Genetic diversity of Salmonella Paratyphi A isolated from enteric fever patients in Bangladesh from 2008 to 2018

BackgroundThe proportion of enteric fever cases caused by Salmonella Paratyphi A is increasing and may increase further as we begin to introduce typhoid conjugate vaccines (TCVs). While numerous epidemiological and genomic studies have been conducted for S. Typhi, there are limited data describing the genomic epidemiology of S. Paratyphi A in especially in endemic settings, such as Bangladesh.Principal findingsWe conducted whole genome sequencing (WGS) of 67 S. Paratyphi A isolated between 2008 and 2018 from eight enteric disease surveillance sites across Bangladesh. We performed a detailed phylogenetic analysis of these sequence data incorporating sequences from 242 previously sequenced S. Paratyphi A isolates from a global collection and provided evidence of lineage migration from neighboring countries in South Asia. The data revealed that the majority of the Bangladeshi S. Paratyphi A isolates belonged to the dominant global lineage A (67.2%), while the remainder were either lineage C (19.4%) or F (13.4%). The population structure was relatively homogenous across the country as we did not find any significant lineage distributions between study sites inside or outside Dhaka. Our genomic data showed presence of single point mutations in gyrA gene either at codon 83 or 87 associated with decreased fluoroquinolone susceptibility in all Bangladeshi S. Paratyphi A isolates. Notably, we identified the pHCM2- like cryptic plasmid which was highly similar to S. Typhi plasmids circulating in Bangladesh and has not been previously identified in S. Paratyphi A organisms.SignificanceThis study demonstrates the utility of WGS to monitor the ongoing evolution of this emerging enteric pathogen. Novel insights into the genetic structure of S. Paratyphi A will aid the understanding of both regional and global circulation patterns of this emerging pathogen and provide a framework for future genomic surveillance studies.     

Typhoidal Salmonella: Distinctive virulence factors and pathogenesis

Although nontyphoidal Salmonella (NTS; including Salmonella Typhimurium) mainly cause gastroenteritis, typhoidal serovars (Salmonella Typhi and Salmonella Paratyphi A) cause typhoid fever, the treatment of which is threatened by increasing drug resistance. Our understanding of S. Typhi infection in human remains poorly understood, likely due to the host restriction of typhoidal strains and the subsequent popularity of the S. Typhimurium mouse typhoid model. However, translating findings with S. Typhimurium across to S. Typhi has some limitations. Notably, S. Typhi has specific virulence factors, including typhoid toxin and Vi antigen, involved in symptom development and immune evasion, respectively. In addition to unique virulence factors, both typhoidal and NTS rely on two pathogenicity‐island encoded type III secretion systems (T3SS), the SPI‐1 and SPI‐2 T3SS, for invasion and intracellular replication. Marked differences have been observed in terms of T3SS regulation in response to bile, oxygen, and fever‐like temperatures. Moreover, approximately half of effectors found in S. Typhimurium are either absent or pseudogenes in S. Typhi, with most of the remaining exhibiting sequence variation. Typhoidal‐specific T3SS effectors have also been described. This review discusses what is known about the pathogenesis of typhoidal Salmonella with emphasis on unique behaviours and key differences when compared with S. Typhimurium.     

Estimating the Burden of Paratyphoid A in Asia and Africa

Despite the increasing availability of typhoid vaccine in many regions, global estimates of mortality attributable to enteric fever appear stable. While both Salmonella enterica serovar Typhi (S. Typhi) and serovar Paratyphi (S. Paratyphi) cause enteric fever, limited data exist estimating the burden of S. Paratyphi, particularly in Asia and Africa.We performed a systematic review of both English and Chinese-language databases to estimate the regional burden of paratyphoid within Africa and Asia. Distinct from previous reviews of the topic, we have presented two separate measures of burden; both incidence and proportion of enteric fever attributable to paratyphoid. Included articles reported laboratory-confirmed Salmonella serovar classification, provided clear methods on sampling strategy, defined the age range of participants, and specified the time period of the study.A total of 64 full-text articles satisfied inclusion criteria and were included in the qualitative synthesis. Paratyphoid A was commonly identified as a cause of enteric fever throughout Asia. The highest incidence estimates in Asia came from China; four studies estimated incidence rates of over 150 cases/100,000 person-years. Paratyphoid A burden estimates from Africa were extremely limited and with the exception of Nigeria, few population or hospital-based studies from Africa reported significant Paratyphoid A burden.While significant gaps exist in the existing population-level estimates of paratyphoid burden in Asia and Africa, available data suggest that paratyphoid A is a significant cause of enteric fever in Asia. The high variability in documented incidence and proportion estimates of paratyphoid suggest considerable geospatial variability in the burden of paratyphoid fever. Additional efforts to monitor enteric fever at the population level will be necessary in order to accurately quantify the public health threat posed by S. Paratyphi A, and to improve the prevention and treatment of enteric fever.     

A thioesterase bypasses the requirement for exogenous fatty acids in the plsX deletion of Streptococcus pneumoniae

PlsX is an acyl‐acyl carrier protein (ACP):phosphate transacylase that interconverts the two acyl donors in Gram‐positive bacterial phospholipid synthesis. The deletion of plsX in Staphylococcus aureus results in a requirement for both exogenous fatty acids and de novo type II fatty acid biosynthesis. Deletion of plsX (SP0037) in Streptococcus pneumoniae did not result in an auxotrophic phenotype. The ΔplsX S. pneumoniae strain was refractory to myristic acid‐dependent growth arrest, and unlike the wild‐type strain, was susceptible to fatty acid synthesis inhibitors in the presence of exogenous oleate. The ΔplsX strain contained longer chain saturated fatty acids imparting a distinctly altered phospholipid molecular species profile. An elevated pool of 18‐ and 20‐carbon saturated fatty acids was detected in the ΔplsX strain. A S. pneumoniae thioesterase (TesS, SP1408) hydrolyzed acyl‐ACP in vitro, and the ΔtesS ΔplsX double knockout strain was a fatty acid auxotroph. Thus, the TesS thioesterase hydrolyzed the accumulating acyl‐ACP in the ΔplsX strain to liberate fatty acids that were activated by fatty acid kinase to bypass a requirement for extracellular fatty acid. This work identifies tesS as the gene responsible for the difference in exogenous fatty acid growth requirement of the ΔplsX strains of S. aureus and S. pneumoniae.     

Overexpression of mltA in Edwardsiella tarda reduces resistance to antibiotics and enhances lethality in zebra fish

Aims: The aim of this study was to investigate the role of membrane‐bound lytic murein transglycosylase A (MltA) in a bacterial fish pathogen Edwardsiella tarda. Methods and Results: An mltA in‐frame deletion mutant (ΔmltA) and an mltA overexpression strain (mltA⁺) of Edw. tarda were constructed through double‐crossover allelic exchange and by transformation of a low‐copy plasmid carrying the intact mltA into the ΔmltA mutant, respectively. Either inactivation or overexpression of MltA in Edw. tarda resulted in elevated sensitivity to β‐lactam antibiotics and lower viability in oligotrophic or high osmotic environment than wild‐type strain. Autolysis induced by EDTA was reduced in ΔmltA strain, while mltA⁺ strain was virtually flimsy, indicating that MltA is responsible for the lysis effect. Moreover, mltA⁺ strain exhibited significant increases in lipopolysaccharide (LPS) biosynthesis and virulence to zebra fish compared with wild‐type strain. Conclusions: The results indicated that MltA plays essential roles in β‐lactam antibiotics and environmental stresses resistance, autolysis, LPS biosynthesis and pathogenicity of Edw. tarda. This is the first report that MltA has a virulence‐related function in Edw. tarda. Significance and Impact of the Study: This study provided useful information for further studies on pathogenesis of Edw. tarda.     

NilD CRISPR RNA contributes to Xenorhabdus nematophila colonization of symbiotic host nematodes

The bacterium Xenorhabdus nematophila is a mutualist of entomopathogenic Steinernema carpocapsae nematodes and facilitates infection of insect hosts. X. nematophila colonizes the intestine of S. carpocapsae which carries it between insects. In the X. nematophila colonization‐defective mutant nilD6::Tn5, the transposon is inserted in a region lacking obvious coding potential. We demonstrate that the transposon disrupts expression of a single CRISPR RNA, NilD RNA. A variant NilD RNA also is expressed by X. nematophila strains from S. anatoliense and S. websteri nematodes. Only nilD from the S. carpocapsae strain of X. nematophila rescued the colonization defect of the nilD6::Tn5 mutant, and this mutant was defective in colonizing all three nematode host species. NilD expression depends on the presence of the associated Cas6e but not Cas3, components of the Type I‐E CRISPR‐associated machinery. While cas6e deletion in the complemented strain abolished nematode colonization, its disruption in the wild‐type parent did not. Likewise, nilD deletion in the parental strain did not impact colonization of the nematode, revealing that the requirement for NilD is evident only in certain genetic backgrounds. Our data demonstrate that NilD RNA is conditionally necessary for mutualistic host colonization and suggest that it functions to regulate endogenous gene expression.     

Antimicrobial Resistance,Virulence Profiles and Molecular Subtypes of Salmonella enterica Serovars Typhi and Paratyphi A Blood Isolates from Kolkata,India during 2009-2013

Enteric fever, caused by Salmonella enterica, remains an unresolved public health problem in India and antimicrobial therapy is the main mode of treatment. The objective of this study was to characterize the Salmonella enterica isolates from Kolkata with respect to their antimicrobial resistance (AMR), virulence profiles and molecular subtypes. Salmonella enterica blood isolates were collected from clinically suspected enteric fever patients attending various hospitals in Kolkata, India from January 2009 to June 2013 and were tested for AMR profiles by standard protocols; for resistance gene transfer by conjugation; for resistance and virulence genes profiles by PCR; and for molecular subtypes by Pulsed Field Gel Electrophoresis (PFGE). A total of 77 Salmonella enterica serovar Typhi (S. Typhi) and 25 Salmonella enterica serovar Paratyphi A (S. Paratyphi A) from Kolkata were included in this study. Although multidrug resistance (resistance to chloramphenicol, ampicillin, co-trimoxazole) was decreasing in S. Typhi (18.2%) and absent in S. Paratyphi A, increased resistance to fluoroquinolone, the current drug of choice, caused growing concern for typhoid treatment. A single, non-conjugative non-IncHI1 plasmid of 180 kb was found in 71.4% multidrug resistant (MDR) S. Typhi; the remaining 28.6% isolates were without plasmid. Various AMR markers (bla _TEM-1, catA, sul1, sul2, dfrA15, strA-strB) and class 1 integron with dfrA7 gene were detected in MDR S. Typhi by PCR and sequencing. Most of the study isolates were likely to be virulent due to the presence of virulence markers. Major diversity was not noticed among S. Typhi and S. Paratyphi A from Kolkata by PFGE. The observed association between AMR profiles and S. Typhi pulsotypes might be useful in controlling the spread of the organism by appropriate intervention. The study reiterated the importance of continuous monitoring of AMR and molecular subtypes of Salmonella isolates from endemic regions for better understanding of the disease epidemiology.     

An AIL family protein promotes type three secretion system‐1‐independent invasion and pathogenesis of Salmonella enterica serovar Typhi

Adhesion and invasion of Intestinal Epithelial Cells (IECs) are critical for the pathogenesis of Salmonella Typhi, the aetiological agent of human typhoid fever. While type three secretion system‐1 (T3SS‐1) is a major invasion apparatus of Salmonella, independent invasion mechanisms were described for non‐typhoidal Salmonellae. Here, we show that T2942, an AIL‐like protein of S. Typhi Ty2 strain, is required for adhesion and invasion of cultured IECs. That invasion was T3SS‐1 independent was proved by ectopic expression of T2942 in the non‐invasive E. coli BL21 and double‐mutant Ty2 (Ty2Δt2942ΔinvG) strains. Laminin and fibronectin were identified as the host‐binding partners of T2942 with higher affinity for laminin. Standalone function of T2942 was confirmed by cell adhesion of the recombinant protein, while the protein or anti‐T2942 antiserum blocked adhesion/invasion of S. Typhi, indicating specificity. A 20‐amino acid extracellular loop was required for invasion, while several loop regions of T2942 contributed to adhesion. Further, T2942 cooperates with laminin‐binding T2544 for adhesion and T3SS‐1 for invasion. Finally, T2942 was required and synergistically worked with T3SS‐1 for pathogenesis of S. Typhi in mice. Considering wide distribution of T2942 among clinical strains, the protein or the 20‐mer peptide may be suitable for vaccine development.     

Differential Epidemiology of Salmonella Typhi and Paratyphi A in Kathmandu,Nepal: A Matched Case Control Investigation in a Highly Endemic Enteric Fever Setting

Background

Enteric fever, a systemic infection caused by the bacteria Salmonella Typhi and Salmonella Paratyphi A, is endemic in Kathmandu, Nepal. Previous work identified proximity to poor quality water sources as a community-level risk for infection. Here, we sought to examine individual-level risk factors related to hygiene and sanitation to improve our understanding of the epidemiology of enteric fever in this setting.

Methodology and principal findings

A matched case-control analysis was performed through enrollment of 103 blood culture positive enteric fever patients and 294 afebrile community-based age and gender-matched controls. A detailed questionnaire was administered to both cases and controls and the association between enteric fever infection and potential exposures were examined through conditional logistic regression. Several behavioral practices were identified as protective against infection with enteric fever, including water storage and hygienic habits. Additionally, we found that exposures related to poor water and socioeconomic status are more influential in the risk of infection with S. Typhi, whereas food consumption habits and migration play more of a role in risk of S. Paratyphi A infection.

Conclusions and significance

Our work suggests that S. Typhi and S. Paratyphi A follow different routes of infection in this highly endemic setting and that sustained exposure to both serovars probably leads to the development of passive immunity. In the absence of a polyvalent vaccine against S. Typhi and S. Paratyphi A, we advocate better systems for water treatment and storage, improvements in the quality of street food, and vaccination with currently available S. Typhi vaccines.     

Non-Protein Coding RNA Genes as the Novel Diagnostic Markers for the Discrimination of Salmonella Species Using PCR

Salmonellosis, a communicable disease caused by members of the Salmonella species, transmitted to humans through contaminated food or water. It is of paramount importance, to generate accurate detection methods for discriminating the various Salmonella species that cause severe infection in humans, including S. Typhi and S. Paratyphi A. Here, we formulated a strategy of detection and differentiation of salmonellosis by a multiplex polymerase chain reaction assay using S. Typhi non-protein coding RNA (sRNA) genes. With the designed sequences that specifically detect sRNA genes from S. Typhi and S. Paratyphi A, a detection limit of up to 10 pg was achieved. Moreover, in a stool-seeding experiment with S. Typhi and S. Paratyphi A, we have attained a respective detection limit of 15 and 1.5 CFU/mL. The designed strategy using sRNA genes shown here is comparatively sensitive and specific, suitable for clinical diagnosis and disease surveillance, and sRNAs represent an excellent molecular target for infectious disease.     

Quorum Sensing Contributes to Seed‐to‐Seedling Transmission of Acidovorax citrulli on Watermelon

Based on the observation that Acidovorax citrulli switches from saprobic to pathogenic growth for seed‐to‐seedling transmission of bacterial fruit blotch of cucurbits (BFB), we hypothesized that quorum sensing (QS) was involved in the regulation of this process. Using aacI (luxI homologue) and aacR (luxR homologue) mutants of AAC00‐1, we investigated the role of QS in watermelon seed colonization and seed‐to‐seedling transmission of BFB. aacR and aacI mutants of AAC00‐1 colonized germinating watermelon seed at wild‐type levels; however, BFB seed‐to‐seedling transmission was affected in a cell density‐dependent manner. There were no significant differences in BFB seedling transmission between watermelon seed infiltrated with approximately 1 × 10⁶ CFU of AAC00‐1, the aacR or aacI deletion mutants (95.2, 94.9 and 98.3% BFB incidence, respectively). In contrast, when seed inoculum was reduced to approximately 1 × 10³ CFU/seed, BFB seed‐to‐seedling transmission declined to 34.3% for the aacI mutant, which was significantly less than the wild type (78.6%). Interestingly, BFB seed‐to‐seedling transmission for the aacR mutant was not significantly different to the wild‐type strain. These data suggest that QS plays a role in regulation of genes involved in seed‐to‐seedling transmission of BFB.     

Microbiological Culture Simplified Using Anti-O12 Monoclonal Antibody in TUBEX Test to Detect Salmonella Bacteria from Blood Culture Broths of Enteric Fever Patients

Definitive diagnosis of infectious diseases, including food poisoning, requires culture and identification of the infectious agent. We described how antibodies could be used to shorten this cumbersome process. Specifically, we employed an anti-Salmonella lipopolysaccharide O12 monoclonal antibody in an epitope-inhibition 10-min test (TUBEX TP) to detect O12⁺ Salmonella organisms directly from routine blood culture broths. The aim is to obviate the need to subculture the broth and subsequently identify the colonies. Thus, blood from 78 young outpatients suspected of having enteric fever was incubated in an enrichment broth, and after 2 or 4 days, broth samplings were examined by TUBEX TP as well as by conventional agar culture and identification. TUBEX TP was performed before the culture results. Eighteen isolates of S. Typhi (15 after 2 days) and 10 isolates of S. Paratyphi A (4 after 2 days) were obtained by conventional culture. Both these Salmonella serotypes, the main causes of enteric fever, share the O12 antigen. In all instances where either of these organisms was present (cultured), TUBEX TP was positive (score 4 [light blue] – to – score 10 [dark blue]; negative is 0 [pink-colored]) i.e. 100% sensitive. Identification of the specific Salmonella serotype in TUBEX-positive cases was achieved subsequently by conventional slide agglutination using appropriate polyclonal antisera against the various serotypes. Twelve Escherichia coli, 1 Alcaligenes spp. and 1 Enterobacter spp. were isolated. All of these cases, including all the 36 culture-negative broths, were TUBEX-negative i.e. TUBEX TP was 100% specific. In a separate study using known laboratory strains, TUBEX TF, which detects S. Typhi but not S. Paratyphi A via the O9 antigen, was found to efficiently complement TUBEX TP as a differential test. Thus, TUBEX TP and TUBEX TF are useful adjuncts to conventional culture because they can save considerable time (>2 days), costs and manpower.     

Invasin of Edwardsiella tarda is essential for its haemolytic activity,biofilm formation and virulence towards fish

Aims

The aim of this study was to investigate the role of invasin in a bacterial fish pathogen Edwardsiella tarda.

Methods and Results

In this study, an in‐frame deletion mutant of invasin (Δinv) in Edw. tarda H1 was constructed through double crossover allelic exchange to explore the function of invasin in virulence to fish. Meanwhile, an invasin overexpression strain (inv⁺) was obtained by electrotransformation of a low‐copy plasmid pACYC184 carrying the intact invasin into the Δinv mutant. Several virulence‐associated characters of the mutants and wild‐type strain were tested. Compared with the wild‐type H1, haemolytic activity and biofilm formation were decreased in Δinv, while increased significantly in inv⁺. In addition, the invasin overexpressing strain inv⁺ exhibited increased internalization into Epithelioma Papulosum Cyprini (EPC) cells. Moreover, in zebrafish model, Δinv showed decreased virulence compared with H1, while inv⁺ restored the virulence of wild type completely.

Conclusions

The results demonstrated that invasin of Edw. tarda plays essential roles in haemolytic activity, biofilm formation, adherence, internalization and pathogenicity of this bacterium.

Significance and Impact of the Study

This study revealed the role of invasin in Edw. tarda infection and provided useful information for further unveiling the pathogenesis of Edw. tarda.     

O:2-CRM197 Conjugates against Salmonella Paratyphi A

Enteric fevers remain a common and serious disease, affecting mainly children and adolescents in developing countries. Salmonella enterica serovar Typhi was believed to cause most enteric fever episodes, but several recent reports have shown an increasing incidence of S. Paratyphi A, encouraging the development of a bivalent vaccine to protect against both serovars, especially considering that at present there is no vaccine against S. Paratyphi A. The O-specific polysaccharide (O:2) of S. Paratyphi A is a protective antigen and clinical data have previously demonstrated the potential of using O:2 conjugate vaccines. Here we describe a new conjugation chemistry to link O:2 and the carrier protein CRM₁₉₇, using the terminus 3-deoxy-D-manno-octulosonic acid (KDO), thus leaving the O:2 chain unmodified. The new conjugates were tested in mice and compared with other O:2-antigen conjugates, synthesized adopting previously described methods that use CRM₁₉₇ as carrier protein. The newly developed conjugation chemistry yielded immunogenic conjugates with strong serum bactericidal activity against S. Paratyphi A.     

Application of attenuated Lactococcus garvieae strain lacking a virulence-associated capsule on its cell surface as a live vaccine in yellowtail Seriola quinqueradiata Temminck and Schlegel

An attenuated Lactococcus garvieae strain lacking a virulence‐associated capsule on its cell surface was evaluated for application as a live vaccine. The attenuated strain (MS93003A) was obtained from the parent strain (MS93003V), which produced a well‐developed capsule, by culturing on an agar medium supplemented with 2,3,5‐triphenyltetrazolium chloride. When live cells of L. garvieae (MS93003A) or formalin‐killed cells (MS93003A) were used as an injectable vaccine, protection against virulent L. garvieae (MS93003V) was conferred on Seriola quinqueradiata. The MS93003A cells did not recover their virulence even after in vivo passages in fish. MS93003A live cells also conferred long‐lasting protective immunity to S. quinqueradiata against virulent L. garvieae infection.     

Cell-associated flagella enhance the protection conferred by mucosally-administered attenuated Salmonella Paratyphi A vaccines

Background

Antibiotic-resistant Salmonella enterica serovar Paratyphi A, the agent of paratyphoid A fever, poses an emerging public health dilemma in endemic areas of Asia and among travelers, as there is no licensed vaccine. Integral to our efforts to develop a S. Paratyphi A vaccine, we addressed the role of flagella as a potential protective antigen by comparing cell-associated flagella with exported flagellin subunits expressed by attenuated strains.

Methodology

S. Paratyphi A strain ATCC 9150 was first deleted for the chromosomal guaBA locus, creating CVD 1901. Further chromosomal deletions in fliD (CVD 1901D) or flgK (CVD 1901K) were then engineered, resulting in the export of unpolymerized FliC, without impairing its overall expression. The virulence of the resulting isogenic strains was examined using a novel mouse LD₅₀ model to accommodate the human-host restricted S. Paratyphi A. The immunogenicity of the attenuated strains was then tested using a mouse intranasal model, followed by intraperitoneal challenge with wildtype ATCC 9150.

Results

Mucosal (intranasal) immunization of mice with strain CVD 1901 expressing cell-associated flagella conferred superior protection (vaccine efficacy [VE], 90%) against a lethal intraperitoneal challenge, compared with the flagellin monomer-exporting mutants CVD 1901K (30% VE) or CVD 1901D (47% VE). The superior protection induced by CVD 1901 with its cell-attached flagella was associated with an increased IgG2a∶IgG1 ratio of FliC-specific antibodies with enhanced opsonophagocytic capacity.

Conclusions

Our results clearly suggest that enhanced anti-FliC antibody-mediated clearance of S. Paratyphi A by phagocytic cells, induced by vaccines expressing cell-associated rather than exported FliC, might be contributing to the vaccine-induced protection from S. Paratyphi A challenge in vivo. We speculate that an excess of IgG1 anti-FliC antibodies induced by the exported FliC may compete with the IgG2a subtype and block binding to specific phagocyte Fc receptors that are critical for clearing an S. Paratyphi A infection.     

The γ‐aminobutyric acid shunt contributes to closing the tricarboxylic acid cycle in Synechocystis sp. PCC 6803

A traditional 2‐oxoglutarate dehydrogenase complex is missing in the cyanobacterial tricarboxylic acid cycle. To determine pathways that convert 2‐oxoglutarate into succinate in the cyanobacterium Synechocystis sp. PCC 6803, a series of mutant strains, Δsll1981, Δslr0370, Δslr1022 and combinations thereof, deficient in 2‐oxoglutarate decarboxylase (Sll1981), succinate semialdehyde dehydrogenase (Slr0370), and/or in γ‐aminobutyrate metabolism (Slr1022) were constructed. Like in Pseudomonas aeruginosa, N‐acetylornithine aminotransferase, encoded by slr1022, was shown to also function as γ‐aminobutyrate aminotransferase, catalysing γ‐aminobutyrate conversion to succinic semialdehyde. As succinic semialdehyde dehydrogenase converts succinic semialdehyde to succinate, an intact γ‐aminobutyrate shunt is present in Synechocystis. The Δsll1981 strain, lacking 2‐oxoglutarate decarboxylase, exhibited a succinate level that was 60% of that in wild type. However, the succinate level in the Δslr1022 and Δslr0370 strains and the Δsll1981/Δslr1022 and Δsll1981/Δslr0370 double mutants was reduced to 20–40% of that in wild type, suggesting that the γ‐aminobutyrate shunt has a larger impact on metabolite flux to succinate than the pathway via 2‐oxoglutarate decarboxylase. ¹³C‐stable isotope analysis indicated that the γ‐aminobutyrate shunt catalysed conversion of glutamate to succinate. Independent of the 2‐oxoglutarate decarboxylase bypass, the γ‐aminobutyrate shunt is a major contributor to flux from 2‐oxoglutarate and glutamate to succinate in Synechocystis sp. PCC 6803.