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Objectives: For low-income countries, data regarding the intestinal colonization with extended-spectrum cephalosporin-resistant (ESC-R) and colistin-resistant (CST-R) Enterobacteriaceae in the community are still scarce. Here, we investigated this phenomenon by analysing hotel employees in Zanzibar., Methods: During June to July 2018, rectal swabs from 59 volunteers were screened implementing selective enrichments and agar plates. Species identification was achieved using MALDI-TOF MS. Strains were characterized using microdilution panels (MICs), microarray, PCRs for mcr-1/-8, repetitive extragenic palindromic-PCR (rep-PCR) and WGS., Results: Colonization prevalence with ESC-R-, CST-R- and mcr-1-positive Enterobacteriaceae were 91.5%, 66.1% and 18.6%, respectively (average: 2.2 strains per volunteer). Overall, 55 ESC-R Escherichia coli (3 also CST-R), 33 ESC-R Klebsiella pneumoniae (1 also CST-R), 17 CST-R E. coli and 21 CST-R K. pneumoniae were collected. The following main resistance genes were found: ESC-R E. coli (blaCTX-M-15-like, 51.0%), ESC-R K. pneumoniae (blaCTX-M-9-like, 42.9%), CST-R E. coli (mcr-1, 55%) and CST-R K. pneumoniae (D150G substitution in PhoQ). ESBL-producing E. coli mainly belonged to ST361, ST636 and ST131, whereas all those that were mcr-1 positive belonged to ST46 that carried mcr-1 in a 33 kb IncX4 plasmid. ESBL-producing K. pneumoniae mainly belonged to ST17, ST1741 and ST101, whereas CST-R strains belonged to ST11., Conclusions: We recorded remarkably high colonization prevalence with ESC-R and/or CST-R Enterobacteriaceae in hotel staff. Further research in the local environment, livestock and food chain is warranted to understand this phenomenon. Moreover, as Zanzibar is a frequent holiday destination, attention should be paid to the risk of international travellers becoming colonized and thereby importing life-threatening pathogens into their low-prevalence countries., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The emergence of the colistin-resistant (COL-R) Enterobacteriaceae represents a worrying health issue. However, only a portion of these strains may carry the plasmid-mediated mcr colistin resistance genes. We evaluated the ability of both ethylenediaminetetraacetic acid (EDTA)-based and dipicolinic acid (DPA)-based broth microdilution (BMD) tests to detect mcr-1 to mcr-5 producers. Of 92 Enterobacteriaceae (85 COL-R), 44 mcr- positive strains (39 Escherichia coli , 3 Klebsiella pneumoniae , and 2 Salmonella spp.) were tested. EDTA (100 μg/mL) was tested in Mueller-Hinton broth (MHB), whereas the DPA (900 μg/mL) was used in cation-adjusted MHB. Results were categorized as positive if in presence of chelator strains exhibited ≥3 two fold MIC decrease compared to the COL MIC alone. The EDTA-based BMD assay detected 41 mcr -positive strains, but 22 false-positive strains (including 12 E. coli and 4 K. pneumoniae ) were recorded (sensitivity [SN], 93.2%; specificity [SP], 54.2%). The DPA-based BMD assay detected 37 mcr- positive strains, with 7 false-negative (2 E. coli , 3 K. pneumoniae , 2 Salmonella spp.) strains (SN, 84.1%; SP, 100%). Overall, the EDTA-based BMD assay is not accurate to detect mcr producers, whereas the DPA-based BMD test ("colistin-MAC test") demonstrated good accuracy, but only when implemented for E. coli strains (SN, 94.9%; SP, 100%). With the aim to prevent the dissemination of mcr -possessing E. coli strains, the COL-MAC test could be implemented by clinical laboratories that are unable to perform molecular tests. Moreover, this assay could be applied to screen large collections of isolates to reveal the expression of new mcr -like genes not yet targeted by the current molecular assays.

We evaluated several EDTA-based combined-disk tests to detect 25 mcr producers among 48 Enterobacteriaceae. Colistin disks plus EDTA (292/584 μg) on MH and CAMH agar were used. Results were positive if with chelator there was an inhibition zone increase ≥3 mm compared to colistin alone. All tests resulted unreliable (sensitivity ≤68%)., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We characterized the genetic environment of mcr-1 in colistin-resistant Escherichia coli strains isolated in Switzerland during 2014 to 2016 from humans ( n = 3) and chicken meat ( n = 6). Whole-genome and plasmid sequencing identified the mcr-1 gene integrated in IncX4 (of which, one strain carried the mcr-1.2 variant), IncI2, IncHI2, and novel IncK2 plasmids (overall, n = 7), as well as in the bacterial chromosome ( n = 2) in single or duplicate copies. Our study supports the easy mobilization of mcr-1 across diverse genetic locations., (Copyright © 2017 American Society for Microbiology.)

Objectives: The aim of this study was to design a rapid and sensitive real-time PCR (rt-PCR) method for colistin resistance mcr-1 gene detection in human faecal samples., Methods: Stools (n=88) from 36 volunteers were analysed. To isolate mcr-1-producing Enterobacteriaceae, samples were enriched overnight in Luria-Bertani (LB) broth containing 2mg/L colistin and were then plated on selective agar plates with 4mg/L colistin. A SYBR ® Green-based rt-PCR targeting mcr-1 was then designed. For method validation and to establish the limit of detection (LOD), total DNA was extracted from mcr-1-negative and mcr-1-positive Escherichia coli. rt-PCR was also performed with DNA extracted from 88 native stools and after enriching them in LB broth containing colistin., Results: Based on the culture approach, three unique volunteers resulted colonised with mcr-1-harboring E. coli strains. For culture isolates, rt-PCR exhibited a LOD of 10 genomic copies/reaction, with both sensitivity and specificity of 100%. Nevertheless, when testing native stools, only two of the three mcr-1-positive specimens were detected. However, after enrichment in LB broth containing colistin, the rt-PCR was strongly positive for all culture-positive samples. The average cycle threshold was 22, granting rapid and confident detection of positive specimens within 30 cycles. No false positives were observed for the remaining 85 culture-negative specimens., Conclusions: A rapid rt-PCR for detection of mcr-1 from stool specimens was developed. The detection rate was increased by testing selective broth enrichments. This approach also has the advantage of concomitant isolation of mcr-1-harboring strains for further antimicrobial susceptibility and genetic testing., (Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

Stool samples from 38 travelers returning from India were screened for extended-spectrum cephalosporin- and carbapenem-resistant Enterobacteriaceae implementing standard selective plates. Twenty-six (76.3%) people were colonized with CTX-M or DHA producers, but none of the strains was colistin resistant and/or mcr-1 positive. Nevertheless, using overnight enrichment and CHROMagar Orientation plates supplemented with colistin, four people (10.5%) were found to be colonized with colistin-resistant Escherichia coli One cephalosporin-susceptible sequence type 10 (ST10) strain carried a 4,211-bp ISApl1-mcr-1-ISApl1 element in an IncHI2 plasmid backbone., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)