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Objectives IMP-type carbapenemases are rarely detected in Europe and limited information is available to guide the treatment of infections caused by carbapenemase-producing Enterobacterales (CPE) producing these carbapenemases. Accurate antimicrobial susceptibility testing (AST) results are essential for optimal antibiotic management. Here we report discrepancies in AST of IMP-producing Enterobacterales (IMP-CPE) complicating the management of severe sepsis. Methods Antimicrobial susceptibilities were analysed by in-house VITEK® 2, Etest and broth microdilution (BMD). Carbapenemase-encoding genes were detected by PCR. Whole-genome sequencing (WGS) was performed using an Illumina MiSeq platform. Results Minimum inhibitory concentrations (MICs) determined by VITEK® 2 for Enterobacter hormaechei and Klebsiella oxytoca blood culture isolates were ≥16 mg/L for meropenem and ≤0.5 mg/L for ertapenem. In contrast, Etest analysis and BMD returned MICs of 2 mg/L and 1 mg/L, respectively. Both isolates tested positive for IMP carbapenemase-encoding genes by PCR. WGS revealed that both isolates carried the same blaIMP-4 gene. Based on VITEK® 2 susceptibilities, initial treatment was with tigecycline and amikacin. After subsequent deterioration, the patient was successfully treated with ertapenem and amikacin. Conclusion This case highlights that automated AST by VITEK® 2 can over-report meropenem resistance for IMP carbapenemase-producers compared with Etest and BMD. Clinicians need to be cautious deciding against carbapenem treatment based on VITEK® 2 susceptibility testing results for IMP-positive Enterobacterales. Tigecycline was inferior to carbapenem treatment for pyelonephritis caused by isolates expressing IMP carbapenemases, however specific evidence guiding the treatment of these infections is lacking.

A Gram-negative rod from the Yersinia genus was isolated from a clinical case of yersiniosis in the United Kingdom. Long read sequencing data from an Oxford Nanopore Technologies (ONT) MinION in conjunction with Illumina HiSeq reads were used to generate a finished quality genome of this strain. Overall Genome Related Index (OGRI) of the strain was used to determine that it was a novel species within Yersinia , despite biochemical similarities to Yersinia enterocolitica . The 16S ribosomal RNA gene accessions are MN434982-MN434987 and the accession number for the complete and closed chromosome is CP043727. The type strain is SRR7544370T (=NCTC 14382T/=LMG 31573T).

In recent years, the concept of bacteria-mediated cancer therapy has gained significant attention as an alternative to conventional therapy. The focus has been on non-typhoidal Salmonella (NTS), particularly S. Typhimurium, for its anti-cancer properties, however, other NTS serovars such as Salmonella Oslo, which are associated with foodborne illnesses could potentially be effective anti-cancer agents. Here, we report the draft genome sequence of Salmonella Oslo isolated from seafood and its laboratory generated auxotrophic mutant.

An antibiotic susceptibility monitoring programme was conducted from 2004 to 2010, resulting in a collection of 143 Escherichia coli cultured from bovine faecal samples (diarrhoea) and milk-aliquots (mastitis). The isolates were subjected to whole-genome sequencing and were distributed in phylogroups A, B1, B2, C, D, E, and G with no correlation for particular genotypes with pathotypes. In fact, the population structure showed that the strains belonging to the different phylogroups matched broadly to ST complexes, however, the isolates are randomly associated with the diseases, highlighting the necessity to investigate the virulence factors more accurately in order to identify the mechanisms by which they cause disease. The antimicrobial resistance was assessed phenotypically, confirming the genomic prediction on three isolates that were resistant to colistin, although one isolate was positive for the presence of the gene mcr-1 but susceptible to colistin. To further characterise the genomic context, the four strains were sequenced by using a single-molecule long read approach. Genetic analyses indicated that these four isolates harboured complex and diverse plasmids encoding not only antibiotic resistant genes (including mcr-1 and bla) but also virulence genes (siderophore, ColV, T4SS). A detailed description of the plasmids of these four E. coli strains, which are linked to bovine mastitis and diarrhoea, is presented for the first time along with the characterisation of the predicted antibiotic resistance genes. The study highlighted the diversity of incompatibility types encoding complex antibiotic resistance elements such as Tn6330, ISEcp1, Tn6029, and IS5075. The mcr-1 resistance determinant was identified in IncHI2 plasmids pCFS3273-1 and pCFS3292-1, thus providing some of the earliest examples of mcr-1 reported in Europe, and these sequences may be a representative of the early mcr-1 plasmidome characterisation in the EU/EEA.

Nontyphoidal Salmonella species are globally disseminated pathogens and are the predominant cause of gastroenteritis. The pathogenesis of salmonellosis has been extensively studied using in vivo murine models and cell lines, typically challenged with Salmonella enterica serovar Typhimurium. Although S. enterica serovars Enteritidis and Typhimurium are responsible for most of the human infections reported to the Centers for Disease Control and Prevention (CDC), several other serovars also contribute to clinical cases of salmonellosis. Despite their epidemiological importance, little is known about their infection phenotypes. Here, we report the virulence characteristics and genomes of 10 atypical S. enterica serovars linked to multistate foodborne outbreaks in the United States. We show that the murine RAW 264.7 macrophage model of infection is unsuitable for inferring human-relevant differences in nontyphoidal Salmonella infections, whereas differentiated human THP-1 macrophages allowed these isolates to be further characterized in a more human-relevant context.

Antimicrobial resistance reported in bacteria of animal origin is considered a major challenge to veterinary public health. In this study, the genotypic and phenotypic characterisation of twelve Escherichia coli isolates of bovine origin is reported. Twelve bacterial isolates of animal origin were selected from a previous study based on their multidrug resistant (MDR) profile. Efflux pump activity was measured using ethidium bromide (EtBr) and the biofilm forming ability of the individual strains was assessed using a number of phenotypic assays. All isolates were resistant to tetracyclines and a number of the isolates expressed resistance to fluoroquinolones which was also confirmed in silico by the presence of a number of these resistance markers. Amino acid substitutions in the quinolone resistance-determining regions were identified in all isolates and the presence of several siderophores were also noted. WGS data showed different STs that were not associated with epidemic STs or virulent clonal complexes. Seven isolates formed biofilms in minimal media with some isolates showing better adaptation at 25 °C while others at 37 °C. The capacity to efflux EtBr was found to be high in 4 isolates and impaired in 4 others.The pathogenicity of three selected isolates was assessed in zebrafish embryo infection models, revealing isolates CFS0355 and CFS0356 as highly pathogenic.These results highlight the application of NGS technologies combined with phenotypic assays in providing a better understanding of E. coli of bovine origin and their adaptation to this niche environment. Department of Agriculture, Food and the Marine Institutional Research Measure (FIRM) Network & Team Building Initiative 2006

Cationic antimicrobial peptides have raised interest as attractive alternatives to classical antibiotics, and also have utility in preventing food spoilage. We set out to enrich cationic antimicrobial peptides from milk hydrolysates using gels containing various ratios of anionic pectin/alginate. All processes were carried out with food-grade materials in order to suggest food-safe methods suited for producing food ingredients or supplements. Hydrolysed caseinate peptides retained in the gel fraction, identified by mass spectrometry, were enriched for potential antimicrobial peptides, as judged by a computational predictor of antimicrobial activity. Peptides retained in a 60:40 pectin:alginate gel fraction had a strong antimicrobial effect against 8 tested bacterial strains with a minimal inhibitory concentration of 1.5–5 mg/mL, while the unfractionated hydrolysate only had a detectable effect in one of the eight strains. Among 110 predicted antimicrobial peptides in the gel fraction, four are known antimicrobial peptides, HKEMPFPK, TTMPLW, YYQQKPVA and AVPYPQR. These results highlight the potential of pectin/alginate food-gels based processes as safe, fast, cost-effective methods to separate and enrich for antimicrobial peptides from complex food protein hydrolysates.

One mcr-1 -carrying Salmonella enterica serovar Indiana strain D90, was identified from 1320 Salmonella enterica isolates from poultry slaughterhouse in 2012 in China. The objective of this study was to verify the transferability of the mcr-1 gene and also completely characterize the sequence of the strain at the whole-genome level. Broth matting assays were carried out to detect the transferability and whole-genome sequencing (WGS) of S. enterica serovar Indiana D90 was performed using the PacBio RS II system. Open reading frames were assigned using Rapid Annotation using Subsystem Technology (RAST) and analysed by BLASTn and BLASTp. Salmonella Pathogenisity Islands (SPIs) were annotated by SPIFinder platform. The complete genome sequence of S. enterica serovar Indiana D90 contained a circular 4,779,514-bp chromosome and four plasmids. Genome analysis and sequencing revealed that 24 multi-drug resistance (MDR) genes were located on plasmids. The largest plasmid pD90-1, was found to be of an IncHI2/HI2A/Q1/N type that encoded a bla CTX-M-65 gene along with 20 additional antimicrobial resistance genes. A 60.5-kbp IncI2 plasmid pD90-2 contained a nikA - nikB - mcr-1 genetic structure, that can be successfully transferred to E. coli and S. enterica serovar Typhimurium at low transfer rates. Interestingly, comparative sequence analysis revealed the plasmids pD90-1 and pD90-2 showed considerable nucleotide similarity to pHNSHP45-2 and pHNSHP45, respectively. Moreover, the genome and the plasmid pD90-2 also showed high similarity to one carbapenem resistant S. enterica serovar Indiana strain, C629 and its plasmid pC629, respectively. This is the first report of the complete nucleotide sequence of one mcr-1 -carrying MDR S. enterica serovar Indiana strain.

A Gram-negative rod from theYersiniagenus was isolated from a clinical case of yersiniosis in the United Kingdom. Long read sequencing data from an Oxford Nanopore Technology (ONT) MinION in conjunction with Illumina HiSeq reads were used to generate a finished quality genome of this strain. Overall Genome Related Index (OGRI) of the strain was used to determine that it was a novel species withinYersinia, despite biochemical similarities toYersinia enterocolitica. The 16S ribosomal RNA gene accessions areMN434982-MN434987and the accession number for the complete and closed chromosome isCP043727.The type strain is CFS3336T(=NCTC 14382T/ =LMGAccession under process).

The factors influencing the virulence of P. aeruginosa in the development of invasive infection remain poorly understood. Here, we investigated the role of the host microenvironment in shaping pathogen virulence and investigated the mechanisms involved. Comparing seven paired genetically indistinguishable clinical bloodstream and peripheral isolates of P. aeruginosa, we demonstrate that isolates derived from bloodstream infections are more virulent than their peripheral counterparts (p = 0.025). Bloodstream and peripheral isolates elicited similar NF-kB responses in a THP-1 monocyte NF-kappaB reporter cell line implicating similar immunogenicity. Proteomic analysis by mass spectrometry identified multiple virulence and virulence-related factors including LecA and RpoN in significantly greater abundance in the bacterial supernatant from the bloodstream isolate in comparison to that from the corresponding peripheral isolate. Investigation by qPCR revealed that control of expression of these virulence factors was not due to altered levels of transcription. Based on these data, we hypothesize a post-transcriptional mechanism of virulence regulation in P. aeruginosa bloodstream infections influenced by surrounding microenvironmental conditions. Science Foundation Ireland

Klebsiella pneumoniae is an important nosocomial pathogen with an extraordinary resistant phenotype due to a combination of acquired resistant-elements and efflux mechanisms. In this study a detailed molecular characterization of 11 K. pneumoniae isolates of clinical origin was carried out. Eleven clinical isolates were tested for their susceptibilities, by disk diffusion and broth microdilution and interpreted according to CLSI guidelines. Efflux activity was determined by measuring the extrusion of ethidium bromide and biofilm formation was assessed following static growth in Müeller-Hinton and minimal media M9 broths at two temperatures and time points. Template DNA from all 11 isolates was extracted and sequenced. The study collection was found to be resistant to several (extended-spectrum beta-lactam) ESBL-type compounds along with several (fluoro)quinolones (FQ). Resistance to tetracycline accounted for 55% of the study collection (n = 6) and three of the 11 isolates were resistance to carbapenems. Genotyping identified blaCTX-M-15 (82%), blaSHV-12 (55%), and blaTEM-1B (45%) ESBL encoding genes and FQ resistance was associated the presence of the oqxAB operon, identified in 10 of the 11 isolates and qnrB gene in one isolate. The polymorphisms detected in the quinolone resistance-determining regions (QRDRs) were associated with isolates of the clonal group CG15. Sequence types (ST) identified were representative of previously described clonal groups including CG258 (n = 7), CG15 (n = 3), and CG147 (n = 1). Plasmid replicon type databases were queried indicating the presence of IncFII and IncFIB replicon types in the majority of the isolates (91%), followed by IncFIA (45%), and IncR (45%). Two of the 11 isolates were found positive for yersiniabactin siderophore-encoding genes. No differences in the ability to efflux ethidium bromide were identified. Biofilm formation was stronger when the isolates were grown under stressed conditions at 37°C for a period up to 96 h. These data confirm the fact that well-recognized clonal groups of K. pneumoniae of importance to human health carries a diverse repertoire of antimicrobial resistance determinants, particularly related to critically important drugs in the ESBL and FQ classes. The capacity of most isolates to form strong biofilms, when stressed under laboratory-simulated conditions, supports the risk to human health associated with nosocomial infections deriving from indwelling medical devices.

The synthesis of several diorganotin(IV) dicarboxylate compounds, including acetates and nicotinates as well as diorganotin(IV) dichloride complexes of the ligands phen, dione and dppz were undertaken. Several difficulties in either the syntheses or complexation reactions with the organic ligands were encountered. The diorganotin(IV) dichloride complexes of the ligands (R2SnCl2·L where R = Me, n-Bu or Ph and L = phen, dione or dppz) were tested against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. The dibutyltin(IV) derivatives exhibited the broadest range of activity in comparison to the dimethyltin(IV) or diphenyltin(IV) derivatives. The addition of the nicotinate group did not promote activity against any of the bacteria. Furthermore, only in the case of Ph2SnCl2·dione was there improved activity compared to the organic ligand itself.

Here, we report the draft genome sequence of the lactose-negative, rifampin-resistant, Escherichia coli strain 26R 793. This isolate has been widely used in conjugation experiments as a general recipient strain.

Identification of Enterobacteriaceae harbouring the plasmid-mediated transferable colistin resistance gene mcr-1 presents a new challenge to public health. The aim of this study was to characterise multidrug-resistant Shiga toxin-producing Escherichia coli (STEC) harbouring the mcr-1 gene on plasmids cultured from pigs in China. Using CHROMagar™ ECC plates combined with stx gene detection by PCR, 93 STEC were recovered from 326 faecal, 351 small intestine content and 326 colon content samples taken from healthy pigs in 2011 and 2012 in China. This study, in which ten colistin-resistant isolates with minimum inhibitory concentrations (MICs) of 8-12 mg/L were identified and found to be positive by PCR for the mcr-1 gene, is a follow-up to an earlier investigation. Plasmid profiling by S1-nuclease digestion followed by pulsed-field gel electrophoresis (PFGE) identified several high-molecular-weight plasmids and these were typed by PCR-based replicon typing (PBRT). Two of the ten isolates, namely STEC-CQ09 (O116:H11/CC23/ST88) and CQ10 (O2:H32/ST3628), were selected for further study as described in this report.

To provide a review of the medical literature and practical experience in the diagnosis, history, and physical examination of the spine with emphasis on the administration of spinal injections and their role in the treatment of painful spinal conditions.

Salmonella enterica (S. enterica) are Gram-negative bacteria that can invade a broad range of hosts causing both acute and chronic infections. This phenotype is related to its ability to replicate and persist within non-phagocytic host epithelial cells as well as phagocytic dendritic cells and macrophages of the innate immune system. Infection with S. enterica manifests itself through a broad range of clinical symptoms and can result in asymptomatic carriage, gastroenteritis, systemic disease such as typhoid fever and in severe cases, death (Gunn et al. 2014). Exposure to S. enterica serovars Typhi and Paratyphi exhibits clinical symptoms including diarrhoea, fatigue, fever and temperature fluctuations. Other serovars such as the non-typhoidal Salmonella (NTS), of which there are over 2,500, are commonly contracted as, but not limited to, food-borne sources causing gastrointestinal symptoms, which include diarrhoea and vomiting. The availability of complete genome sequences for many S. enterica serovars has facilitated research into the genetic determinants of virulence for this pathogen. This work has led to the identification of important bacterial components, including flagella, type III secretion systems, lipopolysaccharides and Salmonella pathogenicity islands, all of which support the intracellular life cycle of S. enterica. Studies focusing on the host-pathogen interaction have provided insights into receptor activation of the innate immune system. Therefore, characterising the host-S. enterica interaction is critical to understand the pathogenicity of the bacteria in a clinically relevant context. This review outlines salmonellosis and the clinical manifestations between typhoidal and NTS infections as well as discussing the host immune response to infection and the models that are being used to elucidate the mechanisms involved on Salmonella pathogenicity.

Building a comprehensive knowledge base of the association of Listeria monocytogenes isolates across national food chains, clinical cases, and environments can play a key role in helping control the incidence of listeriosis. Today, many food chains cross national borders and are often shared by neighboring countries. This study characterized L. monocytogenes isolated from food samples in Northern Ireland and investigated whether similarities in the population and associations of L. monocytogenes strains exist in the neighboring countries of Northern Ireland and the Republic of Ireland, which together constitute the island of Ireland. Listeria monocytogenes isolates were characterized using serotyping and pulsed-field gel electrophoresis subtyping. This data was then interrogated against existing data for the Republic of Ireland, to identify any shared trends in the ecology and contamination patterns of L. monocytogenes strains. The results of this study indicated that contaminated food products often shared L. monocytogenes strains with other products. A total of six different strain subtypes were identified among 18 contaminated products. Overall strain diversity in positive samples was low, with no sample yielding more than one L. monocytogenes strain, as determined by pulsed-field gel electrophoresis subtyping. When comparisons against an Irish strain database were performed, many related strain subtypes were also shared by a variety of sources in the Republic of Ireland. This study highlights the potential benefits that a whole-island surveillance approach may present to food safety and public health in both Northern Ireland and the Republic of Ireland.

Leptospirosis, an emerging zoonotic disease with worldwide distribution, is caused by spirochetes belonging to the genus Leptospira. More than 500,000 cases of severe leptospirosis are reported annually, with >10% of these being fatal. Leptospires can survive for weeks in suitably moist conditions before encountering a new host. Reservoir hosts, typically rodents, exhibit little to no signs of disease but shed large numbers of organisms in their urine. Transmission occurs when mucosal surfaces or abraded skin come into contact with infected urine or urine-contaminated water or soil. In humans, leptospires can cause a variety of clinical manifestations, ranging from asymptomatic or mild fever to severe icteric (Weil's) disease and pulmonary haemorrhage. Currently, little is known about how Leptospira persist within a reservoir host. Prior in vitro studies have suggested that leptospires alter their transcriptomic and proteomic profiles in response to environmental signals encountered during mammalian infection. However, no study has examined gene expression by leptospires within a mammalian host-adapted state. To obtain a more faithful representation of how leptospires respond to host-derived signals, we used RNA-Seq to compare the transcriptome of L. interrogans cultivated within dialysis membrane chambers (DMCs) implanted into the peritoneal cavities of rats with that of organisms grown in vitro. In addition to determining the relative expression levels of “core” housekeeping genes under both growth conditions, we identified 166 genes that are differentially-expressed by L. interrogans in vivo. Our analyses highlight physiological aspects of host adaptation by leptospires relating to heme uptake and utilization. We also identified 11 novel non-coding transcripts that are candidate small regulatory RNAs. The DMC model provides a facile system for studying the transcriptional and antigenic changes associated with mammalian host-adaption, selection of targets for mutagenesis, and the identification of previously unrecognized virulence determinants., Author Summary Leptospirosis, a global disease caused by the unusual bacterium Leptospira, is transmitted from animals to humans. Pathogenic species of Leptospira are excreted in urine from infected animals and can continue to survive in suitable environments before coming into contact with a new reservoir or accidental host. Leptospires have an inherent ability to survive a wide range of conditions encountered in nature during transmission and within mammals. However, we know very little about the regulatory pathways and gene products that promote mammalian host adaptation and enable leptospires to establish infection. In this study, we used a novel system whereby leptospires are cultivated in dialysis membrane chambers implanted into the peritoneal cavities of rats to compare the gene expression profiles of mammalian host-adapted and in vitro-cultivated organisms. In addition to providing a facile system for studying the transcriptional and physiologic changes leptospires undergo during mammalian infection, our data provide a rational basis for selecting new targets for mutagenesis.

The synaptopodin 2 gene can be differently spliced, resulting in three mRNAs with varying 3′ ends coding for different C-termini (De Ganck et al 2008). The calculated molecular weights of these proteins are 117, 119, and 136 kDa. These isoforms require the expression of exons 1–3. However, only a single protein product has been detected in mammalian muscle lacking the product of these first exons.We extracted four fesselin isoforms from avian smooth muscle tissue. These include the first isolated 79 and 103 kDa isoforms (Leinweber et al. 1999). The newly detected isoforms migrated on SDS gels with apparent molecular masses of 140 and 160 kDa. In contrast to our initial assumption that the 79 kDa was a proteolytic product of the 103 kDa protein we now show that they are different spliceforms. The 79 kDa isoform forms the core of synaptopodin 2. The other isoforms have different extensions at either the N- or C-terminal regions.The different isoforms were differentially extracted by different buffers. Extractions were most complete under conditions that depolymerized both actin and intermediate filaments. Surprisingly although fesselin binds to actin and myosin none of the four different isoforms was extracted with the acto-myosin-complex. The extraction data suggest that fesselin functions in actin filament organization rather than in regulation of actin-myosin interactions. In contrast to smooth muscle tissue we detected one isoform of fesselin in skeletal and heart muscle tissue in agreement with the findings in mammalian tissue. In avian skeletal muscle we observed a 79 kDa isoform and in heart muscle a 170 kDa isoform. The reason for the differential expression of fesselin in different muscle types is unknown.