Lack of correlation between growth, stress, and virulence phenotypes in strains of Salmonella enterica serovar Enteritidis, S. Typhimurium DT104, S. 4,12, b:- and S. Liverpool.

Strains of Salmonella Enteritidis (SEnt, n = 10) and S. Typhimurium (STm, n = 11), representing clones with high impact on human health, and strains of S. 4,12: b:- (S412B n = 11) and S. Liverpool (SLiv, n = 4), representing clones with minor impact on human health were characterized for 16 growth, stress, and virulence phenotypes to investigate whether systematic differences exist in their performance in these phenotypes and whether there was correlation between performance in different phenotypes. The term serotype was not found to be predictive of a certain type of performance in any phenotype, and surprisingly, on average, strains of SEnt and STm were not significantly better in adhering to and invading cultured intestinal cells than the less pathogenic types. Forest analysis identified desiccation tolerance and the ability to grow at 42°C with high salt as the characters that separated serovars with low human health impact (S412B/SLiv) from serovars with high human health impact (SEnt/STm). The study showed that variation in phenotypes was high even within serovars and correlation between phenotypes was low, i.e. the way that a strain performed phenotypically in one of the tested conditions had a low predictive value for the performance of the strain in other conditions.

Antimicrobial-induced horizontal transfer of antimicrobial resistance genes in bacteria: a mini-review.

The emergence and spread of antimicrobial resistance (AMR) among pathogenic bacteria constitute an accelerating crisis for public health. The selective pressures caused by increased use and misuse of antimicrobials in medicine and livestock production have accelerated the overall selection of resistant bacteria. In addition, horizontal gene transfer (HGT) plays an important role in the spread of resistance genes, for example mobilizing reservoirs of AMR from commensal bacteria into pathogenic ones. Antimicrobials, besides antibacterial function, also result in undesirable effects in the microbial populations, including the stimulation of HGT. The main aim of this narrative review was to present an overview of the current knowledge of the impact of antimicrobials on HGT in bacteria, including the effects of transformation, transduction and conjugation, as well as other less well-studied mechanisms of HGT. It is widely accepted that conjugation plays a major role in the spread of AMR in bacteria, and the focus of this review is therefore mainly on the evidence provided that antimicrobial treatment affects this process. Other mechanisms of HGT have so far been deemed less important in this respect; however, recent discoveries suggest their role may be larger than previously thought, and the review provides an update on the rather limited knowledge currently available regarding the impact of antimicrobial treatment on these processes as well. A conclusion from the review is that there is an urgent need to investigate the mechanisms of antimicrobial-induced HGT, since this will be critical for developing new strategies to combat the spread of AMR.

Evaluation of a novel multiplex qPCR method for rapid detection and quantification of pathogens associated with calf diarrhoea.

AIMS: Diarrhoea is a common health problem in calves and a main reason for use of antimicrobials. It is associated with several bacterial, viral and parasitic pathogens, most of which are commonly present in healthy animals. Methods, which quantify the causative agents, may therefore improve confidence in associating a pathogen to the disease. This study evaluated a novel commercially available, multiplex quantitative polymerase chain reaction (qPCR) assay (Enterit4Calves) for detection and quantification of pathogens associated with calf-diarrhoea. METHODS AND RESULTS: Performance of the method was first evaluated under laboratory conditions. Then it was compared with current routine methods for detection of pathogens in faecal samples from 65 calves with diarrhoea and in 30 spiked faecal samples. The qPCR efficiencies were between 84%-103% and detection limits of 100-1000 copies of nucleic acids per sample were observed. Correct identification was obtained on 42 strains of cultured target bacteria, with only one false positive reaction from 135 nontarget bacteria. Kappa values for agreement between the novel assay and current routine methods varied between 0.38 and 0.83. CONCLUSION: The novel qPCR method showed good performance under laboratory conditions and a fair to good agreement with current routine methods when used for testing of field samples. SIGNIFICANCE AND IMPACT OF STUDY: In addition to having fair to good detection abilities, the novel qPCR method allowed quantification of pathogens. In the future, use of quantification may improve diagnosis and hence treatment of calf diarrhoea.

Association between antimicrobial usage and resistance in Salmonella from poultry farms in Nigeria.

BACKGROUND: Antimicrobial resistance (AMR) is a global health threat affecting treatment outcome in animals and humans. A pre-requisite for development of AMR reduction strategies is knowledge of antimicrobial use patterns, and how these affect resistance development. The aim of this study was to determine antimicrobial usage (AMU) and whether such usage was associated with AMR in Salmonella from poultry farms in Northwest Nigeria. RESULTS: Fifteen (37%) of antimicrobial products observed contained compounds that are of highest priority and critically important for human medicine. Broilers chicken consumed higher (28 ± 14 mg/kg active ingredients) amounts of antimicrobials compared to layers (13 ± 8 mg/kg) per week (p = 0.0009). Surprisingly, chickens raised under backyard system consumed higher amounts of antimicrobials (34 ± 7 mg/kg) than poultry in other systems (p = 0.02). High levels of resistance to tetracycline (58%), sulphonamides (65%), ciprofloxacin (46%) and gentamicin (42%) correlated with high farm level usage of these antimicrobials, and there was a strong correlation (r = 0.9) between farm usage and resistance of isolates to the same antimicrobials (p = 0.03). CONCLUSION: High AMU, including use of highest priority critically important antimicrobials was observed at poultry farms in Northwest Nigeria. AMU correlated with high levels of resistance. Communication of prudent use of antimicrobials to farmers and regulation to obtain reduction in AMU should be a priority.

Indications for the use of highest priority critically important antimicrobials in the veterinary sector.

BACKGROUND: Among the measures taken to preserve the clinical efficacy of highest priority critically important antimicrobials (HP-CIAs), the WHO has recommended avoiding their use in food-producing animals. Little is known regarding the indications for which different antimicrobial classes are used in animals, even in countries where data on antimicrobial use are available. OBJECTIVES: To outline, in a narrative review, the diseases for which HP-CIAs are used in veterinary medicine, highlighting incongruences with international guidelines and disease conditions where effective alternatives to HP-CIAs are missing. METHODS: Scientific literature, national reports and expert opinion were used to describe the indications for the use of HP-CIAs in the main food-producing (pigs, cattle and poultry) and companion (horses, dogs and cats) animal species. RESULTS: The most common indications for use of HP-CIAs are enteric and respiratory infections in pigs, cattle and poultry, urogenital infections in dogs and cats and respiratory infections in horses. In some instances, no valid and convenient alternatives to colistin and macrolides are available against certain porcine enteric and bovine respiratory pathogens. Effective, legal and convenient alternatives to HP-CIAs are also lacking for managing common infections in cats, for which oral administration is difficult, Rhodococcus equi infections in horses, some enteric and respiratory infections in poultry and MDR infections in all companion animal species. CONCLUSIONS: Future research and stewardship programmes should focus on the disease conditions identified by this review to reduce the use of HP-CIAs in the veterinary sector.

F4- and F18-Positive Enterotoxigenic Escherichia coli Isolates from Diarrhea of Postweaning Pigs: Genomic Characterization.

This study aimed to characterize in silico enterotoxigenic Escherichia coli F4- and F18-positive isolates (n = 90) causing swine postweaning diarrhea, including pathogenic potential, phylogenetic relationship, antimicrobial and biocide resistance, prophage content, and metal tolerance rates. F4 strains belonged mostly to the O149 and O6 serogroups and ST100 and ST48 sequence types (STs). F18 strains were mainly assigned to the O8 and O147 serogroups and ST10, ST23, and ST42. The highest rates of antimicrobial resistance were found against streptomycin, sulfamethoxazole, tetracycline, trimethoprim, and ampicillin. No resistance was found toward ciprofloxacin, cefotaxime, ceftiofur, and colistin. Genes conferring tolerance to copper (showing the highest diversity), cadmium, silver, and zinc were predicted in all genomes. Enterotoxin genes (ltcA, 100% F4, 62% F18; astA, 100% F4, 38.1% F18; sta, 18.8% F4, 38.1% F18; stb, 100% F4, 76.2% F18) and fimbria-encoding genes typed as F4ac and F18ac were detected in all strains, in addition to up to 16 other virulence genes in individual strains. Phage analysis predicted between 7 and 20 different prophage regions in each strain. A highly diverse variety of plasmids was found; IncFII, IncFIB, and IncFIC were prevalent among F4 isolates, while IncI1 and IncX1 were dominant among F18 strains. Interestingly, F4 isolates from the early 1990s belonged to the same clonal group detected for most of the F4 strains from 2018 to 2019 (ONT:H10-A-ST100-CH27-0). The small number of single-nucleotide polymorphism differences between the oldest and recent F4 ST100 isolates suggests a relatively stable genome. Overall, the isolates analyzed in this study showed remarkably different genetic traits depending on the fimbria type.IMPORTANCE Diarrhea in the postweaning period due to enterotoxigenic E. coli (ETEC) is an economically relevant disease in pig production worldwide. In Denmark, prevention is mainly achieved by zinc oxide administration (to be discontinued by 2022). In addition, a breeding program has been implemented that aims to reduce the prevalence of this illness. Treatment with antimicrobials contributes to the problem of antimicrobial resistance (AMR) development. As a novelty, this study aims to deeply understand the genetic population structure and variation among diarrhea-associated isolates by whole-genome sequencing characterization. ST100-F4ac is the dominant clonal group circulating in Danish herds and showed high similarity to ETEC ST100 isolates from China, the United States, and Spain. High rates of AMR and high diversity of virulence genes were detected. The characterization of diarrhea-related ETEC is important for understanding the disease epidemiology and pathogenesis and for implementation of new strategies aiming to reduce the impact of the disease in pig production.

Interaction Differences of the Avian Host-Specific Salmonella enterica Serovar Gallinarum, the Host-Generalist S. Typhimurium, and the Cattle Host-Adapted S. Dublin with Chicken Primary Macrophage.

Most Salmonella serovars cause disease in many host species, while a few serovars have evolved to be host specific. Very little is known about the mechanisms that contribute to Salmonella host specificity. We compared the interactions between chicken primary macrophages (CDPM) and host-generalist serovar Salmonella enterica serovar Typhimurium, host-adapted Salmonella enterica serovar Dublin, and avian host-specific Salmonella enterica serovar Gallinarum. S Gallinarum was taken up in lower numbers by CDPM than S Typhimurium and S Dublin; however, a higher survival rate was observed for this serovar. In addition, S Typhimurium and S Dublin caused substantially higher levels of cell death to the CDPM, while significantly higher concentrations of NO were produced by S Gallinarum-infected cells. Global transcriptome analysis performed 2 h postinfection showed that S Gallinarum infection triggered a more comprehensive response in CDPM with 1,114 differentially expressed genes (DEGs) compared to the responses of S Typhimurium (625 DEGs) and S Dublin (656 DEGs). Comparable levels of proinflammation responses were observed in CDPM infected by these three different serovars at the initial infection phase, but a substantially quicker reduction in levels of interleukin-1ß (IL-1ß), CXCLi1, and CXCLi2 gene expression was detected in the S Gallinarum-infected macrophages than that of two other groups as infections proceeded. KEGG cluster analysis for unique DEGs after S Gallinarum infection showed that the JAK-STAT signaling pathway was top enriched, indicating a specific role for this pathway in response to S Gallinarum infection of CDPM. Together, these findings provide new insights into the interaction between Salmonella and the host and increase our understanding of S Gallinarum host specificity.

Effect of tetracycline treatment regimens on antibiotic resistance gene selection over time in nursery pigs.

BACKGROUND: The majority of antimicrobials given during the production of pigs are given to nursery pigs. The influence of antimicrobial use on the levels of antimicrobial resistant (AMR) genes is important to quantify to be able to assess the impact of resistance on the food chain and risk to human and animal health. RESULTS: This study investigated the response on the levels of nine AMR genes to five different treatment strategies with oxytetracycline, and the dynamics of gene abundance over time by following 1167 pigs from five different farms in Denmark. The results showed no significant difference between treatments and an increase in abundance for the efflux pump encoding tet(A) gene and the genes encoding the ribosomal protection proteins tet(O) and tet(W) tetracycline resistant genes following treatment, while tet(M) showed no response to treatment. However, it was also observed that the levels of tet(O), tet(W), and ermB in some farms would drift more over time compared to a single treatment-course with antibiotic. CONCLUSION: This study underlines the large variation in AMR levels under natural conditions and the need for increased investigation of the complex interactions of antimicrobial treatment and other environmental and managerial practices in swine production on AMR gene abundance.

The Homolog of the Gene bstA of the BTP1 Phage from Salmonella enterica Serovar Typhimurium ST313 Is an Antivirulence Gene in Salmonella enterica Serovar Dublin.

In a previous study, a novel virulence gene, bstA, identified in a Salmonella enterica serovar Typhimurium sequence type 313 (ST313) strain was found to be conserved in all published Salmonella enterica serovar Dublin genomes. In order to analyze the role of this gene in the host-pathogen interaction in S Dublin, a mutant where this gene was deleted (S Dublin ΔbstA) and a mutant which was further genetically complemented with bstA (S Dublin 3246-C) were constructed and tested in models of in vitro and in vivo infection as well as during growth competition assays in M9 medium, Luria-Bertani broth, and cattle blood. In contrast to the results obtained for a strain of S Typhimurium ST313, the lack of bstA was found to be associated with increased virulence in S Dublin. Thus, S Dublin ΔbstA showed higher levels of uptake than the wild-type strain during infection of mouse and cattle macrophages and higher net replication within human THP-1 cells. Furthermore, during mouse infections, S Dublin ΔbstA was more virulent than the wild type following a single intraperitoneal infection and showed an increased competitive index during competitive infection assays. Deletion of bstA did not affect either the amount of cytokines released by THP-1 macrophages or the cytotoxicity toward these cells. The histology of the livers and spleens of mice infected with the wild-type strain and the S Dublin ΔbstA mutant revealed similar levels of inflammation between the two groups. The gene was not important for adherence to or invasion of human epithelial cells and did not influence bacterial growth in rich medium, minimal medium, or cattle blood. In conclusion, a lack of bstA affects the pathogenicity of S Dublin by decreasing its virulence. Therefore, it might be regarded as an antivirulence gene in this serovar.

Putrescine biosynthesis and export genes are essential for normal growth of avian pathogenic Escherichia coli.

BACKGROUND: Avian pathogenic Escherichia coli (APEC) is the infectious agent of a wide variety of avian diseases, which causes substantial economic losses to the poultry industry worldwide. Polyamines contribute to the optimal synthesis of nucleic acids and proteins in bacteria. The objectives of this study were to investigate; i) whether APEC E. coli encodes the same systems for biosynthesis and uptake as described for E. coli K12 and ii) the role of polyamines during in vitro growth of an avian pathogenic E. coli strain (WT-ST117- O83:H4T). RESULTS: Following whole genome sequencing, polyamine biosynthesis and export genes present in E. coli MG1655 (K-12) were found to be identical in WT-ST117. Defined mutants were constructed in putrescine and spermidine biosynthesis pathways (ΔspeB, ΔspeC, ΔspeF, ΔspeB/C and ΔspeD/E), and in polyamines transport systems (ΔpotE, ΔyeeF, ΔpotABCD and ΔpotFGHI). Contrary to what was observed for MG1655, the ΔpotE-ST117 mutant was growth attenuated, regardless of putrescine supplementation. The addition of spermidine or orthinine restored the growth to the level of WT-ST117. Growth attenuation after induction of membrane stress by SDS suggested that PotE is involved in protection against this stress. The ΔspeB/C-ST117 mutant was also growth attenuated in minimal medium. The addition of putrescine or spermidine to the media restored growth rate to the wild type level. The remaining biosynthesis and transport mutants showed a growth similar to that of WT-ST117. Analysis by Ultra-High Performance Liquid Chromatography revealed that the ΔspeB/C mutant was putrescine-deficient, despite that the gene speF, which is also involved in the synthesis of putrescine, was expressed. CONCLUSIONS: Deletion of the putrescine transport system, PotE, or the putrescine biosynthesis pathway genes speB/C affected in vitro growth of APEC (ST117- O83:H4) strain, but not E. coli MG1655, despite the high similarity of the genetic make-up of biosynthesis and transport genes. Therefore, blocking these metabolic reactions may be a suitable way to prevent APEC growth in the host without disturbing the commensal E. coli population.

The impact of inactivation of the purine biosynthesis genes, purN and purT, on growth and virulence in uropathogenic E. coli.

De novo synthesis of purines has been suggested to be an important factor for the pathogenesis of uropathogenic E. coli (UPEC). We analyzed the role of the redundant purine biosynthesis genes purN and purT, responsible for the third step in the purine biosynthesis, during UPEC infection. Growth experiments in M9 (minimal media), MOPS (rich media), filtered urine, and human serum with E. coli UTI89 and ΔpurN, ΔpurT, and ΔpurN/T mutants revealed that UPEC relies on de novo purine synthesis for growth in minimal medium. Mutants in individual genes as well as the double mutant grew equally well as the wild type in urine, rich media, and serum. However, during competition for growth in urine, the wild type UTI89 strain significantly outcompeted the purine auxotrophic ΔpurN/T mutant from late exponential growth phase. Inactivation of purN and/or purT significantly affected UPEC invasion of human bladder cells, but not the intracellular survival. Cytotoxicity levels to bladder cells were also diminished when both purN and purT were deleted, while single gene mutants did not differ from the wild type. When infecting human macrophages, no differences were observed between UTI89 and mutants in uptake, survival or cytotoxicity. Finally, the lack of the pur-gene(s), whether analysed as single or double gene knock-out, did not affect recovery rates after in vivo infection in a mouse model of UTI. These findings suggest that de novo synthesis of purines might be required only when UPEC is fully deprived of nucleotides and when grown in competition with other microorganisms in urine.

Effect of Tetracycline Dose and Treatment Mode on Selection of Resistant Coliform Bacteria in Nursery Pigs.

This study describes the results of a randomized clinical trial investigating the effect of oxytetracycline treatment dose and mode of administration on the selection of antibiotic-resistant coliform bacteria in fecal samples from nursery pigs. Nursery pigs (pigs of 4 to 7 weeks of age) in five pig herds were treated with oxytetracycline for Lawsonia intracellularis-induced diarrhea. Each group was randomly allocated to one of five treatment groups: oral flock treatment with a (i) high (20 mg/kg of body weight), (ii) medium (10 mg/kg), or (iii) low (5 mg/kg) dose, (iv) oral pen-wise (small-group) treatment (10 mg/kg), and (v) individual intramuscular injection treatment (10 mg/kg). All groups were treated once a day for 5 days. In all groups, treatment caused a rise in the numbers and proportions of tetracycline-resistant coliform bacteria right after treatment, followed by a significant drop by the time that the pigs left the nursery unit. The counts and proportions of tetracycline-resistant coliforms did not vary significantly between treatment groups, except immediately after treatment, when the highest treatment dose resulted in the highest number of resistant coliforms. A control group treated with tiamulin did not show significant changes in the numbers or proportions of tetracycline-resistant coliforms. Selection for tetracycline-resistant coliforms was significantly correlated to selection for ampicillin- and sulfonamide-resistant strains but not to selection for cefotaxime-resistant strains. In conclusion, the difference in the dose of oxytetracycline and the way in which the drug was applied did not cause significantly different levels of selection of tetracycline-resistant coliform bacteria under the conditions tested.IMPORTANCE Antimicrobial resistance is a global threat to human health. Treatment of livestock with antimicrobials has a direct impact on this problem, and there is a need to improve the ways that we use antimicrobials in livestock production. We hypothesized that antibiotic resistance development following treatment of diarrhea in nursery pigs could be reduced either by lowering the dose of oxytetracycline or by replacing the commonly used practice of flock treatment with individual or small-group treatments, since this would reduce the number of pigs treated. However, the study showed no significant difference between treatment groups with respect to the number or proportion of tetracycline-resistant coliforms selected. The most important conclusion is that under practical field conditions, there will be no added value, in terms of lowering resistance development, by exchanging flock treatment for individual or small-group treatment of nursery pigs. The reason for the lack of an effect of single-animal treatment is probably that such animals share the environment with treated animals and take up resistant bacteria from the environment.

Genotype variation and genetic relationship among Escherichia coli from nursery pigs located in different pens in the same farm.

BACKGROUND: So far, little is known about the genetic diversity and relatedness among Escherichia coli (E. coli) populations in the gut of swine. Information on this is required to improve modeling studies on antimicrobial resistance aiming to fight its occurrence and development. This work evaluated the genotype variation of E. coli isolated from swine fecal samples at the single pig and pen level, as well as between pens using repetitive extragenic palindromic (REP) PCR fingerprinting and pulsed field gel electrophoresis (PFGE). The genetic diversity of strains collected from media supplemented with ampicillin or tetracycline was also investigated. Besides, the genetic relationship of strains within each pen, between pens, as well as among strains within each group isolated from media with or without antibiotic, was assessed. RESULTS: REP-PCR patterns (N = 75) were generated for all the isolates (N = 720). Two profiles (REP_2 and REP_5) dominated, accounting for 23.7 and 23.3% of all isolates, respectively. At the pig and at the pen level, the number of different strains ranged from two to eight, and from 27 to 31, respectively, and multiple isolates from a single pen were found to be identical; however, in some of the pens, additional strains occurred at a lower frequency. E. coli isolates yielding different REP profiles were subjected to PFGE and led to 41 different genotypes which were also compared. CONCLUSIONS: Despite the presence of dominant strains, our results suggest a high genetic diversity of E. coli strains exist at the pen level and between pens. Selection with antibiotic seems to not affect the genetic diversity. The dominant REP profiles were the same found in a previous study in Denmark, which highlights that the same predominant strains are circulating in pigs of this country and might represent the archetypal E.coli commensal in pigs.

Multistrain models predict sequential multidrug treatment strategies to result in less antimicrobial resistance than combination treatment.

BACKGROUND: Combination treatment is increasingly used to fight infections caused by bacteria resistant to two or more antimicrobials. While multiple studies have evaluated treatment strategies to minimize the emergence of resistant strains for single antimicrobial treatment, fewer studies have considered combination treatments. The current study modeled bacterial growth in the intestine of pigs after intramuscular combination treatment (i.e. using two antibiotics simultaneously) and sequential treatments (i.e. alternating between two antibiotics) in order to identify the factors that favor the sensitive fraction of the commensal flora. Growth parameters for competing bacterial strains were estimated from the combined in vitro pharmacodynamic effect of two antimicrobials using the relationship between concentration and net bacterial growth rate. Predictions of in vivo bacterial growth were generated by a mathematical model of the competitive growth of multiple strains of Escherichia coli. RESULTS: Simulation studies showed that sequential use of tetracycline and ampicillin reduced the level of double resistance, when compared to the combination treatment. The effect of the cycling frequency (how frequently antibiotics are alternated in a sequential treatment) of the two drugs was dependent upon the order in which the two drugs were used. CONCLUSION: Sequential treatment was more effective in preventing the growth of resistant strains when compared to the combination treatment. The cycling frequency did not play a role in suppressing the growth of resistant strains, but the specific order of the two antimicrobials did. Predictions made from the study could be used to redesign multidrug treatment strategies not only for intramuscular treatment in pigs, but also for other dosing routes.

Modeling the growth dynamics of multiple Escherichia coli strains in the pig intestine following intramuscular ampicillin treatment.

BACKGROUND: This study evaluated how dosing regimen for intramuscularly-administered ampicillin, composition of Escherichia coli strains with regard to ampicillin susceptibility, and excretion of bacteria from the intestine affected the level of resistance among Escherichia coli strains in the intestine of nursery pigs. It also examined the dynamics of the composition of bacterial strains during and after the treatment. The growth responses of strains to ampicillin concentrations were determined using in vitro growth curves. Using these results as input data, growth predictions were generated using a mathematical model to simulate the competitive growth of E. coli strains in a pig intestine under specified plasma concentration profiles of ampicillin. RESULTS: In vitro growth results demonstrated that the resistant strains did not carry a fitness cost for their resistance, and that the most susceptible strains were more affected by increasing concentrations of antibiotics that the rest of the strains. The modeling revealed that short treatment duration resulted in lower levels of resistance and that dosing frequency did not substantially influence the growth of resistant strains. Resistance levels were found to be sensitive to the number of competing strains, and this effect was enhanced by longer duration of treatment. High excretion of bacteria from the intestine favored resistant strains over sensitive strains, but at the same time it resulted in a faster return to pre-treatment levels after the treatment ended. When the duration of high excretion was set to be limited to the treatment time (i.e. the treatment was assumed to result in a cure of diarrhea) resistant strains required longer time to reach the previous level. CONCLUSION: No fitness cost was found to be associated with ampicillin resistance in E. coli. Besides dosing factors, epidemiological factors (such as number of competing strains and bacterial excretion) influenced resistance development and need to be considered further in relation to optimal treatment strategies. The modeling approach used in the study is generic, and could be used for prediction of the effect of treatment with other drugs and other administration routes for effect on resistance development in the intestine of pigs.

Apramycin treatment affects selection and spread of a multidrug-resistant Escherichia coli strain able to colonize the human gut in the intestinal microbiota of pigs.

The effect of apramycin treatment on transfer and selection of an Escherichia coli strain (E. coli 912) in the intestine of pigs was analyzed through an in vivo experiment. The strain was sequenced and assigned to the sequence type ST101 and serotype O11. It carried resistance genes to apramycin/gentamicin, sulphonamide, tetracycline, hygromycin B, ß-lactams and streptomycin [aac(3)-IV, sul2, tet(X), aph(4), bla TEM-1 and strA/B], with all but tet(X) located on the same conjugative plasmid. Nineteen pigs were randomly allocated into two inoculation groups, one treated with apramycin (pen 2) and one non-treated (pen 3), along with a non-inoculated control group (pen 1). Two pigs of pen 2 and 3 were inoculated intragastrically with a rifampicin resistant variant of the strain. Apramycin treatment in pen 2 was initiated immediately after inoculation. Strain colonization was assessed in the feces from all pigs. E. coli 912 was shown to spread to non-inoculated pigs in both groups. The selective effect did not persist beyond 3 days post-treatment, and the strain was not detected from this time point in pen 2. We demonstrated that E. coli 912 was able to spread between pigs in the same pen irrespective of treatment, and apramycin treatment resulted in significantly higher counts compared to the non-treated group. This represents the first demonstration of how antimicrobial treatment affects spread of resistant bacteria in pig production. The use of apramycin may lead to enhanced spread of gentamicin-resistant E. coli. Since gentamicin is a first-choice drug for human bacteremia, this is of concern.

Importance of sigma factor mutations in increased triclosan resistance in Salmonella Typhimurium.

BACKGROUND: Salmonella enterica is the second most common foodborne pathogen. The use of biocides is crucial to prevent spread of foodborne pathogens, and it would be devastating for food safety if Salmonella would become resistant to the disinfectants used. Another concern is that exposure to disinfectants might lead to decreased susceptibility to antibiotics. The current study aimed to identify genetic changes causing high level triclosan resistance in S. enterica serovar Typhimurium and evaluate how these affected antibiotic resistance and efflux pump activity. RESULTS: Wild type strains S. Typhimurium 4/74 and DTU3 were adapted to increasing concentrations of the biocide triclosan by serial passage. High level triclosan resistant isolates (MIC > 1000 µg/ml) were obtained. Strains were genome sequenced, and SNPs in fabI, rpoS and rpoD were found to be associated with high level resistance. However, work with defined mutants revealed that a SNP in fabI was not sufficient to obtain high level resistance. This required additional mutations in the sigma factors rpoS or rpoD. The adapted strains showed triclosan-dependent increased efflux, increased fabI expression and reduced susceptibility towards the antibiotics enrofloxacin and sulphamethoxazole/trimethoprim. CONCLUSIONS: Medium level triclosan resistance could be obtained by fabI mutations in S. Typhimurium, however, high level resistance was found to require sigma factor mutations in addition to a fabI mutation. Reduced antibiotic sensitivity was observed for the adapted strains, which could be associated with increased efflux.

Identification of potential drug targets in Salmonella enterica sv. Typhimurium using metabolic modelling and experimental validation.

Salmonella enterica sv. Typhimurium is an established model organism for Gram-negative, intracellular pathogens. Owing to the rapid spread of resistance to antibiotics among this group of pathogens, new approaches to identify suitable target proteins are required. Based on the genome sequence of S. Typhimurium and associated databases, a genome-scale metabolic model was constructed. Output was based on an experimental determination of the biomass of Salmonella when growing in glucose minimal medium. Linear programming was used to simulate variations in the energy demand while growing in glucose minimal medium. By grouping reactions with similar flux responses, a subnetwork of 34 reactions responding to this variation was identified (the catabolic core). This network was used to identify sets of one and two reactions that when removed from the genome-scale model interfered with energy and biomass generation. Eleven such sets were found to be essential for the production of biomass precursors. Experimental investigation of seven of these showed that knockouts of the associated genes resulted in attenuated growth for four pairs of reactions, whilst three single reactions were shown to be essential for growth.

Enumeration of salmonellae in table eggs, pasteurized egg products, and egg-containing dishes by using quantitative real-time PCR.

Salmonellae are a major cause of food-borne outbreaks in Europe, with eggs and egg products being identified as major sources. Due to the low levels of salmonellae in eggs and egg products, direct quantification is difficult. In the present study, enrichment quantitative real-time PCR (qPCR) was employed for enumeration of salmonellae in different matrices: table eggs, pasteurized egg products, and egg-containing dishes. Salmonella enterica serovar Enteritidis and S. enterica serovar Tennessee were used to artificially contaminate these matrices. The results showed a linear regression between the numbers of salmonellae and the quantification cycle (Cq) values for all matrices used, with the exception of pasteurized egg white. Standard curves were constructed by using both stationary-phase cells and heat-stressed cells, with similar results. Finally, this method was used to evaluate the fate of salmonellae in two egg-containing dishes, long egg and tiramisu, at abused refrigeration temperatures, and results indicated the growth of bacteria over a 1-week period. In conclusion, enrichment qPCR was shown to be reliable for enumeration of salmonellae in different egg products.

The role of ClpP, RpoS and CsrA in growth and filament formation of Salmonella enterica serovar Typhimurium at low temperature.

BACKGROUND: Salmonellae are food-borne pathogens of great health and economic importance. To pose a threat to humans, Salmonellae normally have to cope with a series of stressful conditions in the food chain, including low temperature. In the current study, we evaluated the importance of the Clp proteolytic complex and the carbon starvation protein, CsrA, for the ability of Salmonella Typhimurium to grow at low temperature. RESULTS: A clpP mutant was severely affected in growth and formed pin point colonies at 10°C. Contrary to this, rpoS and clpP/rpoS mutants were only slightly affected. The clpP mutant formed cold resistant suppressor mutants at a frequency of 2.5 × 10(-3) and these were found not to express RpoS. Together these results indicated that the impaired growth of the clpP mutant was caused by high level of RpoS. Evaluation by microscopy of the clpP mutant revealed that it formed filamentous cells when grown at 10°C, and this phenotype too, disappered when rpoS was mutated in parallel indicating a RpoS-dependency. A csrA (sup) mutant was also growth attenuated a low temperature. An rpoS/csrA (sup) double mutant was also growth attenuated, indicating that the phenotype of the csrA mutant was independent from RpoS. CONCLUSIONS: The cold sensitivity of clpP mutant was associated with increased levels of RpoS and probably caused by toxic levels of RpoS. Although a csrA mutant also accumulated high level of RpoS, growth impairment caused by lack of csrA was not related to RpoS levels in a similar way.