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Summary Background Inadequately reprocessed reusable surgical instruments (RSIs) may harbour infectious agents which may then be transferred to a suitable site for replication. Aim To determine the cumulative effect of 20 cycles of contamination, cleaning (manual or manual followed by automated) and steam sterilization on high-complex-design RSIs used for orthopaedic surgery. Methods New flexible medullary reamers and depth gauges were contaminated by soaking in tryptone soya broth, containing 5% sheep blood and 109 cfu/mL of Staphylococcus aureus (ATCC 25923), for 5 min. To mimic a worse-case scenario, RSIs were dried 7 h and subjected to either (a) rinsing in distilled water, (b) manual cleaning or (c) manual plus automated cleaning (reference standard), and steam sterilization. The contamination, cleaning, and sterilization cycle was repeated 20 times. Adenosine triphosphate (ATP) was measured after cleaning procedures; microbial load and residual protein were measured following the 10th and 20th reprocessing, in triplicate. Scanning electron microscopy (SEM) was used to confirm soil and biofilm presence on the RSIs after the 20th reprocessing. Findings Manual and manual plus automated cleaning significantly reduced the amount of ATP and protein residues for all RSIs. Viable bacteria were not detected following sterilization. However, SEM detected soil after automated cleaning, and soil, including biofilms, after manual cleaning. Conclusion Soil and/or biofilms were evident on complex-design RSIs following 20 cycles of contamination and reprocessing, even using the reference standard method of cleaning. Although the depth gauges could be disassembled, biological residues and biofilm accumulated in its lumen. The current design of these RSIs prevents removal of all biological soil and this may have an adverse effect on patient outcome.

Summary Background The importance of biofilms to clinical practice is being increasingly realized. Biofilm tolerance to antibiotics is well described but limited work has been conducted on the efficacy of heat disinfection and sterilization against biofilms. Aim To test the susceptibility of planktonic, hydrated biofilm and dry-surface biofilm forms of Staphylococcus aureus, to dry-heat and wet-heat treatments. Methods S. aureus was grown as both hydrated biofilm and dry-surface biofilm in the CDC biofilm generator. Biofilm was subjected to a range of temperatures in a hot-air oven (dry heat), water bath or autoclave (wet heat). Findings Dry-surface biofilms remained culture positive even when treated with the harshest dry-heat condition of 100°C for 60 min. Following autoclaving samples were culture negative but 62–74% of bacteria in dry-surface biofilms remained alive as demonstrated by live/dead staining and confocal microscopy. Dry-surface biofilms subjected to autoclaving at 121°C for up to 30 min recovered and released planktonic cells. Recovery did not occur following autoclaving for longer or at 134°C, at least during the time-period tested. Hydrated biofilm recovered following dry-heat treatment up to 100°C for 10 min but failed to recover following autoclaving despite the presence of 43–60% live cells as demonstrated by live/dead staining. Conclusion S. aureus dry-surface biofilms are less susceptible to killing by dry heat and steam autoclaving than hydrated biofilms, which are less susceptible to heat treatment than planktonic suspensions.

During functionality testing and packaging of reusable surgical instruments (RSI) for sterilization, instruments are frequently touched. There is a lack of standards relating to hand hygiene frequency and use of gloves in the sterilizing service unit packing area.To determine the effect of hand hygiene and glove use on maintenance of RSI cleanliness.Following manual and automated cleaning, Halsted-mosquito forceps were assessed for adenosine triphosphate (ATP), protein and microbial contamination after handling with gloved and ungloved but washed hands using an ATP surface swab test, bicinchoninic acid assay, and standard culture plate/broth, respectively. Gram's stain was used to classify the isolates. RSI contamination was assessed immediately following and 1, 2, and 4 h after washing hands.Packing instruments with hands that had been unwashed for 2 or 4 h resulted in a significant increase in contaminating ATP when compared with all other treatment groups (P 0.05). There was a significant correlation between the time since washing hands, the amount of ATP (r = 0.93; P ≤ 0.001), and the microbial load (r = 0.83; P ≤ 0.001) contaminating the forceps, where the longer the time the hands remained unwashed the higher the contamination. Significantly more contaminating protein was found on forceps handled with ungloved hands that had not been washed for 2 or 4 h (P 0.001).Critical RSI inspection, assembling, lubricating and packing should be performed using either gloves or within 1 h of washing hands.

Summary Background Dry hospital environments are contaminated with pathogenic bacteria in biofilms, which suggests that current cleaning practices and disinfectants are failing. Aim To test the efficacy of sodium hypochlorite solution against Staphylococcus aureus dry-surface biofilms. Methods The Centers for Disease Control and Prevention Biofilm Reactor was adapted to create a dry-surface biofilm, containing 1.36 × 10 7 S. aureus /coupon, by alternating cycles of growth and dehydration over 12 days. Biofilm was detected qualitatively using live/dead stain confocal laser scanning microscopy (CLSM), and quantitatively with sonicated viable plate counts and crystal violet assay. Sodium hypochlorite (1000–20,000parts per million) was applied to the dry-surface biofilm for 10min, coupons were rinsed three times, and residual biofilm viability was determined by CLSM, plate counts and prolonged culture up to 16 days. Isolates before and after exposure underwent minimum inhibitory concentration (MIC) and minimum eradication concentration (MEC) testing, and one pair underwent whole-genome sequencing. Findings Hypochlorite exposure reduced plate counts by a factor of 7 log 10 , and reduced biofilm biomass by a factor of 100; however, staining of residual biofilm showed that live S. aureus cells remained. On prolonged incubation, S. aureus regrew and formed biofilms. Post-exposure S. aureus isolates had MICs and MECs that were not significantly different from the parent strains. Whole-genome sequencing of one pre- and post-exposure pair found that they were virtually identical. Conclusions Hypochlorite exposure led to a 7-log kill but the organisms regrew. No resistance mutations occurred, implying that hypochlorite resistance is an intrinsic property of S. aureus biofilms. The clinical significance of this warrants further study.

Summary Background Hospital-associated infections cause considerable morbidity and mortality, and are expensive to treat. Organisms causing these infections can be sourced from the inanimate environment around a patient. Could the difficulty in eradicating these organisms from the environment be because they reside in dry surface biofilms? Aim The intensive care unit (ICU) of a tertiary referral hospital was decommissioned and the opportunity to destructively sample clinical surfaces was taken in order to investigate whether multidrug-resistant organisms (MDROs) had survived the decommissioning process and whether they were present in biofilms. Methods The ICU had two ‘terminal cleans' with 500ppm free chlorine solution; items from bedding, surrounds, and furnishings were then sampled with cutting implements. Sections were sonicated in tryptone soya broth and inoculated on to chromogenic plates to demonstrate MDROs, which were confirmed with the Vitek2 system. Genomic DNA was extracted directly from ICU samples, and subjected to polymerase chain reaction (PCR) for femA to detect Staphylococcus aureus and the microbiome by bacterial tag-encoded FLX amplicon pyrosequencing. Confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) were performed on environmental samples. Findings Multidrug-resistant bacteria were cultured from 52% (23/44) of samples cultured. S. aureus PCR was positive in 50%. Biofilm was demonstrated in 93% (41/44) of samples by CLSM and/or SEM. Pyrosequencing demonstrated that the biofilms were polymicrobial and contained species that had multidrug-resistant strains. Conclusion Dry surface biofilms containing MDROs are found on ICU surfaces despite terminal cleaning with chlorine solution. How these arise and how they might be removed requires further study.

Human hepatitis B virus (HBV) is an important cause of nosocomial infections and can be transmitted by contaminated instruments. However, tests of the efficacy of sterilization of materials and equipment contaminated by HBV are difficult to perform because the virus cannot be cultured in the laboratory. In this study, we aimed to evaluate the capability of a low temperature, hydrogen peroxide gas plasma sterilizer (Sterrad, Advanced Sterilization Products, Irvine California,) to inactivate duck hepatitis B virus (DHBV). In laboratory efficacy studies using DHBV dried on to glass filter carriers and exposed to one-half of the hydrogen peroxide gas plasma sterilization process, there was a 10(7) or greater decrease in the viral titer, with no infectivity detected on the carriers after treatment. In-use studies were performed using a laparoscope that was experimentally contaminated with DHBV to mimic the possible transmission of infection between successive patients. Following exposure to the hydrogen peroxide gas plasma sterilization process no transmission of DHBV infection from the laparoscope occurred despite obvious visual soiling with blood (N = 8) while the transmission rate for the unprocessed laparoscope (positive control) was 100% (26/26), and that for instruments after a water wash was 63% (7/11). In conclusion the hydrogen gas plasma sterilization process completely inactivates DHBV a representative of the hepadna group of viruses.

Nosocomial transmission of hepatitis B virus (HBV), associated with interventional procedures, has been attributed to its survival on improperly decontaminated instruments. To date, guidelines for chemical disinfection of potentially contaminated heat-sensitive instruments have been based largely on extrapolation of data from in-vitro disinfectant testing. Direct infectivity testing has not been possible for HBV because of the lack of a practical culture assay or susceptible experimental animal model. In this study the related duck hepadnavirus was used to simulate in-vivo transmission of a HBV during surgery, and to evaluate the effectiveness of 2% glutaraldehyde disinfection of surgical laparoscopes. Multiple laparoscopic liver biopsies were performed on 'biohazardous' duck hepatitis B (DHBV) positive ducks. Laparoscopes were then subjected to different disinfection regimes using 2% glutaraldehyde, and residual infectivity tested by placing their tips into the peritoneal cavities of uninfected four-day-old ducklings. Direct transmission of DHBV occurred in all ducks when laparoscopes were not washed. Rinsing with water lowered the transmission rate to 64% and no infection transmission occurred after 5 min of contact time with the disinfectant. In contrast, previous in-vitro studies had shown complete viral inactivation after a shorter period of disinfection. It is postulated that the longer inactivation time observed in our study may be a result of surface interactions of virus and instrument, interfering with disinfectant access or activity. Tests of instrument surface samples for viral DNA by the polymerase chain reaction (PCR) did not correlate with transmission of virus infection in vivo. PCR is an inappropriate test for evaluating the efficacy of disinfectant action despite its sensitivity. This in use method will allow testing of other decontamination procedures and their effectiveness on more complex surgical instruments.