Skip to main content
Erschienen in: Gut Pathogens 1/2020

Open Access 01.12.2020 | Research

Emergence of plasmid-mediated mcr genes from Gram-negative bacteria at the human-animal interface

verfasst von: Humera Javed, Sidrah Saleem, Aizza Zafar, Aamir Ghafoor, Ahmad Bin Shahzad, Hasan Ejaz, Kashaf Junaid, Shah Jahan

Erschienen in: Gut Pathogens | Ausgabe 1/2020

Abstract

Background

The global emergence of plasmid-mediated colistin resistance (Col-R) conferred by mcr genes in gram-negative rods (GNRs) has jeopardized the last treatment option for multidrug-resistant bacterial infections in humans. This study aimed to assess the emergence of mcr gene-mediated Col-R in GNRs isolated from humans and animals in Pakistan.

Methods

Animal and clinical specimens collected from various sources were prospectively analysed using standard microbiological procedures. Pathogens were identified using the API 20E and API 20NE systems (bioMerieux). Minimum inhibitory concentration (MIC) against colistin was determined using the MIC detection methods, and multiplex polymerase chain reaction (PCR) was used to amplify the mcr-1 to mcr-5 genes.

Results

We isolated 126 (88.1%) animal and 17 (11.9%) human Col-R phenotypes, among which there was a significant association (P < 0.01) of Escherichia coli and Proteus mirabilis with animals and of Acinetobacter baumannii with humans. Animal strains exhibited statistically significant (P < 0.05) resistance to co-trimoxazole, chloramphenicol, and moxifloxacin, and the human pathogens exhibited statistically significant (P < 0.05) antibiotic resistance to cephalosporins, carbapenems, and piperacillin-tazobactam. For Col-R strains, MIC50 values were > 6 µg/mL and > 12 µg/mL for human and animal isolates, respectively. mcr genes were detected in 110 (76.9%) bacterial strains, of which 108 (98.2%) were mcr-1 and 2 (1.8%) were mcr-2.

Conclusions

The detection of a considerable number of mcr-1 and mcr-2 genes in animals is worrisome, as they are now being detected in clinical pathogens. The acquisition of mcr genes by colistin-susceptible bacteria could leave us in a post-antibiotic era.
Hinweise

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Abkürzungen
Col-R
Colistin resistance
mcr
Mobile colistin resistance
MIC
Minimum inhibitory concentration
PCR
Polymerase chain reaction
QC
Quality control
XDR
Extensively drug-resistant
ESBLs
Extended-spectrum beta-lactamases
GNR
Gram-negative rods

Background

Continuously emerging antibiotic resistance poses a serious survival challenge to humankind and is leading us into a post-antibiotic era. The emergence of superbugs carrying extended-spectrum beta-lactamases (ESBLs), AmpC beta-lactamases, and metallo-beta-lactamases has reduced therapeutic choices [1]. Colistin is a cyclopeptide antibiotic prescribed as a last resort for the treatment of extensively drug-resistant (XDR) bacteria [2]. This drug was discovered more than seven decades ago and was first introduced in the 1960s for clinical use. It was replaced with other antibiotics in the 1970s because of its nephrotoxic and neurotoxic effects. Since then, it has been introduced into veterinary medicine [3]. Colistin attracted renewed attention and was reintroduced as an emergency solution in the 1990s in response to the escalating prevalence of XDR bacteria [4].
The situation became alarming because of the emergence of mobile colistin resistance (mcr) genes, initially in China, in animals and humans [5]. To date, more than 40 countries have reported mcr variants (mcr-1 to mcr-9) from five different continents across the globe, indicating the epidemicity of the mcr gene [6]. The mcr genes have been reported in seven Asian and nine European countries, and they were recently identified in Pakistan, Iran, Italy, Finland, America, South Africa, and some other territories [710]. The plasmid-borne mcr gene has been found in several enterobacteria, including Escherichia coli, Salmonella, Aeromonas, Enterobacter cloacae, Klebsiella pneumoniae, Escherichia fergusonii, Kluyvera ascorbata, Citrobacter braakii, Cronobacter sakazakii, Klebsiella aerogenes, and, most recently, Raoultella ornithinolytica [6].
Poultry and livestock, including chickens, ducks, pigeons, geese, pigs, and cattle, have been reported to be reservoir hosts for mcr-harbouring bacterial strains [11]. Of particular note, the animal-to-human transmission of mcr-1 colistin resistance (Col-R) has already been established in China, Thailand, Laos, and Denmark, which has raised a serious concern about its possible global dissemination [5, 12, 13]. In addition to their isolation from animals and humans, mcr genes have also been reported in bacteria from sewage, seawater, fresh food products, and seafood [14].
The extensive veterinary use of colistin and the increasing reports of Col-R in food animal strains of enterobacteria are indeed a matter of concern. The large-scale subclinical use of colistin for prophylaxis and growth promotion in livestock is a major cause of resistance [6]. The expansion of Col-R to various countries has led us to evaluate the magnitude of this drug resistance phenomenon in Pakistan. In this study, we aimed to assess the plasmid-mediated Col-R conferred by mcr genes among gram-negative rods (GNRs) isolated from humans and animals. The resistance spectrum of the Col-R and multidrug-resistant GNRs to a variety of antibiotics was elucidated to identify possible therapeutic regimens for combating these superbugs.

Methods

Study design and setting

The study was conducted prospectively over 18 months according to the ethical principles provided by the World Medical Association (WMA) Declaration of Helsinki. The study collaborated with and received ethics approval from the University of Health Sciences and the Children’s Hospital and the Institute of Child Health, Lahore, Pakistan.

Sample collection

A total of 38,500 human clinical specimens were randomly collected from the tertiary care public and private hospitals in Lahore, which treat patients from all over the Punjab province (population of approximately 120 million). The specimens collected from the clinical settings included blood, cerebrospinal fluid, swabs, tracheal secretions, urine, and faeces. Animal meat, chicken faecal, and respiratory secretion specimens (n = 630) were collected from different retail shops. We also collaborated with the University Diagnostic Laboratory (UDL), University of Veterinary and Animal Sciences, Lahore, Pakistan, to collect bacterial strains. UDL analyses animal pathological samples from all over the province.

Microbiological identification

The clinical specimens from sterile sites were processed for culture using blood, chocolate, and MacConkey’s culture media [15]. Urine samples were cultured on cysteine lactose electrolyte deficient (CLED) media, while xylose lysine deoxycholate (XLD) agar was used to culture the faecal specimens. The samples collected from the animal sources were processed on XLD and MacConkey’s agar. The bacterial cultures were identified using standard microbiological techniques, including Gram staining, oxidase production, and the API 20E and API 20NE systems (bioMerieux, France). Only GNRs resistant to colistin were included and processed further in our study.

Minimum inhibitory concentrations (MICs) against colistin

The GNRs recovered from both human and animal sources were tested for Col-R using E-test strips (Liofilchem, Italy), and the selected strains were confirmed with the SensiTest™ Colistin (Liofilchem, Italy). Only the Col-R strains were included in the study for further processing. The minimum inhibitory concentrations (MICs) were determined using an MIC epidemiological cut-off value (ECV) ≤ 2 µg/mL for the wild-type (WT) strains and ≥ 4 µg/mL for the non-wild-type (NWT) strains [16]. In the culture media and disc diffusion techniques, the ATCC 25922 (colistin-sensitive E. coli) and ATCC 25933 (colistin-resistant Proteus mirabilis) strains were used for quality control (QC).

Disc diffusion antibiotic testing

The Col-R phenotypes were assessed further to determine the association with drug resistance in the human and animal strains [16]. Antibacterial drug resistance against 16 other drugs that belong to several classes of antibiotics was tested using the disc diffusion method [16, 17]. The discs primarily used included aminoglycosides, cephalosporins, fluoroquinolones, carbapenems, and beta-lactam combinations.

Molecular detection of mcr genes and data analysis

The bacterial DNA from the freshly cultured GNR strains was thermally extracted by emulsifying 2–3 colonies in 200 µL of Tris EDTA (TE) buffer and boiling for 10 min [18]. Previously described mcr1 to mcr5 primers were used in the multiplex polymerase chain reaction (PCR) [19]. The amplification was performed on a thermal cycler (Biorad, T1000) using 12.5 µL Dream Taq master mix (Thermo Fisher Scientific, USA), 0.5 µL each of the 10 forward and reverse primers (10 µM), 5.5 µL nuclease-free water, and 2 µL of bacterial DNA in a final reaction mixture of 25 µL. The amplification procedure comprised an initial denaturation step at 94 °C for 15 min followed by 25 cycles at 94 °C for 30 s, 58 °C for 90 s, 72 °C for 60 s, and a final extension at 72 °C for 10 min [19]. The amplified mcr gene products were loaded on a horizontal agarose gel electrophoresis apparatus using 6 × loading dye and SYBR Safe DNA gel stain (Invitrogen). A 100 bp ladder was included with each electrophoresis run, and the gene bands were visualized with a gel documentation system (EZ Imager Bio-Rad). GraphPad Prism 7 and SPSS 23 were used for the statistical analysis. Chi-square tests were used to examine the association of antimicrobial drug-resistant microorganisms with the animal and human sources, and a significance threshold was set at P < 0.05.

Results

Association of Col-R in human and animal isolates

A total of 5,893 (15.3%) gram-negative rods from 38,500 human clinical specimens and 630 (49%) from 1,285 animal samples were identified. The distribution of colistin-sensitive (Col-S) and Col-R gram-negative strains isolated from human and animal sources is given in Table 1. We identified 143 gram-negative Col-R phenotypes from the human and animal isolates, of which 126 (88.1%) were isolated from animal and 17 (11.9%) from human sources. Among the animal isolates, 91 (72.2%) E. coli and 20 (15.9%) P. mirabilis strains were significantly associated (P < 0.01) with the animal sources, while 3 (17.6%) Acinetobacter baumannii strains were significantly associated (P < 0.01) with the human sources. There was no significant association of K. pneumoniae and Pseudomonas aeruginosa with any source (Table 2).
Table 1
Distribution of colistin-sensitive (Col-S) and colistin-resistant (Col-R) gram-negative strains isolated from human and animal sources
Organisms
Animal isolates (n = 1285)
Human isolates (n = 5893)
Total
Col-S
Col-R
Total
Col-S
Col-R
E. coli
658
567 (86.2%)
91 (13.8%)
2219
2216 (99.9%)
3 (0.1%)
Klebsiella species
231
219 (94.8%)
12 (5.2%)
1592
1591 (99.9%)
1 (0.1%)
Pseudomonas species
81
78 (96.3%)
3 (3.7%)
995
995 (100%)
0 (0%)
Citrobacter species
89
89 (100%)
0 (0%)
351
351 (100%)
0 (0%)
Acinetobacter species
0
0 (0%)
0 (0%)
318
315 (99.1%)
3 (0.9%)
Enterobacter species
85
85 (100%)
0 (0%)
260
260 (100%)
0 (0%)
Proteus species
20
0 (0%)
20 (100%)
10
0 (0%)
10 (100%)
Salmonella species
109
109 (100%)
0 (0%)
85
85 (100%)
0 (0%)
Sphingomonas paucimobilis
0
0 (0%)
0 (0%)
25
25 (100%)
0 (0%)
Chryseomonas luteola
4
4 (100%)
0 (0%)
19
19 (100%)
0 (0%)
Pantoea species
8
8 (100%)
0 (0%)
19
19 (100%)
0 (0%)
Table 2
Association of colistin resistance (Col-R) in bacteria isolated from human and animal sources (n = 143)
Organism
Col-R animal source n (%)
Col-R human source n (%)
P-value
126 (88.1)
17 (11.9)
E. coli
91 (72.2)
3 (17.6)
< 0.01
K. pneumoniae
12 (9.5)
1 (5.9)
0.62
P. aeruginosa
3 (2.4)
0 (0)
0.52
A. baumannii
0 (0)
3 (17.6)
< 0.01
P. mirabilis
20 (15.9)
10 (58.8)
< 0.01

Distribution of Col-R strains from different specimens

The Col-R bacterial strains were predominantly found in different poultry specimens, including 90 (62.9%) in faecal, 22 (15.4%) in meat, and 14 (9.8%) in secretion samples. From the human sources, 6 (4.2%) Col-R strains were found in urine, and 5 (3.5%) were found in tracheal secretions, while the rest of the strains were found in the other human specimens (Fig. 1). The predominant source was poultry faeces, with 70 samples (77.8%) containing E. coli and 16 (17.8%) containing P. mirabilis, followed by poultry meat, with 18 samples (81.1%) containing E. coli. The frequencies of bacterial isolates from each animal source are shown in Fig. 2.

Drug-resistance spectrum against various antibiotics

For the animal strains, there was a statistically significant association of drug resistance to co-trimoxazole (80.2% vs. 47.1%; P = 0.01), chloramphenicol (73% vs. 52.9%; P = 0.05), and moxifloxacin (71.4% vs. 41.2%; P = 0.03). Meropenem and piperacillin-tazobactam had the lowest number of animal strains resistant to them, each at 10 (7.9%), followed by 12 (9.5%) strains resistant to cefoperazone-sulbactam, 16 (12.7%) to imipenem, and 20 (15.9%) to amikacin (Table 3).
Table 3
Association of antibiotic resistance in animal and human bacterial strains (n = 143)
Antibiotics
Animal isolates
Human isolates
P-value
n = 126 (%)
n = 17 (%)
Co-trimoxazole
101 (80.2)
8 (47.1)
0.01
Chloramphenicol
92 (73)
9 (52.9)
0.05
Moxifloxacin
90 (71.4)
7 (41.2)
0.03
Levofloxacin
41 (32.5)
4 (23.5)
0.59
Ciprofloxacin
42 (33.3)
4 (23.5)
0.69
Co-amoxiclav
37 (29.4)
9 (52.9)
0.05
Cefotaxime
33 (26.2)
10 (58.8)
0.01
Cefixime
32 (25.4)
9 (52.9)
0.02
Cefuroxime
29 (23)
10 (58.8)
0.007
Ceftriaxone
28 (22.2)
11 (64.7)
0.01
Ceftazidime
25 (19.8)
10 (58.8)
< 0.01
Amikacin
20 (15.9)
4 (23.5)
0.73
Imipenem
16 (12.7)
5 (29.4)
< 0.01
Cefoperazone-sulbactam
12 (9.5)
4 (23.5)
0.22
Piperacillin-tazobactam
10 (7.9)
2 (11.8)
0.05
Meropenem
10 (7.9)
5 (29.4)
0.02
For the human pathogens, there was a statistically significant association of antibiotic resistance, primarily to the cephalosporins: ceftriaxone (64.7% vs. 22.2%; P = 0.01), cefuroxime (58.8% vs. 23%; P = 0.007), cefotaxime (58.8% vs. 26.2%; P = 0.01), ceftazidime (58.8% vs. 19.8%; P < 0.01), and cefixime (52.9% vs. 25.4%; P = 0.02). There was a statistically significant difference in resistance between the human isolates and the animal isolates to the beta-lactamase-resistant drugs, which include co-amoxiclav (52.9% vs. 29.4%; P = 0.05), meropenem (29.4% vs. 7.9%; P = 0.02), imipenem (29.4% vs. 12.7%; P < 0.01), and piperacillin-tazobactam (11.8% vs. 7.9%; P = 0.05). The lowest number of strains among the human pathogens resistant to a drug was 2 (11.8%), which was for piperacillin-tazobactam, followed by 4 (23.5%) each for cefoperazone-sulbactam, amikacin, ciprofloxacin, and levofloxacin (Table 3).
Escherichia coli and K. pneumoniae isolated from animal and human sources and P. aeruginosa isolated from animal sources showed more resistance to co-trimoxazole, chloramphenicol, and moxifloxacin than to the other drugs. All of the K. pneumoniae (human sources) and P. aeruginosa (animal sources) strains were also resistant to levofloxacin and ciprofloxacin. A. baumannii strains isolated from human sources showed resistance to all the antibacterial drugs except piperacillin-tazobactam. The detailed antimicrobial resistance profiles of the individual Col-R bacterial strains are presented in Table 4.
Table 4
Antimicrobial resistance profiles of individual colistin-resistant bacterial strains from animal and human sources
Antibiotics
E. coli
K. pneumoniae
P. aeruginosa
A. baumannii
P. mirabilis
Animal
Human
Animal
Human
Animal
Human
Animal
Human
Animal
Human
(n = 91)
(n = 3)
(n =12)
(n = 1)
(n = 2)
(n = 0)
(n = 0)
(n = 3)
(n = 20)
(n = 10)
Co-trimoxazole
64 (70%)
3 (100%)
5 (42%)
1 (100%)
2 (100%)
3 (100%)
8 (40%)
10 (100%)
Chloramphenicol
64 (70%)
3 (100%)
5 (42%)
1 (100%)
2 (100%)
3 (100%)
8 (40%)
4 (40%)
Moxifloxacin
59 (65%)
2(67%)
5 (42%)
1 (100%)
2 (100%)
3 (100%)
3 (15%)
4 (40%)
Levofloxacin
21 (23%)
1 (33%)
3 (25%)
1 (100%)
2 (100%)
2 (75%)
3 (15%)
1 (10%)
Ciprofloxacin
38 (42%)
0 (0%)
2 (17%)
1 (100%)
2 (100%)
3 (100%)
5 (25%)
1 (10%)
Co-amoxiclav
39 (43%)
2 (67%)
1 (8%)
1 (100%)
0 (0%)
3 (100%)
6 (30%)
1 (10%)
Cefotaxime
35 (38%)
1 (33%)
1 (8%)
1 (100%)
0 (0%)
2 (75%)
3 (15%)
1 (10%)
Cefixime
29 (32%)
2 (67%)
3 (25%)
1 (100%)
0 (0%)
3 (100%)
6 (30%)
3 (30%)
Cefuroxime
27 (30%)
2 (67%)
0 (0%)
1 (100%)
0 (0%)
3 (100%)
7 (35%)
3 (30%)
Ceftriaxone
27 (30%)
2 (67%)
1 (8%)
1 (100%)
0 (0%)
3 (100%)
7 (35%)
3 (30%)
Ceftazidime
27 (30%)
1 (33%)
0 (0%)
1 (100%)
0 (0%)
3 (100%)
1 (5%)
3 (30%)
Amikacin
16 (18%)
1 (33%)
3 (25%)
1 (100%)
0 (0%)
2 (75%)
2 (10%)
0 (0%)
Imipenem
12 (13%)
1 (33%)
3 (25%)
1 (100%)
0 (0%)
1 (33%)
0 (0%)
2 (20%)
Cefoperazone-sulbactam
7 (8%)
1 (33%)
2 (17%)
1 (100%)
1 (50%)
2 (67%)
2 (10%)
0 (0%)
Piperacillin-tazobactam
9 (10%)
0 (0%)
0 (0%)
1 (100%)
0 (0%)
0 (0%)
2 (10%)
1 (10%)
Meropenem
9 (10%)
1 (33%)
1 (8%)
1 (100%)
0 (0%)
3 (100%)
2 (10%)
0 (0%)

MICs of colistin against Col-R bacterial strains

The MICs of colistin against bacterial strains (n = 113) from human and animal sources showed MICs of 6, 8, 12, 24, 32, and 64 µg/mL. Because of the intrinsic resistance to colistin, the MICs of all of the P. mirabilis strains were > 264 µg/mL. The MIC distributions of colistin against human pathogens were MIC50 > 6 µg/mL and MIC90 > 12 µg/mL. The MIC50 and MIC90 values were > 12 µg/mL and > 32 µg/mL, respectively, for the animal isolates (Fig. 3).

Occurrence of mcr genes in isolated bacteria

All of the Col-R gram-negative bacterial strains were analysed to determine the presence of mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5. mcr genes were detected in 110 (76.9%) Col-R bacterial strains from animal and human sources, of which 108 (98.2) were mcr-1 and 2 (1.8%) were mcr-2 (Fig. 4). For the animal pathogens, mcr-1 was found in 90 (83.3%) E. coli strains, 12 (11.1%) K. pneumoniae strains, and 1 (0.9%) strain each of P. aeruginosa and P. mirabilis. For the human pathogens, mcr-1 was found in 3 (2.8%) E. coli strains and 1 (0.9%) K. pneumoniae strain. Only 2 (100%) strains of P. mirabilis isolated from the animal sources harboured mcr-2 (Table 5).
Table 5
Distribution of mcr genes in human and animal bacterial strains (n = 110)
Bacterial strains
mcr-1 (n = 108; 98.2%)
mcr-2 (n = 2; 1.8%)
Animal n (%)
Human n (%)
Animal n (%)
Human n (%)
104 (96.3)
4 (3.7)
2 (100)
0 (0)
E. coli
90 (83.3)
3 (2.8)
0 (0)
0 (0)
K. pneumoniae
12 (11.1)
1 (0.9)
0 (0)
0 (0)
P. aeruginosa
1 (0.9)
0 (0)
0 (0)
0 (0)
P. mirabilis
1 (0.9)
0 (0)
2 (100)
0 (0)
A. baumannii
0 (0)
0 (0)
0 (0)
0 (0)

Demographic and clinical data for the human isolates

Analysis of the demographic and clinical data of the patients infected with Col-R pathogens revealed that Col-R strains were isolated from various clinical specimens from patients of different ages and genders. The mcr-1 gene was detected in 3 E. coli (urine specimen) strains and 1 K. pneumoniae (blood specimen) strain, and none of the mcr variants were identified in any other bacterial strain of human origin (Table 6).
Table 6
Demographic and clinical data of patients infected with colistin-resistant pathogens (n = 17)
Gender
Age (Years)
Ward
Organism isolated
Specimen
Colistin MIC µg/mL
mcr gene
Male
1
Medical ICU
K. pneumoniae
Blood
6
mcr-1
Female
38
Outpatient department
E. coli
Urine
6
mcr-1
Female
3
Nephrology Ward
E. coli
Urine
12
mcr-1
Male
67
Cardiac ICU
E. coli
Urine
6
mcr-1
Female
53
Medical ICU
A. baumannii
Tracheal secretions
8
Not detected
Male
3
Neurosurgery ICU
A. baumannii
Tracheal secretions
8
Not detected
Female
61
Medical ICU
A. baumannii
Tracheal secretions
6
Not detected
Female
13
Outpatient department
P. mirabilis
Urine
≥ 264
Not detected
Male
68
Medical Ward
P. mirabilis
Urine
≥ 264
Not detected
Male
51
Medical Ward
P. mirabilis
Stool
≥ 264
Not detected
Female
8
Nephrology Ward
P. mirabilis
Blood
≥ 264
Not detected
Male
3
Medical ICU
P. mirabilis
Tracheal secretions
≥ 264
Not detected
Male
3
Ortho Ward
P. mirabilis
Pus
≥ 264
Not detected
Male
10
General Surgery Ward
P. mirabilis
Pus
≥ 264
Not detected
Male
73
Cardiac ICU
P. mirabilis
Tracheal secretions
≥ 264
Not detected
Female
6
Neurosurgery Ward
P. mirabilis
CSF
≥ 264
Not detected
Female
3
Medical Ward
P. mirabilis
Urine
≥ 264
Not detected

Discussion

The global dissemination of ESBLs, AmpC, and carbapenemase-producing bacteria has narrowed the options for appropriate antibiotics to treat gram-negative bacterial infections. The effectiveness of colistin in the treatment of XDR gram-negative bacterial infections is well known. In this era of antibacterial drug resistance, a new debate has started following the emergence of Col-R bacterial strains isolated from humans and animals. These strains can disseminate the mcr genes to other susceptible bacterial strains [8]. Here, we found 143 GNR Col-R phenotypes, of which 88.1% were isolated from animals and 11.9% from human sources.
In our study, Col-R was predominantly observed in strains isolated from poultry faecal samples and in uropathogens isolated from hospitalized patients. Col-R is frequently observed in animal faecal strains, indicating the intestinal colonization of Col-R bacteria in these animals [20]. Col-R has been found and reported in different countries in samples from humans, animals, and the environment [5, 2123]. The predominant Col-R strains found were E. coli (72.2%) and P. mirabilis (15.9%) from animal sources. E. coli (17.6%) and P. mirabilis (58.8%) were also the predominant strains from human sources, although the total number of Col-R strains was not high. These findings corroborate previous studies on Col-R E. coli, which reported rates of 8% from broiler chicken and 37.5% from pig rectal swabs [9, 24].
The treatment of infections caused by ESBL- and AmpC beta-lactamase-producing strains remains a major concern [25, 26]. The emergence of NDM-1 during the past few years has made treating these infections challenging [27]. Polymyxin B and colistin have saved patients’ lives and are considered a vital regimen for treating XDR bacterial infections [12]. Other studies have reported the use of colistin, aminoglycosides, co-trimoxazole, piperacillin-tazobactam, cefoperazone-sulbactam, and tigecycline to treat multidrug-resistant bacteria [18]. Unfortunately, Col-R isolates have emerged worldwide because of the injudicious use of colistin, particularly in veterinary medicine [5].
Here, we analysed the antibacterial activity of aminoglycosides, cephalosporins, fluoroquinolones, carbapenems, beta-lactam, and other combinations against Col-R phenotypes. We found MIC90 values > 12 µg/mL and > 32 µg/mL for the clinical and animal isolates, respectively. It is important to accurately determine the MICs of colistin and the detection of mcr genes provides valuable information to better understand the mechanism of resistance in borderline or resistant cases [19]. In our study, the animal strains were significantly resistant to co-trimoxazole, chloramphenicol, and moxifloxacin, which is consistent with the findings of a Thai study [28]. Interestingly, we noticed lower resistance to carbapenems, piperacillin-tazobactam, cefoperazone-sulbactam, and amikacin. These antibiotics are currently used to combat bacterial infections. Nevertheless, this raises the question of what alternatives would be left if these organisms were found to harbour ESBL, AmpC, NDM-1, and Col-R together. We can speculate that this could lead us into the post-antibiotic era, where we would have no remaining options to treat XDR strains.
The prevalence of the mcr-1 gene has been reported in different animals from 28 countries [29]. The mcr gene is more frequently isolated from animal strains than from human bacterial strains [5]. We found that 98.2% of our bacterial strains from both the animal and human sources contained mcr-1. Two cases (1.8%) of mcr-2 were found, both in P. mirabilis isolated from animal sources, while none of the other mcr variants were found in our study. The presence of plasmid-mediated mcr resistance has been reported in different regions around the world [5, 2123]. A study in Argentina reported 149 (49%) cases of Col-R in E. coli isolated from poultry, and all of them harboured the mcr-1 gene [30]. The coexistence of mcr-1 genes from animal, clinical, and environmental sources has also been reported in several Asian countries [31]. The mcr-1 gene is primarily found in E. coli and K. pneumoniae of human origin, which is in line with our study. The plasmid-mediated Col-R possibly developed in animals and was ultimately transmitted to humans [5, 32, 33].
The four human isolates which harboured mcr-1 in our study were isolated from one septic ICU patient and three patients with urinary tract infections from different wards. We did not find any history of travel or previous use of colistin for these patients. The exact source of mcr-1 could also not be established in an Egyptian and a Polish study; however, some evidence implicated the community exposure of the patients [34, 35]. The detection of mcr-1 and mcr-2 in P. mirabilis in our study may be the first report of mcr genes identified in an intrinsically Col-R organism. This finding may not be significant as far as antibacterial resistance is concerned, and mcr genes are probably not usually searched for in an intrinsically resistant organism. However, this finding indicates the potential danger of the dissemination of mcr-mediated drug resistance to susceptible bacterial strains.
This study had few limitations. One limitation of our study is that we were not able to perform the broth microdilution test on all Col-R strains because of financial and time limitations. Second, we could not establish a definite route for acquiring mcr genes in clinical settings. Moreover, in the statistical comparison of drug resistance, having fewer bacterial isolates in one category could have affected the statistical analysis.

Conclusions

The plasmid-mediated Col-R in GNRs among poultry is a significant emerging problem. The transfer of mcr genes to human bacterial strains represents a danger for patients with XDR infections. The use of colistin to promote growth in animals and increase agriculture production and its indiscriminate use in clinical settings are potential reasons for the dissemination of plasmid-mediated Col-R. We identified considerable animal reservoirs harbouring mcr genes that could be transferred to environmental and human strains, leading to acquired Col-R. A crucial finding of this study was the detection of the mcr-2 gene in intrinsically Col-R P. mirabilis, as it could lead to the uncontrolled spread of mcr genes among animals and human microbiota. The rationale for the use of colistin and its availability for livestock use without a prescription should be critically reviewed to decrease the dissemination of Col-R bacteria in humans and animals.

Acknowledgements

We thank the Higher Education Commission of Pakistan, University of Health Sciences Lahore, University of Veterinary and Animal Sciences Lahore, and The Children’s Hospital, Lahore Pakistan, for their support of this study.
The University of Health Sciences and the Children’s Hospital and Institute of Child Health Lahore, Pakistan, provided ethics approval for the study. The study did not include any interventional human or animal procedures.
Not applicable.

Competing interests

The authors declare that they have no competing interests.
Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creativecommons.​org/​licenses/​by/​4.​0/​. The Creative Commons Public Domain Dedication waiver (http://​creativecommons.​org/​publicdomain/​zero/​1.​0/​) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Literatur
1.
Zurück zum Zitat Shad A. MCR-1 colistin resistance in Escherichia coli wildlife: a continental mini-review. J Drug Metab Toxicol. 2018;9:243.CrossRef Shad A. MCR-1 colistin resistance in Escherichia coli wildlife: a continental mini-review. J Drug Metab Toxicol. 2018;9:243.CrossRef
2.
Zurück zum Zitat Coetzee J, Corcoran C, Prentice E, Moodley M, Mendelson M, Poirel L, et al. Emergence of plasmid-mediated colistin resistance (MCR-1) among Escherichia coli isolated from South African patients. S Afr Med J. 2016;106:35–6.CrossRef Coetzee J, Corcoran C, Prentice E, Moodley M, Mendelson M, Poirel L, et al. Emergence of plasmid-mediated colistin resistance (MCR-1) among Escherichia coli isolated from South African patients. S Afr Med J. 2016;106:35–6.CrossRef
3.
Zurück zum Zitat Liu Y, Liu JH. Monitoring colistin resistance in food animals. An urgent threat. Expert Rev Anti Infect Ther. 2018;16:443–6.CrossRef Liu Y, Liu JH. Monitoring colistin resistance in food animals. An urgent threat. Expert Rev Anti Infect Ther. 2018;16:443–6.CrossRef
4.
Zurück zum Zitat Dandachi I, Chabou S, Daoud Z, Rolain JM. Prevalence and emergence of extended-spectrum cephalosporin-, carbapenem- and colistin-resistant gram negative bacteria of animal origin in the Mediterranean Basin. Front Microbiol. 2018;9:2299.CrossRef Dandachi I, Chabou S, Daoud Z, Rolain JM. Prevalence and emergence of extended-spectrum cephalosporin-, carbapenem- and colistin-resistant gram negative bacteria of animal origin in the Mediterranean Basin. Front Microbiol. 2018;9:2299.CrossRef
5.
Zurück zum Zitat Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R, Spencer J, et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis. 2016;16:161–8.CrossRef Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R, Spencer J, et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis. 2016;16:161–8.CrossRef
6.
Zurück zum Zitat Luo Q, Wang Y, Xiao Y. Prevalence and transmission of mobilized colistin resistance (mcr) gene in bacteria common to animals and humans. Biosaf Health. 2020;2020:71–8.CrossRef Luo Q, Wang Y, Xiao Y. Prevalence and transmission of mobilized colistin resistance (mcr) gene in bacteria common to animals and humans. Biosaf Health. 2020;2020:71–8.CrossRef
7.
Zurück zum Zitat Carretto E, Brovarone F, Nardini P, Russello G, Barbarini D, Pongolini S, et al. Detection of mcr-4 positive Salmonella enterica serovar Typhimurium in clinical isolates of human origin, Italy, October to November 2016. Euro Surveill. 2018;23:17–00821.CrossRef Carretto E, Brovarone F, Nardini P, Russello G, Barbarini D, Pongolini S, et al. Detection of mcr-4 positive Salmonella enterica serovar Typhimurium in clinical isolates of human origin, Italy, October to November 2016. Euro Surveill. 2018;23:17–00821.CrossRef
8.
Zurück zum Zitat Grondahl-Yli-Hannuksela K, Lonnqvist E, Kallonen T, Lindholm L, Jalava J, Rantakokko-Jalava K, et al. The first human report of mobile colistin resistance gene, mcr-1, in Finland. APMIS. 2018;126:413–17.CrossRef Grondahl-Yli-Hannuksela K, Lonnqvist E, Kallonen T, Lindholm L, Jalava J, Rantakokko-Jalava K, et al. The first human report of mobile colistin resistance gene, mcr-1, in Finland. APMIS. 2018;126:413–17.CrossRef
9.
Zurück zum Zitat Lv J, Mohsin M, Lei S, Srinivas S, Wiqar RT, Lin J, et al. Discovery of a mcr-1-bearing plasmid in commensal colistin-resistant Escherichia coli from healthy broilers in Faisalabad, Pakistan. Virulence. 2018;9:994–99.CrossRef Lv J, Mohsin M, Lei S, Srinivas S, Wiqar RT, Lin J, et al. Discovery of a mcr-1-bearing plasmid in commensal colistin-resistant Escherichia coli from healthy broilers in Faisalabad, Pakistan. Virulence. 2018;9:994–99.CrossRef
10.
Zurück zum Zitat Moosavian M, Emam N. The first report of emerging mobilized colistin-resistance (mcr) genes and ERIC-PCR typing in Escherichia coli and Klebsiella pneumoniae clinical isolates in southwest Iran. Infect Drug Resist. 2019;12:1001–10.CrossRef Moosavian M, Emam N. The first report of emerging mobilized colistin-resistance (mcr) genes and ERIC-PCR typing in Escherichia coli and Klebsiella pneumoniae clinical isolates in southwest Iran. Infect Drug Resist. 2019;12:1001–10.CrossRef
11.
Zurück zum Zitat Zhang J, Chen L, Wang J, Butaye P, Huang K, Qiu H, et al. Molecular detection of colistin resistance genes (mcr-1 to mcr-5) in human vaginal swabs. BMC Res Notes. 2018;11:143.CrossRef Zhang J, Chen L, Wang J, Butaye P, Huang K, Qiu H, et al. Molecular detection of colistin resistance genes (mcr-1 to mcr-5) in human vaginal swabs. BMC Res Notes. 2018;11:143.CrossRef
12.
Zurück zum Zitat Olaitan AO, Chabou S, Okdah L, Morand S, Rolain JM. Dissemination of the mcr-1 colistin resistance gene. Lancet Infect Dis. 2016;16:147.CrossRef Olaitan AO, Chabou S, Okdah L, Morand S, Rolain JM. Dissemination of the mcr-1 colistin resistance gene. Lancet Infect Dis. 2016;16:147.CrossRef
13.
Zurück zum Zitat Roer L, Hansen F, Stegger M, Sönksen UW, Hasman H, Hammerum AM. Novel mcr-3 variant, encoding mobile colistin resistance, in an ST131 Escherichia coli isolate from bloodstream infection, Denmark, 2014. Euro Surveill. 2017;22:22846.CrossRef Roer L, Hansen F, Stegger M, Sönksen UW, Hasman H, Hammerum AM. Novel mcr-3 variant, encoding mobile colistin resistance, in an ST131 Escherichia coli isolate from bloodstream infection, Denmark, 2014. Euro Surveill. 2017;22:22846.CrossRef
14.
Zurück zum Zitat Chen K, Chan EW, Xie M, Ye L, Dong N, Chen S. Widespread distribution of mcr-1-bearing bacteria in the ecosystem, 2015 to 2016. Euro Surveill. 2017;22:17–00206.CrossRef Chen K, Chan EW, Xie M, Ye L, Dong N, Chen S. Widespread distribution of mcr-1-bearing bacteria in the ecosystem, 2015 to 2016. Euro Surveill. 2017;22:17–00206.CrossRef
15.
Zurück zum Zitat Javed H, Ejaz H, Zafar A, Rathore AW. Metallo-beta-lactamase producing Escherichia coli and Klebsiella pneumoniae: a rising threat for hospitalized children. J Pak Med Assoc. 2016;66:1068–72.PubMed Javed H, Ejaz H, Zafar A, Rathore AW. Metallo-beta-lactamase producing Escherichia coli and Klebsiella pneumoniae: a rising threat for hospitalized children. J Pak Med Assoc. 2016;66:1068–72.PubMed
16.
Zurück zum Zitat Clinical and Laboratory Standard Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing. 27th edi. CLSI Supplement M100. Wayne: Clinical and Laboratory Standard Institute; 2017. Clinical and Laboratory Standard Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing. 27th edi. CLSI Supplement M100. Wayne: Clinical and Laboratory Standard Institute; 2017.
17.
Zurück zum Zitat Clinical and Laboratory Standard Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing. 28th edi. CLSI Supplement M100. Wayne: Clinical and Laboratory Standard Institute; 2018. Clinical and Laboratory Standard Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing. 28th edi. CLSI Supplement M100. Wayne: Clinical and Laboratory Standard Institute; 2018.
18.
Zurück zum Zitat Ejaz H, Alzahrani B, Hamad MFS, Abosalif KOA, Junaid K, Abdalla AE, et al. Molecular analysis of the antibiotic resistant NDM-1 gene in clinical isolates of Enterobacteriaceae. Clin Lab. 2020;66:409–17.CrossRef Ejaz H, Alzahrani B, Hamad MFS, Abosalif KOA, Junaid K, Abdalla AE, et al. Molecular analysis of the antibiotic resistant NDM-1 gene in clinical isolates of Enterobacteriaceae. Clin Lab. 2020;66:409–17.CrossRef
19.
Zurück zum Zitat Rebelo AR, Bortolaia V, Kjeldgaard JS, Pedersen SK, Leekitcharoenphon P, Hansen IM, et al. Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes. Euro Surveill. 2018;23:17–00672.CrossRef Rebelo AR, Bortolaia V, Kjeldgaard JS, Pedersen SK, Leekitcharoenphon P, Hansen IM, et al. Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes. Euro Surveill. 2018;23:17–00672.CrossRef
20.
Zurück zum Zitat El Garch F, de Jong A, Bertrand X, Hocquet D, Sauget M. mcr-1-like detection in commensal Escherichia coli and Salmonella spp. from food-producing animals at slaughter in Europe. Vet Microbiol. 2018;213:42–6.CrossRef El Garch F, de Jong A, Bertrand X, Hocquet D, Sauget M. mcr-1-like detection in commensal Escherichia coli and Salmonella spp. from food-producing animals at slaughter in Europe. Vet Microbiol. 2018;213:42–6.CrossRef
21.
Zurück zum Zitat Mulvey MR, Mataseje LF, Robertson J, Nash JH, Boerlin P, Toye B, et al. Dissemination of the mcr-1 colistin resistance gene. Lancet Infect Dis. 2016;16:289–90.CrossRef Mulvey MR, Mataseje LF, Robertson J, Nash JH, Boerlin P, Toye B, et al. Dissemination of the mcr-1 colistin resistance gene. Lancet Infect Dis. 2016;16:289–90.CrossRef
22.
Zurück zum Zitat Shen Z, Wang Y, Shen Y, Shen J, Wu C. Early emergence of mcr-1 in Escherichia coli from food-producing animals. Lancet Infect Dis. 2016;16:293.CrossRef Shen Z, Wang Y, Shen Y, Shen J, Wu C. Early emergence of mcr-1 in Escherichia coli from food-producing animals. Lancet Infect Dis. 2016;16:293.CrossRef
23.
Zurück zum Zitat Xavier BB, Lammens C, Ruhal R, Kumar-Singh S, Butaye P, Goossens H, et al. Identification of a novel plasmid-mediated colistin-resistance gene, mcr-2, in Escherichia coli, Belgium, June 2016. Euro Surveill. 2016;21:1–6.CrossRef Xavier BB, Lammens C, Ruhal R, Kumar-Singh S, Butaye P, Goossens H, et al. Identification of a novel plasmid-mediated colistin-resistance gene, mcr-2, in Escherichia coli, Belgium, June 2016. Euro Surveill. 2016;21:1–6.CrossRef
24.
Zurück zum Zitat Stoesser N, Mathers AJ, Moore CE, Day NP, Crook DW. Colistin resistance gene mcr-1 and pHNSHP45 plasmid in human isolates of Escherichia coli and Klebsiella pneumoniae. Lancet Infect Dis. 2016;16:285–6.CrossRef Stoesser N, Mathers AJ, Moore CE, Day NP, Crook DW. Colistin resistance gene mcr-1 and pHNSHP45 plasmid in human isolates of Escherichia coli and Klebsiella pneumoniae. Lancet Infect Dis. 2016;16:285–6.CrossRef
25.
Zurück zum Zitat Ejaz H, Wang N, Wilksch JJ, Page AJ, Cao H, Gujaran S, et al. Phylogenetic Analysis of Klebsiella pneumoniae from Hospitalized Children, Pakistan. Emerg Infect Dis. 2017;23:1872–75.CrossRef Ejaz H, Wang N, Wilksch JJ, Page AJ, Cao H, Gujaran S, et al. Phylogenetic Analysis of Klebsiella pneumoniae from Hospitalized Children, Pakistan. Emerg Infect Dis. 2017;23:1872–75.CrossRef
26.
Zurück zum Zitat Younas S, Ejaz H, Zafar A, Ejaz A, Saleem R, Javed H. AmpC beta-lactamases in Klebsiella pneumoniae: An emerging threat to the paediatric patients. J Pak Med Assoc. 2018;68:893–97.PubMed Younas S, Ejaz H, Zafar A, Ejaz A, Saleem R, Javed H. AmpC beta-lactamases in Klebsiella pneumoniae: An emerging threat to the paediatric patients. J Pak Med Assoc. 2018;68:893–97.PubMed
27.
Zurück zum Zitat Heinz E, Ejaz H, Bartholdson Scott J, Wang N, Gujaran S, Pickard D, et al. Resistance mechanisms and population structure of highly drug resistant Klebsiella in Pakistan during the introduction of the carbapenemase NDM-1. Sci Rep. 2019;9:2392.CrossRef Heinz E, Ejaz H, Bartholdson Scott J, Wang N, Gujaran S, Pickard D, et al. Resistance mechanisms and population structure of highly drug resistant Klebsiella in Pakistan during the introduction of the carbapenemase NDM-1. Sci Rep. 2019;9:2392.CrossRef
28.
Zurück zum Zitat Strom G, Halje M, Karlsson D, Jiwakanon J, Pringle M, Fernstrom LL, et al. Antimicrobial use and antimicrobial susceptibility in Escherichia coli on small- and medium-scale pig farms in north-eastern Thailand. Antimicrob Resist Infect Control. 2017;6:75.CrossRef Strom G, Halje M, Karlsson D, Jiwakanon J, Pringle M, Fernstrom LL, et al. Antimicrobial use and antimicrobial susceptibility in Escherichia coli on small- and medium-scale pig farms in north-eastern Thailand. Antimicrob Resist Infect Control. 2017;6:75.CrossRef
29.
Zurück zum Zitat Al-Tawfiq JA, Laxminarayan R, Mendelson M. How should we respond to the emergence of plasmid-mediated colistin resistance in humans and animals? Int J Infect Dis. 2017;54:77–84.CrossRef Al-Tawfiq JA, Laxminarayan R, Mendelson M. How should we respond to the emergence of plasmid-mediated colistin resistance in humans and animals? Int J Infect Dis. 2017;54:77–84.CrossRef
30.
Zurück zum Zitat Dominguez JE, Faccone D, Tijet N, Gomez S, Corso A, Fernández-Miyakawa ME, et al. Characterization of Escherichia coli carrying mcr-1-plasmids recovered from food animals from Argentina. Front Cell Infect Microbiol. 2019;9:41.CrossRef Dominguez JE, Faccone D, Tijet N, Gomez S, Corso A, Fernández-Miyakawa ME, et al. Characterization of Escherichia coli carrying mcr-1-plasmids recovered from food animals from Argentina. Front Cell Infect Microbiol. 2019;9:41.CrossRef
31.
Zurück zum Zitat Skov RL, Monnet DL. Plasmid-mediated colistin resistance (mcr-1 gene): three months later, the story unfolds. Euro Surveill. 2016;21:30155.CrossRef Skov RL, Monnet DL. Plasmid-mediated colistin resistance (mcr-1 gene): three months later, the story unfolds. Euro Surveill. 2016;21:30155.CrossRef
32.
Zurück zum Zitat Pishnian Z, Haeili M, Feizi A. Prevalence and molecular determinants of colistin resistance among commensal Enterobacteriaceae isolated from poultry in northwest of Iran. Gut Pathog. 2019;11:2.CrossRef Pishnian Z, Haeili M, Feizi A. Prevalence and molecular determinants of colistin resistance among commensal Enterobacteriaceae isolated from poultry in northwest of Iran. Gut Pathog. 2019;11:2.CrossRef
33.
Zurück zum Zitat Johura FT, Tasnim J, Barman I, Biswas SR, Jubyda FT, Sultana M, et al. Colistin-resistant Escherichia coli carrying mcr-1 in food, water, hand rinse, and healthy human gut in Bangladesh. Gut Pathog. 2020;12:5.CrossRef Johura FT, Tasnim J, Barman I, Biswas SR, Jubyda FT, Sultana M, et al. Colistin-resistant Escherichia coli carrying mcr-1 in food, water, hand rinse, and healthy human gut in Bangladesh. Gut Pathog. 2020;12:5.CrossRef
34.
Zurück zum Zitat Elnahriry SS, Khalifa HO, Soliman AM, Ahmed AM, Hussein AM, Shimamoto T, et al. Emergence of plasmid-mediated colistin resistance Gene mcr-1 in a clinical Escherichia coli isolate from Egypt. Antimicrob Agents Chemother. 2016;60:3249–50.CrossRef Elnahriry SS, Khalifa HO, Soliman AM, Ahmed AM, Hussein AM, Shimamoto T, et al. Emergence of plasmid-mediated colistin resistance Gene mcr-1 in a clinical Escherichia coli isolate from Egypt. Antimicrob Agents Chemother. 2016;60:3249–50.CrossRef
35.
Zurück zum Zitat Izdebski R, Baraniak A, Bojarska K, Urbanowicz P, Fiett J, Pomorska-Wesołowska M, et al. Mobile MCR-1-associated resistance to colistin in Poland. J Antimicrob Chemother. 2016;71:2331–3.CrossRef Izdebski R, Baraniak A, Bojarska K, Urbanowicz P, Fiett J, Pomorska-Wesołowska M, et al. Mobile MCR-1-associated resistance to colistin in Poland. J Antimicrob Chemother. 2016;71:2331–3.CrossRef
Metadaten
Titel
Emergence of plasmid-mediated mcr genes from Gram-negative bacteria at the human-animal interface
verfasst von
Humera Javed
Sidrah Saleem
Aizza Zafar
Aamir Ghafoor
Ahmad Bin Shahzad
Hasan Ejaz
Kashaf Junaid
Shah Jahan
Publikationsdatum
01.12.2020
Verlag
BioMed Central
Erschienen in
Gut Pathogens / Ausgabe 1/2020
Elektronische ISSN: 1757-4749
DOI
https://doi.org/10.1186/s13099-020-00392-3

Weitere Artikel der Ausgabe 1/2020

Gut Pathogens 1/2020 Zur Ausgabe

Leitlinien kompakt für die Innere Medizin

Mit medbee Pocketcards sicher entscheiden.

Seit 2022 gehört die medbee GmbH zum Springer Medizin Verlag

Update Innere Medizin

Bestellen Sie unseren Fach-Newsletter und bleiben Sie gut informiert.