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J Med Microbiol 57 (2008), 1122-1128; DOI: 10.1099/jmm.0.2008/001552-0
© 2008 Society for General Microbiology
ISSN 1473-5644

Penicillin resistance in the intestinal spirochaete Brachyspira pilosicoli associated with OXA-136 and OXA-137, two new variants of the class D β-lactamase OXA-63

Sheila M. Mortimer-Jones, Nyree D. Phillips, Tom La, Ram Naresh and David J. Hampson

School of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, Western Australia 6150, Australia

Correspondence
David J. Hampson
d.hampson{at}murdoch.edu.au

Received 27 February 2008
Accepted 28 April 2008

Penicillin resistance mediated by β-lactamase activity has been reported previously in the anaerobic intestinal spirochaete Brachyspira pilosicoli, and a novel class D β-lactamase (OXA-63) hydrolysing oxacillin was described recently in a resistant human strain from France. In the current study, 18 B. pilosicoli strains from Australia and Papua New Guinea were tested for ampicillin and oxacillin susceptibility, and investigated for the presence of the class D β-lactamase gene blaOXA-63 using PCR. PCR products were amplified from seven human and four porcine strains that were penicillin resistant, but not from seven penicillin-sensitive strains. Sequence analysis of the whole gene amplified from seven of the resistant strains from humans and pigs revealed only minor nucleotide differences among them, but there were significant differences compared with blaOXA-63. The predicted amino acid sequence of the enzyme from all seven strains had the same key structural motifs as the previously reported OXA-63, but two variants with 94–95 % identity with OXA-63 were identified. OXA-136 had an additional amino acid and 12 other consistent amino acid substitutions compared with OXA-63. OXA-137 had the same differences compared with OXA-63 as OXA-136, but had an additional amino acid substitution at position 16. No structures consistent with integrons or transposons were found in the nucleotide sequences in the vicinity of blaOXA-136 in partially sequenced B. pilosicoli strain 95/1000, and the GC content (25.2 mol%) of the gene was similar to that of the whole genome. The gene encoding OXA-136 from B. pilosicoli strain Cof-10 conferred penicillin resistance on Escherichia coli. This study shows that penicillin resistance in human and porcine B. pilosicoli strains from Australia is associated with the production of two variants of OXA-63, and that susceptible strains lack the genes encoding OXA-63 or the variants.

Abbreviations: PNG, Papua New Guinea.

The GenBank/EMBL/DDBJ accession numbers for the β-lactamase genes from strains WesB, 95/1000, Cof-10, Gap 51.2, H/A1, H/A2 and H/A3 are EU086830–EU086836, respectively.


   INTRODUCTION
TOP
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The anaerobic intestinal spirochaete Brachyspira pilosicoli (formerly Serpulina pilosicoli) colonizes the large intestine of a range of species, including humans, pigs, chickens, ducks, dogs and horses (Hampson et al., 2006a). The spirochaete is transmitted via the faecal–oral route, but probably also indirectly in drinking water contaminated by human or animal faeces (Oxberry et al., 1998; Margawani et al., 2004; Munshi et al., 2004). B. pilosicoli is considered to be potentially zoonotic (Hampson et al., 2006a). Once established in the large intestine, the spirochaete may attach by one cell end to the colorectal epithelium to form a ‘false brush border’, in a condition called intestinal spirochaetosis (Harland & Lee, 1967; Mikosza & Hampson, 2001; Hampson & Duhamel, 2006). A similar histological picture occurs following colonization with the related spirochaete Brachyspira aalborgi (Mikosza & Hampson, 2001). In humans, colonization with these spirochaetes can be associated with a variety of non-specific problems, including chronic diarrhoea, abdominal discomfort and failure to thrive in children (Douglas & Crucioli, 1981; Brooke et al., 2006; Esteve et al., 2006). Infection with B. pilosicoli occurs commonly in intensively farmed pigs and chickens, in which it is considered to be an important enteric pathogen, and also in people living in crowded and unhygienic conditions in developing countries (Trott et al., 1997a; Margawani et al., 2004; Munshi et al., 2004). Although colonization of humans is rare among the general population in developed countries, in Australia up to 30 % of faecal specimens from rural Aboriginal Australians may contain B. pilosicoli (Lee & Hampson, 1992; Brooke et al., 2001, 2006) and the spirochaete has been isolated from the faeces or from rectal biopsies of 30–50 % of homosexual males and individuals with human immunodeficiency virus (Law et al., 1994; Trivett-Moore et al., 1998). Spirochaetaemias with B. pilosicoli have been reported in individuals who are debilitated for reasons such as chronic alcoholism or neoplastic conditions (Fournié-Amazouz et al., 1995; Trott et al., 1997b; Kanavaki et al., 2002).

Penicillin has been used to treat intestinal spirochaetosis (Kanavaki et al., 2002; Lima et al., 2005), but resistance to penicillin has been reported among B. pilosicoli isolates from humans, pigs and chickens (Tompkins et al., 1987; Brooke et al., 2003; Dassanayake et al., 2005; Hampson et al., 2006b). Furthermore, by using nitrocefin, β-lactamase activity has been identified in penicillin-resistant strains of B. pilosicoli (Tompkins et al., 1987; Brooke et al., 2003). Clavulanic acid enhances the activity of penicillin, ampicillin and amoxicillin against resistant B. pilosicoli (Tompkins et al., 1987; Brooke et al., 2006), and this initially suggested the presence of a class A β-lactamase (Ambler, 1980; Bush et al., 1995). However, analysis of near-complete (~90 %) genomic sequence data from the ampicillin-resistant porcine strain 95/1000 has failed to identify class A β-lactamase genes (T. La & D. J. Hampson, unpublished data). Furthermore, class A genes have not been found in any spirochaete species to date. Recently, a novel membrane-bound group VI class D β-lactamase that hydrolyses oxacillin, called OXA-63, has been described in B. pilosicoli strain BM4442 isolated from a human in France (Meziane-Cherif et al., 2008; GenBank accession nos AY619003 and AAU88145 for the nucleotide and amino acid sequences, respectively).

The purpose of the current study was to look for blaOXA-63, the gene encoding OXA-63, in a collection of B. pilosicoli strains from humans in Australia and Papua New Guinea (PNG), and in strains from pigs, and to determine to what extent it is associated with penicillin resistance in B. pilosicoli.


   METHODS
TOP
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Spirochaete strains. Eighteen strains of B. pilosicoli from the culture collection held at the Reference Centre for Intestinal Spirochaetes at Murdoch University, Australia, were used. These included six strains isolated from the faeces of Australian Aborigines, one strain from an Australian homosexual male, seven strains from villagers in the highlands of PNG and four strains from Australian pigs. Five of the human strains from Aborigines, the strain from the homosexual male and two of the porcine strains have been shown in a previous study to be resistant to amoxicillin, to have this susceptibility restored by clavulanic acid and to have β-lactamase activity as assessed using nitrocefin discs (Brooke et al., 2003).

Medium, culture conditions and antimicrobial susceptibility testing. B. pilosicoli strains were subcultured on trypticase soy agar (TSA; BBL Microbiology Systems) plates containing 5 % defibrinated ovine blood. The cells were grown for 1 week at 37 °C in an anaerobic jar, with an atmosphere of 94 % H2 and 6 % CO2 generated using a GasPak Plus gas generator envelope (BBL). A zone of haemolysis around the inoculated culture indicated growth, which was confirmed by resuspending surface growth in PBS and examining for motile spirochaetes using a phase-contrast microscope at a magnification of x400.

All of the strains were tested for susceptibility to ampicillin, and eight resistant strains (five from Aboriginal Australians, one from a homosexual male and two porcine strains) were also tested with ampicillin plus clavulanic acid, oxacillin, and oxacillin plus clavulanic acid. Doubling dilutions of ampicillin were used with concentrations of 1–128 µg ml–1; clavulanic acid was tested in combination with ampicillin at a ratio of 4 : 1. Oxacillin was tested at 0.06 and 0.125 µg ml–1 and then at doubling dilutions up to 128 µg ml–1; clavulanic acid was added to oxacillin at a ratio of 4 : 1. Antimicrobial susceptibility was assessed using the agar-dilution method, with all tests conducted in quadruplicate. The test plates consisted of TSA containing 5 % defibrinated ovine blood and the appropriate antibiotic concentration. Control plates did not include antibiotics.

Inocula were prepared by adding 1 ml sterile PBS to plates containing pure cultures and resuspending the cells. These were counted using a haemocytometer chamber viewed under a phase-contrast microscope, diluted in PBS and 105 cells of each strain were stab-inoculated onto the test and control plates (CLSI, 2007). The plates were incubated for 5 days at 37 °C in anaerobic jars, with an atmosphere of 94 % H2 and 6 % CO2, and then observed for haemolysis. The first sensitive colony zone and the last resistant colonies were checked for spirochaete growth using a phase-contrast microscope at a magnification of x400. The MIC was reported as the lowest concentration of antimicrobial that inhibited growth.

Presence of blaOXA-63 in B. pilosicoli 95/1000. The partial (~90 %) genome sequence of porcine B. pilosicoli strain 95/1000 was examined for the presence of blaOXA-63 using BLAST homology analysis with international nucleotide and protein databases as well as conserved domain searches using the NCBI Conserved Domains Database (www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml). The genome sequence in the regions flanking the gene also was examined for motifs consistent with transposable elements, such as transposons and integrons, which may show evidence of horizontal gene transfer.

DNA isolation and manipulations. Basic recombinant DNA procedures were carried out according to standard protocols (Sambrook et al., 1989). Chromosomal DNA was prepared from bacterial cells using a DNeasy Tissue kit (Qiagen) and plasmid DNA was purified using a QIAprep Spin Miniprep kit (Qiagen).

PCR. For PCR assays, the amplification mixture consisted of 1x PCR buffer (containing 1.5 mM MgCl2), 0.5 U Taq DNA polymerase (Fisher Biotech), 0.2 mM each dNTP (Promega), 0.5 µM of the primer set and 50–100 ng chromosomal template DNA in a total volume of 50 µl. Cycling conditions involved an initial denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and primer extension at 72 °C for 2 min. The PCR products were separated by electrophoresis in 1.5 % (w/v) agarose in Tris/acetate buffer (40 mM Tris/acetate, 1 mM EDTA), stained with a 1 µg ethidium bromide ml–1 solution and viewed using UV light.

PCR amplifications, and sequencing of blaOXA-63. Three pairs of oligonucleotide primers that annealed to two regions internal to and one external to the 807 bp blaOXA-63 coding sequence in the whole genomic sequence of B. pilosicoli strain 95/1000 were designed using the Primer3 program (Rozen & Skaletsky, 2000) and synthesized by Geneworks. The three PCRs were optimized for the detection of blaOXA-63 using chromosomal DNA from all 18 strains of B. pilosicoli. The external primer set consisted of Bp-oxa-US (5'-TTTGGTTTTCAAGGCTCAACAG-3') and Bp-oxa-DS (5'-GCTTTAGATATTGCTTTTTTGGC-3'), which annealed to complementary sequences adjacent to the coding region of blaOXA-63. The two internal primer sets consisted of Bp-lac1-F192 (5'-AAGATTTTATCCAGCATCAAC-3') and Bp-lac1-R634 (5'-ATCCAGTTTTTCCATGAAGC-3'), which amplified a 450 bp region within the coding region of blaOXA-63, and Bp-oxa-F484 (5'-CCTTTGGAAATAAGTGCGATGGAGCAAG-3') and Bp-oxa-R692 (5'-TCAAGCCAGCCTACGAACCAACC-3'), which amplified a 209 bp region within the coding region.

PCR products generated by the external primer set were purified using an UltraClean PCR Clean-up kit (Mo Bio Laboratories). The products from seven ampicillin-resistant strains of B. pilosicoli were sequenced in both directions using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Mix (PE Applied Biosystems), using the same primers. The sequence results were edited and compiled using VectorNTI Advance 10 (Invitrogen). The sequences were aligned using CLUSTAL_X (Sun et al., 2003) and compared for similarity among themselves and with known class D β-lactamase genes in GenBank.

Cloning of the B. pilosicoli β-lactamase gene into Escherichia coli JM109. The entire 807 bp coding region of the β-lactamase gene was amplified from B. pilosicoli strain Cof-10 using the primers Bp-oxa-F-EcoRI (5'-CAAGGAATTCCAGATGGAAGATACTATATTGCTATG-3') and Bp-oxa-R-XhoI (5'-TTTTCTCGAGTTTAGATATTGCTTTTTTGGC-3') and cloned into plasmid pBK-CMV (Stratagene), which was digested with EcoRI and XhoI. The recombinant plasmid (designated pBK-blaBP-1) was transformed into E. coli JM109 (Promega) and plated onto Luria–Bertani agar supplemented with 50 µg kanamycin ml–1. Plates were incubated at 37 °C overnight. The plasmids from two ampicillin-resistant clones were purified using a QIAprep Spin Miniprep kit (Qiagen) and sequenced as described above using the T3 (5'-AATTAACCCTCACTAAAGGG-3') and T7 (5'-CGGGATATCACTCAGCATAATG-3') vector primers.


   RESULTS
TOP
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Antimicrobial susceptibility

The results of the in vitro antimicrobial susceptibility tests are presented in Table 1Go. All strains grew on the control plates containing TSA and 5 % defibrinated ovine blood. The CLSI (2007) guidelines do not give MIC breakpoints for ampicillin for B. pilosicoli, but an MIC >2 µg ml–1 is considered to indicate resistance in Gram-negative anaerobes. Using this definition, all seven strains from PNG villagers were sensitive to ampicillin, whereas all six Aboriginal strains, the strain obtained from a homosexual male and the four porcine strains were clearly resistant. The ampicillin-resistant strains that were tested had reduced MICs following the addition of clavulanic acid. The strains tested for ampicillin/clavulanic acid sensitivity were also tested with oxacillin. Again, there are no guidelines for MIC breakpoints for Brachyspira species against oxacillin; however, all eight ampicillin-resistant strains tested had MICs of >128 µg ml–1 for oxacillin, which indicates significant resistance. In five out of eight cases, the addition of clavulanic acid to oxacillin reduced the MIC.


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  		Table 1. B. pilosicoli strains investigated and the results of antimicrobial susceptibility tests

CA, Clavulanic acid; NT, not tested.

 
PCR detection of blaOXA-63

PCR products of the correct predicted size were generated using the two PCRs with primers internal to blaOXA-63 and a PCR using primers external to the gene from all 11 strains that were resistant to ampicillin. No products were generated from the seven ampicillin-sensitive strains.

Sequence of blaOXA-63

The 807 bp nucleotide sequences of the β-lactamase genes in the seven penicillin-resistant strains were very similar to each other, differing by only 1–3 bp. The blaOXA-63 gene from French human strain BM4442 was previously reported to have a coding sequence of 807 bp (Meziane-Cherif et al., 2008), but this should have been recorded as 804 bp, as the sequence included a stop codon. The seven sequences obtained in the current study had between 8 and 17 bp differences compared with the blaOXA-63 sequence in strain BM4442, including four consistent insertions and one deletion that resulted in frame-shifts (Table 2Go).


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  		Table 2. Sites of nucleotide differences in blaOXA-63 from French B. pilosicoli strain BM4442 and seven Australian strains

 
Structure of OXA-63 and the new variants

Consistent with a previous report (Meziane-Cherif et al., 2008), the 804 bp blaOXA-63 gene of French B. pilosicoli strain BM4442 was predicted to encode a 268-amino-acid protein with the structural features of a class D β-lactamase. The enzymes in the seven sequenced strains in the current study were predicted to represent two new variants of OXA-63. They all had 12 amino acid differences from OXA-63 in strain BM4442, and had one additional amino acid, such that they both comprised 269 amino acids (Fig. 1Go). The common differences from OXA-63 occurred in three regions (OXA-63 numbering): (i) in amino acids 96–98, where they had Ile–Asn–Gly rather than Leu–Met–Ala, followed by an insertion of a new amino acid (Glu), and a substitution of Val for Ser at amino acid 100; (ii) in amino acid 182, where they had Pro instead of Gln; (iii) in amino acids 196–202, where they had Leu–Glu–Gln–Thr–Tyr–Asn–Tyr rather than Thr–Arg–Ala–Asn–Leu–His–Ile. These differences did not affect the key structural sites on the enzyme. In addition to this form of variant, designated OXA-136, which was found in strains Gap51.5, 95/1000, Cof-10 and WesB, strains H/A1, H/A2 and H/A3 also differed at amino acid position 16 (OXA-63 numbering), where they had Thr rather than an Ile (Fig. 1Go). This second variant was designated OXA-137. The predicted molecular mass and pI values of OXA-63 and the two variants are presented in Table 3Go.


Figure 1
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  		Fig. 1. Amino acid sequence alignment of OXA-1 from E. coli (Sun et al., 2003) and class D enzymes of French human B. pilosicoli strain BM4442 (OXA-63) (Meziane-Cherif et al., 2008), porcine B. pilosicoli strains 95/1000 and Cof-10, and the five human B. pilosicoli strains Gap 51.2, Wes-B, H/A1, H/A2 and H/A3. All position numbers are based on the OXA-1 enzyme (Sun et al., 2003).

 

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  		Table 3. Predicted molecular mass, pI and percentage identity of OXA-85 from F. nucleatum subsp. polymorphum and OXA-63, OXA-136 and OXA-137 from B. pilosicoli

 
Cloning of blaOXA-136 into E. coli

Cloning of blaOXA-136 from B. pilosicoli strain Cof-10 conferred ampicillin resistance on E. coli JM109.

Analysis of the contig containing blaOXA-136 in the B. pilosicoli 95/1000 genomic sequence

The blaOXA-136 gene was identified as a single copy in the B. pilosicoli strain 95/1000 genome sequence, confirming its chromosomal location. The contig containing blaOXA-136 had no integrase or tRNA genes in the regions from ~4500 bp upstream to ~10 000 bp downstream from the gene. The GC content of the 95/1000 blaOXA-136 gene was 25.2 mol%, whilst the mean GC content for the whole contig was 27 mol% (both values were similar to that of the whole genome content: 24.6 mol%). Putative –10 (TATACT) and –35 (TTGACA) promoter sites were located at positions –31 and –53 from blaOXA-136, whilst a ribosome-binding site (AAGGA) was present 6 bp upstream from an ATG initiation codon, leading to an 807 bp coding sequence.


   DISCUSSION
TOP
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study builds on previous work, confirming that some strains of B. pilosicoli are susceptible to penicillin, whilst others are not (Brooke et al., 2003). The fact that penicillin susceptibility was restored with clavulanic acid indicates the presence of a β-lactamase. No class A β-lactamase genes were identified in the genomic sequence of strain 95/1000, but the primers designed to amplify the newly described class D β-lactamase OXA-63 produced specific products, which were found in all of the ampicillin- and oxacillin-resistant strains. In contrast, using three separate PCRs targeting sequences internal and external to blaOXA-63, there was no evidence of this gene being present in the seven penicillin-susceptible strains (all from PNG villagers). This initial finding was consistent with penicillin resistance in the B. pilosicoli strains being due to the presence of the class D β-lactamase OXA-63.

A description of OXA-63 and the gene encoding it from the French human strain BM4442 has been given previously (Meziane-Cherif et al., 2008). The gene and the β-lactamase were contrasted with those of their nearest ancestral relations, blaFUS-1 and FUS-1 (OXA-85), respectively, from Fusobacterium nucleatum subsp. polymorphum, as well as with other OXA-type enzymes (Voha et al., 2006). OXA-63 shares 54 % sequence identity with OXA-85 (Table 3Go).

The translated amino acid sequences from the seven B. pilosicoli strains investigated all showed consistent differences from that of the original OXA-63 in strain BM4442. Nucleotide substitutions, deletions and insertions were such that the translated enzymes shared only 94 or 95 % identity with OXA-63. OXA-136 had 12 amino acid substitutions and one additional amino acid compared with OXA-63, whilst OXA-137 had these same differences as well as another amino acid substitution at position 16. All seven strains containing the variants came from Australia, with OXA-136 being found in strains from one homosexual male, one Aboriginal individual and two pigs, whilst OXA-137 was found in three strains from Aboriginal people. From an epidemiological standpoint, it would be extremely valuable to examine a range of other B. pilosicoli isolates from different sources to determine how widespread these variants are in different host species and geographical regions.

Analysis of the contig on which blaOXA-136 resides in the genome of B. pilosicoli 95/1000 failed to identify any structures reminiscent of transposons or integrons. A similar finding was made for blaOXA-63 in the French strain BM4442 (Meziane-Cherif et al., 2008). Furthermore, the GC content of blaOXA-136 in strain 95/1000 was similar to that of the whole contig and the whole genome. Hence if the gene has been acquired by horizontal gene transfer, this is likely either to have been an ancient event or to have been a more recent acquisition from another Brachyspira species or B. pilosicoli strain with a similar GC content, using a different transfer mechanism. The related spirochaete Brachyspira hyodysenteriae contains an unusual prophage-like gene transfer agent named VSH-1 that transfers random 7.5 kb fragments of spirochaetal DNA between cells, where it may undergo recombination (Matson et al., 2005). A similar transfer agent appears to be present in other Brachyspira species, including the B. pilosicoli strain WesB (penicillin-resistant) that was used in the current study (Stanton et al., 2003). The occurrence of this form of gene transfer would help to explain the presence of β-lactamase genes in resistant strains, without the need to infer that such strains had a different ancestral background to the susceptible strains that apparently lack these genes. Consistent with this, results from multilocus enzyme electrophoresis studies of B. pilosicoli have revealed considerable genetic diversity among strains, and do not obviously separate penicillin-sensitive strains from strains with β-lactamase genes (Lee & Hampson, 1994; Trott et al., 1998; data not shown). Once a functional gene is acquired, penicillin use would select for these resistant strains. As pointed out by Brooke et al. (2003), villagers from the highlands of PNG who were carrying susceptible strains are unlikely to have been exposed extensively to penicillins and, due to their isolation, they may not have encountered resistant strains of B. pilosicoli or other Brachyspira species from other sources. On the other hand, despite also being relatively isolated, Australian Aboriginal people in rural settlements are likely to have had exposure to β-lactams in the course of their treatment for the many health problems that occur within this population (Williams et al., 1997), thus selecting for resistant strains that may have been introduced. Resistance may then have spread to different strains that circulate in these communities (Lee & Hampson, 1992, 1994; Brooke et al., 2001). Interestingly, among B. pilosicoli isolates from homosexual males and recent migrants to Australia from developing countries, approximately half were found to be resistant to amoxicillin and half were susceptible (Brooke et al., 2003). This ratio is presumably governed by the opportunity to acquire β-lactamase genes from other Brachyspira strains or species, and the extent of pressure for selection of resistant strains caused by penicillin use. Although all four porcine strains in the current study were resistant to penicillin, susceptible strains from pigs have been reported previously (Brooke et al., 2003). Penicillins are frequently used to treat porcine infections, and again this use within a population would select for any resistant strains that are present.

Further work is required to investigate the presence, variation and source of the genes encoding OXA-63, OXA-136 and OXA-137 in B. pilosicoli strains from different regions and host species, to examine the potential distribution of these genes in other Brachyspira species, and to determine whether they can be transferred between strains using the Brachyspira prophage-like gene transfer agent.


   ACKNOWLEDGEMENTS
 
Thanks are due to Professor Tom Riley for supplying substrates for the antimicrobial sensitivity testing and to Dr George Jacoby for assistance with establishing the OXA numbers of the variant enzymes.


   REFERENCES
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 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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