J Med Microbiol 57 (2008), 1273-1276; DOI: 10.1099/jmm.0.2008/001271-0
© 2008 Society for General Microbiology
ISSN 1473-5644
Plasmid-borne armA methylase gene, together with blaCTX-M-15 and blaTEM-1, in a Klebsiella oxytoca isolate from China
Ying Zhang1,
Hua Zhou1,
Xiao-qiang Shen2,
Ping Shen1,
Yun-song Yu1 and
Lan-juan Li1
1 State Key Laboratory for Diagnosis and Treatment of Infectious Disease, First Affiliated Hospital, College of Medicine, Zhejiang University, No. 79, Qing Chun Road, Hangzhou, Zhejiang 310003, PR China
2 Department of Respiratory Disease, Hospital of Hangzhou Red Cross Association, No. 208, Huan Cheng Dong Road, Hangzhou, Zhejiang 310006, PR China
Correspondence
Yun-song Yu
yvys119{at}163.com
Received 13 February 2008
Accepted 23 June 2008
An armA-producing Klebsiella oxytoca isolate, strain 157, was detected after screening of 447 extended-spectrum β-lactamase-producing Enterobacteriaceae isolates in China. K. oxytoca 157 was resistant to aminoglycosides, ciprofloxacin and most β-lactams. Resistance to aminoglycosides and β-lactams could be transferred to recipient Escherichia coli by conjugation. armA, blaCTX-M-15 and blaTEM-1 genes were detected in K. oxytoca 157 and transconjugant E. coli strain 600(pEC157). Mutation of aa 87 in GyrA was found in K. oxytoca 157. A plasmid of 
55 kb was extracted from K. oxytoca 157(pKO157) and E. coli 600(pEC157). Southern blot hybridization confirmed that the armA, blaCTX-M-15 and blaTEM-1 genes were all located on this conjugative plasmid (pEC157). PCR mapping was also performed to investigate the genetic environment of armA. The armA gene was found to be flanked by the same putative transposable elements as reported previously in E. coli, Klebsiella pneumoniae and Citrobacter freundii isolates from different countries.
Abbreviations: ESBL, extended-spectrum β-lactamase.
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INTRODUCTION
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Aminoglycoside antibiotics are widely used in clinical settings, particularly for the treatment of life-threatening infections caused by Gram-negative bacteria. The 16S rRNA methylase confers high-level resistance to 4,6-substituted deoxystreptamines, including arbekacin, amikacin, kanamycin, tobramycin and gentamicin, by post-transcriptional methylation of 16S rRNA leading to loss of affinity for aminoglycosides (Yamane et al., 2005). The armA gene was first isolated from an isolate of Klebsiella pneumoniae (Galimand et al., 2003). It has been reported that the armA gene is flanked by putative transposable elements (González-Zorn et al., 2005). In previous studies, most of the armA-positive isolates of Escherichia coli and K. pneumoniae have been found to have CTX-M-type enzymes (Yan et al., 2004). Klebsiella oxytoca is an opportunistic pathogen involved in antibiotic-associated diarrhoea and nosocomial infections (Decré et al., 2004). The chromosome of wild-type K. oxytoca carries the blaOXY gene. This gene is constitutively expressed at low levels, which usually confers low-level resistance to amino- and carboxypenicillins but no significant resistance to other β-lactams (Livermore, 1995).
The aim of this study was to determine the prevalence of the 16S rRNA methylase gene in clinical extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae isolates and to characterize the genetic environment of these genes.
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METHODS
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Bacterial isolates.
Clinical isolates of ESBL-producing Enterobacteriaceae isolates were collected from six provinces in China (Beijing, Zhejiang, Xinjiang, Henan, Jiangsu and Hubei) from September 1998 to November 2002 (Yu et al., 2007a). These strains were identified using the API 20E system (bioMérieux). E. coli strain 600 (resistant to rifampicin) was used as the recipient for conjugation, armA-producing Acinetobacter baumannii and rmtB-producing Pseudomonas aeruginosa were used as positive controls for PCR, and E. coli ATCC 25922 was used as the control strain in susceptibility tests.
Screening for 16S rRNA methylase-producing isolates and detection of resistance genes and the genetic environment.
16S rRNA methylase genes (armA, rmtA, rmtB, rmtC, rmtD and npmA) were detected by PCR using primers and conditions described previously (Yokoyama et al., 2003; Doi et al., 2004; Yamane et al., 2005; Wachino et al., 2007; Yu et al., 2007b). For the 16S rRNA methylase-producing isolates, amplification and identification of ESBL-encoding genes were performed with previously described primers (Yu et al., 2007a) for blaTEM, blaSHV, blaCTX-M-1-like, blaCTX-M-2-like, blaCTX-M-8-like and blaCTX-M-9-like genes. The blaOXY, parC and gyrA genes were detected as reported previously (Maurin et al., 2001; Decré et al., 2004). PCR mapping experiments were performed to investigate the gene environment of the armA gene. Primers were designed according to the reported sequence of K. pneumoniae strain BM4536 (GenBank accession no. AY220558). All primers used are listed in Table 1
. PCR products were sequenced on an ABI 3730 automatic sequencer.
Susceptibility testing of 16S rRNA methylase-producing isolates.
The MICs of 20 antimicrobial agents for 16S rRNA methylase-producing isolates were determined using an Etest (AB Biodisk) according to the manufacturer’s instructions. Results were interpreted according to published recommendations (CLSI, 2006). The breakpoint of netilmicin was used for isepamicin (Gomez-Flores et al., 2004).
Conjugation and MIC determination of transconjugants.
Conjugation experiments were performed as described previously (Yan et al., 2001), with rifampicin-resistant E. coli 600 as the recipient. Mueller–Hinton agar plates supplemented with rifampicin (256 µg ml–1) and amikacin (128 µg ml–1) were used to select transconjugants. The MICs of 20 antimicrobial agents for transconjugants were determined by Etest according to the manufacturer’s instructions.
Plasmid extraction and Southern blot hybridization analysis of the 16S rRNA methylase and β-lactamase genes.
A Plasmid Midi kit (Qiagen) was used to extract plasmids from the donor isolate and the transconjugant. For Southern blot hybridization, plasmids were transferred from electrophoresis gels to nylon membranes (Bio-Rad) and then hybridized with armA, blaCTX-M-15 or blaTEM-1 gene fragments labelled with [
-33P]dCTP (DuPont). After washing, the membrane was compressed with a storage phosphor screen (Kodak) for 48 h and scanned for positive hybridization.
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RESULTS
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Description of clinical isolates
Among the 447 Enterobacteriaceae isolates screened, 27 harboured blaSHV-type ESBL, 341 isolates were positive for blaCTX-M-9 group ESBLs and 112 isolates were positive for blaCTX-M-1 group ESBLs (Yu et al., 2007a). Twenty-one isolates were positive for blaCTX-M-14 and blaCTX-M-3, two isolates were positive for blaCTX-M-3 and blaSHV-12, one isolate was positive for blaCTX-M-9 and blaSHV-12, four isolates were positive for blaCTX-M-14 and blaSHV-12, one isolate was positive for blaCTX-M-24 and blaSHV-12, one isolate was positive for blaCTX-M-3, blaCTX-M-14 and blaSHV-12, and one isolate was positive for blaCTX-M-3, blaCTX-M-14 and blaSHV-43 (Yu et al., 2007a).
Screening of 16S rRNA methylase genes
All 447 isolates were negative for rmtA, rmtB, rmtC, rmtD and npmA genes by PCR tests. Only one isolate was positive for armA. Sequencing analysis confirmed that the PCR product had 100 % nucleotide identity to the armA gene of K. pneumoniae strain BM4536 (GenBank accession no. AY220558). This isolate, named strain 157, was identified as K. oxytoca.
Characterization of resistance genes in K. oxytoca strain 157
K. oxytoca 157 was negative for blaSHV, blaCTX-M-1 group, blaCTX-M-2 group and blaCTX-M-8 group ESBL genes, but positive for blaTEM and blaCTX-M-9 group genes. Sequencing analysis confirmed the PCR products as blaTEM-1 and blaCTX-M-15. The PCR products of blaOXY showed 100 % nucleotide identity to blaOXY-2 (GenBank accession no. Z49084). One substitution of Asp87
Asn in GyrA was identified in K. oxytoca 157.
Transfer of resistance
Resistance to aminoglycosides and most of the β-lactams could be transferred by conjugation. The conjugative transfer frequency was 1.5x10–6 transconjugants per recipient. The resistance characteristics of the transconjugant, named E. coli 600(pEC157), are shown in Table 2. PCR product analysis confirmed that the armA, blaCTX-M-15 and blaTEM-1 genes were co-transferred by conjugation.
Susceptibility profile of K. oxytoca 157 and the transconjugate
K. oxytoca 157 showed an extraordinarily high level of resistance (MIC >1024 µg ml–1) to the five tested aminoglycosides. It was also resistant to ciprofloxacin, most of the β-lactams except the carbapenems and ceftazidime/clavulanic acid. Tigecycline and polymyxin E had good activity against K. oxytoca 157, as shown in Table 2
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Plasmid extraction and Southern blot analysis of the armA, blaCTX-M-15 and blaTEM-1 genes
A plasmid of the same size (55 kb) was extracted from both K. oxytoca 157 and E. coli 600(pEC157) (Fig. 1). The plasmid DNA showed a hybridization pattern represented by 55 kb bands for both K. oxytoca 157 and transconjugate E. coli 600(pEC157) using a labelled armA gene fragment (Fig. 1). When labelled blaCTX-M-15 or blaTEM-1 gene fragments were used for hybridization, the plasmid DNA also showed a positive hybridization pattern (data not shown).
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Fig. 1. Southern blot analysis of the armA gene. Left three panels: plasmid DNA extracted from K. oxytoca 157(pKO157) and transconjugate E. coli 600(pEC157). Right panels: DNA was transferred to a membrane and hybridized with an armA probe. V517, plasmid size markers. The results showed that the armA gene is located on the 55 kb plasmid.
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Exploration of the regions surrounding the armA gene
PCR mapping and sequencing experiments revealed that the sequences upstream and downstream of the armA gene in K. oxytoca 157 were identical to the genetic environment of armA genes in isolates of K. pneumoniae in France (GenBank accession no. AY220558), E. coli in Japan and Spain (GenBank accession nos AB117519 and AY522431, respectively) and Citrobacter freundii in Poland (GenBank accession no. AF550415).
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DISCUSSION
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In China, an armA-producing A. baumannii clone was found to be widely spread in different hospitals (Yu et al., 2007b) and rmtB-producing E. coli and Enterobacter cloacae have been isolated from the commensal flora of healthy, food-producing animals (Chen et al., 2007). Here, we have described for the first time the occurrence of plasmid-borne 16S rRNA methylases among ESBL-producing Enterobacteriaceae isolates in China; this is the first report of an ArmA-producing K. oxytoca isolate.
Transfer of high-level aminoglycoside resistance to E. coli has been successful among armA-containing E. coli, K. pneumoniae, C. freundii, Salmonella enterica and Shigella flexneri isolates from Taiwan (Yan et al., 2004) and France (Galimand et al., 2005), and co-transfer with blaCTX-M-3 and blaTEM-1 genes has also been observed (Yan et al., 2004). In this study, the armA, blaCTX-M-15 and blaTEM-1 genes were readily co-transferred from K. oxytoca 157 to E. coli 600.
Descriptions of the genetic basis of armA in clinical and animal isolates have given insight into the spread of this methylase (Galimand et al., 2005; González-Zorn et al., 2005). Our results confirm that the armA genetic background of K. oxytoca 157 in China is the same as that of Enterobacteriaceae isolates of clinical and animal origin from other countries. This is an important indication that this gene may soon be detected in other species in other regions worldwide.
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ACKNOWLEDGEMENTS
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This work was supported by a research grant from the Program for Excellent Talents of the Health Bureau of Zhejiang, a grant from funding for Doctor of the Ministry of Education of China (no. 20070335188) and a grant from the Health Bureau of Hangzhou (no. 2007B0032). We are grateful to Dr Wang Hui, Peking Union Medical College Hospital, PR China, for kindly providing rifampicin-resistant E. coli 600.
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INTRODUCTION
METHODS
