Supplemental nutrients stimulate the amplification of carbapenemase-producing Klebsiella pneumoniae (CPKP) in a sink drain in vitro biofilm reactor model.

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.

Colonization of carbapenem-resistant Klebsiella pneumoniae in a sink-drain model biofilm system.

BACKGROUND: Sink drains in healthcare facilities may provide an environment for antimicrobial-resistant microorganisms, including carbapenemase-producing Klebsiella 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 ß and γ Proteobacteria, as determined using 16S rRNA community analysis. Following biofilm development CBRs were inoculated with either K. pneumoniae ST45 (ie, strain CAV1016) or K. pneumoniae ST258 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.

Bacteriophage Infections of Biofilms of Health Care-Associated Pathogens: Klebsiella pneumoniae.

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.

A microbiological survey of handwashing sinks in the hospital built environment reveals differences in patient room and healthcare personnel sinks.

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.

Bacteriophage treatment of carbapenemase-producing Klebsiella pneumoniae in a multispecies biofilm: a potential biocontrol strategy for healthcare facilities.

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.

Droplet- Rather than Aerosol-Mediated Dispersion Is the Primary Mechanism of Bacterial Transmission from Contaminated Hand-Washing Sink Traps.

An alarming rise in hospital outbreaks implicating hand-washing sinks has led to widespread acknowledgment that sinks are a major reservoir of antibiotic-resistant pathogens in patient care areas. An earlier study using green fluorescent protein (GFP)-expressing Escherichia coli (GFP-E. coli) as a model organism demonstrated dispersal from drain biofilms in contaminated sinks. The present study further characterizes the dispersal of microorganisms from contaminated sinks. Replicate hand-washing sinks were inoculated with GFP-E. coli, and dispersion was measured using qualitative (settle plates) and quantitative (air sampling) methods. Dispersal caused by faucet water was captured with settle plates and air sampling methods when bacteria were present on the drain. In contrast, no dispersal was captured without or in between faucet events, amending an earlier theory that bacteria aerosolize from the P-trap and disperse. Numbers of dispersed GFP-E. coli cells diminished substantially within 30 minutes after faucet usage, suggesting that the organisms were associated with larger droplet-sized particles that are not suspended in the air for long periods.IMPORTANCE 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.

Hydrogel delivery of lysostaphin eliminates orthopedic implant infection by Staphylococcus aureus and supports fracture healing.

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.

Centers for Disease Control and Prevention Guideline for the Prevention of Surgical Site Infection, 2017.

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.

Bacteriophage-mediated control of a two-species biofilm formed by microorganisms causing catheter-associated urinary tract infections in an in vitro urinary catheter model.

Microorganisms 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 of Pseudomonas aeruginosa and Proteus mirabilis bacteriophages on the development of single- and two-species biofilms in a multiday continuous-flow in vitro model 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 targeting P. aeruginosa and P. mirabilis were designed based on the biofilm inhibition screens. Hydrogel-coated catheters were pretreated with one or both cocktails and challenged with approximately 1×10(3) CFU/ml of the corresponding pathogen(s). The biofilm growth on the catheter surfaces in AUM was monitored over 72 to 96 h. Phage pretreatment reduced P. aeruginosa biofilm counts by 4 log10 CFU/cm2 (P≤0.01) and P. mirabilis biofilm counts by >2 log10 CFU/cm2 (P≤0.01) over 48 h. The presence of P. mirabilis was 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.

Bacteriophage K antimicrobial-lock technique for treatment of Staphylococcus aureus central venous catheter-related infection: a leporine model efficacy analysis.

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.

Microbial biofilms on needleless connectors for central venous catheters: comparison of standard and silver-coated devices collected from patients in an acute care hospital.

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.

A biofilm model developed to investigate survival and disinfection of Mycobacterium mucogenicum in potable water.

Water in healthcare environments can be a source for healthcare-associated infections (HAI). However, information on the exposure risk to opportunistic pathogens in potable water distribution systems (PWDS) is lacking. Laboratory studies characterizing the interaction of opportunistic pathogens with biofilms are needed to understand their role in water systems within healthcare facilities. A stable, repeatable, PWDS multi-species biofilm model comprising Sphingomonas paucimobilis, Methylobacterium sp., Delftia acidovorans, and Mycobacterium mucogenicum was developed in the CDC Biofilm Reactor (CBR), reaching 6 log(10) CFU cm(-2) within 6 days. The model was used to investigate the interaction of the opportunistic pathogen M. mucogenicum with the other species, and to determine the efficacy of monochloramine (NH(2)Cl) as a disinfectant against 2-week-old biofilms. Addition of 1 or 2 mg l(-1) NH(2)Cl resulted in the same or an increased log density of viable M. mucogenicum in the biofilm while inactivating some of the Proteobacteria. Although M. mucogenicum preferentially resided in the biofilm, NH(2)Cl exposure caused release of viable M. mucogenicum from the biofilm into the water. Additional studies with this model should determine if sodium hypochlorite has a comparative effect and if other nontuberculous mycobacteria (NTM) respond to NH(2)Cl similarly.

Biofilm elimination on intravascular catheters: important considerations for the infectious disease practitioner.

The presence of biofilms on intravascular catheters and their role in catheter-related bloodstream infections is well accepted. The tolerance of catheter-associated biofilm organisms toward systemic antimicrobial treatments and the potential for development of antimicrobial resistance in the health care environment underscores the importance of alternative treatment strategies. Biofilms are microbial communities that exhibit unique characteristics that must be considered when evaluating the potential of biofilm prevention or control strategies. Because biofilm-associated infections do not respond consistently to therapeutically achievable concentrations of many antimicrobial agents, treatments that are more effective against slowly growing biofilm cells or combination treatments that can penetrate the biofilm matrix may be more effective. Alternative strategies that do not incorporate antimicrobial drugs have also been investigated. These approaches have the potential to prevent or eradicate biofilms on indwelling intravascular catheters and prevent or resolve catheter-related infections.

Bacteriophage cocktail for the prevention of biofilm formation by Pseudomonas aeruginosa on catheters in an in vitro model system.

Microorganisms develop biofilms on indwelling medical devices and are associated with device-related infections, resulting in substantial morbidity and mortality. This study investigated the effect of pretreating hydrogel-coated catheters with Pseudomonas aeruginosa bacteriophages on biofilm formation by P. aeruginosa in an in vitro model. Hydrogel-coated catheters were exposed to a 10 log(10) PFU ml(-1) lysate of P. aeruginosa phage M4 for 2 h at 37 degrees C prior to bacterial inoculation. The mean viable biofilm count on untreated catheters was 6.87 log(10) CFU cm(-2) after 24 h. The pretreatment of catheters with phage reduced this value to 4.03 log(10) CFU cm(-2) (P < 0.001). Phage treatment immediately following bacterial inoculation also reduced biofilm viable counts (4.37 log(10) CFU cm(-2) reduction; P < 0.001). The regrowth of biofilms on phage-treated catheters occurred between 24 and 48 h, but supplemental treatment with phage at 24 h significantly reduced biofilm regrowth (P < 0.001). Biofilm isolates resistant to phage M4 were recovered from catheters pretreated with phage. The phage susceptibility profiles of these isolates were used to guide the development of a five-phage cocktail from a larger library of P. aeruginosa phages. The pretreatment of catheters with this cocktail reduced the 48-h mean biofilm cell density by 99.9% (from 7.13 to 4.13 log(10) CFU cm(-2); P < 0.001), but fewer biofilm isolates were resistant to these phages. These results suggest the potential of applying phages, especially phage cocktails, to the surfaces of indwelling medical devices for mitigating biofilm formation by clinically relevant bacteria.

Structural analysis of biofilm formation by rapidly and slowly growing nontuberculous mycobacteria.

Mycobacterium avium complex (MAC) and rapidly growing mycobacteria (RGM) such as M. abscessus, M. mucogenicum, M. chelonae, and M. fortuitum, implicated in health care-associated infections, are often isolated from potable water supplies as part of the microbial flora. To understand factors that influence growth in their environmental source, clinical RGM and slowly growing MAC isolates were grown as biofilm in a laboratory batch system. High and low nutrient levels were compared, as well as stainless steel and polycarbonate surfaces. Biofilm growth was measured after 72 h of incubation by enumeration of bacteria from disrupted biofilms and by direct quantitative image analysis of biofilm microcolony structure. RGM biofilm development was influenced more by nutrient level than by substrate material, though both affected biofilm growth for most of the isolates tested. Microcolony structure revealed that RGM develop several different biofilm structures under high-nutrient growth conditions, including pillars of various shapes (M. abscessus and M. fortuitum) and extensive cording (M. abscessus and M. chelonae). Although it is a slowly growing species in the laboratory, a clinical isolate of M. avium developed more culturable biofilm in potable water in 72 h than any of the 10 RGM examined. This indicates that M. avium is better adapted for growth in potable water systems than in laboratory incubation conditions and suggests some advantage that MAC has over RGM in low-nutrient environments.

Preventing biofilms of clinically relevant organisms using bacteriophage.

Biofilms might result in healthcare-associated infections and substantially impact healthcare delivery. Bacteriophage (phage) has been used to treat infectious diseases in humans and there is interest in phage to control biofilms. Phages propagate in their bacterial host and many phages produce depolymerases that hydrolyze biofilm extracellular polymers. Drawbacks of phage to consider include narrow host range, bacterial resistance to phage and phage-encoded virulence genes that can incorporate into the host bacterial genome. The immune system might inactivate phage, and impure phage preparations could contain endotoxin. Phage mixtures or engineered phages could provide effective strategies to overcome these obstacles. Lytic bacteriophages could become a new class of anti-biofilm agents.

RETRACTED: Structural analysis of biofilm formation by rapidly and slowly growing nontuberculous mycobacteria.

This article has been retracted.