• Aucun résultat trouvé

β-lactamase mediated resistance in H. influenzae

The early 1970s witnessed the first detection of β-lactamase-mediated ampicillin resistance in H. influenzae, and in 1975 that β-lactamase was identified as a TEM type (56). In 1981, Rubin et al. revealed the presence of a novel β-lactamase in H. influenzae, later called ROB-1 (57). Both enzymes are plasmid-mediated class A serine β-lactamases that confer resistance to ampicillin and are efficientely inhibited by β-lactamase inhibitors such as clavulanic acid. However, they display clear differences in their isoelectric points (ROB-1 = 8.1, TEM-1 = 5.4) (57). In clinical microbiology laboratories, the detection of β-lactamase-mediated ampicillin resistance is generally easy. Among 14'870 H. influenzae strains isolated in different contries from patients with community-acquired respiratory tract infection included in the PROTEKT study (1999 - 2003), 15% were β-lactamase positive. Among the β-lactamase positive strains, 93.7% were positive for TEM-1, 4.6% for ROB-1, and only 0.04% for both genes. Twenty seven β-lactamase-positive H. influenzae isolates were negative for ROB-1 and TEM-1 genes. The authors explained the absence of both genes in these strains by the possible mutation that occurred in ROB-1 and/or TEM-1 genes, or the presence of other enzymes (58). The prevalence of ROB-1 shows substantial variations according to the studied geographical area, as exemplified by the following rates: Mexico (30%), United States (13.2%) and Canada (9.4%) (58). The isolates positive for either enzyme (TEM type or ROB-1) display ampicillin MICs well above the resistant breakpoint (i.e. MIC90 ≥32 µg/mL), and both enzymes are detected by nitrocefin hydrolysis (58), enabling reliable routine detection. However, only molecular methods can determine which of the two β-lactamases is present in a given isolate.

7.1.1. TEM-type β-lactamases

In the early 1960s the first β-lactamase was identified in Gram-negative bacteria in Greece. Thus, it was named TEM after the Greek patient name (Temoniera) from whom an E. coli strain carring this enzyme has been isolated (59). Afterwards, TEM-2 was discovered. TEM-1 and TEM-2 have identical biochemical properties in spite of the fact that TEM-2 has an amino acid substitution at position 37 (Gln37Lys) corresponding to a single base pair difference (C317A) in the coding region and a single base pair difference (C32T) in the promoter region of the blaTEM-2 gene (60).

These β-lactamases are frequently encountered in Gram-negative bacteria, including Enterobacteriaceae, Pseudomonas aeruginosa, H. influenzae, and Neisseria gonorrhoeae. TEM-1 and TEM-2 hydrolyze penicillins and the first-generation cephalosporins (e.g. cephalothin and cefazolin). However, they are not effective against broad-spectrum cephalosporins, such as ceftriaxone, ceftazidime, or cefepime (61). Large-scale analysis of plasmids identified in H. influenzae revealed that the blaTEM originating from Enterobacteriaceae was transposed through Tn2 or Tn3 onto cryptic plasmids already present in other Haemophilus species (50, 62).

Importantly, blaTEM genes have four differents promoters (P3, Pa/Pb, Prpt, and Pdel).

The promoter of the blaTEM-1A gene which is situated on the first widely used E. coli cloning vectors (plasmid pBR322) and on a Tn3 transposon, is corresponding to the promoter P3 (63). The promoter Pa/Pb is the result of the overlapping of two promoters Pa and Pb deriving from the mutation C32T according to the Sutcliffe numbering system (Figure-11) (63).

Figure-11 (source (63)): Nucleotide sequence of Pa/Pb promoter

Red arrow indicates the mutation C32T which conducted to the generation of promoter Pa/Pb.

Prpt promoter is characterized by the insertion of 54 base pairs. This insertion is composed by a repeat of bp 145T to 198A inclusive, inserted directly after bp 198.

Pdel promoter is defined by the deletion of 135 base pairs from bp G23 through to C157 inclusive, and one substitution (G162T) (50). Among the β-lactamase-positive H. influenzae strains, the distribution of these four promoters shows substantial variations. Using a collection of 104 β-lactamase-positive H. influenzae strains, Tristram et al. identified Pa/Pb promoter in about 60% of the strains. Pdel and Prpt were present in 20% of the strains (64). The TEM β-lactamase-specifying plasmids in H. influenzae represent two groups, non-conjugative (less than10kb) and conjugative (about 40kb). Interestingly, the blaTEM is the only resistance determinant carried by the non-conjugative plasmids (50, 65, 66).

Over the last decades, the blaTEM genes have undergone important variations. Different types of blaTEM genes were reported in association with extended-spectrum β-lactamases (ESBLs) in Gram-negative bacteria. These ESBLs are effective against broad-spectrum cephalosporins such as ceftriaxone, or ceftazidime. In contrast, carbapenems (e.g. imipenem) are generally active against ESBL-producing strains.

In the study conducted by Tristram et al., TEM-type ESBL genes (blaTEM-3, blaTEM-4 and blaTEM-5) were cloned into the reference strain H. influenzae Rd KW20. The analysis of

up to 0.5 µg/mL (as compared to 0.03 µg/mL for the strain control which carry only blaTEM-1), these recombinant strains would still not be classified as resistant according to the current clinical breakpoints (67). Thus, such ESBLs would be missed by the phenotypic screening methods commonly used in clinical microbiology laboratories.

The relative contribution of PBP3 alterations and TEM-1, TEM-15, or ROB-1 β-lactamases on cefotaxime resistance using an isogenic environment of H. influenzae was then reported by A. Søndergaard and N. Nørskov-Lauritsen (68). In 2002 two Haemophilus parainfluenzae isolates collected from two South African patients were shown to produce a TEM-15 ESBL, conferring cefotaxime MICs of >16 µg/mL (69).

H. parainfluenzae and H. influenzae can exchange plasmids carring β-lactamase (70).

It is therefore surprising that TEM-type ESBLs have not yet been detected in H. influenzae.

7.1.2. ROB β-lactamase

The first report of the ROB-1 β-lactamase was in a Hib causing meningitis (57).

Nowdays this enzyme is detected in different related species including Pasteurella multocida and Haemophilus ducreyi (71, 72). It is supposed that ROB-1 β-lactamase has an animal reservoir given the fact that the porcine pathogen Haemophilus pleuropneumoniae also produces this enzyme (73). Thus, its prevalence in animal strains may contribute to the spread of resistance to human pathogens.

7.2. β-lactamase-negative ampicillin-resistant (BLNAR) and altered penicillin