Escherichia fergusonii.

Escherichia fergusonii was introduced in the genus Escherichia almost 65 years later than Escherichia coli after which the genus was named. From then (1985) onwards mainly case reports on E. fergusonii associated with disease in individuals of veterinary or human origin have been reported and only very few more extensive studies became available. This has resulted in very fragmented knowledge on this organism. The aim of this manuscript is to give an overview of what is known on E. fergusonii today and to stimulate more research on this organism so that better insight can be obtained in the role that E. fergusonii plays in human and animal infections.

Extended biofilm susceptibility assay for Staphylococcus aureus bovine mastitis isolates: evidence for association between genetic makeup and biofilm susceptibility.

Staphylococcus aureus is one of the most prevalent causes of bovine mastitis. The antimicrobial treatment of this disease is currently based on antimicrobial susceptibility tests according to Clinical and Laboratory Standards Institute standards. However, various authors have shown a discrepancy between the results of this standard susceptibility test and the actual cure rate of the applied antimicrobial treatment. Increasing evidence suggests that in vivo biofilm formation by Staph. aureus, which is not assessed in the antimicrobial susceptibility tests, is associated with this problem, resulting in disappointing cure rates, especially for infections of longer duration. Previous data obtained with a limited number of strains showed that the extended biofilm antimicrobial susceptibility (EBS) assay reveals differences between strains, which cannot be derived from a standard susceptibility test or from a 24-h biofilm susceptibility test. The objective of this study was to test a collection of Staph. aureus bovine mastitis strains in the EBS assay and to model the effect of antimicrobial exposure, duration of antimicrobial exposure, and genotype profile of the strains on antimicrobial susceptibility. With the results from a previous study with the same collection of strains, the effect of genotype represented by accessory gene regulator gene (agr-type), the presence of insertional sequence 257 (IS257), intercellular adhesion (ica), and the ß-lactamase (blaZ) gene were entered as explanatory factors in a logistic regression model. The agr locus of Staph. aureus controls the expression of most of the virulence factors, represses the transcription of several cell wall-associated proteins, and activates several exoproteins during the post-exponential phase. The IS257 gene has been related to biofilm formation in vitro and was found earlier in 50% of the agr-type 2 strains. The ica gene cluster encodes for the production of an extracellular polysaccharide adhesin, termed polysaccharide intercellular adhesin, which appears to have an important role in pathogenic Staph. aureus infections. The blaZ gene encodes the presence of the penicillin resistance in the strain. The EBS assay together with the logistic regression model revealed that the duration of therapy is the most important factor of therapy outcome in this in vitro model. Furthermore, the effect of genotypic differences seems to be more important for therapy outcome than the antimicrobial used in this model.

Clostridium difficile infection in humans and animals, differences and similarities.

Clostridium difficile is well known as the most common cause of nosocomial infections in human patients. In recent years a change in epidemiology towards an increase in incidence and severity of disease, not only inside the hospital, but also in the community, is reported. C. difficile is increasingly recognized in veterinary medicine as well and is now considered the most important cause of neonatal diarrhea in swine in North America. Research on the presence of C. difficile in production and companion animals revealed a huge overlap with strains implicated in human C. difficile infection (CDI). This has lead to the concern that interspecies transmission of this bacterium occurs. In this review C. difficile infections in humans and animals are compared. The pathogenesis, clinical signs, diagnosis and prevalence of CDI are described and similarities and differences of CDI between humans and animals are discussed.

Biofilm formation and genotyping of Staphylococcus aureus bovine mastitis isolates: evidence for lack of penicillin-resistance in Agr-type II strains.

The increasing evidence for a role of biofilm formation in bovine mastitis caused by Staphylococcus aureus led to further investigations on biofilm formation by S. aureus isolates from mastitis in two growth media (TSBg and bovine milk serum). The ability of 99 S. aureus strains that were recently isolated or obtained from a culture collection (historical strains) to form biofilm, in both growth media as well as the correlation of biofilm formation with the presence of the ica-, bap-, and IS257 genes are described. These genes have been correlated with biofilm formation by human S. aureus isolates. All strains were also genotyped with respect to their Agr-type and -subtype, and for the presence of the antibiotic resistance genes blaZ and smr by PCR. The prevalence of the Agr-types and the investigated genes and their correlation with biofilm formation were statistically evaluated. The Agr-type of a strain had a marked effect on the biofilm formation, by that strain, however in contrast to human isolates no significant effect of ica- and IS257 genes on biofilm formation was observed. The bap gene was not found in any of the investigated strains. The presence of biofilm related genes showed a high correlation with the Agr-type of the strains. The data give evidence for a very strong correlation of Agr-type I strains and penicillin-resistance in the bovine S. aureus mastitis strains; none of the Agr-type II strains was found to harbor penicillin-resistance genes. These data indicate that the most prevalent Agr-types in S. aureus bovine mastitis, Agr-type I and II, can be regarded as different subspecies, with different abilities for the formation of biofilm in bovine milk serum. The very high correlation between Agr-type II and penicillin-susceptibility strongly suggests that these strains are not able to accommodate blaZ genes.

The presence of Arcobacter species in breeding hens and eggs from these hens.

The presence of Arcobacter spp. in 2 breeding hen flocks was determined by examination of the intestinal tract, oviduct magnum mucosa, and ovarian follicles of slaughtered chicken. The bacteria were detected by PCR and cultural isolation in 34 out of 40 intestinal tracts from one flock (A) and 6 out of 30 from the other (B). The strains were Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii. From flock A, arcobacters were recovered from 6 out of 40 oviduct magnum mucosa samples. The majority of isolated strains were A. butzleri. Arcobacter spp. could not be detected, by either PCR or isolation, from 20 eggs collected on the farm of flock A and from 20 eggs still remaining in the vagina of hens in flock B. Furthermore, none of the ovarian follicles from each flock were positive. The results indicate that breeding hens can be infected with Arcobacter spp. in the intestinal tract and oviduct. No evidence was obtained for transmission of Arcobacter spp. from hens to eggs.

Extended antimicrobial susceptibility assay for Staphylococcus aureus isolates from bovine mastitis growing in biofilms.

Staphylococcus aureus is one of the most prevalent causes of bovine mastitis. The antimicrobial treatment of this disease is currently based on antimicrobial susceptibility tests according to CLSI standards. However, various studies have shown that there is a discrepancy between the results of this standard susceptibility test and the actual cure rate of the applied antimicrobial treatment. Increasing evidence suggests that biofilm formation by S. aureus is associated with this problem. The currently available antimicrobial susceptibility assays for bacteria growing in biofilms, are not considered reliable enough for routine application. Therefore, the objective of this study was to further develop a susceptibility test for bacteria growing in biofilm, suitable for routine testing of the antimicrobial susceptibility of S. aureus. With the expansion of the available MBEC assay to an extended biofilm susceptibility test, that comprises 2 and 4 consecutive days of antimicrobial challenge, the antimicrobial susceptibility for S. aureus growing in biofilm was further analysed. The results showed clear differences between strains and various antimicrobial agents with respect to the effect of longer duration of the antimicrobial challenge on the eradication of S. aureus growing in biofilm. The extended biofilm susceptibility test also indicates that each bacterial strain requires a specific duration of antimicrobial therapy, which cannot be derived from a standard susceptibility test or from a 24-h biofilm susceptibility test.

Comparative assessment of the antimicrobial susceptibility of Staphylococcus aureus isolates from bovine mastitis in biofilm versus planktonic culture.

Mastitis is one of the most important diseases in dairy cattle of which Staphylococcus aureus is a major pathogen. Despite an apparently good antimicrobial susceptibility in vitro, the cure of diseased animals from this bacteriological infection is often disappointing, which results in cases of recurrent clinical- and chronic subclinical infections. It has been suggested that these recurrent and chronic Staphylococcus infections can be attributed to the growth of bacteria in biofilm. The objective of this study was to compare the susceptibility for antimicrobial agents of S. aureus isolates obtained from bovine mastitis growing under different conditions. These conditions include a conventional conventional microbroth dilution assay in which minimal inhibitory concentration values are determined, the MBEC assay which measures both the susceptibility in biofilm and the susceptibility of sequester cells released from the biofilm. A comparison of the susceptibility for antimicrobial agents of a number of representative S. aureus isolates grown in broth (representing in vitro growth conditions) or milk (representing in vivo growth conditions) is also made. The results indicate that S. aureus isolates obtained from bovine mastitis are highly resistant to antimicrobial agents when growing in biofilms.

Potential routes of acquisition of Arcobacter species by piglets.

The aim of this study was to determine the prevalence and the transmission routes of Arcobacter spp. in sows and their offspring on a breeding farm. Twelve Arcobacter-positive sows and their litters were studied for this purpose. Analysis of rectal samples showed a high prevalence of Arcobacter spp. among the sows (approximately 42% of the sows carried one or more Arcobacter species). Intermittent excretion of one particular species and shifts in excretion from one species to another were observed in individual animals over time. The detection of Arcobacter spp. in amniotic fluid of the sows and in rectal samples from newborn piglets (ranging from 38.5-83.3% per litter), as well as the high similarity between PFGE profiles of Arcobacter isolates from sows and their respective newborns indicated the existence of an intra-uterine transmission route for Arcobacter spp. Specific antibodies against Arcobacter spp. were detected in colostrum by Western blot. At 2 weeks of age, only a few piglets were positive for Arcobacter. The reappearance of Arcobacter in these piglets at Week 3 and the shift in the Arcobacter species detected (from a prominent presence of A. cryaerophilus at birth to the presence of A. skirrowii and A. butzleri at 3 weeks after birth) showed that a post-natal infection route from their mothers, newcomers or the environment to the piglets existed. Thus, in this manuscript the transmission of Arcobacter spp. (both vertical and horizontal) from carrying sows to their offspring is demonstrated.

Extrahepatic production of acute phase serum amyloid A.

Amyloidosis is a group of diseases characterized by the extracellular deposition of protein that contains non-branching, straight fibrils on electron microscopy (amyloid fibrils) that have a high content of beta-pleated sheet conformation. Various biochemically distinct proteins can undergo transformation into amyloid fibrils. The precursor protein of amyloid protein A (AA) is the acute phase protein serum amyloid A (SAA). The concentration of SAA in plasma increases up to 1000-fold within 24 to 48 h after trauma, inflammation or infection. Individuals with chronically increased SAA levels may develop AA amyloidosis. SAA has been divided into two groups according to the encoding genes and the source of protein production. These two groups are acute phase SAA (A-SAA) and constitutive SAA (C-SAA). Although the liver is the primary site of the synthesis of A-SAA and C-SAA, extrahepatic production of both SAAs has been observed in animal models and cell culture experiments of several mammalian species and chicken. The functions of A-SAA are thought to involve lipid metabolism, lipid transport, chemotaxis and regulation of the inflammatory process. There is growing evidence that extrahepatic A-SAA formation may play a crucial role in amyloidogenesis and enhances amyloid formation at the site of SAA production.

H-NS and Lrp serve as positive modulators of traJ expression from the Escherichia coli plasmid pRK100.

Conjugative transfer of F-like plasmids is a tightly regulated process. The TraJ protein is the main positive activator of the tra operon which encodes products required for conjugative transfer of F-like plasmids. Nucleotide sequence analysis revealed potential Lrp and H-NS binding sites in the traJ regulatory region. Expression of a traJ-lacZ fusion in hns and lrp mutant strains showed that both are positive modulators of traJ expression. Competitive RT-PCR demonstrated that H-NS and Lrp exert their effect at the transcriptional level. Electrophoretic mobility-shift assays showed that H-NS and Lrp proteins bind to the traJ promoter. Conjugative transfer of pRK100 was decreased in hns but not in lrp mutant strains. Together, the results indicate H-NS and Lrp function as activators of traJ transcription.

Distribution of papG alleles among uropathogenic Escherichia coli isolated from different species.

The distribution of alleles I, II and III of the P adhesin gene papG among Escherichia coli isolated from urinary tract infections in humans, dogs and cats was studied by PCR. Allele I was present in 6% and 5% of the human and cat isolates. Allele II as such was present in 30% and 22%, or in association with allele III in 12% and 2% of the human and canine isolates, respectively. Allele III was present in 33% of the human strains and predominated largely over allele II in E. coli isolates from cystitis of animal origin (72% in dog and 95% in cat strains). The three different classes of the PapG adhesin have been suggested to play a role in host specificity, for example human versus canine specificity. Recent studies, however, showed papG III positive human and dog cystitis isolates to be largely indistinguishable. We found the Class II allele in animal isolates and detected for the first time in Europe the Class I allele in a different genetic background than the J96-like clonal group. Our findings show that uropathogenic E. coli isolates from different species can have the same papG alleles and thus may have zoonotic potential.

Codon-usage based regulation of colicin K synthesis by the stress alarmone ppGpp.

The molecular mechanism of the upregulation of Escherichia coli colicin K (Cka) synthesis during stress conditions was studied. Nutrient starvation experiments and the use of relA spoT mutant strains, IPTG-regulated overproduction of ppGpp and lacZ fusions revealed that the stringent response alarmone guanosine 3',5'-bispyrophosphate (ppGpp) is the main positive effector of Cka synthesis. Comparison of the amounts of protein produced (Western blotting) and specific mRNA (Northern blotting) before and after nutrient starvation demonstrated increases in Cka protein with unaltered specific mRNA levels, suggesting a post-transcriptional regulatory mechanism. Reporter (beta-galactosidase) assays using truncated cka of variable length fused to lacZ located the key regulatory region close to the 5' end of the cka mRNA. Closer analysis of this region indicated the presence of several rare codons, including the leucine-encoding codon CUA. Synonymous exchange of the rare codons with more frequently used ones abolished the regulatory effect of ppGpp. Supplementation of the strain with the plasmid CodonPlus carrying several rare tRNA genes yielded similar results, indicating that codon usage (in particular, the fifth codon for the amino acid leucine) and tRNA availability (i.e. tRNAleu) are the key elements of the regulatory function of ppGpp. We conclude that ppGpp regulates Cka synthesis via a novel post-transcriptional mechanism that is based on rare codon usage and variable cognate tRNA availability.

Bacterial virulence: can we draw the line?

The molecular approach to microbial pathogenesis has resulted in an impressive amount of data on bacterial virulence genes. Bacterial genome sequences rapidly add candidate virulence genes to electronic databases. The interpretation of this overwhelming information is obscured because every gene involved in pathogenicity is called a virulence gene, regardless of its function in the complex process of virulence. This review summarizes the changing concept of bacterial virulence and the detection and identification strategies followed to recognize virulence genes. A refined definition of virulence genes is proposed in which the function of the gene in the virulence process is incorporated. We propose to include the life-style of bacteria in the assessment of their putative virulence genes. A universal nomenclature in analogy to the EC enzyme numbering system is proposed. These recommendations would lead to a better insight into bacterial virulence and a more precise annotation of (putative) virulence genes, which would enable more efficient use of electronic databases.

Virulence genes and P fimbriae PapA subunit diversity in canine and feline uropathogenic Escherichia coli.

In this study, a total of 118 Escherichia coli strains isolated from dogs (93) and cats (25) with urinary tract infection (UTI) were tested in a multiplex polymerase chain reaction for the presence of adhesin-encoding genes (pap, sfa, and afa), hemolysin encoding genes (hly), cytotoxic necrotizing factor 1 (cnf1) and aerobactin (aer) genes. Virulence gene frequencies detected in those isolates which had been randomly collected (68 canine strains) were: 43% pap, 57% sfa, 1% afa, 44% hly, 41% cnf1 and 34% aer. These frequencies were much higher in the remaining 50 hemolytic strains of either cat or dog origin. Virulence factor associations in the 80 hemolytic strains studied revealed that 50/80 simultaneously had two adhesin genes (pap and sfa) and two cytotoxin genes (hly and cnf1), and 15/80 in addition had the aer gene. The major structural subunit and antigenic determinant of P fimbriae of uropathogenic E. coli is PapA. Polymorphism in this subunit was studied by an F antigen-specific papA allele polymerase chain reaction in 51 canine and 22 feline pap positive E. coli strains. The most prevalent canine papA alleles were F10 (39%), F15 (37%) and F12 (35%). In feline strains F15 (50%) was more frequent, other allele frequencies were F12 (45%), F14 and F10 (27%) and F16 (23%). Only nine canine and two feline strains were negative for one of the 11 serologically defined F types of P fimbriae. Three copies of the pap operon were found in 16/51 canine and 9/22 feline UTI E. coli pap positive strains. In this study, we show that a particular combination of virulence genes appears with high frequency in dog and cat urinary tract E. coli strains (pap, sfa, hly, and cnf1). In spite of the more frequent presence of F10, F12 and F15 papA alleles in this virulence gene combination, the occurrence of different papA alleles in strains where up to three copies of the pap operon are present accounts for the observed P fimbriae diversity.

Studies about the mechanism of internalization by mammary epithelial cells of Escherichia coli isolated from persistent bovine mastitis.

The purpose of this study was to investigate the interaction between Escherichia coli and primary mammary epithelial cell cultures derived from cows with persistent intramammary infection (IMI). Two strains of E. coli, isolated from the milk of two different cows suffering from persistent E. coli IMI were tested for adhesion to and invasion of three primary mammary epithelial cell cultures derived from mammary biopsies of the two infected cows. Intracellular E. coli were detected during five days post infection in vitro. Both strains of E. coli adhered to and invaded monolayers of all three primary mammary epithelial cell cultures. One strain adhered less but invaded more than the other. Comparison with other mammary pathogens indicated that E. coli invaded the cells less efficiently than Staphylococcus aureus, about as efficiently as Streptococcus dysgalactiae and more efficiently than Streptococcus uberis. The mechanism of E. coli invasion was studied using the cytoskeleton disrupting agents colchicine and cytochalasin D. These compounds inhibited the invasion of E. coli. Invasion of E. coli could also be inhibited by the phosphokinase inhibitors genistein and staurosporin in a dose-dependent fashion. Phorbol-myristyl-acetate (PMA) had no effect on the invasion of E. coli. Histology of mammary tissue revealed chronic inflammatory changes in quarters that were persistently infected by E. coli. Intracellular bacteria were not detected in mammary tissue sections. Polymerase chain reaction (PCR) analysis suggested that the two strains of E. coli lacked genes encoding for bundle-forming pili (bfpA), intimin (eae) and translocated intimin receptor (tir), which are characteristic for enteropathogenic E. coli (EPEC).

Phylogenetic and pathotypic similarities between Escherichia coli isolates from urinary tract infections in dogs and extraintestinal infections in humans.

Seventeen Escherichia coli isolates from dogs with urinary tract infection (UTI) were characterized with respect to phylogenetic background and virulence genotype and were compared with the E. coli reference (ECOR) collection and with human clinical isolates with similar serotypes from patients with diverse extraintestinal infections. Most of the canine urine isolates were from (virulence-associated) E. coli phylogenetic groups B2 or D, expressed papG allele III, and exhibited numerous other putative virulence genes that are characteristic of human extraintestinal pathogenic E. coli (ExPEC). Close phylogenetic and pathotypic correspondence was documented within 5 clonal groups among individual canine and human isolates, including archetypal human ExPEC strains CFT073 (O6:K2:H1), 536 (O6:K15:H31), and J96 (O4:K-:H5). These findings suggest that canine UTI isolates, rather than being dog-specific pathogens, as previously suspected, may pose an infectious threat to humans. Commonality between canine and human ExPEC has potentially important implications for disease prevention, antibiotic resistance avoidance, and studies of pathogenesis.

Virulence factors of Escherichia coli isolated from urine of diabetic women with asymptomatic bacteriuria: correlation with clinical characteristics.

Since Escherichia coli isolated from compromised patients with symptomatic urinary tract infections (UTIs) express fewer virulence factors than those isolated from healthy controls, the question arises whether this is also the case for diabetic patients with asymptomatic bacteriuria (ASB). Polymerase chain reaction (PCR) assays were conducted on 111E. coli strains, isolated from the urine of diabetic women with ASB, using primers for the major subunit A and the G-adhesin (I, II, and III) of P fimbriae, type 1 fimbriae, S fimbriae, afimbrial adhesin, cytotoxic necrotizing factor (CNF), and aerobactin. Phenotypically, hemolysis, mannose-sensitive hemagglutination, mannose-resistant hemagglutination and O:K:H-serotypes were determined. Furthermore, we investigated the associations between virulence factors and patient characteristics (including deterioration of renal function). Type 1 fimbriae were the most prevalent virulence factor (86% by genotyping and 59% phenotypically). Except for a lower prevalence of known uropathogenic O-serotypes, we found the same number of virulence factors in our compromised patient group as listed in the literature in noncompromised patients with ASB. Certain virulence factors (type 1 and S fimbriae and CNF) of the causative E. coli correlated with the risk of a decline in renal function. In conclusion, the number of virulence factors in E. coli isolated from the urine of diabetic women with ASB are comparable with the results found in other (noncompromised) patients with ASB. Furthermore, certain virulence factors of E. coli might contribute to a decline in renal function.

Cytokine secretion is impaired in women with diabetes mellitus.

BACKGROUND: As women with diabetes mellitus (DM) have an increased prevalence of asymptomatic bacteriuria (ASB) and it is known that a correlation exists between the increased prevalence of genitourinary tract infection and impaired cytokine production in women infected with Human Immunodeficiency Virus (HIV), we studied urinary cytokine excretion in diabetic women and compared it with that of nondiabetic controls. MATERIALS AND METHODS: To evaluate the cytokine secretion capacity of women with DM, both whole blood and isolated monocytes of women with and without DM were stimulated in vitro with lipopolysaccharide (LPS). RESULTS: Lower urinary interleukin-8 (IL-8) and interleukin-6 (IL-6) concentrations (P = 0.1 and P < 0.001, respectively) were found in diabetic women than in nondiabetic controls. A lower urinary leukocyte cell count correlated with lower urinary IL-8 and IL-6 concentrations (P < 0.05). Lower tumour necrosis factor-alpha (TNF-alpha) and IL-6, but comparable interleukin-10 (IL-10) concentrations were found in whole blood (P < 0.04) and isolated monocytes (P = 0.03) of women with DM type 1 compared to women without DM. CONCLUSIONS: Diabetic women with ASB have lower urinary IL-6 concentrations than nondiabetic bacteriuric controls. In addition, monocytes of women with DM type 1 secrete lower pro-inflammatory cytokines after stimulation with LPS than monocytes of women without DM. This is not due to an inhibitory effect of the anti-inflammatory cytokine IL-10. This can have important consequences for both host defense, endothelial cell functioning and atherogenesis.