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

<p>Abstract</p> <p>Background</p> <p>Feed contaminated with <it>Salmonella </it>spp. constitutes a risk of <it>Salmonella </it>infections in animals, and subsequently in the consumers of animal products. <it>Salmonella </it>are occasionally isolated from the feed factory environment and some clones of <it>Salmonella </it>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 <it>Salmonella </it>strains from feed- and fishmeal factories. The study included 111 <it>Salmonella </it>strains isolated from Norwegian feed and fish meal factories in the period 1991–2006 of serovar Agona, serovar Montevideo, serovar Senftenberg and serovar Typhimurium.</p> <p>Results</p> <p>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. <it>Salmonella </it>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.</p> <p>Conclusion</p> <p>The results indicate a correlation between persistence and biofilm formation which suggests that biofilm forming ability may be an important factor for persistence of <it>Salmonella </it>in the factory environment.</p

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

<p>Abstract</p> <p>Background</p> <p>Biofilm has been shown to be one way for <it>Salmonella </it>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 <it>Salmonella </it>in the environment. Multicellular behaviour by <it>Salmonella </it>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.</p> <p>The aim of the study was to look into the distribution of morphotypes among feed and fish meal factory strains of <it>Salmonella</it>, with emphasis on potential differences between morphotypes with regards to survival in the feed factory environment.</p> <p>Results</p> <p>When screening a total of 148 <it>Salmonella </it>ser. Agona, <it>Salmonella </it>ser. Montevideo, <it>Salmonella </it>ser. Senftenberg and <it>Salmonella </it>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 <it>Salmonella </it>ser. Agona and 19% of <it>Salmonella </it>ser. Senftenberg displayed the bdar morphology.</p> <p>Inconsistency in Calcofluor binding, indicating expression of cellulose, was found among 25% of all the strains tested, however <it>Salmonella </it>ser. Agona showed to be highly consistent in Calcofluor binding (98%).</p> <p>In biofilm, <it>Salmonella </it>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.</p> <p>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 <it>Salmonella </it>biofilm matrix.</p> <p>Conclusion</p> <p>The bdar morphotype is common among <it>Salmonella </it>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 <it>Salmonella </it>biofilm matrix.</p

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

<p>Abstract</p> <p>Background</p> <p>The presence of <it>Salmonella </it>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 <it>Salmonella </it>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 <it>Salmonella </it>and other bacteria in feed production plants and how they affect survival and biofilm formation of <it>Salmonella </it>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 <it>Salmonella </it>to stress factors typically found in feed processing environments. Moreover, the role of dominant resident flora strains in the biofilm development of <it>Salmonella </it>was determined.</p> <p>Results</p> <p>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 <it>Salmonella</it>. Results showed higher survival properties by resident flora isolates for desiccation, and disinfection compared to <it>Salmonella </it>isolates. Dual-species biofilms favored <it>Salmonella </it>growth compared to <it>Salmonella </it>in mono-species biofilms, with biovolume increases of 2.8-fold and 3.2-fold in the presence of <it>Staphylococcus </it>and <it>Pseudomonas</it>, respectively.</p> <p>Conclusions</p> <p>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 <it>Salmonella</it>. Eliminating the establishment of resident flora isolates in feed industry surfaces is therefore of interest for impeding conditions for <it>Salmonella </it>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 <it>Salmonella </it>in feed processing environments.</p

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

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

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, internalisation, 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 implications for food safety of such adhesion will be discussed.The authors are members of the EU COST Action FA1202 (CGAFA1202): A European Network for Mitigating Bacterial Colonisation and Persistence on Foods and Food Processing Environments (http://www.bacfoodnet.org/) and acknowledge this action for facilitating collaborative networking that assisted with this study. The work was further supported by the Ministry of Education, Youth and Sports of the Czech Republic (project COST LD 14015 and project LO1218 under the NPU I program), the 'Cooperation Scientifique Universitaire (CSU)' France Denmark 2012 from the Embassy of France in Denmark 'Institut Francais du Danemark' (IFD) (no. 14/2012/CSU.8.2.1), the EGIDE Programme Hubert Curien (PHC) France Germany PROCOPE 2013 2015 from the 'Ministere des Affaires Etrangeres et Europeennes' (no. 28297WG) and by the Norwegian Research Council (grant no. 192402)

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

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 &ge;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