Drain Biofilm Research Articles

Handwashing basins and healthcare associated infections: Bacterial diversity in biofilms on faucets and drains

BackgroundIncreasingly, hospital handwashing basins have been identified as a source of healthcare-associated infections. Biofilms formed on the faucet and drains of handbasins can potentially harbour pathogenic microbes and promote the dissemination of antimicrobial resistance. However, little is known about the diversity of these biofilm communities and the routes of contamination. AimThe aim of this paper was to use 16S rRNA gene amplicon sequencing to investigate the diversity of prokaryote communities present in faucet and drain biofilm samples taken from hospital and residential handbasins. FindingsThe biofilm prokaryotes communities were diverse, with high abundances of potentially corrosive, biofilm forming and pathogenic genera, including those that are not typically waterborne. The β-diversity showed statistically significant differences in the variation of bacterial communities on the basis on building type (hospital vs residential p = 0.0415). However, there was no statistically significant clustering based on sampling site (faucet vs drain p = 0.46). When examining the β-diversity between individual factors, there was a significant difference between drain biofilms of different buildings (hospital drain vs residential drain p = 0.0338). ConclusionThis study demonstrated that biofilms from hospital and residential handbasins contain complex and diverse microbial communities that differ significantly by building type. It also showed biofilms formed on the faucet and drain of a hospital's handbasins were not significantly different. Future research is needed to understand the potential mechanisms of transfer between drains and faucets of hospital handbasins. This information will inform improved infection control guidelines to control this underrecognized source of infections.

Drain biofilm microbiota inside a cheese processing facility: Comparison of Listeria positive and Listeria negative drains

Listeria monocytogenes was identified as one of the most dangerous food-borne pathogens of the 21st century, not only due to its high mortality rate but also because of its ability to establish itself as a part of drain biofilms and persist there over long periods of time. The insides of twelve drains in a cheese processing facility were swabbed over a period of five months. Bacterial DNA was isolated and quantified to ascertain the presence and abundance of the biofilm. Presence of Listeria spp. prs gene was found in four of the twelve drains, with one drain testing positive for the prs gene presence on three different occasions. 16S rRNA amplicon sequencing was conducted on the isolated DNA from two Listeria positive and two Listeria negative drains. The microbial community composition was evaluated. Most notably genera Streptococci and unclassified Lactobacillales were significantly different in abundance in the Listeria negative drains. Genera Flavobacterium and unclassified Xanthomonadaceae were found to be significantly more different in abundance in the LisTeria positive drains. The latter two genera were not previously associated with Listeria spp. or reported to be widespread in food processing environments (FPE), however, they help to illustrate the higher diversity that was seen in Listeria positive drains. This study suggests that even though the alpha diversity is not sigN ificantly different, there are differences between the drain biofilm composition between Listeria positive and Listeria negative drains, which can be further researched to ascertain how these organisms affect the presence of Listeria spp. inside drain biofilms.

A novel method using a differential staining fluorescence microscopy (DSFM) to track the location of enteric pathogens within mixed-species biofilms

This study developed a new tool, differential staining fluorescence microscopy (DSFM), to measure the biovolume and track the location of enteric pathogens in mixed-species biofilms which can pose a risk to food safety in beef processing facilities. DSFM was employed to examine the impact of pathogenic bacteria, Escherichia coli O157:H7 and three different Salmonella enterica strains on mixed-species biofilms of beef processing facilities. Fourteen floor drain biofilm samples from three beef processing plants were incubated with overnight BacLight stained enteric pathogens at 7 °C for 5 days on stainless steel surface then counter-stained with FM-1-43 biofilm stain and analyzed using fluorescence microscopy. Notable variations in biovolume of biofilms were observed across the fourteen samples. The introduction of E. coli O157:H7 and S. enterica strains resulted in diverse alterations of biofilm biovolume, suggesting distinct impacts on mixed-species biofilms by different enteric pathogens which were revealed to be located in the upper layer of the mixed-species biofilms. Pathogen strain growth curve comparisons and verification of BacLight Red Stain staining effectiveness were validated. The findings of this study show that the DSFM method is a promising approach to studying the location of enteric pathogens within mixed-species biofilms recovered from processing facilities. Understanding how foodborne pathogens interact with biofilms will allow for improved targeted antimicrobial interventions.

Shotgun-metagenomics reveals a highly diverse and communal microbial network present in the drains of three beef-processing plants.

Multi-species biofilms pose a problem in various environments, especially food-processing environments. The diversity of microorganisms in these biofilms plays a critical role in their integrity and protection against external biotic and abiotic factors. Compared to single-species biofilms, mixed-species biofilms are more resistant to various stresses, including antimicrobials like sanitizers. Therefore, understanding the microbiome composition and diversity in biofilms and their metabolic potential is a priority when developing intervention techniques to combat foodborne pathogens in food processing environments. This study aimed to describe and compare the microbiome profile of 75 drain biofilm samples obtained from five different locations (Hotscale, Hotbox, Cooler, Processing, & Grind room) of three beef-processing plants (Plant A, B & C) taken over two timepoints 2017-18 (T1) and 2021 (T2) by shotgun sequencing. Core microbiome analysis found Pseudomonas, Psychrobacter, and Acinetobacter to be the top three prevalent genera among the plants and locations. Alpha diversity analysis demonstrated a high diversity of microbiome present in all the plants and locations across the time points. Functional analysis showed the high metabolic potential of the microbial community with abundance of genes in metabolism, cell-adhesion, motility, and quorum sensing. Moreover, Quaternary Ammonium Compound (QAC) resistance genes were also observed, this is significant as QAC sanitizers are commonly used in many food processing facilities. Multi-functional genes such as transposases, polymerases, permeases, flagellar proteins, and Mobile Genetic Elements (MGEs) were found suggesting these are dynamic microbial communities that work together to protect themselves against environmental stresses through multiple defense mechanisms. This study provides a framework for understanding the collective microbial network spanning a beef processing system. The results can be used to develop intervention strategies to best control these highly communicative microbial networks.

Cluster of Carbapenemase-Producing Carbapenem-Resistant Pseudomonas aeruginosa Among Patients in an Adult Intensive Care Unit - Idaho, 2021-2022.

Treatment of carbapenemase-producing carbapenem-resistant Pseudomonas aeruginosa (CP-CRPA) infections is challenging because of antibiotic resistance. CP-CRPA infections are highly transmissible in health care settings because they can spread from person to person and from environmental sources such as sink drains and toilets. During September 2021-January 2022, an Idaho hospital (hospital A) isolated CP-CRPA from sputum of two patients who stayed in the same intensive care unit (ICU) room (room X), 4 months apart. Both isolates had active-on-imipenem metallo-beta-lactamase (IMP) carbapenemase gene type 84 (blaIMP-84) and were characterized as multilocus sequence type 235 (ST235). A health care-associated infections team from the Idaho Division of Public Health visited hospital A during March 21-22, 2022, to discuss the cluster investigation with hospital A staff members and to collect environmental samples. CP-CRPA ST235 with blaIMP-84 was isolated from swab samples of one sink in room X, suggesting it was the likely environmental source of transmission. Recommended prevention and control measures included application of drain biofilm disinfectant, screening of future patients who stay in room X (e.g., the next 10 occupants) upon reopening, and continuing submission of carbapenem-resistant P. aeruginosa (CRPA) isolates to public health laboratories. Repeat environmental sampling did not detect any CRPA. As of December 2022, no additional CP-CRPA isolates had been reported by hospital A. Collaboration between health care facilities and public health agencies, including testing of CRPA isolates for carbapenemase genes and implementation of sink hygiene interventions, was critical in the identification of and response to this CP-CRPA cluster in a health care setting.

Environmental sponge sampling: We are only scraping the surface

Drains located in retail deli departments are known harborage points for pathogenic and spoilage microorganisms. Previous studies have used sequencing approaches to study the microbial consortia found in drains and biofilms in various food processing environments. None of these studies, however, have focused on retail. The primary objective of this study was to characterize the microbiome of retail deli drain sponge samples and drain biomass samples through shotgun metagenomic and 16S rRNA amplicon sequencing approaches. Sponge samples were collected from the surfaces of retail deli drain covers and biomass samples were harvested from inside deli drain trenches from fourteen retail delis in three major US cities. All samples were subjected to 16S rRNA gene sequencing, and shotgun metagenomic sequencing was performed on a subset of samples. In the majority of samples, Pseudomonas was the most abundant; the remaining genera differed by sample, city, and sequencing method. Common sanitizer tolerance genes (qacE∆1 and qacE) were observed in all biomass samples which were subjected to shotgun metagenomic sequencing. While mean alpha diversity indices were similar between biomass and sponge samples evaluated via 16S RNA amplicon sequencing, a greater range of diversity was observed between biomass scrapes than sponge samples. Beta diversity differed significantly (P < 0.01) between biomass scrapes and sponges. Additionally, some taxa, such as the opportunistic pathogen Elizabethkingia, were virtually absent from sponge samples, but present at low abundance in biomass scrape samples. While studies traditionally have focused on detection of specific microorganisms of interest, this study indicates that drain trenches may harbor microorganisms that are not observed through traditional methods of environmental sampling (i.e. surface sponge samples). Collection of drain biofilms or biomass scrapes and use of culture-independent approaches would provide a more complete picture of the deli drain microbiome.

Characterization of Salmonella Strains and Environmental Microorganisms Isolated From a Meat Plant With Salmonella Recurrence

Salmonella entericais a leading cause of foodborne illness in the United States. In this study, 3S. enterica strains (serovars Cerro, Montevideo, and Typhimurium) were isolated from a beef processing plant with a history of Salmonella recurrence. Floor drains of the hotbox area and storage cooler of the same plant were sampled for environmental microorganisms. Biofilm formation on stainless steel (SS) by the microorganisms recovered from the drains and surface colonization by the isolated S. enterica strains were assessed. Pathogen survival and community compositions after sanitization (quaternary ammonium compound [QAC]) were examined. Alone the Cerro strain exhibited greater (P&amp;lt;0.05) surface attachment (5.4 log10CFU/SS chip) than the Montevideo or Typhimurium strains (4.6 and 4.8 log10CFU/SS chip, respectively). The Typhimurium strain was the most QAC tolerant (0.3 log reduction), whereas the Cerro and Montevideo strains exhibited similar sensitivities (1.6 log and 1.1 log reduction, respectively). The environmental microorganisms from the drain samples all developed biofilms ranging from 5.7 to 6.0 log10CFU/SS chip. All S. enterica strains were efficiently integrated into the drain biofilms where the Montevideo and Typhimurium strains ranged from 3.8 to 4.7 log10CFU/chip and the Cerro strain 4.0 to 5.8 log10CFU/chip. Enhanced QAC tolerance was only exhibited by the Montevideo strain when integrated into the drain biofilms. Metagenomic analysis of the drain biofilms showed that the most abundant genus was Pseudomonas (57.7%), followed by Brochothrix (28.6%), Serratia (7.2%), Lactococcus (4.2%), and Carnobacterium (1.1%), with all varying significantly among the different biofilm samples and their response to QAC treatment. Thus,various factors underlie the survival advantages of the S. enterica strains, allowing them to tolerate stress as well as out-compete and coexist with environmental companion bacteria for protection, which might lead to pathogen prevalence and recurrent product contamination.

Is your ice machine really clean? Uncovering the presence of opportunistic pathogens in hospital ice machines

Background: Ice is used in healthcare facilities for medical purposes and consumption by the medical staff and the patients, but some studies have revealed significant microbial contamination of ice machines leading to nosocomial outbreaks or pseudooutbreaks and infections by opportunistic pathogens, including the fungi Candida, the bacteria Pseudomonas aeruginosa, and nontuberculous mycobacteria (NTM). Although ice machines are complex devices that are prone to contamination, very little is known about their potential as vectors of infections for populations at risk in hospitals. Only few studies document efficient maintenance regimes, specifically cleaning procedures and microbial indicators that would ensure their safe use. Method: In this prospective study, combined samples of water and ice, and drain biofilm samples were collected from 36 ice and cold-water distribution machines of a recently built hospital, for a total of 72 samples. Physicochemical parameters (total and free chlorine, temperature, etc) were measured in water, and several opportunistic pathogens (ie, Candida spp, P. aeruginosa, NTM) and biological indicators (ie, heterotrophic plate counts (HPCs), total and viable bacteria and enterococci) were monitored in water and ice and biofilm. Culture methods were used for HPCs, Candida spp, P. aeruginosa, and enterococci, and total and viable bacterial populations were estimated using flow cytometry. NTM were monitored by quantitative polymerase chain reaction (qPCR). Results: We observed clear differences between the machines in terms of biological contamination, with frequent detection of NTM and presumed Candida. Thus, NTM were detected in the 36 samples of ice and water with concentrations from 0.5 to 2×104 gene copies/mL. Among the several species of fungi detected in the ice machines, some were identified as C. parapsilosis and C. guilliermondii, which are organisms of concern in healthcare facilities. Factors affecting the level of contamination in ice machines include the location of the machines and water quality (ie, temperature and chlorine residual concentration). Depending on the location in the building and the model of ice machine sampled, the biological indicators measurements indicated more or less significant contamination. No link was established between environmental strains recovered from the machines and clinical infections. Conclusions: Monitoring results showed that ice machines, while subject to few regulations and controls, can be reservoirs of unsuspected opportunistic pathogens that could lead to nosocomial infections of vulnerable patients. Cleaning procedures should be based on the disinfection of resistant opportunistic pathogens, such as Candida and NTM, and the use of general indicators, such as HPCs, should be questioned.Funding: NoneDisclosures: None

It's a trap! The development of a versatile drain biofilm model and its susceptibility to disinfection

Pathogens in drain biofilms pose a significant risk for hospital-acquired infection. However, the evidence of product effectiveness in controlling drain biofilm and pathogen dissemination are scarce. A novel in-vitro biofilm model was developed to address the need for a robust, reproduceable and simple testing methodology for disinfection efficacy against a complex drain biofilm. Identical complex drain biofilms were established simultaneously over 8 days, mimicking a sink trap. Reproducibility of their composition was confirmed by next-generation sequencing. The efficacy of sodium hypochlorite 1000 ppm (NaOCl), sodium dichloroisocyanurate 1000 ppm (NaDCC), non-ionic surfactant (NIS) and peracetic acid 4000 ppm (PAA) was explored, simulating normal sink usage conditions. Bacterial viability and recovery following a series of 15-min treatments were measured in three distinct parts of the drain. The drain biofilm consisted of 119 mixed species of Gram-positive and -negative bacteria. NaOCl produced a >4 log10 reduction in viability in the drain front section alone, while PAA achieved a >4 log10 reduction in viability in all of the drain sections following three 15-min doses and prevented biofilm regrowth for >4 days. NIS and NaDCC failed to control the biofilm in any drain sections. Drains are one source of microbial pathogens in healthcare settings. Microbial biofilms are notoriously difficult to eradicate with conventional chemical biocidal products. The development of this reproducible in-vitro drain biofilm model enabled understanding of the impact of biocidal products on biofilm spatial composition and viability in different parts of the drain.

Wastewater drains: epidemiology and interventions in 23 carbapenem-resistant organism outbreaks.

For many years, patient-area wastewater drains (ie, sink and shower drains) have been considered a potential source of bacterial pathogens that can be transmitted to patients. Recently, evolving genomic epidemiology tools combined with new insights into the ecology of wastewater drain (WWD) biofilm have provided new perspectives on the clinical relevance and hospital-associated infection (HAI) transmission risks related to these fixtures. To further clarify the clinical relevance of WWD-associated pathogen transmission, reports of outbreaks attributed to WWDs were selected for review that (1) investigated the outbreak epidemiology of WWD-associated transmission of bacterial pathogens, (2) utilized advanced microbiologic methods to establish clonality of outbreak pathogens and/or resistance genes, or (3) described interventions implemented to mitigate transmission of the outbreak pathogens from WWDs. These reports were collated, compared, and analyzed, and the results are presented here.

Investigation using whole genome sequencing of a prolonged restaurant outbreak of Salmonella Typhimurium linked to the building drainage system, England, February 2015 to March 2016.

Following notification of a Salmonella enterica serovar Typhimurium gastroenteritis outbreak, we identified 82 cases linked to a restaurant with symptom onset from 12 February 2015 to 8 March 2016. Seventy-two cases had an isolate matching the nationally unique whole genome sequencing profile (single nucleotide polymorphism (SNP) address: 1.1.1.124.395.395). Interviews established exposure to the restaurant and subsequent case–control analysis identified an association with eating carvery buffet food (adjusted odds ratios (AOR): 20.9; 95% confidence interval (CI): 2.2 – ∞). Environmental inspections, food/water testing, and a food trace-back investigation were inconclusive. Repeated cycles of cleaning were undertaken, including hydrogen peroxide fogging, however, transmission continued. After 7 months of investigation, environmental swabbing identified 106 isolates from kitchen surfaces and restaurant drains matching the outbreak profile. We found structural faults with the drainage system and hypothesised that a reservoir of bacteria in drain biofilm and underfloor flooded areas may have sustained this outbreak. Ineffective drain water-traps (U-bends) may have also contributed by allowing transmission of contaminated aerosols into the kitchen environment. These findings suggest that routine swabbing of sink drain points and inspection of drainage systems should be considered in future outbreak scenarios.

Formulation of Biocides Increases Antimicrobial Potency and Mitigates the Enrichment of Nonsusceptible Bacteria in Multispecies Biofilms.

The current investigation aimed to generate data to inform the development of risk assessments of biocide usage. Stabilized domestic drain biofilm microcosms were exposed daily over 6 months to increasing concentrations (0.01% to 1%) of the biocide benzalkonium chloride (BAC) in a simple aqueous solution (BAC-s) or in a complex formulation (BAC-f) representative of a domestic cleaning agent. Biofilms were analyzed by culture, differentiating by bacterial functional group and by BAC or antibiotic susceptibility. Bacterial isolates were identified by 16S rRNA sequencing, and changes in biofilm composition were assessed by high-throughput sequencing. Exposure to BAC-f resulted in significantly larger reductions in levels of viable bacteria than exposure to BAC-s, while bacterial diversity greatly decreased during exposure to both BAC-s and BAC-f, as evidenced by sequencing and viable counts. Increases in the abundance of bacteria exhibiting reduced antibiotic or BAC susceptibility following exposure to BAC at 0.1% were significantly greater for BAC-s than BAC-f. Bacteria with reduced BAC and antibiotic susceptibility were generally suppressed by higher BAC concentrations, and formulation significantly enhanced this effect. Significant decreases in the antimicrobial susceptibility of bacteria isolated from the systems before and after long-term BAC exposure were not detected. In summary, dose-dependent suppression of bacterial viability by BAC was enhanced by formulation. Biocide exposure decreased bacterial diversity and transiently enriched populations of organisms with lower antimicrobial susceptibility, and the effects were subsequently suppressed by exposure to 1% BAC-f, the concentration most closely reflecting deployment in formulated products.IMPORTANCE Assessment of the risks of biocide use has been based mainly on the exposure of axenic cultures of bacteria to biocides in simple aqueous solutions. The current investigation aimed to assess the effects of formulation on the outcome of biocide exposure in multispecies biofilms. Formulation of the cationic biocide BAC significantly increased antimicrobial potency. Bacteria with lower antimicrobial susceptibility whose populations were enriched after low-level biocide exposure were more effectively suppressed by the biocide at in-use concentrations (1% [wt/vol]) in a formulation than in a simple aqueous solution. These observations underline the importance of simulating normal deployment conditions in considering the risks and benefits of biocide use.

A study on microbiological contamination on air quality in hospitals in Egypt

This study aims to evaluate airborne environmental bacteria (environmental origin), mesophilic bacteria (human related bacteria) and fungi at governmental (GH) and private (PH) hospitals, in addition to assess sink drain biofilms and dust accumulated on air conditioning filters and floor surfaces to determine if they constitute potential sources of airborne microbial biodiversity. Samples were collected from intensive care unit (ICU), operating theatre (OT), admission department (AD) and outdoor for comparison. Airborne bacteria and fungi were collected using an Andersen two-stage impactor sampler which divides particles into fine (≤8 µm) and coarse (≥8 µm) size ranges. Airborne environmental bacteria, mesophilic bacteria and fungi concentrations, were respectively in the range of 0.0–2638; 0.0–1654.9 and 11.7–566.5 CFU/m3 indoors and 155–32436; 11.7–5554 and 35–664 CFU/m3 outdoors. Bacillus atrophaeus was commonly isolated in all locations. However, Alloiococcus otitis was only found at the GH and Bacillus licheniformis at the PH. Aspergillus and Penicillium were the common fungal types in the air state and dust. Dust was accumulated on air conditioning filters and floor surfaces and these would constitute important sources of airborne bacteria and fungi inside these hospitals.

Metagenomic discovery of novel enzymes and biosurfactants in a slaughterhouse biofilm microbial community.

DNA derived from environmental samples is a rich source of novel bioactive molecules. The choice of the habitat to be sampled predefines the properties of the biomolecules to be discovered due to the physiological adaptation of the microbial community to the prevailing environmental conditions. We have constructed a metagenomic library in Escherichia coli DH10b with environmental DNA (eDNA) isolated from the microbial community of a slaughterhouse drain biofilm consisting mainly of species from the family Flavobacteriaceae. By functional screening of this library we have identified several lipases, proteases and two clones (SA343 and SA354) with biosurfactant and hemolytic activities. Sequence analysis of the respective eDNA fragments and subsequent structure homology modelling identified genes encoding putative N-acyl amino acid synthases with a unique two-domain organisation. The produced biosurfactants were identified by NMR spectroscopy as N-acyltyrosines with N-myristoyltyrosine as the predominant species. Critical micelle concentration and reduction of surface tension were similar to those of chemically synthesised N-myristoyltyrosine. Furthermore, we showed that the newly isolated N-acyltyrosines exhibit antibiotic activity against various bacteria. This is the first report describing the successful application of functional high-throughput screening assays for the identification of biosurfactant producing clones within a metagenomic library.

Microbial communities in dairy processing environment floor-drains are dominated by product-associated bacteria and yeasts

Floor-drains in food production environments harbour diverse microbes and can be contamination sources. To prevent cross-contamination with spoilage organisms or pathogens, sanitation protocols are regularly applied in food processing facilities. Along with product-associated samples, floor-drain water serves as an important indicator in hygiene monitoring in food processing facilities. However, knowledge about the microbial communities in floor-drains is still low. In this study, the microbial communities in floor-drain water and biofilm samples from an Austrian dairy plant were analysed and revealed that floor-drain microbial communities are dominated by product-associated microbes. DNA was extracted of drain water and drain biofilm samples from three different areas (n = 6) of an Austrian cheese production facility. To characterise the bacterial and eukaryotic communities, 16S rRNA gene pyrosequencing as well as cloning and sequencing of 18S rRNA genes were used. Floor-drain communities were dominated by product-associated bacterial (e.g. Lactobacillus kefiranofaciens, Streptococcus thermophilus) and eukaryotic phylotypes (e.g. Debaryomyces hansenii, Saccharomyces unisporus). In addition, putative drain water-derived phylotypes (e.g. Psychrilyobacter atlanticus, Cobetia marina) and ciliates were identified.

Detection of human pathogenicFusariumspecies in hospital and communal sink biofilms by using a highly specific monoclonal antibody

The fungus Fusarium is well known as a plant pathogen, but has recently emerged as an opportunistic pathogen of humans. Habitats providing direct human exposure to infectious propagules are largely unknown, but there is growing evidence that plumbing systems are sources of human pathogenic strains in the Fusarium solani species complex (FSSC) and Fusarium oxysporum species complex (FOSC), the most common groups infecting humans. Here, a newly developed Fusarium-specific monoclonal antibody (mAb ED7) was used to track FSSC and FOSC strains in sink drain biofilms by detecting its target antigen, an extracellular 200 kDa carbohydrate, in saline swabs. The antigen was detectable in 52% of swab samples collected from sinks across a University campus and a tertiary care hospital. The mAb was 100% accurate in detecting FSSC, FOSC, and F. dimerum species complex (FDSC) strains that were present, as mixed fungal communities, in 83% of sink drain biofilms. Specificity of the ELISA was confirmed by sequencing of the internally transcribed spacer 1 (ITS1)-5.8S-ITS2 rRNA-encoding regions of culturable yeasts and molds that were recovered using mycological culture, while translation elongation factor (TEF)-1α analysis of Fusarium isolates included FSSC 1-a, FOSC 33, and FDSC ET-gr, the most common clinical pathotypes in each group.

Bacterial diversity of floor drain biofilms and drain waters in a Listeria monocytogenes contaminated food processing environment

Sanitation protocols are applied on a daily basis in food processing facilities to prevent the risk of cross-contamination with spoilage organisms. Floor drain water serves along with product-associated samples (slicer dust, brine or cheese smear) as an important hygiene indicator in monitoring Listeria monocytogenes in food processing facilities. Microbial communities of floor drains are representative for each processing area and are influenced to a large degree by food residues, liquid effluents and washing water. The microbial communities of drain water are steadily changing, whereas drain biofilms provide more stable niches. Bacterial communities of four floor drains were characterized using 16S rRNA gene pyrosequencing to better understand the composition and exchange of drain water and drain biofilm communities. Furthermore, the L. monocytogenes contamination status of each floor drain was determined by applying cultivation-independent real-time PCR quantification and cultivation-dependent detection according to ISO11290-1. Pyrosequencing of 16S rRNA genes of drain water and drain biofilm bacterial communities yielded 50,611 reads, which were clustered into 641 operational taxonomic units (OTUs), affiliated to 16 phyla dominated by Proteobacteria, Firmicutes and Bacteroidetes. The most abundant OTUs represented either product- (Lactococcus lactis) or fermentation- and food spoilage-associated phylotypes (Pseudomonas mucidolens, Pseudomonas fragi, Leuconostoc citreum, and Acetobacter tropicalis). The microbial communities in DW and DB samples were distinct in each sample type and throughout the whole processing plant, indicating the presence of indigenous specific microbial communities in each processing compartment. The microbiota of drain biofilms was largely different from the microbiota of the drain water. A sampling approach based on drain water alone may thus only provide reliable information on planktonic bacterial cells but might not allow conclusions on the bacterial composition of the microbiota in biofilms.

Molecular analysis of dominant species in Listeria monocytogenes-positive biofilms in the drains of food processing facilities.

Listeria monocytogenes exhibits symbiotic codependence with the dominant commensal bacteria, which may help it avoid being removed or inactivated by disinfectants in local environments. In this study, we investigated L. monocytogenes-positive biofilms at food production facilities, and the dominant bacterial species of the biofilms were identified to determine the properties of the microbiological background. For this purpose, the ISO 11290 method was used for the detection and isolation of L. monocytogenes, and the species were further identified based on 16S rRNA and hly genes. 16S rRNA gene-based cloning, terminal restriction fragment length polymorphism, and denaturing gradient gel electrophoresis were combined to evaluate the dominant bacteria of the drain biofilms. Out of 100 drain samples, 8 were naturally contaminated with L. monocytogenes. Three molecular methods consistently showed that Pseudomonas psychrophila, Pseudomonas sp., and Klebsiella oxytoca were dominant species in 3Q, 5Q, and 6Q samples; Aeromonas hydrophila and Klebsiella sp. were significantly dominant in 1-2, 1-3, and 3-2 samples; A. hydrophila and K. oxytoca were dominant in the 2-3 sample; and A. hydrophila and Pseudomonas sp. were prominent in the 3-3 sample. Different biofilms from the same plant shared common bands, suggesting that similar bacteria can be found and can be dominant in different biofilms. This study provides a better understanding of the dominant compositions in these bacterial communities. Further studies to determine the mechanism of co-culture with L. monocytogenes will be of critical importance in predicting effective disinfection strategies.

Contamination of raw poultry meat by airborne Listeria originating from a floor drain

Poultry processing plants can become colonized with Listeria monocytogenes, resulting in long-term residence in floor drains. Earlier work showed that water spray into a contaminated floor drain causes airborne dissemination of a nonvirulent surrogate, Listeria innocua. The objective of the current study was to determine the extent to which a hose spray can result in the transfer of L. innocua from an established floor drain biofilm to raw poultry meat. Model floor drains were inoculated and subjected to a 2-s water spray during which broiler breast fillets were left uncovered on a table 2.4 m away. Other fillets were indirectly exposed by placement on the table 10 min after the drain spray. The number of Listeria on each fillet was determined on the day of exposure and after 4 d of refrigerated storage. An average of approximately 18 Listeria cells was detected per air-exposed filet; about half that number was detected on indirectly exposed fillets. The number of Listeria did not change substantially during 4 d of cold storage. An inadvertent 2-s spray into a contaminated floor drain could result in transfer of low numbers of Listeria to raw meat on a work surface.

Phylogenetic relationships among members of the Fusarium solani species complex in human infections and the descriptions of F. keratoplasticum sp. nov. and F. petroliphilum stat. nov.

Fusarium species are frequently associated with mycotic keratitis and, to a lesser extent, cases of localized and disseminated infections. The Fusarium solani species complex (FSSC) is the most common group of fusaria associated with human infectious diseases. Several studies to date have revealed dozens of strongly supported phylogenetic species within this important evolutionary clade, though little work has been done to improve the taxonomy and understanding of the reproductive mode and phenotypes of the predominant clinically relevant species. Here we described Fusarium keratoplasticum sp. nov., and Fusarium petroliphilum stat. nov., two phylogenetic species that are among the most frequently isolated fusaria in plumbing drain biofilms and outbreaks of contact lens-associated mycotic keratitis. F. keratoplasticum isolates were highly variable and showed a range of morphological characteristics typical for most classical concepts of ‘F. solani.’ Many isolates failed to produce sporodochia and macroconidia. Although most attempts to sexually cross F. keratoplasticum isolates failed, a heterothallic sexual stage typical for the FSSC was discovered by pairing isolates of opposite mating type on V-8 agar, the ascospores of which showed molecular evidence of recombination. Secondary metabolite profiles of FSSC species defined through molecular data were compared for the first time and revealed the production of bioactive compounds including cyclosporines and several novel compounds of unknown function. We speculate that the inferred phenotypic variability in these species is the result of the almost entirely anthropogenic sources from which they are derived, including biofilms on plumbing systems.