Use of antimicrobials in veterinary medicine and ...€¦ · 202 S. Schwarz, E. Chaslus-Dancla 1. USE OF ANTIMICROBIALS 1.1. Different aims for the use of antibiotics in food-producing

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Use of antimicrobials in veterinary medicine andmechanisms of resistance

Stefan Schwarz, Elisabeth Chaslus-Dancla

To cite this version:Stefan Schwarz, Elisabeth Chaslus-Dancla. Use of antimicrobials in veterinary medicine and mecha-nisms of resistance. Veterinary Research, BioMed Central, 2001, 32 (3-4), pp.201-225. �10.1051/ve-tres:2001120�. �hal-00902704�

https://hal.archives-ouvertes.fr/hal-00902704

https://hal.archives-ouvertes.fr

Review article

Use of antimicrobials in veterinary medicine and mechanisms of resistance

Stefan SCHWARZa*, Elisabeth CHASLUS-DANCLAb

a Institut für Tierzucht und Tierverhalten, Bundesforschungsanstalt für Landwirtschaft (FAL),Dörnbergstr. 25–27, 29223 Celle, Germany

b Institut National de la Recherche Agronomique, Pathologie Aviaire et Parasitologie, 37380 Nouzilly, France

(Received 18 December 2000; accepted 14 February 2001)

Abstract – This review deals with the application of antimicrobial agents in veterinary medicineand food animal production and the possible consequences arising from the widespread and multi-purpose use of antimicrobials. The various mechanisms that bacteria have developed to escape theinhibitory effects of the antimicrobials most frequently used in the veterinary field are reported in detail.Resistance of bacteria to tetracyclines, macrolide-lincosamide-streptogramin antibiotics, β-lactam antibi-otics, aminoglycosides, sulfonamides, trimethoprim, fluoroquinolones and chloramphenicol/florfenicolis described with regard to enzymatic inactivation, decreased intracellular drug accumulation and mod-ification/protection/replacement of the target sites. In addition, basic information is given aboutmobile genetic elements which carry the respective resistance genes, such as plasmids, transposons,and gene cassettes/integrons, and their ways of spreading via conjugation, mobilisation, transduction,and transformation.

antibiotic therapy / growth promotion / resistance mechanism / resistance gene / gene transfer

Résumé – Utilisation d’agents antimicrobiens en médecine vétérinaire et mécanismes de résis-tance. Cette revue présente les différents buts pour lesquels les agents antimicrobiens sont utilisés enmédecine vétérinaire, dans les élevages d’animaux entrant dans la chaîne alimentaire et les pos-sibles conséquences de cette large utilisation. Une synthèse est faite des différents mécanismes de résis-tance développés par les bactéries, comme l’inactivation enzymatique, la diminution de la concen-tration intracellulaire de l’antibiotique, les modification/protection/déplacement de cible, qui permettentd’échapper à l’action des antibiotiques les plus fréquemment utilisés dans le domaine vétérinaire : tétra-cyclines, macrolide-lincosamide-streptogramine, β-lactamines, aminosides, sulfamides, trimétho-prime, fluoroquinolones et chloramphénicol/florfénicol. Le rôle d’éléments génétiques mobiles

Vet. Res. 32 (2001) 201–225 201© INRA, EDP Sciences, 2001

* Correspondence and reprintsTel.: (49) 5141 384673; fax: (49) 5141 381849; e-mail: [email protected]

S. Schwarz, E. Chaslus-Dancla202

1. USE OF ANTIMICROBIALS

1.1. Different aims for the use of antibiotics in food-producing animals

Unlike in human medicine, antibiotics infood-producing animals are used for twodifferent purposes: (a) prevention and con-trol of bacterial infections and (b) growthpromotion [63].

The control and prevention of bacterialinfections is achieved by either therapeutic,metaphylactic or prophylactic applicationof antimicrobials. For this, substances ofmainly the same classes as used in humanmedicine are available for the treatment offood-producing animals – the antimicro-bials available in the different European

countries are listed in Table I. The purposeof therapy is to treat a declared infection.According to the number of animals pre-sent and the type of production, these treat-ments may be individual as in pet and com-panion animals, dairy cattle, horses andsows, and given by oral or parenteral ways.Nevertheless, in most cases, when largegroups of animals have to be treated, as inpoultry or swine production, they are appliedvia water or feed. With such mass produc-tion, when a limited number of animals havebeen identified as infected, rapid treatmentof all animals of the respective group/herd/flock is necessary to prevent further exten-sion of the infection. This is referred to asmetaphylaxis [63]. In addition to these inter-ventions, prophylaxis is a solely preventivemeasure, given individually or to groups ofanimals, which appears unavoidable under

portant les gènes de résistance tels que les plasmides, les transposons ou les cassettes/intégrons, et leurmode de diffusion par conjugaison, mobilisation ou transduction sont présentés.

traitement antibiotique / promoteur de croissance / mécanisme de résistance / gène de résistance / transfert de gène

Table of contents

1. Use of antimicrobials ...............................................................................................................2021.1. Different aims for the use of antibiotics in food-producing animals...............................2021.2. Volumes of antimicrobials used for animals in Europe...................................................2041.3. Particularities of usage in animals and classes of antibiotics used in animals.................206

2. Origins of antimicrobial resistance...........................................................................................2073. Transfer of resistance genes .....................................................................................................209

3.1. Elements involved in the horizontal transfer of resistance genes....................................2093.2. Gene transfer mechanisms...............................................................................................210

4. Resistance mechanisms............................................................................................................2115. Resistance to antimicrobials used in veterinary medicine .......................................................213

5.1. Resistance to tetracyclines ..............................................................................................2135.2. Resistance to macrolides, lincosamides, and streptogramins (MLS) .............................2165.3. Resistance to β-lactam antibiotics ...................................................................................2185.4. Resistance to aminoglycosides ........................................................................................2195.5. Resistance to sulfonamides and trimethoprim.................................................................2195.6. Resistance to fluoroquinolones........................................................................................2205.7. Resistance to chloramphenicol and florfenicol ...............................................................221

6. Conclusion................................................................................................................................222

Antimicrobial use and mechanisms of resistance 203T

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certain circumstances and in some key peri-ods of animal life, such as surgery, vacci-nation, transport and mixing of animals,weaning of pigs, and the end of lactation indairy cows. During such periods, animalsare generally recognised as more susceptibleto infections and long-term experience withthe current animal production systemsrequires the application of antimicrobials atsuch times to avoid the onset of infections.Without these preventive treatments, sub-sequent clinical infections would occur morefrequently and would require more thera-peutic interventions for an efficient control.However, prophylactic application of antimi-crobials is criticised for its possible involve-ment in the selection of resistant bacteriaand the promotion of the spread of resis-tance genes [63].

The second purpose of the use of antibi-otics, growth promotion, is specific to food-producing animals. A specified number ofsubstances licensed as growth promotersare given at low concentrations to improvegrowth during the entire growth period ofanimals. The available molecules and con-ditions of use are clearly defined in licencesspecifying the target animals, duration, anddosage. In Europe, since 1975, no β-lac-tams or tetracyclines have been used asgrowth promoters whereas they are still inuse in the USA. Recent discussions at theEuropean level have resulted in a limitedlist of four drugs available for this purpose.Indeed, all drugs similar or closely related tothose used for human therapy, such as theglycopeptide avoparcin, the macrolidestylosin and spiramycin, the streptograminvirginiamycin, and the polypeptide Zn-bac-itracin, were banned or withdrawn in 1998and 1999. In addition, the quinoxalines car-badox and olaquindox, were withdrawn in1999 for possible toxicological effects. Cur-rently, the four available molecules areflavophospholipol, monensin-Na, salino-mycin-Na, and avilamycin. Only two ofthem (flavophospholipol, avilamycin) havea real antibiotic activity. The remaining twosubstances are used for their coccidiostatic

effects [63]. The situation regarding avil-amycin was critical as it is structurallyrelated to everninomycin that was then beingdeveloped for human therapy. However,information was given in May 2000 by thecompany, Schering-Plough, concerning theending of further development of evernino-mycin. In the present situation, all theantimicrobial growth promotors used areexpected to develop no cross-resistance withmolecules used for human medicine.

1.2. Volumes of antimicrobials used for animals in Europe

In most countries except Denmark, Swe-den and Finland, no legal obligation existsfor pharmaceutical companies to supply dataon antibiotic sales. Thus it is very difficult tohave a precise knowledge of the volumesof antibiotics sold in Europe. Such data forthe year 1997 were available for the firsttime from the European federation for ani-mal health (Fedesa) after being requestedby the European Commission to provideinformation on the actual usage of antibi-otics in the EU including Switzerland [6].Extrapolation factors were needed for eachcountry to obtain the total amount of antibi-otics sold taking into account estimationsof sales by non-Fedesa members. Therespective data for 1997 are available on theinternet (http://www.fedesa.be/eng/PublicSite/xtra/dossiers/doss9/).

The world-wide use of antibiotics for ani-mal health purposes in 1996 was estimatedat 27 000 tonnes of which about 25% wasused throughout the EU. In the EU, an esti-mated distribution was given as 50% fortherapeutic purposes, 25% feed additiveusage and the 25% remaining for ionophorefeed additives to prevent coccidiosis in poul-try [6]. Ninety percent of all the antibiotics(including those used for growth promotionand those for therapy) produced in the worldfor animal use are distributed via feed. Theyare mainly used for pigs (60%), poultry and

Antimicrobial use and mechanisms of resistance 205

rabbits (20%), ruminants (18%), fish (1%),and pets (1%) [7].

In Europe, in 1997, the total sales vol-umes of antibiotics was 10 493 tonnes ofactive ingredients, which can be subdividedinto 5 400 t for human health usage (52%),3 494 t for animal health use (33%), and1 599 t for growth promotion (15%).

High differences in percentages of drugsused for therapy or growth promotion exist

between the different countries in relationto the type of animal production, whetherintensive or not (Tabs. II and III).

It was interesting to further analyse rela-tionships between the amounts of antibi-otics used in each class of livestock and thenumber of animals produced per country.For 1997, this comparison was difficult asthe animal production data were availablefor 1996 only, as sales data for 1997 were

Table II. Sales volumes of antimicrobial agents (therapeutics and growth promoters) in differentEU member states in 1997 (adapted from [22]).

Country Sales Salesof growth promoters of therapeutics

Tonnes of % of the EU Tonnes of % of the EU active market active market

substances substances

Austria 23 1 8 <1 Belgium-Luxembourg 110 7 125 4 Denmark 75 5 60 2 Finland <1 <1 12 <1 France 339 21 492 14 Germany 255 16 488 14 Greece 15 1 110 3 Ireland 34 2 22 <1 Italy 100 6 389 11 The Netherlands 226 14 300 9 Portugal 24 2 44 1 Spain 198 12 616 18 Sweden <1 <1 20 <1 UK 191 12 788 23

Table III. Sales volumes of antimicrobial agents in the EU and Switzerland in 1997 (adapted from[22]).

Classes of antimicrobials Sales as estimated by FEDESA

Tonnes of active substances % of total

Tetracyclines 2294 66 Macrolides 424 12 Penicillins 322 9 Aminoglycosides 154 4 Trimethoprim/sulfonamides 75 2 Fluoroquinolones 43 1 Other classes 182 5


estimated. Nevertheless, animal productionin 1997 was expected to be similar to 1996.Another difficulty was that data on farmanimal production numbers are availablefrom an annual consensus within all memberstates and represent calculations of animalnumbers on a certain day and not the totalnumber of animals raised in one year. Incontrast, production data (slaughtered ani-mals, milk/egg production) are calculatedon a yearly basis. Carcass weights of par-ticular animals at slaughter considerably dif-fer in the countries of the EU according tothe type of production and consumer pref-erences.

Although based on an estimation, theresults of comparisons made it possible toclassify countries into three groups: coun-tries with the highest level of antibiotics(therapeutic and growth promotion) usedby tonne of live weight of slaughtered ani-mal, which are the UK, Greece, Spain andThe Netherlands, countries with an inter-mediate position such as Belgium, France,Italy, Germany and Portugal, and finallycountries with a low ratio such as Sweden,Denmark, Finland. This classification largelyreflects the type of husbandry system.

Another relevant analysis could be thecomparison of the consumption of antibi-otics in human and veterinary medicine. Asthis comparison could not be performed onthe basis of a calculation per head and peryear as the lives of several animals areshorter than one year, the sales volumes andbody mass were compared instead [72].From the evaluation of the animal produc-tion data obtained for 1996, including ani-mals at slaughter and animals for milk andegg production, the body mass of 6.1 bil-lion farm animals could be calculated as51.5 billion tonnes to which the consumptionof 54 mg antibiotic per kg can be related.Similar data were expected for the year1997. In 1997, the European population wasof 373 million inhabitants with a mean indi-vidual body weight of 60 kg to which the54 000 tonnes used in human medicinecould be related. The corresponding calcu-

lated dose was of 241 mg per kg bodyweight which is 4.5-fold higher than in vet-erinary medicine [72]. Nevertheless, this isonly a rough estimation, as different param-eters appear to be under- or overestimated,for instance, in human medicine not all theantibiotics sold are totally consumed.

1.3. Particularities of usage in animalsand classes of antibiotics used in animals

An important aspect which is specific toveterinary medicine is the problem ofresidues in carcasses at slaughter. This prob-lem has been taken into account and Euro-pean legislation was recently revisited inorder to provide safe products to consumers.For each antibiotic used, the MaximumResidue Level (MRL) had to be definedbefore licensing, that is the maximum levelof antibiotic residue acceptable in carcassesat slaughter without any adverse effect onpublic health [79] . In toxicological andmicrobiological studies, this non-observedeffect level (NOEL) is generally taken asthe dose at and below which adverse effectsdo not occur. EU legislation was introducedin two steps: as from January 1992, newactive ingredients or pharmaceutically activeexcipients could be introduced onto the mar-kets of the Member States after definitionof a Community MRL, and the substancesalready in use at that date were subject to asystematic call-up in order to establish theMRL. For these molecules already in use,the initial deadline, 1997, had to be post-poned. In such conditions, CommunityMRLs for veterinary drugs were establishedfor different purposes: (a) as a guarantee forconsumers of safe foodstuffs, (b) as a basisfor the definition of withdrawal periods(periods between the end of a treatment andthe arrival of carcasses at slaughter), and(c) as standards for residue surveillance andtrade.

The calculation of MRL was based on the NOEL found in toxicological and


microbiological studies on sensitive animalspecies. Extrapolation to humans (standardhuman body weight of 60 kg) was based onthe acceptable daily intake or daily dose,which was calculated from the NOEL bydividing it by a safety factor (100 to 1000).Distribution of the daily dose in the differ-ent foods was then evaluated taking intoaccount the average consumption of the rep-resentative European man of 60 kg, which isstated to include: meat (300 g), liver (100 g),fat (50 g), milk (1.5 L), and eggs (100 g).As a consequence, some drugs such as nitro-imidazoles (1993), chloramphenicol (1994),and furazolidones (1995) were withdrawnat the European level for use in food-pro-ducing animals.

Although antibiotics used for ther-apy/methaphylaxis or for prophylaxis belongto the same classes as those used for humantherapy, relevant differences exist withinclasses and some particularities of the vet-erinary pharmacopoeia compared to thehuman pharmacopoeia can be listed:

(i) In veterinary medicine, the cost of acourse of treatment has to be taken intoaccount. For economic reasons, old butstill efficient molecules are largely usedsuch as penicillins and tetracyclines. In1997, among the 3494 tonnes of antibi-otics (active ingredients) used for ther-apy in animals, the top three were tetra-cyclines, macrolides, and penicillins(Tab. III).

(ii) In veterinary medicine, some familiesare under-developed in comparison totheir relatives currently in human use.In France, among cephalosporins, onlytwo molecules of third generation, cef-tiofur and cefquinome, are used in cat-tle in injections or intra-mammary infu-sions.

(iii) A very limited number of newmolecules have been introduced intoveterinary medicine over the lastdecades, including tiamuline, florfeni-col, a fluorinated derivative of chlo-ramphenicol (1995), and fluoro-

quinolones. Quinolones represent 1%of the antibiotics used. As regards oraluse in all types of animals, nalidixicacid is maintained in three countriesonly – Italy, Spain, and Portugal – andflumequin or oxolinic acid are licensedin most countries except Germany. Therecently introduced fluoroquinolones,enrofloxacin, marbofloxacin, difloxacinor danofloxacin are used in most coun-tries. Sarafloxacin is limited to fish andhas been introduced in the UK and Ire-land only, and orbifloxacin used exclu-sively in pets in the UK [22].

(iv) Some molecules have been introducedinto veterinary medicine only, such asthe aminoglycoside apramycin, flor-fenicol, tylosin, tilmicosine and tiamu-line.

(v) Some molecules recently introduced inhuman medicine have not been intro-duced in animal therapy, as for instance,the third generation of cephalosporins,amikacin, or minocyline. In addition,promising new classes of antimicro-bials, such as ketolides, glycylcyclinesor oxazolidinones which are currentlyunder development or in clinical trialswill be exclusively reserved for humantherapy.

2. ORIGINS OF ANTIMICROBIALRESISTANCE

Most antimicrobial agents currently usedin human and veterinary medicine are lowmolecular weight substances which inhibitgrowth of bacteria or even kill them at verylow concentrations. The first antimicrobialsused represented substances or close rela-tives of substances which were producedby fungi or soil bacteria and provided aselective advantage to the antimicrobial pro-ducer in the fight for resources and ecolog-ical niches. Thus bacteria have come intocontact with antimicrobial substances a longtime before the first antimicrobial agentswere introduced into clinical use.


Admittedly, this contact between antimi-crobial substances and sensitive microor-ganisms in the pre-antibiotic era occurredat a distinctly lower frequency than nowa-days. Nevertheless, it represented a selec-tive pressure which in return forced microor-ganisms to develop mechanisms to escapethe inhibitory activities of antimicrobialagents. There are mainly three ways inwhich bacteria gain resistance to antimi-crobial agents. One way is for bacteria toacquire resistance genes from the antibioticproducers and modify them with a view tooptimising functionality in the new host.The resistance genes that antibiotic pro-ducers harbour as a mechanism of self-defence from their own products are usu-ally located in the chromosomal DNA. Theirspread to other bacteria must thereforeinvolve the integration of these genes intomobile genetic elements such as plasmidsand transposons, both of which have beendetected in bacteria collected in the pre-antibiotic era. When such resistance genesare transferred across species and even genusborders, they may undergo mutations in theirnew hosts resulting in a wide variety ofstructurally heterogeneous, but functionallyhomologous resistance determinants. Exam-ples of such a divergent evolution from acommon ancestor are the efflux proteinsassociated with tetracycline resistance ingram-positive and gram-negative bacteria[52]. The second way to develop resistance

genes is a stepwise mutation of genes whoseproducts play a role in physiological cellmetabolism. As a result, genes are modifiedin a way that the substrate spectrum of theirproducts changes from metabolites ofbiosynthetic or biodegradative pathways tocertain antimicrobial agents only. The var-ious enzymes exhibiting acetyl-, adenyl- orphosphotransferase activities involved inthe inactivation of aminoglycosides or chlo-ramphenicol are believed to have evolvedthis way [19]. A third major way for bacte-ria to gain resistance is to modify their tar-get structures by either single-step (strep-tomycin resistance) or multi-step mutations(fluoroquinolone resistance), so that theybecome resistant to the inhibitory effects ofthe respective antimicrobials [2].

As a result of the exposure of bacteria toantimicrobial agents, a large number of resis-tance genes has developed. The observationthat the introduction of an antimicrobial intoclinical use has been either accompanied orfollowed shortly by the occurrence of bac-teria which are resistant to this particularsubstance (Tab. IV) underlines the extraor-dinary capacity of bacteria to quickly andefficiently respond to the selective pressureimposed by the use of that substance. Inrecent years, bacteria have also developedresistance to completely synthetic substanceswhich have no natural counterpart. Thisfinding confirms the stunning ability of bac-teria to cope with changed environmental

Table IV. Time coincidence between the discovery/production of antimicrobial agents, their intro-duction into clinical use as well as the occurrence of resistant bacteria (modified according to [22]).

Antimicrobial agent Discovery / Introduction into Occurrence of production clinical use resistant bacteria

Penicillin 1940 1943 1940 Streptomycin 1944 1947 1947, 1956 Tetracycline 1948 1952 1956 Erythromycin 1952 1955 1956 Vancomycin 1956 1972 1987 Nalidixic acid 1960 1962 1966 Gentamicin 1963 1967 1970 Fluoroquinolones 1978 1982 1985


conditions and to effectively explore a widevariety of ways to survive even in the pres-ence of toxic substances such as antimicro-bial agents [5]. The exchange of resistancegenes between members of a mixed bacterialpopulation has distinctly accelerated thewidespread occurrence of certain resistancegenes in a large number of pathogenic bac-teria, but also in harmless commensals.Resistance genes were usually first presentin the bacteria in which they had evolvedand were initially only transmitted verti-cally. However when integrated into mobilegenetic elements, the resistance genes werespread by horizontal transfer among bacte-ria of the same and of different species andgenera. Thus the driving forces of emerg-ing antimicrobial resistance are repeatedexposure of the bacteria to antibiotics andaccess of the bacteria to a large resistancegene pool as the latter is available in apolymicrobial environment.

3. TRANSFER OF RESISTANCEGENES

3.1. Elements involved in horizontaltransfer of resistance genes

The rapid spread of antimicrobial resis-tance genes between bacteria of the sameand of different species and genera is mainlythe result of horizontal transfer events ofmobile genetic elements carrying one ormore resistance genes. Among them, plas-mids, transposons and integrons/gene cas-settes play a major role. These three types ofelements are composed of double-strandedDNA, but differ distinctly in their sizes,structures, biological properties as well asways of spreading.

Plasmids are extrachromosomal elementswhich have been detected in virtually allbacterial genera of medical or veterinaryimportance, but also in bacteria which con-stitute the physiological flora of the skinand the various mucosal surfaces in humans

and animals. Their size varies from less than2 kilobase pairs (kbp) to more than 100 kbp.Plasmids are capable of autonomous repli-cation due to their replication systems. Aslong as plasmids belong to different incom-patibility groups, they can stably coexist inthe same bacterial cell. Plasmid-borne prop-erties are not essential for the survival ofthe bacteria under physiological conditions,but may be of benefit for the bacteriumunder specific conditions. These accessoryproperties include resistance to antimicrobialagents, disinfectants, heavy metal cations,anions, nucleic acid binding substances orbacteriocins. In addition to resistance prop-erties, various other traits are known to beplasmid-borne, such as metabolic proper-ties, virulence properties, and fertility func-tions [68]. Plasmids can carry one or moreresistance gene(s) in addition to genes cod-ing for other of the above-mentioned func-tions. Plasmids may form cointegrates withother plasmids, may integrate or be inte-grated, either in part or in toto, into the chro-mosomal DNA or can act as vectors fortransposons and integrons/gene cassettes[5]. Large plasmids can carry genes (tragene complex) which enable them to moveon their own from one host cell to another.Such plasmids are referred to as conjuga-tive plasmids.

In contrast to plasmids, transposons donot possess replication systems and there-fore must integrate for their stable mainte-nance into replication-proficient vectormolecules such as chromosomal DNA orplasmids in the cell. Transposons also varyin size (< 1 kbp → 60 kbp) and structure.The smallest transposons, also known asinsertion sequences, solely carry the genefor a transposase which is responsible forthe movement of the element. Larger trans-posons usually carry one or more additionalgenes, most of which code for antibioticresistance properties. Many transposonshave little or no target specificity and there-fore can insert themselves at various posi-tions in the chromosomal or plasmid DNA.


Large conjugative transposons may also har-bour tra genes [2, 4].

Gene cassettes represent small mobileelements of less than 2 kbp and, to date,have only been detected in gram-negativebacteria [51]. They commonly consist ofonly a specific recombination site and a sin-gle gene which is in most known cases anantimicrobial resistance gene. Gene cas-settes differ from plasmids by the lack ofreplication systems, and from transposonsby the lack of transposition systems. Theymove by site-specific recombination. Theyare usually present at specific sites withinan integron. Integrons most often representintact or defective transposons and com-monly consist of two conserved regions,one of which, the 5’ conserved region, codesfor the integrase that is responsible for thesite-specific insertion of the gene cassettesand also harbours the promoter for theexpression of the cassette-borne genes. The3’ conserved region may represent anotherresistance gene, such as the sulfonamideresistance gene sulI [51]. The role of inte-grons in the diffusion of resistance wasreviewed by Carattoli [10].

3.2. Gene transfer mechanisms

Plasmids, transposons and gene cas-settes/integrons are spread vertically duringthe division of the host cell, but can also betransferred horizontally between bacteria ofthe same or different species and genera viatransduction, conjugation/mobilisation ortransformation [5, 61].

Transduction describes a bacteriophage-mediated transfer process. Bacteriophagesare also referred to as “bacterial viruses”.They infect bacteria by injection of theirDNA. In the new host cell, the phage DNAcan direct the production of new phage par-ticles which includes expression of phage-borne genes, replication of the phage DNAand packaging of this DNA into new phageparticles which are released from the bac-terial cell (lytic cycle). On the other hand,

the phage DNA may integrate into the chro-mosomal DNA of the host cell as a“prophage” and remain there for long peri-ods in an inactive state (lysogenic cycle).External factors such as UV-irradiation canactivate the prophage and initiate a lyticcycle. Chromosomal resistance genes thatare located close to the integration site ofthe prophage may become part of the phagegenome when the prophage is not excisedprecisely from the chromosomal DNA. Inthis case, the resistance genes spread withthe phage particles to new host cells. Duringphage assembly, resistance plasmids mayaccidentally be packaged into phage headsinstead of phage DNA. The resulting “pseu-dophages” are able to infect new host cellsas the regular phages do. However, sincethey lack phage DNA, they can only injectthe plasmid DNA and thus promote thespread of resistance plasmids to new hostcells. The spread of resistance genes viatransduction is strongly influenced by thelimited amount of DNA that can be pack-aged into a phage head and the requirementof specific receptors for phage attachment onthe surface of the new host cell. For staphy-lococci, it has been reported that 45 kbp isthe upper size limit of DNA that can betransduced. While smaller plasmids aretransduced as linear concatemers, largerplasmids cannot be packaged into a phagehead. Since only host cells that are phylo-genetically closely related carry the samereceptors for phage attachment, transduc-tion is commonly observed between bacte-ria of the same species, but rarely seenbetween bacteria of different species andgenera. Transducing phages have beendetected in a wide variety of bacteria [34].

Conjugation describes the self-transferof a conjugative plasmid or transposon froma donor cell to a recipient cell [5]. Closecontact between donor and recipient is oneof the major requirements for efficient con-jugation. The tra gene complex whose geneproducts represent components of the transfer apparatus spans at least 15 kbp ingram-positive bacteria and 30 kbp in


gram-negative bacteria and thus cannot belocated on small resistance plasmids com-monly seen among bacterial pathogens.Small non-conjugative plasmids which co-reside in the same host cell may use thetransfer apparatus provided by the con-jugative element, as long as they have anoriT region (origin of transfer) but possiblyalso possess mobilisation (mob) genes. Thisprocess is known as mobilisation. Conju-gation and mobilisation are believed to be ofmajor importance for the spread of resis-tance genes between bacteria of differentspecies and genera in bacterial mixed pop-ulations as seen on the skin and mucosa ofthe alimentary, respiratory, and genital tractof humans and animals. So far, conjugativeplasmids and transposons carrying one ormore antibiotic resistance genes have beenreported to be present in gram-positive andgram-negative bacterial pathogens [60].

Transformation describes the transfer offree DNA into competent recipient cells.Transformation is the major way of intro-ducing plasmids into new host bacteriaunder in vitro conditions. Under in vivo con-ditions, transformation is considered to playonly a limited role in the transfer of resis-tance genes [5]. On the one hand, free DNAoriginating from lysed bacteria is usuallyrapidly degraded under most environmentalconditions. On the other hand, only a fewbacteria, such as Streptococcus pneumoniaeor Bacillusspp., exhibit a natural ability totake up DNA from their environment.

4. RESISTANCE MECHANISMS

A bacterium is considered to be resistantto an antimicrobial agent when the concen-tration of the antimicrobial agent at the siteof infection is not sufficiently high to eitherinhibit replication of the bacterium or evenkill it [78]. This definition clearly showsthat antimicrobial resistance is not solely amicrobiological problem, but also includespharmacological, pharmacokinetic and clin-ical aspects. Antimicrobial resistance is a

highly flexible property of the bacteriawhich varies with respect to the antimicro-bial agents, the respective bacteria and theresistance mechanism. Up to six differentmechanisms have been described as con-ferring resistance to the same antimicrobialagent. Some resistance mechanisms are dis-tributed among a wide variety of bacteria,while others appear to be specific for cer-tain bacterial species and genera. In con-trast to genus- or species-specific intrinsicresistance properties which are mainly basedon either the lack of or the inaccessibilityof the target sites for the antimicrobialagents, acquired resistance propertiesaccount for most of the resistance problemscurrently encountered in human and veteri-nary medicine. Acquired resistance repre-sents a strain-specific property which may bebased on mutations in certain chromosomal“housekeeping” genes which act as targetsfor antimicrobial agents. Such mutations aremainly based on the exchange of one or afew bases and consequently cause onlyslight changes in the amino acid sequence ofthe corresponding gene product. Thesesequence alterations often have little or noinfluence on the biological activity of thegene products, but render them insensitive tothe inhibitory activities of the respectiveantimicrobial agents [2, 48]. Acquired resis-tance, however, is more often associatedwith the acquisition of mobile genetic ele-ments that carry one or more resistancegenes [2, 5]. Such resistance genes code forproteins which do not usually have a knownfunction in physiological cell metabolism,but mediate resistance to either singleantimicrobial substances or members of thesame class of substances (e.g. tetracyclines).They can also mediate resistance to mem-bers of different classes of antimicrobialswhich, however, have the same target sitewithin the bacterial cell (e.g. macrolides,lincosamides and B-compounds of the strep-togramins). In the case of multidrug trans-porter systems which export toxic metabo-lites from the cell, resistance to structurallyand functionally different antibiotics, often


Tab

le V

.Exa

mpl

es o

f bac

teria

l res

ista

nce

to a

ntim

icro

bial

s by

e

nzy

ma

tic in

act

iva

tion(m

odifi

ed fr

om [6

1]).

Res

ista

nce

Res

ista

nce

to

Via

R

esis

tanc

eG

ene

Bac

teria

R

ef.

mec

hani

sm

gene

(s)

loca

tion

a

chem

ical

mod

ifica

tion

chlo

ram

phen

icol

acet

yltr

ansf

eras

eca

tA, c

atB

P, C

, T, G

Cgr

am+

, gra

m–

, aer

obic

, [4

2, 6

5]an

aero

bic

bact

eria

amin

ogly

cosi

des

acet

yl-,

ade

nyl-

or a

ac,

aa

d (

an

t), a

ph

P, T

, GC

, Cgr

am+

, gra

m–,

[2

0, 4

1, 6

4, 8

0]ph

osph

otra

nsfe

rase

aero

bic

bact

eria

amin

ocyc

litol

sad

enyl

tran

sfer

ase

aa

d(a

nt)

T, G

C, P

gram

+, g

ram

– ba

cter

ia[6

4]

A-c

ompo

unds

of

acet

yltr

ansf

eras

eva

t(A-E

)P

, CS

tap

hyl

oco

ccu

s,

[53]

stre

ptog

ram

ins

En

tero

cocc

us

linco

sam

ides

nucl

eotid

yltr

ansf

eras

eln

u(A

),ln

u(B

)P

Sta

ph

ylo

cocc

us

[53]

mac

rolid

esph

osph

otra

nsfe

rase

mp

h(A

-C)

P, T

, CE

sch

erich

ia, S

hig

ella

, [3

6, 5

3]S

tap

hyl

oco

ccu

s

tetr

acyc

lines

oxire

duct

ase

tet(X

)T

Ba

cte

roid

es

[52,

67]

hydr

olys

isβ-

lact

ams

β-la

ctam

ases

bla

P, T

, Cgr

am+

, gra

m–

, aer

obic

, [9

, 38,

71]

anae

robi

c ba

cter

ia

B-c

ompo

unds

of

lact

one

hydr

olas

esvg

b(A

), v

gb(

B),

sb

hP

Sta

ph

ylo

cocc

us

[53]

stre

ptog

ram

ins

mac

rolid

es

este

rase

ere

(A),

ere

(B)

P, G

Cgr

am+

, gra

m–

bact

eria

[53]

aP

= p

lasm

id; T

= tr

ansp

oson

; GC

= g

ene

cass

ette

; C =

chr

omos

omal

DN

A.


in addition to hydrophobic cations, deter-gents or nucleic acid binding compounds,has been largely observed [44, 47].

There are three major mechanisms bywhich bacteria have become resistant toantimicrobial agents: enzymatic inactiva-tion as well as reduced intracellular accu-mulation of the antimicrobials, but also pro-tection, alteration or replacement of thecellular target sites [2, 48, 61].

A wide variety of enzymes is knownwhich inactivate antimicrobial agents bytransferring acetyl, adenyl or phosphoricgroups to specific sites of the antibiotics,thereby destroying their antimicrobial activ-ity. Other enzymes, such as β-lactamases,hydrolases and esterases directly attack theantimicrobial molecule and destroy it. Thesubstrate spectrum of inactivating enzymesis usually limited to a small number of struc-turally related compounds (Tab. V).

Reduced intracellular accumulation ofantimicrobials can be achieved in principlein two different ways: decreased uptake orincreased removal of the drugs. Decreaseddrug uptake is not usually mediated by resis-tance genes. The outer membrane of gram-negative bacteria may represent a perme-ability barrier for certain antibiotics [59].Mutations are known which cause reducedexpression, structural alteration or even lossof porins by which antibiotics enter the bac-terial cell [48]. A switch in the charge ofthe cell wall lipopolysaccharides (LPS) hasbeen reported in Pseudomonas aeruginosato prevent highly positively charged antibi-otics, such as aminoglycosides, from cross-ing the outer membrane [59]. Resistancegenes which code for a number of mem-brane-associated efflux proteins have beendetected on plasmids, transposons or genecassettes. Such efflux systems mostly exporta narrow range of structurally related sub-strates from the bacterial cell by energy-dependent processes [44]. In contrast, thereis also a large number of multidrug trans-porters known in gram-positive and gram-negative bacteria, most of which export a

wide range of structurally heterogeneoustoxic compounds including antimicrobialagents [44, 47] (Tab. VI).

Chemical modification of the target site,e.g. by methylation, may render the targetsite inaccessible to the antibiotics [35]. Pro-tection of the target sites, such as the ribo-some, by specific protective proteins whichare considered to inhibit binding of antimi-crobials have been reported in connectionwith tetracycline resistance [52]. The over-expression of sensitive target structures, butalso the replacement of sensitive target struc-tures by new targets which exhibit reducedaffinity for – or even insensitivity to – theantimicrobials represent other ways for thebacteria to resist the inhibitory activities ofantimicrobials [48]. Moreover, a number ofmutations in the genes coding for targetstructures which render the correspondinggene products resistant to the inhibitoryeffects of the antimicrobial agents have beenidentified (Tab. VII).

5. RESISTANCE TO ANTIMICROBIALS USED IN VETERINARY MEDICINE

5.1. Resistance to tetracyclines

So far, several different mechanisms oftetracycline resistance have been described,among which active efflux and ribosomeprotection are the most prevalent mecha-nisms among gram-positive and gram-neg-ative pathogens [52].

The energy-dependent efflux of tetracy-clines is mediated by at least two types oftransmembrane proteins, both of whichexchange a proton for a tetracycline-cationcomplex [52]. On the basis of hybridisationexperiments, at least 14 different classes canbe differentiated [37]. Among them, themost intensively studied classes are A, B,C, D, H, K, and L. The genes tet(K) andtet(L) are mainly present in gram-positivebacteria and code for a protein which


Tab

le V

I. E

xam

ples

of b

acte

rial r

esis

tanc

e to

ant

imic

robi

als

by

de

cre

ase

d in

tra

cellu

lar

dru

g a

ccu

mu

latio

n

(mod

ified

from

[61]

).

Res

ista

nce

Res

ista

nce

to

Via

R

esis

tanc

eG

ene

Bac

teria

R

ef.

mec

hani

sm

gene

(s)

loca

tion

a

activ

e ex

port

via

te

trac

yclin

es12

-, 1

4-T

MS

bte

t(A-E

, G, H

, I, J

, K,

P, T

, Cva

rious

gra

m+

and

[3

7, 5

2, 6

7]sp

ecifi

c ex

port

ers

efflu

x sy

stem

L, Z

1),

tetA

(P),

tet3

0gr

am–

bact

eria

chlo

ram

phen

icol

, 12

TM

S e

fflux

sys

tem

p

p-f

lo, f

loR

, cm

lA-lik

eP

, T,G

C, C

Ph

oto

ba

cte

riu

m,

[3, 8

, 17,

32,

flo

rfen

icol

Sa

lmo

ne

lla, E

sch

erich

ia33

, 77]

14-,

15-

mem

bere

d ef

flux

syst

em o

f m

ef(A

)P

, T, C

Str

ep

toco

ccu

s, gra

m+

[5

3]m

acro

lides

the

maj

or fa

cilit

ator

ba

cter

iasu

perf

amily

stre

ptog

ram

in

efflu

x sy

stem

of

vga(

A),

vg

a(B

)P

Sta

ph

ylo

cocc

us

[53]

A-c

ompo

unds

th

e A

BC

fam

ily

activ

e ex

port

via

m

acro

lides

and

ef

flux

syst

emm

sr(A

)P

Sta

ph

ylo

cocc

us

[53,

55]

spec

ific

tran

spor

t st

rept

ogra

min

in

com

bina

tion

with

pr

otei

nsB

-com

poun

dsan

AT

P-b

indi

ng

tran

spor

t pro

tein

activ

e ex

port

via

ch

lora

mph

enic

ol,

12-T

MS

mul

tidru

gb

lt, n

orA

CB

aci

llus,

[2

, 44,

46]

mul

tidru

g flu

oroq

uino

lone

s,

efflu

x pr

otei

n of

Sta

ph

ylo

cocc

us

tran

spor

ters

nucl

eic

acid

bin

ding

th

e m

ajor

faci

litat

or

com

poun

dssu

perf

amily

tetr

acyc

lines

, nuc

leic

4-

TM

S m

ultid

rug

em

rEC

Esc

he

rich

ia[2

, 44]

acid

bin

ding

com

poun

dsef

flux

prot

ein

chlo

ram

phen

icol

, β-la

ctam

s,

mul

tidru

g ef

flux

me

xB-m

exA

-op

rMC

Pse

ud

om

on

as

[2, 4

4, 4

5]m

acro

lides

, flu

oroq

uino

lone

s,

in c

ombi

natio

n a

crA-a

crB-t

olC

CE

sch

erich

ia c

oli,

[2

, 25,

27]

tetr

acyc

lines

, etc

. w

ith s

peci

fic O

MP

’sS

alm

on

ella

a P

= p

lasm

id; T

= tr

ansp

oson

; GC

= g

ene

cass

ette

; C =

chr

omos

omal

DN

A.

bT

MS

= tr

ansm

embr

ane

segm

ents

.


Tab

le V

II. E

xam

ples

of b

acte

rial r

esis

tanc

e to

ant

imic

robi

als

by

alte

ratio

n o

f th

e ta

rge

t (mod

ified

from

[61]

).

Res

ista

nce

Res

ista

nce

to

Via

R

esis

tanc

eG

ene

Bac

teria

R

ef.

mec

hani

sm

gene

(s)

loca

tion

a

prot

ectio

n of

te

trac

yclin

esrib

osom

e pr

otec

tive te

t(M, O

, P, Q

, S, T

) T

, Cva

rious

gra

m+

and

[3

7, 5

2, 6

7, 6

9]

the

targ

et s

ite

prot

eins

gram

– ba

cter

ia

chem

ical

m

acro

lides

, lin

cosa

mid

es,

rRN

A m

ethy

lase

erm

(A, B

, C, F

) P

, T, C

va

rious

gra

m+

bac

teria

,[1

5, 3

5, 5

3, 7

4–76

] m

odifi

catio

n B

-com

poun

d of

E

sch

erich

ia, B

act

ero

ide

s of

the

targ

et s

itest

rept

ogra

min

s

repl

acem

ent o

f a

trim

etho

prim

inse

nsiti

ve

dh

frI-

XV

II,P

, T, G

CE

nte

rob

act

eria

cea

e,

[21,

29,

58,

66,

70]

sens

itive

targ

et

dihy

drof

olat

e re

duct

ase d

frA

, dfr

B, d

frD

Sta

ph

ylo

cocc

us,L

iste

ria

stru

ctur

e by

a

resi

stan

t tar

get

sulfo

nam

ides

inse

nsiti

ve d

ihyd

ropt

eroi

c su

lI, s

ulII

P, G

CE

nte

rob

act

eria

cea

e, [2

9, 4

9, 6

6, 7

0]ac

id s

ynth

etas

eP

ast

eu

rella

cea

e

all ß

-lact

ams

incl

. pe

nici

llin-

bind

ing

me

cAC

Sta

ph

ylo

cocc

us

[24,

26,

31]

ceph

alos

porin

s, c

arba

pene

ms

prot

eins

with

alte

red

and

mon

obac

tam

ssu

bstr

ate

spec

ifici

ty

mut

atio

nal

stre

ptom

ycin

mut

atio

n in

the

gene

-

Cse

vera

l gra

m+

and

[4

8]m

odifi

catio

nco

ding

for

ribos

omal

gr

am–

bact

eria

of th

e ta

rget

site

prot

ein

S12

stre

ptom

ycin

mut

atio

n in

the

16S

rR

NA

-C

Myc

ob

act

eriu

m[4

8]

mac

rolid

esm

utat

ion

in th

e 23

S r

RN

A-

CM

yco

ba

cte

riu

m[4

0]

fluor

oqui

nolo

nes

mut

atio

ns in

the

gene

s -

Cva

rious

gra

m+

[4

, 23,

30,

73]

for

DN

A g

yras

e an

d an

d gr

am–

bact

eria

topo

isom

eras

e

tetr

acyc

lines

m

utat

ion

in th

e 16

S r

RN

A-

CP

rop

ion

iba

cte

riu

m[5

6]

aP

= p

lasm

id; T

= tr

ansp

oson

; GC

= g

ene

cass

ette

; C =

chr

omos

omal

DN

A.


consists of 14 transmembrane regions. TheirTc-inducible expression is regulated by amechanism known as “translational atten-uation”. Genes of classes K and L are fre-quently found on small plasmids which inrare cases may be integrated into other plas-mids or into the chromosomal DNA, butmay also undergo interplasmidic recombi-nation with other resistance plasmids. Thetetracycline efflux proteins present in gram-negative bacteria exhibit only 12 trans-membrane segments. To date, eight differ-ent tet genes for efflux proteins ingram-negative bacteria, tet(A–E, G, H) andtet(J), have been sequenced. Each of these tetstructural genes is accompanied by a spe-cific tetrepressor gene. Tc-inducible expres-sion of these tetgenes is based on the bind-ing of a tetracycline Mg2+ complex to thetetrepressor protein which, in the absence oftetracycline, blocks transcription of the tetstructural gene [52]. The tetgenes of classesC, E, and G are often found on plasmidswhile those of classes A, B, D, and H areassociated with non-conjugative transposonsor transposon-like elements which may alsoreside on plasmids.

The ribosome protective proteins iden-tified so far share considerable homologywith ribosomal elongation factors and alsoexhibit GTPase activity [69]. Until now,eight different classes of tet genes whichcode for ribosome protective proteins, M,O, P, Q, S, T, W as well as otrA, are known[37]. Tc-inducible expression of genestet(M) and tet(O) seems to be regulated atthe transcription level. The tet(M) gene iscommonly found on conjugative transposonswhich exhibit an extremely broad host range.Thus tet(M) genes are known to occur in awide variety of gram-positive and gram-negative bacteria [52]. Genes of class Ohave mainly been detected in Campylobac-ter, Streptococcus, Enterococcus, and thoseof class S are present in Listeria and Ente-rococcus. The tet(Q) gene has been shownto be part of large conjugative transposons inBacteroidesand related genera. The geneof class T has so far only been detected in

Streptococcus pyogenesand that of class Win Butyrivibrio fibrisolvensand other rumi-nal bacteria. The otrA gene which origi-nated from tetracycline-producing Strepto-mycesspp. was also found in mycobacteria[52].

Only the tet gene of class X has beenfound to be involved in the inactivation oftetracyclines. The TetX protein represents acytoplasmic protein that chemically modifiestetracycline in the presence of oxygen andNADPH. Surprisingly, the tetX gene hasnot been detected in bacteria other thananaerobic Bacteroidesspp. where the TetXprotein is inactive [52, 67].

Different types of multidrug transportersmediating resistance to tetracycline in addi-tion to resistance to a number of structurallyunrelated compounds have been described,for instance, in Escherichia coli (EmrE),Salmonella(AcrAB/TolC) and Pseu-domonas aeruginosa(MexAB/OprM;MexCD/OprJ) [44, 47].

A permeability barrier due to the reducedproduction of the OmpF porin by whichtetracyclines cross the outer membrane hasbeen described in Escherichia coli. Muta-tions in the marRAB operon which also reg-ulates OmpF expression may play a role inthis type of tetracycline resistance [48].

A mutation in the 16S rRNA has beenidentified in Propionibacterium acnes[56]as conferring tetracycline resistance. Thismutation consisted of a single base exchange(1058G → 1058C). The position 1058 islocated in a region known as helix 34 whichplays an important role in the terminationof peptide chain elongation as well as in theaccuracy of translation.

5.2. Resistance to macrolides, lincosamides, and streptogramins(MLS)

Many gram-negative bacilli exhibitintrinsic resistance to the therapeuticallyachievable concentrations of macrolides and


lincosamides, based on the reduced perme-ability of the outer membrane to these sub-stances [48]. The mechanisms involved inresistance to macrolides, lincosamides andstreptogramins so far observed mainlyamong gram-positive bacteria include tar-get modification, active efflux and enzy-matic inactivation [35, 36, 53].

Target modification by rRNA methylaseshas been detected in a wide variety of gram-positive but also several gram-negative bac-teria. It is commonly due to the expressionof a plasmid- or transposon-borne ermgenewhose gene product dimethylates a specificadenine residue (A2058) in a conservedregion of the 23S rRNA [35, 74]. Thismethylation confers cross-resistance tomacrolides, lincosamides and B-compoundsof streptogramins (MLSB antibiotics).Expression of the ermgenes may be con-stitutive or inducible via translational atten-uation; the type of expression depends ona regulatory region upstream of the ermgene[75]. Sequence deletions, duplications, andpoint mutations in the regulatory regions ofcertain ermgenes have been found to causea switch from inducible to constitutiveexpression [76]. At least 22 different classesof erm genes have been identified, four ofwhich – A, B, C, and F – also play a role inveterinary pathogens [53]. A summary ofthe genera in which the different ermgeneshave so far been detected has recently beenpublished [53]. Genes of class A and B arepart of non-conjugative or conjugative trans-posons, while genes of class C are com-monly located on small plasmids up to 4 kbp[53]. The erm(F) gene has been describedas part of conjugative transposons in Bac-teroidesspp. [50]. A recent survey on thedistribution and host range of the erm(F)gene has shown that this gene is widely dis-tributed among gram-positive and gram-negative bacteria of medical and veterinaryimportance [15].

At least ten different efflux/transport sys-tems have been described to confer resis-tance to members of the MLS group ofantibiotics, four of which have been iden-

tified among staphylococci and other gram-positive pathogens while the remaining oneshave been detected in soil bacteria of thegenus Streptomyces[53]. These efflux/trans-port systems differ from one another in theirsubstrate spectra. The gene erpA is involvedin the active efflux of 14- and 15-memberedmacrolides. The gene msr(A) and its closerelatives msr(SA), msr(SA)’ and msr(B)code for ATP-binding transport proteinswhich mediate the active efflux of 14-mem-bered macrolides and B-compounds of thestreptogramins. Genes vga(A) and vga(B)also code for ATP-binding transport pro-teins which, however, are involved in theexport of A-compounds of the strep-togramins. Most of the genes for these trans-port proteins are located on plasmids. Sincethe Msr and Vga proteins do not display thetopology of membrane proteins, it isassumed that they interact with a membrane-associated ABC-transporter. One such trans-porter with which MsrA may interact hasbeen identified [55]. In addition to thesetransport proteins, another two closelyrelated genes, mef(A) and mef(E), whichcode for efflux proteins involved in theexport of macrolides were detected in mem-bers of the genera Streptococcus, Entero-coccus, Staphylococcusand Corynebac-terium. The Mef proteins exhibit homologyto the major facilitator family of efflux pro-teins [53].

Enzymatic inactivation of MLS antibi-otics is mediated by a number of differentenzymes, each of which displays a narrowsubstrate spectrum [36, 53]. Three differ-ent types of inactivating enzymes areknown: esterases, hydrolases, and trans-ferases. Among the esterases, two genes,ere(A) and ere(B) are known to occurmainly in Enterobacteriaceae. An esteraseis also believed to confer resistance to 14-and 16-membered macrolides in a clinicalstrain of Staphylococcus haemolyticus. Lac-tone hydrolases, encoded by genes vgb(A)and vgb(B) inactivate B-compounds of thestreptogramins. Acetyltransferases, encodedby genes vat(A-E) inactivate A-compounds


of the streptogramins while nucleotidyl-transferases encoded by lnu(A) and lnu(B)inactivate lincosamides. Phosphotransferasessuch as those encoded by genes mph(A) andmph(B) have been detected in Escherichiacoli, the phosphotransferase encoded bymph(C) in Staphylococcus. Most of thegenes for inactivating enzymes are associ-ated with plasmids.

Mutations in the 23S rRNA associatedwith resistance to macrolides have beendescribed in members of the genusMycobacterium[40].

5.3. Resistance to β-lactam antibiotics

Resistance to β-lactam antibiotics ismainly mediated by a large number ofβ-lactamases which differ in their abilities tohydrolyse the various β-lactam antibiotics[2, 9, 38, 71]. Other resistance mechanismsinclude the acquisition of penicillin-binding proteins (PBPs) with reduced affin-ity to β-lactams, mutations in the PBPs [24,26], but also reduced β-lactam uptake due toalterations in the outer membrane of gram-negative bacteria or export by multidrugtransporters [44, 48].

Enzymatic inactivation of β-lactamantibiotics is achieved by β-lactamases [2, 9,38, 71]. On the basis of their substrate spec-tra and their inhibition by clavulanic acid(CA), β-lactamases are classified into atleast four classes (1–4), one of which,class 2, consists of eight subclasses [9]. Itis noteworthy that single amino acidexchanges may result in changes of the sub-strate spectrum. The most frequently occur-ring β-lactamases are those of classes 1, 2a,2b, and 2be. Class 1 β-lactamases(e.g. AmpC) represent cephalosporinaseswhich are insensitive to inhibition by CA.The respective bla genes are located on plas-mids or in the chromosomal DNA. Theβ-lactamases of classes 2a, 2b (e.g. TEM-1,TEM-2, SHV-1, ROB-1) and 2be(e.g. TEM-3 – 27; SHV-2 – 7; K1) hydrol-yse either penicillins, penicillins and

cephalosporins, or penicillins, cephalosporinsand monobactams, respectively. Members ofthese three subclasses are sensitive to inhibi-tion by CA, and their bla genes are mainlylocated on plasmids. The β-lactamases of class2c (e.g. PSE-1, 3, 4; BRO-1,2) represent CA-sensitive penicillinases. Class 2d β-lactamases(e.g. PSE-2; OXA-1 – 11) exhibit relativeresistance to CA and are capable ofhydrolysing penicillins, cephalosporins andoxacillin. Class 2f (e.g. IMI-1) and class 3(e.g. IMP-1; L1) β-lactamases are both ableto hydrolyse penicillins, cephalosporins,monobactams and carbapenems, however,they differ in their sensitivity to CA: class 2fenzymes are sensitive while class 3 enzymesare resistant. The β-lactamases of class 4hydrolyse penicillins and are resistant toinhibition by CA. Only β-lactamases of class2a are present in gram-positive bacteria, asall other β-lactamases are found mainlyamong gram-negative bacteria. The β-lac-tamases of gram-negative bacteria arereleased into the periplasmic space whileβ-lactamases of gram-positive bacteria aresecreted from the cell. With the exception ofa few class 1 enzymes, most β-lactamases ofgram-negative bacteria are constitutivelyexpressed whereas class 2a β-lactamases ofgram-positive bacteria are usually induciblyexpressed [48].

The acquisition of β-lactam-resistantPBPs which replace β-lactam sensitive PBPsis the cause of methicillin resistance inStaphylococcus aureus [24, 26]. Methicillin-resistant Staphylococcus aureus(MRSA)isolates are resistant not only to all peni-cillins, but also to cephalosporins, car-bapenems, and monobactams. The mecAgene which codes for β-lactam-resistantPBPs is located on a 52 kbp genetic elementdesignated Staphylococcuscassette chro-mosome mec(SSCmec) [31]. PBPs whichexhibit low affinity for β-lactams have alsobeen detected in streptococci and entero-cocci [48].

Reduced uptake of β-lactams as observedin Escherichia colimay be based on thedecreased expression or the structural


alteration of the porins OmpF and OmpCby which β-lactams pass through the outermembrane. In Pseudomonas aeruginosa,resistance to imipenem has been shown to bebased on the loss of the porin OprD [48].

Multidrug transporters such as theMexAB/OprM export system in Pseu-domonas aeruginosa[44], AcrAB/TolC inSalmonellaand E. coli [47] can also medi-ate the excretion of β-lactams.

5.4. Resistance to aminoglycosides

Resistance to aminoglycosides is mainlybased on enzymatic inactivation by amino-glycoside-modifying enzymes [64]. More-over, decreased uptake of aminoglycosidesand chromosomal mutations conferringhigh-level resistance to streptomycin havealso been described [48].

Enzymatic inactivation of aminoglyco-sides is conferred by N-acetyltransferases,O-adenyltransferases or O-phosphotrans-ferases [64]. There are numerous membersof each of these three classes of aminogly-coside-modifying enzymes, most of whichexhibit a specific substrate spectrum. A sum-mary of the known aminoglycoside-modi-fying enzymes and their molecular rela-tionships was published in 1993 [64].However, since then, several new amino-glycoside-inactivating enzymes have beenidentified, some of which are part of inte-grons / gene cassettes [51]. Four classes ofN-acetyltransferases (AACs) are knownwhich acetylate the amino groups at posi-tions 1-, 3-, 2’- and 6’ [20, 41, 64, 80]. Todate, at least 16 different AACs have beenidentified, most of which were found ingram-negative bacteria [80]. Resistance toapramycin used in veterinary medicine onlyemerged after the introduction of this drug[13], in Salmonella andE. coli in animals;later on, a limited diffusion in hospitals wasstudied [12, 14]. All the known AAC vari-ants differ in their substrate spectra. Abifunctional enzyme which codes for acetyl-transferase AAC(6’) and phosphotransferase

APH(2’’) activities was found on transposonTn4001which is widely spread amongstaphylococci and enterococci [57]. Mostaac genes are located on plasmids, trans-posons or integrons. Five classes ofO-adenyltransferases (ANTs) which act atpositions 6, 9, 4’, 2’’, and 3’’ are differen-tiated [20, 41, 64, 80]. The different ANTenzymes also show distinct differences intheir substrate spectra. The various antgenesare mostly associated with either plasmids ortransposons. Among the phosphotrans-ferases (APHs) which phosphorylate thehydroxyl groups at positions 4, 6, 3’, 2’’,and 3’’, at least 11 variants have been iden-tified. Some of these aphgenes are locatedon mobile genetic elements [20, 41, 64, 80].The different APH variants also differ intheir substrate profiles.

Decreased drug uptake of aminoglyco-sides has been described to be based on amutation in LPS phosphates or on a changein the charge of the LPS in Escherichia coliand Pseudomonas aeruginosa, respectively[59]. Since the entry of aminoglycosidesacross the cytoplasmic membrane is mainlybased on the electron transport system,anaerobes and facultative anaerobes exhibitrelative resistance to aminoglycosides [48].

Mutations in either the gene for the ribo-somal protein S12 or 16S rRNA have beendescribed in connection with streptomycinresistance [48].

Efflux systems such as AcrD inEscherichia coli [54] and MexXY in Pseu-domonas [1] have recently been described.

5.5. Resistance to sulfonamides and trimethoprim

Sulfonamides and trimethoprim blockdifferent enzymatic steps in tetrahydrofo-late biosynthesis. Sulfonamides are struc-tural analogs of p-aminobenzoic acid andcompetitively inhibit the enzyme dihy-dropteroic acid synthetase (DHPS) whiletrimethoprim competitively inhibits the


enzyme dihydrofolate reductase (DHFR).While some bacteria are intrinsically resis-tant, acquired resistance may be due to chro-mosomal mutations or to plasmid-encodedDHPS or DHFR enzymes which are resis-tant to sulfonamides and trimethoprim,respectively [2, 21, 48, 70]. A detaileddescription of the molecular basis of resis-tance to trimethoprim and sulfonamides wasgiven by Sköld [66].

Intrinsic resistance to both compoundsby outer membrane impermeability has beenobserved in Pseudomonas aeruginosa. Bac-teria which can utilise exogenous folatessuch as enterococci and lactobacilli, alsoshow intrinsic resistance to trimethoprimand sulfonamides. The DHFR enzymes ofseveral bacterial genera including Clostrid-ium, Neisseria, Brucella, BacteroidesandMoraxella exhibit low affinity for trimetho-prim and thus render their hosts intrinsicallyresistant to trimethoprim [48].

Chromosomal mutations that cause anoverexpression of p-aminobenzoic acidand/or DHFR can result in sulfonamideand/or trimethoprim resistance [48]. Muta-tions in the genes for DHPS and DHFR canreduce the affinity of the respective geneproducts for sulfonamides and trimetho-prim, respectively. Moreover, mutationalinactivation of the thymidylate synthetasewhich causes thymine auxotrophy resultsin resistance to folate pathway antagonists[48].

The replacement of sensitive enzymes byresistant enzymes usually causes high-levelresistance [29]. Two resistant DHPSenzymes encoded by genes sulI and sulIIhave been described in gram-negative bac-teria [2, 29, 48]. Gene sulI is part of class Iintegrons in transposon Tn21which is oftenfound on conjugative plasmids. The sulIIgene occurs together with a streptomycinresistance gene on conjugative or non-con-jugative plasmids [29, 49]. A number of dif-ferent dhfr genes have been describedamong gram-negative bacteria, several ofwhich are part of gene cassettes [29]. In

staphylococci, the composite transposonTn4003has been identified on various mul-tiresistance plasmids [58]. Tn4003is com-posed of a central dfrA gene which codesfor a DHFR enzyme with reduced affinity totrimethoprim, bracketed by copies of IS257.Another two trimethoprim resistance genes,dfrB from S. haemolyticusand dfrD fromL. monocytogenes, have been encountered ingram-positive bacteria [11].

5.6. Resistance to fluoroquinolones

Resistance to fluoroquinolones is basedeither on mutations which render the targetresistant to the drugs or on decreased intra-cellular drug accumulation [2, 23, 27, 48].Enzymatic inactivation has not beenobserved so far. The molecular basis andepidemiology of quinolone resistance inE. coli and Salmonellawas reviewed byCloeckaert and Chaslus-Dancla [16], Webber and Piddock [73] and Bager andHelmuth [4], respectively.

Mutational alteration of the target struc-tures mainly involves genes gyrA, gyrB(coding for DNA gyrase) and parC andparE (coding for DNA topoisomerase IV).A wide variety of mutations has beendetected in the various target genes of a widerange of gram-positive and gram-negativebacteria of human and veterinary impor-tance [16, 23, 27]. The effect of the differentmutations on resistance also differs withrespect to the various fluoroquinolones [30].The gyrA mutations are commonly locatedwithin what is referred to as a quinoloneresistance-determining region of 130 bp[48].

Efflux systems conferring fluoroquinoloneresistance have been identified in variousgram-positive and gram-negative bacteria,such as Ps. aeruginosa(MexAB/OprM,MexCD/OprJ), S. aureus(NorA), S. pneu-moniae(PmrA), B. subtilis(Blt), E. coliandSalmonella(AcrAB/TolC). For reviews, seereferences [16, 45–47]. Many of these effluxsystems represent multidrug transporters


which are able to export, in addition toquinolones, a wide range of other toxic sub-stances from the bacterial cell [16, 44–46].Since the basal level of expression of theseefflux systems is low, upregulation of theirexpression is required to confer resistance tofluoroquinolones and other antimicrobials.

Decreased drug uptake in gram-negativebacteria is due to the MAR-mediated down-regulation of OmpF porin production [48].OmpF is an important porin for the entry ofquinolones into the bacterial cell. Moreover,mutations in different gene loci (cfxB, norB,nfxB, norC or nalB) are also associated withdecreased permeability [28].

The relative involvement of these differ-ent mechanisms in the resistance to fluoro-quinolones is questionable [3, 25, 43].

5.7. Resistance to chloramphenicol and florfenicol

Chloramphenicol resistance in gram-positive and gram-negative bacteria ismainly due to enzymatic inactivation [2,48]. Efflux systems which confer either onlychloramphenicol resistance or combinedresistance to chloramphenicol and florfeni-col have also been described [2, 3, 8, 17,18, 32, 77]. Permeability barriers and mul-tidrug transporters only play a role in certaingram-negative bacteria [44, 48].

Enzymatic inactivation of chloram-phenicol is due to chloramphenicol acetyl-transferases (Cat) which are capable of trans-ferring acetyl groups to the C1 and C3positions of the chloramphenicol molecule;acetylated chloramphenicol derivatives can-not inhibit bacterial protein biosynthesis.Two different types of Cat enzymes areknown: CatA and CatB enzymes. All CatAand CatB variants have a trimeric structurecomposed of three identical subunits, each ofwhich ranges in size between 207 and 238amino acids [42, 65]. The cat gene codesfor a Cat monomer. Expression of the mostlyplasmid-borne catAgenes found in Staphy-

lococcus, Streptococcus, Enterococcus,Bacillus, and Listeria is inducible by chlo-ramphenicol via translational attenuation[39]. Several catAgenes identical or closelyrelated to those of the S. aureusplasmidspC221, pC223/pSCS7 or pC194 have beendetected in various staphylococci, but also inStreptococcus, Bacillusor Listeria, respec-tively [61]. The catAgenes of Clostridiumspp. are constitutively expressed. Three dif-ferent types of constitutively expressed catAgenes, designated I-III, have been detectedin Enterobacteriaceae[65]. Gene catAI islocated on the non-conjugative transposonTn9 and related transposons. The genecatAII has been detected in Haemophilusspp. while the catAIII gene was present inEnterobacteriaceaeand Pasteurella. ThecatBgenes – also referred to as xat (xeno-biotic acetyltransferase) genes – differ dis-tinctly from the catA genes in theirsequences, but appear to be related to othergenes such as vat(A-E), coding for acety-lating enzymes involved in streptograminresistance [42]. The first catB gene wasdetected in Agrobacterium tumefaciens, butothers have been found on transposonTn2424 in E. coli, on transposon Tn840from Morganella morganiiand in the chro-mosome of Ps. aeruginosa. Incompletesequences of further catBgenes from Ser-ratia marcescens, B. sphaericusandS. aureushave been reported [42] and sug-gest a wider distribution of catB genesamong gram-positive and gram-negativebacteria than initially assumed.

Decreased intracellular chlorampheni-col accumulation may result from mutationsthat cause reduced expression of a majorouter membrane protein in Haemophilusinfluenzae and of the OmpF protein inSalmonella typhi[2, 48]. Specific chloram-phenicol exporters as encoded by genescmlA and cmlB have been detected in Pseu-domonas aeruginosaand Rhodococcus fas-cians[44]. The cmlA1 gene which is locatedon transposon Tn1696has also been identi-fied as part of a gene cassette [51]. Mul-tidrug transporter systems in Pseudomonas


aeruginosa, such as MexAB/OprM andMexCD/OprJ, also export chloramphenicol[44]. Genes, such as pp-floand floR– cod-ing for membrane-associated efflux systemswhich export chloramphenicol and flor-fenicol – have been detected on a plasmid inPhotobacterium damselae subsp. piscicida[33] as part of a chromosomal multiresis-tance gene cluster in Salmonella entericaserovars Typhimurium [3, 8] and Agona[18], but also on multiresistance plasmidsof E. coli [17, 32, 77].

Moreover, the plasmid-borne gene, cfr,from Staphylococcus sciurihas been foundto mediate combined resistance to chlo-ramphenicol and florfenicol by a yet uniden-tified mechanism [62].

6. CONCLUSION

The data presented in this review showthat bacteria of the different species andgenera have developed a wide variety ofresistance genes to escape the inhibitoryeffects of antimicrobial agents. The rela-tively short time periods between the intro-duction of an antimicrobial agent into clin-ical use and the occurrence of resistantbacteria confirms that bacteria are able toquickly and efficiently adapt to altered envi-ronmental conditions caused by thewidespread use of antimicrobials. In thisregard, bacteria have also developed highlyefficient ways to transfer resistance genesbetween members of different species andgenera. These transfer systems allow a rapidexchange of resistance genes within bacte-rial mixed populations commonly seen onthe skin but also on the mucosal surfaces ofrespiratory, alimentary and genito-urinarytract of humans and animals. The detectionof the same resistance gene in a wide varietyof bacteria (for an example see the distri-bution of tet genes as described in [52])illustrates that such transfer systems involv-ing mobile genetic elements are effectivelyused under in vivo conditions. In addition,bacteria have also developed a number of

mutations which render their cellular targetsites resistant to the respective antimicro-bial agents. Three key factors with regardto the emergence of antimicrobial resistancehave to be taken into account: (i) the asso-ciation of the resistance gene(s) with mobilegenetic elements, (ii) the close contactbetween bacteria in a polymicrobial envi-ronment, and (iii) the selective pressure asimposed by the use of antimicrobials. Thislatter aspect is the one which can effectivelybe influenced by all those people in humanand veterinary medicine who prescribe anduse antimicrobial agents. Bearing in mindthat no new classes of antimicrobial agentsare to be expected for veterinary use in thenear future, every effort must be undertakento retain the efficacy of those substancescurrently available. Since every use ofantimicrobial agents may select for resis-tant bacteria, resistance development amongbacteria is a physiological stress responseto changes in their environmental condi-tions. We cannot avoid resistance develop-ment, but we can dramatically slow downthe development and spread of resistanceproperties. Therefore, guidelines have beenpublished by numerous national and inter-national boards to assist all those peopleinvolved in the effort towards a prudent andjudicious use of antimicrobials. Sticking tothese guidelines will minimise the risk ofselecting resistant bacteria during the ther-apeutic use of antimicrobials and in factappears to be the only broad scale approachto retain the efficacy of antimicrobial agentsfor the control of bacterial infections in ani-mals.

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