- Email: [email protected]
Plasmid-mediated colistin-resistant Escherichia coli detected from 2014 in Norway
Letters to the Editor / International Journal of Antimicrobial Agents 48 (2016) 220–230
David M. Jacobs * Sara DiTursi Department of Pharmacy Practice, University at Buffalo School of Pharmacy and Pharmaceutical Sciences, 346 Abbott Hall, Buffalo, NY 14214, USA Christine Ruh Department of Pharmacy, Erie County Medical Center, 462 Grider Street, Buffalo, NY 14215, USA Rajnikant Sharma Department of Pharmacy Practice, University at Buffalo School of Pharmacy and Pharmaceutical Sciences, 346 Abbott Hall, Buffalo, NY 14214, USA Jonathan Claus Rashmi Banjade Division of Infectious Diseases, Department of Medicine, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14214, USA Gauri G. Rao ** Department of Pharmacy Practice, University at Buffalo School of Pharmacy and Pharmaceutical Sciences, 346 Abbott Hall, Buffalo, NY 14214, USA * Corresponding author. Tel.: +1 716 829 2134; fax: +1 716 829 3109. E-mail address: [email protected] (D.M. Jacobs) ** Corresponding author. Fax: 716-829-3109. E-mail address: [email protected] (G.G. Rao) 10 April 2016 5 June 2016 http://dx.doi.org/10.1016/j.ijantimicag.2016.06.002
Plasmid-mediated colistin-resistant Escherichia coli detected from 2014 in Norway Sir, Since its first description in China, presence of the plasmidmediated colistin resistance gene mcr-1 has been documented on a large scale from the environment, among community-acquired and hospital-acquired pathogens, and from animals worldwide [1]. It has mainly been isolated from Escherichia coli in food animals, but on several plasmid backbones, suggesting an independent dissemination in distant geographic areas. Here we report the first finding of mcr-1 in Norway. In Norway, all presumptive enteropathogenic E. coli isolated from humans are submitted to the National Reference Laboratory for Enteropathogenic Bacteria (NRL) at the Norwegian Institute of Public
227
Health (Oslo, Norway). At the NRL, isolates undergo species verification, virulence gene screening, serotyping and molecular typing. From 2006–2015, the NRL received a total of 4951 E. coli isolates, of which 359 could not be assigned to an E. coli pathotype and were thus classified as non-enteropathogenic E. coli. For surveillance purposes, 100 of these non-enteropathogenic E. coli (10 from each year from 2006–2015) were retrospectively selected for antibiotic susceptibility testing. The aim was to identify extendedspectrum β-lactamase (ESBL)-producers and colistin-resistant strains. Isolate selection was based on the patient’s travel history, covering visitors to all six of the World Health Organization (WHO) geographic regions. Susceptibility to ampicillin (10 μg), cefotaxime (5 μg), ceftazidime (10 μg), meropenem (10 μg), ciprofloxacin (5 μg), nalidixic acid (30 μg), azithromycin (15 μg), gentamicin (10 μg), chloramphenicol (30 μg) and tetracycline (30 μg) was tested by disk diffusion (BD Sensi-Disc™; Becton Dickinson, Sparks, MD) and to colistin using MIC Test Strip (Liofilchem, Abruzzi, Italy). Interpretation of results was according to clinical breakpoints set by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). A single isolate (IP2-01) displayed phenotypic resistance to colistin [minimum inhibitory concentration (MIC) of 4 μg/mL]. It was isolated in 2014 from a traveller returning from India, symptomatic with traveller’s diarrhoea. In addition to colistin, the isolate was resistant to ampicillin, nalidixic acid and ciprofloxacin. However, it was susceptible to cefotaxime and ceftazidime and was accordingly a non-ESBL-producer. To determine the molecular mechanisms conferring colistin resistance in IP2-01, whole-genome sequencing was performed using an Illumina MiSeq platform (2 × 150-bp paired-end reads). The web tool ResFinder (http://www.genomicepidemiology.org/) was used for initial screening of resistance genes and subsequent detection of mcr-1 [2]. The sequence data were further analysed to investigate the genetic context of mcr-1 in IP2-01. The mcr-1 gene was located on an IncI2 plasmid, named pIP2-01, which displayed 99% similarity to pHNSHP45, the plasmid reported to harbour the mcr-1 gene in China (Fig. 1). To assess the ability of pIP2-01 to transfer, microbroth conjugation experiments were performed to the lactose-non-fermenting streptomycin-resistant E. coli recipient strain IP617. Transconjugants were confirmed by growth on selective media and by determining the presence of mcr-1 by PCR. Transconjugants were recovered with a 4- to 8-fold increase in the MIC for colistin compared with the recipient strain, but they remained susceptible to ampicillin, nalidixic acid and ciprofloxacin, i.e. no other resistance was co-transferred with pIP2-01. These observations are consistent with the findings of Liu et al and substantiate concerns regarding the dissemination of colistin resistance [1]. These observations add to the global and national public health concerns related to the dissemination of mobile colistin resistance. Recovery of mcr-1-positive non-enteropathogenic E. coli suggests not only the undetected presence of this gene in the community, but also the possibility of independent dissemination of mcr1-harbouring plasmids in different bacteria and between human hosts. There should be no selection pressure present in the community for mcr-1 and it is likely that the fitness cost associated with
pHNSHP45
pIP2-01 Fig. 1. Comparison between plasmids pIP2-01 and pHNSHP45. The figure shows a comparison between assembled reads, labelled pIP2-01 (bottom blue line), and the first reported mcr-1-containing plasmid pHNSHP45 (top red line). The white lines indicate nucleotide differences between the sequences. The figure was produced with the Artemis Comparison Tool (ACT; http://www.sanger.ac.uk/science/tools/artemis-comparison-tool-act). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
228
Letters to the Editor / International Journal of Antimicrobial Agents 48 (2016) 220–230
mcr-1-mediated colistin resistance would force strains to rapidly eradicate from the normal gut flora. Previous reports on shortterm colonisation of colistin-resistant E. coli are consistent with this [3]. However, travel and the constant influx of new and better adapted strains is a continuous cause of concern. Presently, ESBL resistance or other co-resistance was not linked to pIP2-01, but as an IncI2 plasmid in E. coli our biggest fear is that ESBLs and mcr-1 evolutionary paths converge and together find their way to nosocomial species, possibly causing severe outbreaks in the future. Analyses made in retrospect indicate that mcr-1 alone or in co-location with ESBLs are present, although undetected, in most continents [4]. So far, the earliest report is of an mcr-1-positive Chinese chicken isolate dating from the 1980s [5]. In conclusion, we report the first detection of plasmid-mediated mcr-1 in Norway. This finding adds to the documentation of the global dissemination of plasmid-mediated mcr-1, including spread via normal flora E. coli. The origin of pIP2-01 remains uncertain, although the patient’s travel history substantiates import from India. This observation establishes the presence of mcr-1-harbouring plasmids in Norway since 2014. Acknowledgments The authors are grateful to Kari-Anne Høiklev and Victoria Kristin Wulff Hansen for technical assistance with antibiotic susceptibility testing. Funding: None. Competing interests: None declared. Ethical approval: Not required.
References [1] 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. [2] Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, Lund O, et al. Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother 2012;67:2640–4. [3] Arcilla MS, van Hattem JM, Matamoros S, Melles DC, Penders J, de Jong MD, et al. Dissemination of the mcr-1 colistin resistance gene. Lancet Infect Dis 2016;16:147–9. [4] Skov RL, Monnet DL. Plasmid-mediated colistin resistance (mcr-1 gene): three months later, the story unfolds. Euro Surveill 2016;21. [5] 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.
Margrete Solheim Jon Bohlin Charlotte R. Ulstad Domain for Infection Control and Environmental Health, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo, Norway Jannice Schau Slettemeås Department of Diagnostic Services, Norwegian Veterinary Institute, P.O. Box 750 Sentrum, 0106 Oslo, Norway Umaer Naseer Domain for Infection Control and Environmental Health, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo, Norway European Programme for Public Health Microbiology Training (EUPHEM), European Centre for Disease Ulf R. Dahle Astrid L. Wester * Domain for Infection Control and Environmental Health, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo, Norway
* Corresponding author. Domain for Infection Control and Environmental Health, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo, Norway. Tel.: +47 2107 6227; fax: +47 2235 3605. E-mail address: [email protected] (A.L. Wester) 31 May 2016 5 June 2016 http://dx.doi.org/10.1016/j.ijantimicag.2016.06.001
Exploring non-hospital-related settings in Angola reveals new Acinetobacter reservoirs for blaOXA-23 and blaOXA-58 Sir, Infections caused by carbapenem-resistant Acinetobacter spp. have a significant impact on patient morbidity and mortality. Carbapenem resistance arises mostly by the production of carbapenemhydrolysing class D β-lactamases (CHDLs), which are not limited to Acinetobacter baumannii but also occur in other Acinetobacter spp. increasingly associated with human infections [1]. However, little is known about the presence of CHDLs and their origins and reservoirs among Acinetobacter from non-clinical sources. Furthermore, in Africa, available data regarding Acinetobacter epidemiology and carbapenem resistance are limited to A. baumannii clinical isolates mainly from northern countries [2]. Angola, a sub-Saharan country with close relations with Europe, Asia and South America, is characterised by poor sanitary conditions that increase the population’s vulnerability, currently reflected by the record numbers of malaria and yellow fever (http://www.cdc.gov/yellowfever/ maps/africa.html). In this study, the occurrence, diversity and antimicrobial resistance, in particular to carbapenems, of Acinetobacter spp. from nonclinical sources from Benguela Province (Angola) was evaluated as part of a larger surveillance project on bacterial resistance to clinically relevant antibiotics [3]. From 63 samples [3] collected in different communes of Benguela Province, 29 revealed the presence of Acinetobacter, including rectal swabs from healthy volunteers (8/18), aquatic environments (river, human drinking water, wastewater; 8/20), rectal swabs from healthy animals (calf/cows; 3/10) and their respective environments (water, feed, floor/walls; 7/8) from four different farms, and drinking water from a wild park (3/3) (Supplementary Table S1). The remaining samples (rectal swabs from monkeys and goats) were negative for Acinetobacter. Presumptive Acinetobacter isolates (n = 73) recovered from CHROMagar™ Orientation with/without cefotaxime (1 mg/L), ceftazidime (1 mg/L) or imipenem (1 mg/L) were further identified using a Bruker Biotyper® matrix-assisted laser desorption/ ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) system as well as rpoB/gyrB analysis [1,4]. Ten different Acinetobacter spp. were identified, with MALDI-TOF/MS results being congruent with rpoB/ gyrB, except for A gerneri, A. bereziniae and A. pittii, first identified as Alcaligenes faecalis, A. guillouiae and A. baumannii, respectively. In addition, MALDI-TOF/MS identified three isolates as A. towneri, however rpoB/gyrB revealed only 92–93% nucleotide similarity with this species, being given the provisional name of A. towneri-like sp. A. baumannii was the most predominant species (26/73) (Supplementary Table S1). Interestingly, some particular clones established by pulsed-field gel electrophoresis (PFGE) with the restriction enzyme ApaI (Supplementary Table S1) were detected both from healthy volunteers and water samples (untreated wastewater, river and drinking water for animals), raising the possibility of a water–human cycle of transmission. A. pittii was the second most prevalent species (17/73) and, to our knowledge, was identified for
David M. Jacobs * Sara DiTursi Department of Pharmacy Practice, University at Buffalo School of Pharmacy and Pharmaceutical Sciences, 346 Abbott Hall, Buffalo, NY 14214, USA Christine Ruh Department of Pharmacy, Erie County Medical Center, 462 Grider Street, Buffalo, NY 14215, USA Rajnikant Sharma Department of Pharmacy Practice, University at Buffalo School of Pharmacy and Pharmaceutical Sciences, 346 Abbott Hall, Buffalo, NY 14214, USA Jonathan Claus Rashmi Banjade Division of Infectious Diseases, Department of Medicine, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14214, USA Gauri G. Rao ** Department of Pharmacy Practice, University at Buffalo School of Pharmacy and Pharmaceutical Sciences, 346 Abbott Hall, Buffalo, NY 14214, USA * Corresponding author. Tel.: +1 716 829 2134; fax: +1 716 829 3109. E-mail address: [email protected] (D.M. Jacobs) ** Corresponding author. Fax: 716-829-3109. E-mail address: [email protected] (G.G. Rao) 10 April 2016 5 June 2016 http://dx.doi.org/10.1016/j.ijantimicag.2016.06.002
Plasmid-mediated colistin-resistant Escherichia coli detected from 2014 in Norway Sir, Since its first description in China, presence of the plasmidmediated colistin resistance gene mcr-1 has been documented on a large scale from the environment, among community-acquired and hospital-acquired pathogens, and from animals worldwide [1]. It has mainly been isolated from Escherichia coli in food animals, but on several plasmid backbones, suggesting an independent dissemination in distant geographic areas. Here we report the first finding of mcr-1 in Norway. In Norway, all presumptive enteropathogenic E. coli isolated from humans are submitted to the National Reference Laboratory for Enteropathogenic Bacteria (NRL) at the Norwegian Institute of Public
227
Health (Oslo, Norway). At the NRL, isolates undergo species verification, virulence gene screening, serotyping and molecular typing. From 2006–2015, the NRL received a total of 4951 E. coli isolates, of which 359 could not be assigned to an E. coli pathotype and were thus classified as non-enteropathogenic E. coli. For surveillance purposes, 100 of these non-enteropathogenic E. coli (10 from each year from 2006–2015) were retrospectively selected for antibiotic susceptibility testing. The aim was to identify extendedspectrum β-lactamase (ESBL)-producers and colistin-resistant strains. Isolate selection was based on the patient’s travel history, covering visitors to all six of the World Health Organization (WHO) geographic regions. Susceptibility to ampicillin (10 μg), cefotaxime (5 μg), ceftazidime (10 μg), meropenem (10 μg), ciprofloxacin (5 μg), nalidixic acid (30 μg), azithromycin (15 μg), gentamicin (10 μg), chloramphenicol (30 μg) and tetracycline (30 μg) was tested by disk diffusion (BD Sensi-Disc™; Becton Dickinson, Sparks, MD) and to colistin using MIC Test Strip (Liofilchem, Abruzzi, Italy). Interpretation of results was according to clinical breakpoints set by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). A single isolate (IP2-01) displayed phenotypic resistance to colistin [minimum inhibitory concentration (MIC) of 4 μg/mL]. It was isolated in 2014 from a traveller returning from India, symptomatic with traveller’s diarrhoea. In addition to colistin, the isolate was resistant to ampicillin, nalidixic acid and ciprofloxacin. However, it was susceptible to cefotaxime and ceftazidime and was accordingly a non-ESBL-producer. To determine the molecular mechanisms conferring colistin resistance in IP2-01, whole-genome sequencing was performed using an Illumina MiSeq platform (2 × 150-bp paired-end reads). The web tool ResFinder (http://www.genomicepidemiology.org/) was used for initial screening of resistance genes and subsequent detection of mcr-1 [2]. The sequence data were further analysed to investigate the genetic context of mcr-1 in IP2-01. The mcr-1 gene was located on an IncI2 plasmid, named pIP2-01, which displayed 99% similarity to pHNSHP45, the plasmid reported to harbour the mcr-1 gene in China (Fig. 1). To assess the ability of pIP2-01 to transfer, microbroth conjugation experiments were performed to the lactose-non-fermenting streptomycin-resistant E. coli recipient strain IP617. Transconjugants were confirmed by growth on selective media and by determining the presence of mcr-1 by PCR. Transconjugants were recovered with a 4- to 8-fold increase in the MIC for colistin compared with the recipient strain, but they remained susceptible to ampicillin, nalidixic acid and ciprofloxacin, i.e. no other resistance was co-transferred with pIP2-01. These observations are consistent with the findings of Liu et al and substantiate concerns regarding the dissemination of colistin resistance [1]. These observations add to the global and national public health concerns related to the dissemination of mobile colistin resistance. Recovery of mcr-1-positive non-enteropathogenic E. coli suggests not only the undetected presence of this gene in the community, but also the possibility of independent dissemination of mcr1-harbouring plasmids in different bacteria and between human hosts. There should be no selection pressure present in the community for mcr-1 and it is likely that the fitness cost associated with
pHNSHP45
pIP2-01 Fig. 1. Comparison between plasmids pIP2-01 and pHNSHP45. The figure shows a comparison between assembled reads, labelled pIP2-01 (bottom blue line), and the first reported mcr-1-containing plasmid pHNSHP45 (top red line). The white lines indicate nucleotide differences between the sequences. The figure was produced with the Artemis Comparison Tool (ACT; http://www.sanger.ac.uk/science/tools/artemis-comparison-tool-act). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
228
Letters to the Editor / International Journal of Antimicrobial Agents 48 (2016) 220–230
mcr-1-mediated colistin resistance would force strains to rapidly eradicate from the normal gut flora. Previous reports on shortterm colonisation of colistin-resistant E. coli are consistent with this [3]. However, travel and the constant influx of new and better adapted strains is a continuous cause of concern. Presently, ESBL resistance or other co-resistance was not linked to pIP2-01, but as an IncI2 plasmid in E. coli our biggest fear is that ESBLs and mcr-1 evolutionary paths converge and together find their way to nosocomial species, possibly causing severe outbreaks in the future. Analyses made in retrospect indicate that mcr-1 alone or in co-location with ESBLs are present, although undetected, in most continents [4]. So far, the earliest report is of an mcr-1-positive Chinese chicken isolate dating from the 1980s [5]. In conclusion, we report the first detection of plasmid-mediated mcr-1 in Norway. This finding adds to the documentation of the global dissemination of plasmid-mediated mcr-1, including spread via normal flora E. coli. The origin of pIP2-01 remains uncertain, although the patient’s travel history substantiates import from India. This observation establishes the presence of mcr-1-harbouring plasmids in Norway since 2014. Acknowledgments The authors are grateful to Kari-Anne Høiklev and Victoria Kristin Wulff Hansen for technical assistance with antibiotic susceptibility testing. Funding: None. Competing interests: None declared. Ethical approval: Not required.
References [1] 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. [2] Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, Lund O, et al. Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother 2012;67:2640–4. [3] Arcilla MS, van Hattem JM, Matamoros S, Melles DC, Penders J, de Jong MD, et al. Dissemination of the mcr-1 colistin resistance gene. Lancet Infect Dis 2016;16:147–9. [4] Skov RL, Monnet DL. Plasmid-mediated colistin resistance (mcr-1 gene): three months later, the story unfolds. Euro Surveill 2016;21. [5] 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.
Margrete Solheim Jon Bohlin Charlotte R. Ulstad Domain for Infection Control and Environmental Health, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo, Norway Jannice Schau Slettemeås Department of Diagnostic Services, Norwegian Veterinary Institute, P.O. Box 750 Sentrum, 0106 Oslo, Norway Umaer Naseer Domain for Infection Control and Environmental Health, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo, Norway European Programme for Public Health Microbiology Training (EUPHEM), European Centre for Disease Ulf R. Dahle Astrid L. Wester * Domain for Infection Control and Environmental Health, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo, Norway
* Corresponding author. Domain for Infection Control and Environmental Health, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo, Norway. Tel.: +47 2107 6227; fax: +47 2235 3605. E-mail address: [email protected] (A.L. Wester) 31 May 2016 5 June 2016 http://dx.doi.org/10.1016/j.ijantimicag.2016.06.001
Exploring non-hospital-related settings in Angola reveals new Acinetobacter reservoirs for blaOXA-23 and blaOXA-58 Sir, Infections caused by carbapenem-resistant Acinetobacter spp. have a significant impact on patient morbidity and mortality. Carbapenem resistance arises mostly by the production of carbapenemhydrolysing class D β-lactamases (CHDLs), which are not limited to Acinetobacter baumannii but also occur in other Acinetobacter spp. increasingly associated with human infections [1]. However, little is known about the presence of CHDLs and their origins and reservoirs among Acinetobacter from non-clinical sources. Furthermore, in Africa, available data regarding Acinetobacter epidemiology and carbapenem resistance are limited to A. baumannii clinical isolates mainly from northern countries [2]. Angola, a sub-Saharan country with close relations with Europe, Asia and South America, is characterised by poor sanitary conditions that increase the population’s vulnerability, currently reflected by the record numbers of malaria and yellow fever (http://www.cdc.gov/yellowfever/ maps/africa.html). In this study, the occurrence, diversity and antimicrobial resistance, in particular to carbapenems, of Acinetobacter spp. from nonclinical sources from Benguela Province (Angola) was evaluated as part of a larger surveillance project on bacterial resistance to clinically relevant antibiotics [3]. From 63 samples [3] collected in different communes of Benguela Province, 29 revealed the presence of Acinetobacter, including rectal swabs from healthy volunteers (8/18), aquatic environments (river, human drinking water, wastewater; 8/20), rectal swabs from healthy animals (calf/cows; 3/10) and their respective environments (water, feed, floor/walls; 7/8) from four different farms, and drinking water from a wild park (3/3) (Supplementary Table S1). The remaining samples (rectal swabs from monkeys and goats) were negative for Acinetobacter. Presumptive Acinetobacter isolates (n = 73) recovered from CHROMagar™ Orientation with/without cefotaxime (1 mg/L), ceftazidime (1 mg/L) or imipenem (1 mg/L) were further identified using a Bruker Biotyper® matrix-assisted laser desorption/ ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) system as well as rpoB/gyrB analysis [1,4]. Ten different Acinetobacter spp. were identified, with MALDI-TOF/MS results being congruent with rpoB/ gyrB, except for A gerneri, A. bereziniae and A. pittii, first identified as Alcaligenes faecalis, A. guillouiae and A. baumannii, respectively. In addition, MALDI-TOF/MS identified three isolates as A. towneri, however rpoB/gyrB revealed only 92–93% nucleotide similarity with this species, being given the provisional name of A. towneri-like sp. A. baumannii was the most predominant species (26/73) (Supplementary Table S1). Interestingly, some particular clones established by pulsed-field gel electrophoresis (PFGE) with the restriction enzyme ApaI (Supplementary Table S1) were detected both from healthy volunteers and water samples (untreated wastewater, river and drinking water for animals), raising the possibility of a water–human cycle of transmission. A. pittii was the second most prevalent species (17/73) and, to our knowledge, was identified for