Biofilm forming properties of quinolone resistant Escherichia coli from the broiler production chain and their dynamics in mixed biofilms.

BACKGROUND: Quinolone resistant Escherichia coli (QREC) have been found in samples from Norwegian broiler chicken, despite quinolones not being administered to poultry in Norway. Biofilm production may be one factor contributing to the observed persistence in the broiler production chain. In the present study, 158 QREC strains from chicken caecal and retail meat samples were screened for biofilm production in microtiter plates, biofilm morphotype on Congo Red (CR) agar plates and phylotype by multiplex PCR. Furthermore, the dynamics in mixed biofilms with strains of different morphotypes were studied on glass slides and on CR agar plates. RESULTS: All strains but one produced biofilm in microtiter plates and/or on CR agar plates at room temperature. There were no differences between strains from chicken caecum and chicken retail meat in the mean amount of biofilm produced in microtiter plates. Furthermore, no differences in biofilm production were observed between phylotypes. However, significant differences in biofilm production were found between biofilm morphotypes. The morphotype RDAR (red dry and rough, which has both curli and cellulose in the matrix, was displayed by 70% of the strains. Mean biofilm production by these strains were significantly higher than by strains with the morphotypes PDAR (pink dry and rough) with only cellulose or BDAR (brown dry and rough) with only curli. Interestingly, the two latter morphotypes produced biofilms with the morphotype RDAR when grown together. None of the strains achieved significantly higher numbers of colony forming units (cfu) in mixed biofilms than in single strain biofilms on glass slides. CONCLUSIONS: The results indicate that QREC can form biofilm reservoirs on both inert and organic surfaces in production environments, as well as on meat. This may contribute to persistence and dissemination of the strains. Strains with both curli and cellulose in the biofilm matrix were significantly better biofilm formers than strains lacking one of these components. However, strains with only one of the components could compensate for this by producing mixed biofilms with strains having the other component, and thereby most likely enhance their probabilities of persistence in the production environment.

Biofilm: A Hotspot for Emerging Bacterial Genotypes.

Bacteria have the ability to adapt to changing environments through rapid evolution mediated by modification of existing genetic information, as well as by horizontal gene transfer (HGT). This makes bacteria a highly successful life form when it comes to survival. Unfortunately, this genetic plasticity may result in emergence and dissemination of antimicrobial resistance and virulence genes, and even the creation of multiresistant "superbugs" which may pose serious threats to public health. As bacteria commonly reside in biofilms, there has been an increased interest in studying these phenomena within biofilms in recent years. This review summarizes the present knowledge within this important area of research. Studies on bacterial evolution in biofilms have shown that mature biofilms develop into diverse communities over time. There is growing evidence that the biofilm lifestyle may be more mutagenic than planktonic growth. Furthermore, all three main mechanisms for HGT have been observed in biofilms. This has been shown to occur both within and between bacterial species, and higher transfer rates in biofilms than in planktonic cultures were detected. Of special concern are the observations that mutants with increased antibiotic resistance occur at higher frequency in biofilms than in planktonic cultures even in the absence of antibiotic exposure. Likewise, efficient dissemination of antimicrobial resistance genes, as well as virulence genes, has been observed within the biofilm environment. This new knowledge emphasizes the importance of biofilm awareness and control.

Effects of norspermidine and spermidine on biofilm formation by potentially pathogenic Escherichia coli and Salmonella enterica wild-type strains.

Polyamines are present in all living cells. In bacteria, polyamines are involved in a variety of functions, including biofilm formation, thus indicating that polyamines may have potential in the control of unwanted biofilm. In the present study, the effects of the polyamines norspermidine and spermidine on biofilms of 10 potentially pathogenic wild-type strains of Escherichia coli serotype O103:H2, Salmonella enterica subsp. enterica serovar Typhimurium, and S. enterica serovar Agona were investigated. We found that exogenously supplied norspermidine and spermidine did not mediate disassembly of preformed biofilm of any of the E. coli and S. enterica strains. However, the polyamines did affect biofilm production. Interestingly, the two species reacted differently to the polyamines. Both polyamines reduced the amount of biofilm formed by E. coli but tended to increase biofilm formation by S. enterica. Whether the effects observed were due to the polyamines specifically targeting biofilm formation, being toxic for the cells, or maybe a combination of the two, is not known. However, there were no indications that the effect was mediated through binding to exopolysaccharides, as earlier suggested for E. coli. Our results indicate that norspermidine and spermidine do not have potential as inhibitors of S. enterica biofilm. Furthermore, we found that the commercial polyamines used contributed to the higher pH of the test medium. Failure to acknowledge and control this important phenomenon may lead to misinterpretation of the results.

Surface adhesins and exopolymers of selected foodborne pathogens.

The ability of bacteria to bind different compounds and to adhere to biotic and abiotic surfaces provides them with a range of advantages, such as colonization of various tissues, internalization, avoidance of an immune response, and survival and persistence in the environment. A variety of bacterial surface structures are involved in this process and these promote bacterial adhesion in a more or less specific manner. In this review, we will focus on those surface adhesins and exopolymers in selected foodborne pathogens that are involved mainly in primary adhesion. Their role in biofilm development will also be considered when appropriate. Both the clinical impact and the implications for food safety of such adhesion will be discussed.

Potentially pathogenic Escherichia coli can form a biofilm under conditions relevant to the food production chain.

The biofilm-producing abilities of potentially human-pathogenic serotypes of Escherichia coli from the ovine reservoir were studied at different temperatures and on different surfaces. A possible influence of the hydrophobicity of the bacterial cells, as well as the presence of two virulence factors, the Shiga toxin-encoding (Stx) bacteriophage and the eae gene, was also studied. A total of 99 E. coli isolates of serotypes O26:H11, O103:H2, and O103:H25 isolated from sheep feces were included. The results show that isolates of all three E. coli serotypes investigated can produce biofilm on stainless steel, glass, and polystyrene at 12, 20, and 37°C. There was a good general correlation between the results obtained on the different surfaces. E. coli O103:H2 isolates produced much more biofilm than those of the other two serotypes at all three temperatures. In addition, isolates of serotype O26:H11 produced more biofilm than those of O103:H25 at 37°C. The hydrophobicity of the isolates varied between serotypes and was also influenced by temperature. The results strongly indicated that hydrophobicity influenced the attachment of the bacteria rather than their ability to form biofilm once attached. Isolates with the eae gene produced less biofilm at 37°C than isolates without this gene. The presence of a Stx bacteriophage did not influence biofilm production. In conclusion, our results show that potentially human-pathogenic E. coli from the ovine reservoir can form biofilm on various surfaces and at several temperatures relevant for food production and handling.

Antimicrobial Resistance in the Terrestrial Environment of Agricultural Landscapes in Norway.

The abundance and diversity of antimicrobial-resistant bacteria (ARB) and antimicrobial resistance genes (ARGs) in agricultural landscapes may be important for the spread of antimicrobial resistance (AMR) in the environment. The aim of this study was to apply screening methods for ARB and ARGs to investigate the impact of farming on the prevalence of AMR in a country with low antibiotic usage. We have analyzed samples (n = 644) from soil and wild terrestrial animals and plants (slugs, snails, mice, shrews, earthworms, and red clover) collected over two years in agricultural fields accompanied by nearby control areas with low human activity. All samples were investigated for the occurrence of 35 different ARGs using high-throughput quantitative PCR (HT-qPCR) on a newly developed DNA array. In addition, samples from the first year (n = 415) were investigated with a culture-based approach combined with whole-genome sequencing (WGS) to identify antimicrobial-resistant E. coli (AREC). ARGs were detected in 59.5% of all samples (2019 + 2020). AREC, which was only investigated in the 2019 samples, was identified in 1.9% of these. Samples collected in the autumn showed more ARGs and AREC than spring samples, and this was more pronounced for organic fields than for conventional fields. Control areas with low human activity showed lower levels of ARGs and a lack of AREC. The use of livestock manure was correlated with a higher level of ARG load than other farming practices. None of the soil samples contained antibiotics, and no association was found between AMR and the levels of metals or pesticides. High qualitative similarity between HT-qPCR and WGS, together with the positive controls to the validation of our 35 ARG assays, show that the microfluid DNA array may be an efficient screening tool on environmental samples. In conclusion, even in a country with a very low consumption of antimicrobials by production animals, our results support the hypothesis of these animals being a source of AREC and ARGs in agricultural environments, primarily through the use of manure.

The Role of Biofilms in the Pathogenesis of Animal Bacterial Infections.

Biofilms are bacterial aggregates embedded in a self-produced, protective matrix. The biofilm lifestyle offers resilience to external threats such as the immune system, antimicrobials, and other treatments. It is therefore not surprising that biofilms have been observed to be present in a number of bacterial infections. This review describes biofilm-associated bacterial infections in most body systems of husbandry animals, including fish, as well as in sport and companion animals. The biofilms have been observed in the auditory, cardiovascular, central nervous, digestive, integumentary, reproductive, respiratory, urinary, and visual system. A number of potential roles that biofilms can play in disease pathogenesis are also described. Biofilms can induce or regulate local inflammation. For some bacterial species, biofilms appear to facilitate intracellular invasion. Biofilms can also obstruct the healing process by acting as a physical barrier. The long-term protection of bacteria in biofilms can contribute to chronic subclinical infections, Furthermore, a biofilm already present may be used by other pathogens to avoid elimination by the immune system. This review shows the importance of acknowledging the role of biofilms in animal bacterial infections, as this influences both diagnostic procedures and treatment.

Evolution of antimicrobial resistance in E. coli biofilm treated with high doses of ciprofloxacin.

The evolution of antimicrobial resistance (AMR) has mainly been studied in planktonic bacteria exposed to sub-inhibitory antimicrobial (AM) concentrations. However, in a number of infections that are treated with AMs the bacteria are located in biofilms where they tolerate high doses of AM. In the present study, we continuously exposed biofilm residing E. coli at body temperature to high ciprofloxacin (CIP) concentrations increasing from 4 to 130 times the minimal inhibitory concentration (MIC), i.e., from 0.06 to 2.0 mg/L. After 1 week, the biofilms were full of CIP resistant bacteria. The evolutionary trajectory observed was the same as described in the literature for planktonic bacteria, i.e., starting with a single mutation in the target gene gyrA followed by mutations in parC, gyrB, and parE, as well as in genes for regulation of multidrug efflux pump systems and outer membrane porins. Strains with higher numbers of these mutations also displayed higher MIC values. Furthermore, the evolution of CIP resistance was more rapid, and resulted in strains with higher MIC values, when the bacteria were biofilm residing than when they were in a planktonic suspension. These results may indicate that extensive clinical AM treatment of biofilm-residing bacteria may not only fail to eradicate the infection but also pose an increased risk of AMR development.

Evaluation of Biofilm Cultivation Models for Efficacy Testing of Disinfectants against Salmonella Typhimurium Biofilms.

Within the European Union, Salmonella is frequently reported in food and feed products. A major route of transmission is upon contact with contaminated surfaces. In nature, bacteria such as Salmonella are often encountered in biofilms, where they are protected against antibiotics and disinfectants. Therefore, the removal and inactivation of biofilms is essential to ensure hygienic conditions. Currently, recommendations for disinfectant usage are based on results of efficacy testing against planktonic bacteria. There are no biofilm-specific standards for the efficacy testing of disinfectants against Salmonella. Here, we assessed three models for disinfectant efficacy testing on Salmonella Typhimurium biofilms. Achievable bacterial counts per biofilm, repeatability, and intra-laboratory reproducibility were analyzed. Biofilms of two Salmonella strains were grown on different surfaces and treated with glutaraldehyde or peracetic acid. Disinfectant efficacy was compared with results for planktonic Salmonella. All methods resulted in highly repeatable cell numbers per biofilm, with one assay showing variations of less than 1 log10 CFU in all experiments for both strains tested. Disinfectant concentrations required to inactivate biofilms were higher compared to planktonic cells. Differences were found between the biofilm methods regarding maximal achievable cell numbers, repeatability, and intra-laboratory reproducibility of results, which may be used to identify the most appropriate method in relation to application context. Developing a standardized protocol for testing disinfectant efficacy on biofilms will help identify conditions that are effective against biofilms.

Evaluation of Disinfectant Efficacy against Biofilm-Residing Wild-Type Salmonella from the Porcine Industry.

Salmonella enterica is a causative pathogen of Salmonellosis, a zoonosis causing global disease and financial losses every year. Pigs may be carriers of Salmonella and contribute to the spread to humans and food products. Salmonella may persist as biofilms. Biofilms are bacterial aggregates embedded in a self-produced matrix and are known to withstand disinfectants. We studied the effect of glutaraldehyde and peracetic acid, two active substances frequently used in disinfectant formulations in the pig industry, on representative biofilm-residing wild-type Salmonella collected from pig housings in the United Kingdom (UK). We screened biofilm production of strains using the microtiter plate (MTP) assay and Congo Red Coomassie Blue (CRCB) agar method. Previously published stainless-steel coupon (SSCA), polyvinylchloride coupon (PCA), and glass bead (GBA) assays were used for disinfection studies. The mean reduction in the tested wild-type strains met the criterion of ≥4 log10 CFU at a disinfectant concentration of 0.05% with SSCA and GBA, and 0.005% with PCA for peracetic acid, along with 0.5% for glutaraldehyde with all three assays on the mean. At these concentrations, both tested disinfectants are suitable for disinfection of pig housings against Salmonella. When evaluating the efficacy of disinfectants, biofilms should be included, as higher disinfectant concentrations are necessary compared to planktonic bacteria.

The Effect of Antimicrobial Resistance Plasmids Carrying blaCMY-2 on Biofilm Formation by Escherichia coli from the Broiler Production Chain.

Extended-spectrum cephalosporin-resistant Escherichia coli (ESCR E. coli) with plasmids carrying the blaCMY-2 resistance gene have been isolated from the Norwegian broiler production chain through the Norwegian monitoring program for antimicrobial resistance in animals, food and feed, NORM-VET. The aim of the present study was to investigate the biofilm forming abilities of these strains, and in particular to see whether these might be influenced by the carriage of blaCMY-2 plasmids. The ESCR E. coli from the broiler production chain displayed relatively low biofilm forming abilities in the crystal violet biofilm assay as compared to quinolone-resistant E. coli (QREC) from the same population (mean ± SD = 0.686 ± 0.686 vs. 1.439 ± 0.933, respectively). Acquisition of two different blaCMY-2 plasmids by QREC strains reduced their biofilm production in microtiter plates, but not their biofilm production on Congo Red agar plates. Furthermore, motility was reduced, but not planktonic growth. We hypothesize that genes carried by these plasmids may have caused the observed reduction in biofilm formation, possibly mediated through changes in flagellar expression or function. Furthermore, this may help explain the different biofilm forming abilities observed between ESCR E. coli and QREC. The results also indicate that the risk of biofilm reservoirs of antimicrobial resistant E. coli on in the broiler production is lower for ESCR E. coli than for QREC.

Exploring Klebsiella pneumoniae in Healthy Poultry Reveals High Genetic Diversity, Good Biofilm-Forming Abilities and Higher Prevalence in Turkeys Than Broilers.

Klebsiella pneumoniae is a well-studied human pathogen for which antimicrobial resistant and hypervirulent clones have emerged globally. K. pneumoniae is also present in a variety of environmental niches, but currently there is a lack of knowledge on the occurrence and characteristics of K. pneumoniae from non-human sources. Certain environmental niches, e.g., animals, may be associated with high K. pneumoniae abundance, and these can constitute a reservoir for further transmission of strains and genetic elements. The aim of this study was to explore and characterize K. pneumoniae from healthy broilers and turkeys. A total of 511 cecal samples (broiler n = 356, turkey n = 155), included in the Norwegian monitoring program for antimicrobial resistance (AMR) in the veterinary sector (NORM-VET) in 2018, were screened for K. pneumoniae by culturing on SCAI agar. K. pneumoniae was detected in 207 (40.5%) samples. Among the broiler samples, 25.8% were positive for K. pneumoniae, in contrast to turkey with 74.2% positive samples (p < 0.01). Antibiotic susceptibility testing was performed, in addition to investigating biofilm production. Whole genome sequencing was performed on 203 K. pneumoniae isolates, and analysis was performed utilizing comparative genomics tools. The genomes grouped into 66 sequence types (STs), with ST35, ST4710 and ST37 being the most prevalent at 13.8%, 7.4%, and 5.4%, respectively. The overall AMR occurrence was low, with only 11.3% of the isolates showing both pheno- and genotypic resistance. Genes encoding aerobactin, salmochelin or yersiniabactin were detected in 47 (23.2%) genomes. Fifteen hypervirulent genomes belonging to ST4710 and isolated from turkey were identified. These all encoded the siderophore virulence loci iuc5 and iro5 on an IncF plasmid. Isolates from both poultry species displayed good biofilm-forming abilities with an average of OD595 0.69 and 0.64. To conclude, the occurrence of K. pneumoniae in turkey was significantly higher than in broiler, indicating that turkey might be an important zoonotic reservoir for K. pneumoniae compared to broilers. Furthermore, our results show a highly diverse K. pneumoniae population in poultry, low levels of antimicrobial resistance, good biofilm-forming abilities and a novel hypervirulent ST4710 clone circulating in the turkey population.

Micro ecosystems from feed industry surfaces: a survival and biofilm study of Salmonella versus host resident flora strains.

BACKGROUND: The presence of Salmonella enterica serovars in feed ingredients, products and processing facilities is a well recognized problem worldwide. In Norwegian feed factories, strict control measures are implemented to avoid establishment and spreading of Salmonella throughout the processing chain. There is limited knowledge on the presence and survival of the resident microflora in feed production plants. Information on interactions between Salmonella and other bacteria in feed production plants and how they affect survival and biofilm formation of Salmonella is also limited. The aim of this study was to identify resident microbiota found in feed production environments, and to compare the survival of resident flora strains and Salmonella to stress factors typically found in feed processing environments. Moreover, the role of dominant resident flora strains in the biofilm development of Salmonella was determined. RESULTS: Surface microflora characterization from two feed productions plants, by means of 16 S rDNA sequencing, revealed a wide diversity of bacteria. Survival, disinfection and biofilm formation experiments were conducted on selected dominant resident flora strains and Salmonella. Results showed higher survival properties by resident flora isolates for desiccation, and disinfection compared to Salmonella isolates. Dual-species biofilms favored Salmonella growth compared to Salmonella in mono-species biofilms, with biovolume increases of 2.8-fold and 3.2-fold in the presence of Staphylococcus and Pseudomonas, respectively. CONCLUSIONS: These results offer an overview of the microflora composition found in feed industry processing environments, their survival under relevant stresses and their potential effect on biofilm formation in the presence of Salmonella. Eliminating the establishment of resident flora isolates in feed industry surfaces is therefore of interest for impeding conditions for Salmonella colonization and growth on feed industry surfaces. In-depth investigations are still needed to determine whether resident flora has a definite role in the persistence of Salmonella in feed processing environments.

The Effect of Disinfectants on Quinolone Resistant E. coli (QREC) in Biofilm.

The aim of disinfection is to reduce the number of microorganisms on surfaces which is a challenge due to biofilms. In the present study, six quinolone resistant Escherichia coli (QREC) strains with three different biofilm matrix compositions were included to assess the log10 colony forming units (CFU) reduction effect of three disinfectants at various exposure times on biofilm of different ages and morphotypes. Biofilm was formed on stainless steel coupons for two and five days before transferred to tubes with Virocid 0, 25%, VirkonS 1%, and TP990 1% and left for various exposure times. The biofilms were scraped off and serial dilutions were spread on blood agar plates where colony forming units (CFU) were counted. A mean log10 CFU reduction ≥4 was seen on two-day-old biofilm with VirkonS and Virocid (30 min) but not on five-day old biofilm. TP990 did not display sufficient effect under the conditions tested. The bactericidal effect was inferior to that reported on planktonic bacteria. The findings of this study should be considered when establishing both disinfectant routines and standard susceptibility tests, which further should accommodate E. coli biofilms and not only Pseudomonas as is the case today.

Bacterial Biofilm and its Role in the Pathogenesis of Disease.

Recognition of the fact that bacterial biofilm may play a role in the pathogenesis of disease has led to an increased focus on identifying diseases that may be biofilm-related. Biofilm infections are typically chronic in nature, as biofilm-residing bacteria can be resilient to both the immune system, antibiotics, and other treatments. This is a comprehensive review describing biofilm diseases in the auditory, the cardiovascular, the digestive, the integumentary, the reproductive, the respiratory, and the urinary system. In most cases reviewed, the biofilms were identified through various imaging technics, in addition to other study approaches. The current knowledge on how biofilm may contribute to the pathogenesis of disease indicates a number of different mechanisms. This spans from biofilm being a mere reservoir of pathogenic bacteria, to playing a more active role, e.g., by contributing to inflammation. Observations also indicate that biofilm does not exclusively occur extracellularly, but may also be formed inside living cells. Furthermore, the presence of biofilm may contribute to development of cancer. In conclusion, this review shows that biofilm is part of many, probably most chronic infections. This is important knowledge for development of effective treatment strategies for such infections.

Persistence of a Stx-Encoding Bacteriophage in Minced Meat Investigated by Application of an Improved DNA Extraction Method and Digital Droplet PCR.

Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens with Shiga toxins as the main virulence factor. Shiga toxins are encoded on Shiga toxin-encoding bacteriophages (Stx phages). Stx phages may exist as free bacteriophages in the environment or in foods or as prophages integrated into the host genome. From a food safety perspective, it is important to have knowledge on the survival and persistence of Stx phages in food products since these may integrate into the bacterial hosts through transduction if conditions are right. Here, we present the results from a study investigating the survival of a Stx phage in minced meat from beef stored at a suboptimal temperature (8°C) for food storage along with modifications and optimizations of the methods applied. Minced meat from beef was inoculated with known levels of a labeled Stx phage prior to storage. Phage filtrates were used for plaque assays and DNA extraction, followed by real-time PCR and digital droplet PCR (ddPCR). The results from the pilot study suggested that the initial DNA extraction protocol was not optimal, and several modifications were tested before a final protocol was defined. The final DNA extraction protocol comprised ultra-centrifugation of the entire phage filtrate for concentrating phages and two times phenol-chloroform extraction. The protocol was used for two spiking experiments. The DNA extraction protocol resulted in flexibility in the amount of DNA available for use in PCR analyses, ultimately increasing the sensitivity of the method used for quantification of phages in a sample. All three quantification methods employed (i.e., plaque assays, real-time PCR, and ddPCR) showed similar trends in the development of the phages during storage, where ddPCR has the benefit of giving absolute quantification of DNA copies in a simple experimental setup. The results indicate that the Stx phages persist and remain infective for at least 20 days under the storage conditions used in the present study. Stx phages in foods might represent a potential risk for humans. Although it can be speculated that transduction may take place at 8°C with subsequent forming of STEC, it can be expected to be a rare event. However, such an event may possibly take place under more optimal conditions, such as an increase in storage temperature of foods or in the gastrointestinal tract of humans.

Bioactive glass S53P4 eradicates Staphylococcus aureus in biofilm/planktonic states in vitro.

Background: Increasing antimicrobial resistance to antibiotics is a substantial health threat. Bioactive glass S53P4 (BAG) has an antimicrobial effect that can reduce the use of antibiotics. The aim of this study was to evaluate the antimicrobial efficacy of BAG in vitro on staphylococci in biofilm and in planktonic form. Secondary aims were to investigate whether supernatant fluid primed from BAG retains the antibacterial capacity and if ciprofloxacin enhances the effect.Methods: BAG-S53P4 granules, <45 µm, primed in tryptic soy broth (TSB) were investigated with granules present in TSB (100 mg/mL) and after removal of granules (100, 200, and 400 mg/mL). The efficacy of BAG to eradicate Staphylococcus aureus biofilm in vitro was tested using 10 different clinical strains and 1 reference strain in three test systems: the biofilm-oriented antiseptic test based on metabolic activity, the biofilm bactericidal test based on culturing surviving bacteria, and confocal laser scanning microscopy (CLSM) combined with LIVE/DEAD staining.Results: Exposure to 48 h primed BAG granules (100 mg/mL) produced bactericidal effects in 11/11 strains (p = 0.001), and CLSM showed reduction of viable bacteria in biofilm (p = 0.001). Supernatant primed 14 days, 400 mg/mL, reduced metabolic activity (p < 0.001), showed bactericidal effects for 11/11 strains (p = 0.001), and CLSM showed fewer viable bacteria (p = 0.001). The supernatant primed for 48 h, or in concentrations lower than 400 mg/mL at 14 days, did not completely eradicate biofilm.Conclusion: Direct exposure to BAG granules, or primed supernatant fluid, effectively eradicated S. aureus in biofilm. The anti-biofilm effect is time- and concentration-dependent. When BAG had reached its full antimicrobial effect, ciprofloxacin had no additional effect.

NarAB Is an ABC-Type Transporter That Confers Resistance to the Polyether Ionophores Narasin, Salinomycin, and Maduramicin, but Not Monensin.

Polyether ionophores are antimicrobial compounds used as feed additives in poultry feed to control diseases caused by coccidia. In addition to the anticoccidial activity of these compounds, polyether ionophores also contain antibacterial properties. Resistance to the polyether ionophore narasin was recently shown to exist on mobile plasmids in Enterococcus faecium and the resistance mechanism was suggested to be associated with a two-gene operon encoding an ABC-type transporter. In this study we demonstrate that the genes encoding the putative narasin resistance mechanism confers reduced susceptibility to the polyether ionophores narasin, salinomycin and maduramicin, but not to monensin and suggest that this resistance mechanism should be referred to as NarAB. Importantly, NarAB does not affect the susceptibility of E. faecium to any of the tested antimicrobial compounds that are used in clinical medicine. However, we show that conjugation in the presence of certain polyether ionophores increases the number of vancomycin resistant E. faecium suggesting that narasin and certain other polyether ionophores can contribute to the persistence of VRE in poultry populations.

Biofilm forming abilities of Salmonella are correlated with persistence in fish meal- and feed factories.

BACKGROUND: Feed contaminated with Salmonella spp. constitutes a risk of Salmonella infections in animals, and subsequently in the consumers of animal products. Salmonella are occasionally isolated from the feed factory environment and some clones of Salmonella persist in the factory environment for several years. One hypothesis is that biofilm formation facilitates persistence by protecting bacteria against environmental stress, e.g. disinfection. The aim of this study was to investigate the biofilm forming potential of Salmonella strains from feed- and fishmeal factories. The study included 111 Salmonella strains isolated from Norwegian feed and fish meal factories in the period 1991-2006 of serovar Agona, serovar Montevideo, serovar Senftenberg and serovar Typhimurium. RESULTS: Significant differences were found between serovars regarding the abilities to form biofilm on polystyrene (microtiter plate assay) and in the air-liquid interface of nutrient broth (pellicle assay). Strains of serovar Agona and serovar Montevideo were good biofilm producers. In Norwegian factories, clones of these serovars have been observed to persist for several years. Most serovar Senftenberg clones appear to persist for a shorter period, and strains of this serovar were medium biofilm producers in our test systems. Strains of the serovar Typhimurium were relatively poor biofilm producers. Salmonella ser. Typhimurium clones have not been observed to persist even though this serovar is resident in Norwegian wild life. When classifying strains according to persistence or presumed non-persistence, persistent strains produced more biofilm than presumed non-persisting strains. CONCLUSION: The results indicate a correlation between persistence and biofilm formation which suggests that biofilm forming ability may be an important factor for persistence of Salmonella in the factory environment.

Survival potential of wild type cellulose deficient Salmonella from the feed industry.

BACKGROUND: Biofilm has been shown to be one way for Salmonella to persist in the feed factory environment. Matrix components, such as fimbriae and cellulose, have been suggested to play an important role in the survival of Salmonella in the environment. Multicellular behaviour by Salmonella is often categorized according to colony morphology into rdar (red, dry and rough) expressing curli fimbriae and cellulose, bdar (brown, dry and rough) expressing curli fimbriae and pdar (pink, dry and rough) expressing cellulose. The aim of the study was to look into the distribution of morphotypes among feed and fish meal factory strains of Salmonella, with emphasis on potential differences between morphotypes with regards to survival in the feed factory environment. RESULTS: When screening a total of 148 Salmonella ser. Agona, Salmonella ser. Montevideo, Salmonella ser. Senftenberg and Salmonella ser. Typhimurium strains of feed factory, human clinical and reference collection origin, as many as 99% were able to express rough morphology (rdar or bdar). The dominant morphotype was rdar (74%), however as many as 55% of Salmonella ser. Agona and 19% of Salmonella ser. Senftenberg displayed the bdar morphology. Inconsistency in Calcofluor binding, indicating expression of cellulose, was found among 25% of all the strains tested, however Salmonella ser. Agona showed to be highly consistent in Calcofluor binding (98%). In biofilm, Salmonella ser. Agona strains with bdar mophology was found to be equally tolerant to disinfection treatment as strains with rdar morphotype. However, rdar morphology appeared to be favourable in long term survival in biofilm in a very dry environment. Chemical analysis showed no major differences in polysaccharide content between bdar and rdar strains. Our results indicate that cellulose is not a major component of the Salmonella biofilm matrix. CONCLUSION: The bdar morphotype is common among Salmonella ser. Agona strains isolated from the factory environment. The rdar and the bdar strains were found to be equally tolerant to disinfectants, while the rdar strain was found to be more tolerant to long-term desiccation and nutrient depletion in biofilm than the bdar strain. Cellulose does not appear to be a major component of the Salmonella biofilm matrix.