Intestinal colonization with multidrug-resistant Enterobacterales: screening, epidemiology, clinical impact, and strategies to decolonize carriers.

The clinical impact of infections due to extended-spectrum ß-lactamase (ESBL)- and/or carbapenemase-producing Enterobacterales (Ent) has reached dramatic levels worldwide. Infections due to these multidrug-resistant (MDR) pathogens-especially Escherichia coli and Klebsiella pneumoniae-may originate from a prior asymptomatic intestinal colonization that could also favor transmission to other subjects. It is therefore desirable that gut carriers are rapidly identified to try preventing both the occurrence of serious endogenous infections and potential transmission. Together with the infection prevention and control countermeasures, any strategy capable of effectively eradicating the MDR-Ent from the intestinal tract would be desirable. In this narrative review, we present a summary of the different aspects linked to the intestinal colonization due to MDR-Ent. In particular, culture- and molecular-based screening techniques to identify carriers, data on prevalence and risk factors in different populations, clinical impact, length of colonization, and contribution to transmission in various settings will be overviewed. We will also discuss the standard strategies (selective digestive decontamination, fecal microbiota transplant) and those still in development (bacteriophages, probiotics, microcins, and CRISPR-Cas-based) that might be used to decolonize MDR-Ent carriers.

Travellers returning from the island of Zanzibar colonized with MDR Escherichia coli strains: assessing the impact of local people and other sources.

OBJECTIVES: Many travellers to low-income countries return home colonized at the intestinal level with extended-spectrum cephalosporin-resistant (ESC-R) and/or colistin-resistant (CST-R) Escherichia coli (Ec) strains. However, nothing is known about the local sources responsible for the transmission of these pathogens to the travellers. METHODS: We compared the ESC-R- and CST-R-Ec strains found in the pre- (n = 23) and post-trip (n = 37) rectal swabs of 37 travellers from Switzerland to Zanzibar with those (i) contemporarily isolated from local people, poultry, retailed chicken meat (n = 31), and (ii) from other sources studied in the recent past (n = 47). WGS and core-genome analyses were implemented. RESULTS: Twenty-four travellers returned colonized with ESC-R- (n = 29) and/or CST-R- (n = 8) Ec strains. Almost all ESC-R-Ec were CTX-M-15 producers and belonged to heterogeneous STs/core-genome STs (cgSTs), while mcr-positive strains were not found. Based on the strains' STs/cgSTs, only 20 subjects were colonized with ESC-R- and/or CST-R-Ec that were not present in their gut before the journey. Single nucleotide variant (SNV) analysis showed that three of these 20 travellers carried ESC-R-Ec (ST3489, ST3580, ST361) identical (0-20 SNVs) to those found in local people, chicken meat, or poultry. Three further subjects carried ESC-R-Ec (ST394, ST648, ST5173) identical or highly related (15-55 SNVs) to those previously reported in local people, fish, or water. CONCLUSIONS: This is the first known study comparing the ESC-R- and/or CST-R-Ec strains obtained from travellers and local sources using solid molecular methods. We showed that for at least one-third of the returning travellers the acquired antibiotic-resistant Ec had a corresponding strain among resident people, food, animal and/or environmental sources.

Rapid Increase of CTX-M-Producing Shigella sonnei Isolates in Switzerland Due to Spread of Common Plasmids and International Clones.

The Swiss Centre for Antibiotic Resistance (ANRESIS) has recently noted an increase of extended-spectrum cephalosporin-resistant (ESC-R) Shigella sonnei isolates nationwide (3.8% in 2016 versus 37.5% in 2019). To understand this phenomenon, we analyzed 25 representative isolates (of which 14 were ESC-R) collected in Switzerland during 2016 to 2019. Whole-genome sequencing was achieved using both the Illumina and the Nanopore platforms. Both ESC-R and extended-spectrum cephalosporin-susceptible isolates belonged to sequence type 152 (ST152). The ESC-R isolates carried blaCTX-M-3 in IncI1-pST57 (n = 5), blaCTX-M-15 in IncFII (F2:A-:B-) (n = 5), blaCTX-M-15 in IncI1-pST16, and blaCTX-M-27, blaCTX-M-55, or blaCTX-M-134 in other IncFII plasmids (n = 1 each). Plasmids having the same bla and Inc group exhibited high degrees of genetic identity to each other but also to plasmids previously reported in other Enterobacterales Core-genome analysis showed that there were 4 main clusters, each of which included strains that differed by <58 single nucleotide variants (SNVs) and that consisted of both blaCTX-M-positive and blaCTX-M-negative isolates. Moreover, most isolates belonging to the same cluster shared an identical core-genome sequence type (cgST). For instance, cluster 1 included 4 isolates of cgST113036, of which only 3 harbored the IncI1-pST57 blaCTX-M-3-positive plasmid. The 25 S. sonnei isolates were also subjected to phylogenetic comparison with deposited international strains. As a result, matching isolates (isolates that had the same cgST and that differed by <8 SNVs) have been reported in the United Kingdom, the United States, France, and the Netherlands. Overall, our results suggest that some common S. sonnei clusters can spread between continents and can be imported into other nations after international trips. Such clusters include, in part, isolates that do not possess blaESBL-harboring plasmids, indicating their tendency to acquire them from other Enterobacterales.

First Clinical Case of In Vivo Acquisition of DHA-1 Plasmid-Mediated AmpC in a Salmonella enterica subsp. enterica Isolate.

A pan-susceptible Salmonella enterica serovar Worthington isolate was detected in the stool of a man returning from Sri Lanka. Under ceftriaxone treatment, a third-generation cephalosporin (3GC)-resistant Salmonella Worthington was isolated after 8 days. Molecular analyses indicated that the two isolates were identical. However, the latter strain acquired a blaDHA-1-carrying IncFII plasmid probably from a Citrobacter amalonaticus isolate colonizing the gut. This is the first report of in vivo acquisition of plasmid-mediated resistance to 3GCs in S. enterica.

Polyclonal gut colonization with extended-spectrum cephalosporin- and/or colistin-resistant Enterobacteriaceae: a normal status for hotel employees on the island of Zanzibar, Tanzania.

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.

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.

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.

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

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.

Investigating the use of bacteriophages as a new decolonization strategy for intestinal carriage of CTX-M-15-producing ST131 Escherichia coli: An in vitro continuous culture system model.

OBJECTIVES: We investigated the use of bacteriophages as a strategy to decolonize intestinal carriers of multidrug-resistant Escherichia coli. METHODS: A fermentor was used as a continuous culture system for 48h. Two different pools of faeces (studies I and II) obtained from volunteers were spiked with a CTX-M-15-producing ST131 E. coli (strain 4901.28) susceptible to bacteriophages and challenged with three doses of INTESTI Bacteriophage cocktail administered at 2, 6 and 10h after the inoculum. Bacterial typing was performed by implementing microdilution panels, spot test, rep-PCR and whole-genome sequencing (including cgMLST and single-nucleotide variant analysis) obtained using Nanopore and Illumina platforms. RESULTS: In study I, bacteriophages decreased the numbers of 4901.28 dramatically (≤101CFU/mL after 6h). In contrast, during study II, a phage-resistant mutant of 4901.28 persisted in the continuous culture (104CFU/mL at 48h). Whole-genome sequencing revealed the presence of two additional plasmids in the mutant as well as 11 single-nucleotide variants, including one chromosomal in a glycosyltransferase family 2 protein that is responsible for the transfer of sugars to polysaccharides and lipids. In both studies, the commensal E. coli population remained unchanged by the phage treatment maintaining itself at 108CFU/mL. CONCLUSIONS: Our data indicates that bacteriophage cocktails may be implemented to decolonize some intestinal carriers. However, the individual microbiota composition may have an impact on the development of phage resistance. Mechanisms underlying this phenomenon are likely to be various and complex. Further in vivo studies and protein expression experiments are needed to confirm our observations and hypotheses.

Poor infection prevention and control standards are associated with environmental contamination with carbapenemase-producing Enterobacterales and other multidrug-resistant bacteria in Swiss companion animal clinics.

BACKGROUND: Intensive medical care in companion animal clinics could pose a risk for the selection and dissemination of multidrug-resistant organisms (MDROs). Infection prevention and control (IPC) concepts are key measures to reduce the spread of MDROs, but data on IPC standards in companion animal clinics is sparse. The study assessed IPC standards in seven companion animal clinics and practices in Switzerland by structured IPC audits and combined results with environmental MDRO contamination and MDRO carriage of the personnel. METHODS: IPC audits were held between August 2018 and January 2019. The observations in 34 IPC areas were scored based on predefined criteria (not fulfilled/partially fulfilled/fulfilled = score 0/1/2). Environmental swabs and nasal and stool samples from veterinary personnel were tested for methicillin-resistant (MR) staphylococci and macrococci and for colistin-resistant, extended-spectrum ß-lactamase- and carbapenemase-producing (CP) Enterobacterales (CPE). Species was identified by MALDI-TOF MS, antimicrobial resistance determined by microdilution and ß-lactam resistance gene detection, and genetic relatedness assessed by REP-/ERIC-PCR and multilocus sequence typing. RESULTS: Of a maximum total IPC score of 68, the institutions reached a median (range) score of 33 (19-55). MDROs were detected in median (range) 8.2% (0-33.3%) of the sampling sites. Clinics with low IPC standards showed extensive environmental contamination, i.e. of intensive care units, consultation rooms and utensils. CPE were detected in two clinics; one of them showed extensive contamination with CP Klebsiella pneumoniae (ST11, blaOXA-48) and MR Staphylococcus pseudintermedius (ST551, mecA). Despite low IPC scores, environmental contamination with MDROs was low in primary opinion practices. Three employees were colonized with Escherichia coli ST131 (blaCTX-M-15, blaCTX-M-27, blaCTX-M-14). Two employees carried CP E. coli closely related to environmental (ST410, blaOXA-181) and patient-derived isolates (ST167, blaNDM-5). MR Staphylococcus aureus (ST225, mecA) and MR S. pseudintermedius (ST551, mecA) of the same sequence types and with similar resistance profiles were found in employees and the environment in two clinics. CONCLUSIONS: The study indicates that IPC standards in companion animal clinics are variable and that insufficient IPC standards could contribute to the evolution of MDROs which can be transferred between the environment and working personnel. The implementation of IPC concepts in companion animal clinics should urgently be promoted.

Evaluation of EDTA- and DPA-Based Microdilution Phenotypic Tests for the Detection of MCR-Mediated Colistin Resistance in Enterobacteriaceae.

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.

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.

Whole-Genome Sequence of the First Extended-Spectrum ß-Lactamase-Producing Strain of Salmonella enterica subsp. enterica Serovar Napoli.

We report here the whole-genome sequence of the first extended-spectrum ß-lactamase (ESBL)-producing strain of Salmonella enterica subsp. enterica serovar Napoli, LC0541/17, isolated from the stools of an ambulatory pediatric patient in northern Italy. The strain was of sequence type 474 (ST474) and possessed a 90-kb IncI1 ST49 plasmid carrying the bla CTX-M-1 ESBL gene.

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%).

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-ß-lactamase (ESBL)-producers (18 CTX-M-15-like, including ST131/ST648 E. coli); 27 plasmid-mediated AmpC ß-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.

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.