Antibiotic resistance begets more resistance: chromosomal resistance mutations mitigate fitness costs conferred by multi-resistant clinical plasmids.

Plasmids are the primary vectors of horizontal transfer of antibiotic resistance genes among bacteria. Previous studies have shown that the spread and maintenance of plasmids among bacterial populations depend on the genetic makeup of both the plasmid and the host bacterium. Antibiotic resistance can also be acquired through mutations in the bacterial chromosome, which not only confer resistance but also result in changes in bacterial physiology and typically a reduction in fitness. However, it is unclear whether chromosomal resistance mutations affect the interaction between plasmids and the host bacteria. To address this question, we introduced 13 clinical plasmids into a susceptible Escherichia coli strain and three different congenic mutants that were resistant to nitrofurantoin (ΔnfsAB), ciprofloxacin (gyrA, S83L), and streptomycin (rpsL, K42N) and determined how the plasmids affected the exponential growth rates of the host in glucose minimal media. We find that though plasmids confer costs on the susceptible strains, those costs are fully mitigated in the three resistant mutants. In several cases, this results in a competitive advantage of the resistant strains over the susceptible strain when both carry the same plasmid and are grown in the absence of antibiotics. Our results suggest that bacteria carrying chromosomal mutations for antibiotic resistance could be a better reservoir for resistance plasmids, thereby driving the evolution of multi-drug resistance.IMPORTANCEPlasmids have led to the rampant spread of antibiotic resistance genes globally. Plasmids often carry antibiotic resistance genes and other genes needed for its maintenance and spread, which typically confer a fitness cost on the host cell observed as a reduced growth rate. Resistance is also acquired via chromosomal mutations, and similar to plasmids they also reduce bacterial fitness. However, we do not know whether resistance mutations affect the bacterial ability to carry plasmids. Here, we introduced 13 multi-resistant clinical plasmids into a susceptible and three different resistant E. coli strains and found that most of these plasmids do confer fitness cost on susceptible cells, but these costs disappear in the resistant strains which often lead to fitness advantage for the resistant strains in the absence of antibiotic selection. Our results imply that already resistant bacteria are a more favorable reservoir for multi-resistant plasmids, promoting the ascendance of multi-resistant bacteria.

Prevalence of colistin resistance and antibacterial resistance in commensal Escherichia coli from chickens: An assessment of the impact of regulatory intervention in South Africa.

BACKGROUND: Antimicrobial resistance (AMR) is a global health problem largely due to the overuse of antimicrobials. In recognition of this, the World Health Assembly in 2015 agreed on a global action plan to tackle AMR. Following the global emergence of the mcr-1-associated colistin resistance gene in the livestock industry in 2016, several countries including South Africa restricted the veterinary use of colistin as the gene threatens the clinical utility of the drug. This study is a follow-up to the restriction in place in order to evaluate the impact of such policy adoption. OBJECTIVE: To assess the prevalence of antibacterial resistance (ABR), and the mcr-1 colistin resistance gene in broiler chicken over a 2-year period, as a follow-up to the veterinary ban on colistin use in South Africa. METHODS: A total of 520 swab samples were obtained during 2019 (March-April) and 2020 (February-March), from healthy broiler chicken carcasses (n = 20) and chicken droppings in transport crates (n = 20) at various poultry abattoirs (N = 7) in the Gauteng province of South Africa. Escherichia coli organisms were isolated and subjected to a panel of 24 antibacterials using the MicroScan machine. Screening for mcr-1 colistin resistance gene was undertaken using PCR. RESULT: Four hundred and thirty-eight (438) E. coli strains were recovered and none demonstrated phenotypic resistance towards colistin, amikacin, carbapenems, tigecycline and piperacillin/tazobactam. The mcr-1 gene was not detected in any of the isolates tested. Resistances to the aminoglycosides (0%-9.8%) and fluoroquinolones (0%-18.9%) were generally low. Resistances to ampicillin (32%-39.3%) and trimethoprim/sulphamethoxazole (30.6%-3.6%) were fairly high. A significant (p < 0.05) increase in cephalosporins and cephamycin resistance was noted in the year 2020 (February-March) when compared with the year 2019 (March-April). CONCLUSION: The absence of mcr-1 gene and colistin resistance suggests that mitigation strategies adopted were effective and clearly demonstrated the significance of regulatory interventions in reducing resistance to critical drugs. Despite the drawback in regulatory framework such as free farmers access to antimicrobials OTC and a dual registration system in place, there is a general decline in the prevalence of ABR when the present data are compared with the last national veterinary surveillance on AMR (SANVAD 2007). To further drive resistance down, mitigation strategies should focus on strengthening regulatory framework, the withdrawal of OTC dispensing of antimicrobials, capping volumes of antimicrobials, banning growth promoters and investing on routine surveillance/monitoring of AMR and antimicrobial consumption.

No fitness cost entailed by type VI secretion system synthesis, assembly, contraction, or disassembly in enteroaggregative Escherichia coli.

IMPORTANCE: Bacteria use weapons to deliver effectors into target cells. One of these weapons, the type VI secretion system (T6SS), assembles a contractile tail acting as a spring to propel a toxin-loaded needle. Due to its size and mechanism of action, the T6SS was intuitively thought to be energetically costly. Here, using a combination of mutants and growth measurements in liquid medium, on plates, and in competition experiments, we show that the T6SS does not entail a growth cost to enteroaggregative Escherichia coli.

Prevalence and In Vivo Assessment of Virulence in Shiga Toxin-Producing Escherichia coli Clinical Isolates from Greater Cairo Area.

Background: Shiga toxin-producing Escherichia coli (STEC) has been identified as an important etiologic agent of human disease in Egypt. Aims: To investigate the occurrence and describe the characterization as well as prevalence of STEC in Greater Cairo hospitals as well as molecular characterization of virulence and resistance genes. Methods: Four hundred seventy E. coli clinical isolates were collected from eight hospitals and analyzed by genotypic and phenotypic methods for STEC, followed by histopathological examination and scoring of different organs lesions. Results: The highest proportion of isolates was from urine (151 isolates), whereas the lowest was from splenic drain (3 isolates). In tandem, when serogrouping was performed, 15 serogroups were obtained where the most prevalent was O157 and the least prevalent was O151. All isolates were positive when screened for identity gene gad A, while only typable strains were screened for seven virulence genes stx1 (gene encoding Shiga toxin 1), stx2 (gene encoding Shiga toxin 2), tsh (gene encoding thermostable hemagglutinin), eaeA (gene encoding intimin), invE (gene encoding invasion protein), aggR (gene encoding aggregative adherence transcriptional regulator), and astA (aspartate transaminase) where the prevalence was 48%, 30%, 50%, 57%, 7.5%, 12%, and 58%, respectively. Of 254 typable isolates, 152 were STEC carrying stx1 or stx2 genes or both. Conclusions: Relying on in vivo comparison between different E. coli pathotypes via histopathological examination of different organs, E. coli pathotypes could be divided into mild virulent, moderate virulent, and high virulent strains. Statistical analysis revealed significant correlation between different serogroups and presence of virulence genes.

Establishment of an In Bacterio Assay for the Assessment of Carbon Storage Regulator A (CsrA) Inhibitors.

Polymicrobial infections involving various combinations of microorganisms, such as Escherichia, Pseudomonas, or Yersinia, can lead to acute and chronic diseases in for example the gastrointestinal and respiratory tracts. Our aim is to modulate microbial communities by targeting the posttranscriptional regulator system called carbon storage regulator A (CsrA) (or also repressor of secondary metabolites (RsmA)). In previous studies, we identified easily accessible CsrA binding scaffolds and macrocyclic CsrA binding peptides through biophysical screening and phage display technology. However, due to the lack of an appropriate in bacterio assay to evaluate the cellular effects of these inhibitor hits, the focus of the present study is to establish an in bacterio assay capable of probing and quantifying the impact on CsrA-regulated cellular mechanisms. We have successfully developed an assay based on a luciferase reporter gene assay, which in combination with a qPCR expression gene assay, allows for the monitoring of expression levels of different downstream targets of CsrA. The chaperone protein CesT was used as a suitable positive control for the assay, and in time-dependent experiments, we observed a CesT-mediated increase in bioluminescence over time. By this means, the cellular on-target effects of non-bactericidal/non-bacteriostatic virulence modulating compounds targeting CsrA/RsmA can be evaluated.

Cost-effective selective deuteration of aromatic amino acid residues produces long-lived solution 1H NMR magnetization in proteins.

Solution NMR studies of large proteins are hampered by rapid signal decay due to short-range dipolar 1H-1H and 1H-13C interactions. These are attenuated by rapid rotation in methyl groups and by deuteration (2H), so selective 1H,13C-isotope labelling of methyl groups in otherwise perdeuterated proteins, combined with methyl transverse relaxation optimized spectroscopy (methyl-TROSY), is now standard for solution NMR of large protein systems > 25 kDa. For non-methyl positions, long-lived magnetization can be introduced as isolated 1H-12C groups. We have developed a cost-effective chemical synthesis for producing selectively deuterated phenylpyruvate and hydroxyphenylpyruvate. Feeding these amino acid precursors to E. coli in D2O, along with selectively deuterated anthranilate and unlabeled histidine, results in isolated and long-lived 1H magnetization in the aromatic rings of Phe (HD, HZ), Tyr (HD), Trp (HH2, HE3) and His (HD2 and HE1). We are additionally able to obtain stereoselective deuteration of Asp, Asn, and Lys amino acid residues using unlabeled glucose and fumarate as carbon sources and oxalate and malonate as metabolic inhibitors. Combining these approaches produces isolated 1H-12C groups in Phe, Tyr, Trp, His, Asp, Asn, and Lys in a perdeuterated background, which is compatible with standard 1H-13C labeling of methyl groups in Ala, Ile, Leu, Val, Thr, Met. We show that isotope labeling of Ala is improved using the transaminase inhibitor L-cycloserine, and labeling of Thr is improved through addition of Cys and Met, which are known inhibitors of homoserine dehydrogenase. We demonstrate the creation of long-lived 1H NMR signals in most amino acid residues using our model system, the WW domain of human Pin1, as well as the bacterial outer membrane protein PagP.

Evaluation of Covalent Organic Frameworks for the low-cost, rapid detection of Shiga Toxin-producing Escherichia coli in ready-to-eat salads.

BACKGROUND: Ready-to-eat products, such as leafy greens, must be carefully controlled as they are directly consumed without any treatment to reduce the presence of potential pathogens. Food industries, especially those that process products with short shelf-life, demand rapid detection of foodborne pathogens such as Shiga Toxin-producing Escherichia coli (STEC). In this sense, molecular methods can fulfill both requirements of turnaround time and consumer safety. The most popular rapid methods are those based on real-time PCR (qPCR) however, vegetables contain inhibitory compounds that may inhibit the amplification reaction thus, there is a need for novel sample preparation protocols. RESULTS: In the current study, a low-cost sample treatment based on sequential filtration steps was developed. This protocol was combined with covalent organic frameworks (COFs), and compared against a chelating resin, to evaluate their performance by multiplex qPCR targeting the major virulence genes of STEC, namely stx1, stx2, and eae, along with the rfbE for the specific identification of serogroup O157 due to its particularly high incidence, and an Internal Amplification Control to assess reaction inhibition. The optimized sample treatment effectively removed vegetable qPCR inhibitory compounds, and it was possible to detect STEC in spiked ready-to-eat salad samples in one working day, roughly 5 h, with an LOD50 of 8.7 CFU/25 g with high diagnostic sensitivity and specificity. The method was also assessed in samples with cold-stressed bacteria with good results, further demonstrating its applicability. SIGNIFICANCE: It was demonstrated for the first time that COFs are suitable for DNA extraction and purification. In addition to this, due to the tunable nature of these materials, it is envisioned that future modifications in terms of pore size or combination with magnetic materials, will allow to further improve their performance. In addition to this, the rapid and low-cost sample treatment protocol developed demonstrated suitable for the rapid screening of STEC vegetable samples.

A trailing ribosome speeds up RNA polymerase at the expense of transcript fidelity via force and allostery.

In prokaryotes, translation can occur on mRNA that is being transcribed in a process called coupling. How the ribosome affects the RNA polymerase (RNAP) during coupling is not well understood. Here, we reconstituted the E. coli coupling system and demonstrated that the ribosome can prevent pausing and termination of RNAP and double the overall transcription rate at the expense of fidelity. Moreover, we monitored single RNAPs coupled to ribosomes and show that coupling increases the pause-free velocity of the polymerase and that a mechanical assisting force is sufficient to explain the majority of the effects of coupling. Also, by cryo-EM, we observed that RNAPs with a terminal mismatch adopt a backtracked conformation, while a coupled ribosome allosterically induces these polymerases toward a catalytically active anti-swiveled state. Finally, we demonstrate that prolonged RNAP pausing is detrimental to cell viability, which could be prevented by polymerase reactivation through a coupled ribosome.

International manufacturing and trade in colistin, its implications in colistin resistance and One Health global policies: a microbiological, economic, and anthropological study.

BACKGROUND: The emergence of colistin-resistant Enterobacterales is a global public health concern, yet colistin is still widely used in animals that are used for food as treatment, metaphylaxis, prophylaxis, and growth promotion. Herein, we investigate the effect of colistin-resistant Enterobacterales in Pakistan, global trade of colistin, colistin use at the farm level, and relevant socioeconomic factors. METHODS: We conducted a microbiological, economic, and anthropological study of colistin-resistant Escherichia coli in humans, animals, and the environment and international trade and knowledge of colistin in Pakistan, Bangladesh, Nigeria, China, India, and Viet Nam. We collected backyard poultry cloacal swabs, commercial broiler cloacal swabs, cattle and buffalo rectal swabs, human rectal swabs, wild bird droppings, cattle and buffalo meat, sewage water, poultry flies, chicken meat, and canal water from 131 sites across Faisalabad, Pakistan, to be tested for mcr-1-positive and mcr-3-positive Escherichia coli. We recruited new patients admitted to Allied Hospital, Faisalabad, Pakistan, with abdominal pain and diarrhoea for rectal swabs. Patients with dysentery and those who were already on antibiotic treatment were excluded. Data for colistin trade between 2017 and 2020, including importation, manufacturing, and usage, were accessed from online databases and government sources in Pakistan, Bangladesh, and Nigeria. We recruited participants from poultry farms and veterinary drug stores in Pakistan and Nigeria to be interviewed using a structured questionnaire. International manufacturing, import, and export data; value analysis; and trade routes of colistin pharmaceutical raw material (PRM), feed additive, and finished pharmaceutical products (FPPs) were accessed from 2017-21 export data sets. FINDINGS: We collected 1131 samples between May 12, 2018, and July 1, 2019: backyard poultry cloacal swabs (n=100), commercial broiler cloacal swabs (n=102), cattle and buffalo rectal swabs (n=188), human rectal swabs (n=200), wild bird droppings (n=100), cattle and buffalo meat (n=100), sewage water (n=90), poultry flies (n=100), chicken meat (n=100), and canal water (n=51). We recruited 200 inpatients at Allied Hospital, Faisalabad, Pakistan, between Nov 15, 2018, and Dec 14, 2018, for rectal swabs. We recruited 21 participants between Jan 1, 2020, and Dec 31, 2020, from poultry farms and drug stores in Pakistan and Nigeria to be interviewed. 75 (7%) of 1131 samples contained mcr-1-positive E coli, including wild bird droppings (25 [25%] of 100), commercial broiler cloacal swabs (17 [17%] of 100), backyard poultry cloacal swabs (one [1%] of 100), chicken meat (13 [13%] of 100), cattle and buffalo meat (two [2%] of 100), poultry flies (eight [8%] of 100), sewage water (six [7%] of 90), and human rectal swabs (three [2%] of 200). During 2017-20, Pakistan imported 275·5 tonnes (68·9 tonnes per year, 95% CI 41·2-96·6) of colistin as PRM, all sourced from China, 701·9 tonnes (175·5 tonnes per year, 140·9-210·1) of colistin as feed additives from China and Viet Nam, and 63·0 tonnes (15·8 tonnes per year, 10·4-21·1) of colistin as FPPs from various countries in Asia and Europe. For Bangladesh and Nigeria, colistin PRM and FPPs were imported from China and Europe. Colistin knowledge and usage practices in Pakistan and Nigeria were unsatisfactory in terms of understanding of the effects on human medicine and usage other than for treatment purposes. China is the major manufacturer of PRM and feed additive colistin and exported a total of 2664·8 tonnes (666·2 tonnes per year, 95% CI 262·1 to 1070·2) of PRM and 2570·2 tonnes (642·6 tonnes per year, -89·4 to 1374·5) of feed additive in 1330 shipments during 2018-21 to 21 countries. INTERPRETATION: Regardless of 193 countries signing the UN agreement to tackle antimicrobial resistance, trading of colistin as PRM, FPPs, and feed additive or growth promoter in low-income and middle-income countries continues unabated. Robust national and international laws are urgently required to mitigate the international trade of this antimicrobial listed on WHO Critically Important Antimicrobials for Human Medicine. FUNDING: Pakistan Agricultural Research Council and INEOS Oxford Institute for Antimicrobial Research TRANSLATION: For the Urdu translation of the abstract see Supplementary Materials section.

Long-term adaptation of ParA, RelE/ParE partition system, replication protein and phage proteins encoding low-cost plasmids of Escherichia species isolated from diarrheic children of North East India.

AIMS: The prevalent distribution of plasmid-mediated ß-lactam resistance is the most pressing global problem in enteric diseases. The current work aims to characterize plasmid-carrying ß-lactam resistant Enterobacteriaceae isolates from North East India for horizontal gene transfer (HGT) and plasmid adaptation study. METHODS AND RESULTS: In vitro transconjugation and transformation showed overall high conjugation frequency (4.11 × 10-1-9.2 × 10-1) and moderate transformation efficiency/µg DNA (1.02 × 102 -1 × 103), and the highest conjugation frequency (9.2 × 10-1) and transformation efficiency (1 × 103) for Escherichia species S-10. Intra/intergenus plasmid transformation efficiency was highest for the transformation of Klebsiella pneumoniae S-2 to Shigellaflexneri S-42 (1.3 × 103) and lowest for Escherichia species S-10 to Escherichia fergusonii S-30 (2 × 102). In the plasmid stability test, S-10 was detected with the highest plasmid carrying frequency (83.44%) and insignificant segregational loss rate (0.0004) until the 60th day with low plasmid cost on the host. The above findings were also validated by whole-plasmid sequencing of Escherichia species S-10. The genome was identified with two plasmids constituting multiple phage proteins, relaxosomal protein NikA, replication protein RepA, and the plasmid maintenance proteins (ParA, RelE/ParE), thus assisting stable plasmid maintenance. CONCLUSIONS: The results thus indicate that the high conjugation ability and low plasmid fitness cost might lead to horizontal gene transfer of the plasmid to the environment due to their prolonged adaptation in nonselective conditions, intensifying the infection's severity.

Disrupting the ArcA Regulatory Network Amplifies the Fitness Cost of Tetracycline Resistance in Escherichia coli.

There is an urgent need for strategies to discover secondary drugs to prevent or disrupt antimicrobial resistance (AMR), which is causing >700,000 deaths annually. Here, we demonstrate that tetracycline-resistant (TetR) Escherichia coli undergoes global transcriptional and metabolic remodeling, including downregulation of tricarboxylic acid cycle and disruption of redox homeostasis, to support consumption of the proton motive force for tetracycline efflux. Using a pooled genome-wide library of single-gene deletion strains, at least 308 genes, including four transcriptional regulators identified by our network analysis, were confirmed as essential for restoring the fitness of TetR E. coli during treatment with tetracycline. Targeted knockout of ArcA, identified by network analysis as a master regulator of this new compensatory physiological state, significantly compromised fitness of TetR E. coli during tetracycline treatment. A drug, sertraline, which generated a similar metabolome profile as the arcA knockout strain, also resensitized TetR E. coli to tetracycline. We discovered that the potentiating effect of sertraline was eliminated upon knocking out arcA, demonstrating that the mechanism of potential synergy was through action of sertraline on the tetracycline-induced ArcA network in the TetR strain. Our findings demonstrate that therapies that target mechanistic drivers of compensatory physiological states could resensitize AMR pathogens to lost antibiotics. IMPORTANCE Antimicrobial resistance (AMR) is projected to be the cause of >10 million deaths annually by 2050. While efforts to find new potent antibiotics are effective, they are expensive and outpaced by the rate at which new resistant strains emerge. There is desperate need for a rational approach to accelerate the discovery of drugs and drug combinations that effectively clear AMR pathogens and even prevent the emergence of new resistant strains. Using tetracycline-resistant (TetR) Escherichia coli, we demonstrate that gaining resistance is accompanied by loss of fitness, which is restored by compensatory physiological changes. We demonstrate that transcriptional regulators of the compensatory physiologic state are promising drug targets because their disruption increases the susceptibility of TetR E. coli to tetracycline. Thus, we describe a generalizable systems biology approach to identify new vulnerabilities within AMR strains to rationally accelerate the discovery of therapeutics that extend the life span of existing antibiotics.

Genome Assessment of Carbapenem- and Colistin-Resistant Escherichia coli from Patients in a Sentinel Hospital in China.

Antimicrobial-resistant (AMR) pathogens are a significant threat to public health worldwide. However, the primary carrier of AMR genes, particularly against last-resort antibiotics, is still only partially studied in Chinese hospitals. In a sentinel hospital in China, we collected 157 E. coli strains from patients between January and July 2021. One blaNDM-1-, nine blaNDM-5-, and one mcr-1-positive E. coli recovered from inpatients were identified as resistant to meropenem and colistin. There are 37 virulence genes discovered in the 11 strains, including astA in strain EC21Z-147 (O128: H4), which belongs to the enteroaggregative E. coli (EAEC). The blaNDM gene is distributed into distinct ST types, including ST48, ST616, ST410, ST711, and ST2003, while the mcr-1 gene was identified in ST117. The conjugative plasmids IncX3, IncI1-I, and IncI2 mediated the blaNDM-5 and mcr-1 genes detected among inpatients. Notably, the youngest age at which mcr-1-positive E. coli has been reported was at one day old, in a child in which the strain is closely related to strains with animal origins. Hospitals are major environments for the spread and dissemination of critical virulence and AMR genes, which requires active monitoring systems at the genome level to surveil the spread of virulence and AMR.

Phenotypic Assessment of Clinical Escherichia coli Isolates as an Indicator for Uropathogenic Potential.

For women in the United States, urinary tract infections (UTIs) are the most frequent diagnosis in emergency departments, comprising 21.3% of total visits. Uropathogenic Escherichia coli (UPEC) causes ~80% of uncomplicated UTIs. To combat this public health issue, it is vital to characterize UPEC strains as well as to differentiate them from commensal strains to reduce the overuse of antibiotics. It has been challenging to determine a consistent genetic signature that clearly distinguishes UPEC from other E. coli strains. Therefore, we examined whether phenotypic data could be predictive of uropathogenic potential. We screened 13 clinical strains of UPEC, isolated from cases of uncomplicated UTI in young otherwise healthy women, in a series of microbiological phenotypic assays using UPEC prototype strain CFT073 and nonpathogenic E. coli strain MG1655 K-12 as controls. Phenotypes included adherence, iron acquisition, biofilm formation, human serum resistance, motility, and stress resistance. By use of a well-established experimental mouse model of UTI, these data were able to predict the severity of the bacterial burden in both the urine and bladders. Multiple linear regression using three different phenotypic assays, i.e., growth in minimal medium, siderophore production, and type 1 fimbrial expression, was predictive of bladder colonization (adjusted R2 = 0.6411). Growth in ex vivo human urine, hemagglutination of red blood cells, and motility modeled urine colonization (adjusted R2 = 0.4821). These results showcase the utility of phenotypic characterization to predict the severity of infection that these strains may cause. We predict that these methods will also be applicable to other complex, genetically redundant, pathogens. IMPORTANCE Urinary tract infections are the second leading infectious disease worldwide, occurring in over half of the female population during their lifetime. Most infections are caused by uropathogenic Escherichia coli (UPEC) strains. These strains can establish a reservoir in the gut, in which they do not cause disease but, upon introduction to the urinary tract, can infect the host and elicit pathogenesis. Clinically, it would be beneficial to screen patient E. coli strains to understand their pathogenic potential, which may lead to the administration of prophylactic antibiotic treatment for those with increased risk. Others have proposed the use of PCR-based genetic screening methods to detect UPEC strains and differentiate them from other E. coli pathotypes; however, this method has not yielded a consistent uropathogenic genetic signature. Here, we used phenotypic characteristics such as growth rate, siderophore production, and expression of fimbriae to better predict uropathogenic potential.

Enzymatic Specificity of Conserved Rho GTPase Deamidases Promotes Invasion of Vibrio parahaemolyticus at the Expense of Infection.

Vibrio parahaemolyticus is among the leading causes of bacterial seafood-borne acute gastroenteritis. Like many intracellular pathogens, V. parahaemolyticus invades host cells during infection by deamidating host small Rho GTPases. The Rho GTPase deamidating activity of VopC, a type 3 secretion system (T3SS) translocated effector, drives V. parahaemolyticus invasion. The intracellular pathogen uropathogenic Escherichia coli (UPEC) invades host cells by secreting a VopC homolog, the secreted toxin cytotoxic necrotizing factor 1 (CNF1). Because of the homology between VopC and CNF1, we hypothesized that topical application of CNF1 during V. parahaemolyticus infection could supplement VopC activity. Here, we demonstrate that CNF1 improves the efficiency of V. parahaemolyticus invasion, a bottleneck in V. parahaemolyticus infection, across a range of doses. CNF1 increases V. parahaemolyticus invasion independent of both VopC and the T3SS altogether but leaves a disproportionate fraction of intracellular bacteria unable to escape the endosome and complete their infection cycle. This phenomenon holds true in the presence or absence of VopC but is particularly pronounced in the absence of a T3SS. The native VopC, by contrast, promotes a far less efficient invasion but permits the majority of internalized bacteria to escape the endosome and complete their infection cycle. These studies highlight the significance of enzymatic specificity during infection, as virulence factors (VopC and CNF1 in this instance) with similarities in function (bacterial uptake), catalytic activity (deamidation), and substrates (Rho GTPases) are not sufficiently interchangeable for mediating a successful invasion for neighboring bacterial pathogens. IMPORTANCE Many species of intracellular bacterial pathogens target host small Rho GTPases to initiate invasion, including the human pathogens Vibrio parahaemolyticus and uropathogenic Escherichia coli (UPEC). The type three secretion system (T3SS) effector VopC of V. parahaemolyticus promotes invasion through the deamidation of Rac1 and CDC42 in the host, whereas the secreted toxin cytotoxic necrotizing factor 1 (CNF1) drives UPEC's internalization through the deamidation of Rac1, CDC42, and RhoA. Despite these similarities in the catalytic activity of CNF1 and VopC, we observed that the two enzymes were not interchangeable. Although CNF1 increased V. parahaemolyticus endosomal invasion, most intracellular V. parahaemolyticus aborted their infection cycle and remained trapped in endosomes. Our findings illuminate how the precise biochemical fine-tuning of T3SS effectors is essential for efficacious pathogenesis. Moreover, they pave the way for future investigations into the biochemical mechanisms underpinning V. parahaemolyticus endosomal escape and, more broadly, the regulation of successful pathogenesis.

Insights into the assessment of highly pathogenic Shiga toxin-producing Escherichia coli in raw milk and raw milk cheeses by High Throughput Real-time PCR.

Current methods for screening Enterohemorrhagic Escherichia coli (EHEC) O157 and non-O157 serogroups in raw milk products typically rely on the molecular detection of stx, eae, and serogroup-specific wzx or wzy genes. As these genetic markers can also be carried by non-EHEC strains, a number of 'false positive' results are obtained during the screening step. The suitability of new EHEC markers (espK, espV, ureD, Z2098, and CRISPRO26:H11) were tested as candidates for a more accurate screening of EHEC in dairy products. High-throughput PCR analysis of 1451 DNA extracts from milk and raw milk cheeses positive for both stx and eae demonstrated that addition of these new markers in the detection scheme resulted in a higher selectivity with a systematic reduction of the number of presumptive positive samples that require further O-group testing and confirmation by strain isolation. This reduction is more important (26% to 52%, depending on the animal production species) in the absence of prior IMS treatment of the enriched culture for the Top7 EHEC serotypes. However, even with prior treatment of the enriched cultures by IMS, the reduction rate varied between 5% and >25%. Analysis of eae-subtype, stx-subtypes indicated strong differences in the STEC (Shiga toxin producing E. coli) flora between animal species (goat, sheep, and cow). This study also pointed toward the possible presence of EHEC O80 (a new emerging EHEC serogroup in Europe) in cow's raw milk cheeses, which warrants further investigations.

Genetic diversity and whole genome sequence analysis data of multidrug resistant atypical enteropathogenic Escherichia coli O177 strains: An assessment of food safety and public health implications.

Atypical enteropathogenic E. coli (aEPEC) strains are emerging pathogens responsible for fatal diarrhoea in humans worldwide. The purpose of this study was to investigate genetic diversity, virulence and antimicrobial resistance profiles of aEPEC O177 strains isolated from faeces of cattle reared in intensive and extensive production systems in South Africa. A total of 96 multidrug resistant (MDR) aEPEC O177 isolates were typed using enterobacterial repetitive intergenic consensus (ERIC) and random amplified polymorphism DNA (RAPD) typing. The resistome, virulome and mobilome of two aEPEC O177 isolates were investigated using WGS analysis. The ERIC typing was efficient and reproducible with a discriminatory index of 0.95. RAPD typing had poor reproducibility with satisfactory discriminatory power of 0.859. The dendrograms constructed based on ERIC and RAPD banding patterns produced 9 and 8 clusters, respectively, which indicate genetic variation among E. coli O177 isolates. WGS analysis revealed that CF-154-A and CF-335-B) isolates belonged to the O177 serotype with H7 and H21, respectively. Both isolates harboured several virulome genes such as intimin (eaeA), haemolysin (hlyA and hlyE), translocated iron receptor (tir), Type III secretion system (eprH, gspL and prgH), bssR and bssS. However, genes encoding shiga toxins were not found in either isolate. Antibiotic resistance genes such as ampC, tet, ermB, sul2, strB AcrD, aph(6)-Ic, aph(6)-Ib, aph(3″)-I, ant (3″)-1a AcrA and acrE were found in the E. coli O177 strains. Furthermore, genome annotation results indicated that both isolates carried plasmids, insertion sequences, prophages and cluster of regularly interspaced short palindromic repeats (CRISPR) type I. Based on in silico multi locus typing (MLST) analysis, the two isolates were assigned to different sequence types (CF-154-A, ST-1308 and CF-335-B, ST-58). Whole genome multi locus typing tree showed that our isolates clustered with E. coli O177:H21 (reference), suggesting the close genomic relatedness among the strains. Overall, these findings showed that cattle carry genetically diverse E. coli O177 strains, which harbour a repertoire of virulome, resistome and mobilome genes. This highlights a need for multidrug resistant E. coli O177 strain surveillance in cattle.

PixR, a Novel Activator of Conjugative Transfer of IncX4 Resistance Plasmids, Mitigates the Fitness Cost of mcr-1 Carriage in Escherichia coli.

The emergence of the plasmid-borne colistin resistance gene mcr-1 threatens public health. IncX4-type plasmids are one of the most epidemiologically successful vehicles for spreading mcr-1 worldwide. Since MCR-1 is known for imposing a fitness cost to its host bacterium, the successful spread of mcr-1-bearing plasmids might be linked to high conjugation frequency, which would enhance the maintenance of the plasmid in the host without antibiotic selection. However, the mechanism of IncX4 plasmid conjugation remains unclear. In this study, we used high-density transposon mutagenesis to identify factors required for IncX4 plasmid transfer. Eighteen essential transfer genes were identified, including five with annotations unrelated to conjugation. Cappable-seq, transcriptome sequencing (RNA-seq), electrophoretic mobility shift assay, and ß-galactosidase assay confirmed that a novel transcriptional regulator gene, pixR, directly regulates the transfer of IncX4 plasmids by binding the promoter of 13 essential transfer genes to increase their transcription. PixR is not active under nonmating conditions, while the expression of the pixR, pilX3-4, and pilX11 genes increased 3- to 6-fold upon contact with recipient Escherichia coli C600. Plasmid invasion and coculture competition assays revealed the essentiality of pixR for spreading and persistence of mcr-1-bearing IncX4 plasmids in bacterial populations. Effective conjugation is crucial for alleviating the fitness cost exerted by mcr-1 carriage. The existence of the IncX4-specific pixR gene increases plasmid transmissibility while promoting the invasion and persistence of mcr-1-bearing plasmids in bacterial populations, which helps explain their global prevalence. IMPORTANCE The spread of clinically relevant antibiotic resistance genes is often linked to the dissemination of epidemic plasmids. However, the underlying molecular mechanisms contributing to the successful spread of epidemic plasmids remain unclear. In this report, we shine a light on the transfer activation of IncX4 plasmids. We show how conjugation promotes the invasion and persistence of IncX4 plasmids within a bacterial population. The dissection of the regulatory network of conjugation helps explain the rapid spread of epidemic plasmids in nature. It also reveals potential targets for the development of conjugation inhibitors.

A multifactorial assessment of the SRP pathway constituent FtsY as a vital mycobacterial constituent.

The signal recognition particle (SRP) system plays an imperative role in transporting the secretory protein to its intended location. The SRP pathway running in Mycobacterium tuberculosis constitutes FtsY (signal receptor), FfH (SRP), and 4.5S RNA in which signal receptor acts in the GTP-dependent manner. In this study, we are rendering the essential facts of FtsY with respect to mycobacterial growth. The growth study experiment showed that downexpressed FtsY slowed the growth of Mycobacterium smegmatis mc2 155 from the initial lag phase to stationary phase. Previously, we have showed that GTPase activity of FtsY is metal ion dependent and showed the maximum activity with 10 mM magnesium. The effect of Mg2+ and Mn2+ on mycobacterial growth showed that Mg2+ did not affect the growth, whereas higher concentration of Mn2+ decreases the bacterial growth. After searching the inhibitor database, 14 GTPase and ATPase inhibitors, Mac0182344, ML141, ITX3, NAV_2729, Br-GTP, Rhosin_HCl, Mac0182099, CCG_50014, CID_1067700, Mac0174809, Nsc_23766, Berberine, Nexinhib20, and EHT1864, were found to interact with FtsY. Further, ML141 and NAV2729 found to decrease the enzymatic activity of FtsY as well as the mycobacterial growth. Therefore, the conclusive statement of the present study can be stated as that the FtsY plays major role in mycobacterial cell survival and ML141 and NAV2729 can be used to constrain the SRP pathway.

Multiplexed assessment of engineered bacterial constructs for intracellular ß-galactosidase expression by redox amplification on catechol-chitosan modified nanoporous gold.

Synthetic biology approaches for rewiring of bacterial constructs to express particular intracellular factors upon induction with the target analyte are emerging as sensing paradigms for applications in environmental and in vivo monitoring. To aid in the design and optimization of bacterial constructs for sensing analytes, there is a need for lysis-free intracellular detection modalities that monitor the signal level and kinetics of expressed factors within different modified bacteria in a multiplexed manner, without requiring cumbersome surface immobilization. Herein, an electrochemical detection system on nanoporous gold that is electrofabricated with a biomaterial redox capacitor is presented for quantifying ß-galactosidase expressed inside modified Escherichia coli constructs upon induction with dopamine. This nanostructure-mediated redox amplification approach on a microfluidic platform allows for multiplexed assessment of the expressed intracellular factors from different bacterial constructs suspended in distinct microchannels, with no need for cell lysis or immobilization. Since redox mediators present over the entire depth of the microchannel can interact with the electrode and with the E. coli construct in each channel, the platform exhibits high sensitivity and enables multiplexing. We envision its application in assessing synthetic biology-based approaches for comparing specificity, sensitivity, and signal response time upon induction with target analytes of interest.

Rapid, Affordable, and Uncomplicated Production of Bacterial Cell-free Lysate.

Cell-free gene expression offers the power of biology without the complications of a living organism. Although many such gene expression systems exist, most are quite expensive to buy and/or require special equipment and finely honed expertise to produce effectively. This protocol describes a method to produce bacterial cell-free lysate that supports high levels of gene expression, using only standard laboratory equipment and requiring minimal processing. The method uses an Escherichia coli strain producing an endolysin that does not affect growth, but which efficiently lyses a harvested cell pellet following a simple freeze-thaw cycle. The only further processing required is a brief incubation followed by centrifugation to clear the autolysate of cellular debris. Dynamic gene circuits can be achieved through heterologous expression of the ClpX protease in the cells before harvesting. An E. coli strain lacking the lacZ gene can be used for high-sensitivity, cell-free biosensing applications using a colorimetric or fluorescent readout. The entire protocol requires as few as 8-9 hours, with only 1-2 hours of hands-on labor from inoculation to completion. By reducing the cost and time to obtain cell-free lysate, this method should increase the affordability of cell-free gene expression for various applications.