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Efficient tools for rapid antibiotic susceptibility testing (AST) are crucial for appropriate use of antibiotics, especially colistin, which is now often considered a last resort therapy with extremely drug resistant Gram-negative bacteria. Here, we developed a rapid, easy and miniaturized colistin susceptibility assay based on microfluidics, which allows for culture and high-throughput analysis of bacterial samples. Specifically, a simple microfluidic platform that can easily be operated was designed to encapsulate bacteria in nanoliter droplets and perform a fast and automated bacterial growth detection in 2 h, using standardized samples. Direct bright-field imaging of compartmentalized samples proved to be a faster and more accurate detection method as compared to fluorescence-based analysis. A deep learning powered approach was implemented for the sensitive detection of the growth of several strains in droplets. The DropDeepL AST method (Droplet and Deep learning-based method for AST) developed here allowed the determination of the colistin susceptibility profiles of 21 fast-growing Enterobacterales (E. coli and K. pneumoniae), including clinical isolates with different resistance mechanisms, showing 100 % categorical agreement with the reference broth microdilution (BMD) method performed simultaneously. Direct AST of bacteria in urine samples on chip also provided accurate results in 2 h, without the need of complex sample preparation procedures. This method can easily be implemented in clinical microbiology laboratories, and has the potential to be adapted to a variety of antibiotics, especially for last-line antibiotics to optimize treatment of patients infected with multi-drug resistant strains., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Plasmids carrying antibiotic resistance genes (ARG) are the main mechanism of resistance dissemination in Enterobacterales. However, the fitness-resistance trade-off may result in their elimination. Chromosomal integration of ARGs preserves resistance advantage while relieving the selective pressure for keeping costly plasmids. In some bacterial lineages, such as carbapenemase producing sequence type ST38 Escherichia coli, most ARGs are chromosomally integrated. Here we reproduce by experimental evolution the mobilisation of the carbapenemase bla OXA-48 gene from the pOXA-48 plasmid into the chromosome. We demonstrate that this integration depends on a plasmid-induced fitness cost, a mobile genetic structure embedding the ARG and a novel antiplasmid system ApsAB actively involved in pOXA-48 destabilization. We show that ApsAB targets high and low-copy number plasmids. ApsAB combines a nuclease/helicase protein and a novel type of Argonaute-like protein. It belongs to a family of defense systems broadly distributed among bacteria, which might have a strong ecological impact on plasmid diffusion., (© 2024. The Author(s).)

OXA-48 has rapidly disseminated worldwide and become one of the most common carbapenemases in many countries with more than 45 variants reported with, in some cases, significant differences in their hydrolysis profiles. The R214 residue, located in the ß5-ß6 loop, is crucial for the carbapenemase activity, as it stabilizes carbapenems in the active site and maintains the shape of the active site through interactions with D159. In this study, we have characterized a novel variant of OXA-48, OXA-933 with a single D159N change. To evaluate the importance of this residue, point mutations were generated (D159A, D159G, D159K, and D159W), kinetic parameters of OXA-933, OXA-48 D159G, and OXA-48 D159K were determined and compared to those of OXA-48 and OXA-244. The bla OXA-933 gene was borne on Tn 2208 , a 2,696-bp composite transposon made of two IS 1 elements surrounded by 9 bp target site duplications and inserted into a non-self-transmissible plasmid pOXA-933 of 7,872 bp in size. Minimal inhibitory concentration values of E. coli expressing the bla OXA-933 gene or of its point mutant derivatives were lower for carbapenems (except for D159G) as compared to those expressing the bla OXA-48 gene. Steady-state kinetic parameters revealed lower catalytic efficiencies for expanded spectrum cephalosporins and carbapenems. A detailed structural analysis confirmed the crucial role of D159 in shaping the active site of OXA-48 enzymes by interacting with R214. Our work further illustrates the remarkable propensity of OXA-48-like carbapenemases to evolve through mutations at positions outside the β5-β6 loop, but interacting with key residues of it., Competing Interests: The authors declare no conflict of interest.

Objectives: In-depth phenotypic and genomic analyses on a carbapenem-resistant Escherichia coli isolate, recovered from the faeces of a farm dog in Lebanon, focusing on its antimicrobial resistance (AMR) patterns and the underlying resistome., Methods: E. coli strain EC-106 was identified using MALDI-TOF-MS. Analyses using Carba NP, immunochromatographic assay NG Carba5, and other antimicrobial susceptibility testing were performed. Whole-genome sequencing (WGS) using the Illumina technology and different software available at the Center of Genomic Epidemiology wwere used to predict the resistome, sequence type (ST), plasmid types, and virulence genes., Results: Susceptibility testing revealed that E. coli EC-106 was multi-drug resistant, including against newer antimicrobials such as imipenem-relebactam (MIC = 16 µg/mL), meropenem-vaborbactam (MIC = 16 µg/mL), and ceftazidime-avibactam (MIC > 32 µg/mL), but remained susceptible to aztreonam (MIC = 0.12 µg/mL), aztreonam-avibactam (MIC = 0.06 µg/mL), and cefiderocol (MIC = 0.5 µg/mL). WGS analyses showed that E. coli EC-106 carried 13 acquired resistance genes associated with resistance to β-lactams (bla NDM-5 and bla TEM-1B ), aminoglycosides (aac(3)-IId, aph(3')-Ia, aadA1, and aadA2), tetracyclines (tetA), amphenicols (partial catA1), macrolides (mphA), sulphonamides (sul1 and sul3), trimethoprim (dfrA12), and quaternary ammonium compounds (partial qacE). The bla NDM-5 was located on an IncX3 plasmid. The isolate was predicted to be a human pathogen (92.9%) and belonged to ST1011., Conclusion: To our knowledge, this is the first report of the detection of an IncX3 plasmid carrying the bla NDM-5 gene in animals in Lebanon, highlighting the severe AMR challenges in the country. Taken together, our current and previous findings suggest that bla NDM-5 might be spreading in different hosts and genetic backgrounds across clinical and non-clinical settings., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objectives: The contamination of fresh surface waters poses a significant burden on human health and prosperity, especially in marginalized communities with limited resources and inadequate infrastructure. Here, we performed in-depth genomic analyses of multidrug-resistant bacteria (MDR-B) isolated from Al-Oueik river water that is used for irrigation of agricultural fields in a disenfranchised area that also hosts a makeshift Syrian refugee camp., Methods: A composite freshwater sample was filtered. Faecal coliforms were counted and extended spectrum cephalosporins and/or ertapenem resistant bacteria were screened. Isolates were identified using MALDI-TOF-MS and analysed using whole-genome sequencing (WGS) to identify the resistome, sequence types, plasmid types, and virulence genes., Results: Approximately 10 6 CFU/100 mL of faecal coliforms were detected in the water. Four drug-resistant Gram-negative bacteria were identified, namely Escherichia coli, Klebsiella pneumoniae, Enterobacter hormaechei, and Pseudomonas otitidis. Notably, the E. coli isolate harboured bla NDM-5 and a YRIN-inserted PBP3, representing an emerging public health challenge. The K. pneumoniae isolate carried bla SHV-187 as well as mutations in the gene encoding the OmpK37 porin. Enterobacter hormaechei and P. otitidis harboured bla ACT-16 and bla POM-1 , respectively., Conclusion: This report provides comprehensive genomic analyses of MDR-B in irrigation water in Lebanon. Our results further support that irrigation water contaminated with faecal material can be a reservoir of important MDR-B, which can spread to adjacent agricultural fields and other water bodies, posing both public health and food safety issues. Therefore, there is an urgent need to implement effective water quality monitoring and management programs to control the proliferation of antibiotic-resistant pathogens in irrigation water in Lebanon., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: The fecal carriage of extended-spectrum β-lactamase-producing Enterobacterales (ESBL-PE) is a major driver of the global spread of these antibiotic resistance determinants. Here we determined the rate of fecal ESBL-PE carriage in pediatric hospitals and community-serving healthcare centers serving adults and children in the Gaza Strip, Palestine., Methods: A total of 373 fecal and rectal samples were collected from different hospitals and clinics in Gaza. The antibiotic susceptibility was determined using the disk diffusion method and interpreted according to CLSI guidelines. The bacterial isolates were tested for ESBL production using phenotypic methods (double disk synergy test and growth on selective chromogenic media). Bla CTX-M , bla SHV , and bla TEM genes were sought by PCR., Results: Out of the 373 isolates tested, 138 (37%) were considered ESBL positive as revealed by phenotypic tests. The prevalence of ESBLs among hospitalized patients was 39.1% (hospital setting) whereas, among outpatients attending community healthcare centers, it was 35.1% (community setting). ESBL production among Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, Proteus mirabilis, and Klebsiella aerogenes isolates was 52.8%, 39.1%, 26.7%, 2.8%, and 2.1% respectively. Meropenem and amikacin were the most effective antibiotics against ESBL producers (68.9% and 73.6% susceptibility, respectively), while only 15.2%, 22.5%, and 24.6% remained susceptible to ceftazidime, cefotaxime, and ceftriaxone, respectively. Out of 138 phenotypically ESBL-positive isolates, 98 randomly chosen were screened for bla CTX-M , bla TEM , and bla SHV genes. The prevalence rate of bla CTX-M was 45.9%, while bla TEM and bla SHV genes were detected in 16.8% and 5.2% of CTX-M-negative isolates (corresponding mostly for K. pneumoniae isolates in the case of SHV-PCR), respectively., Conclusions: The study revealed an alarmingly high prevalence of fecal carriage of ESBL-producing Enterobacterales among hospitalized children but also in the community of the Gaza Strip. In addition, 30% of ESBL-producers were already resistant to carbapenems, the treatment of choice of infections with ESBL-producers., (© 2023. The Author(s).)

Background: Carbapenem- and extended-spectrum cephalosporin-resistant Enterobacterales (CR-E and ESCR-E, respectively) are increasingly isolated worldwide. Information about these bacteria is sporadic in Lebanon and generally relies on conventional diagnostic methods, which is detrimental for a country that is struggling with an unprecedented economic crisis and a collapsing public health system. Here, CR-E isolates from different Lebanese hospitals were characterized., Materials and Methods: Non-duplicate clinical ESCR-E or CR-E isolates ( N  = 188) were collected from three hospitals from June 2019 to December 2020. Isolates were identified by MALDI-TOF, and their antibiotic susceptibility by Kirby-Bauer disk diffusion assay. CR-E isolates ( n  = 33/188) were further analyzed using Illumina-based WGS to identify resistome, MLST, and plasmid types. Additionally, the genetic relatedness of the CR-E isolates was evaluated using an Infrared Biotyper system and compared to WGS., Results: Using the Kirby-Bauer disk diffusion assay, only 90 isolates out of the 188 isolates that were collected based on their initial routine susceptibility profile by the three participating hospitals could be confirmed as ESCR-E or CR-E isolates and were included in this study. This collection comprised E. coli ( n  = 70; 77.8%), K. pneumoniae ( n  = 13; 14.4%), Enterobacter spp. ( n  = 6; 6.7%), and Proteus mirabilis ( n  = 1; 1.1%). While 57 were only ESBL producers the remaining 33 isolates (i.e., 26 E. coli , five K. pneumoniae , one E. cloacae , and one Enterobacter hormaechei ) were resistant to at least one carbapenem, of which 20 were also ESBL-producers. Among the 33 CR-E, five different carbapenemase determinants were identified: bla NDM-5 (14/33), bla OXA-244 (10/33), bla OXA-48 (5/33), bla NDM-1 (3/33), and bla OXA-181 (1/33) genes. Notably, 20 CR-E isolates were also ESBL-producers. The analysis of the genetic relatedness revealed a substantial genetic diversity among CR-E isolates, suggesting evolution and transmission from various sources., Conclusion: This study highlighted the emergence and broad dissemination of bla NDM-5 and bla OXA-244 genes in Lebanese clinical settings. The weak AMR awareness in the Lebanese community and the ongoing economic and healthcare challenges have spurred self-medication practices. Our findings highlight an urgent need for transformative approaches to combat antimicrobial resistance in both community and hospital settings., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Daaboul, Oueslati, Rima, Kassem, Mallat, Birer, Girlich, Hamze, Dabboussi, Osman and Naas.)

Cefiderocol resistance is increasingly reported in New Delhi metallo-β-lactamase-producing Enterobacterales. Genomic and phenotypic analysis of Escherichia coli sequence type 361, a primary clone causing carbapenemase spread in France, revealed mutations leading to cefiderocol resistance. Continued genomic surveillance of carbapenem-resistant Enterobacterales could clarify prevalence of cefiderocol-resistant E. coli in Europe.

Objectives: We isolated a highly colistin-resistant Escherichia coli, strain 58, from fresh chicken wings in Lebanon. Here, we performed in-depth phenotypic and genomic analyses to identify the resistome of the isolate, focusing on the determinants that encoded colistin resistance., Methods: The minimum inhibitory concentration (MIC) of colistin and resistance to other antibiotics were determined using the broth microdilution method and the Kirby-Bauer disk diffusion assay, respectively. Whole-genome sequencing (WGS) and different software available at the Center of Genomic Epidemiology were used to predict the resistome, the sequence type (ST), and the presence of virulence genes and plasmid replicon types., Results: Susceptibility testing revealed that E. coli 58 exhibited multidrug resistance, including against colistin (MIC = 32 µg/mL). Whole-genome sequencing analyses showed that E. coli 58 carried 26 antimicrobial resistance genes associated with resistance to polymyxins (mcr-1.26), β-lactams (blaTEM-1b and blaCMY-2), fosfomycin (fosA4), aminoglycosides (aac(3)-IId, aadA2b, aadA5, partial aadA1, aph(3'')-Ia, aph(3')-Ia, and aph(6)-Id), tetracyclines (tetA and tetM), quinolones (qnrS1), sulphonamides (sul2 and sul3), trimethoprim (dfrA14, dfrA17, and dfrA5), phenicols (floR and cmlA1), macrolides (mphA), lincosamides (lnu(F)), quaternary ammonium compounds (partial qacL and qacE), and peroxides (sitABCD). mcr-1.26 was located on an IncX4 plasmid and induced colistin resistance in otherwise naïve E. coli and Salmonella Enteritidis. Escherichia coli 58 was predicted to be a human pathogen and belonged to ST3107., Conclusion: To our knowledge, this is the first report of mcr-1.26 in poultry meat worldwide. We previously reported mcr-1.26 in an MDR E. coli (ST2207) isolated from a pigeon in Lebanon, which suggests that it might be spreading in different animal hosts and genetic backgrounds., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: OXA-48-producing Enterobacterales have widely disseminated globally with an increasing number of variants identified. Among them, OXA-244 is increasingly reported, despite detection difficulties., Objectives: To determine the steady-state kinetic parameters of OXA-244., Methods: The blaOXA-244 gene was amplified, cloned into plasmids p-TOPO and pET41b+, and transformed into Escherichia coli TOP10 for MIC determination and E. coli BL21 DE3 for purification. Steady-state kinetic parameters and IC50s of clavulanic acid, tazobactam and NaCl were determined using purified OXA-244. Molecular modelling was also performed., Results: A reduction in MICs of temocillin and carbapenems was observed in E. coli expressing OXA-244 as compared with OXA-48. The kinetic parameters revealed a reduced carbapenemase activity of OXA-244 as compared with OXA-48, especially for imipenem, which was 10-fold lower. Similarly, catalytic efficiency (kcat/Km) was reduced by 4-fold and 20-fold for ampicillin and temocillin, respectively. Kinetic parameters for cephalosporins were, however, similar. Molecular modelling studies evidenced the key role of R214 in OXA-48, establishing salt bridges with D159 and with the carboxylate group of the R1 substituent of temocillin. These interactions are not possible with G214 in OXA-244, explaining the reduced affinity of temocillin for this enzyme. The R214G mutation in OXA-244 is also likely to induce changes in the active site's water network that would explain the decrease in the hydrolysis rate of carbapenems., Conclusions: Our data confirm that the R214G mutation (present in OXA-244) results in reduced carbapenem- and temocillin-hydrolysing activity, confirming the crucial role of residue 214 in the hydrolysis of these substrates by OXA-48-like β-lactamases., (© The Author(s) 2021. 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.)

Since 2016, OXA-244-producing Escherichia coli has been increasingly isolated in France. We sequenced 97 OXA-244-producing E. coli isolates and found a wide diversity of sequence types and a high prevalence of sequence type 38. Long-read sequencing demonstrated the chromosomal location of bla OXA-244 inside the entire or truncated Tn51098.

Background: OXA-244, a single amino acid variant of OXA-48, demonstrates weaker hydrolytic activity towards carbapenems and temocillin compared with OXA-48. Of note, these antimicrobials are present in high concentrations in several carbapenemase-producing Enterobacterales (CPE) screening media. As a result, some screening media fail to grow OXA-244-producing isolates, while the prevalence of OXA-244 producers is constantly increasing in France., Methods: Here, we evaluate the performance of three commercially available CPE screening media [ChromID® CARBA SMART (bioMérieux), Brilliance™ CRE (Thermo Fisher) and mSuperCARBA™ (MAST Diagnostic)] for their ability to detect OXA-244 producers (n = 101). As OXA-244 producers may also express an ESBL, two additional ESBL screening media were tested (Brilliance™ ESBL and ChromID® BLSE). MICs of temocillin and imipenem were determined by broth microdilution. The clonality of OXA-244-producing Escherichia coli isolates (n = 97) was assessed by MLST., Results: Overall, the sensitivity of the ChromID® CARBA SMART, Brilliance™ CRE and mSuperCARBA™ media were 14% (95% CI = 8.1%-22.5%), 54% (95% CI = 43.3%-63.4%) and 99% (95% CI = 93.8%-100%), respectively, for the detection of OXA-244 producers. Among the 101 OXA-244-producing isolates, 96% were E. coli and 77%-78% grew on ESBL screening media. MLST analysis identified five main STs among OXA-244-producing E. coli isolates: ST38 (n = 37), ST361 (n = 17), ST69 (n = 12), ST167 (n = 11) and ST10 (n = 8)., Conclusions: Our results demonstrated that the mSuperCARBA™ medium is very efficient in the detection of OXA-244 producers, unlike the ChromID® CARBA SMART medium. The high prevalence of ESBLs among OXA-244 producers allowed detection of 77%-78% of them using ESBL-specific screening media., (© The Author(s) 2020. 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.)

An OXA-181 producing Escherichia coli isolate that went recurrently undetected directly from rectal swab sample using Xpert® Carba-R, was successfully detected using ertapenem supplemented broth enrichment followed by culture-based method. Our data suggest that implementation of culture-based methods plus enrichment might be crucial for the efficient screening of patients considered to be at "high-risk" of colonization with carbapenemase producers and who are colonized at low level., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objectives: Escherichia coli is characterized by three resistance patterns to β-lactams/β-lactamase inhibitors (BLs/BLIs): (i) resistance to ampicillin/sulbactam and susceptibility to amoxicillin/clavulanic acid and piperacillin/tazobactam (RSS); (ii) resistance to ampicillin/sulbactam and amoxicillin/clavulanic acid, and susceptibility to piperacillin/tazobactam (RRS); and (iii) resistance to ampicillin/sulbactam, amoxicillin/clavulanic acid and piperacillin/tazobactam (RRR). These resistance patterns are acquired consecutively, indicating a potential risk of developing resistance to piperacillin/tazobactam, but the precise mechanism of this process is not completely understood., Methods: Clinical isolates incrementally pressured by piperacillin/tazobactam selection in vitro and in vivo were used. We determined the MIC of piperacillin/tazobactam in the presence and absence of piperacillin/tazobactam pressure. We deciphered the role of the blaTEM genes in the new concept of extended-spectrum resistance to BLs/BLIs (ESRI) using genomic analysis. The activity of β-lactamase was quantified in these isolates., Results: We show that piperacillin/tazobactam resistance is induced in E. coli carrying blaTEM genes. This resistance is due to the increase in copy numbers and transcription levels of the blaTEM gene, thus increasing β-lactamase activity and consequently increasing piperacillin/tazobactam MICs. Genome sequencing of two blaTEM-carrying representative isolates showed that piperacillin/tazobactam treatment produced two types of duplications of blaTEM (8 and 60 copies, respectively). In the clinical setting, piperacillin/tazobactam treatment of patients infected by E. coli carrying blaTEM is associated with a risk of therapeutic failure., Conclusions: This study describes for the first time the ESRI in E. coli. This new concept is very important in the understanding of the mechanism involved in the acquisition of resistance to BLs/BLIs., (© 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.)

Escherichia coli ST410 (Ec-ST410) is an emerging, multidrug-resistant clone. Recent investigations of its global epidemiology and evolution have been based almost exclusively on isolates from Europe and North America. It is unclear whether Southeast Asian-origin Ec-ST410 (SEA-Ec-ST410) belong to these same clones or represent regionally disseminated variants. Antimicrobial resistance mechanisms among SEA-Ec-ST410 were characterised, and whether they belonged to regional variants was investigated by contextualising them within a global collection. Seven Ec-ST410 were identified among a recent collection of expanded-spectrum cephalosporin-resistant E. coli recovered from 91 healthy women (stool) and 26 infected patients (blood and urine) living in Phnom Penh, Cambodia. Nine additional Ec-ST410 genomes were identified from Thailand (n = 7) and Vietnam (n = 2) through EnteroBase and PubMed searches. The assembled genomes were characterised and a SNP-based phylogenetic tree was created comparing these 16 SEA-Ec-ST410 with a previously published Ec-ST410 collection, primarily sourced from Europe (97/128) and North America (24/128). SEA-Ec-ST410 belonged to several distinct branches within previously described clonal clades. SEA-Ec-ST410 within the B3/H24Rx sublineage encoded bla CTX-M-55 (8/12) and F18:A-:B1 plasmid replicons (6/12), neither of which were detected among other Ec-ST410 belonging to this clade. Three of four SEA-Ec-ST410 within the B4/H24RxC sublineage lacked both bla OXA-181 and an IncX3 plasmid replicon that were harboured by 97% and 100% of all other Ec-ST410 in this sublineage (n = 64), respectively. In conclusion, Ec-ST410 are present in Southeast Asia following multiple introductions. The unique pattern of antimicrobial resistance elements harboured by SEA-Ec-ST410 suggests independent circulation in the region., (Copyright © 2019 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

With the widespread use and abuse of antibiotics for the past decades, antimicrobial resistance poses a serious threat to public health nowadays. β-Lactams are the most used antibiotics, and β-lactamases are the most widespread resistance mechanism. Class C β-lactamases, also known as cephalosporinases, usually do not hydrolyze the latest and most potent β-lactams, expanded spectrum cephalosporins and carbapenems. However, the recent emergence of extended-spectrum AmpC cephalosporinases, their resistance to inhibition by classic β-lactamase inhibitors, and the fact that they can contribute to carbapenem resistance when paired with impermeability mechanisms, means that these enzymes may still prove worrisome in the future. Here we report and characterize the CMY-136 β-lactamase, a Y221H point mutant derivative of CMY-2. CMY-136 confers an increased level of resistance to ticarcillin, cefuroxime, cefotaxime, and ceftolozane/tazobactam. It is also capable of hydrolyzing ticarcillin and cloxacillin, which act as inhibitors of CMY-2. X-ray crystallography and modeling experiments suggest that the hydrolytic profile alterations seem to be the result of an increased flexibility and altered conformation of the Ω-loop, caused by the Y221H mutation.

We compared extended-spectrum β-lactamase-producing Escherichia coli isolates from meat and fish, gut-colonized women, and infected patients in Cambodia. Nearly half of isolates from women were phylogenetically related to food-origin isolates; a subset had identical multilocus sequence types, extended-spectrum β-lactamase types, and antimicrobial resistance patterns. Eating sun-dried poultry may be an exposure route.

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.

With the dissemination of carbapenemase-producing Enterobacteriaceae (CPE) strains worldwide, carbapenem-hydrolyzing enzymes are increasingly reported among isolates of Escherichia coli , the first hospital and community-acquired opportunistic pathogen. Here, we have performed an epidemiological survey of carbapenemase-producing E. coli (CP- Ec ) isolates received at the French National Reference Centre (F-NRC) in 2012 and 2013. Antimicrobial susceptibilities for last-resort antibiotics and antimicrobial compounds commonly used to treat urinary tract infections were determined by broth microdilution. Clonal relationship was assessed using repetitive sequence-based PCR (rep-PCR) and multilocus sequence typing (MLST). From this collection of 140 carbapenemase-producing E. coli isolates, 74% produced an OXA-48-like carbapenemase and 21% produced an NDM carbapenemase. A link with a foreign country was suspected for 37% of infected/colonized patients. Most of the isolates were from screening (56%) and from urine samples (26%). Colistin, fosfomycin, and nitrofurantoin possessed the most consistent activity, with 100%, 95%, and 96% isolates susceptible, respectively. A wide diversity of carbapenemase-producing E. coli isolates has been found (50 different sequence types [STs]). The most prevalent clones were (i) E. coli sequence type 38 (ST38) producing OXA-48 ( n = 21), a clone linked to Turkey and North African countries, (ii) E. coli ST-90 producing OXA-204 ( n = 9), which was responsible for an outbreak related to a contaminated duodenoscope, and (iii) E. coli ST-410 producing OXA-181 ( n = 5), which was recovered from patients of different geographical origins. These specific clones might be considered high-risk clones for the dissemination of carbapenemases in E. coli The wide diversity of STs, combined with the increasing number of CP- Ec isolates received by the F-NRC, suggests a likely dissemination of CP- Ec isolates in the community., (Copyright © 2018 American Society for Microbiology.)

OXA-244 is a single-point-mutant derivative of OXA-48 displaying reduced carbapenemase activity. Here, we report the microbiological features of seven OXA-244-producing Escherichia coli isolates. Only one isolate grew on ChromID Carba Smart medium (bioMérieux), but six of the seven isolates grew on ChromID extended-spectrum-β-lactamase (ESBL) medium (bioMérieux), as they coproduced an ESBL and/or a plasmid-encoded cephalosporinase. The production of a carbapenemase was detected in 57.1%, 71.4%, 71.4%, and 100% of the E. coli isolates using the Carba NP test, the Rapidec Carba NP test (bioMérieux), a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) hydrolysis assay (Bruker), and the OXA-48 K-SeT assay (Coris BioConcept), respectively. Our results indicate that OXA-244-producing E. coli isolates are difficult to detect, which may lead to their silent spread., (Copyright © 2017 American Society for Microbiology.)

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., (Copyright © 2017 American Society for Microbiology.)

Background: The dissemination of extended-spectrum β-lactamase (ESBL)-producing bacteria presented a great concern worldwide. Gram-negative organisms such as Escherichia coli and Klebsiella pneumoniae are the most frequently isolated pathogens responsible for nosocomial infections., Objectives: The aim of this study was to investigate and to follow the emergence of resistance and the characterization of Extended-Spectrum Beta-Lactamases (ESBL) among broad-spectrum beta-lactam- Escherichia coli clinical isolates recovered from the military hospital and Habib Thameur hospital in Tunisia., Methods: A total of 113 E.coli isolates obtained during the period 2004 through 2012 showed a significant degree of multi-resistance. Among these strains, the double-disk synergy test confirmed the ESBL phenotype in 46 isolates. These included 32(70%) strains from Hospital A and 14(30%) from Hospital B., Results: The ESBL was identified as CTX-M-15. The ESBL resistance was transferred by a 60 kb plasmid CTXM-15-producing isolates were unrelated according to the PFGE analysis and characterization of the regions surrounding the blaCTX-M-15 showed the ISEcp1 elements located in the upstream region of the bla gene and 20 of them truncated by IS26., Conclusion: ESBL producing E. coli strains are a serious threat in the community in Tunisia and we should take into consideration any possible spread of such epidemiological resistance.

Objectives: KPC-producing pathogens exhibit variable carbapenem susceptibility levels, which is probably the result of the genetic environment of the bla KPC genes. Here we determined the transcriptional start sites (TSSs) and the expression of the bla KPC-2 gene in various genetic contexts and in different hosts ( Escherichia coli , Pseudomonas aeruginosa and Acinetobacter baumannii )., Methods: The bla KPC-2 genes along with the upstream sequences derived from Tn 4401b (structure A), Tn 4401b interrupted by Tn 3 /IS 26 (structure B) and Tn 4401b interrupted by Tn 5563 (structure C) were cloned in two E. coli shuttle vectors (pBBR1MCS.3 for expression studies in P. aeruginosa and pIM-arr2 for expression studies in A. baumannii ). MICs were determined by Etests. 5' RACE (where RACE stands for rapid amplification of cDNA ends) and quantitative RT-PCR experiments were performed to determine TSSs and transcription levels, respectively., Results: Depending on the bacterial host, different promoters were used for bla KPC-2 gene expression. The highest transcriptional level was obtained in P. aeruginosa with structure C, described only in P. aeruginosa . Tn 4401b (structure A), harbouring two promoters (P1 and P2), was the most efficient in E. coli and A. baumannii . This structure was also efficient in P. aeruginosa , although the same deduced promoter was not used (P1, instead of P2 used by E. coli and A. baumannii ). Two novel TSSs and putative promoters (P2b and P3b) were identified in structure B. In this structure, P2b and P3b were preferably used in E. coli and in P. aeruginosa , respectively, whereas P1 was used in A. baumannii ., Conclusions: We determined the preferred TSSs of the bla KPC gene in each species and described two novel deduced promoters in structure B., (© The Author 2017. 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 metallo-β-lactamase NDM-1 is among the most worrisome resistance determinants and is spreading worldwide among Gram-negative bacilli. A bleomycin resistance gene, ble MBL , downstream of the bla NDM-1 gene has been associated with resistance almost systematically. Here, we characterized the corresponding protein, BRP MBL , conferring resistance to bleomycin, an antitumoral glycopeptide molecule. We have determined whether the expression of the bla NDM-1 - ble MBL operon is inducible in the presence of carbapenems and/or bleomycin-like molecules using quantitative reverse transcription-PCR (qRT-PCR), determination of imipenem and zeocin MICs, and carbapenemase-specific activity assays. We showed that the bla NDM-1 - ble MBL operon is constitutively expressed. Using electrophoretic mobility shift and DNA protection assays performed with purified glutathione S -transferase (GST)-BRP MBL , we demonstrated that BRP MBL is able to bind and sequester bleomycin-like molecules, thus preventing bleomycin-dependent DNA degradation. In silico modeling confirmed that the mechanism of action required the dimerization of the BRP MBL protein in order to sequester bleomycin and prevent DNA damage. BRP MBL acts specifically on bleomycin-like molecules since cloning and expression of ble MBL in Staphyloccoccus aureus did not confer cross-resistance to any other antimicrobial glycopeptides such as vancomycin and teicoplanin., (Copyright © 2017 American Society for Microbiology.)

Extended-spectrum β-lactamases (ESBLs) of the CTX-M type are increasingly being reported worldwide, with more than 90 known variants. Clinical Escherichia coli isolate Bre-1 was isolated in 2009 and displayed an unusual ESBL phenotype, made of a synergy image between expanded cephalosporins and clavulanic acid discs and susceptibility to penicillins. E. coli Bre-1 harbored a novel CTX-M-encoding gene, designated bla(CTX-M-93). CTX-M-93 differed from CTX-M-27 by only a single L169Q substitution. Compared to CTX-M-27, CTX-M-93 conferred higher MICs of ceftazidime for E. coli (MIC of 8 versus 1.5 μg/ml) and decreased MICs of other expanded-cephalosporins (MIC of cefotaxime of 1 versus 32 μg/ml) and penicillins (MIC of ticarcillin of 0.5 versus >256 μg/ml). A comparison of enzymatic properties revealed that the L169Q substitution led to a decreased Km for ceftazidime (25.5 versus 330 μM) but decreased hydrolytic activity against good substrates, such as cefotaxime (kcat of 0.6 versus 113 s(-1)), probably owing to the alteration of the omega loop positioning during the catalytic process. The blaCTX-M-93 gene was surrounded by the ISEcp1 and IS903 elements and inserted onto a 150-kb non-self-transferrable IncF-type plasmid. E. coli Bre-1 belongs to phylogroup D and is of multilocus sequence type (MLST) 624, a sequence type found only in rare Spanish CTX-M-14-producing E. coli isolates. We have characterized a novel CTX-M variant, CTX-M-93, lacking significant penicillin hydrolysis but with increased ceftazidime hydrolysis.

Cloning, sequencing, and biochemical analysis identified a novel AmpC-type beta-lactamase conferring resistance to extended-spectrum cephalosporins in an Escherichia coli clinical isolate. This enzyme, exhibiting 14 amino acid substitutions compared to a reference AmpC cephalosporinase of E. coli, hydrolyzed ceftazidime and cefepime significantly.

Proteus mirabilis NEL-1 was isolated from a urine sample of a patient hospitalized in a long-term care facility. Strain NEL-1 produced a beta-lactamase with a pI of 5.2 conferring resistance to amoxicillin and amoxicillin-clavulanic acid. Sequencing of a PCR amplicon by using TEM-specific primers revealed a novel bla(TEM) gene, bla(TEM-67). TEM-67 was an IRT-1-like TEM derivative related to TEM-65 (Lys39, Cys244) with an additional Leu21Ile amino acid substitution in the leader peptide. The biochemical properties of TEM-67 were equivalent to those described for TEM-65. Analysis of sequences surrounding bla(TEM-67) revealed that it was located on a transposon, Tn1, which itself was located on a 48-kb non-self-transferable plasmid, pANG-1. Electroporation of plasmid pANG-1 into Escherichia coli DH10B resulted in the integration of bla(TEM-67) into the chromosome, whereas it remained episomal in the P. mirabilis CIP103181 reference strain. Further characterization of pANG-1 revealed the presence of two identical sequences on both sides of Tn1 that contained an IS26 insertion sequence followed by a novel colicin gene, colZ, which had 20% amino acid identity with other colicin genes. The characterization of this novel TEM derivative provides further evidence for the large diversity of plasmid-encoded beta-lactamases produced in P. mirabilis and for their spread to other enterobacterial species through transposable-element-mediated events.

OXA-244, an R214G variant of OXA-48, is silently spreading worldwide likely because of difficulties in detection using classical screening media. Here, we characterized two clinical isolates of Escherichia coli and Citrobacter youngae that displayed reduced susceptibility to carbapenems but were lacking significant carbapenemase activity as revealed by negative Carba NP test results. However, positive test results were seen for OXA-48-like enzymes by lateral flow immunoassays. WGS revealed the presence of a blaOXA-181-like gene that codes for OXA-484, an R214G variant of OXA-181. BlaOXA-484 gene was located on a 58.4-kb IncP1-like plasmid (pN-OXA-484), that upon transfer into E. coli HB4 with impaired permeability, conferred carbapenem and temocillin resistance (MICs > 32 mg/L). E. coli TOP10 (pTOPO-OXA-484) revealed reduced MICs in most substrates as compared to E. coli TOP10 (pTOPO-OXA-181), especially for imipenem (0.25 mg/L versus 0.75 mg/L) and temocillin (16 mg/L versus 1028 mg/L). Catalytic efficiencies of OXA-484 were reduced as compared to OXA-181 for most ß-lactams including imipenem and temocillin with 27.5- and 21.7-fold reduction, respectively. Molecular modeling confirmed that the salt bridges between R214, D159, and the R1 substituent's carboxylate group of temocillin were not possible with G214 in OXA-484, explaining the reduced affinity for temocillin. In addition, changes in active site's water network may explain the decrease in hydrolysis rate of carbapenems. OXA-484 has weak imipenem and temocillin hydrolytic activities, which may lead to silent spread due to underdetection using selective screening media or biochemical imipenem hydrolysis confirmatory tests. [ABSTRACT FROM AUTHOR]

Abscesses represent the most prominent emerging problem in the red meat industry, leading to great economic constraints and public health hazards. Data on etiological agents present in these purulent lesions in Algeria are very scarce. The aim of this study was to identify the bacteria responsible for these abscesses and to determine their antibiotic susceptibility profiles. A total of 123 samples of abscesses from 100 slaughtered sheep and 23 slaughtered cattle were cultured in several media. A total of 114 bacterial isolates were cultured from 103 abscesses. Bacteria were identified using MALDI–TOF, and antibiotic susceptibility was determined by the disk diffusion method on Mueller–Hinton agar. A total of 73.6% (n = 84) corresponded to Enterobacterales, of which four were multidrug-resistant (MDR). These isolates, together with Staphylococcus aureus, coagulase negative Staphylococci, and seven randomly chosen susceptible Escherichia coli isolates, were further characterized using WGS. Resistome analysis of the four MDR Enterobacterales isolates revealed the presence of OXA-48 carbapenemase in two Klebsiella pneumoniae ST985 and one E. coli ST10 isolates and a CTX-M-15 ESBL in one E. coli isolate ST1706. Two coagulase-negative Staphylococci isolates were found to carry the mecA gene. WGS showed the presence of different resistance genes and virulence genes. Our study revealed 5% of MDR Enterobacterales (including ESBLs and carbapenemases) identified from abscesses, thus urging the need for abscess monitoring in slaughterhouses. [ABSTRACT FROM AUTHOR]

This article discusses a study on antimicrobial resistance (AMR) in Lebanon, specifically focusing on the presence of the blaNDM-5 gene in E. coli isolates. The study found that blaNDM-5-harbouring E. coli ST617 was present in Syrian refugees, their animals, and the camp environment, posing a health risk to both the refugees and the hosting community. The study emphasizes the importance of integrated surveillance programs and One Health studies to address AMR in vulnerable populations and highlights the role of war, mass migration, and displacement in the spread of AMR. Funding for the study was provided by various organizations, and ethical considerations were taken into account. [Extracted from the article]

This article discusses the emergence of a multidrug-resistant strain of Enterobacter xiangfangensis (Exn692) in a patient with COVID-19 and diabetes in Lebanon. The strain showed resistance to 37 antibiotics from different classes and carried genes that encoded resistance to these antibiotics. However, it was susceptible to a few antibiotics. The study emphasizes the challenges in treating infections caused by multidrug-resistant bacteria and the importance of effective antimicrobial stewardship. It also highlights the need for monitoring the evolution of Enterobacter xiangfangensis and developing accurate identification tools to understand its epidemiology, pathogenesis, and antibiotic resistance properties. [Extracted from the article]

Here, we characterized the first French NDM-9-producing Acinetobacter baumannii isolate. A. baumannii 13A297, which belonged to the STPas25 (international clone IC7), was highly resistant to β-lactams including cefiderocol (MIC >32 mg/L). Whole genome sequencing (WGS) using both Illumina and Oxford Nanopore technologies revealed a 166-kb non-conjugative plasmid harboring a blaNDM-9 gene embedded in a Tn125 composite transposon. Complementation of E. coli DH5α and A. baumannii CIP70.10 strains with the pABEC plasmid carrying the blaNDM-1 or blaNDM-9 gene, respectively, resulted in a significant increase in cefiderocol MIC values (16 to >256-fold), particularly in the NDM-9 transformants. Interestingly, steady-state kinetic parameters, measured using purified NDM-1 and NDM-9 (Glu152Lys) enzymes, revealed that the affinity for cefiderocol was 3-fold higher for NDM-9 (Km = 53 μM) than for NDM-1 (Km = 161 μM), leading to a 2-fold increase in catalytic efficiency for NDM-9 (0.13 and 0.069 μM−1 . s−1, for NDM-9 and NDM-1, respectively). Finally, we showed by molecular docking experiments that the residue 152 of NDM-like enzymes plays a key role in cefiderocol binding and resistance, by allowing a strong ionic interaction between the Lys152 residue of NDM-9 with both the Asp223 residue of NDM-9 and the carboxylate group of the R1 substituent of cefiderocol. [ABSTRACT FROM AUTHOR]

Piperacillin-tazobactam resistance (P/T-R) is increasingly reported among Escherichia coli isolates. Although in vitro experiments have suggested that bla TEM gene plays a key role in the P/T-R acquisition, no clinical in vivo study has yet confirmed the role of bla TEM or other genes. Therefore, we aimed to identify the mechanisms underlying P/T-R by following up patients with E. coli complicated intra-abdominal infections (cIAI) who experienced P/T treatment failure. Four pairs of strains, clonally related from four patients, were isolated both before and after treatment with P/T dosed at 4 g/0.5 g intravenously. The P/T MIC was tested using broth microdilution, and β-lactamase activity was determined in these isolates. Whole-genome sequencing (WGS) was performed to decipher the role of bl a TEM and other genes associated with P/T-R. Changes in the outer membrane protein (OMP) profile were analyzed using SDS-PAGE, and bla TEM and ompC transcription levels were measured by RT-qPCR. In addition, in vitro competition fitness was performed between each pairs of strains (P/T-susceptible vs. P/T-resistant). We found a higher copy number of bla TEM gene in P/T-R isolates, generated by three different genetic events: (1) IS 26 -mediated duplication of the bla TEM gene, (2) generation of a small multicopy plasmid (ColE-like) carrying bla TEM , and (3) adaptive evolution via reduction of plasmid size, leading to a higher plasmid copy number. Moreover, two P/T-R strains showed reduced expression of OmpC. This study describes the mechanisms involved in the acquisition of P/T-R by E. coli in patients with cIAI. The understanding of P/T-R evolution is crucial for effectively treating infected patients and preventing the spread of resistant microorganisms. • IS26-mediated duplication of the bla TEM gene is involved in the acquisition of P/T-R by E. coli. • Generation of a small multicopy plasmid carrying bla TEM is involved in the acquisition of P/T-R by E. coli. • Adaptive evolution via plasmid size reduction, leading to a higher plasmid copy number is involved in the P/T-R by E. coli. • Reduction of OmpC expression is associated with P/T-R in E. coli. [ABSTRACT FROM AUTHOR]

Background: Despite the fact that carbapenem-resistant Enterobacterales (CRE) mostly cause urinary tract infections (UTIs), only few studies have focused on the efficacity of mecillinam against these CRE.Objectives: To evaluate the mecillinam susceptibility of a huge collection of CRE, including carbapenemase-producing Enterobacterales (CPE) and non-CPE (ESBL and AmpC producers with decreased permeability of the outer membrane).Methods: A total of 8310 non-duplicate clinical CRE, including 4042 OXA-48-like producers, 1094 NDM producers, 411 VIM producers, 174 KPC producers, 42 IMI producers, 153 multiple-carbapenemase producers and 45 isolates producing other types of carbapenemases (such as IMP-like enzymes or GES-5), were included in the study. WGS was performed on all CPE using Illumina technology. Categorization of susceptibility to mecillinam was performed using disc diffusion (mecillinam discs at 10 μg; I2A, France) according to EUCAST recommendations. The results were interpreted according to EUCAST guidelines (S ≥15 mm).Results: Significantly higher susceptibility rates were observed for carbapenem-resistant Proteus spp. (85%) and carbapenem-resistant Escherichia coli (84%), which are the two most common species responsible for UTIs, than for Klebsiella pneumoniae (67%), Enterobacter cloacae complex (75%), Citrobacter spp. (65%), Serratia spp. (34%) and Morganella morganii (12%). Susceptibility rates were 84%, 71% and 91% for OXA-48-like, NDM and IMI producers and 70% for non-CPE CRE. Mecillinam was less active against VIM and KPC producers (14% and 0%, respectively).Conclusions: Mecillinam might be an alternative for the treatment of infections due to CRE, particularly UTIs, except for VIM and KPC producers and for M. morganii and Serratia spp species. [ABSTRACT FROM AUTHOR]

The differential expression of VIM-1 in Atlantibacter hermannii WEB-2 and Enterobacter hormaechei ssp. hoffmannii WEB-1 clinical isolates from a rectal swab of a hospitalized patient in France was investigated. A. hermannii WEB-2 was resistant to all β-lactams except carbapenems. It produced ESBL SHV-12, but the Carba NP test failed to detect any carbapenemase activity despite the production of VIM-1. Conversely, E. hormaechei WEB-1, previously recovered from the same patient, was positive for the detection of carbapenemase activity. The bla VIM–1 gene was located on a plasmid and embedded within class 1 integron. Both plasmids were of the same IncA incompatibility group and conferred the same resistance pattern when electroporated in Escherichia coli TOP10 or Enterobacter cloacae CIP7933. Quantitative RT-PCR experiments indicated a weaker replication of pWEB-2 in A. hermannii as compared to E. hormaechei. An isogenic mutant of A. hermannii WEB-2 selected after sequential passages with increased concentrations of imipenem possessed higher MICs for carbapenems and cephalosporins including cefiderocol, higher levels of the bla VIM–1 gene transcripts, and detectable carbapenemase activity using the Carba NP test. Assessment of read coverage demonstrated that a duplication of the region surrounding bla VIM–1 gene occurred in the A. hermannii mutant with detectable carbapenemase activity. The lack of detection of the VIM-1 carbapenemase activity in A. hermannii WEB-2 isolate was likely due to a weak replication of the IncA plasmid harboring the bla VIM–1 gene. Imipenem as selective pressure led to a duplication of this gene on the plasmid and to the restoration of a significant carbapenem-hydrolyzing phenotype. [ABSTRACT FROM AUTHOR]

CTX-M-producing Escherichia coli are spreading since 1999 both in clinical and in community settings. Environmental samples such as rivers have also been pointed out as being vectors for ESBL producers. In this report, we have investigated the presence and the diversity of CTX-M-producing E. coli isolates in two samplings of the Seine River (next to Notre Dame), Paris France, performed in June 2016 and 2017. The total number of bacteria growing on the selective ChromID ESBL agar was 3.1 × 105 cfu/L (23.8% of all growing bacteria) in 2016, whereas it was 100-fold lower in 2017 (3 × 103 cfu/L; 8.3% of all growing bacteria). However, among them, the prevalence of ESBL-producing E. coli increased from <0.1 to 1.1% in one-year. ESBLs were exclusively of the CTX-M-type: CTX-M-1 (n = 5), CTX-M-15 (n = 7), CTX-M-14 (n = 1), and CTX-M-27 (n = 2). The isolates belonged to several multi locus sequence types, and a wide diversity of incompatibility groups of plasmids were identified in those E. coli isolates. The occurrence and diversity of E. coli isolates belonging to many clones and producing many CTX-M-variants have been identified in our study. The presence of these bacteria in rivers that are open again for recreational usage (swimming) is worrying as it may contribute to further dissemination of ESBL producers in the community. [ABSTRACT FROM AUTHOR]

Background: Carbapenem-resistant Enterobacteriaceae are considered by WHO as "critical" priority pathogens for which novel antibiotics are urgently needed. The dissemination of carbapenemase-producing Escherichia coli (CP-Ec) in the community is a major public health concern. However, the global molecular epidemiology of CP-Ec isolates remains largely unknown as well as factors contributing to the acquisition of carbapenemase genes. Methods: We first analyzed the whole-genome sequence and the evolution of the E. coli sequence type (ST) 410 and its disseminated clade expressing the carbapenemase OXA-181. We reconstructed the phylogeny of 19 E. coli ST enriched in CP-Ec and corresponding to a total of 2026 non-redundant isolates. Using the EpiCs software, we determined the significance of the association between specific mutations and the acquisition of a carbapenemase gene and the most probable order of events. The impact of the identified mutations was assessed experimentally by genetic manipulations and phenotypic testing. Results: In 13 of the studied STs, acquisition of carbapenemase genes occurred in multidrug-resistant lineages characterized by a combination of mutations in ftsI encoding the penicillin-binding protein 3 and in the porin genes ompC and ompF. Mutated ftsI genes and a specific ompC allele related to that from ST38 inducing reduced susceptibility to diverse β-lactams spread across the species by recombination. We showed that these mutations precede in most cases the acquisition of a carbapenemase gene. The ompC allele from ST38 might have contributed to the selection of CP-Ec disseminated lineages within this ST. On the other hand, in the pandemic ST131 lineage, CP-Ec were not associated with mutations in ompC or ftsI and show no signs of dissemination. Conclusions: Lineages of CP-Ec have started to disseminate globally. However, their selection is a multistep process involving mutations, recombination, acquisition of antibiotic resistance genes, and selection by β-lactams from diverse families. This process did not yet occur in the high-risk lineage ST131. [ABSTRACT FROM AUTHOR]

Background: KPC-like carbapenemases have spread worldwide with more than 30 variants identified that differ by single or double amino-acid substitutions.Objectives: To describe the steady-state kinetic parameters of KPC-28, which differs from KPC-2 by a H274Y substitution and the deletion of two amino acids (Δ242-GT-243).Methods: The blaKPC-2, blaKPC-3, blaKPC-14 and blaKPC-28 genes were cloned into a pTOPO vector for susceptibility testing or into pET41b for overexpression, purification and subsequent kinetic parameter (Km, kcat) determination. Molecular docking experiments were performed to explore the role of the amino-acid changes in the carbapenemase activity.Results: Susceptibility testing revealed that Escherichia coli producing KPC-28 displayed MICs that were lower for carbapenems and higher for ceftazidime and ceftazidime/avibactam as compared with KPC-2. The catalytic efficiencies of KPC-28 and KPC-14 for imipenem were 700-fold and 200-fold lower, respectively, than those of KPC-2, suggesting that Δ242-GT-243 in KPC-28 and KPC-14 is responsible for reduced carbapenem hydrolysis. Similarly, the H274Y substitution resulted in KPC-28 in a 50-fold increase in ceftazidime hydrolysis that was strongly reversed by clavulanate.Conclusions: We have shown that KPC-28 lacks carbapenemase activity, has increased ceftazidime hydrolytic activity and is strongly inhibited by clavulanate. KPC-28-producing E. coli isolates display an avibactam-resistant ESBL profile, which may be wrongly identified by molecular and immunochromatographic assays as the presence of a carbapenemase. Accordingly, confirmation of carbapenem hydrolysis will be mandatory with assays based solely on blaKPC gene or gene product detection. [ABSTRACT FROM AUTHOR]

Objectives: Available commercial tools (molecular methods or immunochromatographic assays) usually allow the detection of the five most prevalent carbapenemases (KPC, NDM, VIM, IMP and OXA-48-like), but miss minor carbapenemases. Here, we characterize two enterobacterial isolates with reduced susceptibility to carbapenems and negative for the most commonly encountered carbapenemase genes.Methods: Enterobacter hormaechei and Citrobacter freundii isolates were recovered from a bile sample and rectal screening, respectively. Both isolates were investigated by WGS. Resistance genes were detected using ResFinder. The blaTMB-1-harbouring plasmid was reconstructed using CLC genomic workbench 10.0 and was annotated using the RAST tool. Transfer frequency was determined by conjugation experiments using the laboratory strain Escherichia coli J53.Results: The two isolates were resistant to broad-spectrum cephalosporins and carbapenems. WGS revealed the presence of blaTMB-1, which has previously only been described in non-fermenters. blaTMB-1 was located within an ISKpn19-based composite class 1 transposon. Comparative genomics revealed that this structure was carried on a conjugative IncN-type plasmid within an integration hotspot. Conjugation experiments revealed high transfer frequencies of ∼1 × 10-3.Conclusions: To the best of our knowledge, this study corresponds to the first report of Tripoli MBL 1-producing Enterobacteriaceae. Despite always being described as likely to be chromosomally located in non-fermenters, the blaTMB-1 gene is now found to be carried by a conjugative plasmid among Enterobacteriaceae, raising concern about the possible dissemination of this carbapenemase. The blaTMB-1 gene should now be suspected when PCRs targeting the main carbapenemases remain negative. [ABSTRACT FROM AUTHOR]

Objectives: To describe a novel plasmid-borne class D carbapenemase (CHDL) named OXA-427 identified in several Enterobacteriaceae clinical isolates from nine patients in one Belgian hospital.Methods: OXA-427-producing isolates were analysed by an electrochemical imipenem hydrolysis method (BYG Carba test), Carba NP test, conventional phenotypic assays and by molecular methods (PCR, whole sequencing of the OXA-427-encoding plasmid and cloning). The antimicrobial resistance profile of OXA-427 was analysed by expression of the cloned gene in Escherichia coli DH10B and J53.Results: Eleven OXA-427-producing Enterobacteriaceae isolates of various species were identified from clinical specimens of nine patients between March 2012 and June 2014. OXA-427 shares only 22%-29% amino acid identity with OXA-48-like enzymes and other acquired CHDL (e.g. OXA-23, -24/40 and -58 of Acinetobacter spp.). Conversely, it appeared closely related to the chromosomal class D β-lactamase of Aeromonas media, Aeromonas hydrophila and Aeromonas sobria (99%, 89% and 77% of identity, respectively). When expressed in E. coli, OXA-427 hydrolysed imipenem and conferred resistance to extended-spectrum cephalosporins (mostly ceftazidime), penicillins including temocillin, and reduced susceptibility to carbapenems. The blaOXA-427 gene was located in a 45 kb resistance island on a 177 kb IncA/C plasmid.Conclusions: OXA-427 is a novel CHDL most closely related to chromosomal class D β-lactamase of A. media WS. It confers resistance to penicillins, ceftazidime and aztreonam and in some instances to carbapenems. OXA-427, which is not detectable by classical molecular tests, caused a protracted outbreak in one university hospital over a 2 year period. [ABSTRACT FROM AUTHOR]

Objectives KPC-producing Pseudomonas aeruginosa are increasingly isolated in the Americas and in the Caribbean islands. Here, we determined the whole-plasmid sequence of two plasmids carrying the blaKPC-2 gene from multidrug-resistant P. aeruginosa clinical isolates from Colombia. Methods The two plasmids, pCOL-1 and pPA-2, were transferred to Escherichia coli recipient strain TOP10 and completely sequenced using high-throughput pyrosequencing for pCOL-1 and classical Sanger sequencing for pPA-2. Results Both plasmids could be transferred to E. coli by transformation and displayed no other resistance marker besides KPC. Plasmid pCOL-1 was 31 529 bp in size, contained 31 open reading frames (ORFs) and belonged to the IncP-6 replicon group. It exhibited genes involved in replication, mobilization and partitioning, but none involved in conjugation. Plasmid pPA-2 was 7995 bp in size and contained seven ORFs. It exhibited a replicase gene of IncU, but was lacking genes involved in mobilization, partitioning and conjugation. Only 2072 bp matched Tn4401, including the blaKPC-2 gene, part of ISKpn6 and a 73 bp segment located upstream of the blaKPC-2 gene, containing the P1 promoter. Sequence identity was interrupted by a Tn3 transposon, itself interrupted by an IS26 element inserted within the β-lactamase blaTEM-1 gene. Conclusions Here we present the genetic features of the very first plasmids carrying the blaKPC-2 gene from P. aeruginosa. The emergence of the blaKPC-2 gene on unrelated plasmids, differing in size and in incompatibility group, and harbouring different genetic structures containing the blaKPC-2 genes in P. aeruginosa isolates suggests that this resistance trait may follow a dissemination scheme in P. aeruginosa similar to that seen in Enterobacteriaceae. [ABSTRACT FROM PUBLISHER]

Discusses the characterization of the genetic environment of the gene encoding the Escherichia coli extended-spectrum beta-lactamase that revealed the presence of a plasmid-located class 1 integron, In53. Condition showing that In53 is a truncated integron located on a composite transposon named Tn2000.

Studies the variations in DNA sequence insertion and compares the restriction fragment length polymorphism (RFLP) of subclones made from individual viable cells recovered from a stab culture of the resting Escherichia coli K-12 strain W3110. Nature of polymorphism; Pedigree of the old stab culture; Physiological studies with genetic variants; Nature of DNA rearrangements.

Escherichia coli is the most frequent Gram-negative bacilli involved in intra-abdominal infections. However, despite high mortality rates associated with biliary tract infections due to E. coli, there is no study focusing on this pathogen. In this study, we have characterized a group of 15 E. coli isolates obtained from 12 patients with biliary tract infections. Demographic and clinical data of the patients were recovered. Phylogeny, resistome, and virulome analysis through whole genome sequencing and biofilm formation were investigated. Among the 15 E. coli isolates, no predominant sequence type (ST) was identified, although 3 of them belonged to unknown STs (20%). Resistance to ampicillin, amoxicillin/clavulanic acid, cotrimoxazole, and quinolones was more present in these isolates; whereas, third and fourth generation cephalosporins, carbapenems, amikacin, tigecycline, and colistin were highly active. Moreover, high diversity of virulence factors has been found, with sfa, fimH, and gad the most frequently detected genes. Interestingly, 26.6% of the E. coli isolates were high biofilm-producers. Altogether, our data characterized for the first time E. coli isolates associated with biliary tract infections in terms of genomic relationship, resistome, and virulome. [ABSTRACT FROM AUTHOR]