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We examined the emergence and characteristics of oxacillinase-484–producing Enterobacterales in France during 2012–2023. Genomic analysis identified 2 predominant sequence types in Escherichia coli: ST410 and ST1722. Plasmid analysis revealed that blaOXA-484 genes were carried mostly on an IncX3-type plasmid associated with genetic elements including insertion sequences IS3000 and ISKpn19.

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 Bacterial evolution is affected by mobile genetic elements like phages and conjugative plasmids, offering new adaptive traits while incurring fitness costs. Their infection is affected by the bacterial capsule. Yet, its importance has been difficult to quantify because of the high diversity of confounding mechanisms in bacterial genomes such as anti-viral systems and surface receptor modifications. Swapping capsule loci between Klebsiella pneumoniae strains allowed us to quantify their impact on plasmid and phage infection independently of genetic background. Capsule swaps systematically invert phage susceptibility, revealing serotypes as key determinants of phage infection. Capsule types also influence conjugation efficiency in both donor and recipient cells, a mechanism shaped by capsule volume and conjugative pilus structure. Comparative genomics confirmed that more permissive serotypes in the lab correspond to the strains acquiring more conjugative plasmids in nature. The least capsule-sensitive pili (F-like) are the most frequent in the species’ plasmids, and are the only ones associated with both antibiotic resistance and virulence factors, driving the convergence between virulence and antibiotics resistance in the population. These results show how traits of cellular envelopes define slow and fast lanes of infection by mobile genetic elements, with implications for population dynamics and horizontal gene transfer.

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.

As bloodstream infections and associated septic shock are common causes of mortality in hospitals, rapid antibiotic susceptibility testing (AST) performed directly on positive blood cultures is needed to implement an efficient therapy in clinical settings. We evaluated the Reveal® rapid AST system on a collection of 197 fully characterized carbapenem-resistant Enterobacterales, including 177 carbapenemase producers (CPE) spiked in blood culture bottles. The clinical categorization based on the Minimal Inhibitory Concentration (MIC) determination of eighteen antimicrobial molecules was compared to the clinical categorization based on the disk diffusion assay as a reference. The Reveal AST system provided results within a mean time to result of 5 h. Overall, the categorical agreement (CA) between the two techniques was 94.1%. The rates of very major errors (VMEs), major errors (MEs) and minor errors (mEs) were 3.8%, 3.7% and 5.6%, respectively. Imipenem was the antimicrobial with the lowest CA rate (78.7%), with rates of 15% VMEs and 10.7% MEs, but the performances were better when considering only the non-CPE category (CA of 89%). On this resistant collection of Enterobacterales with numerous acquired β-lactamases, the Specific Reveal assay proved to be useful for a rapid determination of AST compatible with a quick adaptation of the patient’s antimicrobial treatment.

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: Objectives: Ceftolozane-tazobactam (C/T) proved its efficacy for the treatment of infections caused by non-carbapenemase producing Pseudomonas aeruginosa and Enterobacterales. Here, we aimed to provide susceptibility data on a large series of Enterobacterales since the revision of EUCAST categorization breakpoints in 2020. Methods: First, C/T susceptibility was determined on characterized Enterobacterales resistant to third generation cephalosporins (3GCs) (extended spectrum β-lactamase [ESBL] production or different levels of AmpC overexpression) (n = 213) and carbapenem-resistant Enterobacterales (CRE) (n = 259), including 170 carbapenemase producers (CPE). Then, 1632 consecutive clinical Enterobacterales responsible for infection were prospectively collected in 23 French hospitals. C/T susceptibility was determined by E-test® (biomérieux) and broth microdilution (BMD) (Sensititre™, Thermo Scientific) to perform method comparison. Results: Within the collection isolates, 88% of 3GC resistant strains were susceptible to C/T, with important variation depending on the resistance mechanism: 93% vs. 13% susceptibility for CTX-M and SHV-ESBL producers, respectively. Only 20% of the CRE were susceptible to C/T. Among CPE, 80% of OXA-48-like producers were susceptible to C/T, whereas all metallo-β-lactamase producers were resistant. The prospective study revealed that 95.6% of clinical isolates were susceptible to C/T. Method comparison performed on these 1632 clinical isolates demonstrated 99% of categorization agreement between MIC to C/T determined by E-test® in comparison with the BMD (reference) and only 74% of essential agreement. Conclusion: Overall, C/T showed good activity against wild-type Enterobacterales, AmpC producers, and ESBL-producing Escherichia coli but is less active against ESBL-producing Klebsiella pneumoniae, and CRE. E-test® led to an underestimation of the MICs in comparison to the BMD reference.

ABSTRACTCarbapenemase-producing Enterobacterales have emerged in the last decades as one of the main threats in modern medicine. Enterobacter is a genus increasingly recognized as a key player in carbapenemase diffusion besides Escherichia coli and Klebsiella pneumoniae, but information regarding Citrobacter spp. remained scarce. A collection of 803 isolates of Citrobacter spp. recovered from the French National Center for carbapenem resistance over a 2-year period during 2019–2020 was analyzed. A total of 15 different carbapenemases were identified of which OXA-48 followed by NDM-1 and OXA-181 represented the main enzymes. Phylogenetic analysis of this collection revealed that Citrobacter freundii was the main species among which three main clones are circulating in France, being ST8, ST22, and ST91. ST22 and ST8 were distributed in all regions whereas ST91 demonstrated a regional spread. Analysis of the species diversity revealed that among the C. freundii complex, Citrobacter portucalensis and Citrobacter braakii were also disseminated. We revealed the dissemination of a clone carrying both blaDHA-15 (β-lactamase-encoding gene rarely reported in Enterobacterales) and blaOXA-48 in the north of France and allowed to identify an outbreak not previously investigated. This study analyzed the largest collection of carbapenemase-producing Citrobacter spp. and allowed deciphering of molecular epidemiology of Citrobacter spp. in France.IMPORTANCEThe emergence of carbapenemase producers in Enterobacterales mostly involves Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae complex species. However, in France, we observed the emergence and the rapid dissemination of carbapenemase in Citrobacter spp. In this study, we demonstrated that a wide variety of carbapenemases is produced by many different species of Citrobacter spp. However, we clearly identify three high-risk clones of Citrobacter freundii, ST8, ST22, and ST91 that drive the spread of carbapenemase in France. This epidemiological study paves the way of further analysis that would aim to identify the virulence factors involved in this pellicular ability of these three clones to disseminate at the hospital.

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.

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 Antibiotic resistance is rapidly spreading via the horizontal transfer of resistance genes in mobile genetic elements. While plasmids are key drivers of this process, few integrative phages encode antibiotic resistance genes. Here, we find that phage-plasmids, elements that are both phages and plasmids, often carry antibiotic resistance genes. We found 60 phage-plasmids with 184 antibiotic resistance genes, providing resistance for broad-spectrum-cephalosporins, carbapenems, aminoglycosides, fluoroquinolones, and colistin. These genes are in a few hot spots, seem to have been cotranslocated with transposable elements, and are often in class I integrons, which had not been previously found in phages. We tried to induce six phage-plasmids with resistance genes (including four with resistance integrons) and succeeded in five cases. Other phage-plasmids and integrative prophages were coinduced in these experiments. As a proof of concept, we focused on a P1-like element encoding an extended spectrum β-lactamase, blaCTX-M-55. After induction, we confirmed that it is capable of infecting and converting four other E. coli strains. Its reinduction led to the further conversion of a sensitive strain, confirming that it is a fully functional phage. This study shows that phage-plasmids carry a large diversity of clinically relevant antibiotic resistance genes that they can transfer across bacteria. As plasmids, these elements seem plastic and capable of acquiring genes from other plasmids. As phages, they may provide novel paths of transfer for resistance genes because they can infect bacteria that are distant in time and space from the original host. As a matter of alarm, they may also mediate transfer to other types of phages. IMPORTANCE The dissemination of antimicrobial resistance is a major threat to global health. Here, we show that a group of temperate bacterial viruses (phages), termed phage-plasmids, commonly encode different and multiple types of resistance genes of high clinical importance, often in integrons. This is unexpected, as phages typically do not carry resistance genes and, hence, do not confer upon their hosts resistance via infection and genome integration. Our experiments with phage-plasmids isolated from clinical settings confirmed that they infect sensitive strains and render them antibiotic resistant. The spread of antibiotic resistance genes by phage-plasmids is worrisome because it dispenses cell-to-cell contact, which is necessary for canonical plasmid transfer (conjugation). Furthermore, their integrons become genetic platforms for the acquisition of novel resistance genes.

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 Matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry (MALDI‐TOF MS) has become a staple in clinical microbiology laboratories. Protein‐profiling of bacteria using this technique has accelerated the identification of pathogens in diagnostic workflows. Recently, lipid profiling has emerged as a way to complement bacterial identification where protein‐based methods fail to provide accurate results. This study aimed to address the challenge of rapid discrimination between Escherichia coli and Shigella spp. using MALDI‐TOF MS in the negative ion mode for lipid profiling coupled with machine learning. Both E. coli and Shigella species are closely related; they share high sequence homology, reported for 16S rRNA gene sequence similarities between E. coli and Shigella spp. exceeding 99%, and a similar protein expression pattern but are epidemiologically distinct. A bacterial collection of 45 E. coli, 48 Shigella flexneri, and 62 Shigella sonnei clinical isolates were submitted to lipid profiling in negative ion mode using the MALDI Biotyper Sirius® system after treatment with mild‐acid hydrolysis (acetic acid 1% v/v for 15 min at 98°C). Spectra were then analyzed using our in‐house machine learning algorithm and top‐ranked features used for the discrimination of the bacterial species. Here, as a proof‐of‐concept, we showed that lipid profiling might have the potential to differentiate E. coli from Shigella species using the analysis of the top five ranked features obtained by MALDI‐TOF MS in the negative ion mode of the MALDI Biotyper Sirius® system. Based on this new approach, MALDI‐TOF MS analysis of lipids might help pave the way toward these goals.

Abstract Background Klebsiella pneumoniae is a leading cause of intractable hospital-acquired multidrug-resistant infections and carbapenemase-producing K. pneumoniae (CPKp) are particularly feared. Most of the clinical isolates produce capsule as a major virulence factor. Recombination events at the capsule locus are frequent and responsible for capsule diversity within Klebsiella spp. Capsule diversity may also occur within clonal bacterial populations generating differences in colony aspect. However, little is known about this phenomenon of phenotypic variation in CPKp and its consequences. Results Here, we explored the genetic causes of in vitro switching from capsulated, mucoid to non-mucoid, non-capsulated phenotype in eight clinical CPKp isolates. We compared capsulated, mucoid colony variants with one of their non-capsulated, non-mucoid isogenic variant. The two colony variants were distinguished by their appearance on solid medium. Whole genome comparison was used to infer mutations causing phenotypic differences. The frequency of phenotypic switch was strain-dependent and increased along with colony development on plate. We observed, for 72 non-capsulated variants that the loss of the mucoid phenotype correlates with capsule deficiency and diverse genetic events, including transposition of insertion sequences or point mutations, affecting genes belonging to the capsule operon. Reduced or loss of capsular production was associated with various in vitro phenotypic changes, affecting susceptibility to carbapenem but not to colistin, in vitro biofilm formation and autoaggregation. Conclusions The different impact of the phenotypic variation among the eight isolates in terms of capsule content, biofilm production and carbapenem susceptibility suggested heterogeneous selective advantage for capsular loss according to the strain and the mutation. Based on our results, we believe that attention should be paid in the phenotypic characterization of CPKp clinical isolates, particularly of traits related to virulence and carbapenem resistance.

The worldwide spread of Klebsiella pneumoniae carbapenemase–producing Klebsiella pneumoniae (KPC-Kp) isolates was reported to be caused by dissemination of 1 clonal complex (i.e., clonal group [CG] 258, which includes sequence types [STs] 258 and 512). We conducted whole-genome sequencing and epidemiologic analysis of all KPC-Kp isolates in France in 2018 and found that new successful high-risk clones of ST147, ST307, ST231, and ST383 are now the main drivers of blaKPC genes. The blaKPC genes were mostly carried by Tn4401a and Tn4401d structures and a new non–Tn4401 element. Our epidemiologic investigations showed that the emergence of these non-CG258 KPC-Kp isolates in France was linked to dissemination of these clones from Portugal. Thus, KPC-Kp epidemiology has changed in Europe, at least in several non–KPC-endemic countries of western Europe, such as France and Portugal, where CG258 is not the most prevalent clone.

Abstract In Enterobacterales, the most common carbapenemases are Ambler’s class A (KPC-like), class B (NDM-, VIM- or IMP-like) or class D (OXA-48-like) enzymes. This study describes the characterization of twenty-four OXA-23 or OXA-58 producing-Proteus mirabilis isolates recovered from human and veterinary samples from France and Belgium. Twenty-two P. mirabilis isolates producing either OXA-23 (n = 21) or OXA-58 (n = 1), collected between 2013 and 2018, as well as 2 reference strains isolated in 1996 and 2015 were fully sequenced. Phylogenetic analysis revealed that 22 of the 24 isolates, including the isolate from 1996, belonged to a single lineage that has disseminated in humans and animals over a long period of time. The bla OXA-23 gene was located on the chromosome and was part of a composite transposon, Tn6703, bracketed by two copies of IS15∆II. Sequencing using Pacbio long read technology of OXA-23-producing P. mirabilis VAC allowed the assembly of a 55.5-kb structure encompassing the bla OXA-23 gene in that isolate. By contrast to the bla OXA-23 genes, the bla OXA-58 gene of P. mirabilis CNR20130297 was identified on a 6-kb plasmid. The acquisition of the bla OXA-58 gene on this plasmid involved XerC-XerD recombinases. Our results suggest that a major clone of OXA-23-producing P. mirabilis is circulating in France and Belgium since 1996.

β-Lactams are among the most prescribed antibiotics worldwide, mainly due to their weak toxicity and good efficacy [...]

ABSTRACT Carbapenemase-producing Escherichia coli (CP-Ec) represents a major public health threat with a risk of dissemination in the community as has occurred for lineages producing extended-spectrum β-lactamases. To characterize the extent of CP-Ec spread in France, isolates from screening and infection samples received at the French National Reference Center (F-NRC) laboratory for carbapenemase-producing Enterobacterales were investigated. A total of 691 CP-Ec isolates collected between 2012 and 2015 and 22 isolates collected before 2012 were fully sequenced. Analysis of their genome sequences revealed some disseminating multidrug-resistant (MDR) lineages frequently acquiring diverse carbapenemase genes mainly belonging to clonal complex 23 (CC23) (sequence type 410 [ST410]) and CC10 (ST10 and ST167) and sporadic isolates, including rare ST131 isolates (n = 17). However, the most represented sequence type (ST) was ST38 (n = 92) with four disseminated lineages carrying blaOXA-48-like genes inserted in the chromosome. Globally, the most frequent carbapenemase gene (n = 457) was blaOXA-48. It was also less frequently associated with MDR isolates being the only resistance gene in 119 isolates. Thus, outside the ST38 clades, its acquisition was frequently sporadic with no sign of dissemination, reflecting the circulation of the IncL plasmid pOXA-48 in France and its high frequency of conjugation. In contrast, blaOXA-181 and blaNDM genes were often associated with the evolution of MDR E. coli lineages characterized by mutations in ftsI and ompC. IMPORTANCE Carbapenemase-producing Escherichia coli (CP-Ec) might be difficult to detect, as MICs can be very low. However, their absolute number and their proportion among carbapenem-resistant Enterobacterales have been increasing, as reported by WHO and national surveillance programs. This suggests a still largely uncharacterized community spread of these isolates. Here, we have characterized the diversity and evolution of CP-Ec isolated in France before 2016. We show that carbapenemase genes are associated with a wide variety of E. coli genomic backgrounds and a small number of dominant phylogenetic lineages. In a significant proportion of CP-Ec, the most frequent carbapenemase gene blaOXA-48, was detected in isolates lacking any other resistance gene, reflecting the dissemination of pOXA-48 plasmids, likely in the absence of any antibiotic pressure. In contrast, carbapenemase gene transfer may also occur in multidrug-resistant E. coli, ultimately giving rise to at-risk lineages encoding carbapenemases with a high potential of dissemination.

Antimicrobial resistance and multidrug-resistant organisms currently constitute a severe public health problem [...]

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.

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 blaVIM–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 blaVIM–1 gene transcripts, and detectable carbapenemase activity using the Carba NP test. Assessment of read coverage demonstrated that a duplication of the region surrounding blaVIM–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 blaVIM–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.

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.

Gram-negative bacteria, especially Enterobacterales, have emerged as major players in antimicrobial resistance worldwide. Resistance may affect all major classes of anti-gram-negative agents, becoming multidrug resistant or even pan-drug resistant. Currently, β-lactamase-mediated resistance does not spare even the most powerful β-lactams (carbapenems), whose activity is challenged by carbapenemases. The dissemination of carbapenemases-encoding genes among Enterobacterales is a matter of concern, given the importance of carbapenems to treat nosocomial infections. Based on their amino acid sequences, carbapenemases are grouped into three major classes. Classes A and D use an active-site serine to catalyze hydrolysis, while class B (MBLs) require one or two zinc ions for their activity. The most important and clinically relevant carbapenemases are KPC, IMP/VIM/NDM, and OXA-48. However, several carbapenemases belonging to the different classes are less frequently detected. They correspond to class A (SME-, Nmc-A/IMI-, SFC-, GES-, BIC-like…), to class B (GIM, TMB, LMB…), class C (CMY-10 and ACT-28), and to class D (OXA-372). This review will address the genetic diversity, biochemical properties, and detection methods of minor acquired carbapenemases in Enterobacterales.

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

Resistance to polymyxins in most Gram-negative bacteria arises from chemical modifications to the lipid A portion of their lipopolysaccharide (LPS) mediated by chromosomally encoded mutations or the recently discovered plasmid-encoded mcr genes that have further complicated the landscape of colistin resistance. Currently, minimal inhibitory concentration (MIC) determination by broth microdilution, the gold standard for the detection of polymyxin resistance, is time consuming (24 h) and challenging to perform in clinical and veterinary laboratories. Here we present the use of the MALDIxin to detect colistin resistant Salmonella enterica using the MALDxin test on the routine matrix-assisted laser desorption ionization (MALDI) Biotyper Sirius system.

In the context of increasing antimicrobial resistance in Enterobacterales, the management of these UTIs has become challenging. We retrospectively assess the prevalence of antimicrobial resistance in Enterobacterales isolates recovered from urinary tract samples in France, between 1 September 2017, to 31 August 2018. Twenty-six French clinical laboratories provided the susceptibility of 134,162 Enterobacterales isolates to 17 antimicrobials. The most frequent species were E. coli (72.0%), Klebsiella pneumoniae (9.7%), Proteus mirabilis (5.8%), and Enterobacter cloacae complex (2.9%). The overall rate of ESBL-producing Enterobacterales was 6.7%, and ranged from 1.0% in P. mirabilis to 19.5% in K. pneumoniae, and from 3.1% in outpatients to 13.6% in long-term care facilities. Overall, 4.1%, 9.3% and 10.5% of the isolates were resistant to cefoxitin, temocillin and pivmecillinam. Cotrimoxazole was the less active compound with 23.4% resistance. Conversely, 4.4%, 12.9%, and 14.3% of the strains were resistant to fosfomycin, nitrofurantoin, and ciprofloxacin. However, less than 1% of E. coli was resistant to fosfomycin and nitrofurantoin. We identified several trends in antibiotics resistances among Enterobacterales isolates recovered from the urinary tract samples in France. Carbapenem-sparing drugs, such as temocillin, mecillinam, fosfomycin, cefoxitin, and nitrofurantoin, remained highly active, including towards ESBL-E.

Since the first description of OXA-48, more than forty variants have been recovered from Enterobacterales isolates. Whereas some OXA-48-related enzymes have been reported as conferring similar resistance patterns, namely, the hydrolysis of carbapenems and penicillins with very weak or almost no activity against expanded-spectrum cephalosporins, some have reduced carbapenem and temocillin hydrolysis, and others hydrolyze expanded-spectrum cephalosporins and carbapenems only marginally. With such drastic differences in the hydrolytic profile, especially of carbapenems, it becomes urgent to establish hydrolytic cutoffs in order to determine when an OXA-48-like enzyme may be considered as a carbapenemase or not. With this aim, the coefficient of activity for imipenem (kcat/Km) was determined for a total of 30 enzymes, including OXA-48, OXA-48-like natural variants, and OXA-48 synthetic mutants. In addition, six different methods for the detection of carbapenemase-producers were performed. The coefficients of activity for imipenem for all the different enzymes went from 550 mM−1·s−1 to 0.02 mM−1·s−1. In order to match the coefficient of activity results with the biochemical confirmatory tests, we suggest the value of 0.27 mM−1·s−1 as the cutoff above which an OXA-48 variant may be considered a carbapenem-hydrolyzing enzyme.

Infections caused by extended-spectrum β-lactamase-producing Klebsiella pneumoniae (ESBL-KP) are constantly rising worldwide and are often reported as causative agent of outbreaks in intensive care units (ICUs). During the first wave of the COVID-19 pandemic, bacterial cross-transmission was thought unlikely to occur due to the reinforcement of hygiene measures and prevention control. However, we report here an ESBL-producing K. pneumoniae (ST394) isolate responsible for a nosocomial outbreak in an ICU dedicated to COVID-19 patients.

Proteus spp. are commensal Enterobacterales of the human digestive tract. At the same time, P. mirabilis is commonly involved in urinary tract infections (UTI). P. mirabilis is naturally resistant to several antibiotics including colistin and shows reduced susceptibility to imipenem. However higher levels of resistance to imipenem commonly occur in P. mirabilis isolates consecutively to the loss of porins, reduced expression of penicillin binding proteins (PBPs) PBP1a, PBP2, or acquisition of several antibiotic resistance genes, including carbapenemase genes. In addition, resistance to non-β-lactams is also frequently reported including molecules used for treating UTI infections (e.g., fluoroquinolones, nitrofurans). Emergence and spread of multidrug resistant P. mirabilis isolates, including those producing ESBLs, AmpC cephalosporinases and carbapenemases, are being more and more frequently reported. This review covers Proteus spp. with a focus on the different genetic mechanisms involved in the acquisition of resistance genes to multiple antibiotic classes turning P. mirabilis into a dreadful pandrug resistant bacteria and resulting in difficult to treat infections.

The linear reverse blotting assays are valid methods for accurate human papillomavirus (HPV) typing required to manage women at risk of developing cervical cancer. However, some samples showed a positive signal in HPV lines but failed to display a positive signal in subsequent typing lines (designated as HPV-X), which indicate that certain types were not available on the respective typing blots. The aim of this study is to elucidate the types or variants of HPV through the high-throughput sequencing (HTS) of 54 ASCUS cervical samples in which the viruses remained untypeable with INNO LiPA HPV® assays. Low-risk (LR)-HPV types (HPV6, 30, 42, 62, 67, 72, 74, 81, 83, 84, 87, 89, 90 and 114), high-risk (HR)-HPV35 and possibly (p)HR-HPV73 were detected among HPV-X. Individual multiple infections (two to seven types) were detected in 40.7% of samples. Twenty-two specimens contained variants characterised by 2–10 changes. HPV30 reached the maximal number of 17 variants with relative abundance inferior or equal to 2.7%. The presence of L1 quasispecies explains why linear reverse blotting assays fail when variants compete or do not match the specific probes. Further studies are needed to measure the LR-HPV quasispecies dynamics and its role during persistent infection. Keywords: Human papillomavirus, Variants, Genotyping, High-throughput sequencing, Quasispecies

We report the case of a 67-year old man with a right knee prosthetic joint infection due to extensively drug-resistant Enterobacter hormaechei. The resistance phenotype was due to the overproduction of the intrinsic cephalosporinase (ACT-5) associated with the production of three acquired β-lactamases (CTX-M-15, TEM-1B and OXA-1), and a putative membrane decreased permeability. He was first treated with colistin-tigecyclin due to adverse drug reactions; treatment was switched to cefiderocol for a 12-week antibiotic duration, with a favorable outcome.

The diffusion of KPC-2 carbapenemase is closely related to the spread of Klebsiella pneumoniae of the clonal-group 258 and linked to IncFIIK plasmids. Little is known about the biology of multi-drug resistant plasmids and the reasons of their successful dissemination. Using E. coli TOP10 strain harboring a multi-replicon IncFIIK-IncFIB blaKPC−2-gene carrying plasmid pBIC1a from K. pneumoniae ST-258 clinical isolate BIC-1, we aimed to identify basal gene expression and the effects of imipenem exposure using whole transcriptome approach by RNA sequencing (RNA-Seq). Independently of the antibiotic pressure, most of the plasmid-backbone genes were expressed at low levels. The most expressed pBIC1a genes were involved in antibiotic resistance (blaKPC−2, blaTEM and aph(3′)-I), in plasmid replication and conjugation, or associated to mobile elements. After antibiotic exposure, 34% of E. coli (pBIC1a) genome was differentially expressed. Induction of oxidative stress response was evidenced, with numerous upregulated genes of the SoxRS/OxyR oxydative stress regulons, the Fur regulon (for iron uptake machinery), and IscR regulon (for iron sulfur cluster synthesis). Nine genes carried by pBIC1a were up-regulated, including the murein DD-endopeptidase mepM and the copper resistance operon. Despite the presence of a carbapenemase, we observed a major impact on E. coli (pBIC1a) whole transcriptome after imipenem exposure, but no effect on the level of transcription of antimicrobial resistance genes. We describe adaptive responses of E. coli to imipenem-induced stress, and identified plasmid-encoded genes that could be involved in resistance to stressful environments.

Beta-Lactamase Database (BLDB) is a comprehensive, manually curated public resource providing up-to-date structural and functional information focused on this superfamily of enzymes with a great impact on antibiotic resistance. All the enzymes reported and characterised in the literature are presented according to the class (A, B, C and D), family and subfamily to which they belong. All three-dimensional structures of β-lactamases present in the Protein Data Bank are also shown. The characterisation of representative mutants and hydrolytic profiles (kinetics) completes the picture and altogether these four elements constitute the essential foundation for a better understanding of the structure-function relationship within this enzymes family. BLDB can be queried using different protein- and nucleotide-based BLAST searches, which represents a key feature of particular importance in the context of the surveillance of the evolution of the antibiotic resistance. BLDB is available online at http://bldb.eu without any registration and supports all modern browsers.

The regulation of RNA decay is now widely recognized as having a central role in bacterial adaption to environmental stress. Here we present an overview on the diversity of ribonucleases (RNases) and their impact at the posttranscriptional level in the human pathogen Staphylococcus aureus. RNases in prokaryotes have been mainly studied in the two model organisms Escherichia coli and Bacillus subtilis. Based on identified RNases in these two models, putative orthologs have been identified in S. aureus. The main staphylococcal RNases involved in the processing and degradation of the bulk RNA are (i) endonucleases RNase III and RNase Y and (ii) exonucleases RNase J1/J2 and PNPase, having 5′ to 3′ and 3′ to 5′ activities, respectively. The diversity and potential roles of each RNase and of Hfq and RppH are discussed in the context of recent studies, some of which are based on next-generation sequencing technology.