Class C ß-Lactamases: Molecular Characteristics.

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

ResFinder 4.0 for predictions of phenotypes from genotypes.

OBJECTIVES: WGS-based antimicrobial susceptibility testing (AST) is as reliable as phenotypic AST for several antimicrobial/bacterial species combinations. However, routine use of WGS-based AST is hindered by the need for bioinformatics skills and knowledge of antimicrobial resistance (AMR) determinants to operate the vast majority of tools developed to date. By leveraging on ResFinder and PointFinder, two freely accessible tools that can also assist users without bioinformatics skills, we aimed at increasing their speed and providing an easily interpretable antibiogram as output. METHODS: The ResFinder code was re-written to process raw reads and use Kmer-based alignment. The existing ResFinder and PointFinder databases were revised and expanded. Additional databases were developed including a genotype-to-phenotype key associating each AMR determinant with a phenotype at the antimicrobial compound level, and species-specific panels for in silico antibiograms. ResFinder 4.0 was validated using Escherichia coli (n = 584), Salmonella spp. (n = 1081), Campylobacter jejuni (n = 239), Enterococcus faecium (n = 106), Enterococcus faecalis (n = 50) and Staphylococcus aureus (n = 163) exhibiting different AST profiles, and from different human and animal sources and geographical origins. RESULTS: Genotype-phenotype concordance was ≥95% for 46/51 and 25/32 of the antimicrobial/species combinations evaluated for Gram-negative and Gram-positive bacteria, respectively. When genotype-phenotype concordance was <95%, discrepancies were mainly linked to criteria for interpretation of phenotypic tests and suboptimal sequence quality, and not to ResFinder 4.0 performance. CONCLUSIONS: WGS-based AST using ResFinder 4.0 provides in silico antibiograms as reliable as those obtained by phenotypic AST at least for the bacterial species/antimicrobial agents of major public health relevance considered.

A Structure-Based Classification of Class A ß-Lactamases, a Broadly Diverse Family of Enzymes.

For medical biologists, sequencing has become a commonplace technique to support diagnosis. Rapid changes in this field have led to the generation of large amounts of data, which are not always correctly listed in databases. This is particularly true for data concerning class A ß-lactamases, a group of key antibiotic resistance enzymes produced by bacteria. Many genomes have been reported to contain putative ß-lactamase genes, which can be compared with representative types. We analyzed several hundred amino acid sequences of class A ß-lactamase enzymes for phylogenic relationships, the presence of specific residues, and cluster patterns. A clear distinction was first made between dd-peptidases and class A enzymes based on a small number of residues (S70, K73, P107, 130SDN132, G144, E166, 234K/R, 235T/S, and 236G [Ambler numbering]). Other residues clearly separated two main branches, which we named subclasses A1 and A2. Various clusters were identified on the major branch (subclass A1) on the basis of signature residues associated with catalytic properties (e.g., limited-spectrum ß-lactamases, extended-spectrum ß-lactamases, and carbapenemases). For subclass A2 enzymes (e.g., CfxA, CIA-1, CME-1, PER-1, and VEB-1), 43 conserved residues were characterized, and several significant insertions were detected. This diversity in the amino acid sequences of ß-lactamases must be taken into account to ensure that new enzymes are accurately identified. However, with the exception of PER types, this diversity is poorly represented in existing X-ray crystallographic data.

Structure-based classification of class A beta-lactamases, an update.

Beta-lactamase (EC 3.5.2.6) synthesis, particularly in Gram-negative bacilli, is a major mechanism of natural and acquired resistance to beta-lactams, sometimes accompanied by impermeability and/or active efflux. These enzymes have been classified into four molecular classes (A-D). The serine enzymes of class A, which may be encoded by the bacterial chromosome or transferable elements and are susceptible to clinically available inhibitors (clavulanic acid, sulbactam, tazobactam, avibactam), are prevalent considering other molecular classes (B,C,D). The continual rapid development of genomic approaches and tremendous progress in automatic sequencer technology have resulted in the accumulation of massive amounts of data. A structure-based classification of class A beta-lactamases based on specific conserved motifs involved in catalytic mechanisms and/or substrate binding (S70XXK, S130DN, K234TG), together with E166 (Ambler numbering) and at least 24 other amino-acid residues or analogs such as G45, F66, V80, L81, L91, L101, P107, A134, L138, G143, G144, G156, L169, T181, T182, P183, was validated on 700 amino-acid sequences, including 132 representative types, but mostly probable enzyme sequences, many produced by environmental bacteria. Two subclasses (A1, A2), six major clusters or groups (e.g. natural limited-spectrum beta-lactamases (LSBL), wider spectrum beta-lactamases (WSBL), and various other clusters were identified on the basis of conserved (> 90%) and specific motifs, and residues such as S70TFKAL, S130DNTAANL, R164XEXXLN, V231GDKTG for subclass A1, S70VFKFH, S130DNNACDI,E166XXM, and V231AHKTG for subclass A2, a probable disulfide bridge C77-C123 and G236, A237, G238, and R244 for the LSBL group. This great diversity of primary structures was used as the basis for a structure-based and phylogenetic classification.

[Bacterial zoonoses: emerging concepts]. / Les zoonoses bactériennes emergentes: quelques aspects nouveaux.

Bacterial zoonoses are evolving with changes in society, climate and lifestyles. A hierarchy of non food-borne zoonoses was recently proposed in France, and includes characteristics such as severity criteria and bioterrorism potential. The creation of specific networks and reference centers has provided the means to monitor the emergence (or re-emergence) of zoonoses such as brucellosis and Q fever. Molecular tools have facilitated the detection of bacteria that are transmitted by arthropod vectors (ticks, fleas, etc.) and that cause diseases such as Lyme borreliosis, bartonellosis and ehrlichiosis.

Chromosomal ampC genes in Enterobacter species other than Enterobacter cloacae, and ancestral association of the ACT-1 plasmid-encoded cephalosporinase to Enterobacter asburiae.

The amplification and sequence of ampC genes in Enterobacter asburiae, Enterobacter cancerogenus, Enterobacter dissolvens, Enterobacter hormaechei and Enterobacter intermedius bring the number of known cephalosporinase sequences from the genus Enterobacter to seven. Expression in Escherichia coli of the ampC genes from E. asburiae, E. hormaechei and E. intermedius established the functional nature of these genes. ampC from E. asburiae shows 96.5% identity to bla(ACT-1) encoding a plasmid-borne cephalosporinase previously believed to derive from Enterobacter cloacae. The reassignment of ACT-1 ancestry to E. asburiae is confirmed by the 95.5% identity between ampR upstream of bla(ACT-1) and ampR from E. asburiae.

BUT-1: a new member in the chromosomal inducible class C beta-lactamases family from a clinical isolate of Buttiauxella sp.

An atypical Enterobacteriaceae strain with a beta-lactam susceptibility pattern of inducible cephalosporinase was isolated in Tenon Hospital (Paris, France) from a patient's skull wound infection. Identifications by the API-50CHE biochemical system and 16S rRNA gene sequencing concluded that it was a member of the Buttiauxella genus. The bla gene was cloned and sequenced. The deduced translated product was a 383-amino acid protein (BUT-1) with 75-78% identity with the chromosomal AmpC beta-lactamases of Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae and Escherichia coli. The isoelectric point of 9.0 and the kinetic constants of BUT-1 were comparable with results described for other Ambler class C enzymes. bla(BUT-1) and the associated ampR transcriptional regulator gene were divergently transcribed from a common intercistronic region, a genetic organization already described for other inducible class C beta-lactamases. The deduced amino acid sequence of AmpR shared 85% and 81% identity with AmpR from E. cloacae and C. freundii respectively.

Diversity of CTX-M beta-lactamases and their promoter regions from Enterobacteriaceae isolated in three Parisian hospitals.

Nine clinical isolates of Enterobacteriaceae (six Escherichia coli and three Proteus mirabilis) isolated in three Parisian hospitals between 1989 and 2000 showed a particular extended-spectrum cephalosporin-resistance profile characterized by resistance to cefotaxime and aztreonam but not to ceftazidime. CTX-M-1, CTX-M-2, CTX-M-9, CTX-M-14 and two novel plasmid-mediated CTX-M beta-lactamases (CTX-M-20, and CTX-M-21) were identified by polymerase chain reaction and isoelectric focusing (pI>8) and were associated in eight cases with TEM-1 (pI=5.4) or TEM-2 (pI=5.6) beta-lactamases. We used internal ISEcp1 and IS26 forward primers and the CTX-M consensus reverse primer to characterize the CTX-M beta-lactamase promoter regions and showed their high degree of structure diversity. We found upstream of some bla(CTX-M) genes, a 266-bp sequence 100% identical to the sequence upstream of the Kluyvera ascorbata beta-lactamase gene, suggesting that this chromosomal enzyme is the progenitor of the CTX-M-2/5 cluster.

Beta-lactamases of Kluyvera ascorbata, probable progenitors of some plasmid-encoded CTX-M types.

Kluyvera ascorbata produces a beta-lactamase that results in an atypical susceptibility pattern, including low-level resistance to penicillins, cephalothin, and cefuroxime, but this resistance is reversed by clavulanate. Ten nucleotide sequences of the corresponding gene, bla(KLUA), were obtained and were found to have minor variations (96 to 100%). Otherwise, bla(KLUA) was found to be similar (95 to 100%) to some plasmid-encoded CTX-M-type beta-lactamases. Finally, mobilization of bla(KLUA) on a plasmid was found to be mediated probably by a genetic mobile element like ISEcp1.

Molecular and biochemical characterization of a novel class A beta-lactamase (HER-1) from Escherichia hermannii.

Escherichia hermannii showed a low level of resistance to amoxicillin and ticarcillin, reversed by clavulanate, and a moderate susceptibility to piperacillin but was susceptible to all cephalosporins. A bla gene was cloned and encoded a typical class A beta-lactamase (HER-1, pI 7.5), which shares 45, 44, 41, and 40% amino acid identity with other beta-lactamases, AER-1 from Aeromonas hydrophila, MAL-1/Cko-1 from Citrobacter koseri, and TEM-1 and LEN-1, respectively. No ampR gene was detected. Only penicillins were efficiently hydrolyzed, and no hydrolysis was observed for cefuroxime and broad-spectrum cephalosporins. Sequencing of the bla gene in 12 other strains showed 98 to 100% identity with bla(HER-1).

Characterization of CMY-type beta-lactamases in clinical strains of Proteus mirabilis and Klebsiella pneumoniae isolated in four hospitals in the Paris area.

We isolated five clinical strains (three Proteus mirabilis and two Klebsiella pneumoniae) with beta-lactam resistance phenotypes consistent with production of an AmpC-type beta-lactamase. The predicted amino acid sequences of the enzymes were typical of class C beta-lactamases. The enzymes were identified as CMY-2, CMY-4 and a new CMY-variant beta-lactamase, CMY-12. The AmpC beta-lactamases from the two K. pneumoniae isolates were found to be encoded on self-transferable plasmids. The genes encoding the AmpC-type beta-lactamase produced by the three P. mirabilis isolates were chromosomal. Four of the five clinical isolates were from patients transferred from Greece, Algeria and Egypt; one of the K. pneumoniae strains was recovered from a French patient. PFGE analysis and rep-PCR fingerprinting showed that the two P. mirabilis isolates from Greek patients were closely related.