Lipases have been implicated to be of importance in the life cycle development, virulence, and transmission of a variety of parasitic organisms. Potential functions include the acquisition of host resources for energy metabolism and as simple building blocks for the synthesis of complex parasite lipids important for membrane remodeling and structural purposes. Using a molecular approach, we identified and characterized the structure of an LdLip3-lipase gene from the primitive trypanosomatid pathogen of humans, Leishmania donovani. The LdLip3 encodes a ~ 33 kDa protein, with a well-conserved sub-strate-binding and catalytic domains characteristic of members of the serine lipase-protein family. Further, we showed that LdLip3 mRNA is constitutively expressed by both the insect vector (i.e., promastigote) and mammalian (i.e., amastigote) life cycle developmental forms of this protozoan parasite. Moreover, a homologous episomal expression system was used to express an HA epitope-tagged LdLip3 chimeric construct (LdLip3∷HA) in these parasites. Expression of the LdLip3 chimera was verified in these transfectants by Western blots and indirect immunofluorescence analyses. Results of coupled immuno-affinity purification and enzyme activity experiments demonstrated that the LdLip3∷HA chimeric protein was secreted/released by transfected L. donovani parasites and that it possessed functional lipase enzyme activity. Taken together these observations suggest that this novel secretory lipase might play essential role(s) in the survival, growth, and development of this important group of human pathogens.

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.

Handwashing sinks and their associated premise plumbing are an ideal environment for pathogenharboring 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 culturedependent 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 log 10 cfU/cm 2 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 log 10 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. From 2014 to 2017, 21.6% of Centers for Disease Control and Prevention (CDC), Division of Healthcare Quality Promotion consultations involved water-related healthcare-associated infections (HAI). Among these investigations, 29.9%, 18.7%, and 10.4% were attributed to nontuberculous mycobacteria (NTM), Pseudomonas spp., and members of the Enterobacteriaceae family, respectively 1. Plumbing fixtures, such as sinks, showers and toilets, with which patients, visitors, and healthcare personnel (HCP) interact are known reservoirs for pathogenic and opportunistic organisms 2-4. Previous work has documented the role that these plumbing fixtures have played in outbreaks, many of which have involved gram-negative rods, such as Pseudomonas spp. and carbapenem-resistant or extended-spectrum β-lactamase producing (ESBL) Enterobacteriaceae 5-9 .

20An alarming rise in hospital outbreaks implicating hand-washing sinks has led to widespread 21 acknowledgement that sinks are a major reservoir of antibiotic resistant pathogens in patient-care

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 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. 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.