This work reports on five novel β-lactamases and provides the first description of TEM-24 in P. mirabilis
. TEM-57 (CF579) is a novel penicillinase related to TEM-1 (Gln-39), which is characterized by the Gly→Asp substitution at position 92 and which constitutes a new polymorphic position of TEM-type enzymes. TEM-66 (CF669) is a novel ESBL that harbors the amino acid substitutions that have been described for TEM-3 (36
) and that are associated with the substitution Gly-92→Asp, as in TEM-57.
The substitution at position 92 of the neutral glycine residue in TEM-1 and TEM-3 by the negatively charged aspartate residue in TEM-57 and TEM-66 could have accounted for the decrease in pI from 5.4 (TEM-1) to 5.2 (TEM-57) and from 6.3 (TEM-3) to 6.0 (TEM-66). Crystallographic analysis (18
) shows that amino acid 92, located in the loop that joins helices H2b and H3, is far from the active site of the enzyme. Hence, it probably has no effect on the intrinsic activity of the enzyme, as suggested by the kinetic constants, which were similar to those of TEM-1 for TEM-57 and similar to those of TEM-3 for TEM-66. Although TEM-57 and TEM-66 had the same substitution, Gly-92→Asp, it seems unlikely that the latter derives from the former. Hence, nucleotide position 477, at which a nucleotide substitution led to the amino acid substitution at position 92, could be a potential hot spot of mutation.
TEM-65 (IRT-16) (CF659) is a novel IRT-1-type (Cys-244) (38
) β-lactamase. This enzyme is related to TEM-2 (Lys-39), as in the previously described IRT-2-type (Ser-244) (38
) enzyme TEM-44 (5
). TEM-73 (IRT-18) (CF739) and TEM-74 (IRT-19) (CF749) are two novel IRT enzymes that harbor both the substitutions Leu-21→Phe and Thr-265→Met that were previously reported in TEM-like and ESBL β-lactamases. These two enzymes differ by the amino acid substitution at position 244: Cys in TEM-73 (like IRT-1) and Ser in TEM-74 (like IRT-2).
The study of nucleotide substitutions at positions 32, 226, 263, 317, 346, 436, 604, 682, and 925 of the TEM-24 and TEM-44 genes showed a nucleotide pattern identical to that of gene blaTEM-2
. The nucleotide sequence patterns of the other genes differed from that of gene blaTEM-2
by only one or two nucleotides at position 317, 682, or 925. These other genes could therefore be considered TEM-2-like genes (8
). The blaTEM
genes described here could result from a complex evolutionary process that originated in gene blaTEM-2
and/or could reflect the diversity of the genes that encode the TEM penicillinases.
For all enzymes reported, the nucleotide sequence showed a T at position 32, as observed in strong promoters (12
). Despite these strong promoters, the IRTs in P. mirabilis
conferred low levels of resistance to ticarcillin compared to the levels of resistance of E. coli
). Likewise, ESBL expression was weak, requiring a modified synergy test for routine detection (35
). When the ESBL-encoding plasmids of P. mirabilis
were transferred to E. coli
, the level of resistance to broad-spectrum cephalosporins was higher in E. coli
than in P. mirabilis
, suggesting the existence of a factor that leads to weak expression of β-lactam resistance, despite the presence of a strong promoter. The reasons for this discrepancy are not known. It could be the result, as suggested by Wu et al. (41
), of regulational phenomena such as mRNA transcription attenuation, protease, or export problems. The promoter of TEM-57 is singular in that it has an adenine immediately under the thymidine at position 32. This insertion may not affect the strength of the promoter that directs the enzyme gene since a level of resistance similar to that conferred by TEM-2 was observed.
The IRT-encoding plasmids were not transferable by conjugation under our conditions and were distinctly smaller (42 to 70 kb) than ESBL-encoding plasmids (170 to 180 kb), which can easily conjugate. Thus, ESBL-producing P. mirabilis
could be a reservoir of β-lactamase-encoding plasmids. In contrast, IRT-producing P. mirabilis
, as suggested previously for E. coli
), may have been selected after mutations of the TEM gene as a result of pressure from penicillin-inhibitor associations instead of the acquisition of IRT-encoding plasmids.
In P. mirabilis
strains, TEM-3, TEM-24, and TEM-66 were encoded by large plasmids which were similar in size, which were associated with similar resistance markers, and which had similar hybridization patterns. The same plasmid was observed in TEM-3-producing K. pneumoniae
CF34, whereas previously it was reported that TEM-3 and TEM-24 are encoded by 85-kb plasmids (13
In our hospital, a 2-year survey (1997 to 1998) revealed that 14.3% of the amoxicillin-resistant P. mirabilis
strains produce an ESBL identified as TEM-3 in 73 of 74 isolates (9
). In the present study, ESBLs TEM-24 and TEM-66 were encountered for the first time in this species. The findings of TEM-10 (27
), TEM-26 (28
), TEM-8 (26
), and TEM-21 (14
) in previous studies and our results show the great diversity of ESBLs in P. mirabilis
Two IRTs related to TEM-2, TEM-44 (IRT-13) (5
) and TEM-65 (IRT-16), have been found in P. mirabilis
. The frequency of TEM-2 in P. mirabilis
is high: 32.7% of penicillinase-producing strains (9
). This high frequency could explain why the two IRTs derived from TEM-2 have been characterized in the species.
The description of TEM-24 in P. mirabilis
and the characterization of five new TEM mutants, TEM-57 (TEM-like), TEM-66 (ESBL), and TEM-65, TEM-73, and TEM-74 (IRTs), show the diversity of TEM mutants in P. mirabilis
. The chromosomal β-lactamase CMY-3 (6
) and the plasmid-mediated β-lactamases CEP-1 (4
), CTX-M-2 (3
), PER-2 (2
), and CMY-4 (39
) have also been observed in this species. This diversity of β-lactamases associated with the high frequency of resistance to β-lactam antibiotics raises the fear that P. mirabilis
-resistant strains could become entrenched in hospitals and could be involved in nosocomial infections, in which case surveillance of this species for susceptibility to β-lactam antibiotics would be warranted.