Diversity of TEM-52 extended-spectrum ß-lactamase-producing non-typhoidal Salmonella isolates in Korea

Kyungwon Lee1,2,3, Dongeun Yong1,2, Jong Hwa Yum2,3, Ho Hoon Kim2 and Yunsop Chong1,2,*

1 Department of Laboratory Medicine, 2 Research Institute of Bacterial Resistance, 3 Brain Korea 21 Medical Sciences, Yonsei University College of Medicine, Seoul, 120-752, Korea

Received 25 February 2003; returned 8 May 2003; revised 22 June 2003; accepted 24 June 2003


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives: Extended-spectrum ß-lactamase (ESBL)-producing non-typhoidal Salmonella (NTS) isolates in Korea were characterized.

Patients and methods: Five isolates of ESBL-producing NTS were isolated from stool specimens of three infants and two adults with diarrhoea. Two infants acquired the infection in the community, and three other infections were hospital acquired.

Results: The isolates were one each of serovars Saintpaul, Stanley and Agona, and two Enteritidis. Cell sonicates of the isolates hydrolysed cefotaxime more efficiently than ceftazidime, and had ß-lactamase bands of approximate isoelectric points 6.0 and 7.4. Sequencing revealed that the ß-lactamases were TEM-52 and an OXA type. The blaOXA gene was located on a class 1 integron. Cefotaxime resistance, associated with TEM-52, was transferred by conjugation. Identical pulsed-field gel electrophoresis patterns of XbaI-digested genomic DNA were observed in initially ß-lactam-susceptible serovar Agona isolates and subsequent ESBL-producing isolates from an infant, and in two isolates of serovar Enteritidis from two different patients.

Conclusions: This study suggests that TEM-52-producing NTS is spreading both clonally and horizontally in Korea.

Keywords: enteritis, class 1 integron, OXA-type ß-lactamase


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Non-typhoidal Salmonella (NTS) is one of the most important enteric pathogens worldwide. Antimicrobial treatment is required for NTS gastroenteritis only when the patients are of extreme ages, or when they have underlying diseases.1 Extended-spectrum ß-lactamase (ESBL) production is a particular concern, as expanded-spectrum cephalosporins are the drugs of choice for children because they cannot be treated with fluoroquinolones. Moreover, the ESBL genes reside on conjugative plasmids and have the potential to spread to other bacteria. TEM- and SHV-type ESBLs were first reported in Korea in 1997,2 and the prevalent type was TEM-52.3 The aim of this study was to determine the phenotypic and genetic characteristics of ESBL-producing NTS strains isolated in Korea.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Strains

NTS strains were isolated from stool specimens during 1995–1997 from five in-patients with diarrhoea at a tertiary-care hospital. The species were identified by conventional methods and the serovar was determined by the National Institute of Health, Korea.

Antimicrobial susceptibility testing and ß-lactamase investigation

The disc diffusion test4 was performed using commercial discs and Mueller–Hinton agar (Becton Dickinson, Cockeysville, MD, USA). ESBL production was determined by the double disc synergy test and confirmed using the NCCLS broth dilution method.4

MICs of ß-lactams were determined using an agar dilution test4 with ampicillin and cefalothin (Sigma Chemical, St Louis, MO, USA), piperacillin and tazobactam (Wyeth, Pearl River, NY, USA), cefotaxime (Aventis, Frankfurt, Germany), ceftazidime and clavulanic acid (GlaxoSmithKline, Greenford, UK), aztreonam (Bristol-Myers Squibb, Princeton, NJ, USA), and cefoxitin and imipenem (Merck, Sharp & Dohme, Rahway, NJ, USA).

Isoelectric points (pI) of ß-lactamases were determined by loading cell sonicates on pre-cast gels and separating them by use of a ThermoFlow Electrophoresis Temperature Control System (Novel Experimental Technology, San Diego, CA, USA). The bands were visualized using nitrocefin solution. ß-Lactamase activities of the cell sonicates were determined using a model UV-2401 spectrophotometer (Shimadzu, Tokyo, Japan) with 100 µM solutions of ß-lactams in 50 mM phosphate buffer (pH 7.0) at 30°C. Results were expressed as relative activity versus that observed with penicillin G.5

PCR amplification and sequencing

Alleles of blaTEM and blaSHV were detected using previously reported primers and reaction conditions.2 For blaTEM sequencing, a PCR product of 1080 bp was amplified using primers: TEM-SF, 5'-ATAAAATTCTTGAAGACGAAA-3' and TEM-SR, 5'-GACAGTTACCAATGCTTAATC-3'. A PCR product from the class 1 integron was obtained using previously reported primers and reaction conditions.6 The nucleotide sequence was determined by direct sequencing on an ABI 3700 Automatic sequencer (Perkin-Elmer, Foster City, CA, USA). Both strands were sequenced twice with independent amplicons.

Plasmid study and pulsed-field gel electrophoresis (PFGE)

Plasmids were isolated by the alkaline lysis method, and after electrophoresis their sizes were estimated by comparison with those of Escherichia coli strain V517. Conjugation was performed by the broth mating method using rifampicin-resistant E. coli strain RG 488 and nalidixic acid-resistant E. coli strain RG 176 as recipients.

Genomic DNA of the NTS isolates was digested using XbaI, and the bands were separated using a CHEF-DRII system according to the manufacturer’s instruction (Bio-Rad, Hercules, CA, USA). The band patterns were compared visually.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Clinical findings

Two infants with no underlying diseases were admitted because of diarrhoea, but one infant and two adults developed diarrhoea during hospitalization to treat underlying diseases. One strain each of Salmonella serovars Saintpaul, Stanley and Agona were isolated from three patients, and two strains of serovar Enteritidis were isolated from two different patients (Table 1). Sporadic or nosocomial outbreaks of ESBL-producing NTS infections have been reported in many countries. It was reported that four of 26 consecutive isolates of community-acquired serovar Typhimurium in Turkey produced ESBLs.7


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Table 1. Clinical features of the patients with ESBL-producing NTS isolation from stool
 
Except for the initial isolates of serovar Agona, all other isolates produced ESBLs. Strains of serovar Agona, which were susceptible to ß-lactams, aminoglycosides, co-trimoxazole and ofloxacin, were isolated initially from one infant, and persisted until the eighteenth hospital day culture. The patient was treated with cefaclor and ESBL-producing strains of the same serovar were isolated from a specimen taken 41 days after the initial isolation of the susceptible strains.

Follow-up stool culture from three infants showed persistence of the same serovar for 1–7 weeks. The carriage was probably prolonged because all of the patients were treated with antimicrobial agents for other diseases, none of which was recommended for the treatment of NTS enteritis.

Antimicrobial resistance and ß-lactamase investigation

In our study, all ESBL-producing isolates and the transconjugants were resistant to multiple ß-lactams, but susceptible to cefoxitin, imipenem and ofloxacin. Susceptibility to aminoglycosides and co-trimoxazole was variable: isolates and the transconjugants of serovars Saintpaul and Enteritidis were resistant to amikacin, netilmicin and tobramycin, but susceptible to gentamicin; serovars Stanley and Agona were resistant to gentamicin and tobramycin, but susceptible to amikacin and netilmicin. All isolates and the transconjugants except serovar Stanley were resistant to co-trimoxazole.

The MICs of cefotaxime were equal to or lower than those of ceftazidime for all the ESBL-producing isolates tested, but cefotaxime was hydrolysed more efficiently than ceftazidime, as reported in other TEM-52-producing isolates.8 The MICs of all ß-lactams were decreased by the addition of clavulanic acid, but those of piperacillin did not decrease significantly after addition of tazobactam in three isolates.

Isoelectric focusing showed ß-lactamase bands of pI ~5.4 and ~6.0 in two isolates, suggesting TEM-type enzymes, but in three isolates with high MICs of piperacillin-tazobactam, bands of pI ~6.0 and ~7.4 were observed indicating the presence of other ß-lactamases (Table 2).


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Table 2. Characteristics of ESBL-producing NTS isolates and their transconjugants
 
Genetic characteristics

By PCR analysis, alleles of blaTEM were detected in all of the strains, and those of blaOXA in three strains with a ß-lactamase band of pI ~7.4 (Table 2). Sequencing of the blaTEM amplicon showed deduced amino acid changes of Glu-104->Lys, Met-182->Thr and Gly-238->Ser from TEM-1, which correspond to TEM-52 ß-lactamase (GenBank accession no. AF126444).

Presence of a class 1 integron was detected by PCR in strains with ß-lactamase band of pI~7.4. Sequencing showed that the integron carried aacA4, an unknown orf, and blaOXA1 (GenBank accession no. AY220520). blaOXA1 (not blaOXA-1) was identical to that reported from a sludge sample (GenBank accession no. AY139600). Class 1 integrons, carrying various resistance gene cassettes, were reported in 76% of serovar Typhimurium DT104 and 48% of the non-phage-typeable strains in Spain.9

Our isolates had plasmids of >120 MDa (data not shown). The plasmids carrying ESBL genes are usually large in size, i.e. >=80 kb, although one study showed that blaTEM-52 gene was carried on a mobil-izable plasmid of 13.5 kb in a Klebsiella pneumoniae isolate.8 The resistance to expanded-spectrum cephalosporins was co-transferred to recipients together with resistance to aminoglycosides and co-trimoxazole, if present. These results indicate that blaTEM-52-carrying plasmids with diverse genetic characteristics had been spreading among the NTS.

Epidemiological features

ESBL-producing salmonellae are extremely rare, but recently their prevalence was reported to be 3.4% in western Pacific countries.10 TEM-52 ESBL in NTS was first found in a Yugoslavian patient. Detection of TEM-52-producing salmonellae is not unusual in Korea, as TEM-52-producing E. coli and K. pneumoniae are prevalent.3 However, it is interesting that the five isolates belonged to four different serovars, although the most frequently isolated serovars were Typhimurium and Enteritidis (data not shown).

Two serovar Enteritidis strains isolated from two different patients 4 months apart had an identical antimicrobial resistance pattern and an identical PFGE pattern, suggesting that they belonged to an identical clone. The ESBL-producing and -non-producing strains of serovar Agona from a patient had an identical PFGE pattern, suggesting acquisition of resistance.

In conclusion, isolation of five strains of TEM-52 ESBL-producing NTS with four different serovars, three different resistance patterns and different genetic characteristics suggests that resistance is spreading both clonally and horizontally to NTS in Korea, not only in hospital, but also in the community.


    Acknowledgements
 
We would like to thank National Institute of Health, Korea for determining the serovar of the isolates and Yonghee Seo and Chasoon Lee for their technical support. This study was supported in part by the Brain Korea 21 Project for Medical Sciences, Yonsei University, in 2003.


    Footnotes
 
* Correspondence address. Department of Laboratory Medicine, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-ku, Seoul 120-752, Korea. Tel: +82-2-361-5866; Fax: +82-2-313-0908; E-mail: whonetkor{at}yumc.yonsei.ac.kr Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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