AbstractBackground:Sink drains in healthcare facilities may provide an environment for antimicrobial-resistant microorganisms, including carbapenemase-producingKlebsiella pneumoniae(CPKP).Methods:We investigated the colonization of a biofilm consortia by CPKP in a model system simulating a sink-drain P-trap. Centers for Disease Control (CDC) biofilm reactors (CBRs) were inoculated with microbial consortia originally recovered from 2 P-traps collected from separate patient rooms (designated rooms A and B) in a hospital. Biofilms were grown on stainless steel (SS) or polyvinyl chloride (PVC) coupons in autoclaved municipal drinking water (ATW) for 7 or 28 days.Results:Microbial communities in model systems (designated CBR-A or CBR-B) were less diverse than communities in respective P-traps A and B, and they were primarily composed of β and γ Proteobacteria, as determined using 16S rRNA community analysis. Following biofilm development CBRs were inoculated with eitherK. pneumoniaeST45 (ie, strain CAV1016) orK. pneumoniaeST258 KPC+ (ie, strain 258), and samples were collected over 21 days. Under most conditions tested (CBR-A: SS, 7-day biofilm; CBR-A: PVC, 28-day biofilm; CBR-B: SS, 7-day and 28-day biofilm; CBR-B: PVC, 28-day biofilm) significantly higher numbers of CAV1016 were observed compared to 258. CAV1016 showed no significant difference in quantity or persistence based on biofilm age (7 days vs 28 days) or substratum type (SS vs PVC). However, counts of 258 were significantly higher on 28-day biofilms and on SS.Conclusions:These results suggest that CPKP persistence in P-trap biofilms may be strain specific or may be related to the type of P-trap material or age of the biofilm.

Liquid wastes (LW) disposed in hospital handwashing sinks may affect colonization of sink P-traps by carbapenemase-producing Klebsiella pneumoniae (CPKP), causing CPKP dispersal into the patient care environment. This study aimed to determine the effect of LW on biofilm formation and CPKP colonization in a P-Trap model (PTM). PTMs containing polymicrobial biofilms grown in autoclaved municipal tap water (ATW) supplemented with 5% dextrose in water (D5W), nutritional shake (Shake), sugar-based soft drink (Soda), or ATW were inoculated with K. pneumoniae ST258 KPC+ (ST258) or K. pneumoniae CAV1016 (CAV1016) and sampled after 7, 14, and 21 d. Biofilm bio-volume, mean thickness, and heterotrophic plate counts were significantly reduced and roughness coefficient significantly increased by Soda compared with D5W, Shake, or ATW. CPKP were significantly reduced by Soda but significantly amplified by D5W (ST258; CAV1016, 7 d) and Shake (ST258) suggesting that reducing LW disposal in sinks may reduce CPKP dispersal into patient care environments.

Handwashing sinks and their associated premise plumbing are an ideal environment for pathogen-harboring biofilms to grow and spread throughout facilities due to the connected system of wastewater plumbing. This study was designed to understand the distribution of pathogens and antibiotic resistant organisms (ARO) within and among handwashing sinks in healthcare settings, using culture-dependent methods to quantify Pseudomonas aeruginosa, opportunistic pathogens capable of growth on a cefotaxime-containing medium (OPP-C), and carbapenem-resistant Enterobacteriaceae (CRE). Isolates from each medium identified as P. aeruginosa or Enterobacteriaceae were tested for susceptibility to aztreonam, ceftazidime, and meropenem; Enterobacteriaceae were also tested against ertapenem and cefotaxime. Isolates exhibiting resistance or intermediate resistance were designated ARO. Pathogens were quantified at different locations within handwashing sinks and compared in quantity and distribution between healthcare personnel (HCP) and patient room (PR) sinks. ARO were compared between samples within a sink (biofilm vs planktonic samples) and between sink types (HCP vs. PR). The drain cover was identified as a reservoir within multiple sinks that was often colonized by pathogens despite daily sink cleaning. P. aeruginosa and OPP-C mean log10 CFU/cm2 counts were higher in p-trap and tail pipe biofilm samples from HCP compared to PR sinks (2.77 ± 2.39 vs. 1.23 ± 1.62 and 5.27 ± 1.10 vs. 4.74 ± 1.06) for P. aeruginosa and OPP-C, respectively. P. aeruginosa and OPP-C mean log10 CFU/ml counts were also higher (p < 0.05) in HCP compared to PR sinks p-trap water (2.21 ± 1.52 vs. 0.89 ± 1.44 and 3.87 ± 0.78 vs. 3.21 ± 1.11) for P. aeruginosa and OPP-C, respectively. However, a greater percentage of ARO were recovered from PR sinks compared to HCP sinks (p < 0.05) for Enterobacteriaceae (76.4 vs. 32.9%) and P. aeruginosa (25.6 vs. 0.3%). This study supports previous work citing that handwashing sinks are reservoirs for pathogens and ARO and identifies differences in pathogen and ARO quantities between HCP and PR sinks, despite the interconnected premise plumbing.

Background: Handwashing sinks in healthcare environments are reservoirs for healthcare pathogens and antibiotic-resistant microorganisms (ARO). We investigated the distribution of HCP and ARO within and among handwashing sinks in healthcare settings. To do this, we determined the differences in the number of ARO between samples within a sink (biofilm vs planktonic samples), between sink types (healthcare worker [HCW] vs patient room sinks), and between hospitals in the same city. Methods: Tap water, sink surface, drain cover, tail pipe, p-trap water and p-trap samples were collected from 2 patient room sinks and 2 HCW sinks over 11 months in 2 acute-care hospitals. Suspected pathogens were isolated from selective media (Pseudosel, Chromagar KPC, and MacConkey with 2 mg/L cefotaxime) and identified via MALDI-ToF. Isolates confirmed to be healthcare pathogens were characterized via disk diffusion to determine their antibiotic susceptibility according to CLSI guidelines. Isolates not susceptible to carbapenems (meropenem or ertapenem) were tested further via the modified carbapenem inactivation method to detect carbapenemase production. Results:Pseudomonas aeruginosa and Enterobacteriaceae (Enterobacter spp, Klebsiella spp, and Citrobacter spp) were the most frequently isolated pathogens. Among these isolates (195 P. aeruginosa and 42 Enterobacteriaceae isolates), 28.5% of P. aeruginosa and 85.7% of Enterobacteriaceae were nonsusceptible to 1 or more of the antibiotics tested. Of the isolates that were nonsusceptible to a carbapenem (46 of 237; 19%), none displayed phenotypic carbapenemase production. Other mechanisms of resistance have not been confirmed. There was no significant difference in the percentage of nonsusceptible HCP isolated from biofilm samples (from p-trap and tail pipe) compared to planktonic (p-trap water) samples (P > .05 for P. aeruginosa and Enterobacteriaceae). A greater percentage of resistant or intermediate isolates was recovered from patient room sinks than from HCW sinks (P < .05) for both P. aeruginosa and Enterobacteriaceae isolates (76.4 vs 32.9% for Enterobacteriaceae, 25.6 vs 0.3% for P. aeruginosa). We detected no significant difference in percentage of nonsusceptible isolates between the 2 hospitals sampled (P > .05). Conclusions: This survey of healthcare sinks supports previous work citing that they are reservoirs for HCP and ARO. This work further examines the distribution of HCP and ARO within and among sinks in these environments. Our findings thus far in the 2 hospitals studied reveal a higher percentage of ARO in patient sinks than in HCW sinks. This finding may suggest a higher input of ARO from patient use or greater selective pressure in patient room sinks. Disclosures: None Funding: Lauren Franco, Centers for Disease Control and Prevention

The p-traps of hospital handwashing sinks represent a potential reservoir for antimicrobial-resistant organisms of major public health concern, such as carbapenemase-producing KPC+ Klebsiella pneumoniae (CPKP). Bacteriophages have reemerged as potential biocontrol agents, particularly against biofilm-associated, drug-resistant microorganisms. The primary objective of our study was to formulate a phage cocktail capable of targeting a CPKP strain (CAV1016) at different stages of colonization within polymicrobial drinking water biofilms using a CDC biofilm reactor (CBR) p-trap model. A cocktail of four CAV1016 phages, all exhibiting depolymerase activity, were isolated from untreated wastewater using standard methods. Biofilms containing Pseudomonas aeruginosa, Micrococcus luteus, Stenotrophomonas maltophilia, Elizabethkingia anophelis, Cupriavidus metallidurans, and Methylobacterium fujisawaense were established in the CBR p-trap model for a period of 28 d. Subsequently, CAV1016 was inoculated into the p-trap model and monitored over a period of 21 d. Biofilms were treated for 2 h at either 25 °C or 37 °C with the phage cocktail (109 PFU/ml) at 7, 14, and 21 d post-inoculation. The effect of phage treatment on the viability of biofilm-associated CAV1016 was determined by plate count on m-Endo LES agar. Biofilm heterotrophic plate counts (HPC) were determined using R2A agar. Phage titers were determined by plaque assay. Phage treatment reduced biofilm-associated CAV1016 viability by 1 log10 CFU/cm2 (p < 0.05) at 7 and 14 d (37 °C) and 1.4 log10 and 1.6 log10 CFU/cm2 (p < 0.05) at 7 and 14 d, respectively (25 °C). No significant reduction was observed at 21 d post-inoculation. Phage treatment had no significant effect on the biofilm HPCs (p > 0.05) at any time point or temperature. Supplementation with a non-ionic surfactant appears to enhance phage association within biofilms. The results of this study suggest the potential of phages to control CPKP and other carbapenemase-producing organisms associated with microbial biofilms in the healthcare environment.

Members of the family Enterobacteriaceae , such as Klebsiella pneumoniae , are considered both serious and urgent public health threats. Biofilms formed by these health care-associated pathogens can lead to negative and costly health outcomes. The global spread of antibiotic resistance, coupled with increased tolerance to antimicrobial treatments in biofilm-associated bacteria, highlights the need for novel strategies to overcome treatment hurdles. Bacteriophages (phages), or viruses that infect bacteria, have reemerged as one such potential strategy. Virulent phages are capable of infecting and killing their bacterial hosts, in some cases producing depolymerases that are able to hydrolyze biofilms. Phage therapy does have its limitations, however, including potential narrow host ranges, development of bacterial resistance to infection, and the potential spread of phage-encoded virulence genes. That being said, advances in phage isolation, screening, and genome sequencing tools provide an upside in overcoming some of these limitations and open up the possibilities of using phages as effective biofilm control agents.

Orthopedic implant infections are a significant clinical problem, with current therapies limited to surgical debridement and systemic antibiotic regimens. Lysostaphin is a bacteriolytic enzyme with high antistaphylococcal activity. We engineered a lysostaphin-delivering injectable PEG hydrogel to treat Staphylococcus aureus infections in bone fractures. The injectable hydrogel formulation adheres to exposed tissue and fracture surfaces, ensuring efficient, local delivery of lysostaphin. Lysostaphin encapsulation within this synthetic hydrogel maintained enzyme stability and activity. Lysostaphin-delivering hydrogels exhibited enhanced antibiofilm activity compared with soluble lysostaphin. Lysostaphin-delivering hydrogels eradicated S. aureus infection and outperformed prophylactic antibiotic and soluble lysostaphin therapy in a murine model of femur fracture. Analysis of the local inflammatory response to infections treated with lysostaphin-delivering hydrogels revealed indistinguishable differences in cytokine secretion profiles compared with uninfected fractures, demonstrating clearance of bacteria and associated inflammation. Importantly, infected fractures treated with lysostaphin-delivering hydrogels fully healed by 5 wk with bone formation and mechanical properties equivalent to those of uninfected fractures, whereas fractures treated without the hydrogel carrier were equivalent to untreated infections. Finally, lysostaphin-delivering hydrogels eliminate methicillin-resistant S. aureus infections, supporting this therapy as an alternative to antibiotics. These results indicate that lysostaphin-delivering hydrogels effectively eliminate orthopedic S. aureus infections while simultaneously supporting fracture repair.

Lung infections associated with pneumonia, or cystic fibrosis caused by Pseudomonas aeruginosa or other bacteria, result in significant morbidity and mortality, in part owing to the development of multidrug resistance, also against last-resort antibiotics. Lytic bacteriophages (that is, viruses that specifically kill bacteria) can reduce lung-associated infections, yet their clinical use is hindered by difficulties in delivering active phages to the deep lung. Here, we show that phage-loaded polymeric microparticles deposit throughout the lung via dry powder inhalation and that they deliver active phages. Phage-loaded microparticles effectively reduced P. aeruginosa infections and the associated inflammation in wild-type and cystic fibrosis transmembrane-conductance-regulator knockout mice, and rescued the mice from pneumonia-associated death. These polymeric microparticles might constitute a clinically translatable therapy for eradicating hospital-acquired lung infections and infections associated with cystic fibrosis.

Among the possible environmental reservoirs in a patient care environment, sink drains are increasingly recognized as a potential reservoir to hospitalized patients of multidrug-resistant health care-associated pathogens. With increasing antimicrobial resistance limiting therapeutic options for patients, a better understanding of how pathogens disseminate from sink drains is urgently needed. Once this knowledge gap has decreased, interventions can be engineered to decrease or eliminate transmission from hospital sink drains to patients. The current study further defines the mechanisms of transmission for bacteria that colonize sink drains.

Importance The human and financial costs of treating surgical site infections (SSIs) are increasing. The number of surgical procedures performed in the United States continues to rise, and surgical patients are initially seen with increasingly complex comorbidities. It is estimated that approximately half of SSIs are deemed preventable using evidence-based strategies. Objective To provide new and updated evidence-based recommendations for the prevention of SSI. Evidence Review A targeted systematic review of the literature was conducted in MEDLINE, EMBASE, CINAHL, and the Cochrane Library from 1998 through April 2014. A modified Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) approach was used to assess the quality of evidence and the strength of the resulting recommendation and to provide explicit links between them. Of 5759 titles and abstracts screened, 896 underwent full-text review by 2 independent reviewers. After exclusions, 170 studies were extracted into evidence tables, appraised, and synthesized. Findings Before surgery, patients should shower or bathe (full body) with soap (antimicrobial or nonantimicrobial) or an antiseptic agent on at least the night before the operative day. Antimicrobial prophylaxis should be administered only when indicated based on published clinical practice guidelines and timed such that a bactericidal concentration of the agents is established in the serum and tissues when the incision is made. In cesarean section procedures, antimicrobial prophylaxis should be administered before skin incision. Skin preparation in the operating room should be performed using an alcohol-based agent unless contraindicated. For clean and clean-contaminated procedures, additional prophylactic antimicrobial agent doses should not be administered after the surgical incision is closed in the operating room, even in the presence of a drain. Topical antimicrobial agents should not be applied to the surgical incision. During surgery, glycemic control should be implemented using blood glucose target levels less than 200 mg/dL, and normothermia should be maintained in all patients. Increased fraction of inspired oxygen should be administered during surgery and after extubation in the immediate postoperative period for patients with normal pulmonary function undergoing general anesthesia with endotracheal intubation. Transfusion of blood products should not be withheld from surgical patients as a means to prevent SSI. Conclusions and Relevance This guideline is intended to provide new and updated evidence-based recommendations for the prevention of SSI and should be incorporated into comprehensive surgical quality improvement programs to improve patient safety.

Microbial Biofilms provides an overview of the formation, structure/architecture, cell-to-cell interactions, and dispersal of fungal and bacterial biofilms. The target audience is biofilm researchers, but this second edition of the book should also be useful for healthcare practitioners seeking a better understanding of microbial biofilms in healthcare delivery. The number of published papers pertaining to microbial biofilms in healthcare and public health has continued to grow since the publication of the first edition of Microbial Biofilms in 2004. For example, a search using PubMed for 2004–2016 identified 488 publications when using the search terms “biofilm and healthcare-associated infection” and 7,303 publications using the search terms “biofilm and public health,” compared to 73 and 1,459 publications, respectively, for 1992–2003. The current edition addresses this level of interest; several of the contributions in this book specifically focus on the role of biofilms in disease processes (Chapters 6, 7, 8, 14, and 19) or biofilm susceptibility to antimicrobial agents (Chapters 2 and 13). Chapter 1 provides a balanced comparison of static and continuous flow methods for growing biofilms that should be beneficial for researchers investigating biofilm development or dispersion, and for applied studies evaluating new treatment strategies for biofilm prevention and control. This beginning can enable those new to the field to evaluate the benefits and drawbacks of different biofilm testing methods for specific applications. Chapter 1 also provides a helpful but brief discussion of the use of “omic” approaches (i.e., genomic, metagenomic, transcriptomic, and proteomic) in the study and characterization of biofilms. Protocols to evaluate biofilm control strategies in vivo are needed, since in vitro methods may not predict performance under the more robust conditions provided in an animal model (1). Published animal model protocols for evaluating biofilm control are few. Chapter 3 provides several animal models for the evaluation of fungal biofilms (primarily Candida spp.), including vascular catheter, urinary catheter, and subcutaneous implant model systems. However, animal model systems for the characterization of bacterial biofilms are not described. Several recent papers have used culture-independent methods to characterize biofilms on indwelling medical devices (2–4). With the exception of a brief discussion in Chapter 1, very little information is provided on the benefits and drawbacks of culture-independent methods to characterize clinically relevant biofilm communities. Chapter 13 provides an excellent overview of antimicrobial tolerance in biofilms, with a good summary of the factors that can influence susceptibility. I found particularly helpful the use of tolerance factors to compare reduced susceptibility of different biofilm-associated organisms toward biocides, antiseptics, and antibiotic drugs. Tolerance factors were plotted as a function of antimicrobial agent molecular weight, substratum material, and biofilm density, providing the reader a method for quickly visualizing these patterns for a wide range of organisms. This information can be very helpful when developing experimental approaches to evaluate biofilm control strategies. In summary, Microbial Biofilms is a useful compendium suitable for students and a practical guide for researchers investigating new biofilm treatment strategies. The emphasis on the role of biofilms in the pathogenesis of various microbial diseases, as well as discussions of biofilm tolerance and antimicrobial resistance should also be helpful and interesting to anyone working in the field of healthcare delivery.

Following graduation from Virginia Tech with a B.S. in biology, it was my great fortune to be chosen to participate in a multinational, NSF-funded research project in Antarctica as an environmental...

ABSTRACTMicroorganisms from a patient or their environment may colonize indwelling urinary catheters, forming biofilm communities on catheter surfaces and increasing patient morbidity and mortality. This study investigated the effect of pretreating hydrogel-coated silicone catheters with mixtures ofPseudomonas aeruginosaandProteus mirabilisbacteriophages on the development of single- and two-species biofilms in a multiday continuous-flowin vitromodel using artificial urine. Novel phages were purified from sewage, characterized, and screened for their abilities to reduce biofilm development by clinical isolates of their respective hosts. Our screening data showed that artificial urine medium (AUM) is a valid substitute for human urine for the purpose of evaluating uropathogen biofilm control by these bacteriophages. Defined phage cocktails targetingP. aeruginosaandP. mirabiliswere designed based on the biofilm inhibition screens. Hydrogel-coated catheters were pretreated with one or both cocktails and challenged with approximately 1 × 103CFU/ml of the corresponding pathogen(s). The biofilm growth on the catheter surfaces in AUM was monitored over 72 to 96 h. Phage pretreatment reducedP. aeruginosabiofilm counts by 4 log10CFU/cm2(P≤ 0.01) andP. mirabilisbiofilm counts by >2 log10CFU/cm2(P≤ 0.01) over 48 h. The presence ofP. mirabiliswas always associated with an increase in lumen pH from 7.5 to 9.5 and with eventual blockage of the reactor lines. The results of this study suggest that pretreatment of a hydrogel urinary catheter with a phage cocktail can significantly reduce mixed-species biofilm formation by clinically relevant bacteria.

ABSTRACT Microorganisms may colonize needleless connectors (NCs) on intravascular catheters, forming biofilms and predisposing patients to catheter-associated infection (CAI). Standard and silver-coated NCs were collected from catheterized intensive care unit patients to characterize biofilm formation using culture-dependent and culture-independent methods and to investigate the associations between NC usage and biofilm characteristics. Viable microorganisms were detected by plate counts from 46% of standard NCs and 59% of silver-coated NCs (P = 0.11). There were no significant associations (P > 0.05, chi-square test) between catheter type, side of catheter placement, number of catheter lumens, site of catheter placement, or NC placement duration and positive NC findings. There was an association (P = 0.04, chi-square test) between infusion type and positive findings for standard NCs. Viable microorganisms exhibiting intracellular esterase activity were detected on >90% of both NC types (P = 0.751), suggesting that a large percentage of organisms were not culturable using the conditions provided in this study. Amplification of the 16S rRNA gene from selected NCs provided a substantially larger number of operational taxonomic units per NC than did plate counts (26 to 43 versus 1 to 4 operational taxonomic units/NC, respectively), suggesting that culture-dependent methods may substantially underestimate microbial diversity on NCs. NC bacterial communities were clustered by patient and venous access type and may reflect the composition of the patient's local microbiome but also may contain organisms from the health care environment. NCs provide a portal of entry for a wide diversity of opportunistic pathogens to colonize the catheter lumen, forming a biofilm and increasing the potential for CAI, highlighting the importance of catheter maintenance practices to reduce microbial contamination.

19 Microorganisms from a patient or their environment may colonize indwelling urinary catheters, 20 forming biofilm communities on catheter surfaces and increasing patient morbidity and 21 mortality. This study investigated the effect of pre-treating hydrogel-coated silicone catheters 22 with mixtures of Pseudomonas aeruginosa and Proteus mirabilis bacteriophages on the 23 development of singleand two-species biofilms in a multi-day, continuous-flow in vitro model 24 using artificial urine. Novel phages were purified from sewage, characterized, and screened for 25 their ability to reduce biofilm development by clinical isolates of their respective hosts. 26 Screening data showed that Artificial Urine Medium (AUM) is a valid substitute for human urine 27 for the purpose of evaluating uropathogen biofilm control by these bacteriophages. Defined 28 phage cocktails targeting each of P. aeruginosa and P. mirabilis were designed based on biofilm 29 inhibition screens. Hydrogel-coated catheters were pre-treated with one or both cocktails and 30 challenged with approximately 1×10 CFU/mL of the corresponding pathogen(s). Biofilm 31 growth on catheter surfaces in AUM was monitored over 72 to 96 h. Phage pre-treatment 32 reduced P. aeruginosa biofilm counts by 4 log10CFU/cm (p<0.01) and P. mirabilis biofilm 33 counts by > 2 log10 CFU/cm (p <0.01) over 48 h. The presence of P. mirabilis was always 34 associated with an increase in lumen pH from 7.5 to 9.5, and with eventual blockage of the 35 reactor lines. Results of this study suggest that pretreatment of a hydrogel urinary catheter with a 36 phage cocktail can significantly reduce mixed species biofilm formation by clinically relevant 37 bacteria. 38

PURPOSE:To determine whether a bacteriophage antimicrobial-lock technique can reduce bacterial colonization and biofilm formation on indwelling central venous catheters in a rabbit model. MATERIALS AND METHODS:Cuffed central venous catheters were inserted into the jugular vein of female New Zealand White rabbits under image guidance. Catheters were inoculated for 24 hours with broth culture of methicillin-sensitive Staphylococcus aureus. The inoculum was aspirated, and rabbits were randomly assigned to two equal groups for 24 hours: (i) untreated controls (heparinized saline lock), (ii) bacteriophage antimicrobial-lock (staphylococcal bacteriophage K, propagated titer > 10(8)/mL). Blood cultures were obtained via peripheral veins, and the catheters were removed for quantitative culture and scanning electron microscopy. RESULTS:Mean colony-forming units (CFU) per cm(2) of the distal catheter segment, as a measure of biofilm, were significantly decreased in experimental animals compared with controls (control, 1.2 × 10(5) CFU/cm(2); experimental, 7.6 × 10(3); P = .016). Scanning electron microscopy demonstrated that biofilms were present on the surface of five of five control catheters but only one of five treated catheters (P = .048). Blood culture results were not significantly different between the groups. CONCLUSIONS:In a rabbit model, treatment of infected central venous catheters with a bacteriophage antimicrobial-lock technique significantly reduced bacterial colonization and biofilm presence. Our data represent a preliminary step toward use of bacteriophage therapy for prevention and treatment of central venous catheter-associated infection.

A repeatable and sensitive method to evaluate the effect of three antiseptics and two disinfection techniques on viable micro-organisms on luer-activated catheter needleless connectors (NCs) was developed. NCs were inoculated with Staphylococcus epidermidis or Klebsiella pneumoniae and disinfected with 315% chlorhexidine gluconate+70% isopropanol (CGI), 70% isopropanol (IPA) or 10% PVP povidone-iodine (PI) antiseptic pads using: (i) scrubbing the NC septum and threaded external surfaces or (ii) wiping only the surface of the septum. Treatments were also evaluated against NCs pretreated with human serum and exposed for 18h to Staph.epidermidis prior to testing. Viable cells were quantified by plate count. The method for inoculation and recovery of luminal micro-organisms was repeatable (SD, 031; n=28). IPA disinfection provided an approximate 3log(10)CFU reduction; CGI and PI provided 3-4 log(10) reductions. PI and CGI were more effective than IPA (P<005), but differences between CGI and PI were not significant for either disinfection method. IPA, but not CGI and PI was also less effective (P<005) against NCs inoculated with Kl.pneumoniae than Staph.epidermidis. Pretreatment with serum and prolonged Staph.epidermidis inoculation removed the advantage seen with CGI and PI; log(10) reductions were 180, 173 and 250 for CGI, PI and IPA, respectively. PI or CGI may be more effective than IPA for NC disinfection but effectiveness may be reduced on NCs contaminated with blood or serum.Significance and Impact of the Studysensitive and repeatable protocol was developed to evaluate antiseptics for disinfecting catheter needleless connectors (NCs). Povidone-iodine (PI) and chlorhexidine gluconate plus isopropanol (CGI) were more effective than isopropanol (IPA) for reducing Staphylococcus epidermidis contamination of NCs. The effectiveness of PI and CGI was reduced on NCs pre-exposed to human serum and prolonged bacterial inoculation. IPA was also less effective against NCs contaminated with Klebsiella pneumoniae.