-IIc	PHT	813	AM743169	2377498..2378310	Stenotrophomonas
aph(3′)-III	PHT	795	M26832	604..1398	Bacillus, Campylobacter, Enterococcus, Staphylococcus, Streptococcus
aph(3′)-IV	PHT	789	X03364	277..1065	Bacillus
aph(3′)-Va	PHT	807	K00432	307..1113	Streptomyces
aph(3′)-Vb	PHT	792	M22126	373..1164	Streptomyces
aph(3′)-Vc	PHT	795	S81599	282..1076	Micromonospora
aph(3′)-Va	PHT	780	X07753	103..882	Acinetobacter, Pseudomonas
aph(3′)-VIb	PHT	780	AJ627643	4934..5713	Alcaligenes
aph(3′)-VIIa	PHT	753	M29953	131..1036	Campylobacter
aph(3′)-VIII	PHT	804	AF182845	1..804	Streptomyces
aph(3′)-XV	PHT	795	Y18050	4758..5552	Achromobacter, Citrobacter, Pseudomonas
aph(3′′)-Ia	PHT	819	M16482	501..1319	Streptomyces
aph(3′′)-Ib	PHT	801	AB366441	11310..12110	Enterobacter, Escherichia, Klebsiella, Pasteurella, Pseudomonas, Salmonella, Shigella, Yersinia, Vibrio
aph(4)-Ia	PHT	1,026	V01499	231..1256	Escherichia
aph(4)-Ib	PHT	999	X03615	232..1230	Streptomyces
aph(6)-Ia	PHT	924	AY971801	1..924	Streptomyces
aph(6)-Ib	PHT	924	X05648	382..1305	Streptomyces
aph(6)-Ic	PHT	801	X01702	485..1285	Escherichia, Pseudomonas, Salmonella
aph(6)-Id	PHT	837	M28829	866..1702	Enterobacter, Escherichia, Klebsiella, Pasteurella, Pseudomonas, Salmonella, Shigella, Yersinia, Vibrio
aph(7″)-Ia	PHT	999	X03615	232..1230	Streptomyces
aph(9)-Ia	PHT	996	U94857	151..1146	Legionella
aph(9)-Ib	PHT	993	U70376	7526..8518	Streptomyces
apmA	ACT	822	FN806789	2858..3682	Staphylococcus
armA	MET	774	AY220558	1978..2751	Acinetobacter, Citrobacter, Enterobacter, Escherichia, Klebsiella, Salmonella, Serratia
npmA	MET	660	AB261016	3069..3728	Escherichia
rmtA	MET	756	AB120321	6677..7432	Pseudomonas
rmtB	MET	756	AB103506	1410..2165	Enterobacter, Escherichia, Klebsiella, Pseudomonas, Serratia
rmtC	MET	846	AB194779	6903..7748	Proteus, Salmonella
rmtD	MET	744	DQ914960	8889..9632	Klebsiella, Pseudomonas
rmtD2	MET	744	HQ401565	14139..14882	Citrobacter, Enterobacter
rmtE	MET	822	GU201947	55..876	Escherichia
spc	MET	783	X02588	331..1113	Enterococcus, Staphylococcus
sph	NUT	801	X64335	6557..7354	Escherichia, Pseudomonas, Salmonella
str	NUT	849	X92946	18060..18908	Enterococcus, Staphylococcus, Lactococcus
sat2A	ACT	525	X51546	518..1042	Acinetobacter, Enterobacter, Escherichia, Klebsiella, Proteus, Pseudomonas, Salmonella, Shigella, Vibrio
sat3A	ACT	543	Z48231	221..763	Escherichia
sat4A	ACT	543	X92945	38870..39412	Campylobacter, Enterococcus, Staphylococcus, Streptococcus

This table was adapted from: Elbourne and Hall (.

ACT, Acetyltransferase; MET, Methyltransferase; NUT, Nucleotidyltransferase; PHT, Phosphotransferase.

AAlthough the .

The major encountered aminoglycoside resistance mechanism is the modification of enzymes. These proteins are classified into three major classes according to the type of modification: AAC (acetyltransferases), ANT (nucleotidyltransferases or adenyltransferases), APH (phosphotransferases; Shaw et al., 1993; Wright and Thompson, 1999; Magnet and Blanchard, 2005; Wright, 2005; Ramirez and Tolmansky, 2010). Within these classes, an additional subdivision can be made based on the enzymes different region specificities for aminoglycoside modifications: i.e., there are four acetyltransferases: AAC(1), AAC(2′), AAC(3), and AAC(6′); five nucleotidyltransferases: ANT(2″), ANT(3″), ANT(4′), ANT(6), and ANT(9) and seven phosphotransferases: APH(2″), APH(3′), APH(3″), APH(4), APH(6), APH(7″), and APH(9). Furthermore, there also exists a bifunctional enzyme, AAC(6′)–APH(2″), that can acetylate and phosphorylate its substrates sequentially (Shaw et al., 1993; Kotra et al., 2000). Table 1 displays the currently known aminoglycoside resistance genes. The action mechanisms of the determinants, the variety in gene lengths, accession numbers, and the distribution are all indicated. As can be deduced from the second column of Table 1, inconsistencies arose in the nomenclature of genes for aminoglycoside-modifying enzymes (Vakulenko and Mobashery, 2003). In some cases, genes were named according to the site of modification, followed by a number to distinguish between genes. Using a different nomenclature, for example, the genes for AAC(6′)-Ia and AAC(3)-Ia are referred to as aacA1 and aacC1, respectively. The nomenclature proposed by Shaw et al. (1993), who utilize the identical names for the enzymes and the corresponding genes, but the names of genes are in lowercase letters and italicized will be used in this review (see Table 1). According to this more convenient nomenclature, the genes for the AAC(6′)-Ia and AAC(3)-Ia enzymes are termed aac(6′)-Ia and aac(3)-Ia, respectively.

β-Lactam

As already mentioned before, the first antibiotic discovered was a β-lactam, i.e., penicillin. The Scottish scientist Alexander Flemming accidentally noticed the production of a substance with antimicrobial properties by the mold Penicillium notatum (Flemming, 1929). Over the last 30 years, many new β-lactam antibiotics have been developed. By definition, all β-lactam antibiotics have a β-lactam nucleus in their molecular structure. The β-lactam antibiotic family includes penicillins and derivatives, cephalosporins, carbapenems, monobactams, and β-lactam inhibitors (Williams, 1987; Bush, 1989; Petri, 2006; Queenan and Bush, 2007).

The core compound of penicillin, 6-aminopenicillanic acid (6-APA) is used as the main starting point for the preparation of numerous semi-synthetic derivatives. Although the cephalosporins are often thought of as new and improved derivatives of penicillin, they were actually discovered as naturally occurring substances (Petri, 2006). They can be grouped in first, second, third, and forth generation cephalosporins according to their spectrum of activity and timing of the agent’s introduction. In general, first generation agents have good Gram-positive activity and relatively modest coverage for Gram-negative organisms; second generation cephalosporins have increased Gram-negative and somewhat less Gram-positive activity; third generation antimicrobials have improved Gram-negative and variable Gram-positive activity; forth generation β-lactams have good true broad spectrum activity against both Gram-negatives and Gram-positives (Williams, 1987; Marshall et al., 2006). The second generation cephamycins are sometimes also grouped among the cephalosporins.

Because carbapenems diffuse easily in bacteria they are considered as broad spectrum β-lactam antibiotic. Imipenem and meropenem are well known representative. Even though monobactams do not contain a nucleus with a fused ring attached, they still belong to the β-lactam antibiotics. The β-lactamase inhibitors, like clavulanic acid, do contain the β-lactam ring, but they exhibit negligible antimicrobial activity and are used in combination with β-lactam antibiotics to overcome resistance in bacteria that secrete β-lactamase, which otherwise inactivates most penicillins.

The β-lactam antibiotics work by inhibiting the cell wall synthesis by binding to so-called penicillin-binding proteins (PBPs) in bacteria and interfering with the structural cross linking of peptidoglycans and as such preventing terminal transpeptidation in the bacterial cell wall. As a consequence it weakens the cell wall of the bacterium and finally results in cytolysis or death due to osmotic pressure (Kotra and Mobashery, 1998; Andes and Craig, 2005).

The β-lactamase inhibitors can be classified as either reversible or irreversible and the latter are considered more effective in that they eventually result in the destruction of enzymatic activity. Not surprisingly the inhibitors in clinical use, i.e., clavulanic acid, sulbactam, and tazobactam are all examples of irreversible β-lactamase inhibitors (Bush, 1988; Drawz and Bonomo, 2010).

The first bacterial enzyme reported to destroy penicillin was an AmpC β-lactamase of E. coli (Abraham and Chain, 1940). Nowadays, bacterial resistance against β-lactam antibiotics is increasing at a significant rate and has become a common problem. There are several mechanisms of antimicrobial resistance to β-lactam antibiotics. The most common and important mechanism through which bacteria can become resistant against β-lactams is by expressing β-lactamases, for example extended-spectrum β-lactamases (ESBLs), plasmid-mediated AmpC enzymes, and carbapenem-hydrolyzing β-lactamases (carbapenemases; Bradford, 2001; Jacoby and Munoz-Price, 2005; Paterson and Bonomo, 2005; Poirel et al., 2007; Queenan and Bush, 2007; Jacoby, 2009).

The β-lactamase family has been subdivided either based on functionality or molecular characteristics. Initially, before genes were routinely sequenced various biochemical parameters were determined of the different β-lactamases which allowed classification of this AR determinants family into four groups (Bush et al., 1995; Wright, 2005). Groups 1, 2, and 4 are serine-β-lactamases, while members of group 3 are metallo-β-lactamases. Classification based on molecular characteristics, i.e., amino acid homology has also resulted in four major groups, the so-called Ambler classes A–D, which correlate well with the functional scheme but lack details concerning the enzymatic activity. Ambler classes A, C, and D include the β-lactamases with serine at their active site, whereas Ambler class B β-lactamases are all metallo-enzymes who require zinc as a metal cofactor for their catalytic activities (Ambler, 1980; Bradford, 2001; Paterson and Bonomo, 2005; Wright, 2005; Poirel et al., 2007, 2010; Bush and Jacoby, 2010; Drawz and Bonomo, 2010). In this review the Ambler classification will be used (Table 2).

Table 2

β-Lactamases and ESBLs families.

Amber class A β-lactamases and ESBLs	Number of variants*	Amber class B β-lactamases and MBLs	Number of variants*	Amber class C β-lactamases and ESBLs	Number of variants*	Amber class D β-lactamases and ESBLs	Number of variants*
blaACI	1	blaB	13	blaACCa	4	ampH	1
blaAER	1	blaCGB	2	blaACTa	9	ampS	1
blaAST	1	blaDIM	1	blaBIL	1	blaLCR	1
blaBEL	3	blaEBR	1	blaBUT	2	blaNPS	1
blaBES	1	blaGIM	1	blaCFEa	1	blaOXAa	219
blaBIC	1	blaGOB	18	blaCMG	1	loxA	1
blaBPS	5	blaIMPa	30	blaCMYa	72
blaCARB	8	blaINDa	7	blaDHAa	8
blaCKO	5	blaJOHN	1	blaFOXa	10
blaCME	2	blaMUS	1	blaLATa	1
blaCTX-Ma	119	blaNDM	6	blaLENc	24
blaDES	1	blaSPM	1	blaMIRa	5
blaERP	1	blaTUS	1	blaMOR	1
blaFAR	2	blaVIMa	30	blaMOXa	8
blaFONA	6	cepA	7	blaOCH	7
blaGESa,b	17	cfiA	16	blaOKP-Ac	16
blaHERA	8	cphA	8	blaOKP-Bc	20
blaIMI	3	imiH	1	blaOXYc	23
blaKLUAd	12	imiS	1	blaTRU	1
blaKLUCd	2			blaZEG	1
blaKLUG	1			cepH	1
blaKLUY	4
blaKPCa	11
blaLUT	6
blaMAL	2
blaMOR	1
blaNMC-A	1
blaPERa	7
blaPME	1
blaPSE	4
blaRAHN	2
blaROB	1
blaSED	1
blaSFC	1
blaSFO	1
blaSHVa	141
blaSMEa	3
blaTEMa	187
blaTLA	1
blaTOHO	1
blaVEBa	7
blaZ	1
cdiA	1
cfxA	6
cumA	1
hugA	1
penA	1

*Last update: June 17th, 2011.

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In addition to the production of β-lactamases resistance can also be due to possession of altered PBPs. Since β-lactams cannot bind as effectively to these altered PBPs, the antibiotic is less effective at disrupting cell wall synthesis. The PBPs are thought to be the ancestors of the naturally occurring chromosomally mediated β-lactamase in many bacterial genera (Bradford, 2001).

Although plasmid-encoded penicillinase arose much earlier in Gram-positives in Staphylococcus aureus, due to the use of penicillin (Aarestrup and Jensen, 1998), the first plasmid-mediated β-lactamase, TEM-1, was described in the early 1960s in Gram-negatives (Datta and Kontomichalou, 1965). Currently over 1,150 chromosomal, plasmid, and transposon located β-lactamases are currently known (Bush and Jacoby, 2010; Drawz and Bonomo, 2010; Table 2).

Based on their activity to hydrolyze a small number or a variety of β-lactams the enzymes can be subdivided into narrow-, moderate-, broad-, and ESBLs. A commonly used definition specifies that broad spectrum β-lactamases are capable to provide resistance to the penicillins and cephalosporins and are not inhibited by inhibitors such as clavulanic acid and tazobactam. The ESBLs confer resistance to the penicillins, first-, second-, and third-generation cephalosporins and aztreonam, but not to carbapenems and are inhibited by β-lactamase inhibitors. In recent years acquired AR genes encoding ESBLs have become a major concern (Bradford, 2001). In time the parent enzymes blaTEM-1, blaTEM-2, and blaSHV-1 have undergone amino acid substitutions (point mutations) evolving to the ESBLs, starting with blaTEM-3 and blaSHV-2 (Bradford, 2001). Additional mutations at critical amino acids important for catalysis resulted in over 140 currently known SHV and TEM ESBL variants. In addition, plasmid-encoded class C β-lactamases or AmpC determinants, like blaCMY have also caught people’s awareness (Jacoby, 2009). Furthermore, in the past decade CTX-M enzymes have become very prevalent ESBLs, both in nosocomial and in community settings (Cantón and Coque, 2006).

Table 2 illustrates the size and diversity of the group of β-lactamases and ESBLs. The vast and still increasing number of (broad spectrum) β-lactamases and ESBLs has become a problem for the nomenclature for novel genes. Names have been assigned according to individual preference rather than according to systematic procedures (Bush, 1989). Fortunately, an authoritative website has been constructed on the nomenclature of ESBLs hosted by Jacoby and Bush.

Chloramphenicol

In 1947, the first chloramphenicol, originally referred to as chloromycetin, was isolated from Streptomyces venezuelae (Ehrlich et al., 1947). Probably because chloramphenicol is a molecule with a rather simple structure only a small number of synthetic derivates have been synthesized without adverse effects on antimicrobial activity (Schwarz et al., 2004). In azidamfenicol two chlorine atoms (−Cl2) are replaced by an azide group. Substitution of the nitro group (−NO2), by a methyl–sulfonyl residue (−SO2CH3) resulted in the synthesis of thiamphenicol, whereas in the fluorinated thiamphenicol derivative florfenicol the hydroxyl group (−OH) is replaced with fluorine (−F).

Chloramphenicol is a highly specific and potent inhibitor of protein synthesis through its affinity for the peptidyltransferase of the 50S ribosomal subunit of 70S ribosomes (Schwarz et al., 2004). Due to its binding to this enzyme the antibiotic prevents peptide chain elongation. The substrate spectrum of chloramphenicol includes Gram-positive and Gram-negative, aerobic and anaerobic bacteria. Chloramphenicol analogs including the fluorinated derivative florfenicol have a similar spectrum of activity.

Resistance mechanism

The first and still most frequently encountered mechanism of bacterial resistance to chloramphenicol is enzymatic inactivation by acetylation of the drug via different types of chloramphenicol acetyltransferases (CATs; Murray and Shaw, 1997; Schwarz et al., 2004; Wright, 2005). CATs are able to inactivate chloramphenicol as well as thiamphenicol and azidamfenicol, however, due to its structural modification florfenicol is resistant to inactivation by these enzymes. Consequently, chloramphenicol resistant strains, in which resistance is exclusively based on the activity of CAT, are susceptible to florfenicol. There are two defined types of genes coding CATs which distinctly differ in their structure: i.e., the classical catA determinants and the novel, also known as xenobiotic CATs, encoded by catB variants (Table 3). Besides the inactivating enzymes, there are also reports on other chloramphenicol resistance systems, such as inactivation by phosphotransferases, mutations of the target site, permeability barriers, and efflux systems (Schwarz et al., 2004). Of the latter mechanism, cmlA and floR are the most commonly known (Bissonnette et al., 1991; Briggs and Fratamico, 1999). The presence of a cmlA gene will result in resistance to chloramphenicol, but susceptibility to florfenicol. In contrast, floR will give rise to a chloramphenicol and florfenicol resistance phenotype. Inconsistencies in the nomenclature arose, like with many other AR genes, due to the increasing number of chloramphenicol resistance determinants. Schwarz et al. (2004) suggested a unified nomenclature. Table 3 represents the currently known chloramphenicol/florfenicol resistance genes. Some characteristics which are mentioned in Table 3 are mechanism of action, diverse gene lengths, accession numbers, and the distribution.

Table 3

Acquired chloramphenicol resistance genes.

Group	Gene	Gene(s) included	Mechanism	Length (nt)	Accession number	Coding region	Genera
Type A-1	catA1	cat, catI, pp-cat	Inactivating enzyme	660	V00622	244..903	Acinetobacter, Escherichia, Klebsiella, Salmonella, Serratia, Shigella
Type A-2	catA2	cat, catII	Inactivating enzyme	642	X53796	187..828	Aeromonas, Agrobacterium, Escherichia, Haemophilus, Photobacterium, Salmonella
Type A-3	catA3	cat, catIII	Inactivating enzyme	642	X07848	272..913	Actinobacillus, Edwardsiella, Klebsiella, Mannheimia, Pasteurella, Shigella
Type A-4	Cat		Inactivating enzyme	654	M11587	880..1533	Proteus
Type A-5	Cat		Inactivating enzyme	663	P20074*	1002758..1003420	Streptomyces
Type A-6	cat86		Inactivating enzyme	663	K00544	145..807	Bacillus
Type A-7	cat(pC221)	cat, catC	Inactivating enzyme	648	X02529	2267..2914	Bacillus, Enterococcus, Lactobacillus, Staphylococcus, Streptococcus
Type A-8	cat(pC223)	cat	Inactivating enzyme	648	AY355285	1000..1647	Enterococcus, Lactococcus, Listeria, Staphylococcus, Streptococcus
Type A-9	cat(pC194)	cat, cat-TC	Inactivating enzyme	651	NC_002013	1260..1910	Bacillus, Enterococcus, Lactobacillus, Staphylococcus, Streptococcus
Type A-10	Cat		Inactivating enzyme	687	AY238971	1055..1741	Bacillus
Type A-11	catP	catD	Inactivating enzyme	624	U15027	2953..3576	Clostridium, Neisseria
Type A-12	catS		Inactivating enzyme	492§	X74948	1..492	Streptococcus
Type A-13	Cat		Inactivating enzyme	624	M35190	309..932	Aeromonas, Campylobacter
Type A-14	Cat		Inactivating enzyme	651	S48276	479..1129	Listonella, Photobacterium, Proteus
Type A-15	catB		Inactivating enzyme	660	M93113	145..804	Clostridium
Type A-16	catQ		Inactivating enzyme	660	M55620	459..1118	Clostridium, Streptococcus
Type B-1	catB1	cat	Inactivating enzyme	630	M58472	148..777	Agrobacterium
Type B-2	catB2		Inactivating enzyme	633	AF047479	5957..6589	Acinetobacter, Aeromonas, Bordetella, Escherichia, Klebsiella, Pasteurella, Pseudomonas, Salmonella
Type B-3	catB3	catB4, catB5, catB6, catB8	Inactivating enzyme	633	AJ009818	883..1515	Acinetobacter, Aeromonas, Bordetella, Enterobacter, Escherichia, Klebsiella, Kluyvera, Morganella, Pseudomonas, Salmonella, Serratia, Shigella
Type B-4	catB7		Inactivating enzyme	639	AF036933	177..815	Pseudomonas
Type B-5	catB9		Inactivating enzyme	630	AF462019	27..656	Vibrio
Type B-6	catB10		Inactivating enzyme	633	AF878850	1197..1829	Pseudomonas
Type E-1	cmlA1	cmlA, cmlA2, cmlA4, cmlA5, cmlA6, cmlA7, cmlA8, cmlA10, cmlB	Efflux	1,260	M64556	601..1860	Acinetobacter, Aeromonas, Arcanobacterium, Enterobacter, Escherichia, Klebsiella, Pseudomonas, Salmonella, Serratia, Staphylococcus
Type E-2	cml		Efflux	903	M22614	427..1335	Escherichia
Type E-3	floR	cmlA-like, flo, pp-flo, cmlA9	Efflux	1,215	AF071555	4445..5659	Acinetobacter, Aeromonas, Bordetella, Pasteurella, Salmonella, Stenotrophomonas, Vibrio
Type E-4	fexA		Efflux	1,428	AJ549214	177..1604	Bacillus, Staphylococcus
Type E-5	cml		Efflux	1,179	X59968	508..1686	Corynebacterium, Pseudomonas
Type E-6	cmlv		Efflux	1,311	U09991	28..1338	Staphylococcus
Type E-7	cmrA	cmr	Efflux	1,176	Z12001	993..2168	Uncultured
Type E-8	cmr	cmx	Efflux	1,176	U85507	3518..4693	Acinetobacter, Escherichia, Klebsiella, Salmonella, Serratia, Shigella
	cfr		Inactivating enzyme	1,050	AJ579365	6290..7339	Aeromonas, Agrobacterium, Escherichia, Haemophilus, Photobacterium, Salmonella
	pexA		Efflux	1,248	HM537013	24055..25302	Actinobacillus, Edwardsiella, Klebsiella, Mannheimia, Pasteurella, Shigella

Adapted from Partridge et al. (.

Glycopeptide

In the late 1950s, the first glycopeptide, vancomycin was introduced in a clinical setting. Vancomycin was isolated as a fermentation product from a soil bacterium, Streptomyces orientalis, displaying antimicrobial activity (McCormick et al., 1956). Nearly 30 years later followed another glycopeptide antibiotic, teicoplanin (Parenti et al., 1978). Currently, four groups of glycopeptides are recognized, i.e., vancomycin type, avoparcin type, ristocetin type, and teicoplanin type. (Yao and Crandall, 1994). Among them, vancomycin and teicoplanin are the only two therapeutics currently used against Gram-positive microorganisms. During the 1990s, an association between the use of avoparcin and the occurrence of glycopeptide-resistant enterococci (GRE), more commonly designated VRE, in farm animals was demonstrated (Aarestrup, 1995; Klare et al., 1995). As a consequence avoparcin was banned as a growth promoter in all European Union countries in 1997.

Glycopeptides have an unusual mode of action. Instead of inhibiting an enzyme, they bind to a substrate. To be more specific, the molecular target of these glycopeptide antibiotics is the d-alanyl–d-alanine (d-Ala–d-Ala) terminus of the cell wall peptidoglycan precursor. After the glycopeptides are bound to their target, they inhibit the subsequent transglycosylation reaction by steric hindrance. (Gao, 2002; Klare et al., 2003).

The introduction of antibiotics into clinical setting is usually followed by the fairly rapid emergence of resistant bacteria. In this respect, vancomycin was somewhat atypical, because for almost 30 years following its introduction, resistance to this glycopeptide was reported only rarely and appeared to have little clinical significance. However, in the late 1980s, the emergence of acquired glycopeptides resistance was recognized for the first time (Leclercq et al., 1988; Johnson et al., 1990). This vancomycin resistance resulted from the production of modified peptidoglycan precursors ending in d-Ala–d-Lac (VanA, VanB, and VanD) or d-Ala–d-Ser (VanC, VanE, and VanG), to which glycopeptides exhibit low binding affinities. Classification of glycopeptide resistance is based on the primary sequence of the structural genes for the resistance-mediating ligases. The vanA and vanB operons are located on plasmids or on the chromosome, whereas the vanC1, vanC2/3, vanD, vanE, and vanG have so far been found exclusively on the chromosome (Gao, 2002; Klare et al., 2003; Depardieu et al., 2007). Currently, resistance to the glycopeptides, vancomycin, and teicoplanin or both, has been detected in six, all Gram-positive bacterial genera: Enterococcus, Erysipelothrix, Lactobacillus, Leuconostoc, Pediococcus, and Staphylococcus (Woodford et al., 1995).

Macrolide–Lincosamide–Streptogramin B

The first macrolide, erythromycin A, was discovered in the early 1950s (McGuire et al., 1952). The main structural component of this molecule is a large lactone ring to which amino and/or neutral sugars are attached by glycosidic bonds. To address the limitations of erythromycin, like chemical instability, poor absorbance, and bitter taste, newer 14-, 15-, and 16-membered ring macrolides such as clarithromycin and the azalide, azithromycin, have been developed (Kirst, 2002; Roberts, 2002).

Macrolides have a similar mode of antibacterial action and comparable antibacterial spectra as two other antibiotic classes, i.e., lincosamides and streptogramins B. Consequently, these antibiotics, although chemically distinct, have been clustered together as Macrolide–Lincosamide–Streptogramin B (MLS) antibiotics (Roberts, 2002). Nowadays this class of antibiotics should even be extended due to the development of various synthetic drugs. The ketolides (Zhanel et al., 2002; Ackermann and Rodloff, 2003) and oxazolidinones (Diekema and Jones, 2000) can be grouped together with the MLS antimicrobial agents which results in the MLSKO family of antibiotics (Roberts, 2008).

Macrolides, lincosamides, and streptogramins B all inhibit protein synthesis by binding to the 50S ribosomal subunit of bacteria (Weisblum, 1995; Roberts, 2002).

Shortly after the introduction of erythromycin into clinical setting in the 1950s, bacterial resistance to this antibiotic was reported for the first time in staphylococci (Weisblum, 1995). Since then a large number of bacteria have been identified that are resistant to MLS due to the presence of various different genes. The AR determinants responsible include rRNA methylases, efflux, and inactivating genes (Roberts et al., 1999; Roberts, 2008). The latter group can be further subdivided in esterases, lyases, phosphorylases, and transferases (Table 4).

Table 4

Acquired macrolide–lincosamide–streptogramin B (MLS) resistance genes.

Gene	Gene(s) included	Mechanism	Length (nt)	Accession number	Coding region	Genera
car(A)		Efflux	1,656	M80346	411..2066	Streptomyces
cfr		rRNA methylase	1,050	AM408573	10028..11077	Staphylococcus
cmr		Other	1,380	U43535	646..2025	Corynebacterium
ere(A)		Inactivating enzymeA	1,221	AY183453	2730..3950	Citrobacter, Enterobacter, Escherichia, Klebsiella, Pantoea, Providencia, Pseudomonas, Serratia, Staphylococcus, Stenotrophomonas, Vibrio
ere(B)		Inactivating enzymeA	1,260	X03988	383..1642	Acinetobacter, Citrobacter, Enterobacter, Escherichia, Klebsiella, Proteus, Pseudomonas, Staphylococcus
ere(C)		Inactivating enzymeA	1,257	FN396877	943..2199	Klebsiella
erm(A)	erm(TR)	rRNA methylase	732	X03216	4551..5282	Aggregatibacter, Bacteroides, Enterococcus, Helcococcus, Peptostreptococcus, Prevotella, Staphylococcus, Streptococcus
erm(B)	erm(2), erm(AM), erm(AMR), erm(BC), erm(BP), erm(BZ), erm(IP), erm(P)	rRNA methylase	738	M36722	714..1451	Aggregatibacter, Acinetobacter, Aerococcus, Arcanobacterium, Bacillus, Bacteroides, Citrobacter, Corynebacterium, Clostridium, Enterobacter, Escherichia, Eubacterium, Enterococcus, Fusobacterium, Gemella, Haemophilus, Klebsiella, Lactobacillus, Micrococcus, Neisseria, Pantoea, Pediococcus, Peptostreptococcus, Porphyromonas, Proteus, Pseudomonas, Ruminococcus, Rothia, Serratia, Staphylococcus, Streptococcus, Treponema, Wolinella
erm(C)	erm(IM), erm(M)	rRNA methylase	735	M19652	988..1722	Aggregatibacter, Actinomyces, Bacillus, Bacteroides, Corynebacterium, Eubacterium, Enterococcus, Haemophilus, Lactobacillus, Micrococcus, Neisseria, Prevotella, Peptostreptococcus, Staphylococcus, Streptococcus, Wolinella
erm(D)	erm(J), erm(K)	rRNA methylase	864	M29832	430..1293	Bacillus, Salmonella
erm(E)	erm(E2)	rRNA methylase	1,146	X51891	190..1335	Bacteroides, Eubacterium, Fusobacterium, Ruminococcus, Shigella, Streptomyces
erm(F)	erm(FS), erm(FU)	rRNA methylase	801	M14730	241..1041	Aggregatibacter, Actinomyces, Bacteroides, Clostridium, Corynebacterium, Eubacterium, Enterococcus, Fusobacterium, Gardnerella, Haemophilus, Lactobacillus, Mobiluncus, Neisseria, Porphyromonas, Prevotella, Peptostreptococcus, Ruminococcus, Shigella, Selenomonas, Staphylococcus, Streptococcus, Treponema, Veillonella, Wolinella
erm(G)		rRNA methylase	735	M15332	672..1406	Bacillus, Bacteroides, Catenibacterium, Lactobacillus, Prevotella, Porphyromonas, Staphylococcus
erm(H)	car(B)	rRNA methylase	900	M16503	244..1143	Streptomyces
erm(I)	mdm(A)	rRNA methylase		–		Streptomyces
erm(N)	tlr(D)	rRNA methylase	876	X97721	160..1035	Streptomyces
erm(O)	lrm, srm(A)	rRNA methylase	783	M74717	40..822	Streptomyces
erm(Q)		rRNA methylase	774	L22689	262..1035	Aggregatibacter, Bacteroides, Clostridium, Staphylococcus, Streptococcus, Wolinella
erm(R)		rRNA methylase	1,023	M11276	333..1355	Arthrobacter
erm(S)	erm(SF), tlr(D)	rRNA methylase	960	M19269	460..1419	Streptomyces
erm(T)	erm(GT), erm(LF)	rRNA methylase	735	M64090	168..902	Enterococcus, Lactobacillus, Streptococcus
erm(U)	lrm(B)	rRNA methylase	837	X62867	361..1197	Streptomyces
erm(V)	erm(SV)	rRNA methylase	780	U59450	397..1176	Eubacterium, Fusobacterium, Streptomyces
erm(W)	myr(B)	rRNA methylase	936	D14532	1039..1974	Micromonospora
erm(X)	erm(CD) erm(Y)	rRNA methylase	855	M36726	296..1150	Arcanobacterium, Bifidobacterium, Corynebacterium, Propionibacterium
erm(Y)	erm(GM)	rRNA methylase	735	AB014481	556..1290	Staphylococcus
erm(Z)	srm(D)	rRNA methylase	849	AM709783	2817..3665	Streptomyces
erm(30)	pikR1	rRNA methylase	1,011	AF079138	1283..2293	Streptomyces
erm(31)	pikR2	rRNA methylase	969	AF079138	154..1122	Streptomyces
erm(32)	tlr(B)	rRNA methylase	843	AJ009971	1790..2632	Streptomyces
erm(33)		rRNA methylase	732	AJ313523	163..894	Staphylococcus
erm(34)		rRNA methylase	846	AY234334	355..1200	Bacillus
erm(35)		rRNA methylase	801	AF319779	33..833	Bacteroides
erm(36)		rRNA methylase	846	AF462611	186..1031	Micrococcus
erm(37)	erm(MT)	rRNA methylase	540	AE000516	2229013..
2229552	Mycobacterium
erm(38)		rRNA methylase	1,161	AY154657	63..1223	Mycobacterium
erm(39)		rRNA methylase	741	AY487229	2153..2893	Mycobacterium
erm(40)		rRNA methylase	756	AY570506	2035..2790	Mycobacterium
erm(41)		rRNA methylase	522	EU590124	258..779	Mycobacterium
erm(42)	erm(MI)	rRNA methylase	906	FR734406	1..906	Pasteurella, Photobacterium
lmr(A)		Efflux	1,446	X59926	318..1763	Streptomyces
lnu(A)	lin(A)	Inactivating enzymeC	486	M14039	413..898	Clostridium, Lactobacillus, Staphylococcus
lnu(B)	lin(B)	Inactivating enzymeC	804	AJ238249	127..930	Clostridium, Enterococcus, Staphylococcus, Streptococcus
lnu(C)		Inactivating enzymeC	495	AY928180	1150..1644	Streptococcus
lnu(D)		Inactivating enzymeC	495	EF452177	19..513	Streptococcus
lnu(F)	lin(F), lin(G)	Inactivating enzymeC	822	EU118119	1030..1851	Escherichia, Salmonella
lsa(A)	abc-23	Efflux	1,497	AY225127	41..1537	Enterococcus
lsa(B)	orf3	Efflux	1,479	AJ579365	4150..5628	Staphylococcus
lsa(C)		Efflux	1,479	HM990671	5193..6671	Gardnerella, Streptococcus
mdf(A)		Other	1,233	Y08743	1..1233	Escherichia, Shigella
mdt(A)		Other	1,257	X92946	10534..11790	Lactococcus
mef(A)		Efflux	1,218	U70055	314..1531	Acinetobacter, Bacteroides, Citrobacter, Clostridium, Corynebacterium, Enterococcus, Enterobacter, Escherichia, Fusobacterium, Gemella, Klebsiella, Lactobacillus, Micrococcus, Morganella, Neisseria, Pantoea, Providencia, Proteus, Ralstonia, Pseudomonas, Salmonella, Serratia, Staphylococcus, Streptococcus, Stenotrophomonas
mef(B)		Efflux	1,230	FJ196385	11084..12313	Escherichia
mef(E)		Efflux	1,218	U83667	1..1218	Enterococcus, Fusobacterium, Gemella, Granulicatella, Staphylococcus, Streptococcus
mef(G)		Efflux	1,218	DQ445270	1..1218	Streptococcus
mph(A)	mph(K)	Inactivating enzymeD	906	D16251	1626..2531	Aeromonas, Escherichia, Citrobacter, Enterobacter, Klebsiella, Pantoea, Pseudomonas, Proteus, Serratia, Shigella, Stenotrophomonas
mph(B)	mph(B)	Inactivating enzymeD	909	D85892	1159..2067	Escherichia, Enterobacter, Proteus, Pseudomonas
mph(C)	mph(BM)	Inactivating enzymeD	900	AF167161	5665..6564	Staphylococcus, Stenotrophomonas
mph(D)		Inactivating enzymeD	840§	AB048591	1..840	Escherichia, Klebsiella, Pantoea, Proteus, Pseudomonas, Stenotrophomonas
mph(E)	mph, mph1, mph2	Inactivating enzymeD	884	AY522431 AF550415 DQ839391	22181..23064	Citrobacter, Escherichia
mre(A)		Efflux	936	U92073	119..1054	Streptococcus
msr(A)	msr(B), msr(SA)	Efflux	1,467	X52085	343..1809	Corynebacterium, Enterobacter, Enterococcus, Gemella, Pseudomonas, Staphylococcus, Streptococcus
msr(C)		Efflux	1,479	AY004350	496..1974	Enterococcus
msr(D)	mel, orf5	Efflux	1,464	AF274302	2462..3925	Acinetobacter, Bacteroides, Citrobacter, Clostridium, Corynebacterium, Enterococcus, Enterobacter, Escherichia, Gemella, Fusobacterium, Klebsiella, Morganella, Neisseria, Proteus, Providencia, Pseudomonas, Ralstonia, Staphylococcus, Streptococcus, Serratia, Stenotrophomonas
msr(E)	mel	Efflux	1,476	AY522431	20650..22125	Citrobacter, Escherichia
ole(B)		Efflux	1,710	L36601	1421..3130	Streptomyces
ole(C)		Efflux	978	L06249	1528..2505	Streptomyces
srm(B)		Efflux	1,653	X63451	558..2210	Streptomyces
tlc(C)		Efflux	1,647	M57437	277..1923	Streptomyces
vat(A)		Inactivating enzymeC	660	L07778	258..917	Staphylococcus
vat(B)		Inactivating enzymeC	639	U19459	67..705	Enterococcus, Staphylococcus
vat(C)		Inactivating enzymeC	639	AF015628	1307..1945	Staphylococcus
vat(D)	sat(A)	Inactivating enzymeC	630	L12033	162..791	Enterococcus
vat(E)	sat(G), vat(E-3)– vat(E-8)	Inactivating enzymeC	645	AF139725	63..707	Enterococcus, Lactobacillus
vat(F)		Inactivating enzymeC	666	AF170730	70..735	Yersinia
vat(G)		Inactivating enzymeC	651	GQ205627	3037..3687	Enterococcus
vga(A)	vga	Efflux	1,569	M90056	909..2477	Staphylococcus
vga(A)LC	vga	Efflux	1,569	DQ823382	1..1569	Staphylococcus
vga(B)		Efflux	1,659	U82085	629..2287	Enterococcus, Staphylococcus
vga(C)	vga(D)	Efflux	1,578	GQ205627	1394..2971	Enterococcus
vgb(A)	vgb	Inactivating enzymeB	900	M20129	641..1540	Enterococcus, Staphylococcus
vgb(B)		Inactivating enzymeB	888	AF015628	399..1286	Staphylococcus

Adapted from .

The most common mechanism of MLS resistance is due to the presence of rRNA methylases, encoded by the erm genes. These enzymes methylate the adenine residue(s) resulting in MLS resistance. The methylated adenine prevents the binding of the drugs from binding to the 50S ribosomal subunit. The other two mechanisms efflux pumps and inactivating genes are encoded by msr and ere determinants, respectively.

Because currently over 60 MLS resistance genes are recognized a nomenclature for naming these genes has been proposed that considers the same rules developed for identifying and naming new tetracycline resistance genes (see below; Roberts et al., 1999; Roberts, 2008). Table 4 represents the MLS acquired resistance genes. The genes included, the resistance mechanism, diverse gene lengths and accession number, and their distribution are displayed in this table.

Quinolone

In 1962, during the process of synthesis and purification of chloroquine (an antimalarial agent), a quinolone derivative, nalidixic acid, was discovered which possessed bactericidal activity against Gram-negatives (Lescher et al., 1962). The second generation quinolones arose when it became clear that the addition of a fluoride atom at position 6 of a quinolone molecule, creating a fluoroquinolone, greatly enhanced its biological activity. During the 1980s, various fluoroquinolones were developed, e.g., ciprofloxacin, norfloxacin, and ofloxacin. These fluoroquinolones demonstrated a broadened antimicrobial spectrum, including some Gram-positives (Wolfson and Hooper, 1989; Hooper, 2000; King et al., 2000).

In the 1990s, further alterations resulted in the third-generation (fluoro)quinolones, e.g., levofloxacin and sparfloxacin, showing potent activity against both Gram-negative and Gram-positive microbes. The new compounds, such as trovafloxacin, also show promising activity against anaerobic bacteria (Hooper, 2000; King et al., 2000).

Quinolones inhibit the action of DNA gyrase and topoisomerase IV, two enzymes essential for bacterial DNA replication and as a result the microbes are killed. (Hooper, 1995, 2000). DNA gyrase is a tetrameric enzyme composed of 2 GyrA and 2 GyrB subunits. The topoisomerase IV has a similar structure, comprised of 2 A and 2 B subunits, encoded by parC and parE, respectively. The four genes coding for the subunits of these enzymes are the targets for resistance mutations (see below).

For decades, the mechanisms of resistance to quinolones were believed to be only chromosome-encoded, however, recently three plasmid-mediated resistance mechanisms have been reported (Robicsek et al., 2006a; Courvalin, 2008; Martínez-Martínez et al., 2008). The chromosome-encoded resistance result in either a decreased outer-membrane permeability related to porin loss, to the (over)expression of naturally occurring efflux pumps or mutations of the molecular targets DNA gyrase and topoisomerase IV (Hooper, 2000; Ruiz, 2003; Jacoby, 2005). In the latter case mutations occur at specific “quinolone resistance determining regions” (QRDR) in the genes gyrA, gyrB, parC, and parE encoding the subunits of DNA gyrase and topoisomerase IV. Not surprisingly this QRDR is situated on the DNA-binding surface of the enzymes (Jacoby, 2005).

Although the possibility of the existence of plasmid-mediated resistance was already suggested in 1990 (Courvalin, 1990), the first actually identified plasmid-mediated quinolone resistance gene, a qnr determinant, which encodes for a protein that protects DNA gyrase and type IV topoisomerase from quinolone inhibition, was reported nearly a decade later (Martínez-Martínez et al., 1998).

Currently five families of qnr genes have been reported; qnrA (7), qnrB (39), qnrC (1), qnrD (1), and qnrS (4). The number in between brackets indicates the variants known of each type (Jacoby et al., 2008; Cattoir and Nordmann, 2009; Cavaco et al., 2009; Strahilevitz et al., 2009; Torpdahl et al., 2009). Because of the increasing number of qnr genes a database has been constructed and will be maintained to assign further allele numbers to novel variants. Very recently an additional family has been described, qnrAS in the fish pathogen Aliivibrio salmonicida (Sun et al., 2010). Table 5 describes all known qnr families and their variants, together with the gene lengths, accession numbers, and in which bacterial genera they have been identified so far.

Table 5

Acquired quinolone resistance genes.

Gene*	Length (nt)	Accession number	Coding region	Genera
qepA	1,536	AB263754	7052..8587	Escherichia
qepA2	1,536	EU847537	1672..3207	Escherichia
qnrA1	657	AY070235	303..959	Citrobacter, Enterobacter, Escherichia, Klebsiella, Shigella
qnrA2	657	AY675584	1..657	Klebsiella, Shewanella
qnrA3	657	DQ058661	1..657	Shewanella
qnrA4	657	DQ058662	1..657	Shewanella
qnrA5	657	DQ058663	1..657	Shewanella
qnrA6	657	DQ151889	1..657	Proteus
qnrA7	657	GQ463707	1..657	Shewanella
qnrAS	657	FM178379	1699484..1700140	Aliivibrio
qnrB1	645	DQ351241	37..681	Enterobacter, Escherichia, Klebsiella
qnrB2	645	DQ351242	1..645	Citrobacter, Enterobacter,, Klebsiella, Salmonella
qnrB3	645	DQ303920	37..681	Escherichia
qnrB4	645	DQ303921	4..648	Citrobacter, Enterobacter, Escherichia, Klebsiella
qnrB5	645	DQ303919	37..681	Enterobacter, Salmonella
qnrB6	645	EF520349	37..681	Enterobacter, Escherichia, Klebsiella, Pantoea
qnrB7	645	EU043311	1..645	Enterobacter, Klebsiella
qnrB8	645	EU043312	1..645	Citrobacter, Enterobacter
qnrB9	645	EF526508	1..645	Citrobacter
qnrB10	645	DQ631414	37..681	Citrobacter, Enterobacter, Escherichia, Klebsiella
qnrB11	645	EF653270	4..648	Citrobacter
qnrB12	645	AM774474	2435..3079	Citrobacter
qnrB13	645	EU273756	37..681	Citrobacter
qnrB14	645	EU273757	37..681	Citrobacter
qnrB15	645	EU302865	37..681	Citrobacter
qnrB16	645	EU136183	37..681	Citrobacter
qnrB17	645	AM919398	37..681	Citrobacter
qnrB18	645	AM919399	37..681	Citrobacter
qnrB19	645	EU432277	1..645	Escherichia, Klebsiella, Salmonella
qnrB20	645	AB379831	37..681	Escherichia, Klebsiella
qnrB21	645	FJ611948	1..645	Escherichia
qnrB22	645	FJ981621	37..681	Citrobacter
qnrB23	645	FJ981622	37..681	Citrobacter
qnrB24	645	HM192542	37..681	Citrobacter
qnrB25	645	HQ172108	1..645	Citrobacter
qnrB26	645	HM439644	1..645	Citrobacter
qnrB27	645	HM439641	1..645	Citrobacter
qnrB28	645	HM439643	1..645	Citrobacter
qnrB29	645	HM439649	37..681	Citrobacter
qnrB30	645	HM439650	37..681	Citrobacter
qnrB31	645	HQ418999	1..681	Klebsiella
qnrB32– qnrB39 not public yet
qnrC	666	EU917444	1717..2382	Proteus
qnrD	645	EU692908	1..645	Salmonella
qnrS1	657	AB187515	9737..10393	Enterobacter, Escherichia, Klebsiella, Proteus, Salmonella, Shigella
qnrS2	657	DQ485530	1..657	Aeromonas, Salmonella
qnrS3	657	EU077611	1..656	Escherichia
qnrS4	657	FJ418153	1..657	Salmonella

*Last update: June 17th 2011.

The second type of plasmid located quinolone resistant gene is a cr variant of aac(6′)-Ib, aac(6′)-Ib-cr, responsible for low-level ciprofloxacin resistance. It encodes an aminoglycoside acetyltransferase, called AAC(6′)-Ib-cr which has two amino acid changes, Trp102Arg and Asp179Tyr. These substitutions are responsible for the enzyme’s ability to acetylate ciprofloxacin (Park et al., 2006; Robicsek et al., 2006b; Strahilevitz et al., 2009).

The third mechanism is qepA, a plasmid-mediated efflux pump which can extrude hydrophilic fluoroquinolones, e.g., ciprofloxacin and enrofloxacin (Périchon et al., 2007; Yamane et al., 2007). A variant of this resistance pump, QepA2, was identified in an E. coli isolate from France (Cattoir et al., 2008).

Streptothricin

In the early days of the antibiotics era screening for new compound resulted in the discovery of a Streptomyces lavendulae isolate which inhibited growth of Gram-negative as well as Gram-positive bacteria. Isolation of the active antimicrobial substance resulted in the identification of streptothricin (Waksman and Woodruff, 1942). Delayed toxicity prevents streptothricin’s use in man, but it is effective in preventing animal infections.

Streptothricins consist of three moieties: gulosamine, streptolidin, and a β-lysine peptide chain. Since, the discovery of the streptothricin, six analogs have been reported, streptothricin A–F. The analogs differ from the parent molecule in the number of β-lysine residues (Keeratipibul et al., 1983; Tschäpe et al., 1984).

The streptothricins are potent inhibitors of bacterial protein synthesis, via direct binding to ribosomes. They also cause misreading of mRNA codons, although they are unrelated to other drugs that cause translational ambiguity, like the aminoglycosides (Tschäpe et al., 1984).

Since streptothricin is inactivated by acetylation in its producer it is not surprising that the identified resistance mechanisms are acetyltransferases. The first streptothricin resistant bacterium identified was an E. coli isolate from a rectal swab of pigs under streptothricin F treatment. The AR gene was localized on a transferable plasmid (Tschäpe et al., 1984). Currently three different streptothricin acetyltransferases are recognized, sat2–sat4 (Partridge and Hall, 2005; see Table 1).

Sulfonamide

Sulfonamides belong to the oldest introduced synthetic drugs. They were first used in 1932 (Domagk, 1935; Sköld, 2001). A number of different sulfonamides have been developed of which the most commonly used nowadays is sulfamethoxazole. Moreover, since 1968, the combination of trimethoprim and sulfamethoxazole (called co-trimoxazole) has been used extensively because a combination of both drugs at certain concentrations has a synergetic bactericidal effect, it reduces selection of AR to either drug and associated costs (Roberts, 2002; Grape, 2006).

A sulfonamide, with its structural analogy to p-aminobenzoic acid, which is involved in the biosynthetic pathway leading to folic acid, competitively inhibits the enzyme dihydropteroate synthase (DHPS). This protein is part of the next to last step of the folate biosynthetic pathway that is required for thymine production and bacterial cell growth (Sköld, 2000, 2001; Roberts, 2002).

Resistance to sulfonamide among pathogenic bacteria appeared quite soon after its introduction into clinical practice in the 1930s (Sköld, 2001). Since sulfonamides are synthetic antibacterial agents, naturally occurring enzymes degrading, or modifying this drug were not to be expected. However, chromosomal sulfonamide resistance occurs, mostly low level, by mutations in the folP gene encoding DHPS (Huovinen et al., 1995; Sköld, 2000, 2001; Grape, 2006).

Acquired sulfonamide resistance was discovered in the 1960s, but the plasmid-mediated genes were characterized later on in the 1980s as sul1 and sul2 (Swedberg and Sköld, 1983; Rådström and Swedberg, 1988; Sundström et al., 1988). Currently three plasmid-borne drug-resistant variants of the DHPS enzymes are known; besides the two genes mentioned above also sul3 has been identified (Perreten and Boerlin, 2003).

Tetracycline

The first tetracycline antibiotic was characterized in 1948 as chlortetracycline from Streptomyces aureofaciens (Chopra et al., 1992; Chopra and Roberts, 2001). In consecutive decades additional tetracyclines were identified either as naturally occurring molecules mostly in Streptomyces species (e.g., oxytetracycline, tetracycline) or products of semi-synthetic approaches (e.g., doxycycline, minocycline; Chopra et al., 1992; Hunter and Hill, 1997; Chopra and Roberts, 2001).

Tetracyclines were the first major group to which the term “broad spectrum” was applied (Chopra and Roberts, 2001). Because of this spectrum of activity, their relative safety, and low cost, tetracyclines have been used widely throughout the world and are second after penicillin in world consumption. This class of antibiotic can be separated into two groups, typical, (e.g., chlortetracycline, doxycycline, minocycline, oxytetracycline, and tetracycline) and atypical tetracyclines (e.g., anhydrotetracycline and 6-thiatetracycline), see below (Rasmussen et al., 1991; Oliva and Chopra, 1992; Chopra and Roberts, 2001).

Initially, it was thought that tetracyclines and most of its derivatives are antimicrobial agents only because they inhibit the growth of microbes by entering the bacterial cell, interacting with the ribosomes, and consequently blocking protein synthesis, the so-called typical tetracyclines (Speer et al., 1992; Roberts, 2002). However, Oliva and Chopra (1992) suggested an additional mode of action. Certain tetracycline derivatives are poor inhibitors of protein synthesis and appear to bind ribosomes inefficiently or not at all, in stead they interact with the bacterial membrane (Rasmussen et al., 1991; Chopra, 1994).

Prior to the mid-1950s, the majority of commensals and pathogens were susceptible to tetracycline. However, in 1953 the first tetracycline resistant bacteria were isolated (Watanabe, 1963). The resistance mechanisms for the tetracycline class of antibiotics fall in three categories; energy-dependent efflux pumps, ribosomal protection proteins (RPPs), or enzymatic inactivation.

A novel tetracycline resistance determinant is identified as unique if it shares <79% amino sequence identity with all previously described genes. Initially, letters of the Roman alphabet have been used to name tetracycline resistance determinants. However, the number of tet genes has reached the end of the alphabet and to accommodate new genes, a nomenclature employing numerals for future determinants was introduced (Levy et al., 1999). Moreover, also naturally occurring hybrid tetracycline resistance genes exist. A simple, descriptive nomenclature for these mosaic tet determinants has been proposed incorporating the designations of the known tet genes classes forming the hybrid, e.g., tet(O/W) and tet(O/W/O; Levy et al., 2005; Stanton et al., 2005; van Hoek et al., 2008).

There are currently over 40 different acquired tetracycline resistance determinants recognized, i.e., 38 tet (tetracycline resistance) and 3 otr (oxytetracycline resistance) genes, additionally 1 tcr gene has been identified (Roberts, 1996, 2005; Brown et al., 2008; see Table 6). Among these 25 of the tet, 2 of the otr genes and the only tcr determinant code for efflux pumps, whereas 10 tet and 1 otr code for a RPP. The enzymatic inactivation mechanism can be attributed to 3 tet genes. The tet(U) determinant represents an unknown tetracycline resistance mechanism since its sequence does not appear to be related to either efflux or RPPs, nor to the inactivation enzymes (Table 6). The efflux and RPP encoding genes are found in members of Gram-positive, Gram-negative, aerobic, as well as anaerobic bacterial species. In contrast the enzymatic tetracycline inactivation mechanism has so far only been identified in Gram-negatives (Table 6). The tet(M) has the broadest host range of all tetracycline resistance genes, whereas tet(B) gene has the widest range among the Gram-negative microbes. In recent years published data indicate that there are increasing numbers of Gram-negative bacteria that carry “Gram-positive tet genes” (Roberts, 2002).

Table 6

Acquired tetracycline resistance genes.

Gene	Mechanism	Length (nt)	Accession number	Coding region	Genera
otr(A)	Ribosomal protection	1,992	X53401	349..2340	Mycobacterium, Streptomyces
otr(B)	Efflux	1,692	AF079900	40..1731	Mycobacterium, Streptomyces
otr(C)	Efflux	1,056	AY509111	324..1379	Streptomyces
tcr	Efflux	1,539	D38215	516..2054	Streptomyces
tet(A)	Efflux	1,200	X00006	1328..2527	Acinetobacter, Aeromonas, Bordetella, Chryseobacterium, Citrobacter, Edwardsiella, Enterobacter, Escherichia, Flavobacterium, Klebsiella, Laribacter, Plesiomonas, Proteus, Pseudomonas, Salmonella, Serratia, Shigella, Variovorax, Veillonella, Vibrio
tetA(P)	Efflux	1,263	L20800	1063..2325	Clostridium
tet(B)	Efflux	1,206	J01830	1608..2813	Acinetobacter, Actinobacillus, Aeromonas, Aggregatibacter, Brevundimonas, Citrobacter, Enterobacter, Erwinia, Escherichia, Haemophilus, Klebsiella, Mannheimia, Moraxella, Neisseria, Pantoea, Pasteurella, Photobacterium, Plesiomonas, Proteus, Providencia, Pseudomonas, Roseobacter, Salmonella, Serratia, Shigella, Treponema, Vibrio, Yersinia
tetB(P)	Ribosomal protection	1,959	L20800	2309..4267	Clostridium
tet(C)	Efflux	1,191	X01654	86..1276	Aeromonas, Bordetella, Chlamydia, Citrobacter, Enterobacter, Escherichia, Francisella, Halomonas, Klebsiella, Proteus, Pseudomonas, Roseobacter, Salmonella, Serratia, Shigella, Vibrio
tet(D)	Efflux	1,185	X65876	1521..2705	Aeromonas, Alteromonas, Citrobacter, Edwardsiella, Enterobacter, Escherichia, Halomonas, Klebsiella, Morganella, Pasteurella, Photobacterium, Proteus, Salmonella, Shewanella, Shigella, Vibrio, Yersinia
tet(E)	Efflux	1,218	L06940	21..1238	Aeromonas, Alcaligenes, Escherichia, Flavobacterium, Plesiomonas, Proteus, Providencia, Pseudomonas, Roseobacter, Serratia, Vibrio
tet(G)	Efflux	1,128	AF071555	6644..7771	Acinetobacter, Brevundimonas, Escherichia, Fusobacterium, Mannheimia, Ochrobactrum, Pasteurella, Proteus, Providencia, Pseudomonas, Roseobacter, Salmonella, Shewanella, Vibrio
tet(H)	Efflux	1,203	U00792	716..1918	Acinetobacter, Actinobacillus, Mannheimia, Moraxella, Pasteurella
tet(J)	Efflux	1,197	AF038993	1084..2280	Escherichia, Morganella, Proteus
tet(K)	Efflux	1,380	M16217	305..1684	Bacillus, Clostridium, Enterococcus, Eubacterium, Haemophilus, Lactobacillus, Listeria, Mycobacterium, Nocardia, Nocardia, Peptostreptococcus, Staphylococcus, Streptococcus, Streptomyces
tet(L)	Efflux	1,377	D00006	189..1565	Acinetobacter, Actinobacillus, Actinomyces, Bacillus, Bifidobacterium, Citrobacter, Clostridium, Enterobacter, Enterococcus, Escherichia, Flavobacterium, Fusobacterium, Geobacillus, Kurthia, Lactobacillus, Listeria, Mannheimia, Morganella, Mycobacterium, Nocardia, Ochrobactrum, Oceanobacillus, Paenibacillus, Pasteurella, Pediococcus, Peptostreptococcus, Proteus, Pseudomonas, Rahnella, Salmonella, Sporosarcina, Staphylococcus, Streptococcus, Streptomyces, Variovorax, Veillonella, Virgibacillus
tet(M)	Ribosomal protection	1,920	U08812	1981..3900	Abiotrophia, Acinetobacter, Actinomyces, Aerococcus, Aeromonas, Afipia, Arthrobacter, Bacillus, Bacterionema, Bacteroides, Bifidobacterium, Brachybacterium, Catenibacterium, Clostridium, Corynebacterium, Edwardsiella, Eikenella, Enterobacter, Enterococcus, Erysipelothrix, Escherichia, Eubacterium, Flavobacterium, Fusobacterium, Gardnerella, Gemella, Granulicatella, Haemophilus, Kingella, Klebsiella, Kurthia, Lactobacillus, Lactococcus, Listeria, Microbacterium, Mycoplasma, Neisseria, Paenibacillus, Pantoea, Pasteurella, Peptostreptococcus, Photobacterium, Prevotella, Pseudoalteromonas, Pseudomonas, Ralstonia, Selenomonas, Serratia, Shewanella, Staphylococcus, Streptococcus, Streptomyces, Ureaplasma, Veillonella, Vibrio
tet(O)	Ribosomal protection	1,920	M18896	207..2126	Actinobacillus, Aerococcus, Anaerovibrio, Bifidobacterium, Butyrivibrio, Campylobacter, Clostridium, Enterococcus, Eubacterium, Fusobacterium, Gemella, Lactobacillus, Megasphaera, Mobiluncus, Neisseria, Peptostreptococcus, Psychrobacter, Staphylococcus, Streptococcus
tet(Q)	Ribosomal protection	1,926	Z21523	362..2287	Anaerovibrio, Bacteroides, Capnocytophaga, Clostridium, Eubacterium, Fusobacterium, Gardnerella, Lactobacillus, Mitsuokella, Mobiluncus, Neisseria, Peptostreptococcus, Porphyromonas, Prevotella, Ruminococcus, Selenomonas, Streptococcus, Subdoligranulum, Veillonella
tet(S)	Ribosomal protection	1,926	L09756	447..2372	Enterococcus, Lactobacillus, Lactococcus, Listeria, Staphylococcus, Streptococcus, Veillonella
tet(T)	Ribosomal protection	1,956	L42544	478..2433	Lactobacillus, Streptococcus
tet(U)	Unknown	318	U01917	413..730	Enterococcus, Staphylococcus, Streptococcus
tet(V)	Efflux	1,260	AF030344	462..1721	Mycobacterium
tet(W)	Ribosomal protection	1,920	AJ222769	3687..5606	Acidaminococcus, Actinomyces, Arcanobacterium, Bacillus, Bacteroides, Bifidobacterium, Butyrivibrio, Clostridium, Fusobacterium, Lactobacillus, Megasphaera, Mitsuokella, Neisseria, Porphyromonas, Prevotella, Roseburia, Selenomonas, Staphylococcus, Streptococcus, Streptomyces, Subdoligranulum, Veillonella
tet(X)	Enzymatic	1,167	M37699	586..1752	Bacteroides, Sphingobacterium
tet(Y)	Efflux	1,176	AF070999	1680..2855	Aeromonas, Escherichia, Photobacterium
tet(Z)	Efflux	1,155	AF121000	11880..13034	Corynebacterium, Lactobacillus
tet(30)	Efflux	1,185	AF090987	1130..2314	Agrobacterium
tet(31)	Efflux	1,233	AJ250203	1651..2883	Aeromonas
tet(32)	Ribosomal protection	1,920	DQ647324	181..2100	Enterococcus, Eubacterium, Clostridium, Streptococcus
tet(33)	Efflux	1,224	AJ420072	22940..24163	Corynebacterium
tet(34)	Enzymatic	465	AB061440	306..770	Aeromonas, Pseudomonas, Serratia, Vibrio
tet(35)	Efflux	1,110	AF353562	2213..3322	Stenotrophomonas, Vibrio
tet(36)	Ribosomal protection	1,923	AJ514254	2534..4456	Bacteroides, Clostridium, Lactobacillus
tet(37)	Enzymatic	327	AF540889	1..327	Uncultured
tet(38)	Efflux	1,353	AY825285	1..1353	Staphylococcus
tet(39)	Efflux	1,188	AY743590	749..1936	Acinetobacter
tet(40)	Efflux	1,221	AM419751	14211..15431	Clostridium
tet(41)	Efflux	1,182	AY264780	1825..3006	Serratia
tet(42)	Efflux	1,287	EU523697	687..1973	Bacillus, Microbacterium, Micrococcus, Paenibacillus, Pseudomonas, Staphylococcus
tet(43)	Efflux	1,560	GQ244501	60..1619	Uncultured
tet(44)	Ribosomal protection	1,923	FN594949	25245..27167	Campylobacter

Adapted from http://faculty.washington.edu/marilynr/

Trimethoprim

Trimethoprim has been available since 1962 and is considered the last truly new antibacterial agent introduced into clinical practice (Roth et al., 1962). All later developed agents have been variations of older antibiotics, that is, belonging to families of agents, within which cross-resistance is common (Sköld, 2001; Roberts, 2002). Trimethoprim is a completely synthetic drug, belonging to the diaminopyrimidine group of compounds, i.e., 5-benzyl-2,4-diamino-pyrimidine (Huovinen, 1987).

Trimethoprim inhibits the enzyme dihydrofolate reductase (DHFR) by competitively binding to its active site. DHFR catalysis the NAHPH-dependent reduction of dihydrofolate acid to the active co-enzyme tetrahydrofolate. As such trimethoprim can be regarded as an antifolate, a structural analog of folic acid. DHFR, like DHPS is part of the folate biosynthetic pathway (Sköld, 2001; Grape, 2006; see section Sulfonamides).

Because trimethoprim like sulfonamide is a synthetic antibacterial agent, naturally occurring enzymes degrading, or modifying it are unlikely. However, resistance, mostly low level, can for example occur via non-allelic and drug-resistant variants of the chromosomal folA gene encoding the bacterial DHFR (Huovinen et al., 1995; Sköld, 2001; Grape, 2006).

High-level resistance is generally achieved by a bypass mechanism through the action of an acquired gene which is a non-allelic and drug-insusceptible variant of a chromosomal DHFR. These plasmid-mediated DHFRs emerged in Gram-negative bacteria within several years of the clinical introduction of the drug (Fleming et al., 1972; Huovinen and Toivanen, 1980; Amyes and Towner, 1990).

Initially, the acquired DHFRs fell into two quite distinct families, dfrA and dfrB genes (Howell, 2005). Members of the dfrA group are at least 474 nucleotides (nt) long (157 amino acids, aa), whereas the dfrB genes are 237 nt in length (78 aa). Currently six plasmid-mediated families can be distinguished with relatively few dfr determinants originating from Gram-positive bacteria. (Table 7). The dfrK gene is the newest addition to the trimethoprim resistance determinant family (Kadlec and Schwarz, 2009). In contrast to the latest reported DHFRs, the oldest families, dfrA and dfrB, each contain several members (Roberts, 2002; Levings et al., 2006). For example, the dfrA group accommodates over 30 genes. Determinant dfrA27 is the newest reported DHFR gene among Gram-negatives (Wei et al., 2009), although a newer, however unpublished, dfrA variant is present in the public DNA library and some genes apparently have changed nomenclature (Table 7). Among this family two sub-families can be distinguished (Adrian et al., 2000). The dfrA1-group with 12 different genes share 64–90% identity on amino acids level. The dfrA12-group, with five members, display 84% amino acid identity and similar trimethoprim-inhibition profiles. The additional dfrA genes are less related to each other, some have even less than 25% amino acid sequence identity. In contrast to the dfrA family, the dfrB group is somewhat smaller, with only eight reported genes (Levings et al., 2006; Partridge et al., 2009).

Table 7

Acquired trimethoprim resistance genes.

Gene	Sub-family	Gene(s) included	Length (nt)	Accession number	Coding region	Genera
dfrA1	dfrA1-group	dhfrIb, dfr1, dhfrI	474	X00926	236..709	Actinobacter, Enterobacter, Escherichia, Klebsiella, Morganella, Proteus, Pseudomonas, Salmonella, Serratia, Shigella, Vibrio
dfrA3			489	J03306	103..591	Salmonella
dfrA5	dfrA1-group	dhfrV, dfrV	474	X12868	1306..1779	Aeromonas, Enterobacter, Escherichia, Klebsiella, Salmonella, Vibrio
dfrA6	dfrA1-group	dfrVI	474	Z86002	336..809	Escherichia, Proteus, Vibrio
dfrA7	dfrA1-group	dhfrVII, dfrVII, dfrA17	474	X58425	594..1067	Actinobacter, Escherichia, Proteus, Salmonella, Shigella
dfrA8			510	U10186	711..1220	Shigella
dfrA9			534	X57730	726..1259	Escherichia
dfrA10			564	L06418	5494..6057	Actinobacter, Escherichia, Klebsiella, Salmonella
dfrA12	dfrA12-group	dhfrXII, dfr12	498	Z21672	310..807	Actinobacter, Aeromonas, Enterobacter, Enterococcus, Citrobacter, Klebsiella, Pseudomonas, Serratia, Salmonella, Staphylococcus
dfrA13	dfrA12-group		498	Z50802	718..1215	Escherichia
dfrA14	dfrA1-group	dhfrIb	474	Z50805	72..545	Achromobacter, Aeromonas, Escherichia, Klebsiella, Salmonella, Vibrio
dfrA15	dfrA1-group	dhfrXVb	474	Z83311	357..830	Enterobacter, Klebsiella, Morganella, Proteus, Pseudomonas, Salmonella, Vibrio
dfrA16	dfrA1-group	dhfrXVI, dfr16	474	AF077008	115..588	Aeromonas, Escherichia, Salmonella
dfrA17	dfrA1-group	dhfrXVII, dfr17	474	AB126604	98..571	Actinobacter, Enterobacter, Klebsiella, Pseudomonas, Salmonella, Serratia, Shigella, Staphylococcus
dfrA18		dfrA19	570	AJ310778	7004..7573	Enterobacter, Klebsiella, Salmonella
dfrA20			510	AJ605332	1304..1813	Pasteurella
dfrA21	dfrA12-group	dfrxiii	498	AY552589	1..498	Klebsiella, Salmonella
dfrA22	dfrA12-group	dfr22, dfr23	498	AJ628423	325..822	Escherichia, Klebsiella
dfrA23			561	AJ746361	6743..7303	Salmonella
dfrA24			558	AJ972619	83..640	Escherichia
dfrA25	dfrA1-group		459	DQ267940	54..512	Citrobacter, Salmonella
dfrA26			552	AM403715	303..854	Escherichia
dfrA27	dfrA1-group	dfr	474	EU675686	2543..3016	Escherichia
dfrA28	dfrA1-group		474	FM877476	116..589	Aeromonas
dfrA29		dfrVII, dfrA7	472	AM237806	615..1086	Salmonella
dfrA30		dhfrV	474	AM997279	705..1178	unknown
dfrA31		dfr6	474	AB200915	1832..2305	Vibrio
dfrA32	dfrA1-group		474	GU067642	535..1008	Laribacter, Salmonella
dfrA33	dfrA12-group		498	FM957884	88..585	Unknown
dfrB1		dhfrIIa, dfr2a	237	U36276	717..953	Aeromonas, Bordetella, Escherichia, Klebsiella
dfrB2		dhfrIIb, dfr2b	237	J01773	809..1045	Escherichia
dfrB3		dhfrIIc, dfr2c	237	X72585	5957..6193	Aeromonas, Enterobacter, Escherichia, Klebsiella
dfrB4		dfr2d	237	AJ429132	69..305	Aeromonas, Escherichia, Klebsiella
dfrB5		dfr2e	237	AY943084	2856..3092	Pseudomonas
dfrB6			237	DQ274503	394..630	Salmonella
dfrB7			237	DQ993182	244..480	Aeromonas
dfrB8			249	GU295656	1048..1296	Aeromonas
dfrC		dfrA	486	Z48233	337..822	Staphylococcus
dfrD			489	Z50141	94..582	Listeria, Staphylococcus
dfrG			498	AB205645	1013..1510	Enterococcus, Staphylococcus
dfrK			492	FM207105	2788..3279	Staphylococcus

Partly adapted from Grape (.

Mobile Genetic Elements

Acquired AR genes are frequently contained within mobile DNA which can be loosely defined as any segment of DNA that is capable of translocation from one part of a genome to another or between genomes. This definition includes a wide range of distinct mobile elements. The major players in HGT are the conjugative and mobilizable elements, the former contain all the genetic information required to transfer from one bacterium to another whilst the latter use the conjugation functions of co-resident conjugative elements (conjugative plasmids or conjugative transposons) to transfer to another host. Bacteriophages also play a role in the spread of DNA between bacteria, they do this by a process called transduction in which bacterial DNA, rather than phage DNA, is packaged into the phage head and injected into the recipient bacterium. There are also elements which are capable of translocation to new sites in the genome but are not themselves capable of transfer to a new host (of course if they transpose to a conjugative element they can be moved to new hosts). These include the transposons and the mobile introns.

Bacteria can also acquire AR genes by transformation. The process occurs in both Gram-positive and Gram-negative bacteria. Bacteria capable of taking up DNA from the environment are termed “competent.” Some microorganisms, such as many streptococci, are competent at a specific stage in their growth whilst others have no obvious competence window. Some bacteria have specific sequence requirements to successfully take up DNA such as Neisseria (Smith et al., 1999), while others like Bacillus subtilis have no obvious such requirements. In this process naked DNA is taken up by the recipient bacteria and either incorporated into the host genome by homologous recombination or transposition. Alternatively the DNA molecule may be able to replicate autonomously, e.g., plasmids. Mobile genetic elements are often acquired by transformation as well as by conjugation. For a recent review of the mechanisms of transformation see (Kovács et al., 2009; Aune and Aachmann, 2010; Burton and Dubnau, 2010).

Conjugative elements (plasmids)

Typically plasmids are extra chromosomal elements that contain their own origin of replication. They have been found in almost all bacterial genera and the simplest of these elements just contain an origin of replication and genes encoding replication functions, e.g., see Chambers et al. (1988). Plasmids also commonly contain an origin of transfer and genes encoding functions that allow them to transfer to new hosts via conjugation (Smillie et al., 2010). Plasmids that harbor conjugation genes are called conjugative and plasmids that only contain an origin of transfer (oriT) but no conjugation genes are called mobilizable as they can make use of the conjugation functions of conjugative plasmids to transfer to a new host.

In addition to functions involved in replication and transfer plasmids commonly encode resistance to antibiotics. If a resistance gene is on a conjugative or mobilizable plasmid then it has the potential to transfer to new hosts. Some plasmids have a broad host range and can transfer between different species whereas others have a much narrower host range and are confined to one genus or species. There are also plasmids that have the capability of transferring to a particular host but cannot replicate in the new host or do not replicate well. In these circumstances the plasmid may be lost, however if it contains a resistance gene on a transposon this genetic element can translocate to the bacterial chromosome and be maintained in the absence of the plasmid. Therefore a plasmid does not necessarily need to be maintained in a particular host in order to contribute to the spread of resistance.

Both circular and linear plasmids have been described. Circular plasmids have in general been more intensively investigated then linear plasmids. This probably reflects the relative ease which they can be separated from the bacterial chromosome. Nonetheless linear plasmids have now been relatively well characterized and have been shown to convey advantageous phenotypes on the host. Like circular plasmids linear plasmids are often capable of conjugation (Meinhart et al., 1997; Chaconas and Kobryn, 2010).

Some (resistance) plasmid types cannot coexist in a microbial cell and this fact gave rise to the division into incompatibility groups (Couturier et al., 1988). Four major groups have been defined on the basis of genetic relatedness and pilus structure: IncF group (containing IncC, IncD, IncF, IncJ, and IncS), IncI group (including IncB, IncI, and IncK), IncP group (consisting of IncM, IncP, IncU, and IncW), and Ti.

Conjugative elements (integrative)

The integrative conjugative elements (ICE), also called conjugative transposons (Roberts et al., 2008), like the conjugative plasmids contain an origin of transfer and the genes required to make the conjugation apparatus. Unlike plasmids these elements do not contain an origin of replication and have to integrate into a replicon in order to be maintained. This replicon can be either plasmid or chromosome. This gives them an advantage over plasmids as they do not have to have replication machinery that is compatible with the host so tend to have a larger host range than plasmids.

Integrative conjugative elements are a highly heterogeneous group of genetic elements with different properties and host ranges. However in general they do have a modular organization, i.e., a conjugation, recombination, regulation, and accessory modules. The latter commonly contains genes encoding AR.

There are also integrative elements that do not contain the conjugation region but can by mobilized by co-resident conjugative ICE or conjugative plasmids. Again these can mediate the spread of AR. There have been a number of comprehensive reviews in this area (Roberts and Mullany, 2009; Frost and Koraimann, 2010; Wozniak and Waldor, 2010).

Transduction

There have been examples of AR genes, and even entire mobile genetic elements, being mobilized by transduction (Willi et al., 1997; Del Grosso et al., 2011). Transduction is a process in which the phage particles are packaged with bacterial DNA instead of phage. There are two type of transduction, generalized in which any segment of bacterial DNA can be packaged into the phage head, and specialized, in which the DNA adjacent to the phage insertion site is packaged.

Translocation within genomes

The simplest of the mobile genetic elements are insertion sequence (IS). These elements just consist of the gene required for element mobility and the inverted repeat at the ends of the element. IS elements can be as short as 1Kb (Siguier et al., 2006). When these elements contain accessory genes not involved in element translocation they are called transposons. A simple transposon will contain an accessory gene (often encoding AR) together with the transposase (for examples of each type of element see Roberts et al., 2008). There are more complex classes of transposons that move using different mechanisms including class II transposons.

The transposons mentioned above are not capable of conjugal transfer to other bacteria and in order for them to be disseminated they need to be contained within a conjugative element. However some of ICE elements as well as being able to transfer to new hosts (see above) are also able to transpose to new genomic sites. Their ability to use different integration sites in the chromosomes depends on the type of recombinases they contain. For example Tn916 can use a large number of different integration sites in most hosts (reviewed in Roberts and Mullany, 2009). However some elements are highly site-specific such as Tn916 (Wozniak and Waldor, 2010). Presumably elements like Tn916 have evolved to use different integration sites in order to increase their host range. Elements that can only use a particular number of insertion sites are limited in the hosts they can use if the site is mutated or occupied.

Gene capture elements

Integrons are genetic elements that include components of a site-specific recombination system enabling them to capture and mobilize genes, in particular AR determinants (Stokes and Hall, 1989; Rechia and Hall, 1995; Fluit and Schmitz, 1999; Depardieu et al., 2007). They harbor an intI gene, encoding a site-specific integrase of the tyrosine recombinase family that carries out recombination between two distinct target sites, i.e., an attI recombination site and a 59-base element (attC site) where attI is the target site for cassette integration and a promoter (Hall and Stokes, 1993; Hall and Collis, 1995; Rechia and Hall, 1997; Mazel, 2006). In contrast to transposons integrons are not flanked by repeat sequences, in addition they do not include any genes encoding proteins that catalyze their movement. HGT of integrons to other bacteria is mostly mediated by plasmids or transposons.

The intI genes have been used as a basis for grouping integrons into ”classes.” Currently, four classes are recognized; those carrying intI1 are defined as class 1, intI2 as class 2, intI3 as class 3, and intI4 as class 4 (Carattoli, 2001; Partridge et al., 2009).

Factors influencing acquisition of mobile genetic elements

The ability of mobile genetic elements containing AR genes to spread is modulated by a range of factors including, selective pressures in the environment, host factors, and properties of the genetic elements themselves. Each of these factors will be examined in turn in the next sections.

Specific host encoded factors

Bacteria have a number of systems that protect them from incoming DNA, including restriction/modification systems and CRISPR-Cas systems (Makarova et al., 2011). These systems although mechanistically very different have the same end point of identifying and destroying foreign DNA. Restriction systems work by identifying particular sequences in the incoming DNA that have not been protected by methylation and digesting them. CRISPRs act as a memory of past infection by a mobile element and can destroy that element if the bacterium encounters it again. Both these systems can be effective in stopping the spread of phage, ICE, and plasmids.

A specific host factor that attracts mobile elements has been documented in the pheromone responsive systems, in which a plasmid less recipient secrets a pheromone to which plasmids containing strains respond and transfer their plasmid to the recipients (Palmer et al., 2010).

None specific host factors

Some none specific factors that can act as barriers to HGT have been eluded to above such as not having the target site for a particular ICE or having incompatible replication systems that stop plasmids replicating in a particular host. Also the architecture of the cell surface my not allow the conjugation systems of all mobile elements to work productively. Additionally one member of a mating pair may produce inhibitory substances. Bacteria produce a number of antimicrobial products the most common being the peptide antibiotics. The best understood are the colicins produced by E. coli. Gram-positive bacteria also produce a diverse array of antimicrobial peptides (Riley and Wertz, 2002).

Genetic element encoded factors

Mobile genetic elements have a plethora of ways to overcome bacterial defense systems. Many plasmids and ICE encode anti-restriction proteins that as the name suggests inactivate the host restriction system allowing the element to enter the new host and survive. Also many mobile genetic elements do not have many restriction enzyme recognition sites so that they avoid the attention of the restriction enzymes. Some, including the common Tn916-like family of conjugative transposons, encode anti-restriction proteins which have been shown to mimic DNA and are recognized by the restriction enzyme. The anti-restriction protein ArdA from Tn916 is one of the best characterized (McMahon et al., 2009).

Many transposons and ICE can transpose into essential genes. If this happens the host will die, to get around this some mobile elements are site-specific or preferentially target inter-genic regions (Cookson et al., 2011). Also most transposable elements (including ICE) are tightly regulated so that they only transpose at low frequency or transpose when the bacteria are stressed, such as antibiotics in their environment (reviewed in Roberts and Mullany, 2009; Wozniak and Waldor, 2010). For example members of the CTndot family of ICE transfer at a much higher frequency in the presence of tetracycline (the antibiotic to which they encode resistance). This is an advantageous response for both the element and the host bacteria (Moon et al., 2005).

Environmental factors

All the factors outlined in the previous sections are important in modulating the spread of AR but obviously if antibiotics are present in the environment there is strong selective pressure for spread of resistance and those factors that promote the spread of resistance will be selected for and those stopping the spread of mobile elements selected against.

Gene transfer is also more likely in environments where bacteria are in close proximity to each other and in relatively high density such as the gut and oral cavity. In order to control the spread of resistance it is important to have an understanding of the molecular biology of the different mobile genetic elements and of the ecology of the environments in which spread is likely.

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.