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International Journal of Antimicrobial Agents 56 (2020) 106 127
Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents
journal homepage: www.elsevier.com/locate/ijantimicag
Phenotypic and WGS-derived antimicrobial resistance profiles of
clinical and non-clinical Acinetobacter baumannii isolates from
Germany and Vietnam
Gamal Wareth
a , b , ∗
, Jörg Linde
a
, Philipp Hammer
c
, Ngoc H. Nguyen
d , g
, Tuan N.M. Nguyen
d
,
Wolf D. Splettstoesser
e
, Oliwia Makarewicz
b , f
, Heinrich Neubauer
a
, Lisa D. Sprague
a
,
Mathias W. Pletz
b , f
a
Friedrich-Loeffler-Institut, Institute of Bacterial Infections and Zoonoses (IBIZ), Jena, Germany
b
Institute for Infectious Diseases and Infection Control, Jena University Hospital, Jena, Germany
c
Department of Safety and Quality of Milk and Fish Products, Max Rubner-Institut, Kiel, Germany
d
The Center of Training and Direction of Healthcare Activities, General Hospital of Phutho, Vietnam
e
Department of Microbiology & Hygiene, LADR GmbH, Medical Laboratory Braunschweig, Germany
f
Research Campus Infectognostics, Jena, Germany
g
Department of Health, General Hospital of Phutho, Phutho, Vietnam
a r t i c l e i n f o
Article history:
Received 20 March 2020
Accepted 29 July 2020
Keywo rds:
Acinetobacter baumannii
Intrinsic resistance
Multidrug-resistant
Whole genome sequencing
Germany
Vietnam
a b s t r a c t
Objectives: This study aimed to combine in vitro phenotyping analysis and whole-genome-sequencing
(WGS) to characterise the phenotype and genetic determinants associated with intrinsic resistance in 100
clinical and non-clinical Acinetobacter baumannii strains originating from Germany and Vietnam. More-
over, it aimed to assess whether powdered milk as a food source functions as a potential reservoir of
antibiotic resistance and possesses similar antimicrobial resistance (AMR) genes as in clinical strains iso-
lated from Germany.
Methods: Antimicrobial susceptibility testing was performed using the broth microdilution method and
the minimum inhibitory concentration (MIC) was determined for 18 antibiotics. The WGS data from all
isolates were mapped to intrinsic genes known to be associated with phenotypic AMR.
Results: The highest resistance frequency was observed for chloramphenicol (100%), followed by fos-
fomycin (96%) and cefotaxime (95%). The lowest resistant rates were observed for colistin (3%), trimetho-
prim/sulfamethoxazole (17%), tigecycline (19%), and amikacin (19%). Thirty-five percent of tested strains
displayed resistance to at least one of the carbapenems. Resistance to fluoroquinolones, aminoglycosides,
tigecycline, penicillins, trimethoprim/sulfamethoxazole, and fourth-generation cephalosporins was deter-
mined only in human strains. About one-quarter of isolates (24%) was multidrug-resistant (MDR) and all
were of human origin. Among them, 16 isolates were extensively drug resistant (XDR) and 10 from those
16 isolates showed resistance to all tested antibiotics except colistin. In silico detection of intrinsic AMR
genes revealed the presence of 36 β-lactamases and 24 non- β-lactamase resistance genes. Two colistin-
resistant and 10 ertapenem-resistant strains were isolated from powdered milk produced in Germany.
Thirty-eight AMR genes associated with resistance to antibiotics were found in isolates recovered from
milk powder. Several resistance mechanisms towards many classes of antibiotics existed in A. baumannii
including β-lactamases, multidrug efflux pumps and aminoglycoside-modifying enzymes.
Conclusion: The use of WGS for routine public health surveillance is a reliable method for the rapid de-
tection of emerging AMR in A. baumannii isolates. Milk powder poses a risk to contain MDR Acinetobacter
strains or resistance genes in Germany.
©2020 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.
∗Corresponding Author at: Friedrich-Loeffler-Institut, Institute of Bacterial Infec-
tions and Zoonoses (IBIZ), Jena, Germany. E-mail address: gamal.wareth@fli.de (G. Wareth).
https://doi.org/10.1016/j.ijantimicag.2020.106127
0924-8579/© 2020 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.
2 G. Wareth, J. Linde and P. Hammer et al. / International Journal of Antimicrobial Agents 56 (2020) 10 6127
1. Introduction
Acinetobacter baumannii ( A. baumannii ) is a Gram-negative op-
portunistic nosocomial pathogen. It is responsible for approxi-
mately 7.7% of all infections in intensive care units [1] , and the ma-
jority ( > 90 %) of A. baumannii isolates in intensive care units are
multidrug-resistant (MDR) and challenging to treat. A. baumannii
can cause a multitude of severe nosocomial infections of the skin
and soft tissue, urinary and respiratory tracts, and bloodstream.
It is also associated with secondary meningitis and ventilator-
associated pneumonia in humans [2-5] . In animals it causes masti-
tis, pneumonia, sepsis, bloodstream, wound and urinary tract in-
fections, septicaemia, bronchopneumonia, neonatal encephalopa-
thy, and eye infections [6-9] . It has been found in humans, an-
imals, foods and the environment. The bacterium is always as-
sociated with severe infection, high mortality rates and massive
economic loss [10] . The World Health Organisation (WHO) has
classified carbapenem-resistant A. baumannii as a priority critical
pathogen [11] regarding the development of novel antibiotics. It is
also a member of the ESKAPE pathogens [12] , a group of bacte-
rial pathogens commonly associated with antibiotic-resistant infec-
tions worldwide: Enterococcus faecium , Staphylococcus aureus , Kleb-
siella pneumoniae , Acinetobacter baumannii , Pseudomonas aerugi-
nosa , and Enterobacter spp. [13] . Spread and dissemination of MDR
A. baumannii has become a public health concern in both, devel-
oping and developed countries, and the prevalence of resistance
towards the last-resort antibiotics such as carbapenems and even
colistin is increasing globally [14] .
Since the late 20
th century, A. baumannii has emerged as a sig-
nificant nosocomial pathogen in Germany [15-17] . The rapid spread
of A. baumannii resistance to carbapenems [18] and to the last-
resort antimicrobial compounds such as colistin and tigecycline
[19] has been reported nationwide and become a major cause of
concern in the modern healthcare system [20] . The prevalence of
MDR A. baumannii has also dramatically increased in other Euro-
pean countries from 15.4% in 2004 to 48.5% in 2014 [21] . Vietnam
has the highest prevalence of resistance amongst Gram-negative
pathogens in the Asia-Pacific region [22] . It is considered one of
the hottest spots of MDR A. baumannii prevalence in Asia. The
prevalence of carbapenem non-susceptible and MDR A. baumannii
has been found to be 80–90% among inpatients [ 23 , 24 ]. A. bauman-
nii possesses the ability to develop intrinsic resistance via reduc-
ing membrane permeability, efflux pump activity and production
of wide varieties of β-lactamases [25] . It is also able to acquire re-
sistance via mutational changes in chromosomal structure as well
as through horizontal gene transfer [25] . Moreover, it presents high
genetic plasticity, which contributes to the accumulation of these
resistance determinants and leads to the MDR pattern [26] .
Dissemination and infection rates of A. baumannii are on the
rise. However, several aspects of resistance development and
pathogenesis are not fully understood. The reservoir for A. bauman-
nii is increasing, not only in the hospitals but also in the commu-
nity and the environment. Its prevalence has gradually increased in
dairy farms and food-producing animals [ 27 , 28 ]. However, it seems
to be neglected in veterinary medicine and food chains, and the
possible risk of human infection concerning animal contact, food
consumption and environmental contamination is unknown.
The current study aimed to combine in vitro phenotyping anal-
ysis and whole-genome-sequencing (WGS) to characterise the phe-
notype and genetic determinants associated with intrinsic resis-
tance in 100 clinical and non-clinical A. baumannii strains origi-
nating from Germany and Vietnam. Moreover, it aimed to assess
whether powdered milk as a food source functions as a potential
reservoir of antibiotic resistance and possesses similar antimicro-
bial resistance (AMR) genes as in clinical strains isolated from Ger-
many.
2. Materials and Methods
2.1. Bacterial isolates and identification
This study used 10 0 non-repetitive A. baumannii strains: 89
isolated in Germany (14 from humans, 71 from milk powder,
two from animals, and two reference strains of A. baumannii
DSM30 0 07 and DSM105126 of German preferential origin), and 11
clinical strains isolated from humans in Vietnam. Human and an-
imal strains were isolated between 2015–2018, while strains ob-
tained from powdered milk were isolated between 2005–2012. The
strains were submitted to the Institute of Bacterial Infections and
Zoonoses (IBIZ, Jena) for confirmation and typing. All strains had
been identified at species level using a combination of Matrix-
Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-
TOF MS) and the bla OXA-51-like-PCR [29] . WGS was used to con-
firm the identity of the strains by Kraken [30] and Average Nu-
cleotide Identity (ANI) approach [31] .
2.2. Antimicrobial susceptibility testing
The minimum inhibitory concentration (MIC) was determined
via the broth microdilution method in Mueller Hinton II broth us-
ing an automated MICRONAUT-S system (Micronaut, MERLIN Diag-
nostics GmbH, Bornheim-Hersel Germany) according to the man-
ufacturer’s instructions. Briefly, susceptibility testing via this sys-
tem is based on rehydration of antibiotic compounds by adding
a standardized bacterial suspension, and the growth is anal-
ysed using the plate reader after incubation at 37 °C for 18–
24 hours. The results were automatically evaluated into sus-
ceptible, intermediate and resistant, with the built-in MICRO-
NAUTS software. Two different plates were used: the Micronaut-
S β-Lactamases and the Micronaut-S-MDR-MRGN-Screening 3
plates. The MIC values were interpreted according to the Clin-
ical and Laboratory Standards Institute (CLSI) breakpoint guide-
lines available for A. baumannii. For A. baumannii, 18 of 26 an-
tibiotics were interpretable by the software. The sensitivity of
all 100 isolates was determined for a panel of the 18 an-
tibiotics: ciprofloxacin (CIP), levofloxacin (LEV), amikacin (AMK),
colistin (COL), chloramphenicol (CMP), fosfomycin (FOS), tigecy-
cline (TGC), trimethoprim/sulfamethoxazole (T/S), piperacillin (PIP),
piperacillin/tazobactam (PIT), cefotaxime (CTX), ceftazidime (CAZ),
ceftazidime/avibactam (CAA), ceftolozane/tazobactam (CTA), ce-
fepime (CEP), imipenem (IMP), meropenem (MER), and ertapenem
(ERT).
2.3. WGS and in silico detection of genes coding for antimicrobial
resistance factors
The genomic DNA was extracted using the High Pure PCR Tem-
plate Preparation Kit (Roche Diagnostics GmbH, Mannheim, Ger-
many) according to the manufacturer’s instructions. The sequenc-
ing library was prepared with the Nextera XT DNA Library Prep
Kit (Illumina, Inc., San Diego, CA, USA) followed by paired-end
sequencing on an Illumina MiSeq sequencer (Illumina, USA). The
Linux-based pipeline WGSBAC that had been developed at the
FLI was used to analyse raw sequencing data ( https://gitlab.com/
FLI _ Bioinfo/WGSBAC ). The pipeline performs quality control of raw
sequencing data by calculating raw sequencing coverage and by us-
ing FastQC (v. 0.11.5) [32] . Results of FastQC were summarised with
MutltiQC [33] . For assembly, the pipeline used SPAdes (v. 3.12.0)
[34] together with the Bayse hammer read correction mode (op-
tion - carefull) and checks the quality of assembled genomes us-
ing QUAST (v. 4.3) [35] . To characterise the assembled genomes,
WGSBAC used ABRIcate (v 0.8.10) ( https://github.com/tseemann/
abricate ). In silico detection of intrinsic AMR genes was performed
G. Wareth, J. Linde and P. Hammer et al. / International Journal of Antimicrobial Agents 56 (2020) 10 6127 3
by using the Resistance Gene Identifier (RGI) based on the Compre-
hensive Antibiotic Resistance Database (CARD) [36] and the NCBI
databases (Bio Project PRJNA313047). The β-lactamase variants and
non- β-lactamase genes with 100% identity using the A. baumannii
reference genome (Accession ASM74664v1) as input were selected
for further analysis. A few genes with identity < 100% were re-
moved from the analysis.
3. Results
3.1. The phenotypic characteristics and antibiotic susceptibility testing
The phenotypic analysis and antibiotic susceptibility testing
(AST) of the 100 isolates to the panel of 18 antibiotics are shown
in Table 1 . The non-susceptible pattern (resistance and interme-
diate) was identified in isolates of human and animal origins,
and in isolates obtained from milk powder. The highest resis-
tance frequency was found for chloramphenicol (100% of the tested
isolates), followed by fosfomycin (96%) and the third-generation
cephalosporins cefotaxime (95%). Thirty-five isolates (35%) dis-
played resistance to at least one of the carbapenems. Among them,
24 isolates (24%) of human origin from Germany and Vietnam
were resistant to imipenem, meropenem and ertapenem. Addi-
tionally, one reference strain (DSM30 0 07) and ten isolates (10%)
obtained from milk powder produced in Germany were resis-
tant only to ertapenem. About one quarter (25%) of the isolates
displayed resistance to the broad-spectrum β-lactams piperacillin
either alone or with the β-lactamase inhibitor tazobactam. A
quarter of the isolates (24%, 25%) displayed resistance to fluoro-
quinolones, levofloxacin and ciprofloxacin, respectively. The lowest
resistance among the isolates was observed towards colistin (3%),
trimethoprim/sulfamethoxazole (17%), the tetracycline derivative
tigecycline (19%), and the aminoglycoside amikacin (19%). Three
colistin-resistant strains originating from Germany were found.
One of them was a human strain isolated in 2016 that showed
resistance to all tested antibiotics, including colistin and tigecy-
cline, while still sensitive only to trimethoprim/sulfamethoxazole.
The other two strains were isolated from milk powder in 2006
and 2016, and showed resistance only to colistin, fosfomycin,
chloramphenicol, and cefotaxime. Resistance to fluoroquinolones,
aminoglycosides, tigecycline, trimethoprim/sulfamethoxazole, and
to broad-spectrum β-lactam piperacillin and the fourth-generation
cephalosporins was found only in human strains isolated from both
Germany and Vietnam. The 71 strains isolated from milk powder
produced in Germany showed resistance against chloramphenicol
(n = 71), fosfomycin (n = 67), the third-generation cephalosporin
cefotaxime (n = 66), the carbapenem ertapenem (n = 10) , and col-
istin (n = 2). The metadata of the 100 isolates and the antibiotic
profiles are shown in Supplementary Table 1.
3.2. The phenotypic characteristics of MDR and XDR strains
According to the standardised international terminology created
by the European Centre for Disease Control (ECDC) and the Centre
for Disease Control (CDC) and Prevention [37] , A. baumannii is con-
sidered MDR when it is resistant to at least one compound in three
or more antimicrobial categories: aminoglycosides (amikacin), flu-
oroquinolones (ciprofloxacin or levofloxacin), cephalosporins (ce-
fotaxime, ceftazidime, ceftazidime or cefepime), and carbapenems
(imipenem, meropenem or ertapenem). Moreover, the isolate is
considered extensively drug-resistant (XDR) when it is resistant
to at least one agent in all but two or fewer antimicrobial cat-
egories. As shown in Table 2 , 24 isolates (24%) were MDR. All
were of human origin and isolated from Germany (n = 14) and
Vietnam (n = 10). All MDR strains were resistant to carbapen-
ems, fluoroquinolones, chloramphenicol, fosfomycin, piperacillin,
Tabl e 1
Phenotypic analysis of all tested Acinetobacter baumannii isolates (n = 10 0) based on detection of MIC using microdilution methods.
No. Antibiotic Antibiotic class
Tota l number and % of susceptible (S), intermediate
(I) and resistant (R) strains Number of non-susceptible strains based on country and source
Vietnam Germany
S I R
Human
(n = 11 )
Human
(n = 14)
Milk powder
(n = 71)
Animal
(n = 2)
Two DSMZ
reference
1 CIP Fluoroquinolones 75 (75%) 1 (1%) 24 (24%) 10 14 - - 1
2 LEV Fluoroquinolones 76 (76%) 0 24 (24%) 10 14 - - -
3 AMK Aminoglycosides 81 (81%) 0 19 (19%) 9 10 - - -
4 COL Polymyxin B 97 (97%) 0 3 (3%) - 1 2 - -
5 CMP Chloramphenicol 0 5 (5%) 95 (95%) 11 14 71 2 2
6 FOS Produced by Streptomyces 4 (4%) 0 96 (96%) 11 14 67 2 2
7 TGC Tetrac yclines 81 (81%) 7 (7%) 12 (12%) 9 10 - - -
8 T/S Sulfonamides 83 (85%) 0 17 (17%) 9 6 - - 2
9 PIP β-lactams 75 (75%) 0 25 (25%) 10 14 - - 1
10 PIT β-lactams 76 (76%) 0 24 (24%) 10 14 - - -
11 CTX 3
rd
-generation cephalosporins 5 (5%) 14 (14%) 81 (81%) 11 14 66 2 2
12 CAZ 3
rd
-generation cephalosporins 76 (76%) 0 24 (24%) 10 14 - - -
13 CAA 3
rd
-generation cephalosporins 79 (79%) 0 21 (21%) 10 11 - - -
14 CTA 3
rd
-generation cephalosporins 76 (76%) 0 24 (24%) 10 14 - - -
15 CEP 4
th
-generation cephalosporins 76 (76%) 2 (2%) 22 (22%) 10 13 - 1 -
16 IMP Carbapenems 76 (76%) 0 24 (24%) 10 14 - - -
17 MER Carbapenems 76 (76%) 0 24 (24%) 10 14 - - -
18 ERT Carbapenems 65 (65%) 0 35 (35%) 10 14 10 - 1
Abbreviations: CIP, ciprofloxacin; LEV, levofloxacin; AMK, amikacin; COL, colistin; CMP, chloramphenicol; FOS, fosfomycin; TGC, tigecycline; T/S, trimethoprim/sulfamethoxazole; PIP, piperacillin; PIT, piperacillin/tazobactam; CTX,
cefotaxime; CAZ, ceftazidime; CAA, ceftazidime/avibactam; CTA, ceftolozane/tazobactam; CEP, cefepime; IMP, imipenem; MER, meropenem; ERT, ertapenem; DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen
(German Collection of Microorganisms and Cell Cultures).
4 G. Wareth, J. Linde and P. Hammer et al. / International Journal of Antimicrobial Agents 56 (2020) 10 6127
Tabl e 2
Antibiotic resistance profile of MDR and XDR Acinetobacter baumannii strains toward a panel of 18 antibiotics based on detection of the MIC using microdilution methods.
No. ID Year of isolation Country Origin CIP LEV AMK COL CMP FOS TGC T/S PIP PIT CTX CAZ CAA CTA CEP IMP MER ERT
1 17Y0437 Ab MVZ 2015 Germany Human R R R S R R S R R R R R S R R R R R
2 17Y0438 Ab MVZ 2015 Germany Human R R S S R R R R R R R R R R R R R R
3 17Y0439 Ab MVZ 2015 Germany Human R R R S R R R S R R R R R R R R R R
4 17Y0440 Ab MVZ 2016 Germany Human R R R R R R R S R R R R R R R R R R
5 17Y0441 Ab MVZ 2016 Germany Human R R R S R R I R R R R R S R R R R R
6 17Y0442 Ab MVZ 2016 Germany Human R R R S R R S R R R R R R R I R R R
7 17Y0444 Ab MVZ 2016 Germany Human R R S S R R S S R R R R R R R R R R
8 17Y0446 Ab MVZ 2016 Germany Human R R R S R R I S R R R R S R S R R R
9 17Y0447
Ab MVZ 2016 Germany Human R R S S R R S S R R R R R R R R R R
10 17Y0448 Ab MVZ 2017 Germany Human R R R S R R R R R R R R R R R R R R
11 18Y0258_Ab_TLV 2018 Germany Human R R R S R R I R R R R R R R R R R R
12 18Y0259_Ab_TLV 2018 Germany Human R R R S R R I S R R R R R R R R R R
13 18Y0260_Ab_TLV 2018 Germany Human R R S S R R I S R R R R R R R R R R
14 18Y0261_Ab_TLV 2018 Germany Human R R R S R R R S R R R R R R R R R R
15 18Y0059 Ab HVU 2017 Vietnam Human R R R S R R R R R R R R R R R R R R
16 18Y0060 Ab HVU 2017 Vietnam Human R R R S R R R R R R R R R R R R R R
17 18Y0061 Ab HVU 2017 Vietnam Human R R R S R R R R R R R R R R R R R R
18 18Y0064 Ab HVU 2017 Vietnam Human R R R S R R R R R R R R R R R R R R
19 18Y0065 Ab HVU 2017 Vietnam Human R R R S R R R S R R R R R R R R R R
20 18Y0066 Ab HVU 2017 Vietnam Human R R R S R R I R R R R R R R R R R R
21 18Y0067 Ab HVU 2017 Vietnam Human R R R S R R R R R R R R R R R R R R
22 18Y0072 Ab HVU 2017 Vietnam Human R R S S R R S S R R R R R R R R R R
23 18Y0074 Ab HVU 2017 Vietnam Human R R R S R R I R R R R R R R R R R R
24 18Y0075 Ab HVU 2017 Vietnam Human R R R S R R R R R R R R R R R R R R
Abbreviations: R, resistant; I, intermediate; S, susceptible; CIP, ciprofloxacin; LEV, levofloxacin; AMK, amikacin; COL, colistin; CMP, chloramphenicol; FOS, fosfomycin; TGC, tigecycline; T/S, trimetho-
prim/Sulfamethoxazole; PIP, piperacillin; PIT, piperacillin/tazobactam; CTX, cefotaxime; CAZ, ceftazidime; CAA, ceftazidime/avibactam; CTA, ceftolozane/tazobactam; CEP, cefepime; IMP, imipenem; MER, meropenem;
ERT, ertapenem.
piperacillin/tazobactam, cefotaxime, ceftazidime, and the novel an-
tibiotics ceftolozane/tazobactam. One of them was sensitive to the
fourth-generation cephalosporin cefepime and five were still sus-
ceptible to amikacin. Sixteen isolates (16%) were classified as XDR
A. baumannii. Among them, ten isolates (10/16) were resistant to
all tested antibiotics except colistin. One human isolate (1/16) from
Germany was resistant to all tested antibiotics, including colistin,
while it was still sensitive only to trimethoprim/sulfamethoxazole.
Five strains (5/16) were resistant to all tested antibiotics and
still sensitive to colistin (n = 5), trimethoprim/sulfamethoxazole
(n = 3), ceftazidime/avibactam (n = 3), tigecycline (n = 2), and
cefepime (n = 1). Metadata of the MDR strains and resistance pro-
files are shown in Table 2 .
3.3. Analysis of intrinsic AMR determinants
The WGS approach identified different AMR-related genes,
among which at least 60 represented intrinsic resistance genes and
51 acquired resistance genes (data not shown). In silico detection
of intrinsic AMR genes using CARD and NCBI databases succeeded
in identifying 36 β-lactamases and ESBLs intrinsic AMR genes
( Table 3 ), as well as 24 non- β-lactamase AMR genes ( Table 4 ).
3.4. Resistance to β-lactams
As shown in Table 3 , three Ambler classes of β-lactamases
(i.e. classes A, C and D) were identified in the current study. At
least three, four and 29 different variants of class A, D and C β-
lactamases were identified, respectively. The members of Ambler
class C β-lactamases were the most predominant genes and en-
compassed 28 Acinetobacter -derived cephalosporinase bla ADC vari-
ants and one A. baumannii AmpC β-lactamase gene. The gene
bla ADC-6 was present in 33 isolates (33%), followed by bla ADC-2
in 24 isolates (24%), bla ADC-52 in 21 isolates (21%), bla ADC-16 in
13 isolates (13%), bla ADC-155 in 12 isolates (12%), bla ADC-156 in
11 isolates (11%), bla ADC-80 in 10 isolates (10%), and bla ADC-73
in nine isolates (9%). The nucleotide sequence of the chromosomal
cephalosporinase gene, which encodes an AmpC β-lactamase, was
found in one strain obtained from milk powder in Germany. Four
AMR genes belonging to the Ambler class D β-lactamases were
identified, the bla OXA-65 gene was present in four isolates (4%),
followed by bla OXA-104, bla OXA.180, and bla OXA.200 ( Table 3 ).
Three AMR genes belonging to Ambler class A β-lactamases were
identified including those of bla TEM-12, which was present in nine
strains (9%), followed by carbenicillin hydrolysing β-lactamase,
bla CARB-16, and bla CARB-14 which were present in three (3%)
and one strain (1%), respectively. Nineteen isolates (19%) had both,
bla ADC-6 and bla ADC-52 of the same Ambler class C, and five iso-
lates (5%) had both, bla ADC-73 and bla TEM-12 of different Ambler
classes. A. baumannii isolates collected from powdered milk sam-
ples from Germany showed wide varieties of β-lactamase AMR
genes from Ambler classes C and D. No AMR genes belonging to
Ambler class B β-lactamase were found. All information regarding
the number of AMR genes, source and origin of strains, as well as
the mechanism of resistance are shown in Table 3 .
3.5. Resistance to aminoglycosides
Among the 100 tested strains, 19 showed resistance to
amikacin. Five aminoglycoside-modifying enzymes (AMEs) genes
were detected. The new subclass of intrinsic aminoglycoside nu-
cleotidyltransferase, ANT(3")-IIa, was widely distributed in human,
animal and milk powder samples. It was found in all tested strains
(100%) followed by the intrinsic aminoglycoside nucleotidyltrans-
ferases aad A and aad A1, which were present in nine strains (9%),
G. Wareth, J. Linde and P. Hammer et al. / International Journal of Antimicrobial Agents 56 (2020) 10 6127 5
Tabl e 3
β-lactamase and ESBL families intrinsic resistance genes identified in A. baumannii isolates (n = 100) based on WGS data using CARD and NCBI databases.
AMR genes No. (%) Mechanism Source Origin Database
bla ADC.2 23 (23%)/
1 (1%)
Ambler class C
β-lactamases
Milk powder/ human Germany/
Vietnam
CARD
bla ADC.3 1 (1%) Milk powder Germany CARD
bla ADC.4 1 (1%) Animal Germany CARD
bla ADC.5 1 (1%) Animal Germany CARD
bla ADC. 6 33 (33%) Human, milk powder Germany CARD
bla ADC.26 6 (6%) Milk powder Germany NCBI
bla ADC.30 4 (4%) Human Germany NCBI
bla ADC.32 4 (4%) Milk powder Germany NCBI
bla ADC.33 1 (1%) Human Germany NCBI
bla ADC.52 21 (21%) Human, milk powder Germany NCBI
bla ADC.73 9 (9%) Human Germany, Vietnam NCBI
bla ADC.76 4 (4%) Milk powder Germany CARD
bla ADC.78 1 (1%) Human Germany CARD
bla ADC.79 3 (3%) Milk powder Germany CARD
bla ADC.80 10 (10%) Milk powder Germany CARD
bla ADC.81 2 (2%) Human Germany CARD
bla ADC.82 4 (4%) Human Vietnam CARD
bla ADC.90 1 (1%) Milk powder Germany NCBI
bla ADC.107 1 (1%) Milk powder Germany NCBI
bla ADC.154 1 (1%) Animal Germany NCBI
bla ADC.155 12 (12%) Milk powder Germany NCBI
bla ADC.156 10 (10%)/
1 (1%)
Milk powder/ human Germany/
Vietnam
NCBI
bla ADC.157 1 (1%) Milk powder Germany NCBI
bla ADC.158 3 (3%) Animal, milk powder Germany NCBI
bla ADC.165 1 (1%) Milk powder Germany NCBI
bla ADC.16 13
(13%) Milk powder Germany NCBI
bla ADC.169 1 (1%) Milk powder Germany NCBI
blaADC.176 2 (2%) Human Germany NCBI
Ab AmpC_ 1 (1%) Milk powder Germany CARD
bla OXA. 104 2 (2%) Ambler
class D
β-lactamases
Animal, milk powder Germany NCBI
bla OXA.180 2 (2%) Milk powder Germany CARD
bla OXA. 200 1 (1%) Human Germany CARD
bla OXA. 65 4 (4%) Milk powder Germany CARD
bla TEM.12 9 (9%) Ambler
class A
β-lactamases
Human Germany, Vietnam CARD
bla CARB.14 1 (1%) Human Germany CARD
bla CARB.16 3 (3%) Human Germany, Vietnam CARD
Abbreviations: CARD, The Comprehensive Antibiotic Resistance Database; NCBI, National Center for Biotechnology Information.
and aadA2 that was present in one strain (1%). Moreover, the in-
trinsic aminoglycoside acetyltransferase aac A34 was found in one
human isolate from Vietnam ( Table 4 ).
3.6. Antibiotic efflux pumps
Four categories of efflux pumps were found in the current
study, including the multidrug and toxic compound extrusion
(MATE) family, the small multidrug-resistance (SMR) family trans-
porters, the resistance-nodulation-division (RND) superfamily, and
the major facilitator superfamily (MFS). Within these different
pumps the RND ( ade FGH, ade IJK and ade L), MATE ( abe M) and SMR
( abe S) were most frequent and found in 99–100% of isolates. The
RND ( ade N, ade R, ade S, and ade AB) was found in 97%, 76%, 71%, and
66% of strains, respectively. The tetC gene of the MFS transporter
family and the adeC gene of the RND were the genes with the low-
est abundance and were found in one and 22 isolates, respectively.
A. baumannii collected from milk powder showed to have wide va-
rieties of multidrug efflux pump genes ( Table 4 ).
3.7. Resistance to phenicols, glycopeptides and macrolides
Among the 100 tested isolates, one human isolate from Ger-
many harboured the catI gene encoding resistance to pheni-
cols, and one human isolate from Vietnam harboured the gene
brp (MBL) encoding resistance to glycopeptides (e.g. bleomycin).
The mphD gene encoding resistance to macrolide-lincosamide-
streptogramin B (MLS) was found in nine strains (9%) of human
origin from both countries, Germany and Vietnam ( Table 4 ).
4. Discussion
Acinetobacter baumannii has become a notorious pathogen in
human medicine and is implicated in nosocomial and community-
acquired infections worldwide [38] . The general distribution and
reservoirs of A. baumannii are increasing in hospitals, communities,
food chains and the environment [39] . Data on the widespread dis-
tribution of A. baumannii in the farm animal population and foods
is becoming alarming in Germany [40] . However, the risk of human
infection regarding animal contact, food consumption and environ-
mental contamination is unknown. To assess whether milk pow-
der is an essential route for dissemination of resistance in A. bau-
mannii , 71 strains isolated from milk powder obtained from two
powdered milk producers in Germany were included in the study.
Among them, two strains were resistant to colistin and ten strains
were resistant to ertapenem. Both antibiotics are considered last-
resort antibiotics. None of the isolates recovered from milk pow-
der showed an MDR pattern. In total, 38 AMR genes associated
with resistance to antibiotics (22 β-lactamases, 15 multidrug ef-
flux pumps and one AMEs) were found in strains obtained from
powdered milk ( Tables 3 and 4 ). It could not be defined whether
the strains originated from animals, humans or the environment
during milk powder manufacturing. Most of the strains were iso-
lated from end products. As milk is pasteurised before spray dry-
ing and subsequent heat treatment is applied during concentration
6 G. Wareth, J. Linde and P. Hammer et al. / International Journal of Antimicrobial Agents 56 (2020) 10 6127
Tabl e 4
Non-
β-lactamases intrinsic resistance genes identified in Acinetobacter baumannii isolates (n = 100 ) based on WGS data using CARD and NCBI databases.
Antibiotic family AMR genes No. (%) Mechanism Source Origin Database
AMEs aac A34 1 (1%) ACT Human Vietnam NCBI
aad A 9 (9%) NUT Human Germany, Vietnam NCBI
aad A1 9 (9%) NUT Human Germany, Vietnam CARD
aad A2 1 (1%) NUT Human Vietnam CARD
ANT .3...IIa 100 (100%) NUT Human / animal, milk powder Germany, Vietnam / Germany CARD
MATE Family abe M 99 (99%) Antibiotic efflux Human / animal, milk powder Germany, Vietnam / Germany CARD
SMR family abe S 100 (100%) Human / animal, milk powder Germany, Vietnam / Germany CARD
RND family ade AB 66 (66%) Human / animal, milk powder Germany, Vietnam / Germany CARD
ade
C 22 (22%) Human / milk powder Germany, Vietnam / Germany CARD
ade FGH 99-100 (99-100%) Human / animal, milk powder Germany, Vietnam / Germany CARD
ade IJK 99-100 (99-100%) Human / animal, milk powder Germany, Vietnam / Germany CARD
ade NL 97, 100 (97, 100%) Human / animal, milk powder Germany, Vietnam / Germany CARD
ade R 76 (76%) Human / animal, milk powder Germany, Vietnam / Germany CARD
ade S 71 (71%) Human / animal, milk powder Germany, Vietnam / Germany CARD
MFS family Tetra cyc li ne s tet. C 1 (1%) Human Germany CARD
Phenicols cat I 1 (1%) Enzymes inactivation Human Germany CARD
MLS family mph D 9 (9%) Human Germany, Vietnam CARD
Glycopeptides Determinant of bleomycin resistance 1 (1%) Human Vietnam CARD
Abbreviations: AMEs, aminoglycoside-modifying enzymes; ACT, acetyltransferase; NUT, nucleotidyltransferase; MATE, multidrug and toxic compound extrusi on transporter; SMR, smallest multidrug trans-
porters; RND, resistance-nodulation-cell division antibiotic efflux pump; MFS, major facilitator superfamily; MLS, macrolide-lincosamide-streptogramin B; CARD, The Comprehensive Antibiotic Resistance
Database; NCBI, National Center for Biotechnology Information.
and spray drying, the survival of Acinetobacter is unlikely. However,
processing of contaminated concentrate and rework of filter pow-
der may result in a contaminated end product [41] . In addition, the
powder might be re-contaminated from the environment of the
factory. Acinetobacter exposure can occur via many sources such
as humans, pests, air, packing material, forklift traffic, etc. Conse-
quently, milk powder probably poses a risk for the dissemination
of resistant A. baumannii to humans and further investigations are
required. Powdered infant milk has been identified as a source of
bacterial infection in neonates and infants, especially in hospital
nurseries [41] . The results in the present study are consistent with
previously published observations [ 27 , 42 ].
Clinically, the presence of MDR A. baumannii is of great con-
cern in modern healthcare systems. Detection and characterisation
of AMR genes are gradually moving from PCR to high through-
put identification via sequencing and in silico detection using free-
ware programs and public databases. Although several PCR proto-
cols have been designed to identify resistance genes, these tools
are very limited because they identify only the expected resistance
genes according to the chosen primers. In the current study, the
implementation of high-throughput WGS for the 100 A. baumannii
isolates facilitated rapid and comprehensive AMR detection. In sil-
ico detection of AMR genes based on CARD and NCBI databases re-
vealed the presence of 60 intrinsic resistance genes. Depending on
one database to discriminate the presence of all AMR genes was
insufficient. Moreover, sequencing-based approaches identify only
the known resistance-associated genes but do not provide any in-
formation if these are active. Thus, this study highlights the neces-
sity of combining in vivo phenotyping with in silico detection of
resistance genes from different databases to determine the resis-
tance profiles of A. baumannii isolates.
Different resistance mechanisms towards many classes of an-
tibiotics are known to exist in A. baumannii [43] . Among them, β-
lactamases, multidrug efflux pumps and aminoglycoside-modifying
enzymes were detected in the current study. A. baumannii ex-
hibits intrinsic resistance due to the presence of two chromo-
somally encoded β-lactamases, the bla ADC cephalosporinase and
bla OXA oxacillinase variants, in addition to the natural membrane
impermeability and basal efflux activity [44] . The chromosomally
encoded cephalosporinase gene ( Amp C type) [45] and oxacillinase
bla OXA-69 [46] play an important role in β-lactam resistance in A.
baumannii. Besides, A. baumannii encode 72 further sequence vari-
ants of the intrinsic, non-transferable OXA-51-like enzymes, repre-
senting one of the largest groups of ß-lactamases [47] . In the cur-
rent study, resistance conferred by ß-lactamases was detected by
36 predicted genes; about two-thirds of the genes (n = 22) were
present in strains obtained from milk powder. Inactivation of β-
lactams constitutes an important part of MDR in A. baumannii , es-
pecially for β-lactam antibiotic resistance. All four Ambler classes
of β-lactamases (i.e . classes A, B, C, and D) can be identified in this
pathogen [48] . However, no resistance gene belonging to Ambler
classes B was found. Thirty-six encoding genes initiating resistance
due to β-lactamases were found, and the chromosomally encoded
Acinetobacter -derived cephalosporinase bla ADC gene was predomi-
nant with 28 allelic variants detected. The variants bla ADC-6 and
bla ADC-2 were the most predominant being present in 33% and
24% of the strains, respectively.
Resistance to aminoglycosides in A. baumannii is mainly me-
diated by enzymes, which chemically modify aminoglycosides
(AMEs) [49] . Four-aminoglycoside nucleotidyltransferase (NUT) and
one acetyltransferase (ACT) genes were found. Resistance due to
antibiotic efflux was mediated by 13 RND antibiotic efflux pump-
encoding genes ( adeABC, adeFGH, adeIJK, adeLN and adeRS ). At least
five RND genes were present in 100 % of strains and another four
were present in 99% of strains. The role of RND efflux pump genes
and antimicrobial resistance in A. baumannii is well documented
G. Wareth, J. Linde and P. Hammer et al. / International Journal of Antimicrobial Agents 56 (2020) 10 6127 7
[50] . The RND genes conferring resistance to clinically essential an-
tibiotics were among the majority of MDR strains in A. bauman-
nii [51] , and were prevalent in the current study. RND proteins
are found in both, prokaryotic and eukaryotic cells. They are sec-
ondary transporters and mediate resistance by pumping the an-
tibiotic from the cell [52] . The present study found one MATE
transporter coded by abe M. The protein encoded by this gene uses
the cationic gradient across the membrane as an energy source
to pump antimicrobial agents from cells [53] . The SMR gene abeS ,
which is restricted to prokaryotic cells [54] , and the MFS gene tetC
were found in 10 0% and 1% of isolates, respectively. Efflux pumps
help bacteria to survive in the presence of high concentrations of
antibiotics but may also confer resistance to antiseptics [55] , and
play an important role in development of AMR A. baumannii [56] .
Although no gene associated with fosfomycin resistance was
found, 96% of strains were resistant to fosfomycin in culture . The
mechanism behind the intrinsic resistance to fosfomycin is not
fully understood in A. baumannii. Despite the fact that fosfomycin
is not the first-line treatment of A. baumannii infections, it has
recently been suggested as a possible treatment for XDR Gram-
negative bacteria [57] . Fosfomycin has been used to treat a wide
variety of bacterial infections in humans. It is not widely used
in animals and its use is limited to treat infectious diseases of
broiler chickens and pigs [58] . Fosfomycin resistance is typically
plasmid-mediated in most members of Enterobacteriaceae . How-
ever, mutants in the ampD and anmK genes of the peptidoglycan-
recycling pathway also contributed to the intrinsic resistance to-
wards fosfomycin in A . baumannii [59] . In general, most A . bauman-
nii strains are intrinsically resistant to chloramphenicol and the re-
sistance is mediated by the major facilitator superfamily (MFS) ef-
flux pump [60] . All isolates (100%) in the current study were chlo-
ramphenicol resistant, while the MFS gene was present only in one
strain. The ade IJK is the second RND efflux system described for
A. baumannii and it contributes to intrinsic resistance to chloram-
phenicol [61] . Besides, inactivation of and trans complementation
with the abe S gene confers low-level resistance to chlorampheni-
col. Among sequenced A. baumannii strains, abe S and ade IJK were
found in 100% of isolates. In Germany, strains resistant to chlo-
ramphenicol have been reported in milk powder obtained from
the same source [27] . A. baumannii strains resistant to fosfomycin
have been described in pets [62] , adult moth flies captured in the
vicinity of hospitals [63] and in patients [ 64 , 65 ]. All A. bauman-
nii strains isolated from humans in Germany (n = 14) and Viet-
nam (n = 10/11) were resistant to imipenem, meropenem and
ertapenem. These results support previous data from Vietnamese
hospitals [ 23 , 24 ], where a substantial increase in the rate of isola-
tion of MDR-carbapenem-resistant A. baumannii was demonstrated .
Until 2005, resistance to carbapenems remained low in Germany
[66] , and the first outbreak of carbapenem-resistant A. bauman-
nii (CRAb) was in 2006 [67] . Since then, carbapenem-resistant
strains have been recovered from several outbreaks every year [68-
72] , and in 2011, CRAb was found in 96.3% of tested strains at
the National Reference Laboratory for Gram-negative nosocomial
pathogens [73] .
5. Conclusion
In conclusion, this study exposed the role of milk powder as
a potential reservoir for antibiotic-resistant A. baumannii strains .
Strains obtained from powdered milk possessed similar AMR genes
as those in clinical isolates from Germany and Vietnam. However, a
direct relationship between milk powder isolates and human iso-
lates has not been established until now. Taken together, the re-
sults of this work emphasise the necessity of combining in vivo
phenotyping and implementing high-throughput WGS for AMR de-
tection and profiling. Almost all isolates of human origin investi-
gated in the current study were MDR and carbapenem-resistant.
The high resistance rates found among clinical isolates are of great
concern and pose an essential threat to healthcare systems. The
current study was more concerned about intrinsic resistance genes
rather than acquired ones. Identification of both intrinsic and hor-
izontally transferable resistance genes could enrich the knowledge
about the dynamics of resistance evolution in A. baumannii , par-
ticularly in cases of foodborne transmission. Multilocus sequence
types (MLST) of isolates, presence of acquired resistance genes and
genes associated with motility and biofilm formation can also ex-
tracted from WGS data. Retrieving these kinds of data is essential
to explore the genetic relatedness of isolates, and to understand
survival and resistance development. Comparing the results of clin-
ical isolates with more strains from animals and environmental
sources is advantageous.
Acknowledgements:
We thank Dr Gernot Schmoock, Johannes Solle, Claudia Grosser
and Birgit Schikowski for excellent technical assistance.
Declarations
Funding: This work was supported by internal funding of the
Friedrich-Loeffler-Institut (FLI). OM and MP were co-funded by
grants from the Federal Ministry of Education and Research (Ger-
many), Grant Numbers: ‘13GW0096D and 01KI1501’.
Competing Interests: None to declare.
Ethical Approval: Not required.
Supplementary materials
Supplementary material associated with this article can be
found in the online version at doi:10.1016/j.ijantimicag.2020.
106127 .
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