Horizontal transfer of antibiotic resistance genes on abiotic touch surfaces: implications for public health

Abstract

Horizontal gene transfer (HGT) is largely responsible for increasing the incidence of antibiotic-resistant infections worldwide. While studies have focused on HGT in vivo, this work investigates whether the ability of pathogens to persist in the environment, particularly on touch surfaces, may also play an important role. Escherichia coli, virulent clone ST131, and Klebsiella pneumoniae harboring extended-spectrum-β-lactamase (ESBL) bla(CTX-M-15) and metallo-β-lactamase bla(NDM-1), respectively, exhibited prolonged survival on stainless steel, with approximately 10(4) viable cells remaining from an inoculum of 10(7) CFU per cm(2) after 1 month at 21°C. HGT of bla to an antibiotic-sensitive but azide-resistant recipient E. coli strain occurred on stainless steel dry touch surfaces and in suspension but not on dry copper. The conjugation frequency was approximately 10 to 50 times greater and occurred immediately, and resulting transconjugants were more stable with ESBL E. coli as the donor cell than with K. pneumoniae, but bla(NDM-1) transfer increased with time. Transconjugants also exhibited the same resistance profile as the donor, suggesting multiple gene transfer. Rapid death, inhibition of respiration, and destruction of genomic and plasmid DNA of both pathogens occurred on copper alloys accompanied by a reduction in bla copy number. Naked E. coli DNA degraded on copper at 21°C and 37°C but slowly at 4°C, suggesting a direct role for the metal. Persistence of viable pathogenic bacteria on touch surfaces may not only increase the risk of infection transmission but may also contribute to the spread of antibiotic resistance by HGT. The use of copper alloys as antimicrobial touch surfaces may help reduce infection and HGT.

Importance: Horizontal gene transfer (HGT) conferring resistance to many classes of antimicrobials has resulted in a worldwide epidemic of nosocomial and community infections caused by multidrug-resistant microorganisms, leading to suggestions that we are in effect returning to the preantibiotic era. While studies have focused on HGT in vivo, this work investigates whether the ability of pathogens to persist in the environment, particularly on touch surfaces, may also play an important role. Here we show prolonged (several-week) survival of multidrug-resistant Escherichia coli and Klebsiella pneumoniae on stainless steel surfaces. Plasmid-mediated HGT of β-lactamase genes to an azide-resistant recipient E. coli strain occurred when the donor and recipient cells were mixed together on stainless steel and in suspension but not on copper surfaces. In addition, rapid death of both antibiotic-resistant strains and destruction of plasmid and genomic DNA were observed on copper and copper alloy surfaces, which could be useful in the prevention of infection spread and gene transfer.

FIG 1

Survival of E. coli (NCTC 13441) (●) and K. pneumoniae (NCTC 13443) (○) containing bla_CTX-M-15 and bla_NDM-1, respectively, on stainless steel at 22°C. Approximately 10⁷ CFU in 20 µl were inoculated onto 1-cm² metal coupons in bacteriological medium. Cells were removed and assessed for culturability as described in the text. For both species, a 2-log reduction in viable cell numbers was observed over the first 10 days, gradually declining to a 3- to 4-log reduction over the following month. Viable E. coli bacteria were detected at 100 days. Error bars represent ± SD (standard deviations), and data are from multiple independent experiments.

FIG 2

Comparison of the frequencies of conjugation of donor E. coli (NCTC 13441) and K. pneumoniae (NCTC 13443) containing bla_CTX-M-15 and bla_NDM-1, respectively, versus the recipient E. coli strain (Az^r [F⁻ met pro], sensitive to β-lactam antibiotics) in suspension and on surfaces. Conjugation frequency is expressed as the number of transconjugants per donor cell as described in the text and is shown as the immediate result (initial isolation) or as the actual result following subculture (i.e., stable transconjugants that retain the gene). Transfer of bla_CTX-M15 occurred immediately in the suspension at a frequency of approximately 3 × 10⁻⁵ and did not increase after 2 h. Similar results were observed on stainless steel, but no transconjugants were recovered from copper after 2 h of contact. Transfer of bla_NDM-1 from K. pneumoniae occurred at a lower frequency of approximately 3 × 10⁻⁷, and although transfer did occur immediately when the cells were mixed in suspension and on copper, the transconjugants were not stable and died on subculturing. However, after 2 h, stable transconjugants were produced on stainless steel and in suspension.

FIG 3

Survival of a wet fomite inoculum of extended-spectrum-β-lactamase-producing E. coli (NCTC 13441) containing bla_CTX-M-15 on copper and copper alloys at 22°C inoculated in a range of matrices. Approximately 10⁷ CFU in 20 µl were inoculated onto the following 1-cm² metal coupons in PBS (A), tryptone soy broth (TSB) (B), or brain heart infusion broth (BHIB) (C) (see Table 1 for constituents of coupons): S30400 (●), C28000 (○), C75200 (▾), C26000 (Δ), C70600 (▪), C51000 (□), and C11000 (♦). Cells were removed and assessed for culturability as described in the text. Prolonged survival was observed on stainless steel with no significant reduction in cell viability for all matrices. Cells in PBS died very rapidly, but death was delayed in the other two matrices. However, there was a significant reduction in numbers of viable cells at 2 h for all alloys and matrices (P < 0.05) except cells inoculated in TSB onto C28000, the alloy with the lowest copper content. Error bars represent ± SD, and data are from multiple independent experiments.

FIG 4

Survival of a wet fomite (A) and dry touch surface (B) inoculum of K. pneumoniae (NCTC 13443) containing bla_NDM-1 on copper and copper alloys at 22°C. Approximately 10⁷ CFU in 20 µl (dries in 30 min) or 1 µl (dries in seconds) were inoculated onto the following 1-cm² metal coupons in PBS: S30400 (●), C28000 (○), C75200 (▾), C26000 (Δ), C70600 (▪), C51000 (□), and C11000 (♦). Cells were removed and assessed for culturability as described in the text. Prolonged survival was observed on stainless steel, but rapid death occurred on copper and copper alloys in proportion to the percentage of copper, especially for the dry touch surface contamination, where all cells were dead on copper and copper nickel after 5 min of contact. Similar results were obtained for extended-spectrum-β-lactamase-producing E. coli (not shown). Error bars represent ± SD, and data are from multiple independent experiments.

FIG 5

Degradation of plasmid DNA and a reduction of copy numbers of bla_CTX-M-15 occur in extended-spectrum-β-lactamase-producing E. coli exposed to copper but not stainless steel surfaces; wet fomite inoculum. (A) The plasmid DNAs of untreated cells (lanes 3 and 10), heat-killed cells (lane 11), or cells exposed to metal surfaces (stainless steel, 0, 60, and 120 min in lanes 4, 5, and 6, respectively; copper, 0, 60, and 120 min in lanes 7, 8, and 9, respectively) were purified and separated by agarose gel electrophoresis as described in the text. The DNAs of untreated cells and those exposed to stainless steel demonstrate the same plasmid bands, indicating that no degradation of DNA had occurred. The reduction of fragment size and smearing of plasmid DNA from cells exposed to copper suggest that extensive degradation was occurring which increased with time. Plasmid DNA from K. pneumoniae is represented in lane 2. Control lanes represent Bioline Hyperladder I (lane 1) and Hyperladder II (lane 12). (B) The same plasmid preparations were assessed for concentrations of bla_CTX-M-15 with gene-specific qPCR as described in the text. Approximately 3 to 4 copies of the gene were present in untreated cells and in those exposed to stainless steel. The copy number increased slightly on cells exposed to copper and immediately removed but then diminished to <1 upon longer exposure; i.e., many cells did not contain the gene.

FIG 6

Degradation of naked plasmid DNA (pBR322) on copper surfaces is dependent on temperature and aqueous content. (A) Sixty nanograms of naked plasmid DNA was applied to metal surfaces for 20 min at room temperature in 1 µl, which dried in seconds (“dry”), or in 10 µl (“wet”). The DNA was removed by pipetting and integrity determined by agarose electrophoresis, as described in the text. For the dry inoculum, plasmid DNA recovered from stainless steel (lane 4) produced two multimer bands, the same as plasmid DNA that had not been exposed to metal (B, lane 11), but the DNA completely degraded on copper (lane 6). The wet inoculum was the same on stainless steel (lane 5), and although extensive degradation occurred on copper (lane 7), traces of DNA were still visible. Control lanes (lanes 1 and 8) represent Bioline Hyperladder I. The ladder DNA was applied directly to stainless steel (lane 2) and copper (lane 3) surfaces. No breakdown of the DNA was evident because the chelator, EDTA, and glycerol components used to stabilize DNA for long-term storage protected the DNA from damaging copper ions released from the surface. (B) Plasmid DNA (“dry” inoculum) was applied directly to stainless steel (lanes 2 to 4), cartridge brass (lanes 5 to 7), or copper (lanes 8 to 10) surfaces at 37°C (lanes 2, 5, and 8), 22°C (lanes 3, 6, and 9), or 4°C (lanes 4, 7, and 10) for 20 min. On stainless steel, DNA remained intact at all temperatures. However, on brass and copper, the DNA degraded completely at 37°C and 22°C, but very little degradation occurred at 4°C. (C) Plasmid DNA (“wet” inoculum) was also applied to stainless steel (lanes 3 to 5), cartridge brass (lanes 6 to 8), or copper (lanes 9 to 11) surfaces at 37°C (lanes 3, 6, and 9), 22°C (lanes 4, 7, and 10), or 4°C (lanes 5, 8, and 11) for 45 min. The DNA remained intact on stainless steel the same as it did on the dry inoculum. On brass and copper, results were similar to those seen with dry inoculation, i.e., most DNA broke down at the highest temperature of 37°C, but the effect was not as extensive. The amount of DNA breakdown was proportional to the copper content. Control lanes (lanes 1 and 12) represent Bioline Hyperladder I, and lane 2 is the untreated plasmid.