Antimicrobial resistance and virulence characteristics in 3 collections of staphylococci from bovine milk samples.

Mastitis is a prevalent disease in dairy cattle, and staphylococci are among the most common causative pathogens. Staphylococci can express resistance to a range of antimicrobials, of which methicillin resistance is of particular public health concern. Additionally, Staphylococcus aureus carries a variety of virulence factors, although less is understood about the virulence of non-aureus staphylococci (NAS). The aim of our study was to identify and characterize 3 collections of staphylococcal isolates from bovine milk samples regarding antimicrobial resistance, with emphasis on methicillin resistance, and their carriage of virulence genes typically displayed by Staph. aureus. A total of 272 staphylococcal isolates collected in Norway and Belgium in 2016 were included, distributed as follows: group 1, Norway, 100 isolates; group 2, Flanders, Belgium, 64 isolates; group 3, Wallonia, Belgium, 108 isolates. Species identification was performed by use of MALDI-TOF mass spectrometry. Phenotypic resistance was determined via disk diffusion, and PCR was used for detection of methicillin resistance genes, mecA and mecC, and virulence genes. Antimicrobial resistance was common in Staphylococcus epidermidis and Staphylococcus haemolyticus from all different groups, with resistance to trimethoprim-sulfonamide frequently occurring in Staph. epidermidis and Staph. haemolyticus as well as in Staph. aureus. Resistance to penicillin was most frequently observed in group 1. Ten Belgian isolates (1 from group 2, 9 from group 3) carried the methicillin resistance determinant mecA: 5 Staph. aureus from 2 different farms and 5 NAS from 3 different farms. Almost all Staph. aureus isolates were positive for at least 3 of the screened virulence genes, whereas, in total, only 8 NAS isolates harbored any of the same genes. Our study contributes to the continuous need for knowledge regarding staphylococci from food-producing animals as a basis for better understanding of occurrence of resistance and virulence traits in these bacteria.

Biofilm as an environment for dissemination of stx genes by transduction.

Dissemination of Shiga toxin (Stx)-encoding bacteriophages is the most likely mechanism for the spread of Stx-encoding genes and the emergence of new Stx-producing Escherichia coli (STEC). Biofilm has been reported to be a place where horizontal gene transfer by plasmid conjugation and DNA transformation may occur, and in this study, horizontal gene transfer by transduction has been demonstrated. Transfer of Stx-encoding bacteriophages to potentially pathogenic E. coli in biofilm was observed at both 20°C and 37°C. The infection rates were higher at 37°C than at 20°C. To our knowledge, this study is the first to show lateral gene transfer in biofilm mediated by a temperate bacteriophage. The study shows that the biofilm environment can be suitable for transduction events and can thereby be an environment for the emergence of new pathogenic E. coli.

Pathogenic potential and horizontal gene transfer in ovine gastrointestinal Escherichia coli.

AIMS: To demonstrate that a thorough characterization and virulotyping of Escherichia coli strains isolated from sheep over time leads to new insights into ovine E. coli potentially becoming human pathogens through horizontal gene transfer. METHODS AND RESULTS: One hundred and fifty E. coli isolates from two sheep, sampled over 3 weeks, were characterized by serotyping, virulotyping, genotyping using multiple locus variable number tandem repeats analysis (MLVA) and susceptibility to phage infection in vitro. The 35 MLVA profiles and the serotype and virulotypes of the strains were closely associated. Many MLVA profiles differed in one locus independent of serotypes. Escherichia coli isolates of the same serotype or virulotype had identical or very similar MLVA profiles. No transductants that incorporated the bacteriophages were found in vivo, but six E. coli isolates were susceptible to the phage infection in vitro. Changes in MLVA profiles were seen after acquisition of Stx phages in vitro only. CONCLUSIONS: The sheep carried Stx phage susceptible E. coli that possessed virulence markers associated with human pathogenicity. Changes in bacterial genomes by phage transfer may complicate outbreak source investigations. Serotype has to be taken into account when evaluating strain relationships by MLVA. SIGNIFICANCE AND IMPACT OF THE STUDY: Sheep carry E. coli that encode for virulence markers and belong to serogroups known to be human pathogens. In addition, a selection of isolates was found to be susceptible to horizontal transfer of Shiga toxin genes by means of bacteriophages in vitro, and the transfer resulted in a discernible change of the MLVA patterns of E. coli.

In vitro and in vivo assessment of phage therapy against Staphylococcus aureus causing bovine mastitis.

OBJECTIVE: The aim of this study was to assess the efficacy of lytic bacteriophages on Staphylococcus aureus causing bovine mastitis, by in vitro and in vivo assays using Galleria mellonella and murine mastitis models. METHODS: Between May and December 2016, ten S. aureus (five methicillin-resistant and five methicillin-sensitive) isolates were isolated from milk samples of cattle with mastitis in Belgium and Norway. The isolates were assessed in vitro for their susceptibility to four lytic bacteriophages (Romulus, Remus, ISP and DSM105264) and subsequently in vivo in G. mellonella larvae and in murine mastitis model. RESULTS: Romulus, Remus and ISP showed a lytic activity against the S. aureus isolates in vitro. A larvae survival rate below 50% was observed at 4 days post-inoculation (DPI) in the groups infected with a methicillin-sensitive S. aureus isolate and treated with these three phages in vivo. An incomplete recovery of the mouse mastitis was observed at 48h post-inoculation (HPI) in the groups infected and treated with the ISP phage in vivo. CONCLUSIONS: The observations are much more pronounced statistically between the infected- phosphate buffered saline (PBS)-treated and infected-phage-treated groups in G. mellonella and the murine mastitis model demonstrating an effect of the phages against S. aureus associated with bovine mastitis.

Is lack of susceptible recipients in the intestinal environment the limiting factor for transduction of Shiga toxin-encoding phages?

AIM: To determine whether a Shiga toxin 2 (Stx2)-encoding phage from Escherichia coli O157:H7 could be transmitted to commensal E. coli in a ruminant host without adding a specific recipient strain. METHODS AND RESULTS: Sheep were inoculated with an E. coli O157:H7 strain containing an Stx2-encoding bacteriophage (Phi3538) in which a chloramphenicol-resistant gene, cat, is inserted into stx(2). A total of 149 faecal samples were sampled and analysed for detection and quantification of E. coli O157:H7 and presumptive transductants. Phage Phi3538 (Deltastx(2)::cat) was demonstrated to be transduced to an ovine E. coli O175:H16 at one occasion. CONCLUSIONS: The study demonstrates an in vivo transduction in sheep from an E. coli O157:H7 strain to an ovine E. coli O175:H16. A functional Stx2-encoding phage was incorporated into the host's DNA. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first in vivo stx phage transduction study reported in which a recipient strain was not fed to the test animals. We suggest that the access to susceptible hosts is one main limiting factor for transduction to occur in the intestine.

Diversity of Escherichia coli O157 in a longitudinal farm study using multiple-locus variable-number tandem-repeat analysis.

AIMS: To perform a longitudinal study of the diversity of Escherichia coli O157 from a ruminant pasture/stream environment using multiple-locus variable-number tandem-repeat analysis (MLVA). METHODS AND RESULTS: Samples of faecal droppings from grazing ruminants and from an adjacent stream were tested longitudinally for E. coli O157 by enrichment and immunomagnetic separation (IMS). Using MLVA, 24 different profiles were identified from a total of 231 E. coli O157 isolates, of which 80 were included in a similarity analysis. Four main clusters with several subclusters were observed. Although there was close contact between sheep and cattle during the study period, E. coli O157 was surprisingly not detected from cattle faeces. CONCLUSIONS: The cluster analysis indicated both unrelated and closely related E. coli O157 strains. The choice of loci to target in MLVA is important for the subtyping result, as loci with high diversities are essential for discriminating between closely related isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: There is a lack of data available on the use of MLVA to describe E. coli O157 diversity and changes over time in the animal reservoirs and the environment. Such data are needed in order to further develop MLVA as a typing method.

Tetracycline resistance genes in Kenyan hospital isolates of Salmonella typhimurium.

All 97 strains of Salmonella typhimurium isolated from patients at a hospital in Nairobi, Kenya, during 1988-90 were resistant to tetracycline. The minimum inhibitory concentration (MIC) showed a large distribution range from 1 microgram/ml to 128 micrograms/ml. The strains were heterogeneous with respect to plasmid content, but initially all strains possessed, in addition to other plasmids, a large 60-, 63- or 65-MDa plasmid. The tetracycline resistance genes were characterized using oligonucleotide probes, and 20% of the resistant strains possessed tetracycline type A (tetr A), 6% tetr B, and 4% tetrC genes. Three strains possessed both type A and B tetracycline resistance determinants, which were shown to be located on the large 65-MDa plasmid. There was no correlation between strains isolated from stools, blood, cerebrospinal or epidural fluids, pus, or urine, with respect to the tetracycline genotypes, MIC values or plasmid content.

Persistence of vancomycin-resistant enterococci (VRE) on Norwegian broiler farms.

Five Norwegian broiler farms previously identified as housing broilers carrying vancomycin-resistant enterococci (VRE) were examined for the presence of VRE 4 years after avoparcin was banned. Environmental samples were obtained from empty, cleaned broiler houses. Faecal samples were collected weekly from the flock housed after the environmental sampling. The hatchery from where the chicks originated was also sampled. VRE were found to be present in the farm environment after depopulation and cleanup of the broiler houses. Within 3 weeks after introduction to the farm, all broiler flocks tested positive for VRE. VRE were not isolated from the hatchery.

Immunomagnetic separation of a Norwalk-like virus (genogroup I) in artificially contaminated environmental water samples.

Rabbit polyclonal antibodies were raised against a recombinant capsid protein from a genogroup I Norwalk-like virus (NLV). Magnetic beads coated with these antibodies were used in immunomagnetic separation (IMS) of the NLV. After capture of the NLV and washing of the beads, viral RNA was heat released and detected by RT-PCR. This IMS procedure was shown to have high sensitivity for detection of homologous NLV, while capture of a genogroup II NLV was less efficient. Antigen capture was not influenced by the content of humic acids in the samples. The combination of IMS and heat release was found to be more efficient than organic extraction of RNA from water contaminated with humic acids. The efficacy and simplicity of IMS/heat release render this combination a feasible tool for the preparation of NLV RNA from environmental samples, although the antigenic diversity of NLV may be a complicating factor.

Pathogenic Escherichia coli found in food.

The bacteria constituting the species Escherichia coli are commonly found in the intestinal flora of man and animals, and were until late 1950s recognized as non-pathogenic normal cohabitants. However, certain strains might induce disease, and E. coli should therefore be regarded as a potential pathogenic organism. The pathogenic strains can cause distinct disease syndrome as different diarrheal diseases, wound infections, meningitis, septicemia, artherosclerosis, hemolytic uremic syndrome and immunological diseases such as reactive and rheumatoid arthritis. Several different groups of diarrhea-inducing strains are known. The enterotoxigenic E. coli (ETEC) strains produce one or more of toxins from the heat-labile and the heat-stable enterotoxin families. These strains possess specific adhesion fimbria for intestinal attachment and colonization. Some enteropathogenic E. coli strains (EPEC) produce one or more of the cytotoxins, but adhere also to intestinal cells interfering with the electrolyte transport system. The group of strains possessing invasive properties are designated enteroinvasive E. coli (EIEC). Recently, the enterohemorrhagic E. coli (EHEC) strains have been identified and shown to produce one or more of the cytotoxins (vero-cytotoxins, shiga-like toxins). Food originating from warm-blooded animals may be contaminated with E. coli, but contamination from human sources are more common for food involved in outbreak of disease. In general, strains causing disease in animals do possess other colonization factors than those found on human pathogenic strains. EIEC strains are, like Shigella, only known to induce disease in man. However, both healthy and sick cattle are suspected to be a major reservoir for EHEC strains, and several outbreaks have been associated with consumption of meat or meat products. Cheeses have been the source of outbreaks of both ETEC and EIEC in Europe and the USA, while water seems to be a major source for the different diarrheic E. coli strains affecting children and tourists in the 3rd world. Strains causing non-enteric disease are less known as being transmitted to humans with food as a vector, but the importance of some of these diseases, should implicate further research on what role food plays in spreading these organisms. The recipient of the potential pathogenic E. coli through food, the humans, are also of different risk of contracting diseases.(ABSTRACT TRUNCATED AT 400 WORDS)

High levels of background flora inhibits growth of Escherichia coli O157:H7 in ground beef.

The influence of natural background flora under aerobic and anaerobic incubation on the growth of Escherichia coli O157:H7 in ground beef was investigated. The background flora from eight different commercial ground beef were added to ground beef spiked with E. coli O157:H7 and stored either aerobically or anaerobically at 12 degrees C. The results showed that the presence of a large number of background bacteria in the ground meat inhibited the growth of E. coli O157:H7 both aerobically and anaerobically. Inhibition was more pronounced under anaerobic conditions. The background floras consisted mainly of lactic acid bacteria of which approximately 80% were Lactobacillus sakei. These results show the importance of the natural background flora in meat for inhibition of growth of E. coli O157:H7.

Detection of small round structured viruses in artificially contaminated water using filter adsorption and reverse transcription polymerase chain reaction.

The small round structured viruses (SRSV) are common causes of gastroenteritis worldwide. Fecally contaminated water is an important vehicle for transmission, but detection of SRSV in environmental samples has been hampered by the lack of sensitive detection methods. The present work describes the detection of SRSV in artificially contaminated deionized water and raw drinking water. SRSV-containing fecal extracts were added to water and virus was recovered by filter adsorption-elution, followed by flocculation. RNA was extracted and SRSV were detected by the use of reverse transcription polymerase chain reaction. The sensitivity of the method corresponded to a positive SRSV detection in 500 ml deionized water with an estimated concentration of 0.5-5 virus particles per ml.

VanA-type vancomycin-resistant enterococci (VRE) remain prevalent in poultry carcasses 3 years after avoparcin was banned.

Avoparcin was used as a growth promoting feed additive in Norwegian broiler and turkey production from 1986 until it was banned in 1995, when an association between vancomycin-resistant enterococci (VRE) and avoparcin use became apparent. A recent study regarding faecal samples documented a continuing high prevalence of VRE among Norwegian poultry 3 years after avoparcin was banned. In the present study, carcasses of broilers and turkeys from farms where avoparcin had previously been in use and carcasses of layer chickens from farms where avoparcin had never been used were examined for the presence of VRE. One carcass from each of 150 different farms was included. By a direct plating method, VRE were isolated from 30 of 100 samples of broilers and turkeys, but not from any samples of layer chickens. When an enrichment step was included, VRE were isolated from a total of 81 of the 100 samples of broilers and turkeys and from nine of the 50 samples of layer chickens. All VRE isolated were highly resistant to vancomycin (MIC > or = 256 microg/ml) and possessed the vanA gene. These results correspond to the prevalence of VRE recently documented in faecal samples from Norwegian poultry. The present study reveals a high prevalence of VRE in broiler and turkey carcasses. Consequently, consumers are exposed to VRE when handling raw poultry meat, although the public health significance of such exposure is unclear.

Mould contaminants on Jarlsberg and Norvegia cheese blocks from four factories.

Visible mould from 225 blocks of the Norwegian semi-hard cheeses Jarlsberg and Norvegia from four factories were subcultured and identified. Altogether 23 different fungal species were detected. The two most important contaminating species were Penicillium commune and P. palitans, constituting 21.4% and 17.9% of the total isolates, respectively. The other dominating contaminants were P. roqueforit spp. roqueforti, Geotrichum candidum, P. solitum and P. crustosum. These species, together with P. commune and P. palitans, represented 80.9% of the total isolates. P. commune, P. palitans, P. roqueforti spp. roqueforti and P. solitum were most common contaminants on cheese produced in all four factories, while G. candidum was found to be important on Jarlsberg cheese from only one factory. P. crustosum was one of the dominating species on Norvegia cheese.

Prevalence of serum antibodies to Norwalk virus among Norwegian military recruits.

The prevalence of serum antibodies against Norwalk virus among military recruits in Norway was investigated using an enzyme-linked immunosorbent assay (ELISA). A total of 1017 sera were assayed for anti-Norwalk virus total antibodies (ig), of which 300 (29.5%) were positive. Of 227 positive sera, 10.6 and 15.4% were positive for IgA and IgM, respectively, while 2.2% were positive for both. The prevalence of antibodies against Norwalk virus in south-east Norway was significantly higher than that in northern Norway.

Escherichia coli O157:H7 in faeces from cattle, sheep and pigs in the southwest part of Norway during 1998 and 1999.

During a 2-year period from January 1998 to December 1999, intestinal content from 1541 cattle, 665 sheep and 1976 pigs were analysed for Escherichia coli O157:H7 using the immunomagnetic separation procedure. The animals originated from 848, 605 and 832 herds from the southwest part of Norway, respectively. E. coli O157:H7 was present in three samples from cattle from different herds, giving a herd prevalence of 0.35% and an animal prevalence of 0.19%. From pigs, E. coli O157:H7 was isolated from two pigs from different herds, giving a herd prevalence of 0.24% and an animal prevalence of 0.1%. A follow-up study revealed another positive testing pig from one of these herds. E. coli O157:H7 was not found from any of the 665 investigated sheep. By PCR analysis, all six E. coli O157:H7 isolates were shown to contain the genes encoding Shiga toxin 2 (stx2), the intimin protein (eae) and the H7 flagellum (fliC-H7). One of the cattle isolates also harboured the Shiga toxin 1 encoding (stx1) gene. The six isolates were differentiated into three pulse-field gel electrophoresis profiles. The results indicate that the occurrence of E. coli O157:H7 in cattle, sheep and pigs in the southwest part of Norway is low compared to other European countries.

Characterization of Escherichia coli strains isolated from pigs with edema disease.

Escherichia coli strains belonging to serogroups O 138 and O 139 isolated from pigs with edema disease, were characterized with respect to the presence of genes encoding Shiga-like toxin I, Shiga-like toxin II and Shiga-like toxin IIv (SLT I, SLT II and SLT IIv). Genes coding for the heat-stable and heat-labile enterotoxins (ST I and LT I) were also detected. Plasmid profiling, restriction enzyme digestion of total DNA, and ribotyping were performed for further characterization of the strains. The oligonucleotide probes applied in this study appeared to be useful tools for detecting genes coding cytotoxins and enterotoxins. DNA from 12 of 16 strains hybridized with two SLT II probes, and DNA from two SLT IIv encoding strains also hybridized with the ST I probe. DNA from one SLT IIv negative strain hybridized with the LT I probe. The results from plasmid profiling, restriction enzyme digestion, and ribotyping were compared with serogrouping in attempts to distinguish between the different E. coli edema disease isolates. Fourteen different plasmid profiles were identified, and as restriction patterns barely did, and ribotyping patterns did not, reveal any information useful for differentiation of the strains beyond serogroup level, plasmid profiling seemed to be the most suitable method for discrimination between the edema disease strains investigated here.

Characterization of antibiotic resistance in Streptococcus suis.

Twenty one isolates of Streptococcus suis were screened for antibiotic resistance by growth on antibiotic-containing media, by measuring minimum inhibitory concentrations, by hybridization to specific DNA and oligonucleotide probes for antibiotic resistance genes, and by PCR. The isolates were from a slaughter house survey of respiratory pathogens in Norwegian pigs in 1986. Fifteen isolates were resistant to tetracycline, with MICs ranging from 4-128 micrograms/ml. Genes coding for the Tet O and Tet M determinants were detected in eight and five isolates, respectively. Genes coding for other Gram positive Tet determinants, Tet K, Tet L, and Tet P, were not detected. One isolate was constitutive resistant to erythromycin with MIC of 128 micrograms/ml. Five other isolates carried inducible erythromycin resistance. All these isolates, and five others, were positive in a PCR assay for erythromycin resistance, and hybridized with the Erm C and/or Erm B probes. No resistance against chloramphenicol (5 micrograms/ml) or rifampin (10 micrograms/ml) could be could be detected, but five isolates were resistant to streptomycin (250 micrograms/ml), four isolates were resistant to kanamycin (10 micrograms/ml), and one isolate was resistant to fusicic acid (10 micrograms/ml). In mating experiments with Enterococcus faecalis JH2-2 as recipient, tetracycline, erythromycin, and kanamycin genes were transferred separately to the recipient strain at a rate of 10(-7) transconjugants/recipient cell.

Characterization of tetracycline and erythromycin resistance in Actinobacillus pleuropneumoniae.

Minimum inhibitory concentrations to tetracycline and erythromycin were determined for nineteen isolates of Actinobacillus pleuropneumoniae of Norwegian origin. The isolates were screened for rRNA methylase determinants (Erm genes) and for the tetracycline resistance Tet B determinant, using oligonucleotide probes, polymerase chain reaction and hybridization. Ten isolates (53%) carried the Erm C determinant, two isolates (10%) carried the Erm A determinant, four isolates (21%) carried both the Erm A and the Erm C determinants, and three isolates (16%) carried none of the Erm determinants examined. Eight isolates (45%) carried the Tet B determinant. Selected isolates were shown to transfer the Erm C and Erm A determinants at a frequency of 10(-7)-10(-9) per recipient cell. This is the first description of A. pleuropneumoniae carrying either Erm A, Erm C or/and Tet B determinants.

Occurrence and characterization of Escherichia coli O157 isolated from cattle in Norway.

Faecal samples from 504 imported beef cattle were screened to investigate the occurrence of Escherichia coli O157. The results were compared with those from a previous screening of Norwegian dairy cattle, and the occurrence was found to be higher in the imported beef cattle. The E. coli O157 isolates from the previous and present studies were characterized for the genes encoding for shigatoxin 1 (stx1), shigatoxin 2 (stx2), the intimin protein (eae) and the flagellar protein H7 (fliC) using PCR analysis, pulsed-field gel electrophoresis (PFGE) with the restriction enzyme XbaI, and bacteriophage lambda RFLP analysis using the PvuII restriction enzyme. The isolates from the dairy and beef cattle could be distinguished by the profiles of the toxin genes and by PFGE patterns. Whether the importation of animals in itself should be regarded as a risk factor for the occurrence of E. coli O157, or whether other management factors contribute to the differences in carrier rates compared to the previous study on domestic cattle, is discussed.