Most Klebsiella pneumoniae carry a chromosomally encoded SHV-1 β-lactamase gene. Consequently, they exhibit a low-level of ampicillin resistance with a minimum inhibitory concentration (MIC) of 8–64 μg ml−1.1 Our previous studies have shown that low or no expression of the blaSHV gene in K. pneumoniae isolates resulted in reduced ampicillin resistance levels.2 Rice et al.3 and Corvec et al.4 have reported that the C→A mutation in –10 region of the promoter of the blaSHV gene or deletion of a downstream region of the promoter promotes high expression of the blaSHV gene, resulting in a significant elevation in the MIC of K. pneumoniae to ampicillin or co-amoxiclav, cefalothin and cefoxitin. These data indicate that the resistance level of K. pneumoniae to antibiotics is associated with the expression of the blaSHV gene. However, because of variation in the upstream promoter region of the blaSHV gene that lead to enhanced drug resistance levels are rare, they cannot fully explain the mechanism of enhancement of K. pneumoniae resistance level to ampicillin.

Drug resistance in K. pneumoniae is primarily determined by the presence of the extended-spectrum β-lactamases (ESBLs). The genes encoding the various β-lactamases include the blaSHV, blaTEM and blaCTX-M genes.5, 6 In this study, we analyzed the distribution of the β-lactamase genes in clinical isolates of K. pneumoniae from the Harbin area of China and determined the effects of these genes on the levels of antibiotic resistance. The aim of this study was to clarify the mechanism of how drug resistance level is enhanced in K. pneumoniae. Our results should provide new targets for controlling drug resistance.

Between June and October 2007, isolates of K. pneumoniae were collected from patients at two hospitals in Harbin, the capital city of the Heilongjiang Province. The strains were identified using routine biochemical methods and the API20E system. Fifty-six isolates were then randomly selected for this study. The MICs of each agent were determined by the micro-dilution method according to the protocols recommended by CLSI.7 In brief, 100 μl of appropriate bacterial suspensions (∼106 CFU ml−1) were inoculated to 100 μl of antibiotic-containing micro-plates and incubated for 18–20 h at 37 °C. MIC determinations were performed four times for each strain to ensure the reproducibility of the MICs by using quality control strain Escherichia coli ATCC25922. Strains with MIC values ≥MIC90 were considered to have high levels of antibiotic resistance. Ampicillin, cefotaxime, amikacin and ciprofloxacin were used for testing. The MIC value of ampicillin was generally high (Figure 1). The high-level antibiotic resistance rates to ampicillin (MIC90 >1024 μg ml−1), cefotaxime (MIC90 >1024 μg ml−1), amikacin (MIC90 >1024 μg ml−1) and ciprofloxacin (MIC90 >128 μg ml−1) were 58.92%, 30.35%, 23.21% and 12.5%, respectively.

Figure 1
figure 1

Distribution of MICs of ampicillin, cefotaxime, amikacin and ciprofloxacin (n=56). MICs were determined by the micro-dilution method. The break point of ampicillin, cefotaxime, amikacin and ciprofloxacin were 32, 64, 64 and 4 μg ml−1 (standard based on M100-S19, CLSI, Wayne, PA, USA, 2009), respectively. The resistance rates to ampicillin, cefotaxime, amikacin and ciprofloxacin were 49 (87.50%), 28 (50.00%), 19 (33.93%) and 36 (64.29%), respectively.

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The PCR primers for the blaSHV,3 blaTEM,8 blaCTX-M9, 10 and blaOXA genes11 were designed according to the sequences in GenBank. The PCR products were sequenced directly after the purification with a QIAquick PCR purification kit (Qiagen, Beijing, China). The SHV amino-acid sequences were aligned with those of known blaSHV gene (GenBank accession no. AF124984) and homology analysis of the sequencing results in GenBank was conducted using BLAST. The SHV genotypes were determined according to the standards found at www.lahey.org/studies/webt.stm.

In the 56 K. pneumoniae isolates, the ESBL genes of local K. pneumoniae isolates consisted mainly of the blaSHV gene (69.64%, 39/56) followed by blaCTX-M (30.36%, 17/56) and blaTEM (37.50%, 21/56) genes, and the blaOXA and other genes were not detected (data not shown). Table 1 demonstrates the interrelationship among β-lactamase genes and the MIC for representative antibiotics. The strains that carried blaCTX-M gene exhibited higher MIC50 values for ampicillin (≥1024 μg ml−1), cefotaxime (≥320 μg ml−1) and amikacin (≥128 μg ml−1) than those without the blaCTX-M gene. The results suggested that different types of β-lactamase gene might affect the antibiotic resistance levels. Table 2 shows that the high-level drug resistance rate of isolates carrying the blaCTX-M gene (ampicillin: 90.48%; cefotaxime: 57.14%) were significantly higher than that for strains without the blaCTX-M gene (ampicillin: 40.40%; cefotaxime: 14.29%; P<0.01). However, the resistance level of isolates to amikacin and ciprofloxacin with and without blaCTX-M gene was not significantly different (P>0.05). The results obtained through multivariate analysis also indicated that the blaCTX-M gene was independently associated with the level of ampicillin (OR: 19.522; 95% CI: 3.307–115.259, P<0.01) and cefotaxime (OR: 10.193; 95% CI: 2.341–44.382, P<0.01; data not shown). Additionally, the blaSHV and blaTEM genes did not have significant effects on the high-level drug resistance to ampicillin, cefotaxime, amikacin and ciprofloxacin. These results demonstrated that the blaCTX-M gene independently enhances the drug resistance levels of K. pneumoniae clinical isolates to ampicillin and cefotaxime.

Table 1 Relationship between b-lactamase genes and antibiotics susceptibility of clinical isolates
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Table 2 Comparison of the effects of b-lactamase genes on the antibiotics-resistance level of K. pneumoniae
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Currently, the most common penicillin antibiotics, such as ampicillin, are rarely used to treat K. pneumoniae infections and the strains generally exhibit low-level resistance to ampicillin.1 However, there were significant differences in the ampicillin MIC values in the K. pneumoniae isolates we collected. High-level ampicillin-resistant strains (MIC ≥1024 μg ml−1) accounted for 58.93% of the isolates, suggesting that many K. pneumoniae strains have a high level (high value of MICs) of ampicillin resistance. Retrospective studies on the penicillin resistance of Streptococcus pneumoniae have also shown that although the frequency of penicillin use has decreased in European countries, the level of penicillin resistance has not decreased.12, 13 Thus, the strategies for controlling antibiotic use do not affect the antibiotic resistance levels of bacteria. Instead, the persistence of drug-resistant genes explains the continued resistance. This study took a new perspective and investigated whether the ESBL-related genes affected the antibiotic resistance levels of bacteria. The results showed that the blaCTX-M gene was closely associated with the high-level drug resistance to ampicillin and cefotaxime (Table 2).

Previous studies have shown that the enhanced antibiotic resistance of K. pneumoniae is associated with mutations in the upstream region of the bacterial blaSHV gene promoter. For example, the C→A mutation in the upstream −10 region of the blaSHV gene promoter results in high levels of SHV expression, thus enhancing bacterial resistance to drugs, such as ampicillin, ceftazidime and piperacilline/tazobactam.3 However, our study did not find any mutation in the upstream promoter region of the K. pneumoniae blaSHV gene or other plasmid-encoded SHV mutants; the blaSHV genes (SHV-1, SHV-11 and SHV-33) were all chromosomally encoded.14, 15 Thus, our results suggest that mutations in the upstream promoter region of the K. pneumoniae blaSHV gene that appear to enhance antibiotic resistance may be an accidental phenomenon.

In summary, this study indicates that the blaCTX-M gene promoted the high-level ampicillin resistance of K. pneumoniae (MIC ≥1024 μg ml−1). Our results also indicate a novel and decisive role for the blaCTX-M gene in bacterial drug-resistance levels, a finding that would further elucidate the modes of action of bacterial drug-resistant genes. Owing to the increase in selective pressures for antibiotic resistance and the movement and spread of drug-resistant genes, the epidemiology of drug resistance is constantly changing. Our data demonstrate that there is a relationship between the bacterial blaCTX-M gene and the resistance of K. pneumoniae isolates to ampicillin. Further studies are required to determine if this relationship is unique to K. pneumoniae or is characteristic of other Enterobacteriaceae.