J Antimicrob Chemother

doi:10.1093/jac/dkz161

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The use of aminoglycosides in animals within the EU: development of
resistance in animals and possible impact on human and
animal health: a review
Engeline van Duijkeren1*, Christine Schwarz2, Damien Bouchard3, Boudewijn Catry4,5, Constança Pomba6,
Keith Edward Baptiste7, Miguel A. Moreno8, Merja Rantala9, Modestas Ruzauskas10, Pascal Sanders11,
Christopher Teale12, Astrid L. Wester13, Kristine Ignate14, Zoltan Kunsagi14 and Helen Jukes15

1
National Institute for Public Health and the Environment, Bilthoven, The Netherlands; 2Federal Office of Consumer Protection and
Food Safety, Berlin, Germany; 3French Agency for Food, Environmental, and Occupational Safety, National Agency for Veterinary
Medicinal Products, Fougères, France; 4Sciensano, Brussels, Belgium; 5Faculty of Medicine, Université libre de Bruxelles (ULB), Brussels,
Belgium; 6Faculty of Veterinary Medicine, University of Lisbon, Lisbon, Portugal; 7Danish Medicines Agency, Copenhagen, Denmark;
8
Faculty of Veterinary Medicine, Complutense University, Madrid, Spain; 9Faculty of Veterinary Medicine, University of Helsinki, Helsinki,
Finland; 10Lithuanian University of Health Sciences, Kaunas, Lithuania; 11French Agency for Food, Environmental, and Occupational
Safety, Fougères Laboratory, Fougères, France; 12Animal and Plant Health Agency, Weybridge, UK; 13World Health Organization,
Genève, Switzerland; 14European Medicines Agency, Amsterdam, The Netherlands; 15Veterinary Medicines Directorate, Addlestone, UK

*Corresponding author. Tel: !31 30 2743942; Fax: !31 30 2744409; E-mail: engeline.van.duijkeren@rivm.nl

Aminoglycosides (AGs) are important antibacterial agents for the treatment of various infections in humans and
animals. Following extensive use of AGs in humans, food-producing animals and companion animals, acquired
resistance among human and animal pathogens and commensal bacteria has emerged. Acquired resistance
occurs through several mechanisms, but enzymatic inactivation of AGs is the most common one. Resistance
genes are often located on mobile genetic elements, facilitating their spread between different bacterial species
and between animals and humans. AG resistance has been found in many different bacterial species, including
those with zoonotic potential such as Salmonella spp., Campylobacter spp. and livestock-associated MRSA. The
highest risk is anticipated from transfer of resistant enterococci or coliforms (Escherichia coli) since infections
with these pathogens in humans would potentially be treated with AGs. There is evidence that the use of AGs in
human and veterinary medicine is associated with the increased prevalence of resistance. The same resistance
genes have been found in isolates from humans and animals. Evaluation of risk factors indicates that the prob-
ability of transmission of AG resistance from animals to humans through transfer of zoonotic or commensal
foodborne bacteria and/or their mobile genetic elements can be regarded as high, although there are no quanti-
tative data on the actual contribution of animals to AG resistance in human pathogens. Responsible use of AGs is
of great importance in order to safeguard their clinical efficacy for human and veterinary medicine.

Introduction
Aminoglycosides (AGs), introduced in 1944, are among the oldest
classes of antimicrobials. AGs are bactericidal antimicrobials that
act by impairing bacterial protein synthesis through binding to the
30S ribosomal subunit.1 AGs must penetrate into the bacterium to
exert their effect, and their uptake in to the bacterial cell is an
oxygen-dependent process. Therefore, the spectrum of activity of
AGs is limited to aerobic and facultative anaerobic bacteria under
aerobic conditions. AGs are less potent in hyperosmolar environ-
ments or environments with low pH. In addition, purulent debris at
the infection site can bind to AGs and inactivate them.1 AGs are
hydrophilic molecules and relatively insoluble in lipids. They are
poorly absorbed from the gut, and penetration of the blood–brain
barrier is minimal.1,2
The spectrum of activity of AGs includes Gram-negative bac-
teria, staphylococci, mycobacteria, leptospira and certain proto-
zoa. They have poor efficacy against streptococci, anaerobic
bacteria and intracellular bacteria. Enterococci and streptococci
generally show a degree of intrinsic resistance to AGs due to imper-
meability of their cell wall, although penetration into the bacterial
cell can be enhanced by other antimicrobials that inhibit cell wall
synthesis, such as the b-lactam antibiotics. Therefore, AGs are
often used in combination with b-lactams. This combination also
broadens the spectrum of activity.1

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Review
In veterinary medicine, AGs are used to treat infections in all
major food-producing animals and in companion animal species.
They are categorized as veterinary critically important antimicro-
bials (VCIAs) by the World Organisation for Animal Health (OIE)3
based on the wide range and nature of the diseases they are used
to treat in animals and the limited availability of economic alterna-
tives for some infections. Similarly, AGs are classified by WHO as
critically important antimicrobials (CIAs-high priority) for human
medicine.4 In human medicine in the EU, AGs are important for
treatment of enterococcal endocarditis, infections with MDR
Gram-negative bacteria (especially Enterobacteriaceae and
Pseudomonas spp.) and MDR TB. Due to their importance in human
medicine, the public health impact of the use of AGs in animals
needs to be assessed. Our objective was to critically review the cur-
rent knowledge on the usage of AGs, resistance development and
the potential impact of this resistance on animal and human
health.
The use of AGs in veterinary medicine
AGs are used extensively in veterinary medicine.5 In 2015, the
sales of AGs made up 3.5% of the total sales of antimicrobials for
food-producing species (including horses), in mg/population cor-
rection unit (PCU), aggregated for 30 European countries. AGs are
the sixth most commonly used antimicrobial class after tetracy-
clines, penicillins, sulphonamides, macrolides and polymyxins
(Figure 1).5 There are marked differences in the sales of AGs be-
tween the different EU countries, with the lowest sales in the
Scandinavian countries and the highest in Spain (Figure 2); these
differences are not explained by the differences in overall anti-
microbial use between countries in all cases. The reasons for these
differences should be investigated. The most frequently used AGs
are neomycin, dihydrostreptomycin and spectinomycin (Figure 3).
Other substances from the group that are used in food-producing
species [where maximum residue limits (MRLs) have been estab-
lished] are: apramycin, gentamicin, kanamycin, paromomycin,
neomycin, framycetin and streptomycin.
In EU Member States (MSs) the vast majority of available AGs
are administered to animals for clinical purposes. The most fre-
quent use is therapy for septicaemia, and infections of the digest-
ive tract, respiratory tract and urinary tract in many animal species
including cattle, pigs, poultry, sheep, goats, horses, dogs and cats
(Table S1 available as Supplementary data at JAC Online). In par-
ticular, gentamicin is indicated for Pseudomonas aeruginosa infec-
tions, with few alternative treatments being available.1 AGs have
not been authorized as growth promoters in the EU MSs since
1976. Before this, neomycin and hygromycin-B were authorized to
be added to poultry feed for growth promotion only on a national
level in certain EU MSs.6 Some products containing AGs, especially
those with old marketing authorizations, are approved for general
indications such as the treatment of ‘infections caused by suscep-
tible organisms’ in various animal species.7
Route of administration in animals
In veterinary medicine, AGs are used for parenteral, oral and topic-
al applications. In the EU, approximately half of AG use is as oral
formulations (premix, oral powder or soluble in drinking water)
and about half is as injectable formulations (Figure S1).5
2 of 17
Substances used for parenteral applications include (dihydro)-
streptomycin, gentamicin, kanamycin, framycetin, spectinomycin
and neomycin. (Dihydro)streptomycin, neomycin, apramycin, gen-
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tamicin, paromomycin and spectinomycin are used in oral formu-
lations.5 The majority of oral formulations (oral solution, oral
powder, premix) are used for treatments in pigs, calves, sheep,
poultry and rabbits. They are generally administered in a once daily
treatment regimen as oral drenches (to neonates) or in feed or
drinking water/milk over a period of 3–5 (and in exceptional cases
even 7) days. Individual products are authorized for considerably
longer treatment durations, e.g. apramycin for 21 days or up to
28 days. Local applications include ear drops, eye drops, topical ap-
plication to the skin and intramammary and intrauterine
preparations.5,7
Pharmacokinetic/pharmacodynamic (PK/PD)
relationship and dosing regimens
For concentration-dependent antimicrobial agents, optimal dosing
involves administration of high doses with long dosing intervals.1
PK/PD indices have been proposed from in vitro and in vivo
infection models and subsequently validated in retrospective or
prospective human clinical trials.8 Two PK/PD indices, Cmax/MIC
and 24 h AUC/MIC, are the most important PK/PD predictors for
bacteriological and clinical efficacy of concentration-dependent
antimicrobials.9–11
Most authors have proposed the Cmax/MIC ratio as the PK/PD
index of choice for AGs (gentamicin, tobramycin and amikacin).
A Cmax/MIC ratio of 10 had the strongest association, with a posi-
tive clinical outcome in patients with pneumonia caused by aerobic
Gram-negative rods and with bacteraemia caused by P. aerugi-
nosa.12–14 Furthermore, a Cmax/MIC ratio of 10–12 was determined
to minimize the survival and overgrowth of resistant strains.8 If
this preferable Cmax/MIC ratio is obtained, most susceptible bac-
teria die rapidly, and consequently the effect of the duration of
drug exposure is minimal. Accordingly, in neutropenic and non-
neutropenic models of infection, significantly more animals sur-
vived a potentially lethal challenge of bacteria when treated with a
single large dose of an AG rather than with the same dose given on
an 8 h schedule. A high-dose and infrequent administration of AGs
has also been shown to reduce the rate of nephrotoxicity.15 These
findings, and meta-analyses of different dosing regimens of AGs,
led to a shift in clinical dosing in humans from two to three times
daily administration to once a day treatments.11,16 The actual goal
of AG therapy is to maximize peak concentrations to increase effi-
cacy and, in order to reduce toxicity, to administer once a day to
achieve a low trough concentration, and to reduce treatment
duration.17
In veterinary medicine, the situation is more complex because
of potential interspecies differences in PK and PD as well as differ-
ences in indications and target pathogens.18 In addition, in ani-
mals, AGs are to a large extent administered via the oral route for
the treatment of gastrointestinal infections where they exert their
antibacterial activity in situ without being absorbed. Thus, for veter-
inary purposes, human-derived PK/PD concepts cannot be applied
for oral applications and may be applied for parenteral applications
by approximation only.
When given via the parenteral route, AGs were traditionally
administered every 8–12 h. Recent studies in veterinary patients
Review
2.1% 3.0%
2.8%
3.5%
6.8%
3.2%
7.2%
1.9%
11.8%
Figure 1 Sales of antimicrobial agents by antimicrobial class as a percentage of the total sales for food-producing species (including horses), in mg/
PCU, aggregated by 30 European countries, for 2015.5 *Amphenicols, cephalosporins, other quinolones and other antibacterials (classified as such in
the ATCvet system). This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.
likewise support high-dose, once daily therapy with AGs to avoid
adaptive resistance and to reduce risks of toxicity. The optimal
doses and the ideal drug monitoring strategy are unknown for
most target animal species. Dosages have to be modified in neo-
nates and in animals with impaired liver or kidney function.1
In conclusion, dosing regimens should be re-investigated in vet-
erinary medicine in order to improve the efficacy and safety of AG
use, and to reduce the risk of antimicrobial resistance. Further re-
search should be conducted into the PK/PD surrogate indices for
treatment of veterinary infections with AGs, as validated parame-
ters are predictive of clinical efficacy and would assist optimization
of dosing regimens as well as reducing the toxicity of AGs that are
administered parenterally.
Aminoglycoside use in different animal species
Poultry
In the EU, neomycin, apramycin, spectinomycin and streptomycin
are authorized.7,19,20 Outside the EU, gentamicin is used as a sub-
cutaneous injection in day-old chicks or by in ovo injections. In ovo
injection is a route for administration of Marek’s disease vaccin-
ation in the USA and, to prevent bacterial contamination of the
eggs, gentamicin is injected in combination with the vaccine.21 In
ovo injections or other applications of gentamicin in poultry are not
authorized in the EU as no MRLs exist for gentamicin in this species.
Neomycin and apramycin are authorized for oral treatment of en-
teric infections, e.g. for the treatment of Escherichia coli and
Salmonella spp. infections in young chickens. Antimicrobials are
not permitted to be used for the specific purpose of control of
Salmonella spp., with certain exceptions [Commission Regulation
(EC) No. 1177/2006]. It is noted that colibacillosis is essentially a
systemic infection in poultry and it therefore appears contrary that
JAC
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Tetracyclines
32.8% Penicillins
Sulphonamides
Trimethoprim
Macrolides
Lincosamides
Polymyxins
Aminoglycosides
Pleuromutilins
Fluoroquinolones
Others*
25.0%
it should be treated orally with substances such as apramycin that
are considered to have very low oral bioavailability.22 The rationale
for indications for treatment of systemic diseases with AGs that
have very low oral bioavailability is debatable and the supporting
evidence should be reviewed.
Pigs
In pigs, apramycin, gentamicin, paromomycin and neomycin are
used for oral treatment of colibacillosis and salmonellosis.23 AGs
are an important alternative to colistin for the treatment of post-
weaning diarrhoea caused by E. coli.24 Dihydrostreptomycin in
combination with benzylpenicillin is authorized for respiratory
infections caused by Actinobacillus pleuropneumoniae and/or
Pasteurella multocida, and for the treatment of Glässer’s disease
caused by Haemophilus parasuis.
Cattle
Neomycin, streptomycin, kanamycin and framycetin, in combin-
ation with other antimicrobial agents, are used in preparations for
intramammary administrations to cows with mastitis.7,19,24 In
calves, neomycin and apramycin are used for the treatment of
bacterial enteritis caused by E. coli and salmonellae, and paromo-
mycin is used for the treatment of enteric infections caused by
E. coli.1,7
Gentamicin is used to treat respiratory infections caused
by Mannheimia haemolytica and P. multocida in calves.7
Dihydrostreptomycin or streptomycin is used in the treatment of
leptospirosis in cattle.
Horses
AGs (amikacin, neomycin and gentamicin) are commonly used for
treatment of bacterial septicaemia, respiratory tract infections,
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Figure 2 Spatial distribution of veterinary sales of aminoglycosides [amikacin, apramycin, (dihydro)streptomycin, framycetin, gentamicin, kanamycin
and neomycin] for food-producing animals in mg/PCU in 30 European countries for 2015.5 Sales of spectinomycin and paromomycin are not included
here as they are reported under ‘other antimicrobials’ in the ESVAC report. This figure appears in colour in the online version of JAC and in black and
white in the print version of JAC.

peritonitis, osteomyelitis, meningitis, wound infections, joint infec-
tions and endometritis, often in combination with other antimicro-
bials such as b-lactams.25 For infections in neonatal foals, where
Gram-negative bacteria are isolated from a high proportion of
cases and a bactericidal effect is desirable, there is a preference to
select gentamicin or amikacin as empirical therapy whilst awaiting
culture results.25 Topical application of AGs is recommended for
infections of the eye and uterus.25 Amikacin is authorized in some
MSs for horses that are kept as companion animals and do not
enter the food chain. Amikacin cannot be used in food-producing
animals as no MRL has been established.
Companion animals
Injections of gentamicin or amikacin are licensed for the treatment
of septicaemia and respiratory infections. In textbooks, AGs are
recommended for the treatment of bacterial peritonitis, metritis,
osteomyelitis, leptospirosis and nocardiosis.1 AGs such as
gentamicin, neomycin and framycetin are used as topical treat-
ment for infections of the eye (blepharitis, conjunctivitis, kerato-
conjunctivitis and anterior uveitis), ear (otitis externa) and skin.7,19
Gentamicin and tobramycin are effective topically to manage
otitis externa caused by P. aeruginosa.26 AGs are included for the
treatment of MDR infections in treatment guidelines for respiratory
infections in dogs and cats and for deep pyoderma, as third-line
choice.27,28 They are also suggested in guidelines to treat ESBL-
producing E coli urinary tract infections when there is resistance to
authorized alternatives.29
Combination preparations
AGs are often used in combination with other antimicrobials, such
as b-lactams, in order to achieve a synergistic effect or to broaden
the spectrum of activity. Streptomycin and neomycin are author-
ized in the EU in combination with penicillin for treatment of a
broad range of non-specific indications in livestock and companion

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3%
3%
6%
4%
18%
30%
Figure 3 Sales of aminoglycosides, spectinomycin and paromomycin in food-producing species, as a percentage of total mg/PCU, aggregated for 30
European countries in 2015 (ESVAC, unpublished data). Minor sales (0.7%) of kanamycin and framycetin were also reported in 2015. This figure
appears in colour in the online version of JAC and in black and white in the print version of JAC.
animals.19 AGs are also used in combination with b-lactams and/
or other antimicrobials in intramammary preparations. In pigs and
poultry, spectinomycin/lincomycin combinations are used for the
treatment of various infections.7,19 The rationale for some of these
combinations is disputable. Owing to the widespread resistance of
many bacterial species to streptomycin, streptomycin/penicillin
combinations have very limited extra value. In addition, a synergis-
tic effect of this combination has been shown for only a limited
number of pathogens and only in vitro. The indications for (dihy-
dro)streptomycin mono-products and its combinations should
therefore be re-evaluated. The penicillin/streptomycin combin-
ation was withdrawn from the US market in 1993 and later by
Australia since no evidence of clinical synergy was presented.30,31
Other applications of AGs
AGs are applied in apiculture, aquaculture and in other minor
species such as rabbits, reptiles and birds, although safety and
efficacy have not been established in all cases, with use often
being off-label. Gentamicin is utilized as a sperm diluter and as an
antimicrobial preservative for vaccines.32 Certain AGs are used
as anthelmintics in animals (destomycin A and hygromycin B).
Furthermore, paromomycin, ribostamycin and streptomycin
are used in horticulture as they have antifungal activity.33
Streptomycin is also used to control fire blight caused by Erwinia
amylovora in orchards.34
The use of AGs in human medicine
AGs (e.g. tobramycin, gentamicin, amikacin and netilmicin) are
used systemically for treatment of (MDR) Gram-negative infec-
tions such as those caused by Pseudomonas spp., Acinetobacter
spp. and Enterobacteriaceae. Kanamycin and amikacin are utilized
for the treatment of MDR TB;35 streptomycin was the first AG to be
used against TB, but is nowadays rarely used. Amikacin may also
JAC
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35% Neomycin
Dihydrostreptomycin
Spectinomycin
Paromomycin
Apramycin
Gentamicin
Streptomycin
be used against non-tuberculous mycobacterial infections. AGs
are used for empirical treatment of sepsis, respiratory tract infec-
tions, urinary tract infections and some CNS infections if MDR
Gram-negative bacteria are suspected to be involved.36
Enterococci are intrinsically resistant to low to moderate levels of
AGs, but synergism is generally seen when they are combined with
a cell wall-active antimicrobial agent. Therefore, AGs are used in
combination with a b-lactam or a glycopeptide for the treatment
of endocarditis caused by Gram-positive cocci without high-level
resistance to AGs. AGs are first-line treatment for plague, brucel-
losis and tularaemia.37 Aerosolized tobramycin, amikacin and gen-
tamicin are used to treat Pseudomonas infections in patients with
cystic fibrosis.37,38 Topical applications of various AGs (gentamicin,
tobramycin and neomycin) are utilized for the treatment of ear
and eye infections.36,39 Paromomycin is used to treat AIDS patients
suffering from cryptosporidiosis40 and is an alternative against dif-
ferent parasite diseases (amoebiasis and giardiasis) and some-
times used topically for the treatment of cutaneous leishmaniasis.
Spectinomycin is occasionally used for the treatment of gonor-
rhoea in patients allergic to penicillins (Table 1).
The AGs most used in hospitals are amikacin, gentamicin and
tobramycin.41 The most common route of administration for sys-
temic infections is parenteral, by intravenous or intramuscular injec-
tion. Oral administration is limited to decontamination of the gut
in ICUs, since bioavailability following oral administration is low.42
In the European Surveillance of Antimicrobial Consumption
Network (ESAC-Net) survey, including data from 20 European
countries, details on the consumption of individual AGs are not
reported separately. Available ESAC-Net data from 2015 show
that there are large differences in AG consumption between EU
MSs, consumption being highest in Romania (0.363 DDD per
1000 inhabitants), Italy (0.289 DDD per 1000 inhabitants) and
Bulgaria (0.277 DDD per 1000 inhabitants) whereas consump-
tion is much lower in other countries, e.g. in Sweden (0.015 DDD
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Table 1 Importance of aminoglycosides in human medicine
Bacterial targets in human medicine (for which
availability of class/substance is critically
Aminoglycoside important due to few alternatives)
Kanamycin Used for MDR infections including TB.
Gentamicin Gram-negative infections, enterococcal and
streptococcal endocarditis, brucellosis, tular-
aemia, plague, oral decolonization, impreg-
nated beads to prevent surgical site infections.
Amikacin MDR Gram-negative infections, MDR TB,
Nocardia spp. infections.
Tobramycin Gram-negative infections, Pseudomonas
infections in cystic fibrosis.
(Dihydro)streptomycin MDR tuberculosis, but very rarely used.
Spectinomycin Gonorrhoea in patients allergic to penicillins.
Paromomycin Cryptosporidiosis.
Apramycin No target.
per 1000 inhabitants) and Finland (0.016 DDD per 1000 inhabi-
tants; Figure 4). In a study of data from the initial ESAC project
describing parenteral antimicrobial treatment in outpatients,
AGs were the second most commonly used class (25.27%) after
the cephalosporins (44.58%). At the level of individual mole-
cules, gentamicin (18.53%) was administered more than the in-
dividual cephalosporins (e.g. ceftriaxone, 17.85%; cefazolin,
13.16%).43
Consumption data from European countries as outlined above
are collected by continuous surveillance data44 aggregated by
country, although many countries have their own surveillance pro-
grammes.45,46 Long-term monitoring in the Netherlands has
shown an increase in the systemic use of AGs in Dutch hospitals
during the last decade (from 2.5 to 3.7 DDD/100 patient-days be-
tween 2006 and 2015). In Norway and Denmark, the use was sta-
ble.45,47 Large teaching hospitals tend to have the highest use.41
In addition to continuous surveillance as performed by the
ESAC survey in outpatients and the hospital sector, targeted point
prevalence surveys are done in hospitals and long-term care. The
latest published data show that on average 34.6% of patients re-
ceive antimicrobial therapy in acute care hospitals versus 4.4% in
long-term care facilities.48,49 In these settings, the proportion of
AG use was 4.5% and 1.2%, respectively. Considering all agents
used in acute care, the AGs most used were gentamicin 3.7%, ami-
kacin 1.1%, tobramycin 0.4% and netilmicin 0.1%.48
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Relative frequency
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of use in humans in Hazard of resistance transfer between animals
the EU and humans
low: rarely used, M. tuberculosis is of limited zoonotic relevance,
generally no plasmid-mediated transfer of resistance
not for first-line determinants in M. tuberculosis.
treatment Enterobacteriaceae—high risk of clonal and
horizontal transfer of resistance genes.
Enterococci—risk of clonal (E. faecalis) and
horizontal (E. faecium and E. faecalis) transfer
of resistance genes.
high see above.
high see above.
high see above.
low see above.
low Gonorrhoea is not transmitted to humans from
non-human sources.Transfer of resistance
genes from non-human sources unlikely.
low Cryptosporidium parvum is of zoonotic
relevance.
not used Selects for gentamicin resistance in
Enterobacteriaceae, such as E. coli and
Salmonella spp.
Resistance mechanisms
Following extensive use of AGs in humans, food-producing animals
and companion animals, resistance has emerged. Resistance
occurs through several mechanisms. Resistance genes can be
located on the chromosome, gene cassettes, plasmids, transpo-
sons or other mobile elements, thereby increasing the likelihood of
spread of AG resistance as well as co-resistance to other com-
pounds.50 The three main mechanisms of bacterial resistance to
AGs are the reduction of the intracellular concentration of the anti-
microbial, the enzymatic modification of the drug and the modifi-
cation of the molecular target.51 Resistance mechanisms are
complex and differ between the AG molecules and between bac-
terial species, and generally there is less cross-resistance when
compared with other classes of antimicrobials.
Decreased intracellular concentrations can result from either
reduced drug uptake or active efflux mechanisms. Reduced uptake
can occur in mutants deficient in components of the electron
transport chain and has been described in Pseudomonas spp.,
E. coli and Staphylococcus aureus.52 Gentamicin resistance by in-
activation of an outer membrane porin, which serves as an entry
for gentamicin to the bacterial cell, has also been described.53
AG efflux is a significant mechanism in Pseudomonas spp.,
Burkholderia spp. and Stenotrophomonas spp., but has also been
described in other bacteria such as E. coli, Lactococcus lactis and
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0.35
DDD per 1000 inhabitants and per day

0.3

0.25

0.2

0.15

0.1

0.05

0
Belgium

United kingdom
Bulgaria

Cyprus*

Denmark

Finland
Croatia

Estonia

France

Greece

Hungary

Ireland

Luxembourg
Italy

Latvia

Lithuania

Malta

Netherlands

Norway

Poland

Portugal

Romania*

Slovakia

Slovenia

Sweden
Figure 4 Total consumption of aminoglycosides expressed as DDD per 1000 inhabitants in European countries in 2015 (source: ESAC-Net142).
*Country provided only total care data.

Acinetobacter baumannii.51,53 There are five families of efflux sys- nucleus or the sugar moieties, preventing ribosomal binding.
tems: the major facilitator superfamily, the ATP-binding cassette Within the three major classes of modifying enzymes, a further
family, the resistance-nodulation division family (RND), the small subdivision can be made based on the target site of the
MDR family as well as the multidrug and toxic compound extrusion enzymes.56 To date, there are four AACs: AAC(1), AAC(20 ), AAC(3)
family.54 The majority of AG transporters belong to the RND fam- and AAC(60 ); five ANTs: ANT(200 ), ANT(300 ), ANT(40 ), ANT(6) and
ily.53 Genes coding for AG efflux mechanisms are most often ANT(9); and seven APHs: APH(200 ), APH(30 ), APH(300 ), APH(4), APH(6),
located on the chromosome, but members of the major facilitator APH(700 ) and APH(9).56 Occasionally several subtypes of these
superfamily can also be located on plasmids.54 The ability of bac- enzymes are present in bacteria. The genes coding for all these
teria to survive antimicrobial challenge without mutation is called enzymes are often located on mobile genetic elements.
adaptive resistance and can be caused by a decreased transport of The AACs catalyse the acetylation of NH2 groups in AGs using
the drug into the bacterial cell.1 Adaptive resistance of P. aerugi- acetyl coenzyme A as donor substrate.51 In addition, the bifunc-
nosa has been shown to be associated with the overproduction of tional enzyme AAC(60 )–APH(200 ) can acetylate and subsequently
the RND efflux system MexXY–OprM.55 The clinical significance of phosphorylate its substrate.51 Among the AACs, AAC(60 ) enzymes
adaptive resistance is that frequent dosing or constant infusion is are the most common and can be found in Gram-positive as well
less effective than high-dose, once daily administration since AGs as Gram-negative bacteria. AAC(1), AAC(2) and AAC(3) are mainly
act in a concentration-dependent manner.1 found in Gram-negative bacteria.51 The substrate profile of AAC(1)
enzymes includes neomycin, apramycin and paromomycin and
that of AAC(20 ) enzymes includes gentamicin, kanamycin, tobra-
Enzymatic drug modification mycin, netilmicin and dibekacin. Enzymes of subclass AAC(3)-I
Roberts et al.56 provide an overview of most acquired resistance confer resistance to fortimicin, sisomicin and gentamicin, while
genes. A few novel spectinomycin resistance genes in staphylo- those of subclass AAC(3)-II confer resistance to gentamicin, tobra-
cocci have subsequently been discovered.57–59 Resistance genes mycin, sisomicin, netilmicin and dibekacin, and AAC(3)-IV to apra-
for AG-modifying enzymes are often found on mobile elements. mycin and gentamicin. AAC(60 ) enzymes cause resistance to
The most common mechanism of resistance to AGs in clinical iso- gentamicin and sometimes amikacin. AAC(60 )-Ib-cr is an enzyme
lates is the production of AG-modifying enzymes such as acetyl- that also confers resistance to selected fluoroquinolones such as
transferases (AACs), phosphotransferases (APHs) and ciprofloxacin.51
nucleotidyltransferases (ANTs).56,60,61 These enzymes modify the The ANTs represent the smallest class of AG-inactivating
AG at the hydroxyl or amino groups of the 2-deoxystreptamine enzymes. These enzymes catalyse the reaction between Mg-ATP

7 of 17
Review
and AGs to form the O-adenylated antimicrobial molecule. To
date, there are five classes of ANTs categorized depending on the
position of adenylation on the AG molecule.51 The ANT(200 ) and
ANT(300 ) enzymes are more frequent among Gram-negative bac-
teria, whereas the ANT(40 ), ANT(6) and ANT(9) enzymes are most
often found in Gram-positive bacteria.51,62 ANT(6) enzymes have
streptomycin as their substrate. The ant(6) gene is often found in a
cluster ant(6)-Ia-sat4-aph(30 )-III that specifies resistance to AGs
and streptothricin. ANT(9) causes resistance to spectinomycin.
ANT(40 ) enzymes confer resistance to tobramycin, amikacin and
isepamicin. ANT(200 ) mediates resistance to gentamicin, tobra-
mycin, dibekacin, sisomycin and kanamycin. ANT(300 ) are the most
commonly found ANT enzymes. They specify resistance to spec-
tinomycin and streptomycin.51
APHs catalyse the transfer of a phosphate group to the
AG molecule. They are widely distributed among bacterial
pathogens.51 APH(200 ) plays an important role in Gram-positive
organisms resistant to gentamicin. APH(30 )-IIIa, generally
found in Gram-positive bacteria, confers resistance to a broad
range of AGs including neomycin, paromomycin, kanamycin
and amikacin, but not tobramycin or gentamicin. Isolates
carrying APH(3) group enzymes show a resistance profile most
often including kanamycin, neomycin and paromomycin, and
APH(30 ) also to amikacin. APH(300 ) mediates resistance to
streptomycin. APH(4) mediates resistance to hygromycin and is
not clinically relevant. APH(6) enzymes confer resistance to
streptomycin. APH(700 ) mediates resistance to hygromycin.
APH(9) enzymes confer resistance to spectinomycin.51
Target modification
Target site modification occurs naturally in AG-producing bacteria:
the bacterium protects the target by employing enzymes that add
a methyl group to specific nucleotides in the 16S rRNA that are es-
sential for AG binding, thus inhibiting the antimicrobial action with-
out interfering with other ribosomal functions. This mechanism
has been described mainly in different species of the AG-producing
genera Streptomyces spp. and Micromonospora spp.62 Nowadays,
the methylation of the ribosomal target responsible for high-level
AG resistance is an emerging mechanism of great concern in clinic-
ally relevant Gram-negative bacteria.51,60–65
The first plasmid-mediated gene identified was the 16S rRNA
methylase armA.64 To date, 10 additional genes encoding methyl-
ases have been reported: rmtA, rmtB, rmtC, rmtD, rmtD2, rmtE,
rmtF, rmtG, rmtH and npmA.60,65 The genes encoding these deter-
minants are usually located on mobile genetic elements and have
been associated with genes coding for resistance to other anti-
microbial classes, such as quinolones (Qnr proteins) or b-lactam
antimicrobials (acquired AmpC-b-lactamases or ESBLs). Recently
these methyltransferases have been found in association with car-
bapenemases such as NDM-1.66–68 The genes rmtA, rmtB, rmtC,
rmtD, rmtD2, rmtE, rmtF, rmtG and rmtH confer resistance to gen-
tamicin, tobramycin, kanamycin and amikacin, whereas npmA
confers resistance to gentamicin, tobramycin, kanamycin, amika-
cin, neomycin and apramycin, but not to streptomycin or
spectinomycin.62,69,70
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Resistance mechanisms in relevant Gram-negative
bacteria
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AG resistance in Enterobacteriaceae relies mainly on the AG-
modifying enzymes (APHs, ANTs and AACs). AAC(3)-II/IV and
AAC(6)-Ib are the most frequently encountered AACs among E.
coli of human and animal origin. Among the ANTs, ANT(200 ) and
ANT(300 ) enzymes are most commonly found in Gram-negative
bacteria.70 The emergence of 16S rRNA methylases in bacteria of
animal origin was first identified in Spain in 2005 in an E. coli isolate
of pig origin harbouring the armA gene.71 Since then, different
methylases have been detected in Enterobacteriaceae isolates
from animals in different countries.72–84 The dissemination of the
armA gene is favoured by its location on a transposon.70 In Spain,
seven K. pneumoniae ST11 isolates from dogs and cats were found
to be resistant to AGs, and the armA gene was responsible for this
phenotype.84 Although the rmtA gene is rarely reported from ani-
mals, it has been found in E. coli isolates from giant pandas in
China.80 In China, the rmtB gene was detected in 69 out of 267
Enterobacteriaceae isolates collected from pets. The rmtB gene
was commonly found with ESBL blaCTX-M-9 group genes within the
same incompatibility group (Inc) FII plasmid.74 The rmtB gene was
also found in clinical E. coli isolates originating from bovines in
China and France and from healthy pigs in China.72,82,83, rmtD has
been found in E. coli isolates from diseased chickens in China and a
horse in Brazil.77,81 The RmtE methylase was reported from E. coli
isolates from healthy calves in the USA and from diseased food-
producing animals in China.78,79
To date, 16S rRNA methylases do not appear to be common in
veterinary bacteria in EU MSs, but the use of most AGs would select
for resistance as these enzymes result in resistance to almost all
AGs, especially those of clinical relevance in humans.
AG efflux is a significant mechanism in P. aeruginosa. AG-
modifying enzymes also confer AG resistance in P. aeruginosa.60 In
A. baumannii, besides AG-modifying enzymes, the armA gene,
located on a transposon, is widespread in many countries world-
wide.60 In addition, rmtB has recently been identified in nine
A. baumannii isolates from humans in Vietnam.85 To date, no meth-
ylases have been reported from veterinary Pseudomonas isolates.
Resistance mechanisms in relevant Gram-positive
bacteria
Resistance to various AGs in staphylococci can be mediated by the
genes aac(60 )-Ie-aph(200 )-Ia (kanamycin/gentamicin/tobramycin/
amikacin resistance), ant(40 )-Ia (kanamycin/neomycin/tobramycin
resistance), aph(30 )-III (kanamycin/neomycin/amikacin resist-
ance), apmA (apramycin resistance and decreased susceptibility
to gentamicin) and aadE or str (streptomycin resistance).86–89
Spectinomycin resistance in staphylococci is mostly mediated by
spectinomycin 9-O-adenyltransferase encoded by the spc gene
located on a transposon. Resistance in staphylococci to spectino-
mycin can also be due to the plasmid-associated gene spd and the
chromosomal- or plasmid-located gene spw.57,59
In Mycobacterium tuberculosis, mutations in the genes rpsL and
rrs encoding the ribosomal protein S12 and the 16S rRNA, respect-
ively, are responsible for most of the high-level streptomycin resist-
ance. rrs A1401G is the most frequent mutation conferring
amikacin and kanamycin resistance.90 Overexpression of the AG
Review
acetyltransferase-encoding gene, eis, has mainly been associated
with resistance to kanamycin in M. tuberculosis. EIS is a unique en-
zyme capable of acetylating multiple positions of any given AG
scaffold.91 This overexpression resulted from either point muta-
tions in the promoter region of the eis gene or mutations of the
whiB7 gene, which encodes a putative regulator of the eis gene.92
Although the eis gene has been mainly associated with kanamycin
resistance, amikacin resistance has also been reported.90 The gene
gidB, when mutated, was found to be associated with low-level
streptomycin resistance in M. tuberculosis.93 The gidB gene enco-
des a 7-methylguanosine methyltransferase that specifically
modifies residues in the 16S rRNA (rrs). It is a non-essential gene,
and loss-of-function mutations in gidB result in failure to methy-
late G527 within the 530 loop of the 16S rRNA molecule. Many dif-
ferent gidB mutations, including deletions, are associated with AG
resistance, suggesting that loss of function of this gene confers re-
sistance.90 To date, no plasmid-mediated resistance has been
reported in M. tuberculosis or M. bovis.
Consideration of susceptibility testing
Susceptibility data from national monitoring programmes are
available, and MIC determination via broth microdilution is the
most frequently used method in these programmes.
Methodologies used differ among countries, since they use differ-
ent standards and guidelines (EUCAST, CLSI or country specific),
different antimicrobial agents for the same bacteria, different con-
centration ranges for the same antimicrobial agent and different
interpretative criteria.94 In vitro susceptibility testing for many anti-
microbials including AGs is hampered by the fact that standards
and guidelines for determination of MICs do not include all micro-
organisms. Single AGs cannot be used as representatives for the
whole AG class, since resistance is not a class effect, i.e. there are
numerous resistance genes specifying a wide variety of resistance
mechanisms with different substrate spectra. Resistance to
streptomycin and spectinomycin, for example, is distinct from re-
sistance to gentamicin, kanamycin and/or tobramycin.95
Alternatively, unrelated enzymes, affecting different sites, can
confer the same resistance phenotypes. Despite these difficulties,
the enzymes produced by isolates can sometimes be predicted
from susceptibility testing.96
To date, EUCAST has no veterinary-specific breakpoints.
VetCAST is the EUCAST subcommittee for Veterinary Antimicrobial
Susceptibility Testing and aims to define clinical breakpoints for
antimicrobial drugs used in veterinary medicine in Europe in the fu-
ture.97 CLSI has veterinary-specific breakpoints for amikacin ap-
plicable to E. coli and P. aeruginosa from dogs, foals and adult
horses, Staphylococcus spp. from dogs, S. aureus from foals and
adult horses, Streptococcus spp. from dogs, and Streptococcus equi
subsp. zooepidemicus and subsp. equi from foals and adult
horses.98–100
For Enterococcus spp. (E. faecalis, E. faecium, E. gallinarum/
E. casseliflavus), AGs (except when tested positive for high-level
resistance) may appear to be active in vitro, but are not effective
clinically and should not be reported as susceptible. Anaerobic bac-
teria, such as Clostridium spp., Bacteroides spp. and Fusobacterium
canifelinum, are intrinsically resistant to AGs.99
A recent study demonstrated that results of susceptibility test-
ing for gentamicin for K. pneumoniae resistant to carbapenems
JAC
obtained with Vitek 2 and Etest should be interpreted with caution,
especially if the EUCAST breakpoints were used. False gentamicin
susceptibilities were observed using Vitek 2 and occurred with
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K. pneumoniae isolates carrying armA.101 Susceptibility testing of
Pseudomonas spp. isolates against tobramycin using MALDI-TOF
MS technology has been explored and it was able to distinguish be-
tween resistant and susceptible isolates. Therefore, this technique
has the potential to allow for the susceptibility testing of a much
wider range of antimicrobial substances in the future.102
In conclusion, interpretation of susceptibility testing is impaired
by the lack of veterinary breakpoints for most AGs. Susceptibility
testing should be standardized and veterinary clinical breakpoints
established.
Occurrence of resistance in bacteria from
animals and humans
Isolates from food-producing animals collected under
EU surveillance programmes
Gram-negatives
In the EU monitoring programmes (MARAN, DANMAP and EFSA),
ecological cut-off values (ECOFFs) are often used for the interpret-
ation of antimicrobial susceptibility testing in food-producing ani-
mals, and therefore isolates reported as ‘resistant’ in this
paragraph are not always clinically resistant.103–105 In the
Netherlands in 2017, gentamicin resistance in Salmonella spp. iso-
lates from humans, food-producing animals and food products
was uncommon (3.4%).103 In Campylobacter spp. isolates from
cattle, pigs and poultry, no gentamicin resistance was found. The
level of resistance to streptomycin varied between 0% and 2.6%
for Campylobacter jejuni isolates from broilers, poultry meat and
cattle, and was 8% in Campylobacter coli isolates from broilers and
73% in C. coli isolates from pigs. For E. coli, 5.6%, 0.7%, 0%, 3.8%
and 2.2% of the isolates originating from broilers, pigs, dairy cattle,
white veal calves and rosé veal calves, respectively, were resistant
to gentamicin in the Netherlands.103 In Denmark, 5% and 12% of
Salmonella Typhimurium isolates from pigs and pork, respectively,
were resistant to gentamicin. The level of resistance among
Danish C. jejuni isolates from broilers and cattle to streptomycin
and gentamicin was low (0%–1%).104 The level of resistance to
gentamicin of indicator E. coli in Denmark was low in broilers, pigs
and cattle (0%–3%).104 Data for 2016 from the European surveil-
lance monitoring show that resistance to gentamicin in Salmonella
spp. isolates from broilers, broiler meat, turkeys and turkey meat is
generally low (5.6%, 2.4%, 7.2% and 4.4%, respectively). There are
differences between Salmonella serovars: in S. Kentucky from
broilers and turkeys, resistance to gentamicin was common, 71%
and 85% of isolates being non-susceptible.105 Resistance to genta-
micin in 2016 was rare in C. jejuni from broilers (0.7%), whereas it
was not found in C. coli isolates.105 Levels of streptomycin resist-
ance were 1.7% for C. jejuni and 12% for C. coli. In indicator E. coli
isolates from broilers, broiler meat, turkeys and turkey meat, resist-
ance to gentamicin was 8.9%, 4.1%, 6.2% and 11%, respectively.
Gram-positives
In Denmark, gentamicin resistance in E. faecalis was 7% in isolates
from pigs, but no high-level resistance was detected.104
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Review
Clinical isolates from animals
Gram-negatives
Equine E. coli isolates were generally susceptible to gentamicin,
the resistance rate being 8.8% in Germany.94 A significant increase
in the percentage of E. coli isolates resistant to gentamicin was
identified in equine E. coli isolates from 2007 to 2012 (53.9%) com-
pared with isolates from 1999 to 2004 (28.5%) in the UK.106 In
France, most equine E. coli isolates were susceptible to strepto-
mycin, neomycin, kanamycin, amikacin and gentamicin (60%–
100% susceptibility).107 In France, the percentages of susceptible
feline E. coli were 74% for streptomycin, 92% for kanamycin, 98%
for gentamicin and 92% for neomycin.107 Canine E. coli isolates
from urinary tract infections were generally susceptible to genta-
micin, neomycin and apramycin (.90% of isolates susceptible)
and 71% were susceptible to streptomycin.107 Integron-borne AG
resistance was found frequently among avian pathogenic E. coli
(APEC) in Italy. High levels of resistance were observed for strepto-
mycin (67.2%), whereas resistance against gentamicin (16.7%),
kanamycin (14.7%) and apramycin (3.0%) was lower.108
Resistance in P. aeruginosa isolates from ear infections of compan-
ion animals was found in 25% of the isolates, while only 41% of
the isolates were fully susceptible.94
Gram-positives
Characterization of 227 Streptococcus suis isolates from pigs dur-
ing 2010–13 showed high-level resistance to neomycin (70.0%)
and gentamicin (55.1%), and resistance to AGs was attributed to
aph(30 )-IIIa and aac(60 )Ie-aph(200 )-Ia genes.109 These genes have
rarely been reported in S. suis.109 Coagulase-positive staphylococci
isolated from the udder of bovines in France were often susceptible
to all AGs tested, with 90%–99% of the isolates susceptible to
streptomycin, kanamycin, neomycin and gentamicin.107Among S.
aureus originating from infections in dogs, 66%, 71% and 74%
were susceptible to streptomycin, neomycin and kanamycin, re-
spectively, and 91% were found to be susceptible to gentamicin in
France.107 Susceptibilities of feline staphylococci were similar.
Among equine S. aureus isolates, susceptibility to AGs was 80% for
kanamycin, 82% for gentamicin and 90% for streptomycin.107
Resistance to gentamicin, tobramycin and kanamycin was com-
mon (36%) among MRSA CC398 isolates collected from pigs at
Dutch slaughterhouses.110 Non-susceptibility to gentamicin was
also found among MRSA isolates on broiler farms.88 Non-
susceptibility to gentamicin (40%), neomycin (30%) and amikacin
(1%) was found among 1290 MRSA isolates from pigs, veal calves,
poultry and meat in the Netherlands.111 A high prevalence of non-
susceptibility to AGs has been reported in MSSA CC398 isolates in
Belgium.112 MRSA CC1 isolates from dairy cattle and humans in
Italy were often kanamycin resistant and carried aphA3 and sat
(conferring streptothricin resistance) genes with Tn5405-like ele-
ments, and contained several markers indicating a human
origin.113
In Germany, 96% of canine and feline S. aureus isolates from
ear infections and 84% of S. aureus from skin infection were sus-
ceptible to gentamicin. Gentamicin susceptibility percentages for
Staphylococcus pseudintermedius isolates were 87% for isolates
from ear infections and 74% for isolates from skin infections.94
Among 103 methicillin-resistant S. pseudintermedius isolates from
10 of 17
dogs originating from several countries in Europe, the USA and
Canada, resistance to gentamicin/kanamycin (88.3%), kanamycin
(90.3%), streptomycin (90.3%) and streptothricin (90.3%) was
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very common.114
Human clinical isolates
Data from the ECDC EARS-Net surveillance of human clinical cases
indicate that resistance of Klebsiella pneumoniae isolates to AGs
was below 5% in Scandinavian countries and Austria, between 5%
and 10% in the Netherlands, Germany and the UK, 25%–50% in
France, Italy, Portugal, Lithuania, Hungary, Croatia and the Czech
Republic and between 50% and 75% in Greece, Bulgaria, Romania,
Poland and Slovakia.115 For E. coli, AG resistance was ,5% in
Finland and Iceland, between 5% and 10% in Sweden, Norway,
the Netherlands, Germany, France, Belgium, the UK and Austria
(among others), 10%–25% in Spain, Italy, Ireland, Greece and
many eastern European countries and 35% in Bulgaria. High-level
gentamicin resistance in E. faecalis was between 10% and 25% in
Sweden, Norway, France, Belgium, the Netherlands, Iceland and
Greece; it was between 25% and 50% in most other EU MSs, while
a prevalence of 56% was found in Romania. Even higher prevalen-
ces of high-level gentamicin resistance were reported for
E. faecium, with levels .70% in the Netherlands, Czech Republic,
Romania, Lithuania and Bulgaria.115 Generally, countries with the
highest human consumption of AGs, such as Bulgaria and
Romania, also reported the highest levels of AG resistance.115
Possible links between the use of AGs in animals and
resistance in bacteria of animal origin
A systematic review on the effect of oral antimicrobials on porcine
E. coli found that oral administration of AGs increased the preva-
lence of antimicrobial resistance.116 Sun et al.117 investigated the
effect of treatment of sows with lincomycin, chlortetracycline and
amoxicillin on resistance development in the intestinal microbiota.
The treatment increased the abundance of AG resistance genes,
probably due to co-selection. Apramycin and neomycin fed in sub-
therapeutic concentrations to pigs enhanced transfer of an anti-
microbial resistance-encoding plasmid from commensal E. coli
organisms to Yersinia and Proteus organisms in an infection model
using isolated ligated intestinal loops.118 Apramycin consumption
at farm level in pigs was most probably driving the increasing oc-
currence of apramycin/gentamicin cross-resistant E. coli in dis-
eased pigs and healthy finishers at slaughter in Denmark. The
duration of use and amounts used both had a significant effect on
the prevalence of apramycin/gentamicin cross-resistance in dis-
eased weaning pigs at the national level.119 Another Danish study
investigated the effect of apramycin treatment on transfer and
selection of an MDR E. coli strain in the intestine of pigs, and found
that the use of apramycin enhanced the spread of gentamicin-
resistant E. coli.120 In a study investigating the influence of oral
administration of a fluoroquinolone, an AG and ampicillin on
prevalence and patterns of antimicrobial resistance among E. coli
and Enterococcus spp. isolated from growing broilers, the overall
resistance to all drugs tested reached the highest level among
enterococci after medication with gentamicin. The frequency of re-
sistance to most antimicrobials tested was significantly higher in
E. coli isolated from broilers receiving intermittent antimicrobial
Review
pressure than that from non-medicated broilers.121 On a German
broiler farm, resistance to spectinomycin in E. coli isolates
increased significantly with age in all three production turns, des-
pite the fact that the substance was not used on the farm. A pos-
sible explanation for this phenomenon was co-selection by the use
of other antimicrobials.122 Gentamicin resistance increased in clin-
ical E. coli isolates from broiler chickens in Québec, despite the fact
that this antimicrobial was no longer used. This increase coincided
with the use of a spectinomycin/lincomycin combination for pre-
vention of colibacillosis, including in ovo injection. The major genes
identified for resistance to gentamicin and spectinomycin were
aac(3)-VI and aadA, respectively. The aadA and aac(3)-VI genes
were located on a modified class 1 integron. Therefore, the use of
spectinomycin/lincomycin not only resulted in an increase in spec-
tinomycin resistance, but also co-selected for gentamicin
resistance.123
The use of various antimicrobials, including kanamycin, at con-
centrations in vitro far below the MIC, promoted the selection of an
ESBL-carrying plasmid conferring resistance not only to b-lactams
but also to AGs, tetracycline, trimethoprim, sulphonamide and
erythromycin, as well as biocides and heavy metals.124 These find-
ings suggest that low concentrations of antimicrobials present in
polluted external environments and in the gut of exposed animals
and humans could allow for selection and enrichment of bacteria
with MDR plasmids and thereby contribute to the emergence,
maintenance and transmission of antimicrobial-resistant, disease-
causing bacteria.
In conclusion, there is evidence that the usage of AGs in veterin-
ary medicine is associated with the increased prevalence of resist-
ance to AGs and other antimicrobial classes in bacteria in animals.
Impact of resistance on animal health
Loss of efficacy of AGs could have a serious negative impact on ani-
mal health and welfare. Although AGs are very important antimi-
crobials for treatment of animal infections, they are seldom the
sole treatment option in the EU. In horses, for example, gentamicin
is one of the few available antimicrobials for treating
Gram-negative infections, the alternative treatment options being
trimethoprim/sulphonamide combinations, third- and fourth-
generation cephalosporins and fluoroquinolones.25 Resistance to
trimethoprim/sulphonamide combinations is very common
among Gram-negative bacteria, and the latter two mentioned
classes are included in Antimicrobial Advice Ad Hoc Expert Group
(AMEG) category 2. Category 2 includes human critically important
antimicrobials also used in veterinary medicine where the associ-
ated risk for public health is estimated to be high. The use of cat-
egory 2 antimicrobials should be restricted to conditions where no
alternative treatment options are available.125 In pigs, AGs are im-
portant drugs for the treatment of post-weaning diarrhoea.1,24
Alternatives include tetracycline, trimethoprim/sulphonamide
combinations and ampicillin/amoxicillin, but the prevalence of re-
sistance among E. coli to these antimicrobials is high. Other alter-
natives include colistin or quinolones.7,24 With regard to infections
with Pseudomonas spp., AGs are one of the few treatment
options.3 In companion animals, AGs are used to treat ear and eye
infections caused by Pseudomonas spp. by topical application of
drops or ointments; alternatives include polymyxins and fluoroqui-
nolones.26 For systemic treatment of Pseudomonas infections,
JAC
fluoroquinolones are one of the few other treatment options be-
sides AGs.1,7,24
In conclusion, AGs are very important for treatment of a broad
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range of Gram-negative infections in animals. If AGs were no lon-
ger available for veterinary medicine, then it could be speculated
that other antimicrobials would replace their use. Alternatives to
AGs for the treatment of some MDR Gram-negative infections in
animals include antimicrobials that are critically important for the
treatment of human infections, such as fluoroquinolones and co-
listin.126 The consequences of the use of these alternatives instead
of AGs should be taken into account in risk assessments.
Impact of resistance on human health
All AGs (including streptomycin, neomycin and kanamycin), with
the exception of spectinomycin, are categorized as ‘critically im-
portant’ antimicrobials for human medicine by the WHO, whereas
spectinomycin is categorized as ‘important’.4 AGs are most often
used in combination with b-lactams for the empirical treatment of
a broad range of life-threatening infections in humans.
Nephrotoxicity and ototoxicity and the discovery of less-toxic anti-
microbials in recent decades have limited the use of AGs in human
medicine.36 However, high levels of resistance to other antimicro-
bials and MDR in certain bacteria have resulted in renewed interest
in the AGs.
Usage of AGs in humans is associated with increased preva-
lence of resistance in humans. In human isolates from the
Enterobacteriaceae family, there was a significant effect of selec-
tion pressure of gentamicin in the selection of resistant K. pneumo-
niae and E. coli and of amikacin in the selection for resistant E. coli
and Enterobacter cloacae isolates.127 Another study showed that
the abundance of antimicrobial resistance genes more than
doubled during selective digestive decontamination with colistin,
tobramycin and amphotericin B in ICU patients, mainly due to a
6.7-fold increase in AG resistance genes, in particular aph(200 )-Ib
and an aadE-like gene.128
To date, ESBLs conferring resistance to broad-spectrum cepha-
losporins, carbapenemases conferring resistance to carbapenems,
and 16S rRNA methylases conferring resistance to all clinically rele-
vant AGs are the most important causes of concern. In recent
years, the global dissemination of A. baumannii and
Enterobacteriaceae, including Salmonella spp., that co-produce
16S rRNA methylases and carbapenemases such as NDM-1 MBL is
becoming a serious threat to human health.60 The resistance
genes are often co-located on the same plasmid. In addition to
16S rRNA methylases, resistance to AGs in both Gram-positive and
Gram-negative clinical isolates is often related to the production of
modifying enzymes of several classes.51 It should be noted that a
systematic review assessed mortality, treatment failures and anti-
microbial resistance by comparing b-lactam therapy versus any
combination of a b-lactam with an AG for human cases of blood-
stream infections. The authors concluded that the addition of an
AG to b-lactams for treatment of sepsis should be discouraged,
since mortality rates were not improved and the addition of AGs
considerably increased the risk for nephrotoxicity. The subgroup of
P. aeruginosa infections was underpowered to examine effects.129
Besides infections with MDR Enterobacteriaceae, Pseudomonas
spp. and Acinetobacter spp., MDR TB and Gram-positive endocardi-
tis are among the diseases for which the availability of AGs is
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Review
critically important due to few alternatives.125 For endocarditis
caused by enterococci without high-level AG resistance, ampicillin
combined with gentamicin is considered the regimen of first
choice. During the last decade, alternative treatment options
including high-dose daptomycin and the combination of ampicillin
with ceftriaxone have been explored and have been shown to be
equally effective in certain studies.130–132
In conclusion, AGs are important drugs in human medicine for
the treatment of infections with MDR Gram-negative bacteria,
enterococcal endocarditis and MDR TB, but they are seldom the
only therapeutic option.
Transmission of resistance and determinants between
animals and humans
AGs are frequently used in veterinary and human medicine, and re-
sistance has emerged. Resistance can be due to chromosomal
mutations, but resistance determinants are often located on mo-
bile elements such as transposons, integrons and plasmids. The
same resistance genes and mobile genetic elements have been
found in isolates from animals and humans.88,133–135 In addition,
resistance to AGs has been found in bacteria that can cause food-
borne infections in humans, such as Salmonella spp. and
Campylobacter spp., although AGs are generally not used to treat
Salmonella or Campylobacter infections in humans.
Gram-negatives
Antimicrobial resistance in several Salmonella enterica serovars is
due to genomic islands carrying a class 1 integron, which carries
the relevant resistance genes. Salmonella genomic island 1 (SGI1)
was found in Salmonella enterica serovar Typhimurium Definitive
phage type (DT) 104 ("S. Typhimurium DT 104) isolates, which are
resistant to ampicillin, chloramphenicol, florfenicol, streptomycin,
spectinomycin, sulphonamides and tetracyclines. Several
Salmonella serovars have been shown to harbour SGI1 or related
islands. SGI1 is an integrative mobilizable element and can be
transferred experimentally into E. coli.134 Co-selection of resistance
to all these antimicrobials can potentially result from the use of
AGs if SGI1 is present. ESBL or plasmidic AmpC b-lactamase-pro-
ducing (ESBL/pAmpC) Enterobacteriaceae are widely distributed
among human and animal populations.135 ESBL- and
carbapenemase-encoding plasmids frequently bear resistance
determinants for other antimicrobial classes, including AGs and
fluoroquinolones, a key feature that fosters the spread of MDR in
Enterobacteriaceae.136 Transmission of ESBL/pAmpC E. coli from
animals to humans can potentially occur by direct contact,
through the food chain or the environment.135 Evidence for clonal
transmission of ESBL-producing E. coli between humans and
broilers has been found on conventional broiler farms, and hori-
zontal gene transfer was suspected on both conventional and or-
ganic farms.137,138
Combined apramycin and hygromycin B resistance in
Enterobacteriaceae is mediated by the aac(3)IV and hphB genes.
These genes are part of one resistance gene operon associated
with an insertion sequence, IS140, which is found on plasmids.139
The organization of aac(3)IV and hphB is such that they are co-
transferred.139 Apramycin is used exclusively in animals. The gene
aac(3)IV, conferring cross-resistance to gentamicin, was first
12 of 17
identified in E. coli and S. Typhimurium from animals in France and
the UK.140–142 A high degree of genetic homology between plas-
mids harbouring aac(3)IV and hphB of human and animal origin
Downloaded from https://academic.oup.com/jac/advance-article-abstract/doi/10.1093/jac/dkz161/5475277 by University of South Australia user on 20 April 2019
has been demonstrated.140,141 In a prospective study on a pig
farm, Hunter et al.143 demonstrated a widespread dissemination
of plasmids harbouring aac(3)IV in E. coli from pigs, calves, the
farmer and the environment. K. pneumoniae with a slightly smaller
conjugative plasmid and a similar resistance pattern was isolated
from the farmer’s wife, indicative of animal to human transmission
of resistance genes.
Gram-positives
The risk of transmission of MDR TB from animals to humans is
limited, as to date the main resistance mechanism for mycobacte-
ria is chromosomal mutation. In addition, TB in humans is mostly
caused by Mycobacterium tuberculosis, which is mainly transmit-
ted from humans to humans. Bovine TB (Mycobacterium bovis) is a
reportable disease in EU MSs and has been eradicated in many EU
MSs. During the years 2006–12, the proportion of cattle herds
infected or positive for M. bovis in the EU (all MSs) was at a very low
level, ranging from 0.37% in 2007 to 0.67% in 2012.144 The emer-
gence of MDR M. bovis strains in the EU is regarded as unlikely be-
cause animals infected with TB are not treated but are culled.
Livestock-associated MRSA CC398 (LA-MRSA) isolates from vet-
erinarians in Belgium and Denmark were often resistant to genta-
micin, kanamycin and tobramycin mediated by aac(60 )-aph(2a00 )
or aadC, and LA-MRSA carriage was significantly associated with
contact with livestock.145 This indicates that LA-MRSA resistant to
AGs can be transmitted between animals and humans.
Molecular epidemiological studies based on MLST have
revealed that the vast majority of E. faecium isolates causing clinic-
al infections and nosocomial outbreaks in humans belong to a glo-
bally dispersed polyclonal subpopulation, genotypically different
from E. faecium strains colonizing animals and healthy humans in
the community. There was a significant discrepancy in accessory
gene content between hospital- and community-acquired ampi-
cillin-resistant E. faecium that includes putative virulence and anti-
microbial resistance genes, and indicates that if zoonotic transfer
occurs, it only occurs infrequently.146 Although E. faecium isolates
from animals do not seem to be a direct hazard to human health
by clonal transmission, they could act as donors of resistance
genes to pathogenic enterococci through horizontal gene transfer.
For E. faecalis, the same MLST types can be detected in isolates
from food, animals and patients with clinical infections, and there-
fore the zoonotic potential is higher.147
Generally, the risk of the emergence and transfer of AG resist-
ance resulting from the use of oral products, which are indicated
mostly to treat enteric infections in pigs, chickens and calves, is
anticipated to be much higher than for other formulations as the
former products are used as mass medication and, as AGs are not
absorbed from the gut, the gut flora is exposed to considerable se-
lective pressure.148 The risk for infections with resistant bacteria in
humans resulting from the use of topical products including drops
used to treat eye and ear infections (mainly infections with
Pseudomonas spp.) in companion animals is generally regarded as
low, since treatment involves individual animals and this local
route of administration does not result in a selective pressure on
the gut flora. This also holds for the use of AGs as intramammary
Review
preparations for the treatment of mastitis in cattle, although their
use as dry cow therapy might result in a somewhat higher risk as
more individuals are treated (unless selective treatment is prac-
tised) and longer-acting preparations are used. The risk for the
emergence of antimicrobial resistance from the use of AGs as in-
jectable formulations will generally be lower if animals are treated
individually rather than as a group.
In humans, AGs are mostly used to treat infections caused by
bacteria that are not transmitted via food or contact with animals.
However, they are also used for treatment of zoonotic infections
such as TB (M. bovis), brucellosis and tularaemia. Even bacteria
causing human infections not directly originating from animals
may acquire resistance determinants from commensal bacteria
transmitted from animals, such as Enterobacteriaceae and enter-
ococci, but the extent to which this occurs is unknown.
Conclusions
These data show that the probability of transfer of AG resistance
from animals to humans is high, especially in Enterobacteriaceae,
LA-MRSA and enterococci. Further research is needed to elaborate
on the link between the use of AGs in animals and the impact on
public health. Since very few new and effective antimicrobials for
the treatment of infections due to MDR Gram-negative bacteria
are likely to be launched in the near future, there is an urgent need
to implement strategies that may slow the development of
acquired resistance. Responsible use in both human and veterinary
medicine is of great importance.
Transparency declarations
None to declare.
Supplementary data
Table S1 and Figure S1 are available as Supplementary data at JAC Online.
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