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Class C β-lactamases or cephalosporinases can be classified into two functional groups (1, 1e) with considerable molecular variability (≤20% sequence identity). These enzymes are mostly encoded by chromosomal and inducible genes and are widespread among bacteria, including Proteobacteria in particular. Molecular identification is based principally on three catalytic motifs ( 64 SXSK, 150 YXN, 315 KTG), but more than 70 conserved amino-acid residues (≥90%) have been identified, many close to these catalytic motifs. Nevertheless, the identification of a tiny, phylogenetically distant cluster (including enzymes from the genera Legionella , Bradyrhizobium , and Parachlamydia ) has raised questions about the possible existence of a C2 subclass of β-lactamases, previously identified as serine hydrolases. In a context of the clinical emergence of extended-spectrum AmpC β-lactamases (ESACs), the genetic modifications observed in vivo and in vitro (point mutations, insertions, or deletions) during the evolution of these enzymes have mostly involved the Ω- and H-10/R2-loops, which vary considerably between genera, and, in some cases, the conserved triplet 150 YXN. Furthermore, the conserved deletion of several amino-acid residues in opportunistic pathogenic species of Acinetobacter, such as A. baumannii, A. calcoaceticus, A. pittii and A. nosocomialis (deletion of residues 304-306), and in Hafnia alvei and H. paralvei (deletion of residues 289-290), provides support for the notion of natural ESACs. The emergence of higher levels of resistance to β-lactams, including carbapenems, and to inhibitors such as avibactam is a reality, as the enzymes responsible are subject to complex regulation encompassing several other genes ( amp R, amp D, amp G, etc.). Combinations of resistance mechanisms may therefore be at work, including overproduction or change in permeability, with the loss of porins and/or activation of efflux systems.

Extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E) have been classified in the group of resistant bacteria of highest priority. We determined the prevalence of ESBL-E collected in feces from household and shelter pets in Guadeloupe (French West Indies). A single rectal swab was taken from 125 dogs and 60 cats between June and September 2019. The prevalence of fecal carriage of ESBL-E was 7.6% (14/185, 95% CI: 4.2-12.4), within the range observed worldwide. The only risk factor associated with a higher prevalence of ESBL-E rectal carriage was a stay in a shelter, suggesting that refuges could be hotspots for their acquisition. All but one ( Klebsiella pneumoniae from a cat) were Escherichia coli . We noted the presence of a bla CTX-M -1 /IncI1-Iγ/sequence type (ST3) plasmid in 11 ESBL-producing E. coli isolates belonging to ST328 ( n = 6), ST155 ( n = 4) and ST953 ( n = 1). A bla CTX-M -15 gene was identified in the three remaining ESBL-E isolates. The bla CTX-M -1 and most of the antimicrobial resistance genes were present in a well-conserved large conjugative IncI1-Iγ/ST3 plasmid characterized by two accessory regions containing antibiotic resistance genes. The plasmid has been detected worldwide in E. coli isolates from humans and several animal species, such as food-producing animals, wild birds and pets, and from the environment. This study shows the potential role of pets as a reservoir of antimicrobial-resistant bacteria or genes for humans and underlines the importance of basic hygiene measures by owners of companion animals., 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 © 2022 Gruel, Couvin, Guyomard-Rabenirina, Arlet, Bambou, Pot, Roy, Talarmin, Tressieres, Ferdinand and Breurec.)

The increasing incidence of carbapenemase-producing Gram-negative bacilli (C-PGNB) represents a major public health challenge. Rapid detection of digestive colonization with C-PGNB is fundamental to control their spread. We performed the validation of a rapid protocol for C-PGNB detection directly on rectal swabs. We developed a protocol combining enrichment by a rapid selective subculture of the rectal swab medium and realization of a Resist-4 O.K.N.V. K-SeT test on the bacterial pellet obtained. The limit of detection and performances of this protocol were validated in vitro on 52 C-PGNB strains spiked on a calibrated sample suspension and confirmed in clinical settings on 144 rectal swabs sampled from patients with C-PGNB digestive colonization ( n  = 48) and controls (patients with extended-spectrum beta-lactamase [ESBL] colonization [ n  = 48] and without carbapenemase/ESBL [ n  = 48]). The protocol detected, with 100% sensitivity, the presence of the 15 OXA-48-, 14 KPC-, 13 NDM-, and 10 VIM-producing GNB from 10 3 CFU/ml. The limit of detection was 2 × 10 2 CFU/ml. Among the 48 C-PGNB-containing rectal swabs of the validation cohort, 46 were accurately detected. False negative were observed for 1 NDM-producing Acinetobacter baumannii strain and 1 OXA-48-producing Escherichia coli strain. The 96 control swabs were negative. Sensitivity and specificity for C-PGNB detection were 97.7% (95% confidence interval [CI], 87.7 to 100) and 100% (95% CI, 96.2 to 100). The negative likelihood ratio was 0.04 (95% CI, 0.01 to 0.16). Considering a C-PGNB digestive colonization prevalence between 0.01% and 0.1%, positive and negative predictive values were 100%. Our protocol is a rapid and low-cost method detecting accurately the digestive colonization with carbapenemase-producing Enterobacteriaceae in 4 h without any requirement for specific equipment., (Copyright © 2021 American Society for Microbiology.)

We undertook a comprehensive comparative analysis of a collection of 30 small (<25 kb) non-conjugative Escherichia coli plasmids previously classified by the gene sharing approach into 10 families, as well as plasmids found in the National Center for Biotechnology Information (NCBI) nucleotide database sharing similar genomic sequences. In total, 302 mobilizable (belonging to 2 MOB rep and 5 MOB RNA families) and 106 non-transferable/relaxase-negative (belonging to three ReL RNA families) plasmids were explored. The most striking feature was the specialization of the plasmid family types that was not related to their transmission mode and replication system. We observed a range of host strain specificity, from narrow E. coli host specificity to broad host range specificity, including a wide spectrum of Enterobacteriaceae . We found a wide variety of toxin/antitoxin systems and colicin operons in the plasmids, whose numbers and types varied according to the plasmid family type. The plasmids carried genes conferring resistance spanning almost all of the antibiotic classes, from those to which resistance developed early, such as sulphonamides, to those for which resistance has only developed recently, such as colistin. However, the prevalence of the resistance genes varied greatly according to the family type, ranging from 0 to 100 %. The evolutionary history of the plasmids based on the family type core genes showed variability within family nucleotide divergences in the range of E. coli chromosomal housekeeping genes, indicating long-term co-evolution between plasmids and host strains. In rare cases, a low evolutionary divergence suggested the massive spread of an epidemic plasmid. Overall, the importance of these small non-conjugative plasmids in bacterial adaptation varied greatly according to the type of family they belonged to, with each plasmid family having specific hosts and genetic traits.

Objectives: Biofilm production in extended spectrum β-lactamase (ESBL)-producing Enterobacteriaceae provides a favourable environment for the exchange of antibiotic-resistance genes and could facilitate widespread dissemination. We aimed to assess biofilm development in ESBL-producing E. coli and K. pneumoniae isolates and determine how development relates to microbiological characteristics and clinical outcomes., Methods: 147 ESBL-producing E. coli and 82 K. pneumoniae were genetically characterized. Biofilm formation was measured at 1.5, 4, 6, and 24 h during culture in blood heart infusion using a microbead immobilization assay (BioFilm Ring test ® ). Results were given as biofilm formation index (BFI) with lower values indicating increased presence of biofilm (range = 0-21)., Results: In total, 57.1% of strains were strong producers of biofilm (BFI < 2), whereas 13.4% lacked biofilm production (BFI > 18). Standard biofilm production (BFI < 7) was common in E. coli isolates (61.9%). For E. coli, biofilm production was less frequently observed in ST131 clones (p = 0.03) but more frequently in strains harbouring toxin (p = 0.008) or adhesin (p = 0.008) virulence factor genes. Despite almost all K. pneumoniae having standard biofilm production (90.2%), there was a 2.4-times higher odds of observing biofilm in ST29/147/323 versus other ST-types (p = 0.13). Patients with standard biofilm producing isolates were not at increased risk of transfer to intensive-care (odds-ratio=2.80, 95%CI=0.59-13.21) or death within 12-months (odds-ratio=1.61, 95%CI=0.75-3.43)., Conclusion: In these ESBL-producing strains, biofilm production is linked to certain virulence factors in E. coli and is common in K. pneumoniae. Further exploration of whether biofilm production increases dissemination and risk of severe clinical outcomes is needed in larger collections of isolates., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Objective: To genetically characterize clusters of virulence factors (VFs) among extended spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae and assess whether these clusters are associated with genetic determinants or clinical outcomes., Methods: One hundred forty-eight E. coli and 82 K . pneumoniae clinical isolates were obtained from 213 patients in Paris, France. Isolates underwent ESBL characterization, MultiLocus Sequence Typing (MLST) typing and phylogenetic group identification. Detection of ten E. coli and seven K . pneumoniae VF-encoding genes were assessed, from which a k -medians partition algorithm with Jaccard similarity measure was used to construct clusters., Results: CTX-M was the predominant ESBL and susceptibility to trimethoprim-sulfamethoxazole (32%), ciprofloxacin (22%) and aminoglycosides (32%) was low. In E. coli , there were five identified clusters, with significantly different distributions of ESBL-sequence type ( P <0.001), ST131 ( P <0.001) and phylogenetic group ( P =0.001) between clusters. "Siderophore exclusive", "siderophore exclusive with iroN " and "adhesin sfa/papGIII-rich" clusters had higher 12-month mortality rates compared to others (49% vs 22%, respectively, P =0.02). In K. pneumoniae , three different clusters, with significantly different distributions of aminoglycoside-sensitivity ( P <0.004), MLST-type ( P <0.001) and relaxase plasmids ( P =0.001) were described., Conclusion: Distinct clusters of E. coli and K. pneumoniae VFs are observed within ESBL-producing isolates and are strongly associated with several genetic determinants. Their association with overall morbidity and mortality requires further evidence., Competing Interests: Disclosure LS received a grant from the “Fondation pour la Recherche Médicale” (DEA20140630021). AB received post-doctoral funding from SIDACTION. PB received a Martinson-Luepker Student Travel Award from the University of Minnesota for the work presented in this manuscript. The authors report no other conflicts of interest in this work.

Travelers are at high risk of acquiring multi-drug resistant Enterobacteriaceae (MRE) while traveling abroad. Acquisition of extended spectrum beta-lactamase producing Enterobacteriaceae (ESBL-E) while traveling has been extensively described, but not that of plasmid-mediated cephalosporinase producing Enterobacteriaceae (pAmpC-E). Here, we characterized the pAmpC-E acquired in 574 French travelers to tropical areas enrolled in the VOYAG-R study. Among the 526 MRE isolated at return, 57 (10.8%) from 49 travelers were pAmpC-E. The acquisition rate of pAmpC-E was 8.5% (49/574) ranging from 12.8% (25/195) in Asia, 7.6% (14/184) in Latin America to 5.1% (10/195) in Africa. The highest acquisition rates were observed in Peru (21.9%), India (21.4%) and Vietnam (20%). The carriage of pAmpC-E decreased quickly after return with 92.5% of colonized travelers being negative at one month. Most enzymes were CMY types (96.5%, n = 55, only met in Escherichia coli), including 40 CMY-2 (70.2%), 12 CMY-42 (21.1%), 1 CMY-6 and two new CMY-2 variants. The remaining were two DHA observed in Klebsiella pneumoniae. CMY-2 producing strains were acquired worldwide whereas CMY-42, except for one, were all acquired in Asia. BlaCMY-2 genes were associated with different plasmid types, including IncI1 (45. 2%), IncF (10%), IncF-IncI (7.5%), IncA/C (5%) and IncR (2.5%) whereas blaCMY-42 were all associated with IncI1 plasmids. Even though the pAmpC-E acquisition rate was much lower than that of ESBL-E, it was significant, especially in Asia, showing that pAmpC-E, especially CMY-type producing E. coli have spread in the community settings of tropical regions., Competing Interests: The authors have declared that no competing interests exist.

Objectives: To describe clinical and molecular characteristics of an outbreak due to metallo-β-lactamases (MBLs) producing Klebsiella pneumoniae collected at Charles Nicolle Hospital of Tunis and to analyze the impact of outer membrane porin (OMP) loss on carbapenem resistance levels., Methods: Between 2010 and 2015, 178 carbapenem-resistant Enterobacteriaceae were isolated. Screening for MBL production was performed using combined disk diffusion method, with imipenem and ethylene diamine tetraacetic acid (EDTA) as inhibitors. Resistance genes and virulence factors were identified by polymerase chain reaction (PCR) and sequencing. Genotyping was performed by pulsed-field gel electrophoresis and multilocus sequence typing. Genetic environment of carbapenemase genes was determined by PCR mapping. Conjugation assays were performed, and plasmids were assigned to incompatibility groups by PCR-based replicon typing. OMPs were profiled by sodium dodecyl sulfate-polyacrilamide gel electrophoresis, and porin genes were sequenced., Results: Nineteen K. pneumoniae (10.6%) showing MBL activity were isolated from patients hospitalized on four different wards. NDM-1 was the only MBL identified, in association with bla OXA-48 . All strains lacked at least one OMP, and carbapenem resistance levels were remarkably elevated in strains lacking OmpK35 and OmpK36. bla NDM-1 was located in IncFIA-type conjugative plasmid, with the same genetic context in all strains. The epidemiological diffusion of bla NDM-1 was due to two clones, one major clone belonging to sequence type (ST) 147 (n = 16) and the other clone belonging to ST307 (n = 3)., Conclusions: This study describes an outbreak of NDM-1-producing K. pneumoniae strains, isolated from a Tunisian hospital, caused by two clones belonging to ST147 and ST307; and highlights the role of OMPs loss, in combination with β-lactamase expression, in conferring high carbapenem resistance.

To understand the evolutionary dynamics of extended-spectrum β-lactamase (ESBL)-encoding genes in Escherichia coli, we undertook a comparative genomic analysis of 116 whole plasmid sequences of human or animal origin isolated over a period spanning before and after the use of third-generation cephalosporins (3GCs) using a gene-sharing network approach. The plasmids included 82 conjugative, 22 mobilizable and 9 non-transferable plasmids and 3 P-like bacteriophages. ESBL-encoding genes were found on 64 conjugative, 6 mobilizable, 2 non-transferable plasmids and 2 P1-like bacteriophages, indicating that these last three types of mobile elements also play a role, albeit modest, in the diffusion of the ESBLs. The network analysis showed that the plasmids clustered according to their genome backbone type, but not by origin or period of isolation or by antibiotic-resistance type, including type of ESBL-encoding gene. There was no association between the type of plasmid and the phylogenetic history of the parental strains. Finer scale analysis of the more abundant clusters IncF and IncI1 showed that ESBL-encoding plasmids and plasmids isolated before the use of 3GCs had the same diversity and phylogenetic history, and that acquisition of ESBL-encoding genes had occurred during multiple independent events. Moreover, the blaCTX-M-15 gene, unlike other CTX-M genes, was inserted at a hot spot in a blaTEM-1-Tn2 transposon. These findings showed that ESBL-encoding genes have arrived on wide range of pre-existing plasmids and that the successful spread of blaCTX-M-15 seems to be favoured by the presence of well-adapted IncF plasmids that carry a Tn2-blaTEM-1 transposon.

Objectives: Incidence of extended-spectrum β-lactamase-producing Gram-negative bacilli (ESBL-PE-GNB)-related infections is worryingly increasing worldwide. ESBL-PE-GNB detection directly on bronchial aspirate samples (BAS) performed for suspected pneumonia may help save empirical carbapenems. Our objectives were to optimize β-LACTA™ test (BLT) realization and evaluate BLT performance for ESBL-PE-GNB detection directly on BAS., Methods: We studied BLT technical optimization using BAS of different matrix types spiked with increasing concentrations of CTX-M-15-producing Klebsiella pneumoniae; in vitro validation of BLT diagnostic performance on 17 ESBL enzymes, belonging to CTX-M, SHV, TEM, OXA, GES, VEB and PER groups; and clinical validation of BLT performance on 126 BAS prospectively collected from seven intensive care units., Results: After optimization, BLT detected with 100% sensitivity the presence of CTX-M-15-producing K. pneumoniae spiked in sterile BAS for inoculums upon two or more GNB per field upon microscopic Gram staining examination (MGSE). The BLT accurately detected the 17 ESBLs tested at 10 6  CFU/mL and all ESBLs except Pseudomonas aeruginosa-related OXA-14 at 10 4  CFU/mL. Among the 126 BAS of the validation cohort, 21 (17%) gave positive BLT (ten in BAS positive and 11 in BAS negative on MGSE). All BLT-positive BAS grew with ESBL-PE-GNB, including five hyper-L2-producing Stenotrophomonas maltophilia strains. BLT detected ESBL-PE-GNB directly on clinical BAS positive for GNB on MGSE and/or growing with ≥10 4  CFU/mL with 100% sensitivity, specificity, and positive and negative predictive values., Conclusions: BLT is an accurate tool for ESBL-PE-GNB detection directly on BAS. Further studies are needed to evaluate the impact of BLT-guided early antimicrobial de-escalation strategies., (Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Purpose: Since their emergence at the beginning of the century, OXA-48 carbapenemases have spread in the community and in hospitals. To assess the diversity of OXA-48-producing bacterial strains and plasmids in the hospital setting, we studied the strains isolated from patients in three hospitals in the Paris area., Materials and Methods: All possible OXA-48-like strains were included in the study. OXA-48-like and extended-spectrum beta-lactamase-encoding genes were identified, and fingerprinting analysis was performed for all Escherichia coli and Klebsiella pneumoniae strains. The backbones and close genetic environments of bla OXA-48 were assessed by amplifying genes that were specific to the pOXA-48a plasmid and PCR, encompassing the junctions between bla OXA-48 and its direct genetic environment., Results: Overall, 68 strains from 30 patients were studied. These strains belonged to seven different enterobacterial species. OXA-48, OXA-204, and OXA-401 were identified in 62, 3, and 3 isolates, respectively. Additional broad-spectrum beta-lactamases were identified in 34% (23/68) of the strains. The strain diversity was high between and within patients. Identical patterns were observed only within individual patients or among epidemiologically related patients. Plasmid mapping was performed in the 62 OXA-48-producing strains and the 3 OXA-405-producing strains, resulting in the identification of 5 different patterns., Conclusion: Because of their ability to transfer between strains, OXA-48 carbapenemases have a high risk of dissemination and may become endemic in France.

FosA, a glutathione S-transferase that inactivates fosfomycin, has been reported as the cause of enzymatic resistance to fosfomycin. We show that multiple lineages of FosA-producing extended spectrum β-lactamase Escherichia coli have circulated in France since 2012, potentially reducing the efficacy of fosfomycin in treating infections with antimicrobial drug-resistant gram-negative bacilli.

Although extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae have become a worldwide public health concern, little is known regarding the clinical course of colonized or infected individuals. Our objective was to characterize the determinants of fatal outcomes related to ESBL-producing microorganisms at a large hospital in Paris, France. In 2012-2013, all consecutive patients with clinical samples testing positive for ESBL-producing Enterobacteriaceae at Saint-Antoine Hospital were identified. Patient clinical data were obtained at hospital entry, while information on intensive care unit (ICU) admissions and death were prospectively collected. Risk-factors for fatal 1-year outcomes were assessed using logistic regression. In total, 643/4684 (13%) ESBL-positive samples were observed, corresponding to 516 episodes (n = 206, 40% treated) among 330 patients. Most episodes were nosocomial-related (n = 347/516, 67%) involving Escherichia coli (n = 232/516, 45%) or Klebsiella pneumoniae (n = 164/516, 32%). Empirical antibiotic therapy was adequate in 89/206 (43%) infections, while the median length of hospital stay was 30 days [interquartile range (IQR) = 11-55] and 39/201 (19%) were admitted to the ICU. Overall, 104/241 patients (43%) with available data died within 1 year. In the multivariable analysis, 1-year death was associated with age >80 years (p = 0.01), concomitant comorbidity (p = 0.001), nosocomial-acquired infection (p = 0.002), and being infected rather than colonized (p < 0.001). In this series of patients with identified samples of ESBL-producing Enterobacteriaceae, hospital burden was large and 1-year mortality rates high. Understanding which patients in this setting would benefit from broad-spectrum empirical antibiotic therapy should be further examined.

Background: Rapid diagnostic tests detecting microbial resistance are needed for limiting the duration of inappropriateness of empirical antimicrobial therapy (EAT) in intensive care unit patients, besides reducing the use of broad-spectrum antibiotics. We hypothesized that the betaLACTA® test (BLT) could lead to early increase in the adequacy of antimicrobial therapy., Methods: This was a case-control study. Sixty-one patients with BLT-guided adaptation of EAT were prospectively included, and then matched with 61 "controls" having similar infection characteristics (community or hospital-acquired, and source of infection), in whom EAT was conventionally adapted to antibiogram results. Endpoints were to compare the proportion of appropriate (primary endpoint) and optimal (secondary endpoint) antimicrobial therapies with each of the two strategies, once microbiological sample culture results were available., Results: Characteristics of patients, infections and EAT at inclusion were similar between groups. Nine early escalations of EAT occurred in the BLT-guided adaptation group, reaching 98% appropriateness vs. 77% in the conventional adaptation group (p < 0.01). The BLT reduced the time until escalation of an inappropriate EAT from 50.5 (48-73) to 27 (24-28) hours (p < 0.01). Seventeen early de-escalations occurred in the BLT-guided adaptation group, compared to one in the conventional adaptation group, reducing patients' exposure to broad-spectrum beta-lactam such as carbapenems. In multivariate analysis, use of the BLT was strongly associated with early appropriate (OR = 18 (3.4-333.8), p = 0.006) and optimal (OR = 35.5 (9.6-231.9), p < 0.001) antimicrobial therapies. Safety parameters were similar between groups., Conclusions: Our study suggests that a BLT-guided adaptation strategy may allow early beta-lactam adaptation from the first 24 hours following the beginning of sepsis management.

Objectives: To explore the VagCD toxin-antitoxin (TA) systems encoded on plasmids in multiresistant Klebsiella pneumoniae strains., Methods: Previously sequenced K. pneumoniae plasmids were used for in silico analysis and a collection of 63 resistant K. pneumoniae strains was used for epidemiological study. Functional analysis was done after separate cloning of the toxin gene under the control of the arabinose-inducible promoter of pBAD43 and of the antitoxin gene under the control of the constitutive promoter of pUC19., Results: In silico , two types of VagCD systems, VagCD1 and VagCD2, encoded on K. pneumoniae plasmids could be distinguished, 15% carrying one of these TA systems. Moreover, in a collection of antibiotic-resistant K. pneumoniae strains including ESBL or carbapenemase producers, 17.5% of isolates were found to harbour a VagCD TA system. VagCD1 and VagCD2 were proved functional TA systems, with VagD the toxin and VagC its antitoxin, not only in K. pneumoniae but also in Escherichia coli and other Enterobacteriaceae. Toxin expression was found to induce a significant decrease in a bacterial population resulting from both bactericidal and bacteriostatic effects., Conclusions: The vagCD genes of K. pneumoniae encode a functional broad-spectrum TA system and are conserved on the large multiple antibiotic resistance-conferring plasmids in this species., (© 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.)

We report two cases of bacteremia due to Clostridium difficile from two French hospitals. The first patient with previously diagnosed rectal carcinoma underwent courses of chemotherapy, and antimicrobial treatment, and survived the C. difficile bacteremia. The second patient with colon perforation and newly diagnosed lung cancer underwent antimicrobial treatment in an ICU but died shortly after the episode of C. difficile bacteremia. A review of the literature allowed the identification of 137 cases of bacteremia between July 1962 and November 2016. Advanced age, gastro-intestinal disruption, severe underlying diseases and antimicrobial exposure were the major risk factors for C. difficile bacteremia. Antimicrobial therapy was primarily based on metronidazole and/or vancomycin. The crude mortality rate was 35% (21/60).

Background: Dissemination of multidrug-resistant Escherichia coli is closely associated with the worldwide spread of a single clone ST131, which is the main cause of urinary tract and bloodstream infections in patients from nursing homes and immunocompromised patients. The aim of our study was to determine the prevalence of ST131 clone and the replicons involved in the spread of bla CTX-M genes among O25b-ST131 CTX-M-producing E. coli isolates in Lithuania., Methods: The strains included in this study were screened for CTX-M β-lactamase-encoding genes, phylogenetic groups and ST131 clone by PCR. Bacterial conjugation was performed to identify plasmid replicon types responsible for bla CTX-M genes dissemination., Results: A total of 158 E. coli clinical non-duplicate ESBL isolates were analyzed. Nearly half (n = 67, 42.4%) of the investigated E. coli isolates belonged to phylogenetic group B2. The isolates producing CTX-M-92 β-lactamases were identified to be the ST131 clone more frequently than the non-ST131 clone (11.5% vs. 3.1%, p = .035). The CTX-M-15 isolates were identified as ST131 isolates less frequently than non-ST131 isolates (50.8% vs. 71.1%; p = .015). The ST131 clone isolates contained type L/M and A/C replicons; a fused FII/FIB replicon was found in four isolates (23.5%). Type HI1 replicon was identified in ST131 E. coli isolates producing CTX-M-15 β-lactamases., Conclusions: This study demonstrates the predominance of the ST131 clone among CTX-M β-lactamase-producing E. coli isolates. Dissemination of bla CTX-M genes in ST131 strains can be linked not only to highly adapted IncF plasmids such as FII/FIB and FII, but also to plasmid replicon types A/C, L/M and HI1.

We aimed to detect the extended-spectrum β-lactamases (ESBLs) secreted by clinical strains of Escherichia coli at Fann University Hospital in Dakar and to characterize them molecularly. We identified 32 isolates producing ESBLs. The CTX-M-15 gene was the most frequently detected ESBL gene, detected in 90.63% of the isolates studied.

Over a period of 40 months, plasmid-mediated AmpC β-lactamases were detected in Tunis, Tunisia, in 78 isolates (0.59%) of Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis. In 67 isolates, only one ampC gene was detected, i.e., blaCMY-2-type (n = 33), blaACC (n = 23), blaDHA (n = 6) or blaEBC (n = 5). Multiple ampC genes were detected in 11 isolates, with the following distribution: blaMOX-2, blaFOX-3, and blaCMY-4/16 (n = 6), blaFOX-3 and blaMOX-2 (n = 3), and blaCMY-4 and blaMOX-2 (n = 2). A great variety of plasmids carrying these genes was found, independently of the species and the bla gene. If the genetic context of blaCMY-2-type is variable, that of blaMOX-2, reported in part previously, is unique and that of blaFOX-3 is unique and new., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The dissemination of antimicrobial resistance genes in Enterobacteriaceae has been largely attributed to plasmids, circular DNA molecules capable of autonomous replication. Whereas high-copy-number plasmids primarily rely on passive diffusion for plasmid maintenance, low-copy-number plasmids utilize so-called partition (par) systems. Plasmid partition relies on three structures, i.e. a centromere like DNA site, a centromere-binding protein and an ATPase or a GTPase motor protein for plasmid positioning. Identification and classification of plasmids is essential for tracing plasmids conferring drug resistance. PCR-based replicon typing is currently the standard method for plasmid identification but there are new classification schemes, especially the relaxase gene typing (PRaseT). Here we developed a multiplex PCR set targeting par loci found on the plasmids most frequently encountered in Enterobacteriaceae. This method, called "plasmid partition gene typing" (PAR-T), was validated with 136 transconjugants or transformants harboring various replicon types. The method was tested with 30 multidrug-resistant clinical isolates including Escherichia coli, Klebsiella pneumoniae and Salmonella enterica subsp. enterica carrying 1-4 replicons; all replicons were tested in parallel with PRaseT for comparison. Six multiplex PCRs and one simplex PCR including 18 pairs of primers recognized plasmids of groups IncA/C, FIA, FIB, FIC, FIIk, FII, HI1, HI2, I1, L/M, N, X. Our set of multiplex PCRs showed high specificity for the classification of resistance plasmids except for IncX replicons., (Copyright © 2015. Published by Elsevier B.V.)

A single multiplex PCR assay targeting seven virulence factors and the wzi gene specific for the K1 and K2 capsular serotypes of Klebsiella pneumoniae was developed and tested on 65 clinical isolates, which included 45 isolates responsible for community-acquired severe human infections. The assay is useful for the surveillance of emerging highly virulent strains., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Multidrug-resistant and highly virulent Klebsiella pneumoniae isolates are emerging, but the clonal groups (CGs) corresponding to these high-risk strains have remained imprecisely defined. We aimed to identify K. pneumoniae CGs on the basis of genome-wide sequence variation and to provide a simple bioinformatics tool to extract virulence and resistance gene data from genomic data. We sequenced 48 K. pneumoniae isolates, mostly of serotypes K1 and K2, and compared the genomes with 119 publicly available genomes. A total of 694 highly conserved genes were included in a core-genome multilocus sequence typing scheme, and cluster analysis of the data enabled precise definition of globally distributed hypervirulent and multidrug-resistant CGs. In addition, we created a freely accessible database, BIGSdb-Kp, to enable rapid extraction of medically and epidemiologically relevant information from genomic sequences of K. pneumoniae. Although drug-resistant and virulent K. pneumoniae populations were largely nonoverlapping, isolates with combined virulence and resistance features were detected.

Infections caused by extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli are an important cause of morbidity and mortality, especially in children. We compared 58 epidemiologically unrelated ESBL-producing E. coli strains that caused infections. They were isolated between 2008 and 2012 in two Parisian pediatric hospitals and grouped according to their origin into either community-acquired (CA) (n=37) or nosocomially acquired (NA) (n=21) strains. Molecular characteristics of the ESBLs, phylogenetic traits of the strains including their belonging to clone O25b-ST131, prevalence of associated virulence genes, growth capacities in different media, metabolic phenotype and biofilm formation abilities were studied. ESBL type, associated resistance and distribution of phylogenetic groups were similar in the CA and NA groups. More than 60% of the B2 phylogroup strains in both groups belonged to the ST131 clone. Interestingly, CA strains possessed more genes encoding virulence factors and the distribution of these genes differed significantly between the two groups: fyuA, hlyC, papC and papGII were more frequent in the CA group, whereas iroN was more frequent in the NA group. CA strains also showed enhanced growth capacities in Luria Bertani rich medium. They tended to produce more biofilm but the difference was not significant. This study confirms the wide spread of clone ST131 among infected children, regardless of whether their infections were community- or nosocomially acquired. It highlights genotypic and phenotypic differences according to the origin of the strains that could indicate adaptability of these multi-resistant bacteria to specific environmental and host factors., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

With the β-Lacta test, production of extended-spectrum β-lactamases (ESBLs) was assayed in 200 urine samples showing Gram-negative bacilli during direct microscopic examination. While 168 samples tested negative, all samples yielding ESBL-producing Enterobacteriaceae after culture gave positive (n = 30) or uninterpretable (n = 2) results. The sensitivity and specificity of ESBL detection were 94% and 100%, respectively., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Seventeen Klebsiella pneumoniae isolates producing DHA-1 β-lactamase were collected in an intensive care unit between 2006 and 2010. Molecular analysis revealed the predominance of ST48 and ST1263 clones of K. pneumoniae and the spread of DHA-1-encoding plasmids belonging to incompatibility group IncL/M or IncHI2., (Copyright © 2014 Elsevier Inc. All rights reserved.)

We report here the complete nucleotide sequence of two IncR replicons encoding multidrug resistance determinants, including β-lactam (blaDHA-1, blaSHV-12), aminoglycoside (aphA1, strA, strB), and fluoroquinolone (qnrB4, aac6'-1b-cr) resistance genes. The plasmids have backbones that are similar to each other, including the replication and stability systems, and contain a wide variety of transposable elements carrying known antibiotic resistance genes. This study confirms the increasing clinical importance of IncR replicons as resistance gene carriers., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Antibiotic resistance has become a major public health problem in Algeria. Indeed the past decade, we have seen a significant increase in resistance to antibiotics especially in Gram-negative bacilli. Resistance to β-lactams in enterobacteria is dominated by the production of ESBL CTX-M-3 and CTX-M-15. The strains producing these enzymes are often the cause of potentially serious infections in both hospital and community settings. Identified plasmid cephalosporinases are CMY-2, CMY-12 and DHA-1. The isolation of strains of Enterobacteriaceae and Pseudomonas aeruginosa producing carbapenemases is rare in Algeria. Some Enterobacteriaceae producing OXA-48 or VIM-19 have been reported; so far, only VIM-2 has been identified in P. aeruginosa. However, the situation regarding the strains of Acinetobacter baumannii resistant to carbapenemases seems to be more disturbing. The carbapenemase OXA-23 is the most common and seems to be endemic in the north. The carbapenemase NDM-1 has also been identified. Resistance to aminoglycosides is marked by the identification armA gene associated with blaCTX-M genes in strains of Salmonella sp. Several other resistance genes have been identified sporadically in strains of Enterobacteriaceae, P. aeruginosa and A. baumannii. Resistance genes to fluoroquinolones are more recent identification in Algeria. The most common are the Qnr determinants followed by the bifunctional enzyme AAC[6']-Ib-cr. Resistance to sulfonamides and trimethoprim was also reported in Enterobacteriaceae strains in the west of the country., (Copyright © 2014. Published by Elsevier SAS.)

Plasmids are the main vectors of antimicrobial drug resistance and virulence genes, especially in Enterobacteriaceae. Identification and classification of plasmids is essential for analysis of their distribution. The most widely used typing method is PCR-based replicon typing (PBRT). A new classification scheme based on relaxase gene typing has been described recently. We propose a practical application of this method, with the development of a multiplex PCR set targeting relaxase genes found on plasmids most frequently encountered in Enterobacteriaceae. This method, here called "plasmid relaxase gene typing" (PRaseT), was validated with 60 transconjugants and transformants harboring various replicon types. The method was tested with 39 multidrug-resistant clinical isolates including Escherichia coli, Klebsiella pneumoniae and Salmonella enterica subsp. enterica carrying 1-7 replicons as well as with 17 plasmids non-typeable using PBRT; all replicons were tested in parallel with PBRT for comparison. Six multiplex PCRs and one simplex PCR, including 24 pairs of primers, recognized plasmids of groups A/C, B/O, colE, FIA, FIB, FIC, FV, FIIk, HI1, HI2, I1, K, L/M, N, P1α, Q1, U, W, X1, X2, X3 and X4. There was perfect correlation between PRaseT and PBRT results in 31/39 (79.5%) clinical isolates. Moreover, 11/17 (64.7%) plasmids non-typeable by PBRT could be typed by PRaseT. Our set of multiplex PCRs showed high sensitivity and specificity for the classification of resistance plasmids. It has proved complementary to the widely used PBRT and will improve the monitoring of plasmid distribution in every-day practice., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

For decades, third-generation cephalosporins (3GC) have been major drugs used to treat infections due to Enterobacteriaceae; growing resistance to these antibiotics makes the rapid detection of such resistance important. The βLacta test is a chromogenic test developed for detecting 3GC-resistant isolates from cultures on solid media within 15 min. A multicenter prospective study conducted in 5 French and Belgian hospitals evaluated the performance of this test on clinical isolates. Based on antibiotic susceptibility testing, strains resistant or intermediate to cefotaxime or ceftazidime were classified as 3GC resistant, and molecular characterization of this resistance was performed. The rates of 3GC resistance were 13.9% (332/2,387) globally, 9.4% in Escherichia coli (132/1,403), 25.6% in Klebsiella pneumoniae (84/328), 30.3% in species naturally producing inducible AmpC beta-lactamases (109/360), and 5.6% in Klebsiella oxytoca and Citrobacter koseri (7/124). The sensitivities and specificities of the βLacta test were, respectively, 87.7% and 99.6% overall, 96% and 100% for E. coli and K. pneumoniae, and 67.4% and 99.6% for species naturally producing inducible AmpC beta-lactamase. False-negative results were mainly related to 3GC-resistant strains producing AmpC beta-lactamase. Interestingly, the test was positive for all 3GC-resistant extended-spectrum beta-lactamase-producing isolates (n = 241). The positive predictive value was 97% and remained at ≥96% for prevalences of 3GC resistance ranging between 10 and 30%. The negative predictive values were 99% for E. coli and K. pneumoniae and 89% for the species producing inducible AmpC beta-lactamase. In conclusion, the βLacta test was found to be easy to use and efficient for the prediction of resistance to third-generation cephalosporins, particularly in extended-spectrum beta-lactamase-producing strains.

Objectives: A relapse from Escherichia coli bloodstream infection was observed in a patient with acute leukaemia treated with ceftazidime for 7 days for febrile neutropenia. Whereas the original E. coli isolate was resistant to β-lactam/β-lactamase inhibitor combinations (EC1), the relapse E. coli isolate showed a similar phenotype but with resistance extended to ceftazidime (EC2). We investigated the molecular mechanisms of β-lactam resistance and sought if EC2 could have been selected in vivo from EC1., Methods: EC1 and EC2 isolates were compared for antibiotic MICs, plasmid content, genotyping, β-lactamase genes and their environment. Both isolates were conjugated with E. coli JW4111ΔampC and MICs determined for transconjugants. In addition, ceftazidime-resistant mutants were selected in vitro from EC1., Results: EC1 and EC2 showed identical patterns for genotyping and resistance plasmids. PCR sequencing of blaTEM in EC1 showed the mutations M69L and N276D corresponding to TEM-35, also called inhibitor-resistant TEM (IRT)-4. In EC2, the TEM allele showed an additional mutation, R164S, known to confer resistance to ceftazidime. The combination of these three mutations was previously reported in TEM-158, described as the complex mutant TEM (CMT)-9, associated with resistance to β-lactamase inhibitors and third-generation cephalosporins. In vitro selection of ceftazidime-resistant mutants from EC1 yielded six different CMT alleles, including TEM-158 containing the R164S mutation., Conclusions: This first known report of in vivo selection of CMT from IRT, reproduced in vitro, shows how the evolution of β-lactamase enzymes is easily driven by antibiotic pressure, even during a short antibiotic therapy.

Six multidrug-resistant Klebsiella pneumoniae isolates were recovered from injured Libyan combatants. Production of carbapenemase was screened by using commercial combination tablets from Rosco combined with a temocillin disk. Polymerase chain reaction (PCR) and sequencing were used to detect several carbapenemase genes and to characterize their genetic environment. Genetic support was studied by mating-out assays. Plasmid size was identified by the KADO method. PCR and sequencing allowed characterization of plasmid scaffold. Genotyping was performed by pulse-field gel electrophoresis (PFGE) and multilocus sequence typing. PCR was used to check for the presence of nine genes linked to virulence in K. pneumoniae. No carbapenemase was identified by Rosco disks, but all isolates showed high-level temocillin resistance. All of them harbored blaOXA-48 in the transposon Tn1999.2, on a self-conjugative plasmid of about 60 kb, similar to pOXA-48. PFGE revealed three clusters in which isolates were genetically related: The first comprised FM9 and FM10, and the second comprised FM1, FM4, and FM5. FM2 formed a third distinct clone. Sequence types ST101, ST11, and ST147 were identified in keeping with PFGE results. The entB, ycfM, ybtS, and mrkD genes were detected in all isolates, and kfu gene was present in the three ST101 strains. This work confirms the current and successful spread of blaOXA-48 by horizontal dissemination of a single IncL/M plasmid through different genetic backbones with strong epidemic potential. It also highlights the need for rapid and reliable phenotypic detection methods. Attempts to link virulence factors and the production of this carbapenemase deserve further studies.

Background: Extended-spectrum β-lactamases (ESBLs), particularly CTX-M- type ESBLs, are among the most important resistance determinants spreading worldwide in Enterobacteriaceae. The aim of this study was to characterize a collection of 163 ESBL-producing Escherichia coli collected in Tunisia, their ESBL-encoding plasmids and plasmid associated addiction systems., Results: The collection comprised 163 ESBL producers collected from two university hospitals of Sfax between 1989 and 2009. 118 isolates harbored blaCTX-M gene (101 blaCTX-M-15 gene and 17 blaCTX-M-14 gene). 49 isolates carried blaSHV-12 gene, 9 blaSHV-2a gene and only 3 blaTEM-26 gene. 16 isolates produced both CTX-M and SHV-12. The 101 CTX-M-15-producing isolates were significantly associated to phylogroup B2 and exhibiting a high number of virulence factors. 24 (23.7%) of the group B2 isolates belonged to clonal complex ST131. Pulsed-field gel electrophoresis (PFGE) typing revealed a genetic diversity of the isolates. 144 ESBL determinants were transferable mostly by conjugation. The majority of plasmid carrying blaCTX-M-15 genes (72/88) were assigned to various single replicon or multireplicon IncF types and had significantly a higher frequency of addiction systems, notably the VagCD module., Conclusion: This study demonstrates that the dissemination of CTX-M-15 producing E. coli in our setting was due to the spread of various IncF-type plasmids harboring multiple addiction systems, into related clones with high frequency of virulence determinants.

Seventy-one isolates of Enterobacteriaceae (17 Escherichia coli, 50 Klebsiella pneumoniae, and 4 Enterobacter cloacae) producing extended-spectrum-β-lactamases (ESBLs) were collected between April 2008 and March 2010 in an intensive care unit and surgical ward of Tlemcen Hospital (West of Algeria). Sequencing identified the bla(CTX-M-15) determinant in 69 isolates and bla(CTX-M-3) in 2 isolates. None of the studied strains produced the class D carbapenemase OXA-48. Repetitive Extragenic palindromic polymerase chain reaction showed a high degree of genotypic diversity among E. coli strains and two major clonal populations of K. pneumoniae (CKp1 n=11 and CKp5 n=25) which were further identified as members of the multilocus sequence typing types (ST931) and (ST15), respectively. The ST15 isolates harbored more resistance genes and virulence factors than the ST931 isolates. The characterization of the spacer region between ISEcp1 and bla(CTX-M) for CTX-M-15 producers individualized two populations. One that derived from the CTX-M-3 under Algerian clinical context and one that is universally found. The dissemination of ESBLs in the studied Enterobacteriaceae isolates was mainly due to the epidemic clones of K. pneumoniae and to genetic transit of plasmids among unrelated strains.

Maternal-fetal Escherichia coli infections, such as neonatal bacteremia and meningitis, are important causes of morbidity and mortality. From 2006 to 2010, we studied newborns and their mothers who were colonized with E. coli in a French hospital in order to document (i) the epidemiology and genetic characteristics of extended-spectrum-beta-lactamase (ESBL)-producing E. coli strains, (ii) the prevalence of associated virulence genes, (iii) the prevalence of clone sequence type 131 (ST131), and (iv) the genetic relationship among ESBL-producing strains. Among the 2,755 E. coli cultures recovered from vaginal or neonatal samples, 68 were ESBL producers (2.46%). We found a wide diversity of ESBL genes, with the majority being bla(CTX-M-14), bla(CTX-M-1), and bla(CTX-M-15), distributed among the 4 main phylogenetic groups. Genes encoding virulence factors were found in 90.7% of the isolates, with ≥ 2 virulence genes present in 76% of cases. The prevalence of ST131 among ESBL-producing E. coli isolates was 9.4% (6/64). Five of these 6 ST131 isolates possessed bla(CTX-M-15) enzymes (and also were resistant to quinolones), and one possessed bla(CTX-M-2) enzymes. Two possessed virulence genes, suggesting the presence of pathogenicity island IIJ96 (PAI IIJ96)-like domains. Pulsed-field gel electrophoresis (PFGE) revealed a high level of genomic diversity overall, except for 3 closely related isolates belonging to clonal group ST131. Repetitive PCR showed that the six ST131 isolates were closely related to ST131 control strains (>95% similarity). This study shows a high prevalence of ESBL-producing E. coli strains and clonal group ST131 in the French maternal-fetal population. These results suggest a widespread distribution of ESBL enzymes in the community and highlight the early transmission between mothers and neonates. These findings are worrisome, especially for this particularly vulnerable population.

In head and neck cancer surgery antibiotic prophylaxis is effective in reducing the incidence of surgical site infections (SSI). However, controversies between antibiotic prophylaxis and curative antibiotic therapy exist, particularly when complex and decaying surgeries are performed in risky underlying conditions, with a risk of persisting salivary effusion in the postoperative period, or in the case of reconstruction with myo-cutaneous flaps. We have performed a systematic review of the literature according to PRISMA recommendations to answer the following questions: indications for antibiotic prophylaxis and curative antibiotic therapy, optimal duration, and choice of antibiotics for prophylaxis in head and neck cancer surgery. Literature analysis allows to conclude that patients undergoing Altemeier classes 2 and 3 surgical procedures should receive perioperative antibiotic prophylaxis restricted to the first 24 postoperative hours. No benefit has been shown with its extension beyond these 24 hours. The most adapted combinations of antibiotics in this setting are "amoxicillin+clavulanic acid" and "clindamycin+gentamicin". However, the level of evidence regarding the most decaying surgeries with high risk of SSI is low, making it necessary to perform new high-powered randomized trials in these patients. Eventually, it should be noted that antibiotic prophylaxis should be an integral part of SSI preventive measures, including application of hygiene measures, and postoperative monitoring of SSI clinical signs., (Copyright © 2013 Société française d’anesthésie et de réanimation (Sfar). Published by Elsevier SAS. All rights reserved.)

Background: We investigated the molecular characteristics of multidrug-resistant, extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae isolated in community settings and in hospitals in Antananarivo, Madagascar., Results: Forty-nine E. coli, K. pneumoniae, K. oxytoca and E. cloacae ESBL-producing isolates were studied. In antimicrobial susceptibility analyses, many of the isolates exhibited resistance to aminoglycosides, fluoroquinolones and trimethoprim-sulfamethoxazole. Gene amplification analysis and sequencing revealed that 75.5% (n=37) of the isolates harbored blaCTX-M-15 and 38.7% (n=19) harbored blaSHV-12. The non-ESBLs resistance genes detected were blaTEM-1, blaOXA-1, aac(6')-Ib,aac(6')-Ib-cr, tetA, sul-1, sul-2, qnrA, qnrB and catB-3. We found dfrA and aadA gene cassettes in the class 1 integron variable regions of the isolates, and the combination of dfrA17-aadA5 to be the most prevalent. All blaCTX-M-15 positive isolates also contained the ISEcp1 insertion element. Conjugation and transformation experiments indicated that 70.3% of the antibiotic resistance genes resided on plasmids. Through a PCR based replicon typing method, plasmids carrying the blaSHV-12 or blaCTX-M-15 genes were assigned to either the IncFII replicon type or, rarely, to the HI2 replicon type. All isolates were subtyped by the rep-PCR and ERIC-PCR methods.Phylogenetic grouping and virulence genotyping of the E. coli isolates revealed that most of them belonged to group A1. One isolate assigned to group B2 harbored blaCTX-M-15 and five virulence genes (traT, fyuA, iutA, iha and sfa) and was related to the O25b-ST131 clone., Conclusions: Our results highlight the dissemination of multidrug resistant Enterobacteriaceae isolates in Antananarivo. These findings underline the need for a rational use of antibiotic and for appropriate methods of screening ESBL in routine laboratories in Antananarivo.

We report the complete nucleotide sequence of the pKpS90 plasmid, carrying the bla(KPC-2) and bla(SHV-12) genes. This plasmid was isolated from a sequence type 258 (ST258) Klebsiella pneumoniae strain responsible for an outbreak in a French university hospital in 2009. pKpS90 is a 53,286-bp plasmid that belongs to the IncX incompatibility group. pKpS90 consists of a backbone from IncX plasmids, in which the KPC-2-encoding Tn4401 transposon and a bla(SHV-12)-encoding region have been inserted.

Increasing resistance of Escherichia coli (E. coli) to antibiotics, especially to the third-generation cephalosporins, has prompted studies on widespread resistance genes such as blaCTX-M and differentiation of E. coli to phylogenetic groups. The aim of this study was to determine the associations between the CTX-M type and the phylogenetic group, the site of infection, and coresistance in Lithuanian E. coli isolates producing β-lactamases. MATERIAL AND METHODS. A total of 90 E. coli ESBL strains were recovered from the lower respiratory tract, the urinary tract, sterile body sites, wounds, and other body sites between 2008 and 2012. The E. coli isolates resistant to at least 2 antibiotics with different modes of action along with resistance to cefotaxime were considered as multiresistant. The blaCTX-M, blaTEM, blaOXA-1, and blaSHV genes, the phylogenetic groups, and the resistance profiles were analyzed. RESULTS. Of the 90 isolates, 84 (93.3%) were classified as multiresistant and 6 (6.6%) as resistant. The blaCTX-M-15 gene was the most prevalent gene followed by the blaCTX-M-14 and blaCTX-M-92 genes. The logistic regression analysis revealed the associations between CTX-M-15 and resistance to ceftriaxone, between CTX-M-14 and resistance to cefoxitin, aztreonam, ampicillin/sulbactam, ticarcillin/clavulanic acid, and tobramycin, and between CTX-M-92 and resistance to cefepime, piperacillin/tazobactam, gentamicin, and tobramycin. CONCLUSIONS. The results of this study showed a significant association between CTX-M-15, CTX-M-14, and CTX-M-92 β-lactamases and resistance to some antibiotics as well as CTX‑M-14 β-lactamase and phylogenetic group A in the Lithuanian population. The associations between the CTX-M type and the site of infection were not determined.

Objectives: To determine the complete nucleotide sequence of the VIM-1-encoding plasmid pTC2, which was isolated from a Greek Providencia stuartii multiresistant strain., Methods: The pTC2 plasmid was extracted and sequenced using shotgun and 3 kb paired-end DNA libraries and a 454 sequencing approach. Following assembly into a unique scaffold, gaps were closed by PCR followed by Sanger DNA sequencing. Gene predictions and annotations were performed using the CAAT-Box tool and the Conserved Domain Search service. Sequence comparisons were performed using the Artemis Comparison Tool., Results: Plasmid pTC2 (180,184 bp) was found to be a multireplicon plasmid (IncA/C and IncR), with a large IncA/C backbone and a mosaic multidrug resistance (MDR) region, in which was inserted a 13 kb IncR fragment. Gene annotation allowed the identification of a complete IncA/C-type transfer system and of several putative maintenance modules, both on the IncA/C backbone and on the IncR fragment. The complex MDR region contained 9 insertion sequences (7 IS26, 1 IS1 and 1 IS6100), 10 resistance genes and a mercury resistance operon integrated into unit transposons, composite transposons or integrons., Conclusions: The pTC2 combines a broad host range, transfer and maintenance capacities, plasticity of the MDR region and a wide variety of resistance genes, properties that may contribute to the spreading of resistance determinants.

Clustered, regularly interspaced, short palindromic repeats (CRISPRs) are implicated in defence against foreign DNA in various archaeal and bacterial species. They have also been associated with a slower spread of antibiotic resistance. However, experimental and evolutionary studies raise doubts about the role of CRISPRs as a sort of immune system in Escherichia coli. We studied a collection of 263 natural E. coli isolates from human and animal hosts, representative of the phylogenetic and lifestyle diversity of the species and exhibiting various levels of plasmid-encoded antibiotic resistance. We characterized the strains in terms of CRISPRs, performed replicon typing of the plasmids and tested for class 1 integrons to explore the possible association between CRISPRs and the absence of plasmids and mobile antibiotic resistance determinants. We found no meaningful association between the presence/absence of the cas genes, reflecting the activity of the CRISPRs, and the presence of plasmids, integrons or antibiotic resistance. No CRISPR in the collection contained a spacer that matched an antibiotic resistance gene or element involved in antibiotic resistance gene mobilization, and 79.8 % (210/263) of the strains lacked spacers matching sequences in the 2282 plasmid genomes available. Hence, E. coli CRISPRs do not seem to be efficient barriers to the spread of plasmids and antibiotic resistance, consistent with what has been reported for phages, and contrary to reports concerning other species.

Clustered, regularly interspaced, short palindromic repeats (CRISPRs) are implicated in defence against foreign DNA in various archaeal and bacterial species. They have also been associated with a slower spread of antibiotic resistance. However, experimental and evolutionary studies raise doubts about the role of CRISPRs as a sort of immune system in Escherichia coli. We studied a collection of 263 natural E. coli isolates from human and animal hosts, representative of the phylogenetic and lifestyle diversity of the species and exhibiting various levels of plasmid-encoded antibiotic resistance. We characterized the strains in terms of CRISPRs, performed replicon typing of the plasmids and tested for class 1 integrons to explore the possible association between CRISPRs and the absence of plasmids and mobile antibiotic resistance determinants. We found no meaningful association between the presence/absence of the cas genes, reflecting the activity of the CRISPRs, and the presence of plasmids, integrons or antibiotic resistance. No CRISPR in the collection contained a spacer that matched an antibiotic resistance gene or element involved in antibiotic resistance gene mobilization, and 79.8% (210/263) of the strains lacked spacers matching sequences in the 2282 plasmid genomes available. Hence, E. coli CRISPRs do not seem to be efficient barriers to the spread of plasmids and antibiotic resistance, consistent with what has been reported for phages, and contrary to reports concerning other species.

This study was conducted to identify the molecular mechanisms of imipenem resistance in a Klebsiella pneumoniae (Kp16137) isolate recovered in August 2008 at the University Hospital Sahloul, Sousse, Tunisia. The strain was identified with the API 20E system; antibiotic-containing disks were used for detection of antibiotic susceptibility by a disk diffusion assay. We investigated the presence of β-lactamases by PCR, using specific primers for bla(TEM), bla(SHV), bla(CTX-M), bla(OXA), bla(CMY), bla(ACC), bla(FOX), bla(IMP), bla(KPC), bla(VIM), and by sequencing. Extraction of plasmid DNA from Kp16137 and the transconjugant was performed by the method of Kado. Southern transfer was performed on nylon. The membrane was hybridized with a specific probe for the bla(CMY-2) gene. Outer membrane proteins were isolated and were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 12% polyacrylamide gel. K. pneumoniae Kp16137 was resistant to all available β-lactams, including third generation cephalosporins and carbapenems. The screening of β-lactamases showed the presence of three β-lactamases: TEM-1, SHV-61, and CMY-4. The CMY-4 β-lactamase was located on an 80-kb plasmid. An analysis of the outer membrane proteins of this isolate revealed that it lacked a porin of 42 kDa. The loss of this outer membrane protein band correlated with imipenem resistance in this strain. In K. pneumoniae 16137, synthesis of a plasmid-mediated β-lactamase: AmpC CMY-4, together with alteration in permeability led to resistance to all available β-lactams and carbapenems.