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

Background Carbapenem-resistant Pseudomonas aeruginosa are being increasingly described worldwide. Here, we investigated the molecular mechanisms underlying carbapenem resistance in an extremely drug-resistant P. aeruginosa isolate from a neonatal intensive care unit in Morocco. Materials and methods P. aeruginosa strain O82J1 was identified using MALDI-TOF-MS. Carba NP, immunochromatographic assay NG Carba5 and antimicrobial susceptibility testing using disc diffusion and microbroth were performed. Whole-genome sequencing using the Illumina and MinION technologies and different software packages available at the Center of Genomic Epidemiology were used to predict the resistome, sequence type and plasmid types. Results P. aeruginosa O82J1 co-expressed two metallo-β-lactamases, bla NDM-1 and bla VIM-2, and was susceptible to colistin and apramycin only. It belonged to ST773 that is frequently reported worldwide as a high-risk P. aeruginosa clone. The bla VIM-2 gene was integron-borne on a IncP-2 465-kb plasmid, whereas the bla NDM-1 gene was chromosomally encoded and embedded in an integrative conjugative element, probably at the origin of its acquisition. A total of 23 antimicrobial resistance genes were detected including a bla PER-1 ESBL gene, and an 16S-rRNA methyltransferase gene rmtB. Conclusions The isolation of XDR P. aeruginosa isolates expressing several carbapenemases in a neonatal intensive care unit is of great concern due to the reduced treatment options, relying only on colistin, but not recommended in neonates, and apramycin, not yet approved for human therapy. Concerns were further elevated due to the resistance to cefiderocol and ATM/AVI, two novel and last-resort antibiotics recommended to treat infections caused by Gram-negative bacteria, particularly XDR P. aeruginosa in adults. [ABSTRACT FROM AUTHOR]

We characterized a collection of IMI-like--producing Enterobacter spp. isolates (n = 112) in France. The main clone corresponded to IMI-1--producing sequence type 820 E. cloacae subspecies cloacae that was involved in an outbreak. Clinicians should be aware of potential antimicrobial resistance among these bacteria. [ABSTRACT FROM AUTHOR]

Background VRE are increasingly described worldwide. Screening of hospitalized patients at risk for VRE carriage is mandatory to control their dissemination. Here, we have developed the Bfast [VRE Panel] PCR kit, a rapid and reliable quantitative PCR assay for detection of vanA , vanB , vanD and vanM genes, from solid and liquid cultures adaptable to classical and ultrafast real-time PCR platforms. Methods Validation was carried out on 133 well characterized bacterial strains, including 108 enterococci of which 64 were VRE. Analytical performances were determined on the CFX96 Touch (Bio-Rad) and Chronos Dx (BforCure), an ultrafast qPCR machine. Widely used culture plates and broths for enterococci selection/growth were tested. Results All targeted van alleles (A, B, D and M) were correctly detected without cross-reactivity with other van genes (C, E, G, L and N) and no interference with the different routinely used culture media. A specificity and sensitivity of 100% and 99.7%, respectively, were determined, with limits of detection ranging from 21 to 238 cfu/reaction depending on the targets. The Bfast [VRE Panel] PCR kit worked equally well on the CFX and Chronos Dx platforms, with differences in multiplexing capacities (five and four optical channels, respectively) and in turnaround time (45 and 16 minutes, respectively). Conclusions The Bfast [VRE Panel] PCR kit is robust, easy to use, rapid and easily implementable in clinical microbiology laboratories for ultra-rapid confirmation of the four main acquired van genes. Its features, especially on Chronos Dx, seem to be unmatched compared to other tools for screening of VRE. [ABSTRACT FROM AUTHOR]

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

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

This article discusses the discovery of the OXA-198 carbapenemase gene cassette in the genus Candidatus Accumulibacter. The Candidatus Accumulibacter genus is known for its role in enhanced biological phosphate removal in wastewater treatment. The OXA-198 gene was first identified in clinical Pseudomonas aeruginosa isolates and later in Enterobacterales. The study found that the blaOXA-198-like gene was present in three different regions of Denmark, but unfortunately, the isolates were not cultivated. The genetic contexts of the blaOXA-198-like genes were analyzed, and it was determined that they were not acquired from other sources. The study suggests that Candidatus Accumulibacter may be the progenitor of OXA-198-like carbapenemase-encoding genes. This finding is significant as it provides insight into the origins of carbapenemase genes and may aid in future research and prevention efforts. [Extracted from the article]

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

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

This article discusses the emergence of carbapenem resistance in Enterobacterales, specifically through the dissemination of carbapenemase-producing bacteria. The study characterizes two new variants of the VIM-1 enzyme, called VIM-29 and VIM-86, which were isolated in multidrug-resistant Enterobacterales in France. The isolates were resistant to all β-lactam antibiotics except aztreonam/avibactam. The study also analyzes the genetic environment of the VIM-29 and VIM-86 genes and identifies common resistance determinants. The plasmids carrying these genes were found to be self-conjugative. The hydrolytic abilities of VIM-29 and VIM-86 were compared to other prevalent VIM variants, and it was found that they displayed similar resistance profiles. The study concludes that these new variants contribute to the growing problem of carbapenem resistance. [Extracted from the article]

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

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

The development of novel antibiotics is mandatory to curb the growing antibiotic resistance problem resulting in difficult-to-treat bacterial infections. Here, we have determined the spectrum of activity of cystobactamids and chelocardins, two novel and promising classes of molecules with different modes of action. A panel of 297 clinically relevant Gram-negative and Gram-positive isolates with different antibiotic susceptibility profiles, going from wild type to multi- or even extremely drug resistant (MDR, XDR) and including carbapenem-resistant isolates, were tested using broth microdilution assays to determine the minimal inhibitory concentrations (MICs), MIC50s and MIC90s of two cystobactamids derivatives (CN-861-2 and CN-DM-861) and two chelocardin derivatives (CHD and CDCHD). Cystobactamids revealed potent activities on the majority of tested Enterobacterales (MIC50s ranging from 0.25 to 4 µg/mL), except for Klebsiella pneumoniae isolates (MIC50s is 128 µg/mL). Pseudomonas aeruginosa and Acinetobacter baumannii showed slightly higher MIC50s (4 µg/mL and 8 µg/mL, respectively) for cystobactamids. Chelocardins inhibited the growth of Enterobacterales and Stenotrophomas maltophilia at low to moderate MICs (0.25–16 µg/mL) and the chemically modified CDCHD was active at lower MICs. A. baumannii and P. aeruginosa were less susceptible to these molecules with MICs ranging from 0.5 to 32 µg/mL. These molecules show also interesting in vitro efficacies on clinically relevant Gram-positive bacteria with MICs of 0.125–8 µg/mL for cystobactamids and 0.5–8 µg/mL for chelocardins. Taken together, the cystobactamid CN-DM-861 and chelocardin CDCHD showed interesting antibiotic activities on MDR or XDR bacteria, without cross-resistance to clinically relevant antibiotics such as carbapenems, fluoroquinolones, and colistin. [ABSTRACT FROM AUTHOR]

Background Cefiderocol is a catechol-substituted cephalosporin with potent in vitro activity against carbapenem-resistant (CR) Gram-negative bacteria (GNB). Cefiderocol susceptibility testing is complex because iron concentrations need to be taken into consideration. Here, we assessed the clinical performance of Bruker's UMIC® Cefiderocol and corresponding iron-depleted CAMHB to determine MIC by broth microdilution (BMD) for clinically relevant GNB. Methods MICs of cefiderocol for 283 GN clinical isolates were determined by BMD using iron-depleted CAMHB. Frozen panels were used as a reference. The concentration range of cefiderocol was 0.03–32 mg/L. The isolates, with different degrees of susceptibility to cefiderocol, included Enterobacterales (n  = 180) , Pseudomonas aeruginosa (n  = 49), Acinetobacter baumannii (n  = 44) and Stenotrophomonas maltophilia (n  = 10). Results The rates of categorical agreement (CA), essential agreement (EA) and bias were calculated to evaluate the performance of the UMIC® Cefiderocol, as compared with the reference method. Overall, the UMIC® Cefiderocol showed 90.8% EA (95% CI: 86.9%–93.7%) with a bias of −14.5% and a CA of 90.1% (95% CI: 86.1%–93.1%). For Enterobacterales, the UMIC® Cefiderocol showed 91.7% EA (95% CI: 86.7%–94.9%) with a bias of −25.0% and a CA of 87.8% (95% CI: 82.2%–91.8%). For non-fermenters, the UMIC® Cefiderocol showed 89.3% EA (95% CI: 81.9%–93.9%) (not significantly different from 90.0%, Student t -test) with a bias of −3.9% and a CA of 94.2% (95% CI: 87.7%–97.3%). Conclusions UMIC® Cefiderocol is a valid method for the determination of cefiderocol MICs even if higher than expected discrepancies were observed with NDM-producing Enterobacterales, which presented in most cases MIC values close to the breakpoint. [ABSTRACT FROM AUTHOR]

Background As carbapenemase-producing Enterobacterales are increasingly reported worldwide, their rapid detection is crucial to reduce their spread and prevent infections and outbreaks. Lateral flow immunoassays (LFIAs) have become major tools for the detection of carbapenemases. However, as for most commercially available assays, only the five main carbapenemases are targeted. Objectives Here, we have developed and evaluated an LFIA prototype for the rapid and reliable detection of the increasingly identified GES-type β-lactamases. Methods The GES LFIA was validated on 103 well-characterized Gram-negative isolates expressing various β-lactamases grown on Mueller–Hinton (MH) agar, chromogenic, and chromogenic/selective media. Results The limit of detection of the assay was 106 cfu per test with bacteria grown on MH agar plates. GES LFIA accurately detected GES-type β-lactamases irrespective of the culture media and the bacterial host. The GES LFIA was not able to distinguish between GES-ESBLs and GES-carbapenemases. Because GES enzymes are still rare, their detection as an ESBL or a carbapenemase remains important, especially because extensive use of carbapenems to treat ESBL infections may select for GES variants capable of hydrolysing carbapenems. Conclusions The GES LFIA is efficient, rapid and easy to implement in the routine workflow of a clinical microbiology laboratory for the confirmation of GES-type β-lactamases. Combining it with immunochromatographic assays targeting the five main carbapenemases (KPC, NDM, VIM, IMP and OXA-48) would improve the overall sensitivity for the most frequently encountered carbapenemases and ESBLs, especially in non-fermenters. [ABSTRACT FROM AUTHOR]

Similarly, Carey et al. investigated plasmid-mediated fluoroquinolone and macrolide resistance genes among ESBL-producing I E. coli i isolates from 147 dairy calves [[20]]. The plasmid-encoded I bla i SB OXA-23 sb gene, together with I bla i SB CTX-M-15 sb , I bla i SB OXA-1 sb , and I bla i SB LEN-12 sb genes, were identified in two ST512 isolated from two residents of the same LTCF, underlining further spread of the I bla i SB OXA-23 sb gene beyond I Acinetobacter i spp. and I Proteus mirabilis i . The I bla i SB OXA-900 sb gene was located on an antibiotic resistance island (ARI), together with two ESBL genes ( I bla i SB CTX-M-39 sb and I bla i SB PER-2 sb genes). The IncC plasmid-mediated I bla i SB OXA-900 sb gene was identified in a I Citrobacter freundii i isolate that displayed significant carbapenem hydrolysis using -Carba (BioRad, Marnes-la-Coquette, France) and a modified CIM test, but it was not detected using commercially available DNA-based methods. [Extracted from the article]

Methicillin-resistant Staphylococcus aureus (MRSA) are spreading worldwide in hospital and community settings, thus posing a serious public health problem. Panton-Valentine Leukocidin (PVL), an important virulence factor of S. aureus, is a marker of community-acquired MRSA. Here we determined the prevalence of pvl genes among S. aureus isolates from different hospitals in the Gaza Strip, Palestine. A total of 285 S. aureus isolates were collected from five different hospitals in the Gaza Strip. All isolates were characterized for their susceptibility patterns to available antimicrobial agents and by using multiplex PCR for the detection of mecA and pvl genes. The overall prevalence of MRSA in Gaza hospitals was 70.2% (range: 76.3% to 65.5%) and that of pvl among S. aureus isolates was 29.8% (range: 32.9% to 26.2%). The pvl gene was equally prevalent among MRSA isolates (30.5%) and MSSA isolates (28.2%). The most effective antibiotics were rifampicin, vancomycin, and clindamycin, with susceptibility rates of 91.2%, 88.7%, and 84.6%, respectively. The highest percentage of strains were observed to be resistant to penicillin and amoxicillin with clavulanic acid—96.1% and 73.6%, respectively. Our results showed a high prevalence of MRSA and pvl-positive isolates in Gaza Strip hospitals, which likely reflects the situation in the community. It is mandatory to implement systematic surveillance of both hospital and community isolates, together with interventions (such as increased hand hygiene, use of hydroalcoholic solutions, and isolation of carriers) to limit their spread. [ABSTRACT FROM AUTHOR]

Carbapenem-resistant Enterobacterales (CRE) remain a significant public health threat, and, despite recent approvals, new antibiotics are needed. Severe infections caused by CRE, such as nosocomial pneumonia and bloodstream infections, are associated with a relatively high risk of morbidity and mortality. The recent approval of ceftazidime–avibactam, imipenem–relebactam, meropenem–vaborbactam, plazomicin, eravacycline and cefiderocol has broadened the armamentarium for the treatment of patients with CRE infections. Cefiderocol is a siderophore cephalosporin with overall potent in vitro activity against CRE. It is taken up via iron transport channels through active transport, with some entry into bacteria through traditional porin channels. Cefiderocol is relatively stable against hydrolysis by most serine- and metallo-beta-lactamases, including KPC, NDM, VIM, IMP and OXA carbapenemases—the most frequent carbapenemases detected in CRE. The efficacy and safety of cefiderocol has been demonstrated in three randomised, prospective, parallel group or controlled clinical studies in patients at risk of being infected by multidrug-resistant or carbapenem-resistant Gram-negative bacteria. This paper reviews the in vitro activity, emergence of resistance, preclinical effectiveness, and clinical experience for cefiderocol, and its role in the management of patients with CRE infections. [ABSTRACT FROM AUTHOR]

Culture media is fundamental in clinical bacteriology for the detection and isolation of bacterial pathogens. However, in-house media preparation could be challenging in low-resource settings. InTray® cassettes (Biomed Diagnostics) could be a valid alternative as they are compact, ready-to-use media preparations. In this study, we evaluate the use of two InTray media as a subculture alternative for the diagnosis of bloodstream infections: the InTray® Müller-Hinton (MH) chocolate and the InTray® Colorex™ Screen. The InTray MH chocolate was evaluated in 2 steps: firstly, using simulated positive blood cultures (reference evaluation study), and secondly, using positive blood cultures from a routine clinical laboratory (clinical evaluation study). The Colorex Screen was tested using simulated poly-microbial blood cultures. The sensitivity and specificity of the InTray MH chocolate were respectively 99.2% and 90% in the reference evaluation study and 97.1% and 88.2% in the clinical evaluation study. The time to detection (TTD) was ≤20 h in most positive blood cultures (99.8% and 97% in the two studies, respectively). The InTray® MH Chocolate agar showed good performance when used directly from clinical blood cultures for single bacterial infections. However, mixed flora is more challenging to interpret on this media than on Colorex™ Screen, even for an experienced microbiologist. [ABSTRACT FROM AUTHOR]

Background: Avibactam, relebactam and vaborbactam are β-lactamase inhibitors that proved their efficiency against KPC-producing Enterobacterales. Regarding their inhibitor activity towards Ambler's class A extended spectrum β-lactamases (ESBL) and Ambler's class C cephalosporinase (AmpC), they should be active on most of the carbapenem-resistant non-carbapenemase-producing Enterobacterales (CR non-CPE). Objectives: Determine the in vitro activity of ceftazidime-avibactam, imipenem-relebactam and meropenem-vaborbactam and comparators against CR non-CPE. Methods: MICs to ceftazidime/avibactam, imipenem/relebactam, meropenem/vaborbactam, but also temocillin, ceftolozane/tazobactam, ertapenem, colistin, eravacycline and tigecycline were determined by broth microdilution (ThermoFisher) on a collection of 284 CR non-CPE (inhibition zone diameter < 22 mm to meropenem). Whole genome sequencing was performed on 90 isolates to assess the genetic diversity as well as resistome. Results: According to EUCAST breakpoints, susceptibility rates of ceftazidime, imipenem, meropenem and ertapenem used at standard dose were 0.7%, 45.1%, 14.8% and 2.5%, respectively. Increased exposure of ceftazidime, imipenem and meropenem led to reach 3.5%, 68.3% and 67.7% susceptibility, respectively. Using the EUCAST clinical breakpoints, susceptibility rates of ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam were 88.4%, 81.0% and 80.6%, respectively. Susceptibility rates of temocillin, ceftolozane/tazobactam, tigecycline, eravacycline, and colistin were 0%, 4.6%, 27.8%, 54.9% and 90.1%. MICs distributions with and without the presence of the inhibitor demonstrated a better ability of avibactam and relebactam compared to vaborbactam to restore susceptibility to the associated β-lactam. Conclusions: This study demonstrated the in vitro efficacy of ceftazidime/avibactam, imipenem/relebactam and to a lesser extent meropenem/vaborbactam against CR non-CPE. Moreover, to test all β-lactams/β-lactamases inhibitors combinations without a priori for CRE, non-CPE is crucial since resistance to one of the β-lactam/β-lactamase inhibitor combinations does not predict resistance to another molecule, depending on the resistance mechanisms involved. [ABSTRACT FROM AUTHOR]

During 2013-2021, increased prevalence of oxacillinase 232-producing Enterobacterales was observed in France, mostly driven by its emergence in Klebsiella pneumoniae. Whole-genome sequencing identified that oxacillinase 232-producing K. pneumoniae belonged to 14 sequence types (STs), among which 2 polyclonal high-risk clones, ST-231 and ST-2096, were overrepresented. [ABSTRACT FROM AUTHOR]

Infections with carbapenem-resistant (CR) Gram-negative (GN) pathogens have increased in many countries worldwide, leaving only few therapeutic options. Cefiderocol (CFDC) is approved in Europe for the treatment of aerobic GN infections in adults with limited treatment options. This study evaluated the in vitro activity of cefiderocol and comparators against multidrug-resistant (MDR) bacteria including meropenem-resistant (MR) or pandrug-resistant (PR) GN clinical isolates from France and Belgium. The minimum inhibitory concentrations (MICs) of CFDC were determined by broth microdilution, using iron-depleted cation-adjusted Mueller–Hinton broth, and were compared to those of 10 last-line antibiotics. The MICs were interpreted according to EUCAST and CLSI breakpoints, and in the absence of species-specific breakpoints, non-species-related pharmacokinetic/pharmacodynamic breakpoints were used. Among the 476 isolates tested, 322 were carbapenemase producers (CP), 58 non-CP-CRs, 52 intrinsically CR, 41 expanded-spectrum cephalosporin resistant and 5 were multi-susceptible. Susceptibility to CFDC was high using EUCAST breakpoints 81%, 99% and 84%, and was even higher using CLSI breakpoints to 93%, 100% and 88% for Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii, respectively. Susceptibility to cefiderocol using non-species-related breakpoints for Stenotrophomonas maltophilia, Achromobacter xylosoxydans and Burkholderia cepacia, was 100%, 100% and 92.3%, respectively. The susceptibility rates were lower with the NDM producers, with values of 48% and 30% using EUCAST breakpoints and 81% and 50% using CLSI breakpoints for Enterobacterales and Acinetobacter spp, respectively. CFDC demonstrated high in vitro susceptibility rates against a wide range of MDR GN pathogens, including MR and PR isolates. [ABSTRACT FROM AUTHOR]

Carbapenem resistance (CR) is an emerging health issue. Epidemiological surveys on carbapenem-resistant Gram-negative bacilli (CR-GNB) in Lebanon remain scarce. In this study, we determined the prevalence of CR-GNB isolated between 2015 to 2019 in three hospitals in northern Lebanon: 311 CR-Enterobacterales (out of 11210; 2.8%), 155 CR-Pseudomonas (out of 1034; 15%) and 106 CR- Acinetobacter (out of 184; 57.6%) were identified. CR mechanisms were determined for 146 randomly chosen isolates: the Carba NP test revealed an enzymatic resistance to carbapenems in 109 isolates (out of 146, 74.7%). Produced carbapenemases were evaluated by the NG-Test Carba5, NG-Test OXA-23 immunochromatographic assays and PCR. Carbapenemase-producing (CP) Enterobacterales expressed blaOXA-48-like, blaNDM-like and blaVIM-like genes and CP-Pseudomonas expressed blaIMP-like and blaVIM-like genes, whereas CP-Acinetobacter expressed blaOXA-23-like genes. The NG-Test Carba5 results were confirmed by PCR sequencing and revealed several variants, such as NDM-19, VIM-62 and OXA-162, never described so far in Lebanon. Isolates with discordant results were sequenced by WGS and highlighted novel variants of the natural oxacillinases of Pseudomonas aeruginosa: blaOXA-50-like genes. Their role in carbapenem resistance should be further studied. Overall, our findings highlight an alarming situation and encourage health care centers to establish performant registration systems that could help in limiting resistance spread. [ABSTRACT FROM AUTHOR]

Background: Lateral flow immunoassays (LFIA) have shown their usefulness for detecting CTX-M- and carbapenemase-producing Enterobacterales (CPEs) in bacterial cultures. Here, we have developed and validated the BL-DetecTool to detect CTX-M enzymes and carbapenemases directly from clinical samples.Methods: The BL-DetecTool is an LFIA that integrates an easy sample preparation device named SPID (Sampling, Processing, Incubation and Detection). It was evaluated in three University hospitals on urine, blood culture (BC) and rectal swab (RS) specimens either of clinical origin or on spiked samples. RS evaluation was done directly and after a 24 h enrichment step.Results: The CTX-M BL-DetecTool was tested on 485 samples (154 BC, 150 urines, and 181 RS) and revealed a sensitivity and specificity of 97.04% (95% CI 92.59%-99.19%) and 99.43% (95% CI 97.95%-99.93%), respectively. Similarly, the Carba5 BL-DetecTool was tested on 382 samples (145 BC, 116 urines, and 121 RS) and revealed a sensitivity and specificity of 95.3% (95% CI 89.43%-98.47%) and 100% (95% CI 98.67%-100%), respectively. While with the Carba5 BL-DetecTool five false negatives were observed, mostly in RS samples, with the CTX-M BL-DetecTool, in addition to four false-negatives, two false-positives were also observed. Direct testing of RS samples revealed a sensitivity of 78% and 86% for CTX-M and carbapenemase detection, respectively.Conclusions: BL-DetecTool showed excellent biological performance, was easy-to-use, rapid, and could be implemented in any microbiology laboratory around the world, without additional equipment, no need for electricity, nor trained personnel. It offers an attractive alternative to costly molecular methods. [ABSTRACT FROM AUTHOR]

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

This study evaluated the performances of immunoassays (LFIA and ELISA) designed for SARS-CoV-2 Antigen (Ag)-detection in nasopharyngeal (NP) and serum samples in comparison to RT-PCR. NP samples from patients with respiratory symptoms (183 RT-PCR-positive and 74 RT-PCR-negative samples) were collected from March to April and November to December 2020. Seroconversion and antigen dynamics were assessed by symptom onset and day of RT-PCR diagnosis. Serum samples from 87 COVID-19 patients were used to investigate the added value of Ag quantification, at diagnosis and during follow-up. The sensitivity of COVID-VIRO-LFIA on samples with Ct ≤ 33, considered as the contagious threshold, was 86% on NPs (CI 95%: 79–90.5) and 76% on serum samples (CI 95%: 59.4–88), with a specificity of 100%. Serum N-Ag was detected during active infection as early as day two from symptom onset, with a diagnostic sensitivity of 81.5%. Within one week of symptom onset, diagnostic sensitivity and specificity reached 90.9% (95% CI, 85.1%–94.6%) and 98.3% (95% CI, 91.1%–99.9%), respectively. Serum N-Ag concentration closely correlated with disease severity. Longitudinal analysis revealed the simultaneous increase of antibodies and decrease of N-Ag. Sensitivities of COVID-VIRO-LFIA and COV-QUANTO-ELISA tests on NP and serum samples were close to 80%. They are suitable COVID-19-laboratory diagnostic tests, particularly when blood samples are available, thus reducing the requirement for NP sampling, and subsequent PCR analysis. ELISA titers may help to identify patients at risk of poor outcomes. [ABSTRACT FROM AUTHOR]

In previous studies, cefiderocol resistance in I E. coli i has been mainly attributed to the increased copy number and expression of the I bla i SB NDM sb -like gene.[2] Thus, we first investigated the gene copy number of I bla i SB NDM sb using the average coverage of reads generated from WGS data normalized to the average coverage of several intrinsic chromosomal genes used as reference ( I bla i SB EC sb -like, I gyrB i , I tonB i , I rpoB i ). Cefiderocol is a novel siderophore-conjugated cephalosporin that is very promising for the treatment of infections caused by MDR bacteria. [Extracted from the article]

The EPIC consortium brings together experts from a wide range of fields that include clinical, molecular and basic microbiology, infectious diseases, computational biology and chemistry, drug discovery and design, bioinformatics, biochemistry, biophysics, pharmacology, toxicology, veterinary sciences, environmental sciences, and epidemiology. The main question to be answered by the EPIC alliance is the following: "What is the best approach for data mining on carbapenemase inhibitors and how to translate this data into experiments?" From this forum, we propose that the scientific community think up new strategies to be followed for the discovery of new carbapenemase inhibitors, so that this process is efficient and capable of providing results in the shortest possible time and within acceptable time and economic costs. [ABSTRACT FROM AUTHOR]

Early detection of multidrug resistant bacteria is of paramount importance for implementing appropriate infection control strategies and proper antibacterial therapies. We have evaluated a novel real-time PCR assay using fluorescent probes and 3base® technology, the EasyScreenTM ESBL/CPO Detection Kit (Genetic Signatures, Newtown, Australia), for the detection of 15 β-lactamase genes (blaVIM, blaNDM, blaIMP, blaOXA-48, blaKPC, blaOXA-23, blaOXA-51, blaSME,blaIMI, blaGES,blaTEM,blaSHV, blaCTX-M,blaCMY, blaDHA) and colistin resistance mcr-1 gene from 341 bacterial isolates (219 Enterobacterales, 66 P. aeruginosa and 56 A. baumannii) that were grown on Mueller–Hinton (MH) agar plates. One colony was suspended in provided extraction buffer, which lyses and converts the nucleic acids into a 3base®-DNA form (cytosines are converted into uracil, and subsequently thymine during PCR). The converted bacterial DNA is then added to the 6 PCR mixes, with primers for three targets plus one internal control. The EasyScreenTM ESBL/CPO Detection Kit was able to detect the 5-major (NDM, VIM, IMP, KPC, OXA-48) and 2-minor (IMI, Sme) carbapenemases and their variants irrespective of the species expressing them with nearly 100% sensitivity and specificity. With cephalosporinases CMY (82% of sensitivity) and DHA (87% of sensitivity) detection of chromosomally encoded variants was less efficient. Similarly, the chromosomally encoded OXA-51 variants were not consistently detected in A. baumannii. Despite being capable of efficiently detecting blaCTX-M-, blaTEM-, blaSHV- and blaGES-like genes, the EasyScreen™ ESBL/CPO Detection Kit was not able to distinguish between penicillinases and ESBL-variants of TEM and SHV and between GES-ESBLs and GES-carbapenemases. As GES enzymes are still rare, their detection as an ESBL or a carbapenemase remains important. Detection of mcr-1 was efficient, but none of the other mcr-alleles were detected in the 341 bacterial isolates tested. The EasyScreenTM ESBL/CPO Detection Kit is adapted for the detection of the most prevalent carbapenemases encountered in Gram-negatives isolated worldwide. [ABSTRACT FROM AUTHOR]

Easy and robust antimicrobial susceptibility testing (AST) methods are essential in clinical bacteriology laboratories (CBL) in low-resource settings (LRS). We evaluated the Beckman Coulter MicroScan lyophilized broth microdilution panel designed to support Médecins Sans Frontières (MSF) CBL activity in difficult settings, in particular with the Mini-Lab. We evaluated the custom-designed MSF MicroScan Gram-pos microplate (MICPOS1) for Staphylococcus and Enterococcus species, MSF MicroScan Gram-neg microplate (MICNEG1) for Gram-negative bacilli, and MSF MicroScan Fastidious microplate (MICFAST1) for Streptococci and Haemophilus species using 387 isolates from routine CBLs from LRS against the reference methods. Results showed that, for all selected antibiotics on the three panels, the proportion of the category agreement was above 90% and the proportion of major and very major errors was below 3%, as per ISO standards. The use of the Prompt inoculation system was found to increase the MIC and the major error rate for some antibiotics when testing Staphylococci. The readability of the manufacturer's user manual was considered challenging for low-skilled staff. The inoculations and readings of the panels were estimated as easy to use. In conclusion, the three MSF MicroScan MIC panels performed well against clinical isolates from LRS and provided a convenient, robust, and standardized AST method for use in CBL in LRS. [ABSTRACT FROM AUTHOR]

The global spread of antimicrobial resistant (AMR) bacteria represents a considerable public health concern, yet their detection and identification of their resistance mechanisms remain challenging. Optimal diagnostic tests should provide rapid results at low cost to enable implementation in any microbiology laboratory. Lateral flow assays (LFA) meet these requirements and have become essential tools to combat AMR. This review presents the versatility of LFA developed for the AMR detection field, with particular attention to those directly triggering β-lactamases, their performances, and specific limitations. It considers how LFA can be modified by detecting not only the enzyme, but also its β-lactamase activity for a broader clinical sensitivity. Moreover, although LFA allow a short time-to-result, they are generally only implemented after fastidious and time-consuming techniques. We present a sample processing device that shortens and simplifies the handling of clinical samples before the use of LFA. Finally, the capacity of LFA to detect amplified genetic determinants of AMR by isothermal PCR will be discussed. LFA are inexpensive, rapid, and efficient tools that are easy to implement in the routine workflow of laboratories as new first-line tests against AMR with bacterial colonies, and in the near future directly with biological media. [ABSTRACT FROM AUTHOR]

Pathogen-focused, randomized, controlled trials (PF-RCT) are important in the fight against carbapenem-resistant (CR) Gram-negative infections. Some recently approved antibiotics and older generic antibiotics with activity against CR Gram-negative bacteria were investigated in PF-RCTs in a variety of infections. We searched Pubmed, Cochrane database and international clinical trial databases for PF-RCTs for the period between 2005 and 2020 and compared the study designs, patient populations, infection types, pathogens, and Day-28 all-cause mortality (ACM). PF-RCTs are particularly challenging to quantitatively assess and compare due to the heterogeneity in infection types, pathogens, CR mechanism, inclusion/exclusion criteria, and endpoints. Data interpretation is further complicated by lack of formal statistical analysis plans and/or non-inferiority design, and limited power across most PF-RCTs. The studies with new antibiotics (i.e. plazomicin, meropenem/vaborbactam, cefiderocol) ranked lower regarding feasibility, with relatively small sample sizes (analyzed: 37–118) versus the comparative effectiveness studies of older generic drugs (analyzed: 94–406). ACM ranged between 11.8% and 40% for CR Enterobacterales, 17.7% and 57.4% for CR Acinetobacter spp., and 20.0% and 30.8% for CR Pseudomonas aeruginosa. The information gathered must be considered carefully alongside the study limitations and caution should be exercised when making direct comparisons across trials. PLAIN LANGUAGE SUMMARY New antibiotics to treat multidrug-resistant Gram-negative bacterial infections are needed because antimicrobial resistance has become a global threat. In recent years, several pathogen-focused, randomized, controlled clinical trials were conducted to test new antibiotics or combinations of older generic antibiotics in the fight against resistant bacteria. However, these trials were exceptionally challenging and most of them enrolled relatively few patients. These studies were highly heterogeneous in terms of species, antibiotics, infection site, mechanism of resistance, endpoints and patient factors. In these trials, all-cause mortality at Day 28 or Day 30 were numerically lower with the new antibiotics in infections caused by carbapenem-resistant (CR) Enterobacterales. However, in the trials which investigated CR Acinetobacter spp. infections, there was no reduction in all-cause mortality at Day 28 or Day 30 with combinations of older generic antibiotics compared with colistin monotherapy. Limited information was available for CR Pseudomonas aeruginosa. More pathogen-focused, randomized, controlled clinical trials with more feasible design and higher patient numbers are needed to demonstrate clinical benefit in drug-resistant infections. [ABSTRACT FROM AUTHOR]

Objectives: To genetically characterize VIM-producing Enterobacter cloacae complex (ECC) isolates recovered in France from 2015 to 2018.Methods: WGS, species determination, MLST, clonal relationship and genetic characterization were performed on 149 VIM-producing ECC isolates.Results: Among VIM-producing Enterobacterales, the prevalence of ECC increased drastically from 6% in 2012 to 52% in 2018. The most prevalent species were Enterobacter hormaechei subsp. hoffmannii (40.9%), E. hormaechei subsp. steigerwaltii (21.5%), E. hormaechei subsp. xiangfangensis (14.8%) and ECC clade S (17.4%). Major STs were ST-873 (17.5%), ST-66 (12.1%), ST-78 (9.4%), ST-419 (8.1%), ST-145 (4.7%), ST-50 (4.0%), ST-118 (4.0%) and ST-168 (4.0%). Finally, six different integrons were identified, with some being specific to a given blaVIM variant (In916 with blaVIM-1-aacA4'-aphA15-aadA1-catB2 and In416 with blaVIM-4-aacA7-dfrA1b-aadA1b-smr2 genes).Conclusions: This study demonstrated the genetic diversity among VIM-producing ECC isolates, indicating that their spread is not linked to a single clone. [ABSTRACT FROM AUTHOR]

Patients infected with severe acute respiratory syndrome coronavirus 2 might have bacterial and fungal superinfections develop. We describe a clinical case of coronavirus disease with pulmonary aspergillosis associated with Bordetella hinzii pneumonia in an immunocompetent patient in France. B. hinzii infections are rare in humans and develop secondary to immunosuppression or debilitating diseases. [ABSTRACT FROM AUTHOR]

Summary: Early detection of expanded‐spectrum cephalosporin (ESC) resistance is essential not only for an effective therapy but also for the prompt implementation of infection control measures to prevent dissemination in the hospital. We have developed and validated a lateral flow immunoassay (LFIA), called LFIA‐CTX test, for the detection of ESC (cefotaxime) hydrolytic activity based on structural discrimination between the intact antibiotic and its hydrolysed product. A single bacterial colony was suspended in an extraction buffer containing cefotaxime. After a 30‐min incubation, the solution is loaded on the LFIA for reading within 10 min. A total of 348 well‐characterized Gram‐negative isolates were tested. Among them, the 38 isolates that did not express any cefotaxime‐hydrolysing β‐lactamase gave negative results. Of the 310 isolates expressing at least one cefotaxime‐hydrolysing β‐lactamase, all were tested positive, except three OXA‐48‐like producers, which were repeatedly detected negative. Therefore, the sensitivity was 99.1% and the specificity was 100%. The LFIA‐CTX test is efficient, fast, low‐cost and easy to implement in the workflow of a routine microbiology laboratory. This paper details the development and validation of a lateral flow immunochromatographic assay for the detection of hydrolysis activity of extended spectrum cephalosporins. This test, based on the use of monoclonal antibodies specific to intact antibiotics, takes advantage of the LFIA and the universality of biochemical tests (detection of enzymatic activity whatever the enzyme or its variants). Its validation on 348 bacterial strains indicates a sensitivity of 99.1% and a specificity of 100%, making it an efficient, rapid, low‐cost and easy to implement test in the workflow of a routine microbiology laboratory, without additional equipment or trained personnel. [ABSTRACT FROM AUTHOR]

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

While there are numerous variants of CIM that have been proposed [[1]], the "rCIM-A" name has already been published almost a year ago to define a rapid CIM (rCIM) protocol that optimizes results for chromosomal cephalosporinase (AmpC) hyper-producing Enterobacterales [[3]]. The Carbapenem Inactivation Method became "rapid" with the work by Muntean et al. [[6]] with final test results in less than 3 h, a time-to-result similar to those of other rapid CPE confirmation methods (colorimetric, spectrometry, etc.), thus changing the paradigm of CIM testing. As stated by Gauthier et al. interpretation of growth of I E. coli i ATCC 25922 on Mueller-Hinton agar in relation to the antibiotic is not always interpretable at 6 h [[5]]. References 1 Mitteregger D., Wessely J., Barisi'c I., Bedeni'c B., Kosak D., Kundi M. A Variant Carbapenem Inactivation Method (CIM) for Acinetobacter Baumannii Group with Shortened Time-to-Result: RCIM-A. Pathogens. [Extracted from the article]

Objectives: Detection of carbapenemase-producing Enterobacterales (CPEs) is sometimes difficult with AmpC-hyperproducing Enterobacterales (AHEs), as they may falsely be classified as CPEs. Here, we present a rapid Carbapenem Inactivation Method (rCIM) optimized for AmpC producers (rCIM-A) that allows rapid and easy discrimination between AHEs and CPEs.Methods: Enterobacterales (n = 249), including natural AmpC producers, AHEs, CPEs and non-carbapenemase-producing carbapenem-resistant control strains were evaluated, using Carba NP, rCIM and rCIM-A. The rCIM-A differs from the rCIM by the addition of cloxacillin (400 μg/mL) to the initial antibiotic incubation step.Results: The rCIM-A yielded a sensitivity and specificity of 84.26% (95% CI: 76.00%-90.55%) and 99.29% (95% CI: 96.11%-99.98%), respectively, while those of the rCIM were 86.11% (95% CI: 78.13%-92.01%) and 80.85% (95% CI: 73.38%-86.99%), respectively; those of Carba NP were lower at 84.04% (95% CI: 75.05%-90.78%) and 91.37% (95% CI: 85.41%-95.46%), respectively, due to indeterminate results. The rCIM-A was capable of discriminating between AHEs and true CPEs, but still failed to identify OXA-23-producing Proteus mirabilis isolates and remained only partially reliable for identifying IMI-like producers and a few MBL (2 NDM-1, 1 LMB-1, 1 TMB-1 and 1 IMP-13) producers. One chromosomally encoded AmpC variant, MIR-10, gave repeatedly positive results using all three tests and was thus considered a false positive.Conclusions: Specificity for AHEs greatly improved with the rCIM-A without altering the test performance for the other resistance mechanisms. It may replace the rCIM as a cheap, easy, rapid and accurate CPE detection test. [ABSTRACT FROM AUTHOR]

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

Ventilator-associated pneumonia (VAP) is associated with increased hospital stay and high morbidity and mortality in critically ill patients. The aims of this study were to (i) determine the incidence of multidrug-resistant (MDR) pathogens in the first episodes of VAP and to assess potential differences in bacterial profiles of subjects with early- versus late-onset VAP. This was a retrospective cohort study over a period of 18 months including all patients who had a first episode of VAP confirmed by positive bacterial culture. Subjects were distributed into two groups according to the number of intubation days: early-onset VAP (<5 days) or late-onset VAP (≥5 days). The primary endpoint was the nature of causative pathogens and their resistance profiles. Sixty patients were included, 29 men and 31 women, with an average age of 38 ± 16 years. The IGS 2 at admission was 40.5 [32–44] and APACHE was 19 [15–22]. Monomicrobial infections were diagnosed in 77% of patients (n = 46). The most frequently isolated bacteria were A. baumannii, 53% (n = 32); P. aeruginosa in 37% (n = 22); Enterobacterales in 28% (n = 17) and S. aureus in 5% (n = 3). Ninety-seven percent of the bacteria were MDR. The VAP group comprised 36 (60%) episodes of early-onset VAP and 24 (40%) episodes of late-onset VAP. There was no significant difference in the distribution of the bacterial isolates, nor in terms of antibacterial resistances between early- and late-onset VAPs. Our data support recent observations that there is no microbiological difference in the prevalence of potential MDR pathogens or in their resistance profiles associated with early- versus late-onset VAPs, especially in countries with high rates of MDR bacteria. [ABSTRACT FROM AUTHOR]

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

Two AMR isolates were found in the soapy water used for cleaning medical devices: one I Acinetobacter indicus i expressing I bla i SB NDM-1 sb and I bla i SB OXA-235 sb genes and one I Enterobacter chengduensis i expressing a I bla i SB CTX-M-15 sb gene. MDR Gram-negatives constitute a global public health concern, as they may cause severe, difficult-to-treat and often deadly infections, such as bloodstream infections and pneumonia. Carbapenem-resistant (CR) I Acinetobacter i spp. I Pseudomonas i spp. and Enterobacterales are among the most worrisome.[1] Even though these bacteria are increasingly described worldwide, epidemiological studies are very scarce from the African continent, except in Northern countries. [Extracted from the article]

Background: VRE are nosocomial pathogens with an increasing incidence in recent decades. Rapid detection is crucial to reduce their spread and prevent infections and outbreaks.Objectives: To evaluate a lateral flow immunoassay (LFIA) (called NG-Test VanA) for the rapid and reliable detection of VanA-producing VRE (VanA-VRE) from colonies and broth.Methods: NG-Test VanA was validated on 135 well-characterized enterococcal isolates grown on Mueller-Hinton (MH) agar (including 40 VanA-VRE). Different agar plates and culture broths widely used in routine laboratories for culture of enterococci were tested.Results: All 40 VanA-VRE clinical isolates were correctly detected in less than 15 min irrespective of the species expressing the VanA ligase and the medium used for bacterial growth. No cross-reaction was observed with any other clinically relevant ligases (VanB, C1, C2, D, E, G, L, M and N). Overall, the sensitivity and specificity of the assay were 100% for VanA-VRE grown on MH agar plates. NG-Test VanA accurately detects VanA-VRE irrespective of the culture medium (agar and broth). Band intensity was increased when using bacteria grown on vancomycin-containing culture media or on MH close to the vancomycin disc as a consequence of VanA induction. The limit of detection of the assay was 6.3 × 106 cfu per test with bacteria grown on MH plates and 4.9 × 105 cfu per test with bacteria grown on ChromID® VRE plates.Conclusions: NG-Test VanA is efficient, rapid and easy to implement in the routine workflow of a clinical microbiology laboratory for the confirmation of VanA-VRE. [ABSTRACT FROM AUTHOR]

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