1. Introduction Among those appeared in the recent years, the detection of a plasmid-mediated colistin resistance, driven by the mcr-1 gene represents a serious clinical concern, as colistin was considered so far as a last-resort drug against multidrug resistant (MDR) Gram negative bacteria. Since its original isolation, the mcr-1 gene was detected almost globally in a 0.1-2% of isolates from livestocks and patients, in different Gram-negative bacteria. The emergence of mcr-1 in clinical Enterobacteriaceae isolates appears particularly alarming, as it frequently occurs in MDR strains, further limiting current treatment options for lethal infections sustained by carbapenem-resistant Enterobacteriaceae (CRE). In Italy, mcr-1 driven colistin resistance was firstly reported in a E. coli strain in 2016, although colistin resistance had already been reported previously in CRE from different Italian peripheral laboratories. However, so far mcr-1 gene was essentially searched and detected in infected subjects, but its presence in the microbial population persistently contaminating hospital environment is not known. On the other hand, it is accepted that surface contamination can contribute to the onset of the so-called healthcare-associated infections (HAIs), which are often sustained by MDR or even panDR strains. This study was therefore aimed to check the diffusion of the mcr-1 driven colistin resistance in the hospital environment. 2. Materials and methods The presence of mcr-1 gene was searched in a library of 300 Enterobacteriaceae strains collected from the surfaces of eight Italian hospitals between 2016-2017. Surface samples were collected from three points in hospital rooms (floor, bed footboard and sink), grown in Mac Conkey broth to amplify the Enterobacteriaceae population, then total DNA was extracted by the bacterial suspension and analyzed for mcr-1 gene presence by nested PCR. Each DNA sample was also analyzed by a universal pan-bacterial PCR, as a control of DNA amplification. Plasmid pBAD24::mcr-1 was used as a positive PCR control. Each PCR-positive sample was further culturally isolated on Mac Conkey agar plates, confirmed for mcr-1 gene presence by nested PCR, identified by Maldi-Tof typization, and tested for drug susceptibility by disc diffusion and broth microdilution. 3. Results The results showed that 25/300 (8.3%) of the total Enterobacteriaceae isolated from hospital surfaces harboured mcr-1 gene. Bacterial identification results allowed evidencing that different species harbored mcr-1 gene, including Klebsiella pneumonia and oxytoca, E. coli, Acinetobacter, Enterobacter cloacae and agglomerans, Citrobacter freundii, Pseudomonas aeruginosa and putida isolates, suggesting that this gene is silently spreading to many Gram-negative bacteria responsible for infections in the clinical settings. All mcr-1 carrying isolates were colistin resistant (Col-R) by microdilution test, with a MIC varying from 4 mg/L to over 16 mg/L. In addition, as judged by the results obtained by the disc-diffusion method, all Col-R isolates were also resistant to two or more antibiotics among those effective against Enterobacteriaceae, exhibiting a MDR phenotype. 4. Discussion and Conclusions Our data show for the first time that mcr-1 carrying Enterobacteriaceae can be detected with relatively high frequency (compared to clinical isolates) on hospital surfaces, indicating that this plasmid has the ability to spread, not only in vitro, in key human pathogens. Persistent surface contamination in hospitals might favor colistin resistance spread among Gram-negative bacteria, perhaps helped by the selective pressure exerted by some disinfectants (i.e. chlorhexidine). This might represent a potential reservoir of threatening nosocomial pathogens and favor their diffusion in hospitalized patients. Based on this, we suggest that surveillance for mcr-1 driven colistin resistance might include not only clinical samples but also environmental analyses, and all clinically relevant Gram-negative species, to control and counteract the increase of untreatable infections.
Spread of mcr-1 driven Colistin resistance on surfaces of Italian hospitals
IRENE SOFFRITTI;MARIA D’ACCOLTI;SANTE MAZZACANE;ELISABETTA CASELLI
2018
Abstract
1. Introduction Among those appeared in the recent years, the detection of a plasmid-mediated colistin resistance, driven by the mcr-1 gene represents a serious clinical concern, as colistin was considered so far as a last-resort drug against multidrug resistant (MDR) Gram negative bacteria. Since its original isolation, the mcr-1 gene was detected almost globally in a 0.1-2% of isolates from livestocks and patients, in different Gram-negative bacteria. The emergence of mcr-1 in clinical Enterobacteriaceae isolates appears particularly alarming, as it frequently occurs in MDR strains, further limiting current treatment options for lethal infections sustained by carbapenem-resistant Enterobacteriaceae (CRE). In Italy, mcr-1 driven colistin resistance was firstly reported in a E. coli strain in 2016, although colistin resistance had already been reported previously in CRE from different Italian peripheral laboratories. However, so far mcr-1 gene was essentially searched and detected in infected subjects, but its presence in the microbial population persistently contaminating hospital environment is not known. On the other hand, it is accepted that surface contamination can contribute to the onset of the so-called healthcare-associated infections (HAIs), which are often sustained by MDR or even panDR strains. This study was therefore aimed to check the diffusion of the mcr-1 driven colistin resistance in the hospital environment. 2. Materials and methods The presence of mcr-1 gene was searched in a library of 300 Enterobacteriaceae strains collected from the surfaces of eight Italian hospitals between 2016-2017. Surface samples were collected from three points in hospital rooms (floor, bed footboard and sink), grown in Mac Conkey broth to amplify the Enterobacteriaceae population, then total DNA was extracted by the bacterial suspension and analyzed for mcr-1 gene presence by nested PCR. Each DNA sample was also analyzed by a universal pan-bacterial PCR, as a control of DNA amplification. Plasmid pBAD24::mcr-1 was used as a positive PCR control. Each PCR-positive sample was further culturally isolated on Mac Conkey agar plates, confirmed for mcr-1 gene presence by nested PCR, identified by Maldi-Tof typization, and tested for drug susceptibility by disc diffusion and broth microdilution. 3. Results The results showed that 25/300 (8.3%) of the total Enterobacteriaceae isolated from hospital surfaces harboured mcr-1 gene. Bacterial identification results allowed evidencing that different species harbored mcr-1 gene, including Klebsiella pneumonia and oxytoca, E. coli, Acinetobacter, Enterobacter cloacae and agglomerans, Citrobacter freundii, Pseudomonas aeruginosa and putida isolates, suggesting that this gene is silently spreading to many Gram-negative bacteria responsible for infections in the clinical settings. All mcr-1 carrying isolates were colistin resistant (Col-R) by microdilution test, with a MIC varying from 4 mg/L to over 16 mg/L. In addition, as judged by the results obtained by the disc-diffusion method, all Col-R isolates were also resistant to two or more antibiotics among those effective against Enterobacteriaceae, exhibiting a MDR phenotype. 4. Discussion and Conclusions Our data show for the first time that mcr-1 carrying Enterobacteriaceae can be detected with relatively high frequency (compared to clinical isolates) on hospital surfaces, indicating that this plasmid has the ability to spread, not only in vitro, in key human pathogens. Persistent surface contamination in hospitals might favor colistin resistance spread among Gram-negative bacteria, perhaps helped by the selective pressure exerted by some disinfectants (i.e. chlorhexidine). This might represent a potential reservoir of threatening nosocomial pathogens and favor their diffusion in hospitalized patients. Based on this, we suggest that surveillance for mcr-1 driven colistin resistance might include not only clinical samples but also environmental analyses, and all clinically relevant Gram-negative species, to control and counteract the increase of untreatable infections.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
