Harmonisation of in-silico next-generation sequencing based methods for diagnostics and surveillance.

Improvements in cost and speed of next generation sequencing (NGS) have provided a new pathway for delivering disease diagnosis, molecular typing, and detection of antimicrobial resistance (AMR). Numerous published methods and protocols exist, but a lack of harmonisation has hampered meaningful comparisons between results produced by different methods/protocols vital for global genomic diagnostics and surveillance. As an exemplar, this study evaluated the sensitivity and specificity of five well-established in-silico AMR detection software where the genotype results produced from running a panel of 436 Escherichia coli were compared to their AMR phenotypes, with the latter used as gold-standard. The pipelines exploited previously known genotype-phenotype associations. No significant differences in software performance were observed. As a consequence, efforts to harmonise AMR predictions from sequence data should focus on: (1) establishing universal minimum to assess performance thresholds (e.g. a control isolate panel, minimum sensitivity/specificity thresholds); (2) standardising AMR gene identifiers in reference databases and gene nomenclature; (3) producing consistent genotype/phenotype correlations. The study also revealed limitations of in-silico technology on detecting resistance to certain antimicrobials due to lack of specific fine-tuning options in bioinformatics tool or a lack of representation of resistance mechanisms in reference databases. Lastly, we noted user friendliness of tools was also an important consideration. Therefore, our recommendations are timely for widespread standardisation of bioinformatics for genomic diagnostics and surveillance globally.

EUCAST disc diffusion criteria for the detection of mecA-Mediated ß-lactam resistance in Staphylococcus pseudintermedius: oxacillin versus cefoxitin.

OBJECTIVES: Until recently, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommended the cefoxitin disc to screen for mecA-mediated ß-lactam resistance in Staphylococcus pseudintermedius. A recent study indicated that cefoxitin was inferior to oxacillin in this respect. We have re-evaluated cefoxitin and oxacillin discs for screening for methicillin resistance in S. pseudintermedius. METHODS: We included 224 animal and human S. pseudintermedius isolates from Europe (n = 108) and North America (n = 116), of which 109 were mecA-positive. Disc diffusion was performed per EUCAST recommendations using 30-µg cefoxitin and 1-µg oxacillin discs from three manufacturers and Mueller-Hinton agar from two manufacturers. RESULTS: Cefoxitin inhibition zones ranged from 6 to 33 mm for mecA-positive S. pseudintermedius (MRSP) and from 29 to 41 mm for mecA-negative S. pseudintermedius (MSSP). The corresponding oxacillin zone intervals were 6-20 mm and 19-30 mm. For cefoxitin 16% (95% CI 14.8-18.0%) of the isolates were in the area where positive and negative results overlapped. For oxacillin the corresponding number was 2% (1.6-2.9%). For oxacillin a breakpoint of susceptible (S) ≥ 20 mm and resistant (R) <20 mm resulted in only 0.4% and 1.1% very major error and major error rates respectively. CONCLUSIONS: This investigation confirms that the 1-µg oxacillin disc predicts mecA-mediated methicillin resistance in S. pseudintermedius better than the 30-µg cefoxitin disc. For a 1-µg oxacillin disc we propose that 20 mm should be used as cut off for resistance, i.e. isolates with a zone diameter <20 mm are resistant to all ß-lactam antibiotics except those with activity against methicillin-resistant staphylococci.

MRI defecography of the ileal pouch-anal anastomosis-contributes little to the understanding of functional outcome.

PURPOSE: Variability in functional outcome after ileal pouch-anal anastomosis (IPAA) is to a large extent unexplained. The aim of this study was to use MRI to evaluate the morphology, emptying pattern and other pathology that may explain differences in functional outcome between well-functioning and poorly functioning pouch patients. A secondary aim was to establish a reference of normal MRI findings in pelvic pouch patients. METHODS: From a previous study, the best and worst functioning patients undergoing IPAA surgery between 2000 and 2013 had been identified and examined with manovolumetric tests (N = 47). The patients were invited to do a pelvic MRI investigating pouch morphology and emptying patterns, followed by a pouch endoscopy. RESULTS: Forty-three patients underwent MRI examination. We found no significant morphological or dynamic differences between the well-functioning and poorly functioning pouch patients. There was no correlation between urge volume and the volume of the bony pelvis, and no correlation between emptying difficulties or leakage and dynamic MRI findings. Morphological MRI signs of inflammation were present in the majority of patients and were not correlated to histological signs of inflammation. Of the radiological signs of inflammation, only pouch wall thickness correlated to endoscopic pouchitis disease activity index scores. CONCLUSION: It seems MRI does not increase the understanding of factors contributing to functional outcome after ileal pouch-anal anastomosis. Unless there is a clinical suspicion of perianal/peripouch disease or pelvic sepsis, MRI does not add value as a diagnostic tool for pelvic pouch patients. Endoscopy remains the golden standard for diagnosing pouch inflammation.

Norwegian patients and retail chicken meat share cephalosporin-resistant Escherichia coli and IncK/blaCMY-2 resistance plasmids.

OBJECTIVES: In 2012 and 2014 the Norwegian monitoring programme for antimicrobial resistance in the veterinary and food production sectors (NORM-VET) showed that 124 of a total of 406 samples (31%) of Norwegian retail chicken meat were contaminated with extended-spectrum cephalosporin-resistant Escherichia coli. The aim of this study was to compare selected cephalosporin-resistant E. coli from humans and poultry to determine their genetic relatedness based on whole genome sequencing (WGS). METHODS: Escherichia coli representing three prevalent cephalosporin-resistant multi-locus sequence types (STs) isolated from poultry (n=17) were selected from the NORM-VET strain collections. All strains carried an IncK plasmid with a blaCMY-2 gene. Clinical E. coli isolates (n=284) with AmpC-mediated resistance were collected at Norwegian microbiology laboratories from 2010 to 2014. PCR screening showed that 29 of the clinical isolates harboured both IncK and blaCMY-2. All IncK/blaCMY-2-positive isolates were analysed with WGS-based bioinformatics tools. RESULTS: Analysis of single nucleotide polymorphisms (SNP) in 2.5 Mbp of shared genome sequences showed close relationship, with fewer than 15 SNP differences between five clinical isolates from urinary tract infections (UTIs) and the ST38 isolates from poultry. Furthermore, all of the 29 clinical isolates harboured IncK/blaCMY-2 plasmid variants highly similar to the IncK/blaCMY-2 plasmid present in the poultry isolates. CONCLUSIONS: Our results provide support for the hypothesis that clonal transfer of cephalosporin-resistant E. coli from chicken meat to humans may occur, and may cause difficult-to-treat infections. Furthermore, these E. coli can be a source of AmpC-resistance plasmids for opportunistic pathogens in the human microbiota.

Restorative proctocolectomy with two different pouch designs: few complications with good function.

AIM: The object of this study was to compare function and quality of life after restorative proctocolectomy (RPC) with ileal pouch-anal anastomosis (IPAA) surgery having two different pouch designs. METHOD: Patients having RPC in an academic unit from 2000 who had had the loop-ileostomy closed by June 2013 were identified from the hospital medical records. They were sent a questionnaire regarding quality of life and interviewed using a pouch function score (PFS) described by Oresland (score 0-16, higher scores denote worse function). RESULTS: One hundred and three patients underwent surgery, of whom 56 had a J-pouch design and 47 a K-pouch design, this being a double-folded Kock pouch without the nipple valve. No patients have had the pouch removed or defunctioned due to failure at a mean of 8 years. The reoperation rate was 11.6%. The mean PFS was 5.43 and 5.27 for J- and K-pouches, respectively (P = 0.766). More patients with a J-pouch reported a social handicap due to poor bowel function (P = 0.041). Patients with a PFS ≥ 8 had a poorer quality of life. A score of ≥ 8 was reported by 16% of K-pouch and 25% of J-pouch patients (P = 0.29). CONCLUSION: RPC is a safe procedure with a low complication rate and good functional outcome. Small improvements in function have an impact on a patient's quality of life. Although the J-pouch is the most commonly used, the K-pouch has some advantages. Other pouch designs deserve further evaluation.

Complications in surgery for Crohn's disease after preoperative antitumour necrosis factor therapy.

BACKGROUND: The use of biological therapy (biologicals) is established in the treatment of Crohn's disease. This study aimed to determine whether preoperative treatment with biologicals is associated with an increased rate of complications following surgery for Crohn's disease with intestinal anastomosis. METHODS: All patients receiving biologicals and undergoing abdominal surgery with anastomosis or strictureplasty were identified at six tertiary referral centres. Demographic data, and preoperative, operative and postoperative details were registered. Patients who were treated with biologicals within 2 months before surgery were compared with a control group who were not. Postoperative complications were classified according to anastomotic, infectious or other complications, and graded according to the Clavien-Dindo classification. RESULTS: Some 111 patients treated with biologicals within 2 months before surgery were compared with 187 patients in the control group. The groups were well matched. There were no differences between the treatment and control groups in the rate of complications of any type (34·2 versus 28·9 per cent respectively; P = 0·402), anastomotic complications (7·2 versus 8·0 per cent; P = 0·976) and non-anastomotic infectious complications (16·2 versus 13·9 per cent; P = 0·586). In univariable regression analysis, biologicals were not associated with an increased risk of any complication (odds ratio (OR) 1·33, 95 per cent confidence interval 0·81 to 2·20), anastomotic complication (OR 0·89, 0·37 to 2·17) or infectious complication (OR 1·09, 0·62 to 1·91). CONCLUSION: Treatment with biologicals within 2 months of surgery for Crohn's disease with intestinal anastomosis was not associated with an increased risk of complications.

Driving anger and metacognition: the role of thought confidence on anger and aggressive driving intentions.

The present studies examined the self-validating role of anger within provoking driving situations, using a scenario method. Specifically, we predicted that one reason for why individuals higher (rather than lower) in trait driving anger are more likely to aggress when provoked is because these individuals are more confident in their thoughts resulting from the provocation. Higher thought confidence, in turn, may influence the amount of anger experienced and the extent to which the anger translates into aggressive behavior. Study 1 found that participants higher in driving anger were more confident in their thoughts in a provoking situation and their thought confidence mediated the effect of trait driving anger on anger in response to the provocation. Using a manipulation of consistency, Study 2 found that thought confidence mediated the influence of anger on aggressive driving intentions, but only for individuals higher in driving anger. The current research adds to the growing work examining a new mechanism by which emotion (e.g., anger) can affect behavior.

Two outbreaks of diarrhoea in nurseries in Norway after farm visits, April to May 2009.

During a 2009 nationwide outbreak of sorbitolfermenting Escherichia coli O157 in Norway, the Norwegian Institute of Public Health was notified of diarrhoea outbreaks in two nurseries. A link to the nationwide outbreak was suspected and investigated, including retrospective cohort studies. Both nurseries had recently visited farms. Faecal specimens were obtained from symptomatic children as well as from the farm animals and tested for Campylobacter, Salmonella, Yersinia, Shigella and pathogenic E. coli, and isolates were further characterised. Nursery A had 12 symptomatic children, and we found the same strain of C. jejuni in faeces from children and lambs. Nursery B had nine symptomatic children, including one child with bloody diarrhoea carrying enterohaemorrhagic E. coli (EHEC) O26. EHEC O26 with a similar multiple-locus variable number tandem repeat analysis (MLVA)-profile was found in sheep. Five children had enteropathogenic E. coli (EPEC) O76. Animals were not tested for EPEC O76. We found no significant association between illness and risk factors for either nursery. The isolated pathogens differed from the one involved in the nationwide outbreak. In each nursery outbreak, the pathogens isolated from children matched those found in farm animals, implicating animal faeces as the source. Hygiene messages are important to prevent similar outbreaks.

The influence of just-world beliefs on driving anger and aggressive driving intentions.

Decades of research demonstrate that the extent to which one believes the world is just can have important interpersonal consequences. Unfortunately, most of the commonly studied consequences are negative in nature. Guided by previous research demonstrating the buffering effect of just-world beliefs and anger, the present research explores how belief in a just world (BJW) may mitigate anger in the domain of driving anger and examines the limiting conditions of this effect. Study 1 demonstrated the expected negative relation between common measures of BJW and anger expression in a driving context. Study 2 found that the buffering effects of just-world beliefs and driver aggression were greater when BJW was violated (vs. not). Study 3 replicated the effects on aggression and anger and established a mediational role of anger on the buffering effects of just-world beliefs on thoughts and driver aggression.

Persistence of staphylococcal species and genotypes in the bovine udder.

Staphylococci are a major cause of intramammary infections (IMI) in ruminants. The main aim of this study was to investigate staphylococcal IMI in dairy cattle with emphasis on persistence and distribution of staphylococcal species and genotypes. With a sampling interval of 4-8 weeks, over a year, 4030 samples from 206 cows in 4 herds were collected. Coagulase-negative staphylococci (CNS) and Staphylococcus aureus were detected in 13.2% and 4.2% of the samples, respectively. Selected CNS isolates from quarter milk samples were identified to species level using sodA sequencing. Staphylococcus chromogenes (32%) and Staphylococcus simulans (25%) predominated. The proportion of S. chromogenes was greater in primiparous (52%) than in multiparous cows (12%), while the opposite was the case for Staphylococcus epidermidis (6% and 21%, respectively). Isolates from possibly persistent IMI were selected for pulsed-field gel electrophoresis (PFGE). Six staphylococcal species were found to cause persistent IMI; S. aureus, S. chromogenes, S. simulans, S. epidermidis, Staphylococcus haemolyticus and Staphylococcus warneri. It was shown that several pulsotypes (PTs) within each species were associated with persistent infections, but only a few were spread and caused persistent IMI in multiple cows within a herd. Of special interest was the observation that only one, or a few, strains of each species caused persistent IMI in multiple cows within a same herd. This indicates strain differences with respect to transmissibility and pathogenicity.

Potentially human-pathogenic Escherichia coli O26 in Norwegian sheep flocks.

A national survey of Escherichia coli O26 in Norwegian sheep flocks was conducted, using fecal samples to determine the prevalence. In total, 491 flocks were tested, and E. coli O26 was detected in 17.9% of the flocks. One hundred forty-two E. coli O26 isolates were examined for flagellar antigens (H typing) and four virulence genes, including stx and eae, to identify possible Shiga toxin-producing E. coli (STEC) and enteropathogenic E. coli (EPEC). Most isolates (129 out of 142) were identified as E. coli O26:H11. They possessed eae and may have potential as human pathogens, although only a small fraction were identified as STEC O26:H11, giving a prevalence in sheep flocks of only 0.8%. Correspondingly, the sheep flock prevalence of atypical EPEC (aEPEC) O26:H11 was surprisingly high (15.9%). The genetic relationship between the E. coli O26:H11 isolates was investigated by pulsed-field gel electrophoresis (PFGE) and multilocus variable number tandem repeat analysis (MLVA), identifying 63 distinct PFGE profiles and 22 MLVA profiles. Although the MLVA protocol was less discriminatory than PFGE and a few cases of disagreement were observed, comparison by partition mapping showed an overall good accordance between the two methods. A close relationship between a few isolates of aEPEC O26:H11 and STEC O26:H11 was identified, but all the E. coli O26:H11 isolates should be considered potentially pathogenic to humans. The present study consisted of a representative sampling of sheep flocks from all parts of Norway. This is the first large survey of sheep flocks focusing on E. coli O26 in general, including results of STEC, aEPEC, and nonpathogenic isolates.

Factors in fish modifying methylmercury toxicity and metabolism.

We report here some results of a long-term (19 month) study with cats fed methylmercury (MeHg) in nutritionally balanced diets based on fish. By using either freshwater pike (low in Se) or canned tuna (high in Se) as the major protein source, basal diets with low levels of MeHg were prepared having different Se content, all Se being of natural origin. The basal diets produced no signs of toxicity or pathological changes over the l9-month period. In cats fed basal diets spiked with medium or high levels of MeHg, evidence for delayed onset of toxic effects from the added MeHg was observed with the tuna diets compared to pike diets. In brain, muscle, and blood, the activity of GSH peroxidase, a selenoenzyme, was decreased by Hg. In liver, substantial accumulation of Hg with Se occured (molar Hg/Se ratio approximately 1.4 to 1.8) but GSH peroxidase activity was unaffected. We suggest that the coaccumulation of Hg and Se in liver measures the extent to which MeHg has been metabolically transformed by metabolism to Hg++, and inactivated by deposition as a Hg/Se complex of low bioavailability. The accumulation of Hg and Se in liver was much greater in cats fed tuna compared to pike, out of proportion to the relatively small differences in Hg and Se content of the tuna and pike basal diets. Some mechanisms are described by which selenium, vitamin E, and other factors might facilitate MeHg breakdown to inorganic Hg during long term low level exposure to MeHg.

Mechanism of block of the hERG K+ channel by the scorpion toxin CnErg1.

The scorpion toxin CnErg1 binds to human ether-a-go-go related gene (hERG) K(+) channels with a 1:1 stoichiometry and high affinity. However, in contrast to other scorpion toxin-ion channel interactions, the inhibition of macroscopic hERG currents by high concentrations of CnErg1 is incomplete. In this study, we have probed the molecular basis for this incomplete inhibition. High concentrations of CnErg1 had only modest effects on hERG gating that could not account for the incomplete block. Furthermore, the residual current in the presence of 1 microM CnErg1 had normal single channel conductance. Analysis of the kinetics of CnErg1 interaction with hERG indicated that CnErg1 binding is not diffusion-limited. A bimolecular binding scheme that incorporates an initial encounter complex and permits normal ion conduction was able to completely reproduce both the kinetics and steady-state level of CnErg1-hERG binding. This scheme provides a simple kinetic explanation for incomplete block; that is, relatively fast backward compared to forward rate constants for the interconversion of the toxin-channel encounter complex and the blocked toxin-channel complex. We have also examined the temperature-dependence of CnErg1 binding to hERG. The dissociation constant, K(d), for CnErg1 increases from 7.3 nM at 22 degrees C to 64 nM at 37 degrees C (i.e., the affinity decreases as temperature increases) and the proportion of binding events that lead to channel blockade decreases from 70% to 40% over the same temperature range. These temperature-dependent effects on CnErg1 binding correlate with a temperature-dependent decrease in the stability of the putative CnErg1 binding site, the amphipathic alpha-helix in the outer pore domain of hERG, assayed using circular dichroism spectropolarimetry. Collectively, our data provides a plausible kinetic explanation for incomplete blockade of hERG by CnErg1 that is consistent with the proposed highly dynamic conformation of the outer pore domain of hERG.

Microbiological and biochemical assessment of the surface area of breast nipples and breast milk of lactating women in Calabar, southeastern Nigeria.

Microbiological and biochemical analyses of 59 breast nipple swab samples and 15 manually expressed breast milk samples of lactating mothers aged 15 to 40 years, was carried out using standard procedures. The incidence of bacterial species in swab samples was Staphylococus aureus (63.8%), Streptococcus sp (25.5%), Escherichia coli (6.4%) and Klebsiella sp (4.26%), indicating the poor sanitary status of the lactating mothers. S. aureus was recovered from only 1 (6.7%) of the milk samples, indicating that breast milk is relatively sterile. The nutritional values for the breast milk were 22.5 mg/ml (protein), 0.3 mg/ml (calcium), 3.5 mg/ml (sugar) and 300 microg/ml (vitamin A) in age group 15-20 years, and 16.4 mg/ml (protein), 0.16 mg/ml (calcium), 1.8 mg/ml (sugar) and 100 microg/ml (vitamin A) in the age group 36-40 years. In conclusion, the nutritive and antimicrobial properties of breast milk decrease with increasing age of lactating mothers. The need for public health enlightenment of lactating mothers regarding hygiene, and the provision of oral vitamin A supplement to infants, is discussed.

Structural basis for rodlet assembly in fungal hydrophobins.

Class I hydrophobins are a unique family of fungal proteins that form a polymeric, water-repellent monolayer on the surface of structures such as spores and fruiting bodies. Similar monolayers are being discovered on an increasing range of important microorganisms. Hydrophobin monolayers are amphipathic and particularly robust, and they reverse the wettability of the surface on which they are formed. There are also significant similarities between these polymers and amyloid-like fibrils. However, structural information on these proteins and the rodlets they form has been elusive. Here, we describe the three-dimensional structure of the monomeric form of the class I hydrophobin EAS. EAS forms a beta-barrel structure punctuated by several disordered regions and displays a complete segregation of charged and hydrophobic residues on its surface. This structure is consistent with its ability to form an amphipathic polymer. By using this structure, together with data from mutagenesis and previous biophysical studies, we have been able to propose a model for the polymeric rodlet structure adopted by these proteins. X-ray fiber diffraction data from EAS rodlets are consistent with our model. Our data provide molecular insight into the nature of hydrophobin rodlet films and extend our understanding of the fibrillar beta-structures that continue to be discovered in the protein world.

Organization of the antiseptic resistance gene qacA and Tn552-related beta-lactamase genes in multidrug- resistant Staphylococcus haemolyticus strains of animal and human origins.

A part (12 kb) of a plasmid containing the beta-lactamase genes of Tn552, the disinfectant resistance gene qacA, and flanking DNA has been cloned from a Staphylococcus haemolyticus isolate and sequenced. This region was used to map the corresponding regions in six other multiresistant S. haemolyticus isolates of human and animal origin. The organizations of the genetic structures were almost identical in all isolates studied. The beta-lactamase and qacA genes from S. haemolyticus have >99.9% identities at the nucleotide level with the same genes from S. aureus, demonstrating that various staphylococcal species able to colonize animal and human hosts can exchange the genetic elements involved in resistance to antibiotics and disinfectants. The use of antibiotics and disinfectants in veterinary practice and animal husbandry may also contribute to the selection and maintenance of resistance factors among the staphylococcal species. Different parts of the 12-kb section analyzed had high degrees of nucleotide identity with regions from several other different Staphylococcus aureus plasmids. This suggests the contribution of interplasmid recombination in the evolutionary makeup of this 12-kb section involving plasmids that can intermingle between various staphylococcal species. The lateral spread of resistance genes between various staphylococcal species is probably facilitated by the generation of large multiresistance plasmids and the subsequent interspecies exchange of them.

Plasmid-borne smr gene causes resistance to quaternary ammonium compounds in bovine Staphylococcus aureus.

Resistance to quaternary ammonium compounds (QAC) in staphylococci is common in hospital environments and has been described in the food industry. Little is known about staphylococcal QAC resistance associated with animal disease, although such disinfectants are widely used in veterinary medicine. In order to investigate the occurrence of QAC resistance in staphylococci isolated from QAC-exposed animals, 32 penicillin- and tetracycline-resistant and 23 penicillin- and tetracycline-susceptible Staphylococcus aureus isolates collected from milk from cows with mastitis during a 4-year period were selected for QAC susceptibility studies and genetic characterization. The isolates originated from four different herds that used a common pasture with a joint milking parlor in the summer. During the pasture season, a teat cream containing the QAC cetyltrimethylammonium bromide had been used daily for more than 10 years for mastitis control. Three of the penicillin- and tetracycline-resistant isolates, which were recovered from three different cows during a 20-month period, were resistant to QAC. Plasmid analysis, PCR, and DNA sequencing revealed a novel plasmid of 2,239 bp containing the smr gene. The plasmid, designated pNVH99, has similarities to small, smr-containing staphylococcal plasmids previously found in human and food isolates. pNVH99 is a new member of the pC194 family of rolling-circle replication plasmids. The three QAC-resistant isolates, as well as 28 of the 29 remaining penicillin- and tetracycline-resistant isolates, were indistinguishable by pulsed-field gel electrophoresis. The study indicates that the occurrence and spread of QAC-resistant S. aureus among dairy cows may be a problem that needs further investigation.

Self-transmissible multidrug resistance plasmids in Escherichia coli of the normal intestinal flora of healthy swine.

The resistance genes and their surroundings on three self-transmissible plasmids found in Escherichia coli of the enteric normal flora of healthy pigs have been characterized. The resistance elements found are similar to those commonly found in clinical isolates, like the transposon Tn1721 including the Tet A tetracycline resistance determinant, Tn10 with the Tet B determinant, Tn21 including a class 1 integron with the aadA1a cassette inserted, sulII encoding sulfonamide resistance, and the strA-strB genes responsible for streptomycin resistance. The plasmids were able to mobilize into various recipients, including swine pathogens, zoonotic bacteria, and commensals when conjugation experiments were carried out. Transfer of plasmids did not require optimal conditions concerning nutrition and temperature as plasmids were transferred in 0.9% saline at room temperature, suggesting that in vivo transfer might be possible. This study shows that transferable resistance elements appearing in normal flora bacteria from animals are similar to those commonly found in clinical isolates of human origin. The results indicate a probable communication between pathogens and the normal flora with respect to exchange of resistance factors.

Resistance to antibiotics in the normal flora of animals.

The normal bacterial flora contains antibiotic resistance genes to various degrees, even in individuals with no history of exposure to commercially prepared antibiotics. Several factors seem to increase the number of antibiotic-resistant bacteria in feces. One important factor is the exposure of the intestinal flora to antibacterial drugs. Antibiotics used as feed additives seem to play an important role in the development of antibiotic resistance in normal flora bacteria. The use of avoparcin as a feed additive has demonstrated that an antibiotic considered "safe" is responsible for increased levels of antibiotic resistance in the normal flora enterococci of animals fed with avoparcin and possibly in humans consuming products from these animals. However, other factors like stress from temperature, crowding, and management also seem to contribute to the occurrence of antibiotic resistance in normal flora bacteria. The normal flora of animals has been studied with respect to the development of antibiotic resistance over four decades, but there are few studies with the intestinal flora as the main focus. The results of earlier studies are valuable when focused against the recent understanding of mobile genetics responsible for bacterial antibiotic resistance. New studies should be undertaken to assess whether the development of antibiotic resistance in the normal flora is directly linked to the dramatic increase in antibiotic resistance of bacterial pathogens. Bacteria of the normal flora, often disregarded scientifically, should be studied with the intention of using them as active protection against infectious diseases and thereby contributing to the overall reduction of use of antibioties in both animals and humans.