MAST® D72C test: a novel option for ESBL, AmpC and carbapenemase detection.

PURPOSE: The MAST® D72C test is a phenotypical test which can detect ESBL and AmpC production in Enterobacterales. It can also identify the suspected presence of carbapenemase. The aim of the present study was to assess the sensitivity and specificity of this test and to discuss its usefulness in laboratories, especially those that use only an automated AST system. METHOD: The performance of the MAST® D72C test was assessed against a collection of 119 non-redundant Enterobacterales isolates characterized for their content in ß-lactamases, and compared with that of the reference double disk synergy test. ß-lactamase content was established from phenotypic and genotypic analyses to collect a broad diversity of resistance mechanisms and bacterial strains, including 30 ESBL-producing strains, 32 strains overproducing chromosomal AmpC, 10 strains producing plasmid-encoded AmpC, 12 carbapenemase-producing strains, 13 strains combining the production of several ß-lactamases, and 22 strains that produced other ß-lactamases. RESULTS: The sensitivity and specificity for ESBL-detection were comparable with those of the synergy test, 75 versus 72.5%, and 94.9 versus 93.7%, respectively. The sensitivity and specificity for AmpC-detection were 71.7% and 100%, respectively, and sensitivity reached 78.7% if we excluded carbapenem-resistant isolates. Carbapenemase-detection sensitivity was 90%. CONCLUSION: These results show that the MAST® D72C test can be a useful tool for the detection of ESBL- and AmpC-production in clinical laboratories.

Long-term trends of salinity in coastal wetlands: Effects of climate, extreme weather events, and sea water level.

Coastal freshwater ecosystems play major roles as reservoirs of biodiversity and provide many ecosystem services and protection from extreme weather events. While they are of particular importance worldwide, they are affected by a large variety of anthropogenic threats, among which salinization has been less studied, particularly regarding large temporal and spatial data sets based on real case scenarios, while salinity can impact biodiversity and ecosystem functioning. In this study, we investigated the variations of salinity across long-term (1996-2020) and seasonal (monthly records) temporal scales and spatial (varying distance to the coastline) scales in water bodies of two typical temperate coastal wetlands situated on the Atlantic coast of France. We complemented our analyses with models of sea water levels computed at both sites across 2000-2020. Our detailed data set allowed for highlighting that salinity in ponds varied seasonally (higher during summer, due to decreased precipitation and higher temperature), but also spatially (higher closer to the seashore, which pattern increased through time). Over the long term, decreased precipitation but not increased temperature induced increasing salinity. We also highlighted contrasted long-term patterns of salinity changes on these two coastal wetlands, with one site were salinity decreased over time linked to the responses to marine flood, allowing to document the temporal dynamics of salinity following a massive intrusion of sea water. Complementarily, at both sites, water levels at high tides increased through time, a pattern which can induce additional salinization. To our knowledge, our study is the first to investigate long-term changes in salinity in coastal wetlands through natural processes (e.g. seaspray, seasonal variations) and ongoing climate perturbations (e.g. marine surges linked to extreme weather events, increased temperature and decreased precipitations).

A prospective study on the pathogenesis of catheter-associated bacteriuria in critically ill patients.

BACKGROUND: Updating the pathogenesis of catheter-associated bacteriuria (CA-bacteriuria) in the intensive care unit (ICU) is needed to adapt prevention strategies. Our aim was to determine whether the main pathway of CA-bacteriuria in ICU patients was endoluminal or exoluminal. In a prospective study, quantitative urine cultures were sampled from catheter sampling sites, collector bags and the catheter outer surface near the meatus from days 1 to 15 after catheterization. The endoluminal pathway was CA-bacteriuria (defined as 102 CFU/mL) first in collector bags and then in catheters. The exoluminal pathway was CA-bacteriuria first in catheters, on day 1 in early cases and after day 1 in late cases. RESULTS: Of 64 included patients, 20 had CA-bacteriuria. Means of catheterization days and incidence density were 6.81 days and 55.2/1000 catheter-days. Of 26 microorganisms identified, 12 (46.2%) were Gram positive cocci, 8 (30.8%) Gram negative bacilli and 6 yeasts. Three (11.5%) CA-bacteriuria were endoluminal and 23 (88.5%) exoluminal, of which 10 (38.5%) were early and 13 (50%) late. Molecular comparison confirmed culture findings. A quality audit showed good compliance with guidelines. CONCLUSION: The exoluminal pathway of CA-bacteriuria in ICU patients predominated and surprisingly occurred early despite good implementation of guidelines. This finding should be considered in guidelines for prevention of CA-bacteriuria.

Carbapenem Resistance Conferred by OXA-48 in K2-ST86 Hypervirulent Klebsiella pneumoniae, France.

We recovered 2 carbapenem-resistant K2-ST86 hypermucoviscous Klebsiella pneumoniae isolates from patients in France. The isolates had genetic attributes of hypervirulent K. pneumoniae but differed in ability to cause mouse lethality. Convergence of hypervirulent K. pneumoniae toward resistance could cause a health crisis because such strains could be responsible for severe and untreatable infections.

Development and Multicentric Validation of a Lateral Flow Immunoassay for Rapid Detection of MCR-1-Producing Enterobacteriaceae.

Colistin has become a last-resort antibiotic for the treatment of infections caused by highly drug-resistant Gram-negative bacteria. Moreover, it has been widely used in the livestock sector. As a consequence, colistin resistance is emerging worldwide. Among the colistin resistance mechanisms, the spread of the plasmid-encoded colistin resistance gene mcr-1 (mostly in Escherichia coli) is of particular concern due to its increased transferability compared to that of chromosome-encoded resistance. The early detection of MCR-1-producing bacteria is essential to prevent further spread and provide appropriate antimicrobial therapy. Lateral flow immunoassays (LFIAs) were manufactured with selected monoclonal antibodies. A collection of 177 human and 121 animal enterobacterial isolates was tested in a multicentric study. One bacterial colony grown on agar plates was suspended in extraction buffer and dispensed on the cassette. Migration was allowed for 15 min, and the results were monitored by the appearance of a specific band. The positive results showed a pink line resulting in an unambiguous interpretation. All MCR-1-producing isolates were found to be positive by the LFIA, and no false-negative results were observed. Three out of four MCR-2-producing isolates were also found to be positive. Our test does not detect MCR-3-, MCR-4-, or MCR-5-producing isolates. LFIA allows the detection of MCR-1 with 100% sensitivity and 98% specificity. This test is fast, sensitive, specific, easy to use, and cost-effective and can therefore be implemented in any microbiology laboratory worldwide. LFIA is a major tool for the rapid detection and monitoring of MCR-1 producers in humans and animals.

Novel Enterobacter Lineage as Leading Cause of Nosocomial Outbreak Involving Carbapenemase-Producing Strains.

We investigated unusual carbapenemase-producing Enterobacter cloacae complex isolates (n = 8) in the novel sequence type (ST) 873, which caused nosocomial infections in 2 hospitals in France. Whole-genome sequence typing showed the 1-year persistence of the epidemic strain, which harbored a blaVIM-4 ST1-IncHI2 plasmid, in 1 health institution and 2 closely related strains harboring blaCTX-M-15 in the other. These isolates formed a new subgroup in the E. hormaechei metacluster, according to their hsp60 sequences and phylogenomic analysis. The average nucleotide identities, specific biochemical properties, and pangenomic and functional investigations of isolates suggested isolates of a novel species that had acquired genes associated with adhesion and mobility. The emergence of this novel Enterobacter phylogenetic lineage within hospitals should be closely monitored because of its ability to persist and spread.

In Vitro Activity of Ceftolozane-Tazobactam against Enterobacter cloacae Complex Clinical Isolates with Different ß-Lactam Resistance Phenotypes.

The study evaluated the in vitro activity of ceftolozane-tazobactam (C/T) against 94 unique clinical isolates of Enterobacter cloacae complex (ECC). No difference was observed according to the ECC cluster. The in vitro activity greatly varied depending on the ß-lactamase-producing profile: 100%, 67%, and 19% of wild-type, extended-spectrum ß-lactamase (ESBL)-producing, and AmpC-overproducing strains, respectively, were susceptible to C/T. The use of C/T could be of interest for the treatment of some infections caused by ESBL-producing AmpC-nonoverexpressing ECC isolates.

Diversity of DHA-1-encoding plasmids in Klebsiella pneumoniae isolates from 16 French hospitals.

Objectives: To provide new insights into the spread of plasmidic cephalosporinase DHA-1, 16 strains of Klebsiella pneumoniae and a strain of Klebsiella variicola producing DHA-1 were isolated between January 2012 and December 2013 in six regions of France and two French overseas departments and territories. Methods: Disc diffusion assays, isoelectric focusing and PCRs were used to characterize the plasmidic DHA-1 ß-lactamase. Plasmid analysis was performed by the method of Kado and Liu and WGS. Virulence of the strains was studied by biofilm formation and the survival of Drosophila. Results: The strains were of low virulence and had one to three plasmids including one of various sizes (∼40 to 319 kb) mediating DHA-1. Nine strains belonged to ST11 and possessed a pKPS30-type DHA-1 plasmid of the IncR (incompatibility) group. A strain of ST307 possessed pENVA, a DHA-1 plasmid of the IncH-type group. The seven remaining plasmids were unknown. Three belonged to the IncL/M group. They were closely related and their sequences were determined. One of the four remaining strains was chosen for further investigation. This strain of ST16 had two plasmids, a pUUH239.2-related plasmid and a new DHA-1 plasmid of ∼319 kb of IncHI2 type. Conclusions: These findings demonstrate the major role of the pKPS30-type plasmid in the spread of DHA-1 cephalosporinase in France and provide evidence of two new emerging plasmids carrying this enzyme.

Rapid detection and discrimination of chromosome- and MCR-plasmid-mediated resistance to polymyxins by MALDI-TOF MS in Escherichia coli: the MALDIxin test.

Background: Polymyxins are currently considered a last-resort treatment for infections caused by MDR Gram-negative bacteria. Recently, the emergence of carbapenemase-producing Enterobacteriaceae has accelerated the use of polymyxins in the clinic, resulting in an increase in polymyxin-resistant bacteria. Polymyxin resistance arises through modification of lipid A, such as the addition of phosphoethanolamine (pETN). The underlying mechanisms involve numerous chromosome-encoded genes or, more worryingly, a plasmid-encoded pETN transferase named MCR. Currently, detection of polymyxin resistance is difficult and time consuming. Objectives: To develop a rapid diagnostic test that can identify polymyxin resistance and at the same time differentiate between chromosome- and plasmid-encoded resistances. Methods: We developed a MALDI-TOF MS-based method, named the MALDIxin test, which allows the detection of polymyxin resistance-related modifications to lipid A (i.e. pETN addition), on intact bacteria, in <15 min. Results: Using a characterized collection of polymyxin-susceptible and -resistant Escherichia coli, we demonstrated that our method is able to identify polymyxin-resistant isolates in 15 min whilst simultaneously discriminating between chromosome- and plasmid-encoded resistance. We validated the MALDIxin test on different media, using fresh and aged colonies and show that it successfully detects all MCR-1 producers in a blindly analysed set of carbapenemase-producing E. coli strains. Conclusions: The MALDIxin test is an accurate, rapid, cost-effective and scalable method that represents a major advance in the diagnosis of polymyxin resistance by directly assessing lipid A modifications in intact bacteria.

Plasmids carrying DHA-1 ß-lactamases.

The aim of this review is to provide an update on the plasmids mediating DHA-1 cephalosporinase in Klebsiella pneumoniae. These plasmids have been mainly found in this bacterium but not only. The first was isolated from Salmonella sp. in France in the early 1990s. They are currently reported worldwide. BlaDHA-1 beta-lactamase gene is usually co-expressed with many other antibiotic resistance genes such as extended-spectrum ß-lactamases (blaCTX-M-, bla SHV -types), oxacillinases (blaOXA-1, blaOXA-30), penicillinases (bla TEM -type), carbapenemases (bla OXA48 , blaKPC-2), aminoglycosides (aacA, aadA, armA), fluoroquinolones (qnrB4, aac6'-1b-cr), and sulfonamide (sul1) resistance genes. Plasmids carrying DHA-1 cephalosporinase have different sizes (22 to 313 kb), belong to diverse groups of incompatibility (R, L/M, FII(k), FIB, A/C2, HI2, HIB), and are self-transferable or not. The multidrug resistance region consists of a mosaic structure composed of resistance genes, insertion sequences, composite transposon, and integrons.

mcr-1 Colistin Resistance in ESBL-Producing Klebsiella pneumoniae, France.

We report intestinal carriage of an extended-spectrum ß-lactamase-producing Klebsiella pneumoniae strain with high-level resistance to colistin (MIC 24 mg/L) in a patient in France who had been hospitalized for fungal meningitis. The strain had the mcr-1 plasmid gene and an inactivated mgrB gene, which are associated with colistin resistance.

MCR-1 and OXA-48 In Vivo Acquisition in KPC-Producing Escherichia coli after Colistin Treatment.

The spread of mcr-1-encoding plasmids into carbapenem-resistant Enterobacteriaceae raises concerns about the emergence of untreatable bacteria. We report the acquisition of mcr-1 in a carbapenem-resistant Escherichia coli strain after a 3-week course of colistin in a patient repatriated to France from Portugal. Whole-genome sequencing revealed that the Klebsiella pneumoniae carbapenemase-producing E. coli strain acquired two plasmids, an IncL OXA-48-encoding plasmid and an IncX4 mcr-1-encoding plasmid. This is the first report of mcr-1 in carbapenemase-encoding bacteria in France.

IncFIIk plasmid harbouring an amplification of 16S rRNA methyltransferase-encoding gene rmtH associated with mobile element ISCR2.

OBJECTIVES: To investigate the resistance mechanisms and genetic support underlying the high resistance level of the Klebsiella pneumoniae strain CMUL78 to aminoglycoside and ß-lactam antibiotics. METHODS: Antibiotic susceptibility was assessed by the disc diffusion method and MICs were determined by the microdilution method. Antibiotic resistance genes and their genetic environment were characterized by PCR and Sanger sequencing. Plasmid contents were analysed in the clinical strain and transconjugants obtained by mating-out assays. Complete plasmid sequencing was performed with PacBio and Illumina technology. RESULTS: Strain CMUL78 co-produced the 16S rRNA methyltransferase (RMTase) RmtH, carbapenemase OXA-48 and ESBL SHV-12. The rmtH- and blaSHV-12-encoding genes were harboured by a novel ∼115 kb IncFIIk plasmid designated pRmtH, and blaOXA-48 by a ∼62 kb IncL/M plasmid related to pOXA-48a. pRmtH plasmid possessed seven different stability modules, one of which is a novel hybrid toxin-antitoxin system. Interestingly, pRmtH plasmid harboured a 4-fold amplification of an rmtH-ISCR2 unit arranged in tandem and inserted within a novel IS26-based composite transposon designated Tn6329. CONCLUSIONS: This is the first known report of the 16S RMTase-encoding gene rmtH in a plasmid. The rmtH-ISCR2 unit was inserted in a composite transposon as a 4-fold tandem repeat, a scarcely reported organization.

Whole grain in manufactured foods: Current use, challenges and the way forward.

Some countries now incorporate recommendations for increased consumption of whole grain (WG) into local dietary guidelines. Cereal and pseudo-cereal grains are good sources of complex carbohydrates, dietary fiber, proteins, phytochemicals, vitamins and minerals. However, research shows that the large majority of consumers are still falling short of WG consumption goals. To address this, we are actively involved in research to help increase the WG content of processed foods without compromising on taste and texture. In order to ensure consumer trust, the advancement of process technologies in incorporating WG to produce tasty food has to go hand in hand with well designed clinical trials that confirm the health benefits resulting from diets rich in WG.

Small-molecule inhibitors prevent the genotoxic and protumoural effects induced by colibactin-producing bacteria.

OBJECTIVE: Colorectal cancers (CRCs) are frequently colonised by colibactin toxin-producing Escherichia coli bacteria that induce DNA damage in host cells and exhibit protumoural activities. Our objective was to identify small molecules inhibiting the toxic effects induced by these colibactin-producing bacteria. DESIGN: A structural approach was adopted for the identification of a putative ligand for the ClbP enzyme involved in the synthesis of colibactin. Intestinal epithelial cells and a CRC mouse model were used to assess the activity of the selected compounds in vitro and in vivo. RESULTS: Docking experiments identified two boron-based compounds with computed ligand efficiency values (-0.8 and -0.9 kcal/mol/atom) consistent with data expected for medicinal chemistry leads. The crystalline structure of ClbP in complex with the compounds confirmed that the compounds were binding to the active site of ClbP. The two compounds (2 mM) suppressed the genotoxic activity of colibactin-producing E coli both in vitro and in vivo. The mean degree of suppression of DNA damage for the most efficient compound was 98±2% (95% CI). This compound also prevented cell proliferation and colibactin-producing E coli-induced tumourigenesis in mice. In a CRC murine model colonised by colibactin-producing E coli, the number of tumours decreased by 3.5-fold in animals receiving the compound in drinking water (p<0.01). CONCLUSIONS: These results demonstrate that targeting colibactin production controls the genotoxic and protumoural effects induced by this toxin.

Sugar replacers: from technological challenges to consequences on health.

PURPOSE OF REVIEW: Dietary sugars play a role in noncommunicable diseases and represent a clear target for reduction. In this context, product reformulation can have a positive impact on health. Several technological solutions are available to replace sugar, all with benefits and limitations. The goal of this review is to describe the main sugar replacement alternatives and discuss their impact on health and product physicochemical properties. RECENT FINDINGS: Although high intensity sweeteners and polyols have been used for a long time to replace sucrose and despite no clear evidence of harm, the trend is today to look for alternatives such as sweet enhancers or alternative sugars such as allulose or tagatose, which are both low caloric. To replace the physical properties of sugars, new trends are to substitute widely used maltodextrins by dietary fibres to confer added health benefits. SUMMARY: A wide range of solutions is currently available to replace dietary sugars and compensate for the impact on bulking properties and sweetness profile of food products.

Western diet induces dysbiosis with increased E coli in CEABAC10 mice, alters host barrier function favouring AIEC colonisation.

OBJECTIVE: Western diet is a risk factor for Crohn's disease (CD). Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is abnormally expressed in CD patients. This allows adherent-invasive Escherichia coli (AIEC) to colonise the gut mucosa and leads to inflammation. We assessed the effects of a high fat/high sugar (HF/HS) Western diet on gut microbiota composition, barrier integrity and susceptibility to infection in transgenic CEABAC10 mice expressing human CEACAMs. DESIGN: Colonic microbiota composition and susceptibility of CEABAC10 mice to AIEC LF82 bacteria infection were determined in mice fed a conventional or HF/HS diet. Barrier function and inflammatory response were assessed by studying intestinal permeability, tight junction protein and mucin expression and localisation, and by determining histological score and levels of cytokine release. RESULTS: HF/HS diet led to dysbiosis in WT and transgenic CEABAC10 mice, with a particular increase in E coli population in HF/HS-fed CEABAC10 mice. These mice showed decreased mucus layer thickness, increased intestinal permeability, induction of Nod2 and Tlr5 gene transcription, and increased TNFα secretion. These modifications led to a higher ability of AIEC bacteria to colonise the gut mucosa and to induce inflammation. CONCLUSIONS: Western diet induces changes in gut microbiota composition, alters host homeostasis and promotes AIEC gut colonisation in genetically susceptible mice. These results support the multifactorial aetiology of CD and highlight the importance of diet in CD pathogenesis.

Variations of salinity during reproduction and development affect ontogenetic trajectories in a coastal amphibian.

Although coastal ecosystems are naturally submitted to temporal variations of salinity, salinization has been increasing over time threatening coastal biodiversity. Species that exploit such habitats can thus be exposed to brackish water at different life stages. However, the impacts of variations of salinity on wildlife remain poorly understood. This is particularly true for coastal amphibians, due to the strong dependency of early life stages (embryos and larvae) on aquatic environments. In order to investigate the effect of salinity during egg laying and embryonic and larval development of coastal amphibians, we used a full-factorial design to expose reproductive adults, eggs, and larvae of coastal spined toads (Bufo spinosus) to fresh (0 g.l-1) or brackish water (4 g.l-1). At egg laying, we evaluated parental investment in reproduction. During embryonic and larval development, we assessed effects on survival, development, and growth. We highlighted strong effects of environmental salinity on reproduction (reduced egg laying time, marginally reduced egg size, and reduced investment in reproduction). Responses to salinity were highly dependent on the developmental stages of exposure (stronger effects when individuals were exposed during embryonic development). These effects carried over when exposure occurred at egg laying or during embryonic development, highlighting the importance of the environmental conditions during early life on ontogenetic trajectories. We also highlighted partial compensation when individuals were transferred back to freshwater. Whether the magnitude of these responses can allow coastal biodiversity to overcome the observed detrimental effects of salinization remain to be assessed.

Low migratory connectivity and similar migratory strategies in a shorebird with contrasting wintering population trends in Europe and West Africa.

Migratory shorebird populations are declining worldwide, showing an apparent inability to respond to the interplaying challenges emerging along their flyways. Within the East Atlantic Flyway, non-breeding populations show moderate to strong declines in Sub-Saharan Africa, contrasting with stable or increasing trends in Europe. Local factors are insufficient to explain the opposite tendencies and, therefore, investigating migratory strategies and connectivity of these populations may help identifying the drivers of their demography. We followed the migratory journeys of 20 grey plovers (Pluvialis squatarola) from their wintering grounds in Guinea-Bissau (West Africa), Portugal and France (Europe) using tracking devices. Grey plovers wintering in Africa and Europe were found to share breeding grounds at European Russia and Western Siberia, revealing low migratory connectivity in the Eastern Atlantic population. All individuals followed a "skipping" migratory strategy, flying mostly mid-distance bouts, and using an unexpected large network of stopover sites to re-fuel usually for short periods. We identified 66 different stopover sites along the West African, European and Russian/Siberian coasts. All birds stopped at the Wadden Sea in both migratory periods, highlighting the importance of this region and the risk for a potential bottleneck. Low migratory connectivity and similar migratory strategies shared by grey plovers wintering in Europe and West Africa do not support their contrasting population trends.

TEM-187, a new extended-spectrum ß-lactamase with weak activity in a Proteus mirabilis clinical strain.

A Proteus mirabilis clinical strain (7001324) was isolated from urine sample of a patient hospitalized in a long-term-care facility. PCR and cloning experiments performed with this strain identified a novel TEM-type ß-lactamase (TEM-187) differing by four amino acid substitutions (Leu21Phe, Arg164His, Ala184Val, and Thr265Met) from TEM-1. This characterization provides further evidence for the diversity of extended-spectrum ß-lactamases (ESBL) produced by P. mirabilis and for their potential spread to other Enterobacteriaceae due to a lack of sensitive detection methods used in daily practice.