Antibiotic resistance in Campylobacter jejuni and Campylobacter coli isolated from poultry in the South-East Queensland region

Objectives: The aim of this study was to determine the antimicrobial resistance patterns of 125 Campylobacter jejuni and 27 Campylobacter coli isolates from 39 Queensland broiler farms. Methods: Two methods, a disc diffusion assay and an agar-based MIC assay, were used. The disc diffusion was performed and interpreted as previously described (Huysmans MB, Turnidge JD. Disc susceptibility testing for thermophilic campylobacters. Pathology 1997; 29: 209–16), whereas the MIC assay was performed according to CLSI (formerly NCCLS) methods and interpreted using DANMAP criteria. Results: In both assays, no C. jejuni or C. coli isolates were resistant to ciprofloxacin or chloramphenicol, no C. coli were resistant to nalidixic acid, and no C. jejuni were resistant to erythromycin. In the MIC assay, no C. jejuni isolate was resistant to nalidixic acid, whereas three isolates (2.4%) were resistant in the disc assay. The highest levels of resistance of the C. jejuni isolates were recorded for tetracycline (19.2% by MIC and 18.4% by disc) and ampicillin (19.2% by MIC and 17.6% by disc). The C. coli isolates gave very similar results (tetracycline resistance 14.8% by both MIC and disc; ampicillin resistance 7.4% by MIC and 14.8% by disc). Conclusions: This work has shown that the majority of C. jejuni and C. coli isolates were susceptible to the six antibiotics tested by both disc diffusion and MIC methods. Disc diffusion represents a suitable alternative methodology to agar-based MIC methods for poultry Campylobacter isolates.

Effect of Crowding Agents, Signal Peptide, and Chaperone SecB on the Folding and Aggregation of E. coli Maltose Binding Protein

SecB, a soluble cytosolic chaperone component of the Secexport pathway, binds to newly synthesized precursor proteins and prevents their premature aggregation and folding and subsequently targets them to the translocation machinery on the membrane. PreMBP, the precursor form of maltose binding protein, has a 26-residue signal sequence attached to the N-terminus of MBP and is a physiological substrate of SecB. We examine the effect of macromolecular crowding and SecB on the stability and refolding of denatured preMBP and MBP. PreMBP was less stable than MBP (ΔTm =7( 0.5 K) in both crowded and uncrowded solutions. Crowding did not cause any substantial changes in the thermal stability ofMBP(ΔTm=1(0.4 K) or preMBP (ΔTm=0(0.6 K), as observed in spectroscopically monitored thermal unfolding experiments. However, both MBP and preMBP were prone to aggregation while refolding under crowded conditions. In contrast to MBP aggregates, which were amorphous, preMBP aggregates form amyloid fibrils.Under uncrowded conditions, a molar excess of SecB was able to completely prevent aggregation and promote disaggregation of preformed aggregates of MBP. When a complex of the denatured protein and SecB was preformed, SecB could completely prevent aggregation and promote folding of MBP and preMBP even in crowded solution. Thus, in addition to maintaining substrates in an unfolded, export-competent conformation, SecB also suppresses the aggregation of its substrates in the crowded intracellular environment. SecB is also able to promote passive disaggregation of macroscopic aggregates of MBP in the absence of an energy source such as ATP or additional cofactors. These experiments also demonstrate that signal peptide can reatly influence protein stability and aggregation propensity.

Uropathogenic Escherichia Coli engages CD14-dependent signaling to enable bladder-macrophage-dependent control of acute urinary tract infection

Background CD14, a coreceptor for several pattern recognition receptors and a widely used monocyte/macrophage marker, plays a key role in host responses to gram-negative bacteria. Despite the central role of CD14 in the inflammatory response to lipopolysaccharide and other microbial products and in the dissemination of bacteria in some infections, the signaling networks controlled by CD14 during urinary tract infection (UTI) are unknown. Methods We used uropathogenic Escherichia coli (UPEC) infection of wild-type (WT) C57BL/6 and Cd14−/− mice and RNA sequencing to define the CD14-dependent transcriptional signature and the role of CD14 in host defense against UTI in the bladder. Results UPEC induced the upregulation of Cd14 and the monocyte/macrophage-related genes Emr1/F4/80 and Csf1r/c-fms, which was associated with lower UPEC burdens in WT mice, compared with Cd14−/− mice. Exacerbation of infection in Cd14−/− mice was associated with the absence of a 491-gene transcriptional signature in the bladder that encompassed multiple host networks not previously associated with this receptor. CD14-dependent pathways included immune cell trafficking, differential cytokine production in macrophages, and interleukin 17 signaling. Depletion of monocytes/macrophages in the bladder by administration of liposomal clodronate led to higher UPEC burdens. Conclusions This study identifies new host protective and signaling roles for CD14 in the bladder during UPEC UTI.

Mechanically Ventilated Broiler Sheds: a Possible Source of Aerosolized Salmonella, Campylobacter, and Escherichia coli.

This study assessed the levels of two key pathogens, Salmonella and Campylobacter, along with the indicator organism Escherichia coli in aerosols within and outside poultry sheds. The study ranged over a 3-year period on four poultry farms and consisted of six trials across the boiler production cycle of around 55 days. Weekly testing of litter and aerosols was carried out through the cycle. A key point that emerged is that the levels of airborne bacteria are linked to the levels of these bacteria in litter. This hypothesis was demonstrated by E. coli. The typical levels of E. coli in litter were similar to 10(8) CFU g(-1) and, as a consequence, were in the range of 10(2) to 10(4) CFU m(-3) in aerosols, both inside and outside the shed. The external levels were always lower than the internal levels. Salmonella was only present intermittently in litter and at lower levels (10(3) to 10(5) most probable number [MPN] g(-1)) and consequently present only intermittently and at low levels in air inside (range of 0.65 to 4.4 MPN m(-3)) and once outside (2.3 MPN m(-3)). The Salmonella serovars isolated in litter were generally also isolated from aerosols and dust, with the Salmonella serovars Chester and Sofia being the dominant serovars across these interfaces. Campylobacter was detected late in the production cycle, in litter at levels of around 107 MPN g(-1). Campylobacter was detected only once inside the shed and then at low levels of 2.2 MPN m(-3). Thus, the public health risk from these organisms in poultry environments via the aerosol pathway is minimal.

Campylobacter jejuni and Campylobacter coli Genotyping by High-Resolution Melting Analysis of a flaA Fragment.

The highly variable flagellin-encoding flaA gene has long been used for genotyping Campylobacter jejuni and Campylobacter coli. High-resolution melting (HRM) analysis is emerging as an efficient and robust method for discriminating DNA sequence variants. The objective of this study was to apply HRM analysis to flaA-based genotyping. The initial aim was to identify a suitable flaA fragment. It was found that the PCR primers commonly used to amplify the flaA short variable repeat (SVR) yielded a mixed PCR product unsuitable for HRM analysis. However, a PCR primer set composed of the upstream primer used to amplify the fragment used for flaA restriction fragment length polymorphism (RFLP) analysis and the downstream primer used for flaA SVR amplification generated a very pure PCR product, and this primer set was used for the remainder of the study. Eighty-seven C. jejuni and 15 C. coli isolates were analyzed by flaA HRM and also partial flaA sequencing. There were 47 flaA sequence variants, and all were resolved by HRM analysis. The isolates used had previously also been genotyped using single-nucleotide polymorphisms (SNPs), binary markers, CRISPR HRM, and flaA RFLP.flaA HRM analysis provided resolving power multiplicative to the SNPs, binary markers, and CRISPR HRM and largely concordant with the flaA RFLP. It was concluded that HRM analysis is a promising approach to genotyping based on highly variable genes.

Human bladder uroepithelial cells synergize with monocytes to promote IL-10 synthesis and other cytokine responses to Uropathogenic Escherichia coli

Urinary tract infections are a major source of morbidity for women and the elderly, with Uropathogenic Escherichia coli (UPEC) being the most prevalent causative pathogen. Studies in recent years have defined a key anti-inflammatory role for Interleukin-10 (IL-10) in urinary tract infection mediated by UPEC and other uropathogens. We investigated the nature of the IL-10-producing interactions between UPEC and host cells by utilising a novel co-culture model that incorporated lymphocytes, mononuclear and uroepithelial cells in histotypic proportions. This co-culture model demonstrated synergistic IL-10 production effects between monocytes and uroepithelial cells following infection with UPEC. Membrane inserts were used to separate the monocyte and uroepithelial cell types during infection and revealed two synergistic IL-10 production effects based on contact-dependent and soluble interactions. Analysis of a comprehensive set of immunologically relevant biomarkers in monocyte-uroepithelial cell co-cultures highlighted that multiple cytokine, chemokine and signalling factors were also produced in a synergistic or antagonistic fashion. These results demonstrate that IL-10 responses to UPEC occur via multiple interactions between several cells types, implying a complex role for infection-related IL-10 during UTI. Development and application of the co-culture model described in this study is thus useful to define the degree of contact dependency of biomarker production to UPEC, and highlights the relevance of histotypic co-cultures in studying complex host-pathogen interactions.

Genome-wide mapping of cystitis due to Streptococcus agalactiae and Escherichia coli in mice identifies a unique bladder transcriptome that signifies pathogen-specific antimicrobial defense against urinary tract infection

The most common causes of urinary tract infections (UTIs) are Gram-negative pathogens such as Escherichia coli; however, Gram-positive organisms including Streptococcus agalactiae, or group B streptococcus (GBS), also cause UTI. In GBS infection, UTI progresses to cystitis once the bacteria colonize bladder, but the host responses triggered in the bladder immediately following infection are largely unknown. Here, we used genome-wide expression profiling to map the bladder transcriptome of GBS UTI in mice infected transurethrally with uropathogenic GBS that was cultured from a 35 year-old women with cystitis. RNA from bladders was applied to Affymetrix Gene-1.0ST microarrays; qRT-PCR was used to analyze selected gene responses identified in array datasets. A surprisingly small significant gene list of 172 genes was identified at 24h; this compared to 2507 genes identified in a side-by-side comparison with uropathogenic E. coli (UPEC). No genes exhibited significantly altered expression at 2h in GBS-infected mice according to arrays despite high bladder bacterial loads at this early time point. The absence of a marked early host response to GBS juxtaposed with broad-based bladder responses activated by UPEC at 2h. Bioinformatics analyses including integrative systems-level network mapping revealed multiple activated biological pathways in the GBS cystitis transcriptome that regulate leukocyte activation, inflammation, apoptosis, and cytokine-chemokine biosynthesis. These findings define a novel, minimalistic type of bladder host response triggered by GBS UTI, which comprises collective antimicrobial pathways that differ dramatically from those activated by UPEC. Overall, this study emphasizes the unique nature of bladder immune activation mechanisms triggered by distinct uropathogens.

Clinical Escherichia coli isolates utilize alpha-hemolysin to inhibit in vitro epithelial cytokine production

Uropathogenic Escherichia coli is the primary cause of urinary tract infections, which affects over 60% of women during their lifetime. UPEC exhibits a number of virulence traits that facilitate colonization of the bladder, including inhibition of cytokine production by bladder epithelial cells. The goal of this study was to identify the mechanism of this inhibition. We observed that cytokine suppression was associated with rapid cytotoxicity toward epithelial cells. We found that cytotoxicity, cytokine suppression and alpha-hemolysin production were all tightly linked in clinical isolates. We screened a UPEC fosmid library and identified clones that gained the cytotoxicity and cytokine-suppression phenotypes. Both clones contained fosmids encoding a PAI II(J96)-like domain and expressed the alpha-hemolysin (hlyA) encoded therein. Mutation of the fosmid-encoded hly operon abolished cytotoxicity and cytokine suppression. Similarly, mutation of the chromosomal hlyCABD operon of UPEC isolate F11 also abolished these phenotypes, and they could be restored by introducing the PAI II(J96)-like domain-encoding fosmid. We also examined the role of alpha-hemolysin in cytokine production both in the murine UTI model as well as patient specimens. We conclude that E. coli utilizes alpha-hemolysin to inhibit epithelial cytokine production in vitro. Its contribution to inflammation during infection requires further study.

Multidrug-resistant extraintestinal pathogenic Escherichia coli of sequence type ST131 in animals and foods.

Multidrug-resistant Escherichia colt sequence type 131 (51131) has recently emerged as a globally distributed cause of extraintestinal infections in humans. Diverse factors have been investigated as explanations for ST131's rapid and successful dissemination, including transmission through animal contact and consumption of food, as suggested by the detection of ST131 in a number of nonhuman species. For example, ST131 has recently been identified as a cause of clinical infection in companion animals and poultry, and both host groups have been confirmed as faecal carriers of ST131. Moreover, a high degree of similarity has been shown among certain ST131 isolates from humans, companion animals, and poultry based on resistance characteristics and genomic background and human and companion animal ST131 isolates tend to exhibit similar virulence genotypes. However, most ST131 isolates from poultry appear to possess specific virulence genes that are typically absent from human and companion animal isolates, including genes associated with avian pathogenic E. coli. Since the number of reported animal and food-associated ST131 isolates is quite small, the role of nonhuman host species in the emergence, dissemination, and transmission of ST131 to humans remains unclear. Nevertheless, given the profound public health importance of the emergent ST131 clonal group, even the limited available evidence indicates a pressing need for further careful study of this significant question.

Quantitative effects of in-line operations on Campylobacter and Escherichia coli through two Australian broiler processing plants

Campylobacter is an important food borne pathogen, mainly associated with poultry. A lack of through-chain quantitative Campylobacter data has been highlighted within quantitative risk assessments. The aim of this study was to quantitatively and qualitatively measure Campylobacter and Escherichia coli concentration on chicken carcasses through poultry slaughter. Chickens (n = 240) were sampled from each of four flocks along the processing chain, before scald, after scald, before chill, after chill, after packaging and from individual caeca. The overall prevalence of Campylobacter after packaging was 83% with a median concentration of 0.8 log10 CFU/mL. The processing points of scalding and chilling had significant mean reductions of both Campylobacter (1.8 and 2.9 log10 CFU/carcase) and E. coli (1.3 and 2.5 log10 CFU/carcase). The concentration of E. coli and Campylobacter was significantly correlated throughout processing indicating that E. coli may be a useful indicator organism for reductions in Campylobacter concentration. The carriage of species varied between flocks, with two flocks dominated by Campylobacter coli and two flocks dominated by Campylobacter jejuni. Current processing practices can lead to significant reductions in the concentration of Campylobacter on carcasses. Further understanding of the variable effect of processing on Campylobacter and the survival of specific genotypes may enable more targeted interventions to reduce the concentration of this poultry associated pathogen.

Number and proportion of selenocucleosides in the transfer RNA of escherichia-coli

tRNA isolated from escherichia-coli grown in a medium containing [75Se] sodium selenosulfate was converted to nucleosides and analysed for selenonucleosides on a phosphocellulose column. Upon chromatography of the nucleosides on phosphocellulose column, the radioactivity resolved into three peaks. The first peak consisted of free selenium and traces of undigested nucleotides. The second peak was identified as 4-selenouridine by co-chromatographing with an authentic sample of 4-selenouridine. The identity of the third peak was not established. The second and third peaks represented 93% and 7% of the selenium present in nucleosides respectively.