An Official Outbreak Investigation of Acute Haemorrhagic Diarrhoea in Dogs in Norway Points to Providencia alcalifaciens as a Likely Cause

An outbreak investigation was initiated in September 2019, following a notification to the Norwegian Food Safety Authority (NFSA) of an unusually high number of dogs with acute haemorrhagic diarrhoea (AHD) in Oslo. Diagnostic testing by reporting veterinarians had not detected a cause. The official investigation sought to identify a possible common cause, the extent of the outbreak and prevent spread. Epidemiological data were collected through a survey to veterinarians and interviews with dog owners. Diagnostic investigations included necropsies and microbiological, parasitological and toxicological analysis of faecal samples and food. In total, 511 dogs with acute haemorrhagic diarrhoea were registered between 1 August and 1 October. Results indicated a common point source for affected dogs, but were inconclusive with regard to common exposures. A notable finding was that 134 of 325 faecal samples (41%) cultured positive for Providencia alcalifaciens. Whole genome sequencing (WGS) of 75 P. alcalifaciens isolates from 73 dogs revealed that strains from 51 dogs belonged to the same WGS clone. Findings point to P. alcalifaciens as implicated in the outbreak, but investigations are needed to reveal the pathogenic potential of P. alcalifaciens in dogs and its epidemiology.

Structural and Biochemical Analyses Reveal that Chlorogenic Acid Inhibits the Shikimate Pathway

ABSTRACT Chlorogenic acid (CGA) is a phenolic compound with well-known antibacterial properties against pathogens. In this study, structural and biochemical characterization was used to show the inhibitory role of CGA against the enzyme of the shikimate pathway, a well-characterized drug target in several pathogens. Here, we report the crystal structures of dehydroquinate synthase (DHQS), the second enzyme of the shikimate pathway, from Providencia alcalifaciens (PaDHQS), in binary complex with NAD and ternary complex with NAD and CGA. Structural analyses reveal that CGA occupies the substrate position in the active site of PaDHQS, which disables domain movements, leaving the enzyme in an open and catalysis-incompetent state. The binding analyses by isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) show that CGA binds to PaDHQS with KD (equilibrium dissociation constant) values of 6.3 μM and 0.5 μM, respectively. In vitro enzyme inhibition studies show that CGA inhibits PaDHQS with a Ki of 235 ± 21 μM, while it inhibits the growth of Providencia alcalifaciens, Moraxella catarrhalis, Staphylococcus aureus, and Escherichia coli with MIC values of 60 to 100 μM. In the presence of aromatic amino acids supplied externally, CGA does not show the toxic effect. These results, along with the observations of the inhibition of the 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) regulatory domain by CGA in our previous study, suggest that CGA binds to shikimate pathway enzymes with high affinity and inhibits their catalysis and can be further exploited for designing novel drug-like molecules. IMPORTANCE The shikimate pathway is an attractive target for the development of herbicides and antimicrobial agents, as it is essential in plants, bacteria, and apicomplexan parasites but absent in humans. The enzymes of shikimate pathway are conserved among bacteria. Thus, the inhibitors of the shikimate pathway act on wide range of pathogens. We have identified that chlorogenic acid targets the enzymes of the shikimate pathway. The crystal structure of dehydroquinate synthase, the second enzyme of the pathway, in complex with chlorogenic acid and enzymatic inhibition studies explains the mechanism of inhibition of chlorogenic acid. These results suggest that chlorogenic acid has a good chemical scaffold and have important implications for its further development as a potent inhibitor of shikimate pathway enzymes.

Screening for Bio-flocculant Producing Bacterial Strains from Asa River in Ilorin Kwara State

Synthetic flocculants in water treatment have been reported to be detrimental to both human health and the environment. Thus, there is a constant search for bio-flocculants that is safe and addresses the effects of synthetic polymers. This study was aimed at isolating bacteria with bio-flocculating potential, their molecular identification and phylogenetic relatedness, and the optimization of their flocculating abilities from an open water (Asa River) in Ilorin Kwara State. The effect of carbon source (glucose, lactose and starch), cations (FeSO4, KCl and CaCl2) and pH (2 to 12) was evaluated on the bio-flocculating activities of the isolates using kaolin clay. The seven (7) novel (new strains) isolates with their respective accession number from National Center for Biotechnology Information (NCBI) after molecular confirmation are Pseudomonas otitidis MTK01 (MK263227), Aeromonas caviea MTK02 (MK263228), Providencia alcalifaciens MTK03 (MK263229), Providencia sp. MTK05 (MK263230), Alcaligenes sp. MTK06 (MK263231), Klebsiella pneumoniae MTK07 (MK263232) and Klebsiella sp. MTK08 (MK263233) while Raoultella ornithinolytica MTK04 was also identified. Phylogenetic tree of relatedness showed close kin of the isolates to established bacteria sequence deposited at the NCBI GenBank. Although the flocculating rate of each isolates varied with different parameters that was used in the study, glucose was the most supportive followed by lactose and starch, CaCl2 was most supportive cation followed by KCl and FeSO4 while pH 12, 6, 8, 10, 2 and 4 were the order of decreasing flocculating rate of the medium. This study has reported the presence of eight (8) bio-flocculating bacteria (out of which 7 are new strains of bacteria) in an open water which has been further optimized for effective flocculating rate and thus provides an ecofriendly and harmless flocculants source that can be employed in water treatment procedures.

KlebsiellaandProvidenciaemerge as lone survivors following long-term starvation of oral microbiota

It is well-understood that many bacteria have evolved to survive catastrophic events using a variety of mechanisms, which include expression of stress-response genes, quiescence, necrotrophy, and metabolic advantages obtained through mutation. However, the dynamics of individuals leveraging these abilities to gain a competitive advantage in an ecologically complex setting remain unstudied. In this study, we observed the saliva microbiome throughout the ecological perturbation of long-term starvation, allowing only the species best equipped to access and use the limited resources to survive. During the first several days, the community underwent a death phase that resulted in a ∼50–100-fold reduction in the number of viable cells. Interestingly, after this death phase, only three species,Klebsiella pneumoniae,Klebsiella oxytoca, andProvidencia alcalifaciens, all members of the family Enterobacteriaceae, appeared to be transcriptionally active and recoverable.Klebsiellaare significant human pathogens, frequently resistant to multiple antibiotics, and recently, ectopic colonization of the gut by oralKlebsiellawas documented to induce dysbiosis and inflammation. MetaOmics analyses provided several leads for further investigation regarding the ecological success of the Enterobacteriaceae. The isolates accumulated single nucleotide polymorphisms in known growth advantage in stationary phase alleles and produced natural products closely resembling antimicrobial cyclic depsipeptides. The results presented in this study suggest that pathogenic Enterobacteriaceae persist much longer than their more benign neighbors in the salivary microbiome when faced with starvation. This is particularly significant, given that hospital surfaces contaminated with oral fluids, especially sinks and drains, are well-established sources of outbreaks of drug-resistant Enterobacteriaceae.

CexE Is A Virulence Factor ofCitrobacter rodentiumAnd Present In Enteric Pathogens Of Humans

CexE is a 12 kDa protein that was originally reported to be present in just three strains of enterotoxigenicEscherichia coli(ETEC); a frequent causes of diarrheal illnesses worldwide. However, an examination of recently sequenced genomes has revealed that CexE is actually present in a majority of ETEC strains. Homologs of CexE are also present in enteroaggregativeE. coli(EAEC) and other enteric pathogens includingYersinia enterocolitica, Providencia alcalifaciens, andCitrobacter rodentium. CexE and its homologs are expressed within virulence regulons of ETEC, EAEC, andC. rodentium. This, along with its distribution across several species of enteric pathogens, suggest that CexE confers a selective advantage to these pathogens. However, this hypothesis has yet to be testedin vivo. Here we demonstrate that CexE is conditionally secreted to the external leaf of the outer membrane of ETEC. Although CexE does not appear to play a role in adherencein vitro, it does facilitate colonization of murine intestinal tissues byC. rodentium in vivo. In adult mice wild-type bacteria reached significantly higher loads and were shed in higher numbers than acexE::kanmutant. A similar trend was observed in neonatal mice. In addition, all of the neonates infected with the wild-type strain succumbed to infection within 16 days of inoculation. In contrast, 45% of the neonates infected with thecexE::kanstrain survived for the 30 day duration of the experiment. These finding indicate that CexE is a conditionally secreted virulence factor that increases the colonization of hosts by enteric pathogens.

Determination of thermotolerant coliforms present in coconut water produced and bottled in the Northeast of Brazil

Coconut water is considered to be a natural isotonic drink and its marketing is gradually increasing. The objective of the present study was to evaluate the microbiological quality of the coconut water produced and bottled in the Northeast of Brazil. Products form ten industries from different states in the Northeast of Brazil were analyzed. The most probable number (MPN) method was used to quantify the coliforms. Samples showing positive for coliforms were seeded on ChromAgar Orient plates and the bacteria identified from isolated colonies using the automated system Vitek 2 (BioMérieux), according to the manufacturer's instructions for the preparation of the inoculum, incubation, reading and interpretation. The samples showed thermotolerant coliform counts between 6.0×102 and 2.6×104 MPN/100 mL. The presence of Klebsiella pneumoniae, Morganella morganii and Providencia alcalifaciens was observed. The implementation of preventive methods and monitoring of the water quality by the industries is required.