CTX-M-producing Salmonella spp. in Hong Kong: an emerging problem

Salmonella infections remain an important public health problem in Hong Kong. Although all salmonellae isolated in Hong Kong so far have been susceptible to third-generation cephalosporins including cefotaxime and ceftriaxone, the drugs of choice for enteric fevers and invasive salmonellosis caused by the gastroenteric salmonellae, strains resistant to these antimicrobial agents due to the production of extended-spectrum ß-lactamases (ESBLs) have been reported in many parts of the world.

The cefotaximases (CTX-M-type ESBLs) have become the most widespread ß-lactamases over the past few years (Walther-Rasmussen & Hoiby, 2004). These enzymes belong to the class A ß-lactamases and the encoding genes are usually borne on plasmids, although genes on the chromosome have been reported (Rodríguez et al., 2004). They are so called because they hydrolyse cefotaxime more effectively than ceftazidime. The amino acid sequences of these enzymes have less than 40 % identity with the TEM- or SHV-type ß-lactamases (Walther-Rasmussen & Hoiby, 2004). FEC-1, the first cefotaximase reported in 1988 in Japan (Matsumoto et al., 1988), was later found to be related to CTX-M-1, the cefotaximase isolated in 1989 in Germany (Bauernfeind et al., 1990). Over 40 different types of CTX-M enzymes, which are classified into five groups according to their amino acid sequence homology, have since been reported in different enterobacteria (Walther-Rasmussen & Hoiby, 2004; ).

CTX-M-2 was the first cefotaximase to be reported in Salmonella spp. (Bauernfeind et al., 1992). More than ten other CTX-M enzymes have subsequently been reported in different Salmonella serovars, with S. enterica serovar Typhimurium being the most common serovar, producing CTX-M-type enzymes such as CTX-M-5 and CTX-M-15 (Gazouli et al., 1998; Batchelor et al., 2005). Other CTX-M-type ß-lactamase-producing Salmonella serovars include S. enterica serovar Enteritidis (Batchelor et al., 2005; Cheung et al., 2005), S. enterica serovar Infantis (Di Conza et al., 2002); S. enterica serovar Livingston (Bouallègue-Godet et al., 2005), S. enterica serovar London (Yong et al., 2005) and S. enterica serovar Virchow (Batchelor et al., 2005). We isolated the first cefotaxime-resistant strains of Salmonella in Hong Kong in 2003. In this study, we aimed to characterize these cefotaxime-resistant Salmonella spp. by biochemical and molecular methods.

Bacterial isolates. A total of 554 non-duplicate isolates from patients in the New Territories East Cluster hospitals, Hong Kong, were collected during 20032004. All isolates were identified by the API20E system (bioMérieux) and were serotyped using Salmonella-specific O and H antigens by the slide agglutination test. Three isolates that were resistant to cefotaxime were subjected to further study. Escherichia coli K-12 Jp995 (Rif^r) was used as a recipient in conjugation experiments. E. coli ATCC 25922, ATCC 35218 and NCTC 10418 and Pseudomonas aeruginosa ATCC 27853 were used as control strains for antimicrobial susceptibility testing.

Antimicrobial susceptibility testing. This was performed using the agar dilution method (NCCLS, 2003) to determine the MICs of antibiotics as listed in Table 1. ESBL was detected by the reduction of MICs of cefotaxime and ceftazidime in the presence of the ß-lactamase inhibitor clavulanic acid and by the double disk synergy test [CLSI (formerly known as NCCLS), 2005].

Table 1. Antimicrobial susceptibilities of CTX-M-producing Salmonella isolates and their transconjugants MICs indicating resistance are shown in bold. Trc, transconjugant of the corresponding donor.

Detection and identification of ß-lactamase genes and their neighbouring genes. ß-Lactamase genes were detected by PCR amplification and identified by DNA sequence determination. DNA template was obtained by boiling a cell suspension for approximately 10 min. PCR was performed in a total volume of 25 µl containing 1 µl DNA template, one set of primers (12.5 pmol each), 7.5 nmol dNTP, 1x PCR buffer and 0.75 U Taq DNA polymerase on a PTC-225 thermal cycler (MJ Research). The amplification cycle consisted of 5 min denaturation at 95 °C, followed by 35 cycles of 1 min denaturation at 92 °C, annealing and extension. Annealing was done at the temperatures and times reported previously: primer pairs CTX-M-F and CTX-M-R, Weill et al. (2004b); SHV-F and SHV-R, Arlet et al. (1997); ISEcp1U1, Saladin et al. (2002); M9W, Bouallègue-Godet et al. (2005); IntI1-L and IntI1-R, Ng et al. (1999); in-F and in-B and qacEΔ1-F and sul1-B, Zhang et al. (2004); 341_STOP, Sabaté et al. (2002); CTX-M-9 (forward) and CTX-M-9 (reverse), Simarro et al. (2000); TEM-F and TEM-R, Weill et al. (2004a); ampC1 and ampC2, Koeck et al. (1997); CTX-M-1 (forward) and CTX-M-1 (reverse), Batchelor et al. (2005); CTX-2F and CTX-2R and CTX-8F and CTX-8R, Jeong et al. (2005). Extension was at 72 °C for 2 min for primer pairs TEM-F and TEM-R and ampC1 and ampC2, 3 min for in-F and in-B and 1 min for the other primer pairs. The final extension time was 10 min at 74 °C. All PCR products were purified using the GFX PCR DNA and Gel Band Purification kit (Amersham Biosciences) and sequenced by Macrogen. DNA sequences were analysed using CLUSTAL W () and BLAST (). PCR products obtained with primers TEM-F and TEM-R were analysed using BLAST with the TEM-1 gene sequence (GenBank accession no. AB194682). PCR products obtained with primers CTX-M-9 (forward) and CTX-M-9 (reverse) were aligned with CTX-M-9 group sequences: TOHO-2, GenBank accession no. D89862; CTX-M-9, GenBank no. AJ416345; CTX-M-13, GenBank no. AF252623; CTX-M-14, GenBank no. AJ416341; CTX-M-16, GenBank no. AY029068; CTX-M-17, GenBank no. AF454633; CTX-M-19, GenBank no. AF325134; CTX-M-21, GenBank no. AJ416346; CTX-M-24, GenBank no. AY143430; and CTX-M-27, GenBank no. AY156923.

Transferability of resistances. The transferability of resistance genes was tested according to the method of Anderson & Threlfall (1974). Transconjugants were selected on MacConkey agar containing 200 µg rifampicin ml¹ and 2 µg cefotaxime ml¹. Plasmid DNA was extracted and electrophoresed on a 0.7 % agarose gel according to the method of Kado & Liu (1981).

Localization of resistance genes. Plasmid DNA on the gel was transferred to nylon membrane using vacuum blotting equipment and hybridized with the following probes according to standard procedures (Sambrook et al., 1989) but with the following modifications: pre-hybridization was carried out at 55 °C for 1 h for the CTX-M-9 probe and at 48 °C for 1 h for the TEM probe. The appropriate probes were prepared by amplification using primers CTX-M-9 (forward) and CTX-M-9 (reverse) or primers TEM-F and TEM-R and labelled by digoxigenin. Hybridization was carried out overnight at 55 °C for the CTX-M-9 probe and at 48 °C for the TEM probe.

Determination of isoelectric point. The isoelectric point (pI) of the ß-lactamases was determined by isoelectric focusing on a PhastSystem apparatus (Amersham-Pharmacia Biotech) using Ampholine PAGplates (pH 3.59.5; Amersham Biosciences). ß-Lactamases extracted from strains producing TEM-1 (pI 5.4), TEM-3 (pI 6.3), K1 (pI 6.5), SHV-2 (pI 7.6), P99 (pI 7.8) and SHV-5 (pI 8.2) were used as pI markers.

PFGE. Typing was performed by PFGE using XbaI according to a method described previously (Ling et al., 2001).

Antibiotic susceptibilities and ß-lactamase detection
Three isolates with MICs of >4 µg cefotaxime ml¹ were studied. Isolate 2171 was isolated in 2003 from a stool sample of a 4-year-old patient and isolates 2601 and 2927, also from stool samples, were isolated in 2004 from patients aged 3 and 25 years, respectively (Table 2). Two of these isolates were S. Typhimurium and the other was S. Enteritidis.

Table 2. Characteristics of CTX-M-producing Salmonella isolates and their transconjugants Plasmids harbouring the blaCTX-M gene are underlined. Trc, transconjugant of the corresponding donor; NT, not tested.

These three isolates were resistant to ampicillin (MICs >128 µg ml¹), piperacillin (MICs >128 µg ml¹) and ceftriaxone and cefotaxime (MICs 64 µg ml¹), but were susceptible to ceftazidime (MICs=28 µg ml¹) (Table 1). The presence of clavulanic acid reduced the MICs of cefotaxime and ceftriaxone by 256-fold and those of ceftazidime by 8- to 16-fold, and the presence of tazobactam reduced the MICs of piperacillin by 64-fold. The diameter of the zone of inhibition around a 30 µg cefotaxime disc was >5 mm larger than that around a disc containing both cefotaxime (30 µg) and clavulanic acid (10 µg). These results suggested that the isolates produced ESBLs of the CTX-M type. CTX-M enzymes, except for CTX-M-19, usually have a higher activity against cefotaxime than ceftazidime (Poirel et al., 2001). Our isolates were susceptible to cefoxitin (MICs=1 µg ml¹), imipenem (MICs=0.060.12 µg ml¹) and meropenem (MICs 0.03 µg ml¹), indicating that they did not produce AmpC ß-lactamases or carbapenemases. They were resistant to at least one of the non-ß-lactam antibiotics tested (Table 1). Isolate 2171 was resistant to gentamicin, tetracycline and cotrimoxazole; isolate 2601 was resistant to gentamicin and cotrimoxazole; and isolate 2927 was resistant to nalidixic acid. Most of the CTX-M-producing isolates reported so far have also been resistant to non-ß-lactam antibiotics such as nalidixic acid, ciprofloxacin, tetracycline, chloramphenicol, trimethoprim and sulfamethoxazole (Walther-Rasmussen & Hoiby, 2004).

Isoelectric focusing of ß-lactamase extracted from isolate 2171 showed a band at pI 8.1 and that from isolates 2601 and 2927 showed a band at pI 7.9. Isolate 2601 also produced a ß-lactamase of pI 5.4, most probably a TEM-1 enzyme. The pI of CTX-M-9 has been reported to be 7.9, 8.0 or 8.4, whilst that of CTX-M-14 was 7.9, 8.0 and 8.1 (Walther-Rasmussen & Hoiby, 2004; ). Identification of these two enzymes cannot therefore depend on pI values.

Detection and DNA sequence determination of ß-lactamase genes
In order to detect the presence of a ß-lactamase gene(s) in these isolates, DNA was amplified with primers specific for SHV-type, CMY-type and CTX-M-1-, CTX-M-2- and CTX-M-8-group genes. However, no PCR product was obtained except for a 540 bp fragment that was produced when amplification was performed with the universal primers for CTX-M-type genes (CTX-M-F and CTX-M-R). PCR using primers specific for the CTX-M-9 genes [CTX-M-9 (forward) and CTX-M-9 (reverse)] was then carried out and the expected 857 bp product representing the fragment from nt 4 to 860 of the full-length CTX-M-9 gene (876 bp) (Simarro et al., 2000) was produced from DNA from all three isolates. Isolate 2601 also produced a 1080 bp product after amplification with primers TEM-F and TEM-R, which were specific for TEM-type genes. The DNA sequence of the CTX-M gene from isolate 2171 was identical to that of CTX-M-9 and the DNA sequence of the CTX-M gene in isolates 2601 and 2927 was identical to that of CTX-M-14. The DNA sequence of the TEM-type gene from isolate 2601 differed from that of TEM-1 by three nucleotides but did not result in amino acid changes: TTT→TTC (Phe6), GGT→GGC (Gly76) and GCT→GCG (Ala132). The encoding genes of CTX-M-9 and CTX-M-14 differ by a single point mutation leading to a substitution of valine for alanine at aa 231 (Dutour et al., 2002).

CTX-M-9 has been reported in other organisms including E. coli, Klebsiella pneumoniae and Enterobacter spp. (Chanawong et al., 2002), whilst CTX-M-14 has been reported in S. Enteritidis in Japan and Hong Kong, as well as in other enterobacteria (Walther-Rasmussen & Hoiby, 2004; Cheung et al., 2005; ). TEM-1 has been reported previously in Salmonella spp. in Hong Kong; however, the encoding gene was not sequenced (Ling et al., 1991).

The genetic environment of the bla_CTX-M genes was investigated by detecting the presence of ISEcp1 and a class I integron by PCR amplification. A PCR product of 223 bp was obtained after amplification of DNA from CTX-M-14-producing isolates 2601 and 2927 with primers ISEcp1U1 and M9W, indicating the presence of ISEcp1. DNA sequencing of this amplimer indicated that it was identical to the corresponding region of bla_CTX-M-14 (GenBank accession no. AJ972956) and that the inverted repeat between ISEcp1 and bla_CTX-M-14 was present. ISEcp1 is a single-copy insertion sequence responsible for mobilization of bla genes and has been identified upstream of several bla_CTX-M genes (Walther-Rasmussen & Hoiby, 2004).

A PCR product of about 558 bp was obtained after amplification of DNA from CTX-M-9-producing strain 2171 with primers IntI1-L and IntI1-R (for detection of class I integrase), 1900 bp with primers in-F and in-B (for detection of the gene cassette with the class I integron), 800 bp with primers qacEΔ1-F and sul1-B [for detection of the 3' conserved segment (3'-CS) of the class I integron)] and 950 bp with primers 341_STOP and M9W (for detection of sequences upstream of the CTX-M-9 gene). The sequences of the 558, 800 and 950 bp amplimers were identical to those of a class I integrase, the 3'-CS of the class I integron and part of the orf513 region (GenBank accession no. AF174129), respectively. DNA sequencing of the 1900 bp fragment showed that two genes that were identical to dfrA1 (conferring resistance to trimethoprim) (GenBank accession no. AJ884723) and aadA1 (conferring resistance to streptomycin and spectinomycin) (GenBank accession no. AJ884723) were present (Fig. 1). The sequence of dfrA1 was 75 % similar to that of dfrA16, whilst that of aadA1 was 89 % similar to that of aadA2, with both dfrA16 and aadA2 present on In60 (Sabaté et al., 2002). The class I integron is another structure found to be associated with the mobilization of bla_CTX-M and is present upstream of orf513 and the bla_CTX-M gene (Walther-Rasmussen & Hoiby, 2004). CTX-M-2- and CTX-M-9-encoding genes have been reported in unusual class I integrons such as InS21 and In60 (Di Conza et al., 2002; Walther-Rasmussen & Hoiby, 2004). Our bla_CTX-M-9 gene was located downstream of a class I integron-containing gene cassette and orf513 similar to those of In60.

(8K): Fig. 1. Schematic representation of the genetic environment of blaCTX-M-9.

Transferability and localization of resistance genes
All three isolates could transfer their ESBL gene to the E. coli recipient at frequencies ranging from 10² to 10⁷. Although the MICs of cefotaxime and ceftriaxone for all of the transconjugants were reduced to below the respective breakpoints for susceptible strains (CLSI, 2005), the MICs of these drugs in the presence of clavulanic acid were 64-fold lower than those in the absence of clavulanic acid (Table 1). The respective CTX-M genes could be amplified from the transconjugants. The resistance to tetracycline and cotrimoxazole of isolate 2171 was also transferable.

All isolates harboured two or three plasmids ranging from 62 to 130 kb in size (Table 2 and Fig. 2a). The CTX-M-9 probe hybridized with the 62 kb plasmid in isolate 2171, the 70 kb plasmid in isolate 2601 and the 92 kb plasmid in isolate 2927, and with the respective plasmids in their transconjugants (Fig. 2b). Most bla_CTX-M genes have been reported to be on plasmids ranging from 7 kb to >160 kb (Walther-Rasmussen & Hoiby, 2004). The TEM probe hybridized with the 62 kb plasmid of isolate 2601 (result not shown). Resistance to gentamicin and cotrimoxazole was transferable in isolate 2171 but not in isolate 2601, and resistance to nalidixic acid was not transferable in isolate 2927, indicating that the resistance genes were located either on the non-transferable 62 kb plasmid or on the chromosome of isolates 2601 and 2927.

(55K): Fig. 2. (a) Agarose gel electrophoresis of plasmids from cefotaxime-resistant salmonellae. (b) Southern blot hybridization of plasmids with a CTX-M-9 probe. Lanes: 1, isolate 2171; 2, transconjugant of isolate 2171; 3, isolate 2601; 4, transconjugant of isolate 2601; 5, isolate 2927; 6, transconjugant of isolate 2927.

Clonal relatedness
The clonal relatedness of the two S. Typhimurium strains was assessed by PFGE using XbaI digestion. The pattern of isolate 2601 differed from that of 2171 by more than five bands, indicating that they were not related (Fig. 3).

(30K): Fig. 3. PFGE of XbaI-restricted DNA from cefotaxime-resistant S. Typhimurium. Lanes: 1, λ DNA PFGE marker; 2, isolate 2171; 3, isolate 2601.

In summary, this study reports a CTX-M-9-producing Salmonella strain, the first to be isolated in Hong Kong, and the presence of bla_CTX-M-9 and bla_CTX-M-14 in S. Typhimurium. Association of these genes with ISEcp1 and a class I integron, facilitating their spread, is worrying. We thank N. W. S. Lo for assistance with isoelectric focusing experiments. E. coli Jp995 was a gift from Dr B. Rowe, Central Public Health Laboratory, Colindale, London, UK.