The introduction of the antibiotics into clinical practice has significantly reduced the mortality of infectious diseases. Although chromosomally mediated β-lactamase is natural in many genera of bacteria, the intensive use of antibiotics is the main cause for the increasing emergence of new β-lactamases. So far, more than 340 β-lactamases have been identified,1 among which, more than 200 are extended-spectrum β-lactamases (ESBLs).2 The most prevalent β-lactamases are class A enzymes, including SHV and TEM. Genes encoding these enzymes generally located in large transferable plasmids. The dissemination of these plasmids attributes to the increasing incidence and spread of β-lactam resistance. It is important to investigate the prevalence and allelic distribution of genes encoding β-lactamase in the bacterial population in order to prevent the emergence of ESBLs in those bacteria and the spread of ESBLs in the clinical setting.
Phenotype-based antibiotic resistance tests are timeconsuming and complicated. Moreover, we are misled by the results from these tests sometimes.3 Although genotyping can not simultaneously detect the present variants of all antibiotic resistance genes in an individual isolate, it is more accurate and cost-effective than phenotype-based methods.3,4 We previously developed a multiplex PCR to investigate the prevalence of the blaSHV gene, with which we have successfully identified 62 blaSHV gene-containing isolates out of 1320 isolates collected from three hospitals in Shanghai (unpublished). Nevertheless, the 494 bp band of multiplex PCR, indicating the presence of the blaSHV gene, derived from a clinical Staphylococcus epidermidis RJ483 strain was unclear. A diffuse and weak smear of PCR band was found in the position instead. Given that the blaSHV gene shows at least 88% nucleotide identity with blaLEN, blaOPK and blaOHIO genes,5,6 there may be other class A non-SHV β-lactamase gene in the isolate RJ483.
In the present study, we found a new blaLEN gene in S. epidermidis RJ483 isolate and investigated the prevalence of this gene by a multiplex PCR in 1320 isolates, which were collected in three hospitals in Shanghai from July to October, 2004.
We collected 1320 nonreptitive isolates between July and October 2004 from the microbiological laboratories of Ruijin Hospital, Dangfang Hospital and Shanghai No. 1 Hospital (unpublished). All isolates were identified by standard biochemical tests. The antimicrobial susceptibilities of the isolates were determined concomitantly by the Kirby-Bauer disk diffusion test as described in National Committee for Clinical Laboratory Standard guidelines.7
PCR amplification and clone-sequencing
The bacteria genomic DNA was extracted from the clones, which were scraped from culture medium, using DNeasy® Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. To identify the non-blaSHV gene, we used primers SHV-Up and SHV-Low to amplify the fragment from the non-blaSHV gene by a DNA polymerase with proofreading activity. Amplification was carried out in a total volume of 30 μl containing 0.3 mmol/L dNTP, 0.2 μmol/L of each primer, 1× PCR reaction buffer, 2 mmol/L MgCl2, 80 ng DNA and 1.25 U LA Taq (Takara, Shiga, Japan). Cycling conditions were as follows: 94°C for 3 minutes; followed by 8 cycles of 94°C for 30 seconds, 66°C for 1.2 minutes with 0.5°C decrement of the annealing temperature per cycle and 72°C for 1 minute; 30 cycles of 94°C for 30 seconds, 62°C for 1 minutes and 72°C for 55 seconds; and a final extension of 5 minutes at 72°C.
PCR product was purified by QIAquick PCR Purification Kit® (Qiagen, Hilden, Germany) following the manufacturer's recommendations and was then cloned into a vector pGEM-T (Promega, Madison, USA). After transformation, five clones representing for the non-blaSHV gene were sequenced with plasmid primers T7 and SP6 by ABI 3700 sequencer according to the Big-Dye chemistry reaction protocol (Applied Biosystems, CA, USA).
Primers were designed using Primer Premier 5 (Premier Biosoft International, CA, USA) on the basis of published sequences of blaLEN, blaTEM, blaOPK and blaOHIO genes from the GenBank database http://www.ncbi.nlm.nih.gov/).The sequences of primers are listed in Table.
Full-lengthblaLEN gene amplification and sequencing
To identify alleles of the blaLEN gene, full-length blaLEN gene was amplified with primers LEN-Up and LEN-Low. Reaction was performed in a total volume of 30 μl containing 0.3 mmol/L dNTP, 0.2 μmol/L of each primer, 1× PCR reaction buffer, 2 mmol/L MgCl2, 80 ng DNA and 1 U Ex Taq (Takara, Shiga, Japan). Cycling conditions were as follows: 94°C for 3 minutes; followed by 8 cycles of 94°C for 30 seconds, 69°C for 40 seconds with 0.5°C decrement of the annealing temperature per cycle and 72°C for 55 seconds; 30 cycles of 94°C for 30 seconds, 65°C for 40 seconds and 72°C for 55 seconds; and a final extension of 5 minutes at 72°C. PCR product was purified by QIAquick PCR purification kit® (Qiagen, Hilden, Germany) following the manufacturer's recommendations and then was sequenced with primers LEN-f, LEN-r and LEN-S by ABI 3700 sequencer according to the Big-Dye chemistry reaction protocol (Applied Biosystems, CA, USA).
To investigate the prevalence of the blaLEN gene in Shanghai, a multiplex PCR was carried out using primer pairs LEN-f/LEN-r and 23S-f/23S-r. This reaction (30 μl) contained 0.3 mmol/L dNTP, 10 mmol/L Tris-HCl (pH 8.3), 100 mmol/L KCl, 2 mmol/L MgCl2, 0.5 μmol/L of primers LEN-f and LEN-r, 0.16 μmol/L of primers 23S-f and 23S-r, 60 ng of DNA and 1.2 U of Taq (Takara, Shiga, Japan). A denaturation step of 94°C for 3 minutes was followed by 10 cycles of 94°C for 30 seconds, 63°C for 30 seconds with 0.5 °C decrement of the annealing temperature per cycle and 72°C for 40 seconds, then followed by 30 cycles of 94°C for 30 seconds, 58°C for 30 seconds and 72°C for 40 seconds, with a final extension of 5 minutes at 72°C. Five μl of PCR products were then identified by 1.5% agarose gel electrophoresis.
With primer pair SHV-Up and SHV-Low, the fragment was successfully amplified from the non-blaSHV gene using the DNA polymerase with proofreading activity and high polymerization rate. Sequence analysis showed that the PCR band at 884 bp derived from S. epidermidis RJ483 isolate was identical to the blaLEN gene. Since primer SHV-Up and SHV-Low overlapped with the blaLEN gene in the open reading frame, to amplify the full-length blaLEN gene, a new primer pair LEN-Up/LEN-Low was designed. Sequence analysis showed that the new blaLEN variant (GenBank accession No. EF205593) encodes LEN-17, which differs from LEN-2 by two amino acid residues: Val114Thr and Ile119Val. The sequence of the new blaLEN-17 gene and the predicted amino acid sequence were showed in Figure 1. Compared with the published sequence of the blaLEN-17 gene (GenBank accession no. DQ149134.1), the new blaLEN gene only has one base pair change at nucleotide 162 (C162G), which results in a synonymous mutation. The new blaLEN gene is a sub-allele of the blaLEN-17 gene. The new blaLEN-17 gene shows 90% homology with blaSHV (GenBank accession No. AF462396) and blaOKP genes (GenBank accession No. AY825330) (Figure 2).
Prevalence of theblaLEN gene
The rrlA gene is a housekeeping gene. Therefore, a 340 bp fragment amplified from rr1A gene locus with primer pair 23S-f/23S-r serves as an internal control for the multiplex PCR. With primer pair LEN-f and LEN-r, a 467 bp fragment was amplified from the blaLEN gene locus, indicating the presence of the blaLEN gene (Figure 3). The 340 bp fragment was observed in all 1320 isolates,
indicating successful amplification in each sample. As illustrated in Figure 3, no isolates other than S. epidermidis RJ483 were found to carry the blaLEN gene (including 129 clinical isolates of K. pneumoniae). So the incidence of the blaLEN gene is estimated as 0.08% in Shanghai.
Staphylococcus spp. produced a chromosomally encoded penicillin-binding protein 2 (PBP2). Staphylococcus isolates have also been reported to produce β-lactamases.1 In the present study, we identified a new blaLEN-17 gene from S.” epidermidis RJ483 isolate. Although all blaLEN genes were originally found in K. pneumoniae5,8,9 and previous studies of the blaLEN gene typically focused on K. pneumoniae, no such gene was found in 129 clinical isolates in this study. Isolate RJ483, which is collected from the sputum of a patient, showed resistance to penicillin but was susceptible to cefprozil. It is regretful that all attempts to cultivate RJ483 had failed and thus we were unable to re-determine the antimicrobial susceptibility of this isolate. Further investigation is needed to characterize the effect of these mutations on the activity of the enzyme and on antibiotic hydrolysis. Moreover, we have recently identified SHV-12, a SHV-type ESBL,10 from one of the 49 S. epidermidis isolates collected in this study in Shanghai (unpublished). Taken together, the incidence of β-lactamase genes in S. epidermidis is at least 4%. β-lactamases may contribute to part of the emergence of Methicillin-resistant S. epidermidis (MRSE). Since Staphylococcus spp. is not among the species routinely monitored for ESBLs, further investigation is needed for clinical microbiologists to determine the transferability and genetic support of the various ESBL determinants and the clonal diversity of MRSE.
The LEN-1, first identified in a K. pneumoniae in 1986 in Japan,11 is a class A, group 2b β-lactamase12 and is chromosomally encoded. LEN-1 comprises 279 amino acid residues, while LEN-2, LEN-4, LEN-5 and LEN-10 contain 286 amino acid residues. LEN-1 confers resistance to penicillin, but not to extended-spectrum β-lactams. Other chromosomally encoded LEN-type β-lactamases, such as LEN-2 and LEN-10, have been identified in recent years.6,8 The spectrum of β-lactam antibiotics susceptible to hydrolysis of these enzymes remains unknown. LEN-17 comprises 286 amino acid residues. The sequence of LEN-2 and LEN-17 differs in two amino acids, Val114Thr and Ile119Val. Although LEN was considered as a chromosomal β-lactamase,5 recent study showed that LEN-5, differed from LEN-2 in four amino acids (VaI8IIe, Asp24Tyr, VaI88Leu and VaI114Thr), is a plasmid-mediated β-lactamase.9 Furthermore, results of this investigation revealed that K. pneumoniae is not the only species carrying the blaLEN gene. The spread of the blaLEN gene may be underestimated in previous reports.
Prevalence of individual β-lactamase gene is usually detected in ESBL producers by genotyping-based methods. Bacteria identified as susceptible to β-lactam antibiotics in vitro by phenotype-based methods sometimes showed resistance to those in clinic. By a genotyping-based method, Xiao et al13 identified 100 SHV-type ESBL-containing isolates out of 2416 isolates, which were previously determined as non-ESBL producers by antimicrobial susceptibility tests. The incidence of the β-lactamase gene can be underestimated by isoelectric focusing (IEF) and antimicrobial susceptibility tests. Genotyping is the preferential method for genetic epidemiological surveillance of β-lactamase genes.14 In this study, we designed a multiplex PCR, which used amplification of the rrlA gene as a positive control, to detect the presence of the blaLEN gene. No isolate other than S. epidermidis RJ483 was found to carry the blaLEN gene. Except the ESBL producers determined by routine methods, all the strains collected in our study, were screened for the presence of the blaLEN gene, the incidence of the blaLEN gene should reflect the true prevalence of the blaLEN gene in Shanghai. However, the result needs to be further validated in a larger clinical samples.
In conclusion, we identified a new blaLEN-17 gene in a clinical isolate of S. epidermidis, other than K. pneumoniae. We also developed a duplex PCR method to investigate the presence of the blaLEN gene in clinical isolates. The distribution of the blaLEN gene is noteworthy due to the emergence of plasmid-mediated LEN9 and the spread of LEN in both gram-positive and gram-negative bacteria.
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Keywords:© 2008 Chinese Medical Association
β-lactamase; LEN; genotyping; Staphylococcus epidermidis