Comparative genomics of Campylobacter jejuni from clinical campylobacteriosis stool specimens.

BACKGROUND: Campylobacter jejuni is a pervasive pathogen of major public health concern with a complex ecology requiring accurate and informative approaches to define pathogen diversity during outbreak investigations. Source attribution analysis may be confounded if the genetic diversity of a C. jejuni population is not adequately captured in a single specimen. The aim of this study was to determine the genomic diversity of C. jejuni within individual stool specimens from four campylobacteriosis patients. Direct plating and pre-culture filtration of one stool specimen per patient was used to culture multiple isolates per stool specimen. Whole genome sequencing and pangenome level analysis were used to investigate genomic diversity of C. jejuni within a patient. RESULTS: A total 92 C. jejuni isolates were recovered from four patients presenting with gastroenteritis. The number of isolates ranged from 13 to 30 per patient stool. Three patients yielded a single C. jejuni multilocus sequence type: ST-21 (n = 26, patient 4), ST-61 (n = 30, patient 1) and ST-2066 (n = 23, patient 2). Patient 3 was infected with two different sequence types [ST-51 (n = 12) and ST-354 (n = 1)]. Isolates belonging to the same sequence type from the same patient specimen shared 12-43 core non-recombinant SNPs and 0-20 frameshifts with each other, and the pangenomes of each sequence type consisted of 1406-1491 core genes and 231-264 accessory genes. However, neither the mutation nor the accessory genes were connected to a specific functional gene category. CONCLUSIONS: Our findings show that the C. jejuni population recovered from an individual patient's stool are genetically diverse even within the same ST and may have shared common ancestors before specimens were obtained. The population is unlikely to have evolved from a single isolate at the time point of initial patient infection, leading us to conclude that patients were likely infected with a heterogeneous C. jejuni population. The diversity of the C. jejuni population found within individual stool specimens can inform future methodological approaches to attribution and outbreak investigations.

Mitotic timing is differentially controlled by A- and B-type cyclins and by CDC6 associated with a bona fide CDK inhibitor Xic1 in Xenopus laevis cell-free extract.

The timing of the M-phase is precisely controlled by a CDC6-dependent mechanism inhibiting the mitotic histone H1 kinase. Here, we describe the differential regulation of the dynamics of this mitotic kinase activity by exogenous cyclin A or cyclin B in the Xenopus laevis cycling extracts. We show that the experimental increase in cyclin A modifies only the level of histone H1 kinase activity, while the cyclin B increase modifies two parameters: histone H1 kinase activity and the timing of its full activation, which is accelerated. On the other hand, the cyclin A depletion significantly delays full activation of histone H1 kinase. However, when CDC6 is added to such an extract, it inhibits cyclin B-associated histone H1 kinase, but does not modify the mitotic timing in the absence of cyclin A. Further, we show via p9 co-precipitation with Cyclin-Dependent Kinases (CDKs), that both CDC6 and the bona fide CDK1 inhibitor Xic1 associate with the mitotic CDKs. Finally, we show that the Xic1 temporarily separates from the mitotic CDKs complexes during the peak of histone H1 kinase activity. These data show the differential coordination of the M-phase progression by cyclin A- and cyclin B-dependent CDKs, confirm the critical role of the CDC6-dependent histone H1 kinase inhibition in this process, and show that CDC6 acts differentially through the cyclin B- and cyclin A-associated CDKs. This CDC6- and cyclins-dependent mechanism likely depends on the precisely regulated association of Xic1 with the mitotic CDKs complexes. We postulate that: i. the dissociation of Xic1 from the CDKs complexes allows the maximal activation of CDK1 during the M-phase, ii. the switch between cyclin A- and cyclin B-CDK inhibition upon M-phase initiation may be responsible for the diauxic growth of mitotic histone H1 kinase activity.

Genome-Scale Metabolic Model Driven Design of a Defined Medium for Campylobacter jejuni M1cam.

Campylobacter jejuni, the most frequent cause of food-borne bacterial gastroenteritis, is a fastidious organism when grown in the laboratory. Oxygen is required for growth, despite the presence of the metabolic mechanism for anaerobic respiration. Amino acid auxotrophies are variably reported and energy metabolism can occur through several electron donor/acceptor combinations. Overall, the picture is one of a flexible, but vulnerable metabolism. To understand Campylobacter metabolism, we have constructed a fully curated, metabolic model for the reference organism M1 (our variant is M1cam) and validated it through laboratory experiments. Our results show that M1cam is auxotrophic for methionine, niacinamide, and pantothenate. There are complete biosynthesis pathways for all amino acids except methionine and it can produce energy, but not biomass, in the absence of oxygen. M1cam will grow in DMEM/F-12 defined media but not in the previously published Campylobacter specific defined media tested. Using the model, we identified potential auxotrophies and substrates that may improve growth. With this information, we designed simple defined media containing inorganic salts, the auxotrophic substrates, L-methionine, niacinamide, and pantothenate, pyruvate and additional amino acids L-cysteine, L-serine, and L-glutamine for growth enhancement. Our defined media supports a 1.75-fold higher growth rate than Brucella broth after 48 h at 37°C and sustains the growth of other Campylobacter jejuni strains. This media can be used to design reproducible assays that can help in better understanding the adaptation, stress resistance, and the virulence mechanisms of this pathogen. We have shown that with a well-curated metabolic model it is possible to design a media to grow this fastidious organism. This has implications for the investigation of new Campylobacter species defined through metagenomics, such as C. infans.

Ceftriaxone-resistant Salmonella Typhi carries an IncI1-ST31 plasmid encoding CTX-M-15.

PURPOSE: Ceftriaxone is the drug of choice for typhoid fever and the emergence of resistant Salmonella Typhi raises major concerns for treatment. There are an increasing number of sporadic reports of ceftriaxone-resistant S. Typhi and limiting the risk of treatment failure in the patient and outbreaks in the community must be prioritized. This study describes the use of whole genome sequencing to guide outbreak identification and case management. METHODOLOGY: An isolate of ceftriaxone-resistant S. Typhi from the blood of a child taken in 2000 at the Popular Diagnostic Center, Dhaka, Bangladesh was subjected to whole genome sequencing, using an Illumina NextSeq 500 and analysis using Geneious software.Results/Key findings. Comparison with other ceftriaxone-resistant S. Typhi revealed an isolate from the Democratic Republic of the Congo in 2015 as the closest relative but no evidence of an outbreak. A plasmid belonging to incompatibility group I1 (IncI1-ST31) which included blaCTX-M-15 (ceftriaxone resistance) associated with ISEcp-1 was identified. High similarity (90 %) was seen with pS115, an IncI1 plasmid from S. Enteritidis, and with pESBL-EA11, an incI1 plasmid from E. coli (99 %) showing that S. Typhi has access to ceftriaxone resistance through the acquisition of common plasmids. CONCLUSIONS: The transmission of ceftriaxone resistance from E. coli to S. Typhi is of concern because of clinical resistance to ceftriaxone, the main stay of typhoid treatment. Whole genome sequencing, albeit several years after the isolation, demonstrated the success of containment but clinical trials with alternative agents are urgently required.

Frequency and susceptibility pattern of uropathogenic Enterobacteriaceae isolated from patients in Algiers, Algeria.

INTRODUCTION: The frequency of Enterobacteriaceae involved in urinary tract infections (UTI) has increased significantly since the early 1990s, particularly in at-risk facilities such as resuscitation, surgery, urology and nephrology. The objective of this study was to evaluate the antimicrobial susceptibility of Enterobacteriaceae causing urinary tract infections (UTIs)at the University Hospital Centre of Benimessous in Algiers. METHODOLOGY: The study was designed as a retrospective study (between January 1st 2010 and December 31st 2012) and a prospective study (between January 1standApril 30th 2013) on 13,611 urine samples. Antimicrobial resistance phenotyping was conducted on the bacterial isolates using disk-diffusion method. RESULTS: On 13,611 urine samples analysed, 1,790 (13.15%) fulfilled the criteria for urinary tract infection. Enterobacteriaceae were identified in 1,561 analysed samples (87%). Escherichia coli was the dominant uropathogen (66,15%) in both hospitalized and non-hospitalized patients. The other main detected Enterobacteriaceae members were Klebsiella pneumoniae (11,96%) and Proteus mirabilis (5,42%). Analysis of results showed also that women were more prone to UTI than men with sex ratio of 3.76(W/M). The susceptibilities of isolated Enterobacteriaceae to antibiotics revealed that they had acquired resistance to several classes, particularly toward ß-lactams. Resistance frequencies were relatively high to ampicillin and sulfomethoxasole, while being very low to aminoglycosides and furans. Results obtained revealed also that 7% of isolates where resistant to third generation cephalosporins by production of extended spectrum ß-lactamases (ESBL). CONCLUSIONS: The continuous monitoring of antibiotic resistance of uropathogenic Escherichia coli is crucial to guide the clinician to choose the best empiric treatment.

An Algerian perspective on non-typhoidal Salmonella infection.

Non-typhoidal Salmonella (NTS) represents a leading cause of food-borne disease worldwide. It is a global public health concern: more than 94 million cases and 115,000 deaths are reported every year, with a disproportionate impact in developing countries. The prevalence of multi-drug-resistant (MDR) Salmonella strains is another major health concern which affects antimicrobial treatment, as many studies report that infections caused by MDR strains are more severe than those caused by susceptible strains. In Algeria, NTS represent one of the primary causes of salmonellosis in both humans and food animal production, especially poultry. Epidemiological surveillance systems and monitoring programs for Salmonella infections are essential requirements to provide data useful for the effective detection and control of Salmonella outbreaks. The present review will supply a perspective on NTS infection, pathogenesis and antimicrobial resistance with a focus on the epidemiology of salmonellosis in Algeria.