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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.

Background: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.

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 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.

Liquid wastes (LW) disposed in hospital handwashing sinks may affect colonization of sink P-traps by carbapenemase-producing

Sink drains in healthcare facilities may provide an environment for antimicrobial-resistant microorganisms, including carbapenemase-producing Klebsiella pneumoniae (CPKP).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.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 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.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.

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.

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 P. aeruginosa and OPP-C, respectively. However, a greater percentage of ARO were recovered from PR sinks compared to HCP sinks (p 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.

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.

Significance Orthopedic implant infections require long-term antibiotic therapy and surgical debridement to successfully retain the implant; however, therapeutic failure can lead to implant removal. Here an injectable PEG-based hydrogel that adheres to exposed tissue and fracture surfaces is engineered to deliver the antimicrobial enzyme lysostaphin to infected, implant-fixed, mouse femoral fractures. Lysostaphin encapsulation within the hydrogel enhances enzyme stability while providing enhanced antibiofilm activity and serving as a controlled delivery platform. In a preclinical animal model of orthopedic-implant infection, we show that lysostaphin-delivering hydrogels outperform prophylactic antibiotic therapy and soluble lysostaphin, by eradicating infection while promoting bone repair. Importantly, lysostaphin-delivering hydrogels are effective against antibiotic-resistant infections. This lysostaphin delivery platform could be highly effective at treating and preventing implant infections., 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.

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.

An alarming rise in hospital outbreaks implicating hand-washing sinks has led to widespread acknowledgement that sinks are a major reservoir of antibiotic resistant pathogens in patient-care areas. An earlier study using a GFP-expressing Escherichia coli (GFP-E. coli) as a model organism demonstrated dispersal from drain biofilm 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 earlier theory that bacteria aerosolize from P-trap and disperse. Numbers of dispersed GFP-E. coli 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.IMPORTANCEAmong the possible environmental reservoirs in a patient care environment, sink drains are increasingly recognized as potential reservoir of multidrug resistant healthcare-associated pathogens to hospitalized patients. With increasing antimicrobial resistance limiting therapeutic options for patients, 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 colonizing sink drains.

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.

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 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.

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...

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.

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°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.

Candida biofilms are microbial communities, embedded in a polymeric matrix, growing attached to a surface, and are highly recalcitrant to antimicrobial therapy. These biofilms exhibit enhanced resistance against most antifungal agents except echinocandins and lipid formulations of amphotericin B. In this study, biofilm formation by different Candida species, particularly Candida albicans , C. tropicalis , and C. parapsilosis , was evaluated, and the effect of caspofungin (CAS) was assessed using a clinically relevant in vitro model system. CAS displayed in vitro activity against C. albicans and C. tropicalis cells within biofilms. Biofilm formation was evaluated after 48 h of antifungal drug exposure, and the effects of CAS on preformed Candida species biofilms were visualized using scanning electron microscopy (SEM). Several species-specific differences in the cellular morphologies associated with biofilms were observed. Our results confirmed the presence of paradoxical growth (PG) in C. albicans and C. tropicalis biofilms in the presence of high CAS concentrations. These findings were also confirmed by SEM analysis and were associated with the metabolic activity obtained by biofilm susceptibility testing. Importantly, these results suggest that the presence of atypical, enlarged, conical cells could be associated with PG and with tolerant cells in Candida species biofilm populations. The clinical implications of these findings are still unknown.

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.

Glycopeptides such as vancomycin are the treatment of choice for infections due to methicillin-resistant Staphylococcus aureus . This study describes the identification of high-level vancomycin-resistant S. aureus (VRSA) isolates in a polymicrobial biofilm within an indwelling nephrostomy tube in a patient in New York. S. aureus , Enterococcus faecalis , Enterococcus faecium , Micrococcus species, Morganella morganii , and Pseudomonas aeruginosa were isolated from the biofilm. For VRSA isolates, vancomycin MICs ranged from 32 to >128 μg/ml. VRSA isolates were also resistant to aminoglycosides, fluoroquinolones, macrolides, penicillin, and tetracycline but remained susceptible to chloramphenicol, linezolid, rifampin, and trimethoprim-sulfamethoxazole. The vanA gene was localized to a plasmid of ∼100 kb in VRSA and E. faecium isolates from the biofilm. Plasmid analysis revealed that the VRSA isolate acquired the 100-kb E. faecium plasmid, which was then maintained without integration into the MRSA plasmid. The tetracycline resistance genes tet (U) and tet (S), not previously detected in S. aureus isolates, were identified in the VRSA isolates. Additional resistance elements in the VRSA isolate included a multiresistance gene cluster, ermB-aadE-sat4-aphA-3 , msrA (macrolide efflux), and the bifunctional aminoglycoside resistance gene aac(6 ′ )-aph(2")-Ia . Multiple combinations of resistance genes among the various isolates of staphylococci and enterococci, including vanA , tet (S), and tet (U), illustrate the dynamic nature of gene acquisition and loss within and between bacterial species throughout the course of infection. The potential for interspecies transfer of antimicrobial resistance genes, including resistance to vancomycin, may be enhanced by the microenvironment of a biofilm.

Microbial biofilms have been grown in laboratories using a variety of different approaches. A laboratory biofilm reactor system, called the CDC biofilm reactor (CBR) system, has been devised for growing biofilms under moderate to high fluid shear stress. The reactor incorporates 24 removable biofilm growth surfaces (coupons) for sampling and analysing the biofilm. Following preliminary experiments to verify the utility of the CBR system for growing biofilms of several clinically relevant organisms, a standard operating procedure for growing a Pseudomonas aeruginosa biofilm was created. This paper presents the results of a rigorous, intra-laboratory, statistical evaluation of the repeatability and ruggedness of that procedure as well as the results of the experiments with clinically relevant organisms. For the statistical evaluations, the outcome of interest was the density (c.f.u. cm−2) of viable P. aeruginosa. Replicate experiments were conducted to assess the repeatability of the log density outcome. The mean P. aeruginosa log10 density was 7·1, independent of the coupon position within the reactor. The repeatability standard deviation of the log density based on one coupon per experiment was 0·59. Analysis of variance showed that the variability of the log density was 53 % attributable to within-experiment sources and 47 % attributable to between-experiments sources. The ruggedness evaluation applied response-surface design and regression analysis techniques, similar to those often used for sensitivity analyses in other fields of science and engineering. This approach provided a quantitative description of ruggedness; specifically, the amount the log density was altered by small adjustments to four key operational factors – time allowed for initial surface colonization, temperature, nutrient concentration, and fluid shear stress on the biofilm. The small size of the regression coefficient associated with each operational factor showed that the method was rugged; that is, relatively insensitive to minor perturbations of the four factors. These results demonstrate that the CBR system is a reliable experimental tool for growing a standard biofilm in the laboratory and that it can be adapted to study several different micro-organisms.

The survival of two strains of Yersinia pestis (avirulent A1122 and virulent Harbin) on the surfaces of four materials was investigated. Viability was evaluated with epifluorescence microscopy by using the metabolic stain cyanoditolyl tetrazolium chloride and plate counts. Small numbers of cells suspended in phosphate buffer survived 2 to 4 h after visible drying on stainless steel, polyethylene, or glass and beyond 48 h on paper. Cells suspended in brain heart infusion broth (BHI) persisted more than 72 h on stainless steel, polyethylene, and glass. Small numbers of cells suspended in BHI were still viable at 120 h on paper. These data suggest that Y. pestis maintains viability for extended periods (last measured at 5 days) under controlled conditions.

SUMMARYThough biofilms were first described by Antonie van Leeuwenhoek, the theory describing the biofilm process was not developed until 1978. We now understand that biofilms are universal, occurring in aquatic and industrial water systems as well as a large number of environments and medical devices relevant for public health. Using tools such as the scanning electron microscope and, more recently, the confocal laser scanning microscope, biofilm researchers now understand that biofilms are not unstructured, homogeneous deposits of cells and accumulated slime, but complex communities of surface-associated cells enclosed in a polymer matrix containing open water channels. Further studies have shown that the biofilm phenotype can be described in terms of the genes expressed by biofilm-associated cells. Microorganisms growing in a biofilm are highly resistant to antimicrobial agents by one or more mechanisms. Biofilm-associated microorganisms have been shown to be associated with several human diseases, such as native valve endocarditis and cystic fibrosis, and to colonize a wide variety of medical devices. Though epidemiologic evidence points to biofilms as a source of several infectious diseases, the exact mechanisms by which biofilm-associated microorganisms elicit disease are poorly understood. Detachment of cells or cell aggregates, production of endotoxin, increased resistance to the host immune system, and provision of a niche for the generation of resistant organisms are all biofilm processes which could initiate the disease process. Effective strategies to prevent or control biofilms on medical devices must take into consideration the unique and tenacious nature of biofilms. Current intervention strategies are designed to prevent initial device colonization, minimize microbial cell attachment to the device, penetrate the biofilm matrix and kill the associated cells, or remove the device from the patient. In the future, treatments may be based on inhibition of genes involved in cell attachment and biofilm formation.

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.

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.

This chapter aims at determining the susceptibility of biofilm-associated Legionella pneumophila to free chlorine and monochloramine. A biofilm reactor was developed to grow biofilms containing L. pneumophila on 24 replicate stainless-steel surfaces in potable water. The ability of this reactor to produce reproducible biofilms was validated by the fact that the standard deviations of the base biofilm densities on stainless-steel coupons (n = 3) ranged from 0.06 to 0.18 log CFU per coupon. When Legionella containing biofilms were exposed to the same dosages of monochloramine for identical contact periods, the treatments were significantly more effective. In summary, the authors have shown that biofilm-associated L. pneumophila are significantly less susceptible to chlorine than are planktonic L. pneumophila, while susceptibility of planktonic and biofilm-associated L. pneumophila to monochloramine are similar. When monochloramine and free chlorine were compared under identical conditions, monochloramine was significantly more effective, indicating that monochloramine may be an effective disinfectant for the inactivation of L. pneumophila within potable water distribution systems. Further research using open system biofilm reactors and model distribution systems is needed to determine the utility of monochloramine as a disinfectant against biofilm-associated L. pneumophila.

Microorganisms universally attach to surfaces and produce extracellular polysaccharides, resulting in the formation of a biofilm. Biofilms pose a serious problem for public health because of the increased resistance of biofilm-associated organisms to antimicrobial agents and the potential for these organisms to cause infections in patients with indwelling medical devices. An appreciation of the role of biofilms in infection should enhance the clinical decision-making process.

Biofilms are formed by a spectrum of microorganisms, including pathogens, and provide a means for these organisms to protect themselves against antimicrobial agents. Several mechanisms have been proposed to explain this phenomenon of resistance within biofilms, including delayed penetration of the antimicrobial into the biofilm extracellular matrix, slowing of growth rate of organisms within the biofilm, or other physiologic changes brought about by interaction of the organisms with a surface. The practical implications of biofilm formation are that alternative control strategies must be devised both for testing the susceptibility of the organisms within the biofilm and treating the established biofilm to alter its structure. A number of testing protocols have been developed. Effective treatment strategies will incorporate antimicrobials or other agents that have been demonstrated to penetrate and kill biofilm organisms, or treatments that disrupt or target specific components of the biofilm matrix. A better understanding of the role of biofilms in infection and how in vivo biofilms respond to selected treatments requires more study.

Seven species of bacterial select agents were tested for susceptibility to free available chlorine (FAC). Under test conditions, the FAC routinely maintained in potable water would be sufficient to reduce six species by 2 orders of magnitude within 10 min. Water contaminated with spores of Bacillus anthracis spores would require further treatment.

UNLABELLED 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 3·15% 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 18 h 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, 0·31; n = 28). IPA disinfection provided an approximate 3 log10 CFU reduction; CGI and PI provided 3-4 log10 reductions. PI and CGI were more effective than IPA (P

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.

We hypothesized that pretreating urinary catheters with benign Escherichia coli HU2117 plus an antipseudomonal bacteriophage (ΦE2005-A) would prevent Pseudomonas aeruginosa biofilm formation on catheters--a pivotal event in the pathogenesis of catheter-associated urinary tract infection (CAUTI).Silicone catheter segments were exposed to one of four pretreatments (sterile media; E. coli alone; phage alone; E. coli plus phage), inoculated with P. aeruginosa and then incubated up to 72 h in human urine before rinsing and sonicating to recover adherent bacteria. Pseudomonas aeruginosa adherence to catheters was almost 4 log(10) units lower when pretreated with E. coli plus phage compared to no pretreatment (P0.001) in 24-h experiments and more than 3 log(10) units lower in 72-h experiments (P0.05). Neither E. coli nor phage alone generated significant decreases.The combination of phages with a pre-established biofilm of E. coli HU2117 was synergistic in preventing catheter colonization by P. aeruginosa.We describe a synergistic protection against colonization of urinary catheters by a common uropathogen. Escherichia coli-coated catheters are in clinical trials; adding phage may offer additional benefit.

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.

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.

This article has been retracted.

Use of indwelling catheters is often compromised as a result of biofilm formation. This study investigated if hydrogel-coated catheters pretreated with a coagulase-negative bacteriophage would reduceStaphylococcus epidermidisbiofilm formation. Biofilms were developed on hydrogel-coated silicone catheters installed in a modified drip flow reactor. Catheter segments were pretreated with the lyticS. epidermidisbacteriophage 456 by exposing the catheter lumen to a 10-log-PFU/ml culture of the bacteriophage for 1 h at 37°C prior to biofilm formation. The untreated mean biofilm cell count was 7.01 ± 0.47 log CFU/cm2of catheter. Bacteriophage treatment with and without supplemental divalent cations resulted in log-CFU/cm2reductions of 4.47 (P< 0.0001) and 2.34 (P= 0.001), respectively. Divalent cation supplementation without bacteriophage treatment provided a 0.67-log-CFU/cm2reduction (P= 0.053). Treatment of hydrogel-coated silicone catheters with anS. epidermidisbacteriophage in an in vitro model system significantly reduced viable biofilm formation byS. epidermidisover a 24-h exposure period, suggesting the potential of bacteriophage for mitigating biofilm formation on indwelling catheters and reducing the incidence of catheter-related infections.

Indwelling prosthetic joints may become colonized by microbial biofilms, although the biofilm structure, composition of the microbial community, and physiologic activity of the organisms in these devices are not well understood. New approaches that rely on the use of fluorescent stain technology can be used to characterize the structure and community composition in a way that earlier methods, which relied on culturing or scanning electron microscopy, could not. Model systems incorporating parameters relevant for indwelling prosthetic joints also can be designed to evaluate the efficacy of treatments for preventing or eradicating biofilms from these devices. Effectively treating microbial biofilms on indwelling medical devices such as prosthetic joints is a challenging proposition. A clearer understanding of the process in vivo and a defined approach for evaluating treatment strategies provide the best hope for success.

Background:Central venous catheter (CVC)-related bloodstream infections (BSIs) are known to increase rates of morbidity and mortality in both inpatients and outpatients, including hematology-oncology patients and those undergoing hemodialysis or home infusion therapy. Biofilm-associated organisms on the lumens of these catheters have reduced susceptibility to antimicrobial chemotherapy. This study tested the efficacy of tetrasodium EDTA as a catheter lock solution on biofilms of several clinically relevant microorganisms.Methods:Biofilms ofStaphylococcus epidermidis, methicillin-resistantS. aureus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, andCandida albicanswere grown to levels of approximately 1 × 105colony-forming units (CFU)/cm−1on CVC segments in a model system, then subjected to the tetrasodium EDTA lock treatment.Results:Comparisons of biofilms before and after exposure to the 40-mg/mL−1tetrasodium EDTA lock for 21 hours showed that the biofilm viable cell counts of all organisms tested were significantly reduced (P< .05) after exposure to the treatment.Conclusion:Antimicrobial lock treatment using 40 mg/mL−1of tetrasodium EDTA for at least 21 hours could significantly reduce or potentially eradicate CVC-associated bio-films of clinically relevant microorganisms (Infect Control Hosp Epidemiol2005;26:515-519).

Legionella pneumophila will infect biofilm-associated protozoa, and in this way might be protected from disinfectants in potable water systems. A base biofilm containing Pseudomonas aeruginosa, Klebsiella pneumoniae, and Flavobacterium spp. was grown on steel coupons in potable water prior to the addition of L. pneumophila and the protozoan H. vermiformis. After 7 d, coupons were removed and treated with 0.5 mgl(-1) free residual chlorine (FRC) or 0.5 mgl(-1) monochloramine (MCA) for 15, 60, or 180 min or 24 h. In a second experiment, only L. pneumophila and the base biofilm organisms were present but with an identical treatment protocol. Treatment of L. pneumophila for 180 min in a system without H. vermiformis resulted in log reductions of 2.07 and 2.11 for FRC and MCA, respectively. When H. vermiformis was present, however, the treatment resulted in log reductions of 0.67 and 0.81 for FRC and MCA, respectively. A similar pattern was observed for 15 and 60 min contact times. These results indicate that L. pneumophila was less susceptible to MCA or FRC when associated with biofilm-associated H. vermiformis in a model potable water biofilm.

Streptococcus pneumoniae forms biofilms, but little is known about its extracellular polymeric substances (EPS) or the kinetics of biofilm formation. A system was developed to enable the simultaneous measurement of cells and the EPS of biofilm-associated S. pneumoniae in situ over time. A biofilm reactor containing germanium coupons was interfaced to an attenuated total reflectance (ATR) germanium cell of a Fourier transform infrared (FTIR) laser spectrometer. Biofilm-associated cells were recovered from the coupons and quantified by total and viable cell count methods. ATR-FTIR spectroscopy of biofilms formed on the germanium internal reflection element (IRE) of the ATR cell provided a continuous spectrum of biofilm protein and polysaccharide (a measure of the EPS). Staining of the biofilms on the IRE surface with specific fluorescent probes provided confirmatory evidence for the biofilm structure and the presence of biofilm polysaccharides. Biofilm protein and polysaccharides were detected within hours after inoculation and continued to increase for the next 141 h. The polysaccharide band increased at a substantially higher rate than did the protein band, demonstrating increasing coverage of the IRE surface with biofilm polysaccharides. The biofilm total cell counts on germanium coupons stabilized after 21 h, at approximately 10 5 cells per cm 2 , while viable counts decreased as the biofilm aged. This system is unique in its ability to detect and quantify biofilm-associated cells and EPS of S. pneumoniae over time by using multiple, corroborative techniques. This approach could prove useful for the study of biofilm processes of this or other microorganisms of clinical or industrial relevance.

Background. Community-onset methicillin-resistant Staphylococcus aureus (CO-MRSA) reports are increasing, and infections often involve soft tissue. During a CO-MRSA skin infection outbreak in Alaska, we assessed risk factors for disease and whether a virulence factor, Panton-Valentine leukocidin (PVL), could account for the high rates of MRSA skin infection in this region. Methods. We conducted S. aureus surveillance in the outbreak region and a case-control study in 1 community, comparing 34 case patients with MRSA skin infection with 94 control subjects. An assessment of traditional saunas was performed. S. aureus isolates from regional surveillance were screened for PVL genes by use of polymerase chain reaction, and isolate relatedness was determined by use of pulsed-field gel electrophoresis (PFGE). Results. Case patients received more antibiotic courses during the 12 months before the outbreak than did control subjects (median, 4 vs. 2 courses; P = .01) and were more likely to use MRSA-colonized saunas than were control subjects (44% vs. 13%; age-adjusted odds ratio, 4.6; 95% confidence interval, 1.7-12). The PVL genes were present in 110 (97%) of 113 MRSA isolates, compared with 0 of 81 methicillin-susceptible S. aureus isolates (P

Legionellae can infect and multiply intracellularly in both human phagocytic cells and protozoa. Growth of legionellae in the absence of protozoa has been documented only on complex laboratory media. The hypothesis upon which this study was based was that biofilm matrices, known to provide a habitat and a gradient of nutrients, might allow the survival and multiplication of legionellae outside a host cell. This study determined whether Legionella pneumophila can colonize and grow in biofilms with and without an association with Hartmannella vermiformis. The laboratory model used a rotating disc reactor at a retention time of 6·7 h to grow biofilms on stainless steel coupons. The biofilm was composed of Pseudomonas aeruginosa, Klebsiella pneumoniae and a Flavobacterium sp. The levels of L. pneumophila cells present in the biofilm were monitored for 15 d, with and without the presence of H. vermiformis, and it was found that, although unable to replicate in the absence of H. vermiformis, L. pneumophila was able to persist.

Human blood components have been shown to enhance biofilm formation by gram-positive bacteria. We investigated the effect of human blood on biofilm formation on the inner lumen of needleless central venous catheter connectors by several gram-negative bacteria, specifically Enterobacter cloacae, Pseudomonas aeruginosa , and Pantoea agglomerans . Results suggest that a conditioning film of blood components promotes biofilm formation by these organisms in an in vitro system.