Infection, colonization and shedding of Campylobacter and Salmonella in animals and their contribution to human disease: A review

Livestock meat and offal contribute significantly to human nutrition as sources of high‐quality protein and micronutrients. Livestock products are increasingly in demand, particularly in low‐ and middle‐income settings where economies are growing and meat is increasingly seen as an affordable and desirable food item. Demand is also driving intensification of livestock keeping and processing. An unintended consequence of intensification is increased exposure to zoonotic agents, and a contemporary emerging problem is infection with Campylobacter and Salmonella spp. from livestock (avian and mammalian), which can lead to disease, malabsorption and undernutrition through acute and chronic diarrhoea. This can occur at the farm, in households or through the food chain. Direct infection occurs when handling livestock and through bacteria shed into the environment, on food preparation surfaces or around the house and surroundings. This manuscript critically reviews Campylobacter and Salmonella infections in animals, examines the factors affecting colonization and faecal shedding of bacteria of these two genera as well as risk factors for human acquisition of the infection from infected animals or environment and analyses priority areas for preventive actions with a focus on resource‐poor settings

Structure activity relationship of pyrazinoic acid analogs as potential antimycobacterial agents

Tuberculosis (TB) remains a leading cause of infectious disease-related mortality and morbidity. Pyrazinamide (PZA) is a critical component of the first-line TB treatment regimen because of its sterilizing activity against non-replicating Mycobacterium tuberculosis (Mtb), but its mechanism of action has remained enigmatic. PZA is a prodrug converted by pyrazinamidase encoded by pncA within Mtb to the active moiety, pyrazinoic acid (POA) and PZA resistance is caused by loss-of-function mutations to pyrazinamidase. We have recently shown that POA induces targeted protein degradation of the enzyme PanD, a crucial component of the coenzyme A biosynthetic pathway essential in Mtb. Based on the newly identified mechanism of action of POA, along with the crystal structure of PanD bound to POA, we designed several POA analogs using structure for interpretation to improve potency and overcome PZA resistance. We prepared and tested ring and carboxylic acid bioisosteres as well as 3, 5, 6 substitutions on the ring to study the structure activity relationships of the POA scaffold. All the analogs were evaluated for their whole cell antimycobacterial activity, and a few representative molecules were evaluated for their binding affinity, towards PanD, through isothermal titration calorimetry. We report that analogs with ring and carboxylic acid bioisosteres did not significantly enhance the antimicrobial activity, whereas the alkylamino-group substitutions at the 3 and 5 position of POA were found to be up to 5 to 10-fold more potent than POA. Further development and mechanistic analysis of these analogs may lead to a next generation POA analog for treating TB.Ministry of Education (MOE)Research reported in this work was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number R01 AI106398 and the Singapore Ministry of Education (MoE) Academic Research Fund Tier 1 (RG107/20)

Mycobacterium tuberculosis PanD StructureFunction Analysis and Identification of a Potent Pyrazinoic Acid-Derived Enzyme Inhibitor

A common strategy employed in antibacterial drug discovery is targeting of biosynthetic processes which are essential and specific for the pathogen. Specificity in particular avoids undesirable interactions with potential enzymatic counterparts in the human host, and ensures on-target toxicity. Synthesis of pantothenate (Vitamine B5), a precursor of the acyl carrier coenzyme A, is an example of such a pathway. In Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), pantothenate is formed by pantothenate synthase, utilizing D-pantoate and β-Ala as substrates. β-Ala is mainly formed by the decarboxylation of L-aspartate, generated by the decarboxylase PanD, a homo-oliogomer in solution. Pyrazinoic acid (POA), the bioactive form of the TB prodrug pyrazinamide, binds and inhibits PanD activity weakly. Here, we generated a library of recombinant Mtb PanD mutants based on structural information and PZA/POA resistance mutants. Alterations in oligomer formation, enzyme activity and/or POA binding were observed in respective mutants, providing insights into essential amino acids for Mtb PanD’s proper structural assembly, decarboxylation activity and drug interaction. This information provided the platform for the design of novel POA analogs with modifications at position 3 of the pyrazine ring. Analog 2, incorporating a bulky naphthamido group at this position, displayed a 1,000-fold increase in enzyme inhibition compared to POA, along with moderately improved antimycobacterial activity. The data demonstrate that an improved understanding of mechanistic and enzymatic features of key metabolic enzymes can stimulate design of more potent PanD inhibitors

Structural and mechanistic insights into Mycobacterium abscessus as-partate decarboxylase PanD and a pyrazinoic acid-derived inhibitor

Mycobacterium tuberculosis (Mtb) aspartate decarboxylase PanD is required for biosynthesis of the essential cofactor coenzyme A and targeted by the first line drug pyrazinamide (PZA). PZA is a prodrug that is converted by a bacterial amidase into its bioactive form pyrazinoic acid (POA). Employing structure-function analyses we previously identified POA-based inhibitors of Mtb PanD showing much improved inhibitory activity against the enzyme. Here, we performed the first structure-function studies on PanD encoded by the non-tuberculous mycobacterial lung pathogen Mycobacterium abscessus (Mab), shedding light on the differences and similarities of Mab and Mtb PanD. Solution X-ray scattering data provided the solution structure of the entire tetrameric Mab PanD, which in comparison to the structure of the derived C-terminal trun-cated Mab PanD1-114 mutant, revealed the orientation of the four flexible C-termini relative to the catalytic core. Enzymatic studies of Mab PanD1-114 explored the essentiality of the C-terminus for catalysis. A library of recombinant Mab PanD mutants based on structural information and PZA/POA resistant PanD mutations in Mtb, illuminated critical residues involved in the substrate tunnel and enzymatic activity. Using our library of POA analogs, we identified (3-(1-naphthamido)pyrazine-2-car-boxylic acid) (analog 2) as the first potent inhibitor of Mab PanD. The inhibitor shows mainly electrostatic- and hydrogen bonding interaction with the target enzyme as explored by isothermal titration calorimetry and confirmed by docking studies. The observed unfavorable entropy indicates that significant conformational changes are involved in the binding process of analog 2 to Mab PanD. In contrast to PZA and POA, which are whole-cell inactive, analog 2 exerts appreciable antibacterial activity against the three subspecies of Mab.Ministry of Education (MOE)National Research Foundation (NRF)Submitted/Accepted versionResearch reported in this publication is supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Numbers 2R01AI106398-05 (T.D., (T.D., (T.D., C.A.,C.A., G.G.) G.G.) . The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The studies are also supported by the National Research Founda-tion (NRF) Singapore, NRF Competitive Research Programme (CRP), Grant Award Number NRF–CRP18–2017–01 (G.G., T.D.), and the Singapore Ministry of Education (MOE) Academic Research Fund Tier 1 (RG107/20) to G.G

GeneXpert MTB/RIF Assay for the Diagnosis of Tuberculous Lymphadenitis on Concentrated Fine Needle Aspirates in High Tuberculosis Burden Settings

<div><p>Introduction</p><p>The diagnosis of tuberculous lymphadenitis (TBL) remains challenging. The routinely used methods (cytology and smear microscopy) have sub-optimal sensitivity. Recently, WHO recommends GeneXpert to be used as the initial diagnostic test in patients suspected of having extra-pulmonary tuberculosis (EPTB). However, this was a conditional recommendation due to very low-quality evidence available and more studies are needed. In this study we evaluated the performance of Xpert for the diagnosis of TBL on concentrated fine needle aspirates (FNA) in Southwest Ethiopia.</p><p>Methods</p><p>FNA was collected from presumptive TBL cases. Two smears were prepared from each aspirate and processed for cytology and conventional microscopy. The remaining aspirate was treated with N-acetyl-L-cysteine-NaOH and centrifuged for 15minutes at 3000g. The concentrated sediment was used for culture and Xpert test. Capilia TB-Neo test was used to differentiate <i>M</i>. <i>tuberculosis</i> complex (MTBC) from non-tuberculous mycobacteria (NTM). Composite bacteriological methods (culture and/or smear microscopy) were considered as a reference standard.</p><p>Result</p><p>Out of 143 enrolled suspects, 64.3% (92/143) were confirmed TBL cases by the composite reference standard (CRS). Xpert detected <i>M</i>. <i>tuberculosis</i> complex (MTBC) in 60.1% (86/143) of the presumptive TBL cases. The sensitivity of Xpert compared to CRS was 87.8% [95% CI: 81.0–94.5] and specificity 91.1% [95% CI: 82.8–99.4]. The sensitivity was 27.8% for smear microscopy and 80% for cytology compared to CRS. Cytology showed the lowest specificity (57.8%). Xpert was positive in 4 out of 45 culture- and smear-negative cases. Among 47 cytomorphologically non-TBL cases, 15 were positive on Xpert. More than half of Xpert-positive cases were in the range of very low cut-off threshold values (28</p><p>Conclusion</p><p>Xpert test showed a high sensitivity and specificity for the diagnosis of TBL on concentrated FNA samples. In addition, Xpert offered rapid detection of rifampicin-resistant <i>M</i>. <i>tuberculosis</i> strains from lymph node aspirates.</p></div

Comparison of Xpert semi-quantitative result (Ct-value) and AFB smear grade.

<p>AFB = acid-fast bacilli; Ct = cycle threshold</p><p>Comparison of Xpert semi-quantitative result (Ct-value) and AFB smear grade.</p

Comparisons of microscopic cytomorphological features and FNA gross appearance with Xpert test and CRS (culture and/or ZN).

<p>TBL = tuberculous lymphadenitis, FNA = fine needle aspirate, CRS = composite reference standard, ZN = Ziehl-Neelsen.</p><p>Comparisons of microscopic cytomorphological features and FNA gross appearance with Xpert test and CRS (culture and/or ZN).</p

Diagnostic accuracy of Xpert for detection of <i>M</i>. <i>tuberculosis</i> from patients with suspected TBL.

<p>TBL = tuberculous lymphadenitis, FNA = fine needle aspirate, + = positive,— = negative.</p