Clavulanate combinations with mecillinam, cefixime or cefpodoxime against ESBL-producing Enterobacterales frequently associated with blaOXA-1 in a paediatric population with febrile urinary tract infections.

OBJECTIVES: Oral treatment of febrile urinary tract infections (FUTIs) can be impaired by MDR Enterobacterales often combining ESBL and inhibitor-resistant genes. We studied the impact of ß-lactamases and Enterobacterales' genotypes on the cefixime, cefpodoxime and mecillinam ± amoxicillin/clavulanate MICs. MATERIALS AND METHODS: In this multicentric study, we included 251 previously whole-genome-sequenced ESBL-producing Enterobacterales, isolated in French children with FUTIs. The MICs of cefixime, cefpodoxime, mecillinam alone and combined with amoxicillin/clavulanate were determined and analysed with respect to genomic data. We focused especially on the isolates' ST and their type of ß-lactamases. Clinical outcomes of patients who received cefixime + amoxicillin/clavulanate were also analysed. RESULTS: All isolates were cefixime and cefpodoxime resistant. Disparities depending on blaCTX-M variants were observed for cefixime. The addition of amoxicillin/clavulanate restored susceptibility for cefixime and cefpodoxime in 97.2% (MIC50/90 of 0.38/0.75 mg/L) and 55.4% (MIC50/90 of 1/2 mg/L) of isolates, respectively, whatever the ST, the blaCTX-M variants or the association with inhibitor-resistant ß-lactamases (34.2%). All isolates were susceptible to mecillinam + amoxicillin/clavulanate with MIC50/90 of 0.19/0.25 mg/L, respectively. Neither therapeutic failure nor any subsequent positive control urine culture were reported for patients who received cefixime + amoxicillin/clavulanate as an oral relay therapy (n = 54). CONCLUSIONS: Despite the frequent association of ESBL genes with inhibitor-resistant ß-lactamases, the cefixime + amoxicillin/clavulanate MICs remain low. The in vivo efficacy of this combination was satisfying even when first-line treatment was ineffective. Considering the MIC distributions and pharmacokinetic parameters, mecillinam + amoxicillin/clavulanate should also be an alternative to consider when treating FUTIs in children.

Diversity and trends in population structure of ESBL-producing Enterobacteriaceae in febrile urinary tract infections in children in France from 2014 to 2017.

BACKGROUND: The population structure of extraintestinal pathogenic Escherichia coli evolves over time, notably due to the emergence of antibiotic-resistant clones such as ESBL-producing Enterobacteriaceae (ESBL-E). OBJECTIVES: To analyse by WGS the genetic diversity of a large number of ESBL-E isolated from urinary tract infections in children from paediatric centres across France between 2014 and 2017 and collected by the National Observatory of febrile urinary tract infection (FUTI) caused by ESBL-E. METHODS: A total of 40 905 Enterobacteriaceae-positive urine cultures were identified. ESBL-E were found in 1983 samples (4.85%). WGS was performed on 251 ESBL-E causing FUTI. STs, core genome MLST (cgMLST), serotype, fimH allele, ESBL genes and presence of papGII key virulence factor were determined. RESULTS: E. coli and Klebsiella pneumoniae were found in 86.9% (218/251) and 11.2% (28/251) of cases, respectively. Several STs predominate among E. coli such as ST131, ST38, ST69, ST73, ST95, ST405, ST12 and ST1193, while no ST emerged in K. pneumoniae. E. coli ST131, ST38 and ST1193 increased during the study period, with a heterogeneity in papGII prevalence (64.5%, 35% and 20% respectively). Most isolates harboured the CTX-M type (97%) with a predominance of blaCTX-M-15. blaCTX-M-27, an emerging variant in E. coli, is found in various STs. cgMLST enabled discrimination of clusters within the main STs. CONCLUSIONS: The predominance of ST131, and the emergence of other STs such as ST38 and ST1193 combined with ESBL genes deserves close epidemiological surveillance considering their high threat in infectious disease. cgMLST could be a discriminant complementary tool for the analyses.

Bacterial infections in children after liver transplantation: A single-center surveillance study of 345 consecutive transplantations.

BACKGROUND: Infectious complications after pediatric liver transplantation frequently occur and are potentially serious. Data concerning strictly defined bacterial infections and their associated risk factors are lacking. METHODS: For the pediatric liver transplant postoperative period, we analyzed data from the nosocomial infection surveillance (2006-2015). RESULTS: A total of 235 bacterial infections in 162 transplantations (47%) occurred, including 32 bacterial pneumonia cases, 104 surgical site infections, 27 urinary tract infections, and 40 bloodstream infections. Sepsis was diagnosed in 127 cases (54%), severe sepsis in 22 (9%) cases, and septic shock in 41 (17%) cases. Thirty patients (9%) died, and septic shock was the leading cause of death. The carrier status of multi-drug resistant bacteria and a tacrolimus level >20 ng/mL were independent risk factors for surgical site infections and the occurrence of severe sepsis or septic shock. The length of mechanical ventilation was an independent risk factor for pneumonia and surgical site infection. CONCLUSION: Bacterial infections in the early postoperative period after pediatric liver transplantation are associated with high morbidity and mortality. Physicians involved in the medical care of these patients should be aware of the specific risk factors, and further development of prevention programs is highly recommended.

A 4.5-Year Within-Patient Evolution of a Colistin-Resistant Klebsiella pneumoniae Carbapenemase-Producing K. pneumoniae Sequence Type 258.

Background: Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kp) has emerged globally over the last decade as a major nosocomial pathogen that threatens patient care. These highly resistant bacteria are mostly associated with a single Kp clonal group, CG258, but the reasons for its host and hospital adaptation remain largely unknown. Methods: We analyzed the in vivo evolution of a colistin-resistant KPC-Kp CG258 strain that contaminated a patient following an endoscopy and was responsible for a fatal bacteremia 4.5 years later. Whole-genome sequencing was performed on 17 KPC-Kp isolates from this patient; single-nucleotide polymorphisms were analyzed and their implication in antimicrobial resistance and bacterial host adaptation investigated. Results: The patient KPC-Kp strain diversified over 4.5 years at a rate of 7.5 substitutions per genome per year, resulting in broad phenotypic modifications. After 2 years of carriage, all isolates restored susceptibility to colistin. Higher expression of the fimbriae conferred the ability to produce more biofilm, and the isolate responsible for a bacteremia grew in human serum. The convergent mutations occurring in specific pathways, such as the respiratory chain and the cell envelope, revealed a complex long-term adaptation of KPC-Kp. Conclusions: Broad genomic and phenotypic diversification and the parallel selection of pathoadaptive mutations might contribute to long-term carriage and virulence of KPC-Kp CG258 strains and to the dissemination of this clone.

Diversity of Carbapenemase-Producing Escherichia coli Isolates in France in 2012-2013.

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.

Trends in carbapenemase-producing Enterobacteriaceae, France, 2012 to 2014.

In 2014, a total of 2,976 Enterobacteriaceae isolates with decreased susceptibility to carbapenems were received at the French Associated National Reference Center for Antibiotic Resistance (NRC) and were characterised for their molecular resistance mechanism to carbapenems and compared with results obtained during 2012 and 2013.The overall number of enterobacterial isolates with decreased susceptibility to carbapenems received at the NRC rapidly increased (more than twofold in two years) with a growing proportion of carbapenemase producers (23.1% in 2012 vs 28.6% in 2013 vs 36.2% in 2014). Between 2012 and 2014, the main carbapenemase type was OXA-48, with an increase in OXA-48 variants (mostly OXA-181) and NDM producers, whereas the number KPC producers decreased. We identified a potential spread of OXA-181 producers in the tropical region of Africa. Finally, OXA-48 and OXA-48-related enzymes remained the predominant carbapenemases in France. The number of carbapenemase-producing Escherischia coli isolates was multiplied by fivefold between 2012 and 2014, suggesting a possible dissemination in the community.

Analysis of OXA-204 carbapenemase-producing Enterobacteriaceae reveals possible endoscopy-associated transmission, France, 2012 to 2014.

OXA-48-like beta-lactamase producing bacteria are now endemic in several European and Mediterranean countries. Among this carbapenemase family, the OXA-48 and OXA-181 variants predominate, whereas other variants such as OXA-204 are rarely reported. Here, we report the molecular epidemiology of a collection of OXA-204-positive enterobacterial isolates (n = 29) recovered in France between October 2012 and May 2014. This study describes the first outbreak of OXA-204-producing Enterobacteriaceae in Europe, involving 12 isolates of an ST90 Escherichia coli clone and nine isolates of an ST147 Klebsiella pneumoniae clone. All isolates co-produced the cephalosporinase CMY-4, and 60% of them co-produced the extended-spectrum beta-lactamase CTX-M-15. The blaOXA-204 gene was located on a 150-kb IncA/C plasmid, isolated from various enterobacterial species in the same patient, indicating a high conjugative ability of this genetic vehicle.

Spread of Plasmids Carrying Multiple GES Variants.

Five GES-producing Enterobacteriaceae isolates that displayed an extended-spectrum ß-lactamase (ESBL) phenotype harbored two GES variants: GES-7 ESBL and GES-6 carbapenemase. In all isolates, the two GES alleles were located on the same integron that was inserted into an 80-kb IncM1 self-conjugative plasmid. Whole-genome sequencing suggested in vivo horizontal gene transfer of the plasmid along with clonal diffusion of Enterobacter cloacae To our knowledge, this is the first description in Europe of clustered Enterobacteriaceae isolates carrying two GES ß-lactamases, of which one has extended activity toward carbapenems.

Prospective evaluation of an algorithm for the phenotypic screening of carbapenemase-producing Enterobacteriaceae.

OBJECTIVES: The objective of this study was to assess the performance of an algorithm based on the disc diffusion method for the screening of carbapenemase-producing Enterobacteriaceae (CPE) referred to the French National Reference Centre for Antibiotic Resistance. METHODS: From April to June 2014, all isolates of Enterobacteriaceae referred to the French National Reference Centre for Antibiotic Resistance were included. The inhibition zone diameters of imipenem, ticarcillin/clavulanate and temocillin using EUCAST disc diffusion methodology were recorded. All isolates were subjected to the algorithm proposed by the Antibiogram Committee of the French Society of Microbiology (CA-SFM) for the screening of carbapenemase producers. Phenotypic, biochemical and molecular detection of carbapenemases was performed for all isolates. RESULTS: A total of 621 consecutive enterobacterial isolates with decreased susceptibility to carbapenems were tested. They included 213 CPE [OXA-48-like (n = 183), NDM (n = 22), VIM (n = 3), KPC (n = 3) and OXA-48-like + NDM (n = 2)] and 408 non-carbapenemase producers. The CA-SFM algorithm combining cut-off values of 15 mm, 15 mm and 22 mm for ticarcillin/clavulanate, temocillin and imipenem, respectively, perfectly detected 204 isolates (32.8%) as non-carbapenemase producers, leading to a negative predictive value of 100% for this algorithm. CONCLUSIONS: Implementation of the CA-SFM algorithm in clinical microbiology laboratories may avoid additional testing for CPE in one-third of the enterobacterial isolates with decreased susceptibility to carbapenems.

Prospective evaluation of the OXA-48 K-SeT assay, an immunochromatographic test for the rapid detection of OXA-48-type carbapenemases.

OBJECTIVES: There is an urgent need for accurate and fast diagnostic tests to identify MDR bacteria. Here, we evaluated an immunochromatographic assay (the OXA-48 K-SeT assay) to detect OXA-48-like carbapenemase-producing Enterobacteriaceae from culture colonies. METHODS: One hundred and sixty-one collection isolates with characterized ß-lactamase content and 185 non-duplicate consecutive clinical isolates referred to the Associated French National Reference Center between 15 February and 15 March 2015 were used to evaluate the OXA-48 K-SeT assay. Among these 346 isolates, 100 were OXA-48-like carbapenemase producers, 3 were OXA-48-like producers lacking carbapenemase activity and 243 were ESBL, AmpC, oxacillinase and/or non-OXA-48 carbapenemase producers. RESULTS: All 100 OXA-48-like carbapenemase producers were correctly detected by the OXA-48 K-SeT assay, including OXA-48 (n = 73), OXA-181 (n = 18), OXA-162 (n = 1), OXA-204 (n = 4), OXA-232 (n = 2) and OXA-244 (n = 2) variants. The three OXA-48 variants lacking carbapenemase activity, OXA-163 (n = 2) and OXA-405 (n = 1), were not detected. All non-OXA-48 producers gave a negative result with the OXA-48 K-SeT assay. No cross-reaction was evidenced with the carbapenemases (VIM, IMP, NDM and KPC), ESBLs (TEM, SHV and CTX-M), AmpCs (CMY-2, DHA-2 and ACC-1) and oxacillinases (OXA-1, -2, -9 and -10). Overall, the sensitivity and specificity of the assay were 100% for OXA-48-like carbapenemase detection. CONCLUSIONS: The OXA-48 K-SeT assay was efficient, rapid and easy to implement in the routine workflow of a clinical microbiology laboratory for the confirmation of OXA-48-like carbapenemase-producing Enterobacteriaceae. It could complete the existing panel of tests available for the confirmation of OXA-48-like carbapenemases, especially in countries with high OXA-48 prevalence.

Efficient Detection of Carbapenemase Activity in Enterobacteriaceae by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry in Less Than 30 Minutes.

The recognition of carbapenemase-producing Enterobacteriaceae (CPE) isolates is a major laboratory challenge, and their inappropriate or delayed detection may have negative impacts on patient management and on the implementation of infection control measures. We describe here a matrix-assisted laser desorption ionization-time of flight (MALDI-TOF)-based method to detect carbapenemase activity in Enterobacteriaceae. After a 20-min incubation of the isolate with 0.5 mg/ml imipenem at 37°C, supernatants were analyzed by MALDI-TOF in order to identify peaks corresponding to imipenem (300 Da) and an imipenem metabolite (254 Da). A total of 223 strains, 77 CPE (OXA-48 variants, KPC, NDM, VIM, IMI, IMP, and NMC-A) and 146 non-CPE (cephalosporinases, extended-spectrum ß-lactamases [ESBLs], and porin defects), were tested and used to calculate a ratio of imipenem hydrolysis: mass spectrometry [MS] ratio = metabolite/(imipenem + metabolite). An MS ratio cutoff was statistically determined to classify strains as carbapenemase producers (MS ratio of ≥0.82). We validated this method first by testing 30 of our 223 isolates (15 CPE and 15 non-CPE) 10 times to calculate an intraclass correlation coefficient (ICC of 0.98), showing the excellent repeatability of the method. Second, 43 strains (25 CPE and 18 non-CPE) different from the 223 strains used to calculate the ratio cutoff were used as external controls and blind tested. They yielded sensitivity and specificity of 100%. The total cost per test is <0.10 U.S. dollars (USD). This easy-to-perform assay is time-saving, cost-efficient, and highly reliable and might be used in any routine laboratory, given the availability of mass spectrometry, to detect CPE.

Evaluation of the RAPIDEC® CARBA NP, the Rapid CARB Screen® and the Carba NP test for biochemical detection of carbapenemase-producing Enterobacteriaceae.

OBJECTIVES: The objective of this study was the evaluation of the performance of two commercially available biochemical tests for the rapid detection of carbapenemase-producing Enterobacteriaceae compared with a home-made technique. METHODS: A collection of 150 enterobacterial isolates, including 132 isolates with decreased susceptibility to at least one carbapenem molecule, were tested for carbapenemase activity using the RAPIDEC(®) CARBA NP (bioMérieux), the Rapid CARB Screen(®) (Rosco Diagnostica) and the home-made Carba NP test. This strain collection included 55 non-carbapenemase producers, 21 KPC producers, 21 NDM producers, 17 VIM producers, 11 IMP producers, 16 OXA-48 producers and 9 OXA-48-like producers (OXA-162, OXA-181, OXA-204, OXA-232 and OXA-244). RESULTS: The RAPIDEC(®) CARBA NP detected all carbapenemase producers except a single OXA-244 producer. Using the Rapid CARB Screen(®), one KPC-2, two NDM-1, one OXA-48 and five OXA-48 variant producers gave equivocal results and one OXA-244 producer was not detected. Using the Carba NP test, the same OXA-244 producer was not detected and one OXA-181 producer and one OXA-244 producer gave equivocal results. Sensitivity and specificity were 99% (95% CI 94.3%-99.8%) and 100% (95% CI 93.5%-100%), respectively, for the RAPIDEC(®) CARBA NP test, 89.5% (95% CI 81.7%-94.2%) and 70.9% (95% CI 57.9%-81.2%) for the Rapid CARB Screen(®) and 96.8% (95% CI 91.1%-98.9%) and 100% (95% CI 93.5%-100%) for the Carba NP test. The impact of the use of an adequate bacterial inoculum for obtaining the optimal performance with the RAPIDEC(®) CARBA NP was noted. CONCLUSIONS: The RAPIDEC(®) CARBA NP possesses the best performance for rapid and efficient detection of carbapenemase-producing Enterobacteriaceae.

Strategy for rapid detection of carbapenemase-producing Enterobacteriaceae.

A prospective survey was conducted on 862 Enterobacteriaceae isolates with reduced susceptibility to carbapenems. The Carba NP test, UV spectrophotometry, and a DNA microarray were used to detect carbapenemase producers, and the results were compared to those from PCR and sequencing. The 172 carbapenemase producers were detected using the Carba NP test and UV spectrophotometry, whereas the DNA microarray failed to detect IMI producers. The use of the Carba NP test as a first screening, followed by the use of molecular techniques, has been determined to be an efficient strategy for identifying carbapenemase-producing Enterobacteriaceae.

Dissemination of carbapenemase-producing Enterobacteriaceae in France, 2012.

OBJECTIVES: Carbapenem-resistant Enterobacteriaceae isolates (n = 1485) were received at the French Associated National Reference Center for Antibiotic Resistance in 2012 and were characterized for their mechanism of resistance to carbapenems. METHODS: Carbapenemase production was detected using the biochemical-based Carba NP test, based on the detection of in vitro hydrolysis of imipenem. All isolates with a positive Carba NP test result were characterized by PCR and sequencing. RESULTS: Carbapenemase production was identified in 23.1% of the isolates. The main carbapenemase type identified was OXA-48 and derivatives (75.5%). An overseas source was clearly demonstrated for only 27.6% of the isolates. CONCLUSIONS: OXA-48 and derivatives are now the most prevalent carbapenemases in France, with a possible spread of these producers in the community.

Enterobacterales carrying chromosomal AmpC ß-lactamases in Europe (EuESCPM): Epidemiology and antimicrobial resistance burden from a cohort of 27 hospitals, 2020-2022.

INTRODUCTION: The ESCPM group (Enterobacter species including Klebsiella aerogenes - formerly Enterobacter aerogenes, Serratia species, Citrobacter freundii complex, Providencia species and Morganella morganii) has not yet been incorporated into systematic surveillance programs. METHODS: We conducted a multicentre retrospective observational study analysing all ESCPM strains isolated from blood cultures in 27 European hospitals over a 3-year period (2020-2022). Diagnostic approach, epidemiology, and antimicrobial susceptibility were investigated. RESULTS: Our study comprised 6,774 ESCPM isolates. MALDI-TOF coupled to mass spectrometry was the predominant technique for bacterial identification. Susceptibility to new ß-lactam/ß-lactamase inhibitor combinations and confirmation of AmpC overproduction were routinely tested in 33.3% and 29.6% of the centres, respectively. The most prevalent species were E. cloacae complex (44.8%) and S. marcescens (22.7%). Overall, third-generation cephalosporins (3GC), combined third- and fourth-generation cephalosporins (3GC + 4GC) and carbapenems resistance phenotypes were observed in 15.7%, 4.6%, and 9.5% of the isolates, respectively. AmpC overproduction was the most prevalent resistance mechanism detected (15.8%). Among carbapenemase-producers, carbapenemase type was provided in 44.4% of the isolates, VIM- (22.9%) and OXA-48-enzyme (16%) being the most frequently detected. E. cloacae complex, K. aerogenes and Providencia species exhibited the most notable cumulative antimicrobial resistance profiles, with the former displaying 3GC, combined 3GC + 4GC and carbapenems resistance phenotypes in 15.2%, 7.4%, and 12.8% of the isolates, respectively. K. aerogenes showed the highest rate of both 3GC resistant phenotype (29.8%) and AmpC overproduction (32.1%), while Providencia species those of both carbapenems resistance phenotype (42.7%) and carbapenemase production (29.4%). ESCPM isolates exhibiting both 3GC and combined 3GC + 4GC resistance phenotypes displayed high susceptibility to ceftazidime/avibactam (98.2% and 95.7%, respectively) and colistin (90.3% and 90.7%, respectively). Colistin emerged as the most active drug against ESCPM species (except those intrinsically resistant) displaying both carbapenems resistance phenotype (85.8%) and carbapenemase production (97.8%). CONCLUSIONS: This study presented a current analysis of ESCPM species epidemiology in Europe, providing insights to inform current antibiotic treatments and guide strategies for antimicrobial stewardship and diagnostics.

Complete sequence of two KPC-harbouring plasmids from Pseudomonas aeruginosa.

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 ß-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.

Characterization of an extended-spectrum class A ß-lactamase from a novel enterobacterial species taxonomically related to Rahnella spp./Ewingella spp.

OBJECTIVES: To characterize the naturally occurring ß-lactamase gene identified from a clinical isolate belonging to a novel enterobacterial species that is closely related to Rahnella spp. and Ewingella spp. METHODS: Shotgun cloning and expression in Escherichia coli were performed in order to characterize this resistance determinant. Enzymatic activities were measured by UV spectrophotometry after an ion-exchange chromatography purification procedure. RESULTS: A chromosomal gene coding for the extended-spectrum ß-lactamase (ESBL) SMO-1 was identified from a novel enterobacterial species that is taxonomically related to Rahnella aquatilis and Ewingella americana. The ß-lactamase efficiently hydrolysed penicillins and cefotaxime, and shared 75% amino acid identity with the plasmid-mediated ß-lactamase SFO-1 from Serratia fonticola, 74% amino acid identity with the plasmid-mediated ESBL CTX-M-2 originating from Kluyvera spp. and 72% amino acid identity with the chromosomally encoded and intrinsic RAHN-1 from R. aquatilis. CONCLUSIONS: We have identified a novel enterobacterial species recovered from a clinical specimen, constituting another potential source of acquired ESBL. The ESBL shared significant similarities with the CTX-M-type enzymes.

Effectiveness of third-generation cephalosporins or piperacillin compared with cefepime or carbapenems for severe infections caused by wild-type AmpC ß-lactamase-producing Enterobacterales: A multi-centre retrospective propensity-weighted study.

BACKGROUND: The optimal treatment regimen for infections caused by wild-type AmpC ß-lactamase-producing Enterobacterales remains controversial. This study compared the outcomes of bloodstream infections (BSI) and pneumonia according to the type of definitive antibiotic therapy: third-generation cephalosporin (3GC), piperacillin ± tazobactam, cefepime or carbapenem. METHODS: All cases of BSI and pneumonia caused by wild-type AmpC ß-lactamase-producing Enterobacterales over 2 years in eight university hospitals were reviewed. Patients who received definitive therapy consisting of either a 3GC (3GC group), piperacillin ± tazobactam (piperacillin group), or cefepime or a carbapenem (reference group) were included in this study. The primary endpoint was 30-day all-cause mortality. The secondary endpoint was treatment failure due to infection by emerging AmpC-overproducing strains. Propensity-score-based models were used to balance confounding factors between groups. RESULTS: In total, 575 patients were included in this study: 302 (52%) with pneumonia and 273 (48%) with BSI. Half (n=271, 47%) received cefepime or a carbapenem as definitive therapy, 120 (21%) received a 3GC, and 184 (32%) received piperacillin ± tazobactam. Compared with the reference group, 30-day mortality was similar in the 3GC [adjusted hazard ratio (aHR) 0.86, 95% confidence interval (CI) 0.57-1.31)] and piperacillin (aHR 1.20, 95% CI 0.86-1.66) groups. The likelihood of treatment failure was higher in the 3GC (aHR 6.81, 95% CI 3.76-12.4) and piperacillin (aHR 3.13, 95% CI 1.69-5.80) groups. The results were similar when stratifying the analysis on pneumonia or BSI. CONCLUSION: Treatment of included BSI or pneumonia caused by wild-type AmpC ß-lactamase-producing Enterobacterales with 3GC or piperacillin ± tazobactam was not associated with higher mortality, but was associated with increased risk of AmpC overproduction leading to treatment failure compared with cefepime or a carbapenem.

Role of ISKpn7 and deletions in blaKPC gene expression.

The carbapenemase-encoding bla(KPC) gene, which is rapidly spreading in Gram-negative rods, is located on a Tn3-based transposon, Tn4401, which carries a polymorphic region giving rise to five isoforms (a, b, c, d, and e) that is located immediately upstream of the bla(KPC) gene and thus likely involved in its expression. Using 5' rapid amplification of cDNA ends (5'RACE), we identified three potential promoter sequences (P1, P2, and P3) upstream of the bla(KPC) gene, of which only P1 (absent from isoforms c and d) and P2 (present in all isoforms, with a -35 box located inside the right inverted repeat of ISKpn7) were shown to be true promoters involved in expression. One representative of each different promoter combination of Tn4401, i.e., P2 alone (isoform c), P1-P2 (isoform a), and P1-P2-P3 (isoform b), was cloned into an Escherichia coli plasmid vector. Using reverse transcription-PCR (RT-PCR), the highest level of expression was obtained with isoform a (P1 and P2), which is also the most commonly encountered form in enterobacterial clinical isolates, followed by isoforms b (P1, P2, and P3) and c (P2 only). These differences in expression led to slight differences in MIC values of carbapenems. In silico analysis of the DNA sequence of isoform b revealed a stem-loop structure that is likely responsible for strong stops observed in 5'RACE experiments and for decreased expression compared to that with isoform a (P1 and P2). In addition, such structures could also be at the origin for the deletions observed in isoforms a and c. Taken together, these results indicate that the P1 and P2 promoters both contribute to the expression of the bla(KPC) gene and that the construct with the highest level of expression is that possessing isoform a, which is also the most commonly encountered form in clinical isolates.

Evaluation of a DNA microarray for the rapid detection of extended-spectrum ß-lactamases (TEM, SHV and CTX-M), plasmid-mediated cephalosporinases (CMY-2-like, DHA, FOX, ACC-1, ACT/MIR and CMY-1-like/MOX) and carbapenemases (KPC, OXA-48, VIM, IMP and NDM).

OBJECTIVES: Carbapenem-resistant Gram-negative bacilli are reported increasingly and represent an emerging public health concern. Laboratory detection of extended-spectrum ß-lactamase (ESBL), plasmid-mediated cephalosporinase (pAmpC) and carbapenemase producers remains a challenge for microbiology laboratories and is important to avoid clinical failure due to inappropriate antimicrobial therapy and to prevent nosocomial outbreaks. We evaluated a novel microarray, the 'Check-MDR CT103 array' test (Check-Points, Wageningen, The Netherlands), that employs highly specific DNA markers to identify the ß-lactamase genes of ESBLs (TEM, SHV and CTX-M, and discriminates between ESBL and non-ESBL TEM and SHV variants), of pAmpC (CMY-2-like, DHA, FOX, ACC-1, ACT/MIR and CMY-1-like/MOX) and of carbapenemases (KPC, OXA-48, VIM, IMP and NDM). METHODS: One-hundred-and-eighty-seven well-characterized Gram-negative bacilli isolates possessing different bla genes were tested. Total DNAs were extracted using a Qiagen DNA mini kit. The 'Check-MDR CT103 array' was used as recommended by the manufacturer. RESULTS: The system correctly identified representatives of the three ESBL gene families tested, including differentiation between non-ESBL and ESBL TEM and SHV variants. All bla(CTX-M) genes were classified into the appropriate family group (i.e. CTX-M-1 group, CTX-M-2 group, CTX-M-9 group and CTX-M-8/25/26 group). In addition, the clinically relevant plasmid-encoded cephalosporinase and carbapenemase genes were also reliably detected. Specificities and sensitivities of 100% were recorded for most bla genes. CONCLUSIONS: The 'Check-MDR CT103 array' is a powerful high-throughput tool for rapid identification of ESBL, pAmpC and carbapenemase producers in culture. Because of its rapid performance, this platform is a valuable tool for epidemiological or infection control studies.