In the present study, we have tried to analyze a type of resistance gene that is located in the chromosome of K. pneumoniae
. Since the SHV type of resistance has been postulated to be present in all K. pneumoniae
), studies with this type of resistance gene may allow more precise delineation of the correlation or, possibly, the evolution of different SHV-related mutations among all isolates.
MIC determinations showed that all of the isolates were resistant to ampicillin. On the other hand, 72.6% of the isolates were susceptible to amoxicillin-clavulanate, indicating that resistance was inhibited by a β-lactamase inhibitor. Twenty-five (22.1%) isolates were partially inhibited by amoxicillin-clavulanate, and only 6 (5.3%) isolates were not. Among 70 cephalothin-resistant isolates, >48% were resistant to aztreonam, cefotaxime, or ceftazidime. However, most of these isolates were shown to be sensitive to cefoxitin, indicating that the production of β-lactamases among these isolates had no effect on susceptibility to the cephamycin class of antibiotics. Although imipenem resistance due to the presence of a combination of an outer membrane protein and AmpC has previously been reported in Spain (10
), imipenem is still a drug to which isolates were susceptible in this study.
Our study shows agreement with the conclusion that SHV-type resistance genes are ubiquitous in K. pneumoniae
was amplified from all isolates, and seven different types of SHV β-lactamases were identified, including two novel β-lactamases, SHV-25 and SHV-26. blaTEM
was amplified from 32 isolates, and all of these isolates carried the TEM-1 resistance gene. According to their amino acid sequences, SHV β-lactamases in Taiwan may basically come from SHV-1 or SHV-11 and are further subdivided by four possible routes. Through stepwise mutation, SHV-1 may form SHV-2 (mutation at amino acid 238) and then SHV-5 (mutations at amino acids 238 and 240). SHV-12 (mutations at amino acids 35, 238, and 240) may possibly come from either SHV-5 or SHV-11 (Fig. ). These stepwise mutations appear to comprise the evolutionary history for SHV-type ESBL producers in Taiwan. On the other hand, a non-ESBL (SHV-25) and a β-lactamase that results in reduced susceptibility to clavulanic acid (SHV-26) are possibly derived from SHV-11 and SHV-1. Ribotyping revealed that there were a total 49 different ribotypes, suggesting a high degree of genetic polymorphism in our collection. Relationships between isolates could be established in only a few instances. The bacteria with two novel β-lactamases were distributed into two particular ribotypes. No other isolates were found in these ribotypes (Fig. ). Clinically, the patients with a K. pneumoniae
SHV-25 carrier (a patient had underlying cirrhosis of the liver with ascites) and a K. pneumoniae
SHV-26 carrier (a patient who was bedridden due to an old cerebral infarction and diabetes mellitus) were under treatment with gentamicin and/or cefazolin. The patient infected with the SHV-26 carrier died shortly after the treatment was begun (data not shown). The discovery of SHV-26, a β-lactamase with reduced susceptibility to clavulanic acid that renders bacteria intermediately resistant to an inhibitor consisting of a β-lactam, may augur the development of β-lactamase inhibitor resistance apart from ESBL inhibitor resistance.
Genetically, comparison of the sequence of blaSHV-25
with that of blaSHV-1
revealed three different mutations in SHV-25, at positions 18, 35, and 129 (Jacoby and Bush, http://www.lahey.org./studies/webt/htm
). The mutation at position 35 has been described in SHV-11 and has no influence on substrate and inhibitor profiles, the pI value, or the MIC (6
). A previous study has shown that the side chain of β-lactam antibiotics is connected to Asn-132 via a hydrogen bond and therefore stabilizes the catalytically important conserved Ser-Asp-Asn (SDN) loop from positions 130 to 132 (5
). Thus, the modification at position 129, which is closer to SDN, may result in a lowering of the Km
and a change of the pI to 7.5. The influence of the modification at position 18 of the signal peptide in leading the β-lactamase to the periplasm, where it resides, remains unclear. However, our kinetics data showed that relatively low Km
values have been detected for cephalothin and cephaloridine. Theoretically, the MIC will be raised when a low Km
is achieved (6
). The MICs obtained in the present study indicate that the cephalothin MIC for isolates with SHV-25 is similar to those for isolates with SHV-1. A previous study showed that a nucleotide substitution, I8F, in the signal sequence in SHV-7 led to slightly increased cephalosporin MICs, suggesting the more efficient transfer of the enzyme precursor into the periplasmic space. Whether the presence of a modification at position 18 in SHV-25, which is within the signal sequence, leads the β-lactamase to a location other than periplasm is unknown. Further confirmation by site-directed mutagenesis should be conducted. For SHV-26, the threefold increase in the clavulanic acid IC50
compared to those for SHV-1 and SHV-25 resulted in a higher amoxicillin-clavulanate MIC for SHV-26. Mutation at position 187 may involve the interaction with clavulanic acid.
In conclusion, we have described two novel β-lactamases, one of which is a non-ESBL and one of which is an enzyme from an isolate with reduced susceptibility to clavulanic acid. The prevalence of these enzymes is rare in northern Taiwan. Although the results do not provide information on the mutation rate, the results obtained in the present study suggest the routes of evolution of SHV β-lactamase genes among isolates in northern Taiwan. Antibiotic selective pressure may explain the development of ESBL-type resistance as well as inhibitor-resistant β-lactam resistance.