Odette Joëlle Bernasconi

Travelers Can Import Colistin-Resistant Enterobacteriaceae, Including Those Possessing the Plasmid-Mediated mcr-1 Gene

ABSTRACT 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.

A SYBR® Green-based real-time PCR method for improved detection of mcr-1-mediated colistin resistance in human stool samples

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.

Heterogeneous Genetic Location of mcr-1 in Colistin-Resistant Escherichia coli Isolates from Humans and Retail Chicken Meat in Switzerland: Emergence of mcr-1-Carrying IncK2 Plasmids

ABSTRACT 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.

Evaluation of a New Commercial Microarray Platform for the Simultaneous Detection of β-Lactamase and mcr-1 and mcr-2 Genes in Enterobacteriaceae

The worldwide prevalence of infections due to Gram-negative bacteria coresistant to extended-spectrum cephalosporins and carbapenems is worrisome. For these organisms, polymyxins are considered the last-resort antibacterials. Thus, the recent emergence of the plasmid-mediated colistin resistance

In vitro activity of three commercial bacteriophage cocktails against multidrug-resistant Escherichia coli and Proteus spp. strains of human and non-human origin

OBJECTIVES Bacteriophages may represent a therapeutic alternative to treat infections caused by multidrug-resistant (MDR) pathogens. However, studies analysing their activity against MDR Enterobacteriaceae are limited. METHODS The in vitro lytic activity of three commercial bacteriophage cocktails (PYO, INTESTI and Septaphage) was evaluated against 70 Escherichia coli and 31 Proteus spp. of human and non-human origin. Isolates were characterised by phenotypic and genotypic methods and included 82 MDR strains [44 extended-spectrum-β-lactamase (ESBL)-producers (18 CTX-M-15-like, including ST131/ST648 E. coli); 27 plasmid-mediated AmpC β-lactamase (pAmpC)-producers (23 CMY-2-like, including ST131 E. coli); 3 ESBL+pAmpC-producers; and 8 carbapenemase-producers]. Phage susceptibility was determined by the spot test. RESULTS E. coli susceptibility to PYO, INTESTI and Septaphage was 61%, 67% and 9%, whereas that of Proteus spp. was 29%, 39% and 19%, respectively. For the subgroup of ESBL-producing E. coli/Proteus spp., the following susceptibility rates were recorded: PYO, 57%; INTESTI, 59%; and Septaphage, 11%. With regard to pAmpC-producers, 59%, 70% and 11% were susceptible to PYO, INTESTI and Septaphage, respectively. Five of eight carbapenemase-producers and three of four colistin-resistant E. coli were susceptible to PYO and INTESTI. CONCLUSIONS This is the first study analysing the activity of the above three cocktails against well-characterised MDR E. coli and Proteus spp. The overall narrow spectrum of activity observed could be related to the absence of specific bacteriophages targeting these contemporary MDR strains that are spreading in different settings. Therefore, bacteriophages targeting emerging MDR pathogens need to be isolated and integrated in such biopreparations.

Intestinal colonisation with extended-spectrum cephalosporin-resistant Escherichia coli in Swiss pets: molecular features, risk factors and transmission with owners

The overlap of molecular features of extended-spectrum cephalosporin-resistant Enterobacteriaceae (ESC-R-Ent) recovered from humans and animals indicates a possible interaction between these two niches. Frequently these strains carry CTX-M1/-14/-15 and CMY-2 β-lactamases in IncF or IncI1 plasmids in different sequence types (STs), including the high-risk clones ST131, ST648 and those belonging to clonal complex 10 (CC10) [1]. In this study, we aimed to assess the role of the intestine of healthy pets in shaping the epidemiology of ESC-R-Ent, the prevalence of intestinal colonisation by these organisms, and potential lifestyle factors associated with this phenomenon. Thus, between July 2013 and May 2016, 81 pets (44 dogs and 37 cats) and their owners (n = 72) from 66 different Swiss households were enrolled in the analysis. Owners were instructed to collect stools into sterile containers. A questionnaire about their pets was also completed. Stools were enriched overnight in Luria–Bertani broth in different conditions to detect both ESC-R-Ent and colistin-resistant (COL-R) strains [2,3]. Species identification was determined by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) (Bruker Daltonics, Leipzig, Germany). Minimum inhibitory concentrations (MICs) were determined using SensititreTM GNX2F microdilution plates (Trek Diagnostic Systems, East Grinstead, UK) and were interpreted according to 2016 European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints (http://www.eucast.org/clinical_breakpoints/). β-Lactamase genes (bla) were identified using the CT103XL microarray (Check-Points, Wageningen, The Netherlands) with subsequent PCR and sequencing. Clonality was assessed by multilocus sequence typing (MLST) (http://mlst.ucc.ie/mlst/dbs/Ecoli) and phylogenetic group determination [2,3]. A PCR-based replicon typing (PBRT) kit (Diatheva, Fano, PU, Italy) was used to type plasmid incompatibility groups. Statistical analysis was performed to compare lifestyle factors of colonised and non-colonised pets using GraphPad Prism v.7.0 (GraphPad Software Inc., La Jolla, CA). Continuous variables were analysed using unpaired t-test, whereas categorical variables were analysed with Fisher’s exact test. Odds ratios (ORs) and 95% confidence intervals (CIs) were computed for categorical variables. Among the total pets analysed, 4 cats and 3 dogs (overall prevalence, 8.6%; 95% CI 4.3–16.9%) were colonisedwith ESC-R Escherichia coli, whereas COL-R Enterobacteriaceae were not detected. Compared with previous reports, these data indicate that the prevalence is steadily increasing in Switzerland, although it is still low compared with other countries [1]. Reports on cats are scarce, with 4% reported in Kenya, 21% in Tunisia and 0% in The Netherlands [1]. Eight ESC-R E. coli isolates were recovered from seven pets; one dog was co-colonised with two different strains (Table 1). None of the isolates were resistant to carbapenems, and resistance to nonβ-lactams was low with ≤25% of the isolates non-susceptible to trimethoprim/sulfamethoxazole, aminoglycosides, quinolones and tetracyclines. Interestingly, these observations are in contrast to previous Swiss and international studies where higher resistance rates were observed [1,4]. ESC-R-Ent isolates from pets produced CTX-M-type or CMY-2 β-lactamases and carried IncF and/or IncI1 plasmids (Table 1), as reported in other international studies [1,4]. The majority of isolates belonged to phylogenetic group B1 (3/8; 37.5%) that is frequently found in the gut of animals [2]. STs were highly diverse, including CC10, CC155 and CC73, which are also found in pets and wildlife in many geographic regions [1,5]. In addition, some of these STs

Deciphering the Complete Deletion of the MgrB Locus in an Unusual Colistin-Resistant Klebsiella pneumoniae Colonizing the Gut of a Traveler Returning from India

most colistin-resistant (Col-R) Klebsiella pneumoniae strains possess alterations of the two-component systems PhoP/Q and PmrA/B. These systems respond to environmental stimuli increasing the expression of the operon pmrHFIJKLM whose products are responsible for lipid A modifications leading to decreased affinity for polymyxins. This process is regulated by a negative feedback of the mgrB gene that encodes for a small protein repressing the PhoP/Q system. Thus, inactivation of mgrB leads to polymyxin resistance.

In Vitro Activity of 3 Commercial Bacteriophage Cocktails Against Salmonella and Shigella spp. Isolates of Human Origin

Background: Salmonella and Shigella spp. are 2 of the most frequent and deadly enteric bacterial pathogens recorded worldwide. In developing countries Salmonella infections are responsible for many deaths annually and these mortality rates are prone to increase due to the emergence of resistance to antibiotics. In this overall scenario new alternative therapeutic approaches are needed. Methods: For the first time, we investigated the activity of 3 commercial bacteriophage cocktails (INTESTI, Septaphage, PYO) against a collection of contemporary Salmonella spp. (n = 30) and Shigella spp. (n = 20) strains isolated in Switzerland. Phage susceptibility was determined by implementing the spot test. Results: The overall susceptibility of Salmonella spp. to INTESTI and Septaphage was 87% and 77%, respectively. With regard to Shigella spp., the overall susceptibility to INTESTI and Septaphage was 95% and 55%, respectively. PYO was observed to be active against only 10% of Salmonella spp. but against 95% of Shigella spp. Conclusions: Our results seem promising, especially for the INTESTI biopreparation against Salmonella enterica infections. Nevertheless, such speculation should be supported by further in vivo studies to confirm efficacy and safety of the cocktails. We also emphasize the importance of large in vitro screening analyses aimed to assess the activity of such biopreparations against contemporary multidrug-resistant strains that are emerging worldwide.

The EDTA-based disk-combination tests are unreliable for the detection of MCR-mediated colistin-resistance in Enterobacteriaceae

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%).

Letter to the EditorDeciphering the complete deletion of the mgrb locus in an unusual colistin-resistant klebsiella pneumoniae colonizing the gut of a traveler returning from india

most colistin-resistant (Col-R) Klebsiella pneumoniae strains possess alterations of the two-component systems PhoP/Q and PmrA/B. These systems respond to environmental stimuli increasing the expression of the operon pmrHFIJKLM whose products are responsible for lipid A modifications leading to decreased affinity for polymyxins. This process is regulated by a negative feedback of the mgrB gene that encodes for a small protein repressing the PhoP/Q system. Thus, inactivation of mgrB leads to polymyxin resistance.